Light and Biological Rhythms in Man

W e n n e r - G r e n In ternat iona l Series

Vol. 60 N e u r o - i m m u n o l o g y of fever ed. T. Bartfai and D. Ottoson Vol. 61 Funct ional organisat ion of t h e human visual cor tex

ed. B. Gulyas, D. Ottoson and P. E. Roland

New in 1993

Light and biological rhy thms in man ed. L Wetterberg Trophic regulat ion of the basal gangl ia ed. K. Fuxe et al.

Light and Biological Rhythms in Man

E d i t e d b y

L. WETTERBERG Karolinska Institute, Stockholm, Sweden

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Preface T H E OBJECTIVE of this b o o k is to summarize the knowledge of light as a regulator of biological rhy thms in m a n in relation to heal th and disease.

The first scientific meeting on biological rhy thms in Sweden was held in 1937 in the small spa of Ronneby in the sou th of Sweden. Abou t twenty scientists who called themselves "Rhy thms enthusias ts" gathered and established a Society for Biological R h y t h m Research. The second meeting on biological rhy thms in Sweden was held in 1955 in Stockholm, still with a small g roup of scientists.

The presentat ions published in this b o o k upda te wha t has happened dur ing the last four decades and focus in par t icular on how light is influencing biological rhy thms. In 1959 Aaron Lerner and coworkers discovered the t ime keeping substance mela tonin . In one chapter in this book Lerner recounts the history of his efforts for four years to find the melanin tonizing h o r m o n e (melatonin) which blanched frog skin, until they found the substance and it could be said "This is it!". F r o m there on many speculations abou t the media tor of biological rhy thm regulat ion have become scientifically testable hypotheses . The results based on Lerner 's work have led to solid da t a on which much of mode rn biological rhy thm research is based.

Tha t Aa ron Lerner 's historical aspects are included in this volume, as well as all the o ther presentat ions will we hope benefit younger generat ions of s tudents and scientists for w h o m the research frontiers of today have a s t rong bear ing on the clinical practice of t o m o r r o w and well beyond the year 2000.

In different ways research is confronted with the fact tha t m a n y of the fundamental r hy thm processes are no t easily observable and must many times be reconstructed from prel iminary da ta . An interesting model to explain the origin of biological rhy thms is the one of Erik Odeb lad from University of Urnea, Sweden, w h o has p roposed tha t it is based on the presence of collective vibrat ions (phonons) in loop-shaped molecules or molecular aggregates. Vibrat ional waves a long a closed loop will give rise to self-interference as the basis for rhy thms . Fu r the r experiments to measure phonons in tissues and organs are called for to test this hypothesis.

V

v i Preface

As illustrated in this book , interfacing disciplines progress steadily th rough experimentat ion, observat ion and theoretical interpretat ion, each one helping the advances of the other . The biological rhy thms covered in this book range from short (infradian) and abou t 24-hour rhy thms (circadian) to longer rhy thms (ultradian) present in all m a m m a l i a n species.

Dur ing the last decade it has become clear tha t h u m a n s also are in m a n y ways influenced by biological rhy thms. Time structures are present on several levels from popula t ion , individuals, organs (e.g. brain and hear t ) to tissues, cells and subcellular organizat ion. An in t roductory chapter identifies the genetic machinery, including the light influenced proteins , which governs the clocks tha t regulate us all.

The new findings abou t the effect of light on biological rhy thms has already lead to therapeut ic trials in different condit ions. The use of light to regulate menst rual cyclicity may have clinical value for rhy thm contracep-tive me thods and for t rea tment of infertility. The further studies of the effect of light on the menst rual cycle may lead to new views of reproduct ive endocrinology. In some chapters , specialists in light percept ion and light reception have presented da t a on light t rea tment as an ant idepressant in seasonal affective disorder. It is clear tha t clinical testing is needed to determine the opt imal intensities, the t iming and dura t ion of light t rea tment for producing beneficial effects.

In the repor t of placebo-control led studies it is s tated tha t it will require further studies to establish how light t rea tment compares with p lacebo. Such work is also impor t an t to help unders tand the mechanisms and etiology of depression and other condi t ions .

T o unders tand the rhy thm regulating effects of light it is necessary to examine the ocular mechanisms which mediate the phot ic effect t h rough the eye. When light reaches the ret ina, there are individual differences in the sensitivity of the photoreceptivi ty and the ability of the mela tonin rhy thm generat ing system to integrate phot ic stimuli, temporal ly .

The overall a im in the present volume is to provide a basis, b o t h for scientific as well as clinical perspectives to establish the specific modes and measures which mediate therapeut ic and physiologically beneficial effects of light as a regulator of biological rhy thms .

I a m truly indebted to all of the cont r ibutors to this volume and very grateful for the suppor t and help of David Ot to son and J o h a n Beck-Friis. The generous financial cont r ibut ion of the Wenner -Gren Center F o u n d a -tion and the Swedish Medical Research Council is acknowledged.

Stockholm, 1993 L E N N A R T W E T T E R B E R G

1

Biological Rhythms: From Gene Expression to Behavior*

J O S E P H S. T A K A H A S H I

NSF Center for Biological Timing, Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208-3520, USA

Abst rac t

Circad ian r h y t h m s regula te the function of living sys tems at vir tual ly every level of o rgan iza t ion . In the last decade , o u r u n d e r s t a n d i n g of the cellular a n d molecu la r processes involved in the genera t ion a n d regu la t ion of c i rcad ian r h y t h m s has a d v a n c e d cons iderab ly . N e w m o d e l sys tems for s tudy ing c i rcad ian osci l la tors have been deve loped , a po ten t i a l r egu la to ry role for cellular immedia te -ear ly genes in c i rcad ian behav io r has been d iscovered , a n d critical pe r iods of m a c r o m o l e c u l a r synthesis for p rogress ion of the c i rcad ian clock t h r o u g h its cycle have been defined. These findings a re of pa r t i cu la r interest because i ndependen t a p p r o a c h e s suggest tha t an i m p o r t a n t role for m a c r o m o l e c u l a r synthesis exists a t all levels of the c i rcad ian sys tem.

In t roduct ion

Circadian rhy thms regulate the behavior , physiology and biochemistry of most living systems: from cyanobacter ium to m a n .

3 , 1 5 , 3 8'

50 A m o n g

animals , much is k n o w n abou t the physiology of circadian rhy thms , and in all cases, circadian control is exerted by structures that contain circadian pacemakers within the central nervous s y s t e m .

4'

2 4 , 2 9'

5 1'

55 O n the basis of

a wide variety of evidence, the mechanism of the circadian clock appears to be cell a u t o n o m o u s and to involve periodic gene e x p r e s s i o n .

5 1'

5 2'

54 In

addi t ion, signal t ransduct ion pa thways into the clock mechanism are present for conveying environmental information for en t ra inment of the clock. Fu r the rmore , the clock mechanism has diverse ou tpu t pa thways for exerting circadian control at all levels of organismal biology. A number of generalizations can be m a d e abou t circadian biology. Circadian rhy thms are a proper ty of all eukaryot ic and some prokaryot ic organisms and are entrained primari ly by environmenta l cycles of light and tempera ture . Circadian rhy thms are genetically determined and single-gene clock

* This paper is dedicated to Dr Aaron B. Lerner for his seminal work on melatonin.

3

4 Light and Biological Rhythms in Man

mutan t s have been found in six organisms including mammal s . Circadian oscillations are remarkably precise and can have a variat ion in cycle length of less than one par t in a thousand . The period length of the oscillation is tempera ture-compensated and usually varies less than 2 0 % for each 10°C change in tempera ture (Q1Q = 0.8-1.2). Finally, the biochemical ma-chinery responsible for generat ing circadian rhy thms can be expressed at the level of individual cells. Thus , circadian rhythmicity is a fundamental organizing feature of virtually all organisms. The propert ies of these rhy thms are unique and widely conserved a m o n g living systems.

This chapter will be organized in two par ts . First , a brief review of vertebrate circadian organizat ion will be presented. Second, a summary of our own work on phot ic en t ra inment and regulat ion of immediate early genes in m a m m a l s will follow.

Components of c i rcadian systems

All circadian systems conta in at least three elements: (1) an input pa thway or set of input pa thways tha t convey environmenta l information to the circadian pacemaker for en t ra inment ; (2) a circadian pacemaker that generates the oscillation; and (3) an ou tpu t pa thway or set of ou tpu t pa thways by which the pacemaker regulates its various ou tpu t r h y t h m s .

58

In all systems, a phot ic en t ra inment pa thway is present as an input . It is already clear from the diversity of photop igments and pho to t ransduc t ion pa thways that phot ic en t ra inment pa thways differ markedly in different o r g a n i s m s .

38 At the receptor level, the diversity spans the spectrum from

phy tochrome in plants to members of the rhodops in family in animals . There appears to be comparab le diversity at the second messenger level in phot ic signal t ransduct ion pa thways . At the ou tpu t level, diversity is even more extreme. Again, this type of regulat ion spans the clock control of photosynthesis in plants to endocrine and behavioral rhy thms in animals . As I have argued p rev ious ly ,

51 the input and ou tpu t pa thways of the

circadian clock within each organism appear to be specific to each system. Despite these differences in the coupling pa thways (inputs and outputs ) of the circadian clock, the core mechanism of the pacemaker appears to be fundamentally similar in all organisms. Whether these similarities in pacemaker mechanisms will ultimately be found to be functionally ana logous or phylogenetically homologous remains to be seen.

Physiological organizat ion of ve r tebra te c i rcadian systems

Circadian pacemakers , which control the behavior and physiology of animals , have been localized as discrete structures within the nervous s y s t e m .

4 5 , 4 8'

55 At the physiological level, bo th "oscillator" and "pace-

make r " function have been defined. A "circadian oscillator" is a s t ructure

Biological Rhythms: From Gene Expression to Behavior 5

that expresses a self-sustained oscillation under constant condit ions (the absolute min imum number of cycles is two). Circadian oscillators have been localized by isolating the tissue in quest ion in vitro and then demonst ra t ing the persistence of circadian oscillations in the isolated tissue under cons tant condi t ions . In vertebrates, three diencephalic structures have been shown to conta in circadian oscillators, the pineal gland of birds, reptiles and f i sh ,

55 the hypotha lamic suprachiasmat ic

nucleus (SCN) of m a m m a l s ,

2 4'

29 and the retinas of amphib ians and

b i r d s .

4'

37 A "circadian pacemaker" is a circadian oscillator that has been

shown to drive and therefore control some overt rhythmic process such as locomotor behavior . Pacemaker function has been experimentally demon-strated by t ransplant ing the s t ructure in quest ion and then showing that the phase or period of the recipient rhy thm is regulated by the t ransplant . This result has been demons t ra ted in vertebrates for the pineal gland of sparrows and the S C N of hamsters , and in invertebrates for the optic lobes of cockroaches and the brain of Drosophila.

A2>A5

Although an ocular-SCN-pineal axis underlies vertebrate circadian organizat ion, it is difficult to make simple generalizations at the physiological level. The clearest division is a mammal i an versus non-mammal i an d ichotomy. In mammal s , the organizat ion is the simplest. The dominan t circadian pacemaker is located in the S C N , the photoreceptors for ent ra inment are exclusively retinal, and ou tpu t pa thways such as the pineal rhy thm of mela tonin are well de f ined .

24 In non -mammal i an

vertebrates, the organizat ion is more c o m p l e x ;

3 5'

45 and , multiple

oscillators and photoreceptors are involved. The pineal gland, the S C N and the eyes can each play a dominan t role in circadian behavior of birds depending on the species. F o r example, in passerine birds, such as the house spar row, the pineal gland plays a dominan t pacemaker role; whereas, in gallinaceous birds, such as chickens and quail , the pineal gland is not essential. The avian S C N appears to be necessary in all species examined, but its role as a pacemaker has not been determined. Finally, in Japanese quail , the eyes play a dominan t role in circadian o r g a n i z a t i o n .

60

In addi t ion to the multiplicity of oscillatory centers, multiple photorecep-tors for en t ra inment are located in the retina, the pineal gland and the b r a i n .

61 In summary , non -mammal i an vertebrates appear to have

distr ibuted circadian oscillators with local photorecept ive input ; whereas , mammal s appear to have retained only a subset of these componen ts .

The m a m m a l i a n suprachiasmat ic nucleus

A wealth of experimental evidence strongly argues that the hypotha lamic S C N is the site of a circadian pacemaker tha t drives overt rhy thms in m a m m a l s .

2 4'

2 9'

4 5'

48 Six lines of evidence suppor t this conclusion. First ,

the S C N receives direct input from the ret ina th rough a specialized visual


pathway, the re t inohypothalamic tract , which is required for ent ra inment to light cycles. Second, a wide variety of circadian rhythms in rodents are disrupted by S C N lesions, ruling out the possibility of a highly specific, limited effect of lesions on a part icular subsystem, for instance, locomotor behavior . Third , the S C N expresses circadian rhythms of mult i-unit electrical activity that persist after neural isolation in vivo. F o u r th , S C N expiants cont inue to express circadian rhythms of single-unit electrical activity, vasopressin release and metabol ic activity in vitro. Fifth, circadian rhythmicity can be restored to a r rhythmic SCN-lesioned animals by t ransplanta t ion of fetal tissue containing S C N cells. Sixth, t ransplanta t ion of S C N tissue derived from Tau m u t a n t hamsters , which express short period rhy thms , demonst ra tes tha t the genotype of the dono r S C N determines the period of the restored rhy thm. Taken together, these results demons t ra te tha t the S C N plays a dominan t role in the generat ion of circadian rhy thms in m a m m a l s .

Funct ional propert ies of phot ic e n t r a i n m e n t in m a m m a l s

Phot ic en t ra inment in m a m m a l s is mediated by retinal photoreceptors that project to the S C N via the re t inohypothalamic t r a c t .

29 We have used

light-induced phase shifts of the circadian rhy thm of wheel-running activity to measure the phot ic sensitivity of the circadian system of the golden hamster . Previously we showed that the spectral sensitivity function for phase-shifting matches an opsin-based pho top igment with a peak ( A m a x) a round 500 n m .

53 Al though the peak sensitivity is similar to

that of rhodops in , two features of this photorecept ive system are unusual : the threshold of the response is high, especially for a rod-domina ted retina like that of the hamster , and the reciprocal relat ionship between intensity and dura t ion holds for extremely long d u r a t i o n s .

33 Figure 1 shows the

phase-shifting response to 300 sec light pulses at circadian time (CT) 19. The phase-shifting response increases with light intensity and can be fit with a four parameter logistic function. The sensitivity to stimulus duration was assessed by measur ing the magni tude of phase-shift responses to phot ic stimuli of different i rradiance and dura t ion (Figure 2). The hamster circadian system is more sensitive to the i rradiance of longer dura t ion stimuli than to that of briefer stimuli. The system is maximally sensitive to the i rradiance of stimuli of 300 sec and longer in dura t ion (Figure 3A). As shown previously the threshold for phot ic s t imulat ion of the hamster circadian pacemaker is high. The threshold irradiance (the m a x i m u m irradiance necessary to induce statistically significant responses) is approximately 1 0

11 pho tons c m

-2 s e c "

1 for opt imal st imulus dura t ions .

This threshold is equivalent to a luminance at the cornea of abou t 0.1 c d - m ~

2. We also measured the sensitivity of this visual pa thway to the


y/-

' — — • — • — • — . — • — • — · — • — - J Dark 8 9 10 11 12 13 14 15 16 17

I r r a d i a n c e ( l o g p h o t o n s · c m ~ ~ 2 . s— 1 )

F I G . 1. M a g n i t u d e of phase shift of h a m s t e r act ivi ty r h y t h m in response to 300 sec 503 n m light pulses of different i r rad iance at c i rcad ian t ime 19 (CT19) . T h e po in t s represent the m e a n ± S E M . T h e c o n t i n u o u s line is a modified N a k a - R u s h t o n function fitted to the d a t a . ( F r o m Ne l son a n d T a k a h a s h i ,

33 copyr igh t by J.

Physiol. (Lond.).)

Dark 9 10 11 12 13 14 15

L o g ( i r r a d i a n c e )

F I G . 2. M a g n i t u d e of p h a s e shift of h a m s t e r activity r h y t h m in response to 503 n m light pulses of vary ing d u r a t i o n s a n d i r rad iances a t C T 1 9 . O p e n circles a re 3,600 sec; closed circles a re 300 sec; open t r iangles are 30 sec; a n d closed t r iangles a re 3 sec s t imuli . C u r v e s a re best fit modif ied N a k a - R u s h t o n funct ions . ( F r o m N e l s o n

a n d T a k a h a s h i ,

33 copyr igh t by J. Physiol. (Lond.).)

total number of pho tons in a st imulus (Figure 3B). Surprisingly, the system is maximally sensitive to phot ic stimuli between 30 and 3,600 sec in dura t ion . The m a x i m u m q u a n t u m efficiency of phot ic integrat ion occurs in 300 sec stimuli.

To summarize , the phot ic threshold for en t ra inment , even under opt imal condi t ions , is relatively insensitive and corresponds to thresholds reported for cone photoreceptors . In addi t ion, the system can integrate light over very long dura t ions . F r o m a functional perspective, these characteristics are clearly adapt ive. The threshold is jus t above the level of full moonl ight and thus en t ra inment would not be disrupted by this inappropr ia te light source. Fu r the rmore , the op t imum for long dura t ion pulses would also render the system insensitive to very intense but brief


0 1 2 3 4 Stimulus duration (log s)

F I G . 3. Sensitivity curves to i r r ad iance a n d to ta l n u m b e r of p h o t o n s in a pulse m e a s u r e d a t C T 1 9 . A, the relat ive sensitivity to i r r ad iance is p lo t t ed as a funct ion of s t imulus d u r a t i o n . B, the relat ive sensitivity to to ta l p h o t o n s as a function of s t imulus d u r a t i o n . ( F r o m N e l s o n a n d T a k a h a s h i ,

33 copyr igh t by J. Physiol.

(Lond.).)

sources of light such as lightening. Thus , bo th the threshold and integrat ion characteristics of the phot ic ent ra inment pa thway in hamsters make this system optimally tuned to respond to the daily l igh t -dark cycle, while at the same time remaining unresponsive to environmental "noise" that would interfere with stable ent ra inment . The na ture of the photoreceptors mediat ing phot ic ent ra inment remain to be established; however, it is already clear tha t specialized retinal elements must exist. F o r example, experiments using the rd mu ta t ion in mice strongly argue that rod photoreceptors are not essential for the phase-shifting r e s p o n s e .

10 In

addi t ion, recent experiments from our l abora tory show that the spectral sensitivity for phase-shifting in another species, the Djungar ian hamster , has a peak a r o u n d 475 n m which is clearly different from that of r h o d o p s i n .

62 Taken together these results suggest that the "circadian

photorecep tor" is no t a rod and is either a cone or some other unidentified photoreceptor class in the retina.

Role of macromolecu lar synthesis in m a m m a l i a n circadian rhythms

It has been known for a number of years that inhibitors of protein synthesis on 80S r ibosomes produce changes in the period length or phase of


circadian rhy thms of microorganisms and invertebrates, including Eug-lena, Acetabularia, Gonyaulax, Neurospora and Aplysia.

11,18,22,32,46,58,63

M o r e recently, we have demons t ra ted tha t the acute adminis t ra t ion of two protein synthesis inhibitors (anisomycin and cycloheximide), with two different mechanisms of act ion, are capable of inducing p ronounced phase shifts in the circadian clock of hamsters , and that the site of act ion of these inhibitors appears to be on cells within the S C N r e g i o n .

1 6'

5 6'

64

Interestingly, the phase response curves for anisomycin and cycloheximide in hamsters are similar to those measured for protein synthesis inhibitors in microorganisms and invertebrates, suggesting tha t the biochemical mechanisms generat ing circadian oscillations in m a m m a l s may share c o m m o n features with those found in very distantly related phylogenetic groups .

In addi t ion to causing phase shifts by themselves, protein synthesis inhibitors have also been shown to block the phase-shifting effects of light pulses in Aplysia and Neurospora.

20A2 We have also found tha t

l ight-induced phase shifts can be blocked in hamsters suggesting tha t the signal t ransduct ion pa thways for phot ic en t ra inment may involve macromolecular synthesis (Takahashi et al, unpubl ished results). Indeed, as described below, light does induce the expression of a set of genes in the SCN.

Al though a requirement for new R N A synthesis has been inferred from genetic e x p e r i m e n t s ,

6'

1 2'

44 this issue has only recently been addressed with

the use of the reversible R N A synthesis inhibitor , 5,6-dichloro-l-/?-D-ribofuranosylbenzimidazole (DRB), in the Aplysia e y e .

41 Pulses of D R B

caused phase-dependent delays at phases between circadian t ime (CT) 20 and 10, and had no effects from CT10 to CT20. Con t inuous t rea tment with D R B caused a dose-dependent lengthening of the circadian period. Similar effects of D R B on phase and period have been found in chick pineal c e l l s .

36

The D R B experiments suggest that a critical period for t ranscr ipt ion of specific genes involved in the generat ion of circadian rhy thms occurs from CT20 to CT10 in Aplysia, and from CT18 to CT19 in chick pineal cells.

While the inhibi tor pulse experiments suggest that a critical period for macromolecular synthesis exists within the circadian cycle, these experi-ments do not provide evidence that such t rea tments can arrest or s top the mot ion of the circadian pacemaker . Kha l sa et al

23 have addressed this

issue by applying very long (5-44 hours) inhibi tor pulses of cycloheximide to Bulla eyes in vitro. Shorter pulses that do not extend past C T O have minimal effects on the rhy thm. However , longer pulses tha t extend beyond C T O delay the phase of the subsequent rhy thm precisely by the dura t ion that the inhibi tor pulse extends past C T O . The results are consistent with the interpretat ion that the mot ion of the circadian pacemaker is arrested dur ing these long inhibi tor pulses. By extrapola t ing the phases of the rhythms "backwards" towards the end of the inhibi tor pulses, it appears

1 0 Light and Biological Rhythms in Man

that the critical period for protein synthesis in Bulla begins in the late subjective night (near C T O ) . By analogy to work in the cell cycle, the " S T A R T " of the circadian cycle therefore appears to be near C T O . Thus , bo th the inhibi tor phase shift and the circadian cycle progression experiments are concordan t and suggest that the circadian clock requires macromolecular synthesis dur ing a critical period for progression th rough the cycle. In addi t ion to inhibitor experiments, there is now direct evidence tha t the Drosophila per iod gene products express circadian oscillations which appear critical for the expression of behavioral r h y t h m s .

1 3 , 14

Phot ic regulat ion of immedia te -ear ly genes in the S C N

A number of groups have repor ted that the products of the p ro to -oncogene, c-fos, are s t imulated by light in the S C N of r o d e n t s .

25 In the

hamster there is a profound induct ion of Fos i m m u n o r e a c t i v i t y

47 and c-fos

m R N A

26 in the S C N following light exposure dur ing the "subjective"

night. To begin to address whether Fos mediates t ranscript ional events involved in phase shifting of the circadian pacemaker , we have further characterized the phot ic induct ion of c-fos m R N A utilizing in situ hybridizat ion techniques. By using defined light stimuli for which the effects on the hamster ' s circadian rhy thm of activity have been measured, we have compared the phot ic induct ion of c-fos m R N A and the behavioral phase shifts p roduced by l i g h t

2 6. Specifically, we examined whether the

phot ic induct ion of c-fos was quanti tat ively correlated with phase shifting of locomotor activity. Because Fos must act in concert with a member of the Jun family of proto-oncogenes to form the t ranscript ion factor, A P - 1

3 0, we have also examined whether Jun and AP-1 are induced by

l i g h t .

27

Photic induction of c-fos and j u n - B mRNA in the SCN

Light exposure of hamsters dur ing the night, at CT19, causes a d ramat ic induct ion of c-fos m R N A levels in the S C N (Figure 4). The increase in c-fos m R N A levels illustrated here was induced by a 5 minute light pulse, after which the animal was re turned to darkness for 25 minutes before sacrifice. The dark-field pho tomic rograph shows that hybridizat ion to complemen-tary R N A probes for c-fos is localized mainly in the ventrolateral por t ion of each SCN, and extends dorsally from the S C N toward the periventricu-lar region. Similar results are seen with jun-B m R N A leve l s .

27 In contras t

to the dramat ic enhancement of jun-B m R N A expression, light exposure at CT19 causes only a modest increase of c-jun m R N A hybridizat ion in the S C N . N o other effect of light on c-fos or jun-B m R N A was observed in

Biological Rhythms: From Gene Expression to Behavior 1 1

Light

*

* « · - , ·

V., ^ . ' ί Λ

..." * . * ' .·

200 μΜ 50 μΜ

F I G . 4. /rc siiw hybr id iza t ion of c-fos m R N A in the S C N of h a m s t e r s 30 m i n u t e s after the onset of a 300 sec light pulse a t C T 1 9 . T o p pane l s show the S C N region from a d a r k con t ro l an ima l . Left side is a d a r k field p h o t o m i c r o g r a p h of emuls ion a u t o r a d i o g r a p h y . Right side is a h igher magni f ica t ion br igh t field p h o t o m i c r o -g r a p h . Lower pane ls show the S C N region from a h a m s t e r receiving light a t C T 1 9 .

( F r o m K o r n h a u s e r et al.,26 copyr igh t by Cell Press.)

other areas of the brain , including retinorecipient regions such as the intergeniculate leaflet ( IGL) (al though we have detected a modes t phot ic induct ion in the I G L with Fos immunocytochemis t ry) . The elevation of c-fos m R N A levels following light s t imulat ion is rapid and transient , with maximal levels occurr ing abou t 30 minutes after the onset of l i g h t . 26 Similar to c-fos m R N A , the highest levels of jun-B m R N A are detected 30 minutes after the onset of the light pulse, and have re turned nearly to basal levels by 120 m i n u t e s . 27 Clearly, this tempora l correlat ion of the expression of c-fos and of jun-B, suggests tha t Fos and jun-B proteins are available to dimerize with one another to form the t ranscr ipt ion factor, AP-1 (see below).

Photic threshold of c-fos mRNA induction

As described above, the response of the hamster circadian system to varying levels of i l lumination has been characterized. The magni tude of


the phase shift in locomotor activity exhibits a monoton ie , saturable dependence on the irradiance of the light st imulus. Figure 5 illustrates the a m o u n t of behavioral phase shift p roduced by 5 minute light pulses (503 nm wavelength) of varying irradiance occurring at C T 1 9 . 26 T o compare the sensitivity to light of the c-fos m R N A induct ion and of this phot ic response of the circadian system, we performed in situ hybridizat ion following 5 minute light stimuli of different i l lumination levels, and quantified the a m o u n t of specific c-fos hybridizat ion within the S C N . The threshold for induct ion of c-fos is indistinguishable from the threshold for light-induced phase shifts, consistent with the idea c-fos is involved in the ent ra inment mechanism of the p a c e m a k e r . 26

2

0 12 24

T i m e ( h )

F I G . 5 . D e p e n d e n c e of c-fos m R N A induc t ion and phase-shif t ing of the l o c o m o t o r activity r h y t h m on light i r rad iance . Light pulses of increas ing i r rad iance were given at C T 1 9 . Left side shows in situ hybr id iza t ion of c-fos m R N A in the S C N region. Pane l 1 shows n o light (dark con t ro l ) ; panels 2 - 5 show increas ing light i r rad iance . Right side shows the l o c o m o t o r activity r h y t h m s of hams te r s exposed to light pulses at C T 1 9 at the same i r rad iance . ( F r o m K o r n h a u s e r et al.,26

copyr igh t by Cell Press.)


Circadian gating o f c-fos and j u n - Β induction by light

Light exposure dur ing the subjective night of the hamster ' s circadian cycle causes a phase shift in the activity rhy thm of the animal . Light exposure dur ing the subjective day, by contras t , produces no effect on the phase of the rhy thm of locomotor activity (Figure 6A). The phot ic induct ion of c-fos gene expression displays a similar phase-dependence: the ability of

3 9 14 19 21 Circadian t ime (h )

F I G . 6. C i r cad i an phase -dependence of l ight - induced behav io ra l p h a s e shifts (A), c-fos m R N A induc t ion in the S C N (B), a n d jun-Β m R N A induc t ion in the S C N of h a m s t e r s . P h o t i c i nduc t ion of c-fos a n d jun-Β were m e a s u r e d at five phases of the cycle. P h o t i c i n d u c t i o n was seen only d u r i n g the subject ive n igh t a n d was cor re la ted with l ight - induced p h a s e shifts of the act ivi ty r h y t h m . ( F r o m

K o r n h a u s e r et al.,

21 copyr igh t by Science.)

light to st imulate c-fos is restricted to the same times of the circadian cycle when light causes a behavioral phase shift (Figure 6B). In situ hybridiza-t ion studies demons t ra te tha t the induct ion of jun-B m R N A following a 5 minute light pulse are likewise dependent on circadian phase (Figure 6C). Light pulses at CT14, which produce a phase-delay in the hamster ' s activity rhy thm, dramatical ly elevate jun-B m R N A levels; light a t CT19 and at CT21 causes a phase-advance and also induces jun-B m R N A . Dur ing the subjective day, at C T 3 and C T 9 , light neither produces phase shifts nor induces the expression ofjun-B. The expression of c-fos and jun-B m R N A , then, are bo th gated by the circadian pacemaker , and are bo th induced under the same tempora l c o n d i t i o n s .

27


Photic regulation of AP-1 DNA binding activity in the SCN

The co-induction of jun-B and c-fos m R N A by light suggests that levels of the heterodimeric AP-1 complex in the S C N are increased by light s t imulat ion. T o determine whether light indeed alters amoun t s of AP-1 factor capable of specifically binding to a consensus D N A recognit ion site, we performed DNA-b ind ing gel mobili ty shift a s s a y s .

27 Whole-cell

extracts were prepared from microdissections of hamster brain tissue containing the SCN. These S C N extracts were incubated with a 3 2

P - l a b e l e d oligonucleotide containing consensus AP-1 sites and then electrophoresed. A protein complex of re tarded mobili ty was detected using extract prepared from hamsters 60 or 120 minutes after the onset of a 5 minute light pulse, presented at CT19 S C N extracts from hamsters receiving the same handl ing, but no light pulse, contained low levels of AP-1 binding activity measured by this in vitro assay. The time course of AP-1 induct ion was elevated at 1 hou r and peaked at 2 hours after light s t imulat ion. As in the case of c-fos and jun-B mRNA, the induct ion of AP-1 by light was also phase-dependent . Light induced AP-1 at CT14 and CT19, but did not induce AP-1 at CT6 . Thus , the circadian clock also gates the phot ic induct ion of AP-1 activity. These results demons t ra te that light increases levels of AP-1 protein complex implying that phot ic s t imulat ion is coupled to AP-1 regulated changes in gene t ranscript ion in the SCN. Taken together, these experiments suggest tha t phot ic en t ra inment in mammals may involve transcript ionally regulated signal t ransduct ion processes, and that AP-1 may play a role in conveying phot ic information within the circadian system.

Light induces phosphory la t ion of the t ranscr ip t ion fac tor , CREB, in the S C N

In cell culture systems, the signaling pa thways tha t regulate the expression of c-fos have been extensively s t u d i e d .

49 In PC12 cells, the c A M P response

element binding protein, C R E B , mediates the t ranscript ional activation of c-fos in response to stimuli tha t elevate the second messenger c A M P or C a

2+ signaling pa thways . These pa thways converge at the level of C R E B

phosphoryla t ion on S e r

1 33 and this phosphoryla t ion event is necessary for

the t ranscript ional activation of CREB-regula ted target g e n e s .

49 Because

C a

2 + is a likely second messenger in neurot ransmit te r regulated pa thways

such as the re t inohypothalamic input to the S C N , we asked whether C R E B may play a role in the phot ic regulat ion of c-fos. In a very fruitful col laborat ion with David Ginty , Michael Greenberg and co l l eagues ,

11 we

have examined the phosphoryla t ion of C R E B using a novel an t ibody that specifically recognizes the S e r

1 33 site of C R E B . Light exposure of hamsters


at CT19 caused the rapid phosphory la t ion of C R E B in the S C N wi thout any detectable changes in the a m o u n t of " total C R E B " . D N A mobili ty shift assays for C R E B using the C a

2 +/ c A M P response element (Ca-CRE,

sequence T G A C G T T T ) of the c-fos p romote r as a p robe , suggests tha t the major C a - C R E binding factor in the S C N is C R E B rather than ano ther member of the C R E B family such as ATF-1 or C R E M .

11

T o determine whether the l ight-induced phosphory la t ion of C R E B is phase dependent , we examined hamsters dur ing the subjective dayt ime at C T 6 .

11 At this t ime, light fails to cause bo th behavioral phase shifts and

immediate-early gene i n d u c t i o n .

27 Light also failed to induce C R E B

phosphory la t ion in the S C N at CT6 . The absence of C R E B phosphory la-tion was not due to a decrease in C R E B because no changes in the level of total C R E B could be detected in the S C N at different phases of the circadian cycle. Thus , l ight-induced C R E B phosphory la t ion and c-fos induct ion are bo th gated by the circadian clock. These experiments strongly suggest tha t the circadian gating mechanism regulating immedi-ate-early genes acts ups t ream of the phosphory la t ion of C R E B . It will be of great interest to examine kinases responsibility for C R E B phosphory la -t ion, as well as o ther regulatory sites in the c-fos p romote r such as the serum response element and the factors tha t interact with it.

Unresolved issues concern ing Fos/AP-1

Thus far the relat ionship between behavioral phase shifting and Fos /AP-1 induct ion is correlative. Are these two responses causally related, with Fos /AP-1 acting as a step in the phot ic en t ra inment pa thway? Or , alternatively, are these two responses coincidental? At the present t ime it has no t been possible to obta in a definitive answer to this issue. The current state is as follows. The phot ic induct ion of Fos is anatomical ly specific and restricted to the S C N and the I G L . The phot ic induct ion of Fos in the S C N is tightly correlated with the behavioral phase-shifting effects of light under a number of condi t ions . These include phase dependence and phot ic threshold. The Fos response is modal i ty specific and is associated with photically induced but no t non-photical ly induced phase s h i f t s .

1 9'

28 This indicates that Fos is related to the phot ic input

ra ther than to a phase-shifting mechanism per se. The requirement for F o s induct ion has tested by pharmacologica l b lockade by N M D A a n t a g o n i s t s ,

1'

8 , 61 by n o n - N M D A a n t a g o n i s t s

2 ,5 and by m e c a m y l a m i n e .

67

In all three cases, agents tha t blocked behavioral phase shifting to light also a t tenuated F o s induct ion. These experiments are all consistent with the hypothesis tha t Fos is a componen t of the phot ic en t ra inment pa thway in rodents . However , experiments tha t selectively remove Fos by other means such as antisense oligonucleotides or gene knock o u t

21 have no t yet

been completed. Clearly, experiments tha t directly and specifically test the


Conclusions

Although we are far from a concrete unders tanding of the clock mechanism in mammal s , substant ial progress on a number of fronts using diverse approaches suggests the following five generalizations.

7. Vertebrate circadian systems

There is a substantial diversity in the organizat ion of circadian systems at the physiological level, but only a few discrete loci within the nervous systems of animals act as circadian pacemakers . The mammal i an SCN, the avian pineal gland and the amphib ian ret ina define three central elements of the circadian system in vertebrates in which oscillator and pacemaker functions have been d e m o n s t r a t e d .

4 , 2 4'

2 9 , 4 5'

5 5'

59

2. A cellular circadian clock

In spite of the diversity at the physiological level, the circadian clock appears to be a cellular entity even in multicellular organisms. The strongest case for this is seen in chick pineal cells in which a circadian oscillator system with a phot ic en t ra inment pa thway can be studied in cell c u l t u r e .

55 Photorecept ion , clock function and melatonin biosynthesis all

appear to be cellular propert ies of a single cell type, the pinealocyte. Recently the S C N and the retina have also been shown to be capable of expressing circadian rhy thms in cell c u l t u r e .

3 1'

37

3. Coupling pathways

The coupling pa thways (inputs and outputs ) of the circadian clock within the organism are specific to each system, and these specific differences in coupling can explain m a n y apparen t differences in "clock mechanism" reported in various systems. A m o n g vertebrates, the differences between the coupling pa thways in the mammal i an S C N and the chick pineal gland are self-evident. En t ra inment pa thways of S C N cells are neurally c o u p l e d ,

29 whereas those in chick pineal cells are n o t .

55 These differences

in input pa thways can explain, for example, why neurally coupled circadian oscillators such as the S C N are phase-shifted by neurot ransmi t -ters and second m e s s e n g e r s ,

3 9 , 40 while non-neural ly coupled circadian

oscillators such as the chick pineal gland are n o t .

3 4'

65

requirement for Fos /AP-1 in phot ic en t ra inment are impor tan t goals for future work .


4. A unified clock mechanism?

The "core mechanism" of the circadian clock, when str ipped of its inputs and ou tpu ts , will be fundamentally the same in all organisms. This assertion is based more on faith than on fact, but as in the case of the cell cycle, I believe a unified mechanism is likely. At the molecular genetic level, this a rgument is still t enuous because only two clock genes in Drosophila and Neurospora have been well c h a r a c t e r i z e d .

6'

7 , 1 2'

44 Interspecific

homologies in clock genes have been found, h o w e v e r .

6'

12 In addi t ion, an

a rgument can be m a d e at the pharmacological level because inhibitors of macromolecular synthesis have strikingly similar effects in all organisms s t u d i e d .

54

5. Gene expression

The mechanism of the circadian clock requires gene expression on an ongoing (daily) basis for the expression of periodic clock proteins tha t are essential for the generat ion of circadian oscillations. There is now direct evidence for this idea in Drosophila with the per g e n e .

9'

1 3'

1 4'

66 The

convergence upon this theme from independent approaches in Drosophila, molluscan eyes and vertebrates suggests that this idea has some m e r i t .

52

Clearly, the isolation and molecular genetic analysis of circadian clock genes in vertebrate systems are critical goals for the future.

A c k n o w l e d g m e n t s

I thank Drs . Lennar t Wetterberg, David O t to son and J o h a n Beck-Friis for organizing an outs tanding symposium. I also thank Dr . Wet terberg for his patience in the prepara t ion of this manuscr ip t . Research was suppor ted by grants from the N I M H , N E I and N S F to J.S.T.

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40. P rosse r R.A., Mil ler J . D . a n d Hel ler H . C . (1990) A se ro ton in agonis t phase-shifts the c i rcad ian clock in the s u p r a c h i a s m a t i c nuclei in vitro. Brain Res. 5 3 4 , 336 -339 .

4 1 . Raju U . , K o u m e n i s C , N u n e z - R e g u e i r o M . a n d Esk in A. (1991 ) Al te ra t ion of the p h a s e a n d pe r iod of a c i rcad ian osci l la tor by a reversible t r ansc r ip t ion inh ib i to r . Science 2 5 3 , 6 7 3 - 6 7 5 .

42. Raju U . , Y e u n g S.J. a n d Esk in A. (1990) Invo lvemen t of p ro t e ins in light reset t ing ocular c i rcad ian osci l la tors of Aplysia. Am. J. Physiol. 2 5 8 , R 2 5 6 - R 2 6 2 .

43 . R a l p h M.R . , F o s t e r R .G . , Dav i s F . C . a n d M e n a k e r M . (1990) T r a n s p l a n t e d sup rach i a sma t i c nucleus de te rmines c i rcad ian pe r iod . Science 2 4 7 , 9 7 5 - 9 7 8 .

44. R o s b a s h M . a n d Hal l J .C . (1989) T h e molecu la r b io logy of c i rcad ian r h y t h m s . Neuron 3 , 387-398 .

45 . Rosenwasse r A . M . (1988) Behav iora l neu rob io logy of c i rcad ian p a c e m a k e r s : A c o m p a r a t i v e perspect ive . Prog. Psychobiol. Physiol. Psychol. 13 , 155-226 .

46. R o t h m a n B.S. a n d S t rumwasse r F . (1979) P h a s e shifting the c i rcad ian r h y t h m of neura l activity in the isolated Aplysia eye wi th p u r o m y c i n a n d cyc loheximide : E lec t rophys io lo -gical a n d b iochemica l s tudies . J. Gen. Physiol. 6 8 , 359 -384 .

47 . R u s a k B. , R o b e r t s o n H.A. , Wisden W . a n d H u n t S.P. (1990) Light pulses t ha t shift r h y t h m s induce gene express ion in the s u p r a c h i a s m a t i c nuc leus . Science 2 4 8 , 1237-1240.

48 . R u s a k B. a n d Z u c k e r I. (1979) N e u r a l regu la t ion of c i rcadian r h y t h m s . Physiol. Rev. 5 9 , 4 4 9 - 5 2 6 .

49. Sheng M . a n d G r e e n b e r g M . E . (1990) T h e regula t ion a n d function of c-fos a n d o the r i m m e d i a t e early genes in the ne rvous sys tem. Neuron 4 , 4 7 7 - 4 8 5 .

50. Sweeney B . M . a n d Borgese M . B . (1989) A c i rcad ian r h y t h m in cell divis ion in a p r o k a r y o t e , the c y a n o b a c t e r i u m Synechocccus W H 7 8 0 3 . J. Phycol. 2 5 , 183-186.

5 1 . T a k a h a s h i J .S . (1991) C i r cad i an r h y t h m s : F r o m gene express ion to behav io r . Cur. Opin. Neurobiol. 1 , 5 5 6 - 5 6 1 .

52. T a k a h a s h i J .S . (1993) C i r cad i an clock regula t ion of gene express ion . Cur. Opin. Genet. Devel. 3 , 301 -309 .

53 . T a k a h a s h i J .S . , D e C o u r s e y P.J . , B a u m a n L. a n d M e n a k e r M . (1984) Spect ra l


sensitivity of a novel pho to recep t ive sys tem med ia t i ng e n t r a i n m e n t of m a m m a l i a n c i rcadian r h y t h m s . Nature 3 0 8 , 186-188 .

54. T a k a h a s h i J .S. , K o r n h a u s e r J . M . , K o u m e n i s C . a n d Eskin A. (1993) Molecu l a r a p p r o a c h e s to u n d e r s t a n d i n g c i rcad ian osci l la t ions. Annu. Rev. Physiol. 5 5 , 7 2 9 - 7 5 3 .

55. T a k a h a s h i J .S. , M u r a k a m i N . , N i k a i d o S.S., P r a t t B.L. a n d R o b e r t s o n L . M . (1989) T h e av ian p inea l , a ve r t eb ra te m o d e l system of the c i rcad ian osci l la tor : Cel lu lar regu la t ion of c i rcad ian r h y t h m s by light, second messengers , a n d m a c r o m o l e c u l a r synthesis . Rec. Prog. Hormone Res. 4 5 , 2 7 9 - 3 5 2 .

56. T a k a h a s h i J .S . a n d T u r e k F . W . (1987) Ani somyc in , an inh ib i to r of p ro te in synthesis , p e r t u r b s the p h a s e of a m a m m a l i a n c i rcad ian p a c e m a k e r . Brain Res. 4 0 5 , 199 -203 .

57. T a k a h a s h i J .S . a n d Z a t z M . (1982) Regu la t ion of c i rcadian rhy thmic i ty . Science 2 1 7 , 1104-1111 .

58. T a y l o r W.R. , D u n l a p J .C . a n d Has t ings J . W . (1982) Inh ib i to r s of p ro te in synthesis on 80s r i b o s o m e sphase shift the g o n y a l a u x clock. J. Exp. Biol. 9 7 , 121-136 .

59. U n d e r w o o d H . , Bar re t t R . K . a n d Siopes T . (1990) T h e quai l ' s eye: A biological c lock. J. Biol. Rhythms 5 , 2 5 7 - 2 6 5 .

60. U n d e r w o o d H . a n d G r o o s G .A . (1982) Ver t eb ra t e c i rcad ian r h y t h m s : re t inal a n d ex t ra re t ina l p h o t o r e c e p t i o n . Experientia 3 8 , 1113-1121 .

6 1 . V ind l ache ruvu R.R. , Eb l ing F . J . P . , M a y w o o d E .S . a n d Has t ings M . H . (1992) B lockade of g lu t ama te rg ic n e u r o t r a n s m i s s i o n in the sup rach i a sma t i c nuc leus p reven ts cellular a n d behav io ra l responses of the c i rcad ian system to light. Eur. J. Neurosci. 4 , 673 -679 .

62. V i t a t e rna M . H . , Mi le t t e J.J. , T u r e k F . W . a n d T a k a h a s h i J .S . (1993) Spect ra l sensitivity of a pho to recep t ive sys tem regula t ing r ep roduc t ive responses to light in a m a m m a l . (Submi t t ed) .

63 . W a l z B. a n d Sweeney B . M . (1979) Kinet ics of the cyc loheximide- induced p h a s e changes in the biological clock in G o n y a l a u x . Prox. Natl. Acad. Sci. USA 7 6 , 6443-6447 .

64. Wol ln ik F . , T u r e k F . W . , Majewski P . a n d T a k a h a s h i J .S . (1989) P h a s e shifting the c i rcad ian clock wi th cyc loheximide: Response of h a m s t e r s wi th an in tac t or split r h y t h m of l o c o m o t o r activi ty. Brain Res 4 9 6 , 8 2 - 8 8 .

65. Z a t z M . a n d Mul l en D .A. (1988) N o r e p i n e p h r i n e , ac t ing via adeny la t e cyclase, inhibi ts m e l a t o n i n b u t does n o t phase-shift the p a c e m a k e r in cu l tu red chick p ineal cells. Brain Res. 4 5 0 , 137 -143 .

66. Z e r r D . M . , Ha l l J . C , R o s b a s h M . a n d Siwicki K . K . (1990) C i r cad i an f luctuat ions of pe r iod p ro te in i m m u n o r e a c t i v i t y in the C N S a n d the visual sys tem of Drosophila. J. Neurosci. 10 , 2749-2762 .

67. Z h a n g Y., Zee P . C . , K i r b y J . D . , T a k a h a s h i J .S . a n d T u r e k F . W . (1993) A chol inergic an t agon i s t , m e c a m y l a m i n e , b locks l ight - induced F o s immunoreac t i v i t y in specific regions of the h a m s t e r s u p r a c h i a s m a t i c nuc leus . Brain Res. 6 1 5 , 107-112 .

2

Light—Definitions and Measurements R O G E R W I B O M

NIOH, National Institute of Occupational and Health, 17184 So/na, Sweden

T H E SUBJECT in this presentat ion is the modern technology involved with light. M u c h of our information abou t the external world is gained th rough the visual sense. Hence adequa te lighting is of major impor tance in everyday life.

We also know that light has non-visual effects. I think that most of you visiting this conference are even more interested in these effects.

The first pa r t of this presenta t ion begins with a short survey of some of the physical aspects of light and definitions of quant i ta t ive measures . The second par t concerns measurement of light and a few demons t ra t ions .

Light

Light is a form of energy that can pass from one material body to ano ther wi thout the need for any material substance in the intervening space. Such energy transfer has come to be called radia t ion, a te rm which implies tha t the energy flows out in straight lines in all directions from the source, a l though in fact straight-line flow does no t always occur, part icularly when material substance is traversed.

Some forms of radia t ion are best described as particles, for example those which are emit ted by radioactive mater ials , and light was at one time thought to consist of a shower of particles.

Experiments later showed that the behaviour of light rays could be better described in terms of waves, the ray directions being the direction in which the waves are travelling. About one hundred years ago it became clear that light waves are electromagnetic in character and occupy only a very small par t of a huge range of wavelengths that const i tute the electromagnetic spectrum (Figure 1).

2 3


Wavelength in metres

Wavelength in nanometres

F I G . 1. T h e r a d i a n t energy (e lec t romagnet ic) spec t rum.

At the long-wave end of this spectrum there are electromagnetic waves used for radio communica t ions , with wavelengths ranging from tens of kilometres down to a few millimetres. At the other end of the spectrum there are X-rays and gamma-rays .

The wavelength of the electromagnetic spectrum ranges from 1 0 ~ 15 to 1 0 4 metre .

The visible spec t rum

The visible por t ion of the spectrum covers the wavelength range from 380 to 760 n m and the eye discriminates between diiferent wavelengths within this range by the sensation of colour. Violet and blue cor respond to the short wavelengths, yellow and green to the middle and red to the long visible range of wavelengths.

Radia t ion reaching the ear th 's surface from the sun covers a range of wavelengths from abou t 290 n m to 1700 nm, which is considerably wider than the visible spectrum. Solar wavelengths shorter t han 290 n m are absorbed by ozone in the upper level of the earth 's a tmosphere and wavelengths beyond 1700 n m are strongly absorbed by water vapour and carbon dioxide in the lower a tmosphere .

Light—Definitions and Measurements 25

Ul t rav io le t radiat ion

Wavelengths jus t beyond the violet end of the visible spectrum are k n o w n as ultraviolet radia t ion (UVR) and cover the region from 1 n m to 400 nm. The region from 1 nm to 100 nm is vacuum UV, 100 n m to 280 nm is U V C , 280 n m to 315 n m is U V B and the wavelengths between 315 and 400 n m is U V A (Figure 1).

Photomet r i c quant i t ies

The fundamental quant i ty used in pho tomet ry is luminous flux. This is a measure of the rate of flow of radiant energy modified according to the h u m a n spectral sensitivity. In other words , luminous flux is rad iant flux multiplied by the spectral sensitivity of the visual system.

Radian t flux is measured in watts and luminous flux in lumens. Luminous flux is an impor tan t quant i ty in determining the total light

ou tpu t of light sources and luminaires, bu t it is no t the only useful quant i ty . The light dis t r ibut ion from luminaire is described by the luminous intensity. This intensity is the flux emit ted/uni t solid angle in a specified direction. The unit quant i ty is called candela.

Luminous flux and luminous intensity are bo th associated with area measures . The luminous flux falling on the unit area of a surface is called the illuminance. The luminous intensity projected area of a surface in a given direction is the luminance. The unit of i l luminance is l u m e n / m e t r e2, usually called lux and the unit of luminance is candela/metre2 (Figure 2).

Luminance is the physical correlate to the psychological sensation of brightness.

The sensitivity of h u m a n eye is, however, not uniform over the visible spectrum, bu t varies with wavelength as shown in Figure 3. The r ight -hand

L u m i n o u s f lux lumen

F I G . 2. P h o t o m e t r i c quan t i t i e s a n d uni t s .


Violet Blue Green Yellow Orange Red

400 500 600 700

Wavelength (nm)

F I G . 3. Relat ive spect ra l sensitivity of h u m a n eye. T h e spect ra l l u m i n o u s for the C I E * s t a n d a r d observer .

curve is valid for bright viewing condit ions when the cones in ret ina are operat ing. These are start ing to work at 3 c d / m

2 and are the receptors used

in photop ic vision. The left-hand curve is the sensitive of the da rk adap ted eye when the

rods are operat ing. These are sensitive to intensities below 0.1 c d / m

2 and

cause the scotopic vision. F o r visual field luminances between 0.1 and 3 c d / m

2 an intermediate state exists, called mesopic, in which bo th cones

and rods operate . Lighting technology is concerned with relatively high brightness and hence photopic vision at t racts greatest a t tent ion.

M e a s u r e m e n t

The il luminance is invisible to the eye. W h a t the eye can record is the luminance.

The i l luminance decreases with the square of the distance from the light source. Hence, a measurement of i l lumination needs to be accompanied by da t a on the distance from the light source and on the posit ion at which the i l lumination has been measured.

In contrast , the luminance is dependent of the distance between the observer and the object. Thus , it is the unit to use when one relates to the visual sensation.

When light t rea tment is discussed, the quant i ty i l luminance is a lmost exclusively used. Levels between 1,000 to 10,000 lux are often reported.

* C I E = Commission Internationale de l'Eclairage (International Commission on Illumina-tion).

Light—Definitions and Measurements 2 7

The quest ion then is, where have the measurements been made . O n the floor, half a meter above the floor, in the middle of the room, etc.? We are not informed abou t the exposure of the retina.

The i l luminance does no t become visible until it has been reflected on a surface, so we can say noth ing abou t the retinal light exposure unless we know the reflectance of the surface.

T o demons t ra te the difference between i l luminance and luminance, I have b rough t these two boxes.

It is easy to vary the record i l luminance if the detector is moved. In principle you can get any i l luminance you wish by moving the detector from high to low. W h a t value do you want to have?

The type and number of fluorescent tubes is the same in bo th boxes, and thus the i l luminance is the same. Nevertheless, the eye sees the intensity as very different. The reason is tha t the surface of this box has a much lower reflectance.

When we report our experimental of light t rea tment , luminance is a far better quant i ty to use than i l luminance.

We all agree tha t the light must enter the eye, to acheive the therapeut ic effect. The quest ion is how much light shall we give to obta in best results, and which dura t ion and spectrum shall we use.

If we mainta in to measure the i l luminance, the measurement needs to be made of the eye (cornea), so we can est imate how m a n y lux that hit the retina. By recording this, results from different laborator ies can be compared .

You may ask why the i l luminance is so often used in the context of SAD-research? The p robab le reason is tha t i l luminance is the quant i ty most often used in designing lighting at workplaces . This quant i ty is useful to describe how m a n y luminaires you need, bu t when a lighting designer wants to control the vision ergonomics at the workplace he should use the quant i ty luminance.

When planning a workplace the lighting designer now considers the d e m a n d set by our no rma l vision. But, in the future the non-visual effects of light should also be considered when a good work envi ronment is designed. If so, it will be very useful if we talk the same " language".

Another thing is that i l luminance is very easy to measure using cheap and simple ins t ruments .

T o measure luminance is, however, no t much more complicated than to measure i l luminance. The main difference is tha t the measur ing instru-ments are more complicated and much more expensive.

In col laborat ion with Professor Lennar t Wet terberg and Docent Bengt Kjellman, I have built a light t rea tment r o o m at St G o r a n s Hospi ta l in Stockholm. The r o o m is homogeneously i l luminated, all the surface is white and the light from the luminieres is indirect. The boxes I show are a minia ture copy of the room.


The result of studies using this r o o m is presented in Chapte r 19 of this volume.

O n e quest ion I often get abou t this r oom is: H o w many lux are in the r o o m ? The answer is that the i l luminance can be everything between 1,000 to 10,000 lux, depending on where I measure .

W h a t we know exactly is how much light that enters the eye. The luminance at the sur rounding surface in the r o o m is always 350 c d / m

2,

irrespective of where in the r o o m the detector is placed.

References

Boyce P .R . (1981) Human Factors in Lighting. Appl ied Science Pub l i shers , L o n d o n . H o p k i n s o n R . G . (1970) The Ergonomics of Lighting. M a c D o n a l d Technica l a n d Scientific,

L o n d o n .

IES Lighting Handbook (1981) Reference V o l u m e , I l lumina t ing Engineer ing Society of N o r t h Amer ica .

3

Mechanisms in the Eye that Mediate the Biological and Therapeutic Effects of Light in Humans G E O R G E C. B R A I N A R D , J A M E S R. G A D D Y , FEL IX M . B A R K E R , J O H N P. H A N I F I N a n d M A R K D. R O L L A G

Departments of Neurology and Psychiatry, Jefferson Medical College, Philadelphia, PA, Pennsylvania College of Optometry, Philadelphia, PA, Department of Anatomy, Uniformed Services University of Health Sciences, Bethesda, MD, USA

Abst rac t

Research on the therapeut ic potency of "br ight" light has ou tpaced the s tudy of the fundamenta l mechan i sms by which light p roduces its beneficial effects. Original ly, it was t hough t tha t i l luminances equal to or above 2,500 lux were necessary to induce biological change or therapeut ic response . D a t a are presented which show tha t i l luminances as low as 5 lux of m o n o c h r o m a t i c green light o r 100 lux of b r o a d b a n d white light also can p r o d u c e significant suppress ion of me la ton in in n o r m a l h u m a n volunteers . T o unde r s t and h o w such low levels of i l luminat ion can p roduce s t rong biological a n d therapeut ic responses , it is necessary to examine the ocula r mechan i sms which media te the pho t ic effects. Specifically, in h u m a n s it is shown tha t : (1) gaze behavior relative to a light source can significantly alter the a m o u n t of light reaching the ret ina; (2) the age of the ocular lens can significantly modify the ba lance of wavelengths reaching the ret ina; (3) near-ul t raviolet rad ia t ion t radi t ional ly considered outs ide of the visible spec t rum can s t imulate visual responses in y o u n g adul t s a t least up to the age of 25 years; (4) pupi l lary di la t ion modu la t e s the capaci ty of a light s t imulus to suppress mela ton in ; a n d (5) the entire retinal field par t ic ipates in mela ton in regula t ion. Al though the specific pho to recep to r s or p h o t o p i g m e n t s which media te the biological a n d therapeut ic effects of light in h u m a n s presently are no t k n o w n , basic d a t a are presented on the effects of different wavelengths elucidat ing mela ton in suppress ion a n d S A D t rea tmen t . In s u m m a r y , it is useful from b o t h a scientific as well as a clinical perspective to establish the specific mechan i sms in the eye which media te the nonvisual therapeut ic a n d biological effects of light.

In t roduct ion

I N 1980, it was demonstra ted that exposing the eyes of healthy humans to bright light at night causes a strong suppression of plasma melatonin, a

2 9


hormone secreted by the pineal g l a n d .

53 This finding opened the door to

numerous studies on the biological and therapeutic effects of light in humans . Since 1980, investigators have tested bright light as a stimulus for regulating neuroendocrine and circadian physiology and producing therapeutic benefits in patients with d e p r e s s i o n ,

4 7'

5 1'

5 2'

8 0'

8 1 , 9 4 - 96 sleep

d i s o r d e r s ,

3 1'

82 menstrual d i f f i cu l t i e s ,

4 6'

6 9 , 70 as well as problems associated

with jet lag and shift w o rk .

1 2'

2 7'

2 8'

3 0'

3 5'

6 2'

6 3'

8 7'

98 In fact, the use of bright

light stimuli as a therapeutic intervention has far outpaced research of the fundamental mechanisms of how light induces such biological and behavioral changes. The aim of this paper is to examine mechanisms related to the eye that mediate the biological and therapeutic effects of light in humans .

Decades before the discovery that light could influence h u m a n physio-logy, researchers had shown that animals exhibit s trong responses to light stimuli. In most vertebrate species, it is known that light enters the eyes and stimulates the retina. Neural signals are sent from the retina to the visual centers of the brain and permit the sensory capacity of vision. In addit ion, neural signals are sent from the retina into the hypothalamus, a non-visual par t of the brain. Most , but not all, of the fibers projecting from the retina into the hypothalamus terminate specifically in the suprachiasmatic nuclei ( S C N ) .

4 4'

6 1'

71 This part of the brain is considered to be an endogenous

oscillator—a fundamental component of the "biological clock", or circadian system, which regulates the body's physiological and behavioral rhythms. The circadian system is responsible for controlling daily rhythms such as sleep and wakefulness, body temperature and hormona l secretion. It is now clear that light is the primary stimulus for regulating the circadian system, al though other external stimuli such as sound, temperature and social cues may also influence the body's timing f u n c t i o n s .

5'

2 7'

5 2 , 6 0'

1 07

The S C N relay retinal information to many of the major control centers in the nervous sys tem.

61 One nerve pathway that carries non-visual photic

information extends from the S C N to the pineal gland via a multisynaptic pathway with connections being made sequentially in the paraventricular hypothalamus, the upper thoracic intermediolateral cell column, and the superior cervical g a n g l i o n .

4 4'

61 Cycles of light and darkness relayed by the

retina entrain S C N neural activity which, in turn, entrain the rhythmic product ion and secretion of melatonin from the pineal gland. In all vertebrate species studied to date, including humans , high levels of melatonin are secreted during the night and low levels are secreted during the day

4 3'

5 4<

7 4'

7 5'

1 0 1'

1 0 2'

1 05

Light- induced suppression of melatonin in humans

In addit ion to entraining melatonin secretion from the pineal gland, light can have an acute suppressive effect on melatonin. Specifically, exposure of

Ocular Mechanisms that Mediate Therapeutic Light Effects 3 1

the eyes to light during the night can cause a rapid decrease in the high nocturnal synthesis and secretion of melatonin. This acute light-induced suppression of nocturnal melatonin synthesis was first o b s e r v e d

45 in 1972

and has been used in numerous animal studies to help determine the neural and biochemical mechanisms of melatonin r e g u l a t i o n .

3 7'

3 8'

4 4'

79 In addi-

tion, this response has been used as a model for testing the capacity of different light parameters for their relative influence on neuroendocrine and/or circadian p h y s i o l o g y .

8'

1 4 - 1 7 , 2 0'

2 1'

6 5'

7 2'

1 00 Complete dose-response

curves show that exposure to 1.2 lux and 0.06 μψ/cm

2 (0.2 lux) of white light

during the night is sufficient to suppress melatonin in rats and hamsters , r e spec t ive ly .

1 6'

59 Fur ther studies in hamsters demonstra te that even lower

illuminances at 0.022 pW/cm

2 (0.05 lux) of monochromat ic (500 nm) green

light can suppress pineal m e l a t o n i n .

6 5'

72 Other studies testing relatively few

illuminances of white light have shown a wide range of sensitivity for melatonin suppression among different spec ies .

73 Although it has not been

firmly established, this diverse range of sensitivities for melatonin suppres-sion may be related to a species' activity pat tern.

Early h u m a n studies failed to demons t ra te the acute suppressive influence of light on mela tonin tha t previously had been demons t ra ted in other mammal i an s p e c i e s .

2'

4 3'

5 4'

1 0 1'

1 0 2 , 1 05 In 1980, however, Lewy and

co l l eagues

53 demons t ra ted tha t exposure of the eyes of no rma l volunteers

to 2,500 lux of white light dur ing the night induced an 8 0 % decrease in circulating mela tonin within 1 hour . In contras t , volunteers exposed to 500 lux of white light exhibited no significant mela tonin s u p p r e s s i o n .

53 The

earlier a t tempts to suppress mela tonin in h u m a n s failed when investigators used typical indoor light levels repor ted to be between 100 to 800 j u x 3 , 4 3 , 5 4 , 1 0 1 , 1 0 2 , 1 0 5 w h e r e a s such typical r o o m light would be sufficient for suppressing mela tonin in m a n y animal s p e c i e s ,

1 6'

6 5'

7 2'

7 3'

79 and would

be adequa te for h u m a n vision, it was not enough to suppress mela tonin in those experiments . Simply put , it takes m u c h more light to suppress mela tonin in h u m a n s than is required for h u m a n vision or for regulat ing the circadian and neuroendocr ine systems of some other animal species. The discovery that much brighter light is needed to suppress mela tonin in h u m a n s provided the g roundwork for numerous studies on biological responses of h u m a n s to bright artificial light including the confirmation tha t br ight light ( > 2,500 lux) entering the eyes of h u m a n s is a po ten t st imulus for controll ing circadian r h y t h m s .

2 7'

2 8'

3 0'

5 2'

6 0'

1 07 However , the

not ion tha t only "br ight" light can suppress mela tonin or regulate circadian rhy thms in h u m a n s is no t entirely accurate .

Several years after the l andmark study of Lewy and co l l eagues ,

53 a

study was done to determine more precisely the dosages of light needed to suppress mela tonin in no rma l v o l u n t e e r s .

14 In tha t s tudy, six normal

males were exposed to carefully control led intensities of monoch roma t i c green light at 509 n m (10 n m half-peak bandwid th ) for one hou r dur ing the


night after a three hour period of da rk -adap ta t ion . Specifically, the volunteers were cont inuously exposed to the experimental light between 2:00 a.m. and 3:00 a.m. with their pupils fully dilated by a mydriat ic agent, their heads held steady relative to the light source by an ophtha lmic head holder, and with translucent white integrat ing hemispheres covering bo th eyes. This procedure produced a cons tant and uniform i l lumination of the whole ret ina dur ing the entire light exposure. The da ta from this experiment (Figure 1 and Table 1) demons t ra ted that light affects a h u m a n h o r m o n e in a dose-response fashion: i.e. the brighter the phot ic stimulus the greater the suppression of m e l a t o n i n .

14

It is interesting to note that all of the stimuli used in this s tudy activated the visual system—i.e. bo th the volunteers and the experimenters saw each of the different light intensities and accurately reported them to be green. The lower light intensities, however, did not change h o r m o n e levels whereas the higher intensities induced a 6 0 - 8 0 % decrease in this h o r m o n e . Thus , light sufficient to activate vision does no t necessarily cause neuroendocr ine change. It appears to be generally true in bo th animals and h u m a n s tha t much more light is needed for biological effects than for

vision. 2 , 1 4 , 1 6 , 2 7 , 2 8 , 3 0 , 4 3 , 5 2 - 5 4 , 6 0 , 6 5 , 6 6 , 7 2 , 7 3 , 7 9 , 9 2 , 1 0 1 , 1 0 2 , 1 0 5 , 1 0 7

Ζ 100-ο (Ω 2 ω —

80 -LU ζ oc Ο û . Η- 60 " ώ < Q - J

LU 40 -H- 2

40 -Ζ LU <

o s 20 -

DC (fi LU < CL _ | o -

CL Ζ

< -20 -LU

2 -40 -

HUMAN VOLUNTEERS (λ Max = 509nm)

2 4 6 8 10 12

IRRADIANCE UUJ/cm

2)

14 16

F I G . 1. T h e dose- response re la t ionsh ip be tween green m o n o c h r o m a t i c light exposure of n o r m a l vo lunteers eyes a n d suppress ion of the h o r m o n e me la ton in .

D a t a po in t s indica te m e a n + S E M .

14

The demons t ra t ion of the dose-response function for light suppression of melatonin in h u m a n s produced an unexpected result: very bright light is not necessarily needed for melatonin suppression. As demons t ra ted by Table 1, the mean threshold i l luminance for producing a statistically significant melatonin suppression was between 5 and 17 lux in normal volunteers—a level of i l luminance equal to civil twilight and well below typical indoor light. This means tha t under the proper condi t ions, 25 to 100 times less light can suppress mela tonin than originally be l i eved .

14


T A B L E 1.

Radiometric and Photometric Stimuli Used in the Melatonin Dose-Response Curve for 509 nm

14

I r r ad iance Q u a n t a I l luminance % M e l a t o n i n

(μψ/cm

2) ( p h o t o n s / c m

2) (pho top ic lux) (scotopic lux) suppress ion

0.01 9.2 χ 1 0

13 0.03 0.17 - 9 . 6 7

0.30 2.8 χ 1 0

15 1.03 5.25 1.83

1.60 1.5 χ 1 0

16 5.50 27.98 37.33

5.00 4.6 χ 1 0

16 17.18 85.90 51.67

13.00 1.2 χ 1 0

17 44.66 227.37 60.67

It is reasonable to ask why ambient r o o m light at levels much higher than 17 lux did not suppress mela tonin in the earlier a t tempts to suppress melatonin in h u m a n s .

2 , 4 3'

5 4'

1 0 1 , 1 0 2 , 1 05 The answer to this can be found by

examining the ocular mechanisms involved in light s t imulat ion of the circadian system. Specifically, it is impor tan t to consider the na ture of the light st imulus and its relation to the eyes, gaze direction, s tatus of the ocular media , pupil lary dilation, retinal field exposure and photorecep-tors . Each of these factors contr ibute to determining if a phot ic st imulus will or will no t be effective in produc ing a biological or therapeut ic response to light.

In f luence of gaze d i rect ion on l ight e f fec ts

In the early h u m a n studies testing for mela tonin suppres-s i o n ,

2 , 4 3'

5 4'

1 0 1'

1 0 2'

1 05 neither the exposure condit ions nor the light stimuli

were optimized. Often the experimental light st imulus consisted of turning on the overhead light provided with the experimental room. Fu r the rmore , measurement of the experimental i l luminance often consisted of placing a light meter at desk height and aimed directly at the overhead lights. Such a measure may over- or under-est imate the actual corneal i l luminance experienced by the subjects. Fu r the rmore , in a lmost any given room, it is possible to vary the light i l luminance entering the eyes by a factor of 10 simply by changing the direction of gaze. Thus , in a r o o m characterized as having a "typical" i l lumination level of 500 lux, the occupants may be able to see up to 500 lux if they look directly towards the light fixtures, but if they look at the floor or walls, the light reaching their eyes may d r o p to 50 lux or lower. Light entering the eyes can be further reduced if the volunteers close their eyes, squint or gaze into shadowy areas.

Experimental circumstances in which subjects are free to alter gaze and distance from a single light source are subject to loss of 8 0 % to 9 9 % of the putat ive corneal i rradiance when subjects distance themselves and avert their gaze from the s o u r c e .

29 The loss of corneal i l luminance is

compounded by the eye's optics which focus an image of the light source on


the ret ina ra ther than diffusing the light across it. In a recent study which examined the influence of gaze direction relative to specific light sources on corneal i l luminance, subjects were posi t ioned 91 cm (3 feet) in front of a 61 χ 122 cm (24 χ 48 inch) light source which was entirely visible with a s traight-on gaze. A 45° ro ta t ion of the subjects from the straight-on posit ion resulted in a 6 0 % reduct ion of the light source luminant surface image averaged over bo th eyes. A 90° ro ta t ion produced an 8 5 % loss. The degree of loss was more extreme for a 16 χ 24 inch light source (45°: 8 2 % ; 90°: 9 6 % ) .

36 Thus , gaze aversion not only reduces general corneal

i l luminance, it also reduces the total area of the retinal image produced by a discrete light source. Beyond the mot ion of the eye relative to the envi ronment and light sources, there are mechanisms within the eye itself tha t determine effectiveness of phot ic stimuli for inducing biological change.

In f luence of ocular media t ransmission on l ight e f fec ts

An impor tan t ocular variable tha t determines bo th the quant i ty and quali ty of light reaching the ret ina is the t ransmit tance characteristic of the ocular media . It is well documented tha t the cornea, aqueous h u m o r , and vitreous h u m o r of the heal thy h u m a n eye are clear tissues which t rans-mit nearly 100% of visible light and ultraviolet wavelengths down to 300 n m .

1 0 , 4 9'

85 In the heal thy h u m a n , there is little change in the

transmission characteristics of these tissues over age. In contrast , the crystalline lens yellows with age and acts as a filter tha t greatly a t tenuates radiant energy of the shorter wavelength por t ion of the spectrum (i.e. less than 1% transmission between 300 n m and 380 n m after age 30; reduced by 5 0 % at 470 n m at age 75) .

6

T o study the lenticular t ransmission of ultraviolet, visible and infrared wavelengths, pos tmor tem lenses (rc = 288) were collected from h u m a n donors ranging from 0 to 91 years of a g e .

6 The spectral t ransmit tance

characteristics of these lenses were measured between 200 n m and 2500 nm with a Beckman UV-5240 spect rophotometer . Figure 2 illustrates the lenticular t ransmission of lenses collected from two different donors .

The da ta in Figure 2 show how lenticular t ransmission varies greatly over the h u m a n life span. N o t e the progressive loss of ultraviolet and short wavelength visible t ransmission with age. The overall results of this study indicated that longer, infrared wavelength transmission is no t substan-tially different between age groups , while significant, gradually occurring differences do exist between age groups in the shorter wavelength and visible ranges. The averaged percent t ransmit tance da ta from the 20-29 year and the 50-59 year age groups are shown in Figure 3 oppos i t e .

6

A por t ion of the averaged percent t ransmit tance da t a from the 20-29

Ocular Mechanisms that Mediate Therapeutic Light Effects

HUMAN LENS TRANSMITTANCE

WO -,

90 -I

F I G . 2. Lent icu la r t r ansmiss ion of single h u m a n lenses collected from a n e w b o r n and 75 year old d o n o r .

6

HUMAN LENS

AVERAGE TRANSMITTANCE

LU Ο Ζ

<

Ε (Λ Ζ < CE

350 400 450

WAVELENGTH (NM)

• AGE 20-29 (Ν=36) + AGE 50-59 (N=40)

F I G . 3. T h e m e a n % t r a n s m i t t a n c e of visible a n d ul t raviole t wave lengths by p o s t m o r t e m h u m a n lenses from 36 ind iv idua ls aged 20 to 29, a n d from 40

indiv iduals aged 50 to 5 9 .

6


year and the 50-59 year age groups are shown in the Table 2. These da ta were analyzed using a 2-tailed " t" test and probabi l i ty values are no t corrected for multiple compar isons .

These da t a show tha t the age of the h u m a n lens significantly modula tes the balance of wavelengths reaching the retina. As the eye ages, the increased yellowing of the lens diminishes the transmission of shorter wavelength energy. Hence, i l lumination measured at the level of the cornea is not necessarily indicative of retinal i l lumination a m o n g different age groups . In terms of using light as a biological or therapeut ic st imulus, for any given b road spectrum light source, there will be a progressive decrease in energy reaching the ret ina in the short wavelength visible and ultraviolet spectrum as pat ients age.

In examining the da t a in Table 2, it is obvious that there are statistically significant differences in lenticular t ransmission between different age groups—part icular ly in the shorter wavelength ranges. It is reasonable to ask if the very small percentage t ransmit tance of near-ultraviolet radiat ion (an average of less than 1%) in young adults allows an effective phot ic stimulus to reach the retina. In ternat ional s tandards define the visible spectrum as wavelengths of electromagnetic radia t ion between 380 n m and 780 n m .

25 There are, however, da t a demonst ra t ing that the spectrum

of radiat ion visible to the h u m a n eye actually extends further below 380 n m into the near-ultraviolet range. Recent studies have demons t ra ted that the visual system of young h u m a n s is responsive to ultraviolet radiat ion at least as low as 340 n m .

1 3 , 84 In one of those studies, a

dose-response relat ionship between varying irradiances of m o n o c h r o m a -tic UV-A (340 nm) and visual evoked potentials (VEP) in young subjects was established. The V E P is a gross electrical signal generated by the visual cortex in response to repetitive phot ic s t imulat ion. Its waveform, peak latency, and ampl i tude vary according to experimental c o n d i t i o n s .

4 , 7 , 88

T o probe for potential sensitivity to ultraviolet radia t ion at 340 nm, healthy, young adults 20 to 25 years-of-age with normal color vision (n = 10) were t e s t e d .

84 The V E P recording techniques and parameters are

described e l s e w h e r e .

3 , 13 The 340 nm stimulus was administered by a l amp

suspended 15 cm above the right eye and the left eye was covered with a pa tch . Seven different flash irradiances plus an audi tory control were presented to each subject under an ambient photopic condi t ion of overhead white fluorescent light at 550 lux. D a t a from this s tudy were analyzed by A N O V A with statistical significance determined by the Student N e w m a n Keuls multiple range test.

The da ta shown in Figure 4 illustrate a dose-dependent response between irradiance and V E P N 2- P 3 ampl i tude for young a d u l t s .

84 This

illustrates that , as i r radiance decreases, the N 2- P 3 ampl i tude also decreases. At an i rradiance of 0.37 / i W / c m

2, only two of the ten subjects in

Ocular Mechanisms that Mediate Therapeutic Light Effects 37

T A B L E 2

Lenticular transmittance in two age groups (mean and SE)

6

A G E 3 4 0 n m 4 4 0 n m 5 4 0 n m 6 4 0 n m 7 5 0 n m

2 0 - 2 9 X 0 . 3 9 4 5 1 . 9 9 6 4 . 6 3 6 8 . 2 9 6 9 . 7 4

(w = 36) SE 0 . 0 7 7 1 . 6 8 1 . 6 8 1 . 5 6 1 . 4 9

5 0 - 5 9 X 0 2 9 . 6 2 5 9 . 9 2 6 8 . 6 4 7 0 . 9 4

(n = 40) SE 0 1 . 3 8 1 . 3 0 1 . 2 0 1 . 1 5

ρ ( 2 tail) 0 . 0 0 0 1 0 . 0 0 0 1 0 . 0 2 8 0 . 8 5 7 0 . 5 2 1

LU Q

- I CL

<

I YOUNG ADULTS (N=10, 20-25 yrs.)

340 NM

X X χ χ

* T

N . D . N . D . w 3 . 9 0 2 . 3 7 1 . 2 7 0 . 7 2 0 . 4 0 0 . 3 7 0 . 3 5 0 . 3 1

IRRADIANCE ^W/cm

2 )

F I G . 4 . Th is g r a p h i l lustrates the visual evoked response a m p l i t u d e in y o u n g adu l t s exposed to different i r rad iances of near -u l t rav io le t r ad i a t i on at 3 4 0 n m .

84

Asterisks (*) ind ica te stat is t ical significance of a t least ρ < 0 . 0 5 c o m p a r e d to the m a x i m u m N 2- P 3 a m p l i t u d e . T h e dagger ( T ) indica tes t ha t only two of the ten subjects were ab le to detect r ad i a t i on at this i r r ad iance . N . D . = no t de tec ted by

any subject .

this age g roup could detect this radia t ion and at the two lowest irradiances, none of the subjects could detect this radia t ion.

It is known that the lens will fluoresce when i l luminated with ultraviolet wavelengths. In this study, for all but the lowest i rradiances, all subjects were able to clearly discriminate the shape of the filaments suspended within the flash l amp dur ing st imulat ion. Therefore, if only secondary lenticular fluorescence accounted for all the energy reaching the ret ina dur ing s t imulat ion, the subjects would not have been able to focus the image. T a n provides an elegant a rgument against such fluorescence-based U V v i s i o n .

93 Thus , this s tudy demonst ra tes that monochroma t i c nea r -UV

radia t ion can elicit visual evoked potentials in a dose-dependent manne r in the young h u m a n e y e .

84 The study also confirms that the very low

percentage lenticular t ransmission of energy at 340 nm as shown in Table 2 is meaningful in tha t it can induce visual responses.

Given that some ultraviolet energy is t ransmit ted th rough the lenses of


young humans , and that ultraviolet radia t ion can st imulate the visual system, it is useful to quest ion the potential role of ultraviolet radia t ion in biological and therapeut ic responses to light. In several rodent species, ultraviolet radia t ion down to 300 n m is t ransmit ted th rough the lenses and can part icipate in the regulat ion of the circadian and neuroendocr ine s y s t e m s .

8 , 1 3'

1 5'

1 9'

7 2'

9 0 , 91 Indeed, very recent findings indicate that the rat

ret ina may conta in a specific pho top igment tha t has its peak sensitivity in the near-ultraviolet s p e c t r u m .

42 Thus , m a m m a l s may have ultraviolet

photoreceptors similar to those which have been identified in various insect, fish, reptile and bird species.

It is currently unclear whether or no t the h u m a n retina contains a specific ultraviolet photoreceptor or if ultraviolet radia t ion can be an effective circadian st imulus for h u m a n s . Fur the r work is required to determine if detection of ultraviolet radia t ion (UV) by the young h u m a n eye serves some purpose other than vision. A practical issue debated a m o n g researchers concerns the role of U V in light therapy. Mos t of the early studies on seasonal affective disorder (SAD) therapy successfully utilized fluorescent lamps tha t emitted white light containing a por t ion of U V w a v e l e n g t h s .

8 0'

96 Those early results erroneously led to the suggestion

tha t U V wavelengths are necessary for successful therapy. The l i terature, however, shows clearly tha t SAD symptoms can be reduced by lamps which emit little or no U V .

9'

4 8'

5 0 , 5 2'

6 4'

6 8'

1 08 Hence, U V wavelengths do

not appear to be necessary for eliciting positive therapeut ic results. Whether or not U V wavelengths can contr ibute to the op t imum balance of wavelengths for SAD therapy remains an open quest ion. It also must be recalled tha t overexposure to ultraviolet radiat ion—part icular ly wave-lengths below 300 n m — c a n be a hazard to bo th skin and eye t i s s u e s .

4 0'

4 9'

8 5'

9 9'

1 03 Similarly, overexposure to visible light and infrared

radia t ion can also be damaging to these t i s s u e s .

4 9'

8 5'

1 03 Thus , it is

becoming increasingly impor tan t to discern between wavelengths and doses of light tha t are effective therapeutically yet are safe in terms of potential tissue damage .

Clearly, the t ransmit tance characteristics of the clear ocular media, and part icularly the crystalline lens, influence the quality and quant i ty of light reaching the retina. Thus , these tissues are involved in determining the biological impact of a phot ic stimulus in terms of neuroendocr ine and circadian regulat ion. Another potent ial ocular mechanism for modifying a phot ic stimulus before it reaches the ret ina involves the iris and the pupil .

Pupil lary d iameter mediates biological e f fec ts of l ight

The iris of the eye is a heavily pigmented layer of muscle tissue which adjusts the pupil of the eye. Pupi l dilation leads to increased amoun t s of


light reaching the ret ina while pupil constr ict ion results in decreased i l lumination reaching the retina. The h u m a n pupil ranges in size from 2 m m to 9 m m depending on the brightness of light being viewed. Extensive studies have been done on the iris as it relates to enhancing vision, providing visual comfort and protect ing the ret ina from d a m a g e .

7 6'

85

Little has been done , however, to assess the role of the iris and pupil in modera t ing the effects of light stimuli on the therapeut ic or biological effects of light. T o test the capacity of pupil di lat ion to affect l ight-induced melatonin s u p p r e s s i o n ,

38 a g roup of eight heal thy male and female

subjects were studied (mean age + S E M = 24.1 + 0 . 7 4 years). All subjects were in good general heal th and passed the Ishihara test for color blindness. Each subject was tested on three separate nights with at least one week between each night of s tudy.

O n two of the three experimental nights, subjects stared at a uniform visual field delivering a corneal i l luminance of 100 lux of white light. O n one of these exposure nights , pupils were free to constrict while on the other , they were pharmacological ly dilated. The third night was a control condit ion in which the subject remained blindfolded th roughou t the 90 minute period. The order of t rea tment varied systematically between subjects. Each study began at midnight of a given night in a l abora tory which was dimly lit to permit approximate ly a 10 lux exposure while the volunteers talked with each other , listened to a personal stereo, read, or slept. At 01:00 to 01:30 hours , 1-2 d rops of 0 . 5 % cyclopentolate (an anticholinergic pupil lary di lator) or placebo (artificial tears) were placed in the subjects ' eyes. At this t ime, all subjects were blindfolded. Cyclopentolate was used on bo th the night of dilated-pupil light exposure and on the night of no light exposure. P lacebo drops were used on the night of 100 lux exposure when the pupils were free to constrict .

In order to provide a spatially-uniform light st imulus tha t filled the visual field, three G o l d m a n n perimeters were modified by aiming a fiber optic light guide in to the t op of the perimeter. A 150 W quar tz-halogen l amp provided uniformly reflected white light from the perimeter 's interior. Spot sampling of the perimeter 's surface with a luminance meter showed a homogeneous luminance across the visual field. Blood samples were taken just pr ior to the beginning of light exposure at 02:00 hours and again just after light exposure was completed at 03:30 hours . P la sma samples were analyzed for mela tonin using a mod i f i ca t i on

37 of the

rad io immunoassay me thod described by Rollag and N i s w e n d e r .

78

Trea tment with cyclopentolate p roduced a marked dilat ion of the subjects' pupils (mean = 7.3, S E M = 0.3). In contras t , the pupils t reated with placebo drops on the night of light exposure were constricted (mean = 3.3, S E M = 0.1). As shown in Figure 5A, in bo th the dilated and free pupil condi t ions , 100 lux of corneal i l luminance significantly suppressed p lasma mela tonin compared to the control night


(F(2 ,14)= 10.6; ρ < 0.002). The da t a in Figure 5B demons t ra te tha t pupil dilation produced stronger light-induced mela tonin suppression at 100 lux corneal i l luminance as compared to the free pupil condi t ion (i = 2.78; d / = 7 , p = 0.027).

-r-

CONTROL FREE PUPIL DILATED PUPIL

— O)

FREE PUPIL DILATED PUPIL

F I G . 5. ( A ) These d a t a i l lus t ra te m e a n ( + S E M ) change scores from basel ine p l a s m a m e l a t o n i n levels (02:00 h o u r s ) to final m e l a t o n i n levels (03:30 h o u r s ) in eight subjects s tudied o n three sepa ra t e n i g h t s .

38 (B) M e a n ( + S E M ) con t ro l -

adjus ted change scores for p l a s m a m e l a t o n i n a t 100 lux of co rnea l i l luminance in b o t h free-pupil a n d di la ted pupi l cond i t ions . Suppress ion is expressed as the change from basel ine o n the tes t ing n ight less the change from basel ine on the

con t ro l (no l ight) n i g h t .

38

This result suggests tha t the threshold for white l ight-induced melatonin suppression in bo th the free- and dilated-pupil condit ions is somewhere below 100 lux of corneal i l luminance for young, heal thy adul ts . This result also confirms that the pupil is impor tan t in controll ing light-induced mela tonin suppression. When the pupil was dilated, mela tonin suppres-sion increased significantly. This finding parallels the classic visual psychophysical principal tha t , as pupil diameter increases, visual stimula-tion inc reases .

76 These similarities suggest tha t the regulat ion of non visual

effects of light occurring via the retina are accessible to classical psychophysical me thods and tha t applicat ion of these me thods should lead to increased unders tanding of basic mechanisms. In addi t ion, these


results suggest tha t pupil diameter may be a factor in the effectiveness of light stimuli used to shift circadian rhy thms or to treat seasonal depression or sleep disorders .

T o reiterate, the original not ion that only relatively bright white light above 2,500 lux can suppress m e l a t o n i n

53 is not entirely valid. Othe r

studies have shown that as little as 200 to 400 lux of white light can suppress p lasma melatonin levels in h u m a n s

1 1'

3 7'

56 and the da t a

illustrated above show tha t as little as 100 lux of white light can suppress melatonin significantly when gaze direction and exposure condit ions are carefully c o n t r o l l e d .

38 These da ta no t only emphasize the impor tance of

behavioral movement of the eyes and uniformity of light presentat ion but also suggest that the spatial location of retinal photoreceptors has a role in mediat ing the therapeut ic and biological effects of light. Specifically, when the entire visual field is steadily exposed, relatively low levels of i l lumination (5 lux of monoch roma t i c green light or 100 lux of white light) can suppress m e l a t o n i n .

1 4'

38

The finding that relatively low levels of light can suppress mela tonin when gaze behavior is r igorously controlled and the entire retinal field is evenly i l luminated suggests that light s t imulat ion of the entire retinal field leads to a s tronger biological response than when the retinal field is only partially s t imulated. A recent study on normal h u m a n s demons t ra ted tha t when i l luminance measured at the cornea is equal , s t ronger mela tonin suppression is observed when the whole retinal field is i l luminated as opposed to i l lumination of approximate ly half of the retinal field.

37 It

appears tha t there m a y be a mechanism for spatial summat ion of phot ic stimuli at the level of the re t ina—the more retinal area exposed to light, the stronger the resulting biological response. Neuroana tomica l studies help suppor t this concept . Morphologica l evidence from rodent and non-h u m a n pr imates indicates that retinal ganglion cells projecting to the suprachiasmat ic nuclei are dis tr ibuted across the entire r e t i n a .

5 7'

71 This

suggests that photoreceptors from the entire retinal field contr ibute to s t imulat ion of the S C N . Fur the r suppor t for this concept comes from a study which examined melatonin suppression in h u m a n s with 1,000 lux of white light on two occasions: one i l luminating the central visual field 5° from the center of gaze, and one directed to the peripheral visual field 60° lateral to the direction of gaze. Tha t s tudy showed tha t mela tonin suppression did no t differ between central and peripheral i l luminat ion .

1 At

this time, m a n y quest ions remain unanswered abou t the how the retina processes phot ic stimuli for mela tonin regulat ion. D o certain areas of the retina send stronger signals to the S C N than others? Does the superior retinal field have a greater role than the inferior retinal field? A relatively homogenous spread of retinal ganglion cells in the ret ina does no t guarantee a homogenei ty of physiological function. Fu r the r studies are needed to determine more precisely how the physical locat ion of


photoreceptors in the retina mediates the therapeut ic or biological effects of light stimuli.

W a v e l e n g t h sensit iv i ty for l ight e f fec ts

Current ly , there are no definitive da ta that determine what photorecep-tors and photopigments t ransduce light stimuli for the non-visual biolo-gical and therapeut ic effects in h u m a n s . In examining animal studies from many laborator ies , some da ta have indicated that the peak sensitivity of the circadian and neuroendocr ine system is a round 500

nm 8 , i 7 , 2 i , 2 2 , 6 5 , 6 6 , 7 2 , 9 2 , 9 7 T h is s u p p o r ts th e hypothesis that rhodops in or

a rhodopsin-based molecule is the pr imary receptor for circadian and neuroendocr ine regulation. In contras t , o ther da ta have suggested that one or more cone photop igments may be involved in these regulatory e f f e c t s .

8 , 1 5 , 1 7 , 4 1 , 5 8 , 72 In considering the potent ial photoreceptor cell

type(s) that t ransduce phot ic information to the SCN, it should be kept in mind that the rodent ret ina contains bo th cone and rod pho to -r e c e p t o r s .

2 3 , 2 4 , 1 06 The retinally degenerate mouse (rd/rd) , however,

shows normal circadian responses to 515 n m light pulses despite nearly total loss of classical visual p h o t o r e c e p t o r s .

34 Fur ther , in a pilot study on a

single 18 year-old congenitally blind man , melatonin suppression was still achieved with bright light over 6,000 lux. Thus , the neuroendocr ine sensitivity to light remained despite a loss of pupillary reflex, outer retinal functioning as determined by electroret inographic testing or conscious perception of the light s t i m u l u s .

55 Such da ta suggest tha t light detection

for the circadian and neuroendocr ine systems may not rely on the rod or cones used for v i s i o n .

3 4 , 55

It is important to note that while most da ta from rodents show that the highest sensitivity is in the blue-green range, other wavelengths may participate in circadian and neuroendocrine regulation. Fo r example, short wavelengths in the ultraviolet region of the s p e c t r u m

8 , 1 3 , 1 5 , 1 9 , 7 2'

9 0 , 91 and

longer wavelengths in the red port ion of the s p e c t r u m

2 0 , 6 7 , 1 00 are quite

capable of suppressing melatonin, entraining circadian rhy thms and influencing reproductive responses in rodents given a sufficiently high intensity. There is considerable diversity in the cellular s tructure and function of the retina across mammal i an spec ies .

77 Thus , further studies

are required to conclusively identify which specific photoreceptors and photopigments are involved in regulating the circadian and neuroendo-crine systems a m o n g different species. Whereas da ta from n o n - h u m a n species may be helpful in guiding research at the h u m a n level, caut ion should be exercised in generalizing results from rodents to h u m a n s .

T o date , only one study has been done to examine the relative impact of different wavelengths on melatonin synthesis in h u m a n s .

14 After complet-

ing the dose-response work illustrated in Figure 1, a preliminary test was


T A B L E 3

The effects of different monochromatic wavelengths on melatonin suppression in normal volunteers

14

Wave leng th (nm) Subjects (n) % M e l a t o n i n Suppress ion S E M

448 3 - 1 2 . 0 0 22.00 474 2 26.00 8.00 509 3 63.67 6.94 542 3 19.67 13.35 576 3 16.33 14.38 604 2 - 1 1 . 5 0 8.50

These da t a suggest that the peak sensitivity for mela tonin suppression may be in the blue-green range as seems to be the case in some lower mammals . It is p remature , however, to pu t much emphasis on such a conclusion since relatively few subjects were tested in this pilot s tudy and analysis of variance shows no statistical differences between mela tonin suppression and the different wavelength stimuli. Only a more complete action spectrum determined with a larger subject sample is useful for determining which photoreceptors or pho top igments are involved in mediat ing mela tonin regulat ion in h u m a n s .

Theoretically, it should also be possible to determine an action spectrum for the therapeut ic effects of light. Cur ren t evidence suppor t s the hypothesis that light therapy for SAD works by way of light shining into the eyes as opposed to light on the s k i n .

1 04 T o begin characterizing which

ocular photoreceptors or photop igments mediate the therapeut ic benefits of light in winter depression, three consecutive studies have been done to specifically compare different por t ions of the spectrum for clinical efficacy in treating S A D .

1 8'

6 8'

89 In the first s tudy, 18 patients were treated with an

equal p h o t o n dose of white, blue or red light for a per iod of one w e e k .

18

The p h o t o n density of 2.3 χ 1 0

15 pho tons / cm

2/ s ec was selected because

this part icular p h o t o n density of b r o a d spectrum white light has been shown in many previous studies to be clinically effective in one week of t h e r a p y .

8 0 , 96 Pa t ients ' clinical s tatus before and after light therapy was

evaluated by means of the 21-item Hami l ton Depression Rat ing Scale (HDRS) , a s tandard scale for measur ing symptoms associated with dep re s s ion .

39 The results of this s tudy are il lustrated in Figure 6.

done to compare the efficacy of different monoch roma t i c wavelengths at equal pho ton densities for suppressing noc turna l p lasma mela tonin . The same exposure me thod described above was employed and two or three of the same subjects were tested at six different wavelengths. These prel iminary results are shown in Table 3.


SAD PATIENTS (N=18) TREATED WITH 2 5 1 2.3 χ 10

15 photons/cm

2/sec

E3 Pretreatment

• One Week Treatment

RED WHITE BLUE (615-685 nm) (400-760 nm) (430-465 nm)

F I G . 6. T h e ba r s in this g r a p h indicate m e a n + S E M H D R S values for pa t i en t s before t r e a tmen t (ha tched bars ) a n d after 1 week of t r e a tmen t wi th equa l p h o t o n densit ies of different light spec t ra (open bars ) . N u m b e r s in pa ren theses indica te

the half-peak b a n d w i d t h of the light s o u r c e .

18

This study was the first step towards defining an action spectrum of light therapy for winter depression. As shown in Figure 6, one week of light therapy with each of the three light sources produced an improvement in depression symptoms a m o n g the groups of patients tested. Specifically, the percent d rop in mean H D R S scores were 2 6 % , 4 7 % and 2 7 % for the red, white and blue light sources, respectively. Thus , the p h o t o n density emitted from the white light source elicited a significantly s tronger clinical response compared to the results obta ined from an equal p h o t o n density from the blue and red light s o u r c e s .

18 This suggests that b road spectrum

white light at this par t icular pho ton density is superior to restricted band widths of light in the red and blue por t ions of the visible spectrum. Tha t result implies that light sources for S A D light therapy could no t be improved by substi tut ing nar rower bandwidths of blue or red light in place of b r o a d b a n d white light.

A second study was done compar ing green light to red light at 2.3 χ 1 0

15

pho tons / cm

2/ s ec for treating S A D .

68 As in the first study, pat ients ' clinical

status before and after one week of light therapy was followed by means of the 21-item H D R S . The results of this study are shown in Figure 7.

As illustrated in Figure 7, one week of light therapy with either green or red light sources produced an improvement in depression symptoms in the groups of patients tested. The percent reduct ion in mean H D R S scores was 5 1 % and 3 0 % for the green and red light sources, respectively. Hence, at this pho ton density, green light was significantly stronger than the red light for treat ing winter dep re s s ion .

68 Considered alongside the results from the

study compar ing red, white and blue light therapy at the same p h o t o n density (Figure 6), the results of this study (Figure 7) suggest tha t b road


SAD PATIENTS (N=14) TREATED WITH 2.3 χ 1 0

15 photons/cm

2 /sec

Pretreatment


(505-555 nm) (615-685 nm)

F I G . 7. T h e ba r s in this g r a p h ind ica te m e a n + S E M H D R S values for pa t i en t s before t r e a t m e n t (ha tched ba rs ) a n d after 1 week of t r e a t m e n t wi th equa l p h o t o n densit ies of green or red light (open bars ) . N u m b e r s in pa ren theses indica te the

half-peak b a n d w i d t h of the light s o u r c e .

68

spectrum white light and nar rower band green light are equivalent in their capacity to reduce symptoms of SAD. Between the two studies, white and green light t rea tments were associated with a 4 8 % and 5 3 % reduct ion in H D R S scores, respectively. Compar i sons of g roup responses between different studies, however, are no t conclusive.

Are white and green light really equivalent in their pho to therapeu t ic s t rength? T o answer this quest ion, 12 pat ients were given 1 week of light therapy for S A D with either green or white light at an equal p h o t o n d e n s i t y .

89 Since therapy with white and green light appeared to cause

roughly equivalent H D R S reduct ions across the first two studies, the experimental p h o t o n density was lowered to 1.23 χ 1 0

15 p h o to n s / cm

2/ s e c

in the third study. Again, pat ients ' clinical s tatus before and after 1 week of light therapy was followed using the 21-item H D R S . The results of this study are illustrated in Figure 8.

As shown in Figure 8, 1 week of therapy using each of the light sources produced an improvement in depression symptoms . Specifically, the percent d r o p in mean H D R S scores was 2 2 % for the green light and 4 6 % for the white light sources. At this lower p h o t o n density, white light was superior to the green light in t reat ing S A D .

89

Together , these three studies form the g round work for determining the action spectrum for SAD light t h e r a p y .

1 8'

6 8'

89 The t radi t ional app roach

to defining a complete action spectrum, however, requires testing with nar rower bandwid th light stimuli with more tightly controlled light e x p o s u r e s .

2 6 , 86 A thoroughly defined act ion spectrum can guide the

development of light t rea tment devices tha t emit the o p t i m u m balance of wavelengths for t reat ing SAD. Fu r the rmore , an act ion spectrum will yield


Pretreatment


GREEN WHITE

(505-555 nm) (400-760 nm)

F I G . 8. T h e ba r s in this g r a p h indica te m e a n + S E M H D R S values for pa t i en t s before t r e a t m e n t (ha tched ba rs ) a n d after 1 week of t r e a t m e n t wi th equa l p h o t o n densit ies of whi te o r green light (open bars ) . N u m b e r s in pa ren theses indica te the

half-peak b a n d w i d t h of the light s o u r c e .

89

impor tan t information abou t the photosensory mechanism(s) responsible for the beneficial effects of light therapy. Current ly , it is p remature to predict what photopigment(s ) or photoreceptor(s) mediate the ant idepres-sant effects of light.

Several methodological problems will have to be overcome before further progress can be made in defining an action spectrum for SAD light therapy. One complicat ion for nearly all studies on SAD involves the fact that they are done on an outpat ient basis. Hence, pat ient compliance on t rea tment t iming, frequency and dura t ion cannot be closely controlled even with the most cooperat ive subjects. Fu r the rmore , as discussed earlier, very small changes in gaze direction and patient posit ion relative to the light source can cause great variability in the a m o u n t of light t ransmit ted to the pat ients ' e y e s .

2 9 , 36 Did pat ients have different gaze behaviors or

different pat terns of light usage with the different wavelength light sources? The op t imum method of compar ing different wavelengths—or any other phot ic parameter—for SAD therapy is to work with more carefully controlled exposures.

Across the wavelength studies outl ined above, each light t rea tment produced some therapeut ic improvements . Does this indicate that each light was at least partially effective in treat ing SAD symptoms, or are some of the therapeut ic benefits of light therapy due to a non-specific or placebo response? Since patient expectat ions of t rea tment ou tcome are thought to contr ibute significantly to the placebo effect, evaluat ion of expectat ions before t rea tment is one strategy for approaching this quest ion. Pr ior to any light t rea tment , subject expectat ions were systematically p robed in

SAD PATIENTS (N=12) TREATED WITH 1.23 χ 1 0

15 photons/cm

2 /sec


Conclusion

Over the past decade, considerable progress has been m a d e in exploring the potent ial of bright light therapy for affective disorders and distur-bances of the circadian system in h u m a n s . In fact, the research on the therapeut ic potency of light has ou tpaced the work on fundamental mechanisms by which light produces its beneficial effects. It is useful from both a scientific as well as a clinical perspective to establish the specific mechanisms in the eye which mediate the non-visual therapeut ic and biological effects of light. Studies with animal models most certainly can guide the way for an improved unders tanding of h o w light may produce its beneficial effects in m a n . Animal studies alone, however, are no t sufficient and must be matched by direct h u m a n studies to determine the relative impact of gaze behavior , s tatus of the ocular media , pupil lary response, retinal field and photorecep tor sensitivity on the biological and thera-peutic effects of light in h u m a n s .

each of the three wavelength studies. In general , all subjects had positive expectat ions abou t the success of light therapy bu t there were no differences between the expectat ions for the different light spectra in these s t u d i e s .

1 8'

6 8 , 89 This evidence suppor t s the idea tha t some of the

therapeut ic benefit of the different light spectra may have been due to a placebo response but tha t the differential therapeut ic responses to the different light spectra were not merely an extension of the pat ients ' preconceived beliefs.

It has been well documented that pat ients with a wide range of disorders—depression, schizophrenia and anxiety as well as cancer, diabetes and ulcers—can successfully respond to inactive or placebo t r e a t m e n t s .

3 2 , 3 3 , 83 Hence , it would be remarkable if SAD patients did not

show some level of placebo response to light therapy. In fact, therapeut ic improvements are almost always observed with light t rea tments regardless of light intensity, wavelength or d u r a t i o n .

8 0 , 96 Al though it is obvious tha t

light therapy indeed will reduce pat ients ' depression symptoms , the critical quest ion is how much of the pat ients ' response to light therapy is due to a non-specific placebo response versus a genuine clinical response? This remains an open quest ion in the SAD field and has been discussed most insightfully by E a s t m a n .

3 2 , 33 The inability to accurately separate placebo

responses from genuine clinical ant idepressant responses causes an element of "noise" in pho to the rapy da ta which seriously hinders the accurate discrimination of differential wavelength effects in light therapy. Unfortunately, until this quest ion is resolved, a more conclusive action spectrum for S A D pho to the rapy may not be possible.



The au thors would like to thank F . L. Ruberg and D . L. Brainard for their tho rough editorial assistance and manuscr ip t p repara t ion , and J. Georg iou for l ibrary and reference suppor t . This work was suppor ted by the N I H G r a n t # R O 3 - M H - 4 4 8 9 0 - 0 1 A l , the F D A G r a n t #785346, the Na t iona l Electrical Manufacturer ' s Assoc. (#LRI 89:DR:1), the Lighting Research Inst . (#LRI 88:SP:LREF:6) , the Dean ' s Overage Research P r o g r a m of Jefferson Medical College, U S U H S G r a n t C07049, and the Phi ladelphia Chap te r of the I l luminating Engineering Society.

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4

The Mammalian Melatonin Rhythm Generating System D A V I D C. K L E I N

Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

A DAY/NIGHT rhythm in circulating melatonin occurs in all vertebrates, with high values always coinciding with the dark period of the night. An example of this rhythm, as detected in cerebral spinal fluid of a non-human p r i m a t e

6 0'

6 0* ,

is presented in Figure 1. The nocturnal increase in circulating melatonin results from the increased product ion of melatonin by the serotonin -> N-acetylserotonin->melatonin pathway, as outlined in Figure 2. This rhythm is driven by an endogenous ~ 24-hour clock, which is synchronized with the 24-hour day/night cycle; the synchronizing signal is light.

In non-mammal ian species the clock and the photodetect ion system are typically located within the pineal gland itself. However, in mammals these elements are located outside the pineal gland. The clock is located in the suprachiasmatic nucleus of the hypothalamus ( S C N )

32 and light is detected

by the retina. In constant darkness the rhy thm persists because the S C N continue to drive the pineal gland (Figure 1); however, the rhythm becomes slightly longer or shorter than 24 hours . The term "melatonin rhy thm generating system" was coined to emphasize that the normal rhy thm in melatonin in mammals reflects the integrated functioning of the pineal gland, SCN and e y e s .

34 Lesions at any point in the system dramatically disrupt the

rhythm, resulting in either no detectable rhythm or a rhy thm which is not synchronized to the environmental lighting cycle, a circumstance described as free-running (see Figure 1).

The components of the melatonin rhythm generating system are given in Figure 3. In addit ion to the critical functional components of the system— the pineal gland, S C N and eye—other elements of the system function as wiring and relay points.

55


ε co υ

Lighting food del ivery

1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 Clock time (h)

F I G . 1. Dai ly r h y t h m in m e l a t o n i n in the C S F of a n o n - h u m a n p r i m a t e . N igh t is represented by d a r k n e s s . F o r 3 days an ima l s were m a i n t a i n e d in a l igh t /da rk cycle. After t h a t an ima l s were m a i n t a i n e d in c o n s t a n t d a r k n e s s , d u r i n g which t ime the r h y t h m in m e l a t o n i n pers is ted. C S F was con t inua l ly w i t h d r a w n ; m e l a t o n i n was

m e a s u r e d by r a d i o i m m u n o a s s a y (from reference 60a) .

Historical perspective

As is true of many aspects of pineal biochemistry, physiology and pharmacology, the concept of a mult icomponent system which regulates the melatonin rhythm can be traced to the pioneering work of McCord and Allen in 1 9 1 7 ,46 who first demonstra ted that small amounts of extracts of the pineal gland cause frog skin to lighten. This was followed up by Aaron Lerner and his coworkers at Yale University in the late 1 9 5 0 s . 4 3' 44 They extracted large amounts of cow pineal glands and were able to isolate a skin lightening compound and determine its structure. The compound was called melatonin.

The next major step was to demonstra te that melatonin could be synthesized within the pineal gland. This occurred soon after the structure of melatonin became known, as a result of the work of Julie Axelrod and his c o w o r k e r s . 6' 9 1' 92 They obtained bovine pineal tissue preparat ions from

Mammalian Melatonin Rhythm System 57

Sero ton in ( 5 - H y d r o x y t r y p t a m i n e )

( p m o l / m g )

N- ace ty l t rans fe rase ( n m o l p r o d u c t / m g / h )

N- a ce ty l se ro ton in (5-hydroxy- N- a ce ty l t ryp t amine )

Hydroxy indo le -O-me thy l t r ans fe r a se (nmol p r o d u c t / m g / h )

M e l a t o n i n (5 - m e t h o x y - N- a ce ty l t ryp t amine )

( p m o l / m g )

1200 1800 2400 0600 1200

Time

F I G . 2. Dai ly r h y t h m s in the s e r o t o n i n - > m e l a t o n i n p a t h w a y in the p inea l g land . R h y t h m s in the se ro ton in ->7V-acetylserotonin->melatonin p a t h w a y are con-trol led by large changes in the activity of se ro ton in JV-acetyl transferase activity

(after reference 25).

Lerner and using these quickly found that the pineal gland could convert serotonin to 7V-acetylserotonin and 7V-acetylserotonin to melatonin. It had already been known that the pineal gland contained an abundan t amoun t of s e ro ton in .

18 Taken together, these advances established that the pineal

gland could make melatonin. The fact that there were large day/night changes in the amounts of indole

compounds in the pineal gland was recognized by several w o r k e r s ,

1 7'

5 7 - 5 9'

69 who found that the amounts of serotonin and melatonin

in the pineal gland exhibited day/night differences. The next critical finding was our discovery of a very large ( ~ 100-fold)

rhythm in the enzyme which converts serotonin to 7V-acetylserotonin, serotonin 7V-acetyltransferase (arylalkylamine 7V-acetyltransferase E.C.


F I G . 3. Mela ton in r h y t h m genera t ing system: T h e r h y t h m is genera ted in the suprach iasmat ic nuclei ( S C N ) a n d s t imula tory signals a re t ransmi t ted to the pineal g land (P) by the indicated pa thway . Light acts t h rough re t inohypo tha lamic projec-t ions ( R H P ) to adjust the clock by resetting in a n d by adjust ing the du ra t ion of the per iod dur ing which the S C N will s t imulate the pineal g land. Light also acts to block t ransmiss ion of signals from the S C N to the pineal gland. T h e do t ted line represents the m e l a t o n i n f e e d b a c k loop . T h e following abbrevia t ions are used: O C , optic chiasm; S C N , suprach iasmat ic nuclei; P V N , paravent r icu la r nucleus of the h y p o t h a l a m u s ; M F P , medial forebrain bund le ; R F , reticular format ion; I M L , in termediola tera l cell co lumn of spinal cord ; S C G , superior cervical ganglia; I C N ,

internal carot id nerve; N C , nervi conari i (from reference 36).

2 . 3 . 1 . 8 7 ) .

3 , 5 0 , 89 Based on this we proposed that the daily rhythm in

melatonin was generated by the rhythm in serotonin 7V-acetyltransferase a c t i v i t y .

2 5'

39 This rhythm produced a large nocturnal increase in N-

acetylserotonin, which in turn generated a large nocturnal increase in melatonin product ion through a mass action effect mediated by hydroxy-indole-O-methyltransferase; large day/night changes in the activity of this enzyme do not appear to occur.

The discovery of the rhythm in TV-acetyl transferase activity was not only of significant intellectual interest to scientists interested in understanding the molecular basis of the melatonin rhythm. It also was of great practical importance because it provided investigators with a sensitive means of monitoring pineal function, at a time before methods to measure melatonin were available.

These early findings stimulated research on many aspects of the melatonin rhythm: Wha t anatomical structures are involved in generating the rhythm? H o w are they connected? Wha t transmitters control melatonin product ion? Wha t is the biochemical basis of the increased synthesis of melatonin?

Funct ional a n a t o m y

The elements compris ing the mammal i an mela tonin rhy thm generat ing system appear in Figure 3 .

27

The eye

The impor tance of the eye in the melatonin rhy thm generat ing system was demons t ra ted in studies which extended the discovery that the noc turna l


increase in 7V-acetyltransferase activity is suppressed by l i g h t .

38 Experi-

ments determined tha t light does no t act when the eyes were removed or when the optic nerves were severed. This indicated that the eyes mediated the effects of light. It was also found tha t light exposure at night causes a very rapid decrease in TV-acetyltransferase activity, and tha t this effect is also mediated by the e y e s .

39 These findings were subsequently confirmed

in h u m a n s .

66

Peripheral structures

The pineal gland is innervated by postganglionic projections from the superior cervical ganglia (SCG), which course with the inferior carot id nerve and then b ranch off to form the nervi conari i . The latter travel over the cerebellum to the pineal gland. The impor tance of these nerves in regulating mela tonin synthesis came from studies which demons t ra ted that removal of the S C G blocks the large noc turna l increase in N-acetyltransferase activity and in mela tonin p r o d u c t i o n .

3 6'

42

The rhy thm in JV-acetyltransferase activity is no t only dependent u p o n the presence of the S C G , but also u p o n central innervat ion of the ganglia. This was demons t ra ted by the finding tha t the rhy thms in N-acetyl t rans-ferase activity and melatonin p roduc t ion are absent following de-c e n t r a l i z a t i o n .

3 6'

42 These findings indicated that the clock regulating the

pineal gland is not located in the pineal gland or S C G , but elsewhere. Subsequent investigations have indicated that the pineal-controll ing

neuronal circuit coursing th rough the S C G is insulated from general sympathet ic t raff ic .

4 2 15 Stress, for example, has relatively little effect on

pineal f unc t i on .

54

The role of the S C G in control l ing the mela tonin rhy thm in m a n is evident from the observat ion that there is no mela tonin rhy thm in individuals with spinal cord injury blocking central innervat ion of the g a n g l i a .

4 2a

Central structures

With the evidence that central innervat ion regulated pineal function, it was proposed tha t the endogenous clock control l ing the pineal gland was located in the brain . The available experimental evidence pointed to the hypo tha lamus as a likely site of the clock, according to studies by Richter, who had found tha t massive lesions in tha t region disrupted locomotor activity r h y t h m s .

63 O u r interest focused specifically on the S C N , because

of new evidence from amino acid tracing experiments which established that a re t ino-hypothalamic projection terminated t h e r e .

4 7a

The role of the S C N in the mela tonin rhy thm generat ing system was investigated in rats and a n o n - h u m a n pr imate using lesioning


e x p e r i m e n t s .

3 1 , 4 7 , 61 In bo th cases it was found that lesioning the S C N

profoundly altered the rhythms in N-acetyltransferase activity and melatonin . These observat ions and related findings established that the t iming mechanisms regulating the pineal gland are located in the SCN.

The functional impor tance of the re t ino-hypothalamic project in the mela tonin rhy thm generat ing system was also established by lesioning e x p e r i m e n t s .

3 1'

47 These demons t ra ted that cutt ing all other visual inputs

from the ret ina did no t block phot ic regulation of N-acetyltransferase activity, but that lesions which destroyed the re t ino-hypothalamic projection block all effects of light on pineal 7V-acetyltransferase activity. These effects include entraining the clock, modula t ing the dura t ion of the period the clock can st imulate the pineal gland, and suppressing S C N st imulat ion of the pineal gland. (See Chapte r 11.)

The neural elements in the S C N -»pineal circuit which linked the S C N to the S C G appear to include projections from the S C N to the paraventr icu-lar nucleus of the hypo tha lamus (PVN) and monosynapt ic projections from the P V N to the S C G . This was first indicated by tracing experiments which demons t ra ted that S C N P V N and P V N - > S C G projections e x i s t .

8 , 70 These observat ions st imulated experiments which demons t ra ted

that destruct ion of the P V N blocks the rhy thm in 7V-acetyltransferase activity and melatonin p r o d u c t i o n ;

36 and , tha t electrical s t imulat ion of the

P V N stimulates these biochemical end p o i n t s .

95

Pineal signal transduction

Norepinephr ine is contained in the sympathet ic nerves which innervate the pineal gland and is released at n i g h t .

1 0 a'

1 9'

94 Using organ culture

experiments it was found tha t t rea tment with norepinephrine produces the same changes in pineal biochemistry which occur at night, including an increase in A/-acetyltransferase activity and melatonin p r o d u c t i o n .

5'

40

Based on these findings, it appears tha t norepinephrine is the t ransmit ter which controls large changes in mela tonin product ion in the mammal i an pineal gland. Organ cul ture studies together with whole animal studies established that the large day/night changes in mela tonin product ion are due to large changes in the activity of serotonin N-acetyltransferase activity. Accordingly, interest in the molecular basis of how melatonin product ion is regulated usually is focused on TV-acetyltransferase and the mechanisms th rough which receptors control the second messengers which regulate the activity of this enzyme (Figure 4), namely cyclic A M P and C a

+ + .

2 8'

2 9'

96

Norepinephr ine acts on the pineal gland th rough at least three adrenergic receptors, which control interacting processes to regulate melatonin product ion . These interactions are mediated by electronic-like biochemical " A N D " gates. Activation of such gates by any one receptor

Mammalian Melatonin Rhythm System 6 1

ADRENERGIC REGULATION OF PINEAL NAT

β* f Ca2-

Ρ ι ,

F I G . 4. " A N D " G a t e regu la t ion of p inea l 7V-acetyltransferase act ivi ty. Cyclic A M P is con t ro l led by a d u a l r ecep to r m e c h a n i s m . Ful l s t imula t ion of the enzyme a p p e a r s to requi re ac t iva t ion of all th ree recep tors , ^ - ad rene rg ic s t imu la t ion act ivates adenyly la te cyclase(AC) t h r o u g h the G T P b ind ing s t imula to ry p ro t e in (Gsa) . a rA d r e n e r g i c s t imu la t ion elevates [ C a

+ +] i? which resul ts in m a x i m a l

ac t iva t ion of A C t h r o u g h a C a

+ +, p h o s p h o l i p i d d e p e n d e n t p ro t e in k inase ( P K C )

m e c h a n i s m . Cyclic A M P a n d C a

+ + act in concer t to s t imula te W-acetyltransfer-

ase act ivi ty.

has little influence. However , s imul taneous act ivat ion of these gates by two receptors produces d ramat ic responses (Figure 4).

The adrenergic receptors involved belong to the β±- and α x- subgroups . The βχ-adrenergic receptor st imulates adenylate cyclase via a G T P binding regulatory p r o t e i n .

7 , 55 This results in a part ial increase in the

intracellular concentra t ion of cyclic A M P . Activation of this receptor is essential for any s t imulat ion of mela tonin p roduc t ion to occur. The second receptor involved is the αx-adrenergic r e c e p t o r .

7 2'

75 Agonist occupancy of

this receptor has several effects. The mos t rapid is an increase in [ C a

+ +] i .

78 Another is o^-adrenergic s t imulat ion of phosphat idyl inositol

turnover , which generates d i a c y g l y c e r o l .

2 1'

23 Together , the increase in

[ C a

+ +] i and diacylglycerol activates C a

+ + , phosphol ip id


dependent protein kinase ( P K C ) .

2 2 , 74 This activation potently enhances

β- adrenergic s t imulat ion of cyclic A M P produc t ion and N-acetyltransfer-ase a c t i v i t y .

7 7 , 7 9 , 86 Together , these elements form a P K C / G protein

" A N D " gate to control adenylate cyclase. C a

++ also acts downs t ream

with cyclic A M P to st imulate the p roduc t ion of 7V-acetyl transferase, resulting in an increase in melatonin product ion . Mos t recently, a2 D- ad rene rg i c receptors have been identified in the mammal i an pineal g l a n d .

6 7 , 68 The function of these receptors remains u n c l e a r .

6 83

The large changes in iV-acetyltransferase are initiated by the act ion of C a

++ and cyclic A M P ; C a

++ strongly potent iates the effects of cyclic

A M P .

96 O n e effect of adrenergic st imulation is to increase gene

expression, which occurs dur ing a period of abou t 90 minutes after the start of adrenergic s t imulat ion; it is possible that bo th C a

++ and cyclic

A M P are involved in this, a l though this has not been proven. Thereafter, protein synthesis is required for the enzyme to reach full s t i m u l a t i o n .

2 8 , 40

In addi t ion, the s t imulat ion of A^-acetyltransferase activity requires ongoing cyclic AMP-dependen t activation of the e n z y m e .

30 Accordingly,

a rapid decrease in norepinephr ine causes a rapid decrease in cyclic A M P , which results in inactivation of 7V-acetyltransferase and cessation of melatonin synthesis.

In summary , at least three elements are required for s t imulat ion of enzyme activity, t ranscr ipt ion, t ranslat ion and activation. The second messengers involved appear to be cyclic A M P and C a

++ , which act

th rough a cyclic A M P / C a

+ + " A N D " gate to st imulate N-acetyltransfer-

ase ac t iv i ty .

96 The simplest scheme is that cyclic A M P and C a

+ + enhance

generat ion of m R N A encoding serotonin TV-acetyltransferase, which then directs synthesis of the protein; and , tha t the protein undergoes cyclic A M P , and /o r C a

+ +- d e p e n d e n t activation. However , this may not be the

case because the possibility exists tha t 7V-acetyltransferase activity is regulated by an act ivator molecule which is controlled by the scheme outlined above.

In addi t ion to the s t imulatory actions detailed above, inhibitory-negative feedback-actions of norepinephr ine also ex i s t ,

71 involving

protein kinase C (Figure 4). These mechanisms may provide a rapid means of down-regulat ing signal t ransduct ion at the membrane level.

The rapid inactivation of N-acetyltransferase may involve protein thiol:disulfide e x c h a n g e .

3 3 , 4 1 , 51 Cystamine and similar disulfides act

th rough this mechanism to inactivate the enzyme in the intact cell and in broken cell p r e p a r a t i o n s .

8'

5 2 , 53 It is possible that cyclic A M P controls

protein thiol disulf ide exchange by controll ing an enzyme which converts a reservoir of free thiols to potent inactivating disulfides in the cell, thereby triggering inactivation of the enzyme.

It should be noted that cyclic A M P and C a

++ have been found to

control expression of genes in other systems; this is one possible site of


interaction of the cyclic A M P / C a

++ " A N D " gate. O the r possibilities

include the media t ion of cyclic A M P dependent and of C a

+ + dependent

kinases, or of the media t ion of a cyclic A M P / C a

+ + act ivated kinase.

Another interesting possible mechanism th rough which 7V-acetyltrans-ferase is regulated is m e m b r a n e potent ial (46). An intriguing observat ion is that the s t imulat ion of 7V-acetyltransferase activity requires m e m b r a n e hype rpo la r i za t ion ,

55 and tha t this hyperpolar izat ion is p roduced by the

cyclic A M P / C a

++ act ivation of a K

+ c h a n n e l .

13 The biochemical

significance of hyperpolar izat ion is no t yet clear. It might reflect changes in a t r ansmembrane spanning kinase or reductase, whose structure and activity are influenced by t r ansmembrane potent ial . F o r example, hyperpolar izat ion might activate an enzyme which keeps N-acetyl t rans-ferase in a reduced stated.

Al though it is obvious that the rhythmic p roduc t ion of mela tonin is regulated by the rhy thm in 7V-acetyltransferase activity, the impor tance of other steps in the t ryp tophan mela tonin pa thway should no t be u n d e r e s t i m a t e d .

73 Changes in any one of the enzymes and cofactors

required for this conversion could alter the a m o u n t of mela tonin produced when the activity of A^-acetyltransferase is elevated.

The melatonin^SCN feedback loop

Several lines of evidence indicate that mela tonin feeds back into the mela tonin rhy thm generat ing system and tha t the pr imary mela tonin target in the system is the S C N .

12 High affinity mela tonin binding sites

have been identified in the S C N of most m a m m a l s , using [

1 2 5I ] 2 -

iodomela tonin . (See Chap te r 6 . )

14 The concent ra t ion of mela tonin

binding sites in the rat S C N is more than 10-fold higher than in essentially all other tissues in mammal s , except for the neonata l pi tui tary gland and the pars tuberal is .

Mela ton in acts on cells in the S C N , as indicated by electrophysiological analysis of ra t t i s s u e .

19 Based on studies using other cells with mela tonin

binding sites, including melanocytes , cells in the pars tuberalis , and neonata l gonado t rophs , it appears tha t mela tonin acts th rough the same type of high affinity binding site found in the S C N .

4 8'

4 9'

8 0'

8 1'

8 5'

9 0'

93 This

establishes these sites as b o n a fide receptors . Mela ton in receptors are linked to G T P - b i n d i n g regulatory proteins

(G-proteins) which are characterized by sensitivity to pertussis t o x i n .

1 1'

4 8'

4 9'

64 Studies on the neonata l rat pi tui tary cell indicate that a

pr imary act ion of mela tonin is hyperpolar izat ion of the p lasma mem-brane . This can alter cell physiology th rough a variety of voltage-sensitive channels. F o r example, melatonin- induced hyperpolar izat ion blocks the GnRH- induced increase in [ C a

+ +] i in a subpopula t ion of melatonin-


sensitive gonado t rophs by inhibit ing C a

++ influx th rough voltage-

sensitive C a

++ c h a n n e l s .

84 It seems possible tha t hyperpolar izat ion in

other cells could have similar effects on agonist- induced increase in [ C a

+ These features provide reason to place melatonin receptors in a

g roup of receptors whose actions are generally similar, including bo th hyperpolar izat ion and inhibit ion by pertussis tox in .

3

Hyperpolar iza t ion, however, is not the only effect of mela tonin . Mela tonin has also been found to block forskolin-induced increases in c A M P product ion in several systems, p robably th rough an effect on adenylate cyclase mediated by an inhibitory G - p r o t e i n .

4 8'

49 Effects on

other second messenger systems have also been r e p o r t e d .

8 7 , 88 Accord-

ingly, it would seem that mela tonin could act on S C N cells th rough a variety of mechanisms.

The most consistent physiological effect of adminis t ra t ion of mela tonin on biological rhy thms is to modify e n t r a i n m e n t .

3 , 4 5 , 66 This has been found

in bo th experimental animals and in humans , as reviewed in detail in Chapte r 12. The work on h u m a n s has been done in par t with blind subjects and clearly demonst ra tes that mela tonin adminis t ra t ion can serve as a synchronizing cue which prevents free running circadian rhythms. It appears likely that mela tonin is acting directly on the S C N because adminis t ra t ion of mela tonin entrains several circadian rhy thms , which suggests tha t a c o m m o n site of act ion is involved.

4 In addi t ion, mela tonin

adminis t ra t ion produces a phase response curve—advancing the clock at certain times and retarding it at o thers—most likely reflecting S C N i n v o l v e m e n t .

4 5 , 66

The finding that mela tonin acts on ent ra inment is consistent with the view that melatonin is useful in preventing jet l a g .

1 ,2 In addi t ion, it is

thought that melatonin might be used successfully to minimize undesirable effects of phase shifting in circumstances where h u m a n s are subject to frequent changes in working schedules, such as in east-west intercontin-ental travel and shift work; and , it may be of practical value in entraining blind subjects to the environmenta l lighting schedu le .

2a

The m e l a t o n i n - * S C N feedback loop may also be involved in phot ic-induced changes in physiology which occur on a seasonal b a s i s .

1 6 , 2 4 , 97

These changes can be mimicked by adminis t ra t ion of melatonin . Mela tonin only acts dur ing a na r row window of t ime, late in the a f t e r n o o n .

20 The precise details of this have not been worked out .

However , it seems probable that the S C N is involved. T o summarize , a m e l a t o n i n S C N feed back loop appears to exist in

many mammal i an species and probably plays a physiological role in the melatonin rhy thm generating system, perhaps fine tuning the process of ent ra inment . The existence of melatonin sensitivity in the S C N provides investigators and clinicians with the oppor tun i ty of accessing and altering circadian function th rough prudent t ime-dependent use of mela tonin .


Deve lopment

The development of the mela tonin rhy thm generat ing system reflects the ontogeny of each e l e m e n t .

2 6 , 34 O n e of the earliest to develop is clock

function in the S C N .

62 A circadian rhy thm in iV-acetyltransferase is first

detectable in the rat pineal gland at the end of the first week of l i fe .

15 O n e of

the last elements to develop is the enzymatic capacity of the pineal gland to convert 7V-acetylserotonin to m e l a t o n i n .

34 This topic has been covered in

several b o o k s .

2 6'

59 Of special developmental interest is the observat ion

that mela tonin mediates striking seasonal control over reproduct ive development seen in some m a m m a l s (see Chap te r 6 ) .

65

Evolut ion

The evolution of the mammal i an eye S C N -> pineal mela tonin rhy thm generat ing system is beginning to be unders tood . As indicated in the In t roduct ion , the system seems to have evolved late in vertebrate evolution, because it is absent or r edundan t in non -mammal i an verte-brates, which appear to have a mela tonin rhy thm generat ing system contained within their pineal gland. The evolut ion of the m a m m a l i a n melatonin rhy thm generat ing system reflects a distinct improvement—one which centralizes circadian functions—an opt imizat ion which uses the S C N as the single master circadian clock driving all rhy thms.

In considering evolut ionary aspects, it is impor tan t to note that the pineal gland and retina share a number of striking biochemical and anatomical features not found e l s ewhere .

54 These include pho to t r ansduc-

tion molecules and melatonin synthesizing enzymes and photoreceptors . F r o m lower to higher vertebrates there is a gradient of cross-over in function and expression of these features, with bo th mela tonin produc t ion and pho to t ransduc t ion evident in bo th the pineal and ret ina of lower forms; in mammal s , however, mela tonin p roduc t ion is generally restricted to the pineal gland and photorecept ion is limited to the retina. The ability of the pineal gland and ret ina of lower forms to detect light and m a k e melatonin p robab ly reflects the evolution of bo th tissues from a c o m m o n primitive photochemical t ransduct ion system which may function in ancestral vertebrate forms.

The associat ion of mela tonin with darkness m a y have its origins very early in the history of life on ear th . This is based on the finding that the last enzyme in mela tonin synthesis, hydroxyindole-O-methyl transferase ( H I O M T ) , is homologous to an enzyme found in Rhodobacter capsulata, hydroxyneurosporene-O-methyl t ransferase ( H N O M T ) .

35 The latter O-

methylates carotenoids , which function in R. capsulata to absorb light and transfer energy to the photosynthet ic appa ra tus ; they also protect against photo-oxidat ive damage . Within a 180 amino acid-long region of


H I O M T , 55 (31 % ) of the residues are identical and an addi t ional 78 (43%) represent conservative subst i tut ions. The similarity of the pr imary structures of H I O M T and H N O M T indicates bo th enzymes evolved from a c o m m o n ancestral protein.

R. capsulata is thought to be closely related to a primitive endosym-biont , existing 2 to 3 billion years ago, which evolved within the primitive eukaryot ic cell to become the mi tochondr ion .

Relevant to the melatonin—darkness link is the observation that the activity of each enzyme decreases as light intensity increases from low to high levels and vice versa. In the case of R. capsulata the entire photo-synthetic appara tus , including H N O M T , appears to increase in dim light. This is an adaptive survival strategy which increases the efficiency of light energy capture when light intensity is low; such an adapta t ion is an obvious advantage to a photosynthet ic organism, especially several billion years ago when light was the energy source and the a tmosphere was devoid of oxygen.

Based on these observat ions, it would appear tha t phot ic regulat ion of O-methylat ion originated as pa r t of a general adapt ive strategy to increase photosynthet ic efficiency, and was recruited to suppor t mela tonin product ion . Al though there is no evidence that R. capsulata can synthesize melatonin , it is still fascinating that traces of the mela tonin rhy thm generating system are found at the earliest times in the evolution of life closely associated with the phot ic environment .

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95 . Y a n o v s k i J., Wi tche r J., Adler N . , M a r k e y S. a n d Kle in , D . C . (1987) S t imula t ion of the pa raven t r i cu l a r nuc leus a rea of the h y p o t h a l a m u s elevates u r ina ry 6 -hydroxymela ton in du r ing day t ime . Brain Res. Bulletin 19 , 129 -133 .

96. Yu L., Schaad N . a n d Kle in D . C . (1991 ) C a l c i u m po ten t i a t e s cyclic A M P s t imula t ion of p ineal N-ace ty l t ransferase . J. Neurochem. (in press) .

97. Z u c k e r I., Lee T . M . a n d D a r k J. (1991) T h e s u p r a c h i a s m a t i c nucleus a n d a n n u a l r h y t h m s of m a m m a l s . In Suprachiasmatic Nucleus (eds. Kle in D . C , M o o r e R.Y. a n d Reppe r t S.M.) , p p . 246 -259 . Oxford Press , N e w Y o r k .

5

Circadian Photoreception in Mammals and Other Vertebrates R U S S E L L G. F O S T E R a n d M I C H A E L M E N A K E R

Department of Biology and NSF Center for Biological Timing, University of Virginia, Charlottesville, VA 22903, USA

C I R C A D I A N RHYTHMS are driven by endogenous pacemakers tha t have periods close to , bu t rarely exactly, 24 hours . In order to function adaptively in the real world, circadian systems, which include pacemakers and the many rhy thms tha t they control must be synchronized (entrained) with the as t ronomical day—their per iods must be constrained to become exactly 24 hours (at least on average). En t ra inment regulates not only period but more impor tant ly , phase . When entrained, the pacemaker of the circadian system must , of necessity, adop t a determinate phase relat ionship with the as t ronomical day while individual circadian rhy thms may each adop t par t icular and different phase relat ionships depending on the specifics of their coupling with the pacemaker . It is phase relat ionships with the envi ronment and with other rhy thms tha t are adapt ive and have therefore been subject to na tura l selection. Indeed, it is the sole function of the circadian system to regulate the times at which biological events occur; times either in relation to specific features of the daily environmenta l cycle such as dawn or dusk or in relation to other cyclic events within the organism. Viewed from a circadian perspective, an organism is an ensemble of adapt ive phase relationships a m o n g mult iple rhy thms at all levels of organizat ion from the molecular to the behavioral .

Thus the process of en t ra inment is central to circadian function and must be unders tood in detail if we are to unders tand circadian systems. This fact has been recognized for some time, and as a result a good deal of effort has been expended on experiments which have addressed various aspects of en t ra inment . Centra l to a lmost all of these experiments has been the recognit ion tha t the dominan t environmenta l entraining agent is the daily a l ternat ion of light and darkness . Al though other

7 3


environmental cycles (principally temperature) can entrain circadian systems, it is the light cycle which, because of its precision and reliability, has been used by na tura l selection as the major external tempora l reference. Tha t is no t at all surprising, however, many of the most "obvious" assumpt ions abou t the mechanisms tha t underlie phot ic ent ra inment of circadian systems have turned out on experimental examinat ion to be unwar ran ted . The first of these is that , for vertebrates, phot ic ent ra inment is mediated by the eyes; it is this assumpt ion that we examine in detail below.

Al though it had been known for some time that some vertebrates possessed extraretinal , non-visual p h o t o r e c e p t o r s ,

1 8'

48 the first report

that such receptors mediated circadian ent ra inment was the work of M e n a k e r

35 who showed that house sparrows (Passer domesticus) would

entrain to labora tory light cycles even though they had been blinded by bilateral enucleation. In a series of papers tha t followed, Menake r and his colleagues demons t ra ted tha t :

(1) The intensity of light passing th rough the skull and reaching the brain of blinded birds could be manipula ted by the removal of feathers and the subdura l injection of India ink, and that whether or not the birds entrained to a part icular light cycle depended on how much light reached the b r a i n .

36

(2) N o t only ent ra inment to light cycles, bu t also the effect of constant light on circadian period length and the effect of bright constant light in making birds a r rhythmic , as well as the reproduct ive effects of different artificial day lengths (photoper iodism) were partially mediated by extraret inal photoreceptors (see Figure l ) .

3 7'

38

(3) The eyes were involved in three of the above four responses to light but were not involved at all in photoper iodic p h o t o r e c e p t i o n .

31

Fur the rmore , in mediat ing ent ra inment and period length, the effects of light perceived by the eyes were additive with those of light perceived by extraretinal photoreceptors , while for the effects of bright constant light in producing arrhythmici ty there was an absolute requirement for light perceived by the e y e s .

3 2'

3 3'

34

(4) The presence of the pineal gland was no t required for blind birds to entrain to light cycles or to respond reproductively to photoper iod (involvement of the pineal in the effects of constant light could no t be ascertained, as its removal renders sparrows ar rhythmic in either constant light or constant d a r k n e s s ) .

2 0'

39

(5) M a n y of the same results were obta ined when similar experiments were performed on several species of l i z a r d s .

57

These results were extended by several laborator ies to other birds and

F I G . 1. Activity records (double -p lo t t ed) from four h o u s e s p a r r o w s in which low a m p l i t u d e l ight cycles from l ight -emi t t ing d iodes ( L E D ) of different co lors were delivered direct ly to the m i d b r a i n . T h e L E D s were i m b e d d e d in b lack plas t ic in a sha l low stainless steel c u p m o u n t e d o n the skul l , b u t were opt ical ly coup led t o a l ight guide wi th in a stainless steel t ube which was imp lan t ed s tereotact ical ly in the b r a in . T h e L E D s were connec ted by fine wires t h r o u g h a c o m m u t a t o r t o a c lock-cont ro l led p o w e r supply a n d the r o t a t i o n of the c o m m u t a t o r as the b i rd m o v e d was sensed by a n infrared de tec to r d i rec ted a t s tr ipes p a i n t e d o n its surface. T h u s "ac t iv i ty" reflects small m o t i o n s of the h e a d (as little as a t u r n of 3°) as well as gross m o v e m e n t s , r a the r t h a n the m o r e usual ly r ecorded "pe rch h o p p i n g . " Bold rectangles o n the r ight halves of each pane l ind ica te the t imes du r ing which cu r ren t was passed . At o the r t imes the b i rds were in c o n s t a n t d a r k n e s s . A is a con t ro l in which cu r r en t was passed t h r o u g h the L E D , b u t n o light was pe rmi t t ed to escape; C a n d D show, respectively, weak e n t r a i n m e n t a n d weak m a s k i n g by the lights-off t r ans i t ion ; Β is the m o s t in teres t ing record , for it shows clearly t ha t a l t h o u g h the b i rd ' s c i rcad ian sys tem was n o t en t r a ined by the light cycle, the light h a d a s t rong inh ib i to ry effect o n m o v e m e n t ; indeed , the b i rd ha rd ly t u r n e d its h e a d d u r i n g the ent i re 12 h o u r s of l ights-on each day . Th i s one record suggests t ha t there m a y well yet be u n a p p r e c i a t e d effects of ext rare t ina l ly perceived light o n the cent ra l ne rvous sys tem ( M e n a k e r M . a n d

Vaa le r G. , unpub l i shed ) .


reptiles as well as to fish and amphib ians . Representative species from all vertebrate classes, except mammal s , have extraretinal photoreceptors . F o r example, in the eel (Anguilla anguilla) the synchronizat ion of circadian locomotor rhy thms requires extraocular and extrapineal p h o t o r e c e p t o r s .

58 Similar findings have been reported in other fish

spec ies .

53 In the frog (Rana esculenta) electrophysiological responses to

light have been recorded from the basal brain in blinded and pinealecto-mized a n i m a l s ,

5'

6 a l though no studies have so far a t tempted to link these

photoreceptors with circadian physiology. In addi t ion to their role in the circadian system, extraretinal pho to -

receptors have also been shown to play an impor tan t par t in the regulation of seasonal reproduct ive cycles in non -mammal i an verte-brates. The pr imary factor controll ing seasonal breeding in temperate-zone birds is the annua l variat ion in daylength, but birds do not require their eyes to perceive this change. Blinded ducks exposed to long photoper iods grow large t e s t e s .

2'

3 M o r e recent studies, involving

enucleation, sectioning of the optic nerve and shielding light from entering the brain confirmed this original finding in a range of bird spec ies .

11 M a n y of these studies showed that , unlike circadian responses

to light, gonadal growth occurred at maximal rates in the absence of the eyes, suggesting that ocular photoreceptors did no t even contr ibute to the detection of the daylength signal. Initially, the pineal was considered as the pr ime candidate for this extraocular photoreceptor , bu t several studies demons t ra ted that the pineal appeared to play no significant role in avian photoper iodic responses. In quail , for example, i l luminating the pineal with radio luminous paint does not st imulate gonadal g r o w t h

26

and pinealectomy of either blinded or intact quail leaves photoper iodic induct ion u n a f f e c t e d .

5 0'

51 In contras t , i l lumination of the basal brain

with luminous paint or fiber optics in blinded and pinealectomized a n i m a l s

4 2'

4 4'

61 causes gonadal growth at maximal rates. These experi-

ments provided s t rong addi t ional evidence for the existence of a "deep brain pho torecep tor" located within the basal brain of birds.

Taken together, these studies have led to the current , generally held view tha t all non -mammal i an vertebrates have several kinds of non-visual, extraret inal , extraocular photoreceptors and use them for circa-dian ent ra inment as well as a variety of other photically regulated activities. Vertebrate photoreceptors include deep brain photoreceptors , the parietal eye (when present) , the pineal gland (the photorecept ive capacity of which has been clearly and repeatedly demons t ra ted and is probably required for en t ra inment of its own internal circadian oscil-lator) in addi t ion to the eyes themselves. The widespread occurrence of extraretinal photoreceptors in non-mammal i an vertebrates (Figure 2) is in direct contras t to the si tuation in m a m m a l s which use retinal photoreceptors exclusively (but see Torres and L y t l e

5 5) .

Circadian Photoreception in Vertebrates (40) 11

F I G . 2. Schemat ic d r a w i n g of the ve r t eb ra te fore-brain showing the diversi ty of ops in -con ta in ing cells wi th in the re t ina , p ineal complex (pineal a n d par ie ta l o rgans ) a n d basa l b ra in . O p s i n - i m m u n o r e a c t i v e cells can be b r o a d l y classified as follows: 1 Cell wi th a n ou t e r segment resembl ing a re t inal c o n e ; 2 Cell wi th a n ou te r segment wi th "d i so rgan ized" m e m b r a n e whor l s ; 3 Cell wi th an ou t e r segment con t a in ing few or n o m e m b r a n e s t ruc tu res ; 4 Cell wi th n o ou t e r segment ; 5 Ret ina l rod cell; 6 Ret ina l cone cell. These ops in -con ta in ing cells show the following t a x o n o m i c d i s t r ibu t ion : In the par ie ta l o r g a n or p a r a p i n e a l o r g a n of cyc los tomes ( lampreys) a n d lacert i l ians ( l izards), o r frontal o r g a n of a n u r a n s (frogs), the d o m i n a n t cell type resembles 1 . T h e pineal o r g a n is p resen t in some form in a lmos t all ve r tebra tes . In the fish a n d a m p h i b i a = cell type 1 d o m i n a t e s ; repti les = cell types 1 a n d 2 ; b i rds = cell types 2 a n d 3 ; m a m m a l s -= cell type 4 . N o t e t h a t in m a m m a l s , ops in has been identified wi th in p ineal cells, b u t these cells a re no t t h o u g h t to be pho to recep t ive , l ack ing p h o t o p i g m e n t c h r o m o p h o r e .

15 O p s i n i m m u n o r e a c t i v e C S F - c o n t a c t i n g cells have only recently

been identified wi th in the basa l b ra in of l a m p r e y l a r v a e ,

19 l i z a r d s

17 a n d b i r d s .

49

W e pos tu l a t e t ha t these cells a re the deep b ra in p h o t o r e c e p t o r s of the n o n -m a m m a l i a n ver tebra tes . O n the basis of earl ier u l t r a s t ruc tu ra l analysis of C S F -con tac t ing cells, these cells m o s t closely resemble cell type 3 . T h e re t ina of all ve r tebra tes con t a in s ei ther rod or cone p h o t o r e c e p t o r s o r m o r e usual ly s o m e mix tu re of b o t h (cell types 5 a n d 6 ; see O k s c h e a n d H a r t w i g

43 for further

detai ls) .


Ident i f ica t ion of c i rcadian photoreceptors in n o n - m a m m a l i a n ver tebra tes

While it has been relatively easy to demons t ra te that par t icular tissues are photosensit ive by surgical removal , directed i l lumination or isolation in vitro, the identification of the photorecept ive cells within these tissues has proved more difficult because to do so it is necessary to demons t ra te photopigments localized within part icular cells. In general, photopig-ments can be characterized on the basis of the physiological responses they mediate and their characteristic biochemistry. All vertebrate photopig-ments have been found to be remarkably similar, consisting of a type of opsin protein coupled to a ch romophore derived from an 11-ds form of vitamin A r e t i na ldehyde .

60 The character izat ion of circadian photorecep-

tors has involved the identification and localization of these photop igment characteristics (opsin and ch romophore ) within the cells and tissues suspected of circadian photorecept ion.

Action spectrum techniques were particularly helpful in the initial characterizat ion of pineal and deep brain photoreceptors . All opsin and 11-ds ret inaldehyde based photop igments have a characteristic shape to their absorbance and action spectra (even though these photopigments may have a very different wavelength of m a x i m u m sensitivity). In the isolated chick pineal, an action spectrum for suppression of the activity of the enzyme TV-acetyltransferase by light demons t ra ted that the response was mediated by a pho top igment maximally sensitive to 500 nm, and with the characteristic shape of an o p s i n : l l ds-retinal based pho top igmen t .

8

Almost identical results have been demons t ra ted in the pineals of several fish, reptiles and amphib ians using a range of different assays of sensitivity, including melatonin release and electrical ac t iv i ty .

21 An action spectrum

for the deep brain photoreceptors mediat ing photoper iodic responses in quail was strikingly similar to the action spectra of pineal responses a l though the receptors involved were clearly not in the p i n e a l .

1 1'12

These action spectra suggested the presence of opsin and 11 -cis ret inaldehyde within the pineal and basal brain; such photop igment components have now been directly demons t ra ted .

In the pineal, ant ibodies against vertebrate visual pigment opsins have immunolabeled pinealocytes, which in many respects resemble the photoreceptors of the developing retina. These cells are distinguished as having a short outer segment comprising lamellar whorls or membrane stacks. In addi t ion to opsin proteins , many pinealocytes contain other proteins associated with p h o t o t r a n s d u c t i o n .

13 A functional pho top igment

requires a ch romophore , and in the limited number of studies under taken , it has always been possible to identify ch romophore (11-ds ret inoid) within the pineal of non-mammal i an ve r t eb ra t e s .

45 Anatomical and

biochemical studies have also provided evidence that pineals of various


vertebrates conta in multiple types of p h o t o r e c e p t o r s . 14 T o da te , this surprising complexity has either been ignored or has s t imulated only limited speculation.

In contras t to the pineal, the identification of candida te photoreceptors within the basal bra in has been difficult. N u m e r o u s studies over the past 25 years have failed to provide an exact ana tomica l loca l i za t ion .17 However , three recent studies have suggested a locat ion for these cryptic photorecep-tors . Silver et al49 demons t ra ted tha t cerebrospinal fluid (CSF)-contact ing neurons within the septal and tuberal areas of the brain in the ring dove, quail and duck could be labeled with an anti-opsin an t ibody. Conclusive opsin identification was not possible in their studies because immunob lo t analysis of the putat ive photoreceptor -conta in ing brain regions failed to identify opsin-like proteins . The second, and mos t detailed s tudy to da te , has been on the lizard Anolis carolinensis.17 In this species anti-opsin ant iserum was shown to bind to CSF-contac t ing neurons in the septal area of the brain (Figure 3). I m m u n o b l o t analysis

F I G . 3. O p s i n immunoreac t i v i t y in the basa l b ra in of the l izard Anolis carolinensis. A Schemat ic d r a w i n g of a frontal sect ion of the b r a in . O p s i n - i m m u n o r e a c t i v e per ivent r icu lar C S F - c o n t a c t i n g n e u r o n s (dots) were identified wi th in the nuc leus ven t romedia l i s of the s e p t u m . Β P h o t o m i c r o g r a p h of C S F - c o n t a c t i n g n e u r o n s s ta ined with an t i -ops in an t ibod ie s , showing the presence of ap ica l processes (arrows) p r o t r u d i n g in to the la tera l ventr icle (dotted line = surface of the ventr icle) . T h e loca t ion of these cells wi th in the b r a in h a s been ind ica ted by a star in A . W e s t e r n b lo t analysis indica tes t h a t o u r an t i -ops in an t ibod ie s recognize a single 40 k D p ro t e in in an t e r io r b r a in ex t rac ts . In a d d i t i o n this a r ea of t he b r a i n con t a in s re t ino ids assoc ia ted wi th p h o t o t r a n s d u c t i o n (11-ds a n d all-trans re t inoid) . These d a t a suggest t ha t C S F - c o n t a c t i n g n e u r o n s a re pho tosens i t ive , a n d t h a t these cells cou ld be respons ib le for c i rcad ian responses to light in the absence of re t inal a n d p ineal p h o t o r e c e p t o r s . Abbreviations: CSF ce rebrosp ina l fluid-contacting n e u r o n s ; dc do rsa l cor tex ; mc med ia l cor tex ; nacc nuc leus a c c u m b e n s ; nvm nucleus ven t romedia l i s of the s e p t u m ; oc op t ic ch i a sm; ps

p a l e o s t r i a t u m ; F lateral ventricle (from F o s t e r et ai11).


showed that the anti-opsin ant ibodies recognized a single 40 k D protein in ocular, pineal and septal brain areas, but not in other areas of the brain. In addi t ion, the anter ior brain of Anolis was shown to contain specific retinoids associated with pho to t ransduc t ion (11-ds and all-irans-reti-noid) . A third study examined a "primit ive" vertebrate , the larval (Ammocoetes) l a m p r e y .

19 In this species, anti-opsin antisera labeled a

popula t ion of CSF-contac t ing neurons within the basal brain (postoptic commissural nucleus and ventral hypotha lamic nucleus), and in addi t ion, ant ibodies raised against the α-subunit of retinal G protein (oc-transducin) labeled the same cells. Collectively these da ta suggest tha t a subpopula t ion of CSF-contac t ing neurons are photosensory and tha t these cells are likely to be the "deep bra in" photoreceptors that mediate extraocular and extrapineal photorecept ion in non-mammal i an vertebrates. While this interpretat ion requires direct experimental confirmation, at least we now have candidate cells upon which to focus our efforts.

Retinal, pineal and parapineal photoreceptors , and CSF-contac t ing neurons are strikingly similar in tha t they all have specialized ciliated dendrites of a sensory type (9 4- 0) which p ro t rude into a cavity derived from the ven t r ic les .

59 CSF-contac t ing neurons are different from retinal

and pineal photoreceptors in that they seem to lack photoreceptor membrane lamellae or vesiculations. However , laminated membranes are probably not obligatory for p h o t o r e c e p t i o n .

2 3'43 In our studies of lamprey

and Anolis, the ciliated dendri te of CSF-contac t ing neurons was often more strongly labeled than the cell body with anti-opsin and a-transducin ant ibodies . If CSF-contac t ing cells are indeed photosensit ive, then the most obvious organelle responsible for pho to t ransduc t ion would be the ciliated dendri te (intraventricular process), which may be homologous with a photoreceptor outer segment.

It is not clear why previous a t tempts to localize opsin proteins within the brain have f a i l e d .

1 3 , 59 The use of newly obta ined cone-specific ant ibodies

may partially explain the more recent results; however, the selection of part icular species may be equally impor tan t . Using methodologies and antibodies identical to those used to study Anolis, we have failed to identify any opsin immunoposi t ive cells within the brains of either chicken, quail or sparrows in spite of the fact that they are almost certainly p r e s e n t .

17

Ident i f ica t ion of c i rcadian photoreceptors in m a m m a l s

The identification of extraretinal photoreceptors in non -mammal i an vertebrates st imulated a search for similar photoreceptors in mammals . Early studies showed tha t the mammal i an photoper iodic response needed an intact r e t i n a / e y e

9'2 4

'2 5

'54

and that the suppression of pineal melatonin synthesis by light was abolished if the sympathet ic input to the pineal was


severed, isolating the gland from the ret ina and suprachiasmat ic nuclei ( S C N ) .

56 Early studies also showed tha t circadian rhy thms could not be

entrained by light cycles in the absence of the e y e s .

22 A more recent study

by Nelson and Z u c k e r

41 on bo th diurnal and noc turna l rodents housed

ou tdoors and exposed to na tura l day-night cycles, demons t ra ted that blinded animals failed to show any sign of circadian ent ra inment .

Al though the physiological evidence suggests that m a m m a l s use their eyes for all forms of light detection, the mammal i an pineal does contain a range of so-called "photoreceptor proteins" , including rhodops in k i n a s e ,

52 the 48 k D protein S - a n t i g e n ,

2 7'

40 in terphotoreceptor retinoid-

binding protein, cellular ret inal-binding p ro t e in ,

4 and o p s i n .

1 5'

28 The

identification of these photoreceptor proteins within the mammal i an pineal led, not unreasonably , to speculation that pho to t ransduc t ion may take place within this tissue and mediate some unrecognized aspect of the animal 's physiology. T o address this issue F o s t e r

15 investigated whether

11-ds and all-trans ret inaldehyde were present within the hamster pineal, and whether , if present, photoisomer iza t ion of the 11-ds c h r o m o p h o r e to the Sill-trans form could be demons t ra ted . Some 400 pineals were collected and assayed for c h r o m o p h o r e content , but none was found (cf. avian or lizard pineal). These da ta suggest that it is unlikely that the opsin within the mammal i an pineal is coupled to a c h r o m o p h o r e and reinforce the view that the mammal i an pineal is incapable of pho to t ransduc t ion .

Al though there is no evidence that adul t m a m m a l s have extraret inal photoreceptors , there is a s t rong suggestion of extraret inal photorecept ion in neonata l mammal s . In carefully control led experiments Torres and L y t l e

55 have shown that l ight-induced suppression of pineal N-acetyl-

transferase occurs in blinded rat pups 4 days old. In animals older than 6 days this response is lost. Their report extends and confirms earlier studies and suggests that further investigation of this p h e n o m e n o n will be fruitful.

Al though it is clear tha t circadian photorecept ion in adult m a m m a l s is exclusively retinal, we do not yet know which cells within the ret ina are responsible. We have used mice with hereditary retinal disorders and transgenically modified retinas in a t tempts to identify the photoreceptors within the m a m m a l i a n ret ina that mediate circadian responses to light. In mice homozygous for the au tosomal recessive allele rd (retinally degenerate) all rod cells degenerate by 60 days of age, and between 90 and 150 days of age electrophysiological and behavioral visual responses to bright light disappear . In the normal mouse retina, approximate ly 9 7 - 9 8 % of all photoreceptors are rods and a l though all rods degenerate in the rd/rd ret ina, a few cone cells ( 2 - 5 % of the original popula t ion) remain in animals over one year of age. It should be stressed that the remaining cone cells in these aged rd/rd mice lack outer segments, and with increasing age cone cell loss continues to complet ion. In view of this massive


photoreceptor damage we expected circadian responses to light to decline in parallel with the loss of visual function, but this did not o c c u r .

16

We assayed circadian photosensit ivity by measur ing the size of the phase shift in the freerunning locomotor rhy thm produced by a single 15 minute pulse of light (515 nm) (Figure 4). We varied the light intensity (irradiance) of the 15 minute pulse to compare the dynamic range of circadian responses to light in three genotypes of mice (rd/rd, rd/ + , + / + ) from the same C57BL strain (Figure 5). Despite the loss of visual photoreceptors in rd/rd mice, these animals show circadian responses to light that are indistinguishable from those of mice with phenotypically normal retinas (rd/ + , + / + ). The irradiance required to produce bo th saturat ing and half-saturating responses are the same for all groups . This result demonst ra tes no t only tha t some photosensit ivity remains in mice with degenerate ret inas, but that the circadian photosensit ivi ty shown by these animals is not different from the sensitivity of animals with normal ret inas. In addi t ion , the site of circadian photorecept ion must reside within the eye because bilateral enucleation of rd/rd animals abolishes all circadian responses to l i g h t .

16

The progression of photoreceptor degenerat ion in rd/rd mice com-mences early in post nata l development with the loss of rods , followed by a more pro t rac ted loss of cone cell bodies. If the surviving cone cell bodies mediated circadian responses to light, then one would expect the sensitivity of the circadian system to light in rd/rd mice to decline with age in parallel with the loss of cones from the retina. Circadian responses to light of rd/rd and + / + mice up to 800 days of age were examined. The results indicate that the phase shifts of rd/rd and + / + mice remain indist inguishable. Circadian responses to light do not parallel either rod or cone cell l o s s .

46

The light input pa thway to the S C N was examined to determine whether the ana tomy or physiology of the re t inohypothalamic tract is affected by the rd/rd muta t ion . Several laborator ies have shown that activation of the immediate early gene c-fos within the S C N is a useful marker of neural activation of the light input pa thway to the circadian s y s t e m .

2 9'

47 If the

rods and /o r cones of the ret ina contr ibute to light information reaching the S C N , one would expect a reduced expression of c-fos in the S C N of rd/rd mice in response to light. O n the other hand , if circadian photoreceptors are not affected by this muta t ion , then light activated c-fos expression should be the same in the S C N of rd/rd and + / + mice. Using immunocytochemical techniques, we have been unable to detect any differences between the two genotypes in the dis tr ibut ion, number and density of o/os-labeled S C N cells.

7

While all of our studies have shown tha t there is no effect of the rd/rd muta t ion on circadian responses to light, a study by Eb ihara and T s u j i

10

found that rd/rd mice required more light to entra in to a l ight :dark cycle


B) M o u s e w i t h d e g e n e r a t e r e t i n a ( r d / r d )

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F I G . 4. L o c o m o t o r activity (wheel r u n n i n g behav io r ) of C 5 7 B L mice with e i ther : A n o r m a l re t inas a n d visual responses ( + / + ) , o r Β retinally degenera te mice h o m o z y g o u s for the rd gene (rd/rd), wi th mass ive loss of visual funct ion. Whee l r u n n i n g activity is s h o w n for 29 days (1 line = 1 day ) ; an ima l s were housed wi th in a l ight- t ight box . F o r the first 5 days , each m o u s e was exposed to a l igh t :dark cycle. N o t e t ha t the an ima l s ta r ted activity a t the s ame t ime each day . T h e m o u s e was t h e n t ransferred to c o n s t a n t d a r k n e s s , a n d the f ree-running r h y t h m was observed . T h e pe r iod of the free-running r h y t h m for b o t h an ima l s was shor t e r t h a n 24 h o u r s . As a result the activity s ta r t s a little earlier on each subsequen t day . O n the 16th day in c o n s t a n t d a r k n e s s , the m o u s e was r emoved from the light t igh t -box , p laced in a s t a n d a r d c h a m b e r a n d exposed to a 15 m i n u t e l ight pulse a t c i rcad ian t ime 16 (CT 16), s h o w n as filled circles o n the record . By conven t ion , activity onse t u n d e r f ree-running cond i t i ons is des igna ted c i rcad ian t ime 12 (CT 12). As a resul t , C T 16 will be 4 h o u r s after activity onse t . T h e d u r a t i o n , wave leng th a n d a m o u n t of l ight del ivered to the a n i m a l was quan t i t a t ive ly de t e rmined . After a further 8 days the effect of the light pulse (i.e. the size of the phase shift) was de t e rmined . T h e m a g n i t u d e of the p h a s e shift p r o d u c e d by the light pulse was m e a s u r e d as the difference be tween the s teady s ta te phase of the f ree-running r h y t h m before a n d after the pulse . T h e p h a s e shift is s h o w n by a Δ in b o t h r ecords . In b o t h an ima l s the light pulse caused a p h a s e delay (animal s ta r ted

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compared to + / + controls . While the au thors concluded tha t this difference was due to the loss of rods in rd/rd animals their results are open to several interpretat ions . The s tudy compared two strains of mice, C 3 H rd/rd with wild-type C57BL. It is therefore possible tha t the differences observed are the result of mouse strain and have noth ing directly to do with retinal photoreceptors . W e have recently compared the a m o u n t of light required to entra in C 3 H rd/rd and + / + mice with C57BL rd/rd and + / + mice and found no differences in sensitivity between these groups . This would suggest that strain canno t account for the differences in sensitivity tha t Eb iha ra and Tsuji observed. We are currently col laborat-ing with the Eb iha ra labora tory to resolve these quest ions. It is possible tha t the Japanese mice have a different or addi t ional mu ta t ion tha t may modify circadian responses to light. If t rue , this would be a part icularly useful tool in identifying the retinal componen t s that regulate circadian responses.

Another retinal muta t ion , rds (retinally degenerate slow) in the C 3 H mouse strain, has provided a second, somewhat different app roach to the same quest ion. In rds/rds mice, the ret ina undergoes no rma l development and differentiation of cells until the first pos tna ta l week, but at this t ime bo th rod and cone photoreceptors fail to develop outer segments and gradually degenerate. This is in contras t to rd/rd animals which develop outer segments tha t later degenerate . In rds/rds mice approximate ly half of all pho torecep tor cell bodies have degenerated by 3 m o n t h s , and virtually all are gone by 1 year of age. D a t a from these animals , including d a t a from aged animals , suggest tha t circadian light detection is identical in rds/rds, rd/rd and + / + genotypes .

1

We have also examined circadian responses in transgenic mice tha t incorpora ted a genetic construct consisting of the gene coding for d iphther ia toxin Α-chain, control led by the rhodops in p r o m o t o r (mice generously dona ted by Maureen McCal l , University of Wisconsin-Madison and Toshimichi Shinohara , N I H ) . This construct eliminates all rod photoreceptors bu t has no obvious effect on neurons in the outer retinal layers or more central visual s tructures. Cone cells survive for several weeks, a l though they lack outer segments, and gradual ly d isappear from the ret ina. In prel iminary studies, the circadian responses of these animals are indist inguishable from those of no rma l control mice.

Collectively, these da t a suggest tha t circadian photorecept ion can be mainta ined either by a small n u m b e r of cone cells wi thout outer segments, or alternatively by an as yet unrecognized class of retinal photorecept ive cell tha t is unaffected by the rd/rd mu ta t ion , the rds/rds mu ta t ion , or the destruct ion of rod photoreceptors by transgenic modification of the retina. We are a t tempt ing to distinguish between these two alternatives by identifying the circadian pho top igment in reduced ret inas, and then localizing this pho top igment within the retina.


Prel iminary act ion spectrum da t a for phase-shifting the circadian rhy thm in rd/rd and + / + mice suggest that the wavelength of m a x i m u m sensitivity is near 500 n m and tha t the shape of the curve is typical of animal photopigments based u p o n an opsin and 11-ds-ret inaldehyde ch romophore (Provencio I. and Fos ter R .G . unpubl ished) . Using High Performance Liquid C h r o m a t o g r a p h y we have identified significant a m o u n t s of 1 l-cis re t inaldehyde within the dark adap ted eyes of rd/rd mice ( 1 - 2 % of the levels found in + / + individuals). If this ch romophore is associated with the rods or cones of the degenerat ing rd/rd ret ina, then it should decline in parallel with their loss and ultimately disappear . Alternatively, if the c h r o m o p h o r e is associated with some other circadian photoreceptor which does no t decline with age, then its level should remain low but constant . O u r early results indicate that after an initial rise and decline dur ing the first 14 days of life, ch romophore levels remain at constant low levels in the rd/rd e y e .

46

The genes and c D N A s for the rod opsin, blue cone opsin and green/red cone opsin in the + / + mouse have been successfully characterized by Meredi th Applebury 's l abora tory at the University of Chicago (personal communica t ion) . Because circadian responses to light as well as ch romo-phore levels remain stable in aged rd/rd mice we ant icipate tha t any opsin mediat ing circadian responses to light would also remain at a cons tant low level. N o r t h e r n blot analysis using opsin probes made from Applebury 's c D N A s has shown that rod opsin m R N A rapidly disappears from the eye, followed by the slower decline in levels of blue cone opsin and green/red cone opsin m R N A . These da t a are open to two interpreta t ions , either (1) one or more of the k n o w n opsins mediates circadian responses to light and occurs in the degenerate ret ina at very low levels, or (2) none of the known opsins mediates circadian responses to light, and there is a unique "circadian" opsin in the re t ina .

1

The loss of rods and cones in m u t a n t mice produces a parallel decline in visual function, yet circadian responses to light remain unaffected in these animals . Full in terpreta t ion is no t yet possible; however, the facts are consistent with the hypothesis that retinal rods and cones do not normally play a significant par t in the regulat ion of the mammal i an circadian system by light, and that some as yet unidentified retinal photoreceptor performs this role.

Impl icat ions

Early in their evolut ionary history m a m m a l s may well have gone th rough what could be called a "noc turna l bot t leneck". Extraret inal photorecep-tors either in the pineal or the deep brain that may have served well for diurnal animals exposed to bright light for hours at a t ime, were simply no t sensitive enough to define dawn and dusk for primitive m a m m a l s tha t


spent the day hiding in dark caves and holes. As a consequence, brain photoreceptors may have been "moved ou t " into the retina where they had better access to the dim light which il luminates the world of strongly noc turna l creatures. Alternatively and perhaps more reasonably, bo th deep brain and specialized retinal circadian photoreceptors were already present in the diurnal mammal- l ike reptiles and the extraretinal receptors became non-functional and were therefore lost when primitive m a m m a l s entered the nocturna l niche. In either case, those mammal i an species which subsequently and secondarily became diurnal have apparent ly failed to re-evolve extraretinal photorecept ion . The above speculation will of course be difficult to substant ia te experimentally, and , it should be noted, does not address the interesting related quest ion of the adapt ive significance of separat ing the perception and processing of visual from that of circadian photorecept ion .

Correct or not , the reasoning outl ined above has led us to search for retinal cell types that resemble and are perhaps homologous to the C S F -contact ing neurons that are our best candidates for deep brain pho to -receptors. The cell type known as Landol t ' s c l u b s

59 are modified bipolar

cells of u n k n o w n function, widely scattered t h roughou t the retina. Landol t ' s clubs have a ciliated dendrit ic process which extends th rough the outer nuclear layer and terminates in the space between the pigmented epithelium and inner and outer segments of the photoreceptors . Vigh and V i g h - T e i c h m a n n

59 classify these cells as CSF-contac t ing neurons and

have noted their resemblance to pinealocytes. They would seem good candidates for further study.

It is wor th examining the implicat ions for h u m a n beings of what we know abou t circadian photorecept ion in vertebrates in general. There is noth ing to suggest that we are different from other m a m m a l s in the retinal location of our circadian photoreceptors . Like them we probably have specialized retinal photoreceptors that convey information abou t the environmenta l light condi t ions more or less directly to the circadian oscillators in the S C N and not to the higher visual centers. Thus it is entirely possible that our circadian oscillators are kept informed of relevant changes in external light condit ions wi thout this information ever reaching consciousness. If this description is correct, it will have at least two consequences: (1) As a genetic defect, some individuals may lack the specialized circadian photorecep tor cell types wi thout being directly aware of their absence. By analogy with the color blindness tha t results when individuals lack a class of cone cell photoreceptors , such individuals would be "time b l ind"—unable to synchronize their circadian oscillators with the outside world and as a result, drifting with their innate circadian periodicity across the strictly 24-hour day which the rest of us inhabi t . Less severe forms of this defect might result in a reduced number of circadian photoreceptors with consequent increase in the intensity of light required


20 0 4 8 12 16 20

Time of day

F I G . 6. M e l a t o n i n suppress ion by light in a h u m a n subject wi th n o consc ious light pe rcep t ion . E x p o s u r e to 1.5 h o u r s of b r igh t light (open vertical ba r , 6,300 to 8,400 lux in the d i rec t ion of gaze) centered a r o u n d the m i n i m u m of the e n d o g e n o u s core b o d y t e m p e r a t u r e r h y t h m (upper pane l ) du r ing the n igh t t ime induced an i m m e d i a t e a n d comple te suppress ion of c i rcula t ing p l a s m a m e l a t o n i n levels, which r e b o u n d e d t o n o c t u r n a l va lues immedia te ly u p o n r e tu rn t o d a r k n e s s

(stippled a rea , light intensi ty 0.02 lux) (from M a r t e n s et α / .

3 0) .

for en t ra inment and perhaps abnorma l phase relationships with the day-n igh t cycle. (2) Some individuals who are visually blind might retain circadian photorecept ion wi thout being aware of it. These people would entrain to the day-n igh t cycle, have normal sleep pat terns and be temporal ly well integrated in society a l though they might a t t r ibute this to social signals ra ther than light. It goes wi thout saying tha t every effort should be m a d e to protect and enhance circadian photorecept ive function in such cases. In this connect ion, a recent report from Czeisler's l abora tory is of great i n t e r e s t .

30 Figure 6, which is taken from tha t repor t , shows

clearly that in a h u m a n subject with no conscious light perception, p lasma mela tonin levels can be suppressed by light. Since it is well known tha t at least some of the effects of light on melatonin synthesis are mediated by retinal input to the S C N which then suppresses the pineal gland via a mult i-synaptic pa thway, it is ha rd to escape the conclusion tha t at least some of the retinal receptors tha t supply light information to the S C N have been spared in this completely "bl ind" subject and that as a consequence, it might be possible for him to entrain to light cycles.


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35. M e n a k e r M . (1968) Ex t ra re t ina l l ight pe rcep t ion in the s p a r r o w I: E n t r a i n m e n t of the biological clock. Proc. Natl. Acad. Sci. USA 5 9 , 4 1 4 - 4 2 1 .

36. M e n a k e r M . (1968) Light pe rcep t ion by ex t ra re t ina l receptors in the b ra in of the s p a r r o w . Proc. 76th Ann. Conv. Am. Psychol. Assoc., 299 -300 .

37. M e n a k e r M . a n d K e a t t s (1968) Ex t ra re t ina l l ight pe rcep t ion in the s p a r r o w I I : P h o t o p e r i o d i c s t imula t ion of testis g r o w t h . Proc. Natl. Acad. Sci. USA 6 0 , 1 4 6 - 1 5 1 .

38. M e n a k e r M . , Robe r t s R., Ell iot t J .A. a n d U n d e r w o o d H . (1970) Ex t ra re t ina l light pe rcep t ion in the s p a r r o w I I I : T h e eyes d o n o t pa r t i c ipa te in p h o t o p e r i o d i c p h o t o r e c e p t i o n . Proc. Natl. Acad. Sci. USA 6 7 , 320 -325 .

39. M e n a k e r M . a n d U n d e r w o o d H . (1976) Ex t ra re t ina l p h o t o r e c e p t i o n in b i rds . Photochem. Photobiol. 2 3 , 299 -306 .

40. M i r s h a h i M . , F a u r e J .P . , Br isson P . , F a l c o n J. a n d G u e r l o t t e J . P . (1984) S-antigen immunoreac t iv i ty in re t inal rods a n d cones a n d pineal photosens i t ive cells. Biol. Cell. 5 2 , 1 9 5 - 1 9 8 .

4 1 . Ne l son R.J. a n d Z u c k e r I. (1981) Absence of ex t r aocu la r p h o t o r e c e p t i o n in d iu rna l a n d n o c t u r n a l r oden t s exposed to direct sunl ight . Comp. Biochem. Physiol. 6 9 A , 145-148 .

42. Oish i T . a n d K a t o M . (1968) P inea l o r g a n as a possible p h o t o r e c e p t o r ; p h o t o p e r i o d i c test icular responses in J a p a n e s e qua i l . Mem. Fac. Sci. Kyoto Univ. 2 , 12 -18 .

4 3 . O k s c h e A. a n d H a r t w i g H . G . (1975) P h o t o n e u r o e n d o c r i n e sys tems a n d the th i rd ventr icle. In Brain-Endocrine Interactions. II. The Ventricular System, 2nd Int. Symp Shizuoka, 1974, (eds. Knigge K . M . , Scot t D .E . , K o b a y a s h i H . a n d Ishii S.), p p . 4 0 - 5 3 . K a r g e r , Basel.

44. Ol iver J., Ja l lageas M . a n d Baylé J . D . (1979) P l a s m a tes tos te rone a n d L H levels in ma le quai l bea r ing h y p o t h a l a m i c lesions or r a d i o l u m i n o u s imp lan t s . Neuroendocrinology 2 8 , 114-122.

Circadian Photoreception in Vertebrates (40) 9 1

4 5 . P r o v e n c i o I. a n d F o s t e r R . G . (1993) Vi t amin A2- b a s e d p h o t o p i g m e n t s wi th in the p ineal g land of a fully terres t r ia l ve r t ebra te . Neurosci. Lett. 1 5 5 , 2 2 3 - 2 2 6 .

46. P r o v e n c i o L, T e n n a n t G .S . , C a r d J . P . a n d F o s t e r R . G . (1992) C i r cad i an p h o t o r e c e p -t ion in aged ret inal ly degenera te (rd/rd) mice . Soc. Res. Biol. Rhythms Abs. 3 , 59.

47. R u s a k B, R o b e r t s o n H.A. , Wisden W. , H u n t S.P. (1990) Light pulses t h a t shift r h y t h m s induce gene express ion in the sup rach i a sma t i c nuc leus . Science 2 4 8 , 12376-1240.

48 . Schar re r , E . (1928) Die Lichtempfindl ichkei t b l inder Elr i tzen. I. U n t e r s u c h u n g e n uber das Zwischenh i rn der F ische . Z . Vergl. Physiol. 7 , 1-38.

49. Silver R., W i t k o v s k y P . , H o r v a t h P . , Alones V., Ba rns t ab l e C.J. a n d L e h m a n M . N . (1988) Coexpress ion of ops in- a n d VIP- l ike - immunoreac t iv i ty in C S F - c o n t a c t i n g n e u r o n s of the av ian b ra in . Cell Tissue Res. 2 5 3 , 189-198 .

50. S impson S.M., U r b a n s k i H . F . a n d R o b i n s o n J .E . (1983) T h e p ineal g l and a n d the p h o t o p e r i o d i c con t ro l of luteinizing h o r m o n e secret ion in in tac t a n d cas t ra t ed J a p a n e s e quai l . J. Endocrinol. 9 9 , 281 -287 .

5 1 . Siopes T . D . a n d Wi l son W . O . (1974) E x t r a o c u l a r modif ica t ion of p h o t o r e c e p t i o n in in tac t a n d p inea lec tomised Coturnix. Poultry Sci. 5 3 , 2 0 3 5 - 2 0 4 1 .

52. Somers R .L . a n d Kle in D . C . (1985) R h o d o p s i n k inase act ivi ty in the m a m m a l i a n p ineal a n d o the r t issues. Science 2 2 6 , 182-184.

53. T a b a t a M . , M i n h - N y o M . a n d O g u r i M . (1989) T h r e s h o l d s of re t inal a n d ex t ra re t ina l p h o t o r e c e p t o r s m e a s u r e d by p h o t o b e h a v i o r a l r e sponse in catfish, Silurus asotus. J. Comp. Physiol. A. 164 , 7 9 7 - 8 0 3 .

54. T h o m s o n A . P . D . (1954) T h e onse t of oes t rus in n o r m a l a n d b l inded females. Proc. Royal Soc. Lond. B. 142 , 126-135 .

55. T o r r e s G . a n d Lyt le L . D . (1989) Ex t ra re t ina l m e c h a n i s m s med ia t e l igh t - induced changes in n e o n a t a l ra t p ineal g land N-ace ty l t ransferase act ivi ty. J. Pineal Res. 7 , 211-220 .

56. U n d e r w o o d H . a n d G r o o s G . (1982) Ver t eb ra t e c i rcad ian r h y t h m s : Ret ina l a n d ex t ra re t ina l p h o t o r e c e p t i o n . Experientia 3 8 , 1013 -1021 .

57. U n d e r w o o d H . a n d M e n a k e r M . (1976) Ex t r a re t ina l p h o t o r e c e p t i o n in l izards . / . Comp. Physiol. 8 3 , 187-222 .

58. Veen T h . van , H a r t w i g H . - G . a n d Mill ier K . (1976) Light d e p e n d e n t m o t o r act ivi ty a n d p h o t o n e g a t i v e behav io r in the eel (Anguilla anguilla L.). J Comp. Physiol. I l l , 2 0 9 - 2 1 9 .

59. Vigh B. a n d V i g h - T e i c h m a n n I. (1988) C o m p a r a t i v e neu roh i s to logy a n d i m m u n o c y t o -chemis t ry of the p ineal complex wi th special reference to C S F - c o n t a c t i n g n e u r o n a l s t ruc tures . Pineal Res. Rev. 6 , 1-65, 1988.

60. W a l d , G . (1968) M o l e c u l a r basis of visual exc i ta t ion . Science 162 , 2 3 0 - 2 3 9 . 6 1 . Y o k o y a m a , K., O k s c h e Α., D a r d e n T.R. a n d F a r n e r D . S . (1978) T h e sites of encepha l ic

p h o t o r e c e p t i o n in the p h o t o p e r i o d i c i nduc t ion of g r o w t h of the testes in the whi te c r o w n e d s p a r r o w , Zonotrichia leucophrys gambelii. Cell. Tissue Res. 189 , 4 4 1 ^ 6 7 .

6

Maternal Entrainment of a Fetal Biological Clock

S T E V E N M . R E P P E R T a n d D A V I D R. W E A V E R

Laboratory of Developmental Chronobiology, Children's Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA

Abstract

A biological clock oscil lates in the m a m m a l i a n fetus, a n d the fetal c lock is en t r a ined by r e d u n d a n t c i rcad ian signals from the m o t h e r . Recent evidence suggests t ha t a d o p a m i n e system wi th in the fetal h y p o t h a l a m u s is a final c o m m o n p a t h w a y t h r o u g h which m a t e r n a l c i rcadian signals en t r a in the fetus. An en t r a inab le biological clock d u r i n g fetal life helps the deve lop ing m a m m a l m o r e readi ly p r e p a r e for life in the ou ts ide wor ld .

In t roduct ion

D U R I N G FETAL life, studies in several m a m m a l i a n species show tha t the biological clock in the suprachiasmat ic nuclei (SCN) is oscillating in time (phase) with the envi ronmenta l l igh t -dark cycle. En t ra inment of the fetal S C N involves materna l communica t ion of circadian signals to the fetus. This chapter reviews these studies in rodents and discusses recent results which suggest a cellular mechanism th rough which the fetal S C N senses materna l entraining signals.

A biological c lock is osci l lat ing in t h e fe tus

In mammal s , circadian rhy thms in physiology and behavior are not overtly expressed until pos tna ta l l i f e .

34 D e g u c h i

7 was the first to suggest

tha t before rhy thm expression a biological clock might be oscillating in the mammal i an fetus. He determined in rats the phase of a rhy thm moni to red under cons tant condi t ions dur ing the pos tna ta l period to est imate the phase of the biological clock at earlier developmental stages. Because circadian rhy thms persist (free run) under cons tan t condi t ions with a cycle length close to 24 hours , the phase of a rhy thm in the pos tna ta l period can be used to infer the phase of the biological clock at earlier developmental

9 3


s tages .

4 Deguchi 's findings suggested that a circadian clock oscillates at or

before bir th and that its phase is coordinated with the dam. Postnatal paradigms cannot conclusively show that a biological clock

actually functions in utero. It is possible that some rhythmic aspect of the birth process itself could start or set the timing of the developing clock. Demonstra t ing prenatal function of the biological clock requires a method that can measure an intrinsic, functionally relevant property of the clock itself. A method proven useful for monitor ing the oscillatory activity of the SCN in adult rats is

1 4C-labe led 2-deoxyglucose (2-DG) autoradiography

which provides a biochemical means of visualizing the metabolic (func-tional) activity of discrete structures in the central nervous system in vivo.**

The 2 -DG method was used by Reppert and S c h w a r t z

35 to delineate a

circadian rhythm of metabolic activity in the S C N of fetal rats. This study showed that the fetal S C N exhibit a circadian variation of metabolic activity that is "in t ime" (coordinated) with the rhythm in the d a m and with the external lighting cycle (Figure 1). Remarkably, this fetal rhythm can be detected as early as day 19 of gestation (2-3 days before birth; all gestational ages have been standardized to day 0 = day of sperm posi t iv i ty) .

36 Recently,

a rhythm of 2 -DG in the fetal S C N has also been demonstrated in vitro.

46

This finding suggests that the metabolic activity rhythm in SCN is endogenously generated in the fetus and not passively driven by the mother .

Neurogenesis of the rat S C N occurs between days 13 and 16 of g e s t a t i o n .

1'

16 A few synapses first appear in the S C N on day 19 of gestation,

with the vast majority of synapses appearing p o s t n a t a l l y .

2 5'

28 A clear

day-night rhythm of S C N action potentials is first apparent in fetal hypothalamic slices on day 21 of ges t a t i on

45 at a time when the metabolic

activity rhythm is quite prominent . Thus , the S C N begin displaying circadian oscillations in metabolic and electrical activity when the neurons are virtually devoid of neural connections. This early circadian function in the absence of neural connections has led to the not ion that individual S C N neurons may function as individual circadian pacemaking u n i t s .

28

Vasopressin m R N A levels can also be used as an intrinsic marker of the oscillatory activity in the SCN during fetal l i fe .

39 A day-night oscillation in

vasopressin m R N A levels was found in the fetal S C N beginning on day 21 of gestation, the earliest time that vasopressin m R N A levels are detectable in SCN. The fetal rhythm was in phase with the circadian time of the d a m .

39

The maternal S C N transduces circadian in format ion for the fetus

Entrainment of the fetus is due to maternal communicat ion of circadian signal(s) to the fetus (Figure 2). Blind pregnant rats were used to show that environmental lighting acts through the maternal circadian system to entrain the rhythm of fetal S C N metabolic ac t iv i ty ;

35 the fetal rhythm was

Maternal Entrainment of a Fetal Biological Clock

Gestational Age (days) 19 1 20 1 21 1 22 -

95

1900 1900 1900

Clock T ime (hrs)

1900 1900

1900 1900

Clock Time (hrs)

F I G . 1. Deoxyg lucose exper iment showing tha t the fetal S C N manifest a d a y - n i g h t r h y t h m in me tabo l i c act ivi ty. F o u r p r e g n a n t S p r a g u e - D a w l e y ra t s were h o u s e d wi th l ights o n from 07.00 to 19.00 h o u r s unti l ges ta t iona l d a y 19, when the an ima l s were p laced in c o n s t a n t d a r k n e s s in p r e p a r a t i o n for the deoxyglucose injection (exper imenta l p a r a d i g m depic ted in u p p e r pane l ) . T w o an ima l s were injected i.v. wi th 2 - d e o x y [

1 4C ] g l u c o s e d u r i n g the pe r iod w h e n the

l ights wou ld have no rma l ly been off (subjective n igh t ) o n ges ta t iona l d a y 20, a n d the o the r two were injected du r ing the pe r iod w h e n the l ights w o u l d have no rma l ly been o n (subjective d a y ) o n ges ta t iona l d a y 2 1 . At 45 minu te s after injection, the an ima l s were killed a n d four fetal b ra ins from each p r e g n a n t a n i m a l were r a n d o m l y chosen , sect ioned a n d processed for a u t o r a d i o g r a p h y . T h e opt ica l densi ty ( O D ) of each fetal s u p r a c h i a s m a t i c nuc leus was m e a s u r e d a n d the O D of adjacent h y p o t h a l a m u s was used as an in te rna l reference s t a n d a r d for each fetal b ra in . T h e d a t a a re thus expressed as relative O D ( O D of S C N / O D of ad jacent h y p o t h a l a m u s ) . E a c h vert ical b a r in the lower pane l gives m e a n relat ive O D ( ± S E M ) for the S C N of eight fetal b r a in s . T h e fetal S C N exhibi t a clear day -n igh t r h y t h m of me tabo l i c act ivi ty ( p > 0 . 0 1 ) ; the nuclei a re metabol ica l ly act ive d u r i n g the m o t h e r ' s subjective day a n d inact ive d u r i n g the m o t h e r ' s subjective n ight .

(Modif ied from reference 35.)

synchronous with the circadian time of the blind mothers and not affected directly by ambient lighting. Investigators have used a variety of pre- and postnatal rhythms to confirm the prenatal coordinat ion of maternal and fetal rhythms in several s p e c i e s .

4 , 42

The involvement of the maternal S C N in the generation of entraining signals for the fetus has been demonstra ted in rats and hamsters by destroying the maternal S C N early in g e s t a t i o n .

5'

1 3'

1 4'

3 8 , 53 These experi-


M O T H E R

F I G . 2 . C o n c e p t u a l mode l of ma te rna l - f e t a l c o m m u n i c a t i o n of c i rcad ian phase . L ight - induced neura l s ignals a re conveyed to the S C N by her r e t i n o h y p o t h a l a m i c p a t h w a y ( R H P ) , en t r a in ing her c i rcad ian r h y t h m s . M a t e r n a l o u t p u t signals then en t ra in the fetal clock at a t ime when the inne rva t ion of the fetal S C N by the R H P

is incomple te .

merits indicate that the entraining signals depend on the integrity of the maternal S C N (Figure 2); maternal S C N lesions cause desynchronization of the individual fetal clocks within a litter, with circadian rhythms in each animal persisting.

Our laboratory showed that during the postnatal period, rat pups born to SCN-lesioned dams respond appropriately (entrain) to l ight-dark cycles despite the absence of prenatal maternal entrainment (Weaver and Reppert , unpublished data) . Fur thermore , pups from sham-operated and SCN-lesioned dams express free-running rhythms with similar cycle l eng th s .

38

Thus, normal circadian function (e.g. light-dark entrainment and free-running cycle length) in adul thood does not appear to require prenatal maternal entrainment.

W h a t are th e materna l ent ra in ing signal(s)

Because the in utero envi ronment provides a rich source of rhythmic hormones from the mothe r (e.g. prolact in, cort icosterone and melatonin) , studies have focused on the possibility that the materna l signal entraining the fetus is a ho rmone . Mela tonin , the principal ho rmone of the pineal gland, was a pr ime candidate for the materna l signal communica t ing phase information to the f e t u s .

33 There is a robust rhy thm of melatonin in the

materna l circulation, and because melatonin can cross the placenta, a mela tonin rhy thm is reflected in the fetal c i r c u l a t i o n .

1 7 , 2 7'

4 0'

5 7 - 59

However , materna l pinealectomy (which eliminates measurable levels

Maternal Entrainment of a Fetal Biological Clock 97

of m e l a t o n i n

2 6) does not abolish materna l coordina t ion of fetal circadian

phase in r a t s ,

37 hamsters (F .C. Davis , personal communica t ion) , or spiny

mice (Weaver and Repper t , unpubl ished results). Fu r the rmore , removal of the mate rna l adrenals , thyro id-para thyro ids , pi tui tary or ovaries (in separate experiments) does not abolish the clear day-n igh t rhy thm of metabol ic activity in the fetal rat S C N when performed on or before day 7 of gestation (the time when materna l S C N lesions are d i s rup t ive ) .

37 The

materna l eyes, a potent ial source of bo th neural and endocrine signals, are also not necessary for the communica t ion , since dams enucleated on day 2 of gestation synchronize the circadian clocks of their fe tuses .

35

Another app roach to determine which aspect of materna l rhythmici ty entrains the fetus is to artificially restore rhythmicity in an SCN-lesioned d a m (who has no endogenous rhythmici ty) . Normal ly , there is a circadian rhy thm in food consumpt ion ; this rhy thm is disrupted by S C N l e s ions .

29

Restricted access to food in SCN-lesioned rats artificially produces a rhy thm in food consumpt ion and can also induce rhythms in locomotor activity and t e m p e r a t u r e .

2 2'

4 8 , 53 Using a food access restriction pa rad igm

(food cue) in SCN-lesioned pregnant rats , we and others have shown that the rhythmic ingestion of food can entrain fetuses of SCN-lesioned d a m s .

1 2'

53

Rhythmic food ingestion clearly would cause rhythmic fluctuations in nutr ient levels in the blood, and this could in turn induce physiological responses to the presence of nutr ients . In SCN-lesioned dams , food restriction may cause generat ion of a nutr ient-related signal which entrains the fetuses in a manne r parallel to tha t which occurs in intact dams . Alternatively, restricted feeding of SCN-lesioned dams can lead to the generat ion of other rhy thms (e.g. in tempera ture or activity) which may be involved in setting the phase of the fetus.

Because the fetus is exposed to a mul t i tude of materna l rhy thms (behavioral rhy thms , as well as ho rmona l ) , it is also possible that multiple materna l rhy thms act in concert to entra in the fetal biological clock (Figure 2). Thus , el iminating any one of these rhy thms would not be sufficient to disrupt materna l en t ra inment . This redundancy may explain some recent, seemingly contradic tory da ta .

As ment ioned above , the materna l pineal gland is not necessary for materna l en t ra inment of the fetus. However , Davis and M a n n i o n

6 have

convincingly shown that t imed injections of pharmacological doses of mela tonin into SCN-lesioned Syrian hamsters restore fetal synchrony. This finding suppor ts the redundancy hypothesis : while mela tonin is not necessary for fetal synchronizat ion, it is one of several circadian signals capable of synchronizing the fetal biological clock.

Another line of evidence implicating melatonin as an impor tan t media tor of time-of-day information from mothe r to fetus is the recent discovery of putat ive mela tonin receptors in the fetal S C N of several


s p e c i e s .

4 1'

5 4'

56 Thus , an anatomical substrate exists for a direct act ion of

materna l mela tonin on the fetal biological clock.

Discovery of a funct iona l dopamine system w i t h i n the fe ta l S C N

We have begun to address the issue of how diverse materna l signals might act at a cellular level to set the time of the fetal SCN. As a first app roach to address this issue, we have m a p p e d neurot ransmi t te r receptor gene expression in the fetal S C N . We discovered that a high level of D x- d o p a m i n e receptor m R N A is expressed in the fetal S C N .

55 Fur ther -

more , we showed that act ivation of these D x- d o p a m i n e receptors induces a high level of c-fos gene exp res s ion .

55

We examined whether Dx-dopamine receptors in the fetal S C N can activate c-fos gene expression by administer ing cocaine to pregnant rats . Cocaine was used because it acts on m o n o a m i n e release and reuptake at presynaptic t e r m i n a l s

1 9 , 23 so that only receptors receiving an endogenous

monoamine input should be affected by cocaine. Maternal ly administered cocaine (10-30 mg/kg) activated c-fos gene expression in the fetal S C N in a dose-dependent manner . The induct ion of c-fos m R N A in the fetal S C N appeared to be the result of a direct act ion of cocaine within the fetus, because cocaine administered directly to rat fetuses delivered by cesarean section on day 20 of gestation also induced c-fos expression in SCN. Interestingly, the ability of cocaine to induce c-fos expression in the fetal biological clock was not dependent on the time of day of adminis t ra-t ion, unlike the effects of light on S C N c-fos expression in adul t r o d e n t s .

3 , 1 0 , 2 0 , 2 1 , 3 1 , 43

Cocaine induct ion of c-fos expression in the fetal S C N was shown to occur in par t th rough Dx-dopamine receptor a c t i v a t i o n .

55 P re t rea tment

with the D rr e c e p t o r antagonis t S C H 23390 (0.2 mg/kg) blocked ca. 5 0 % of the cocaine-induced induct ion of c-fos m R N A in the fetal SCN. Fur the rmore , the D ^ d o p a m i n e selective agonist S K F 38393 (10 mg/kg) induced high levels of c-fos gene expression in the fetal S C N . An S K F 38393-induced increase in c-fos expression has been recently shown in the SCN of fetal mice and Syrian hamsters , as well as in fetal rats (Figure 3). Thus , D ^ d o p a m i n e receptor-mediated c-fos induct ion occurs in three rodent species in which maternal-fetal en t ra inment has been demon-strated.

The c-fos response to Όλ-dopamine receptor s t imulat ion in the fetal rat S C N is apparen t on G D 18 when the nuclei are immature and devoid of synapses, as discussed above. Nonetheless , neurot ransmiss ion could occur in the absence of convent ional synapses, since monoamines can be released from growth c o n e s .

24 Tyrosine hydroxylase-posit ive neurons and fibers

have been repor ted in the S C N region of the the fetal rat brain and could

Maternal Entrainment of a Fetal Biological Clock 9 9

Hamster

F I G . 3. D ^ d o p a m i n e recep tor ac t iva t ion induces c-fos express ion in the fetal S C N of three r o d e n t species. Dep ic ted a re dark-field p h o t o m i c r o g r a p h s of film a u t o r a d i o g r a m s of c o r o n a l b ra in sect ions t h r o u g h the fetal S C N (ar rows) . D a m s were injected ip wi th the specific Όί-dopamine agonis t S K F 38393 (10 m g / k g ) or vehicle. Fe ta l b ra ins were collected 40 minu te s after injection. Weave r a n d

Repper t , unpub l i shed d a t a .

provide a source of dopamine for Dx- recep to r act ivation within the fetal S C N . 3 2' 5 1- 52

The functional consequences of D^dopamine - r ecep to r -med ia t ed c-fos induct ion in fetal S C N may be substant ial . In the adult S C N , light exposure at night induces expression of c-fos m R N A and Fos p r o t e i n 3' 1 0 , 2 0 , 2 1 , 3 1' 43 and causes phase shifts in circadian r h y t h m s . 18 Fur the rmore , the quant i ta t ive features of l ight-induced c-fos expression and l ight-induced phase shifts are very similar suggesting tha t Fos may be a molecular componen t of the phot ic en t ra inment pa thway for m a m m a l s . 2 0' 21 We recently showed by Western blot analysis that Fos protein is expressed in the fetal hypo tha l amus (unpublished da ta ) . Thus , an activatable fetal dopamine system may represent an impor tan t unexpected mechanism for materna l signals to entra in the fetal S C N (Figure 4). Indeed, a dopamine system in the fetal hypo tha lamus may represent a final c o m m o n pa thway by which diverse entraining stimuli

1 0 0 Light and Biological Rhythms in Man

F I G . 4. H y p o t h e t i c a l m o d e l of a fetal d o p a m i n e sys tem serving as a final c o m m o n p a t h w a y for the ac t ion of m a t e r n a l en t r a in ing signals o n fetal S C N . M a t e r n a l signals act u p o n a d o p a m i n e sys tem wi th in the fetal S C N to regula te local d o p a m i n e release. D o p a m i n e b inds to its D l - r e c e p t o r in S C N a n d via second messenger systems ac t iva tes c-fos a n d o the r i m m e d i a t e early genes . F o s a n d J u n p ro te ins a re t r ans loca ted t o the nuc leus , d imer ize a n d in te rac t wi th A P - 1 b ind ing

sites on ta rge t genes u l t imate ly leading t o a p h a s e shift.

converge upon the fetal S C N . As previously ment ioned, the two identified entraining signals are rhythmic food ingestion and t imed injections of m e l a t o n i n .

6'

53 The rhythmic ingestion of food by the mo the r could

provide a rhythmic precursor pool of tyrosine, driving rhy thm fetal bra in dopamine p r o d u c t i o n ;

2'

11 maternal ly derived melatonin could directly

modula te the release of dopamine in the fetal S C N as demons t ra ted in other s y s t e m s .

8'

60 Studies to determine whether D ^ d o p a m i n e - r e c e p t o r -

st imulants can actually entra in the fetal S C N are currently underway.

Postnatal materna l e n t r a i n m e n t

Dur ing the pos tna ta l period, the materna l circadian system of altricial rodents continues to coordina te the t iming of the developing circadian s y s t e m .

49 It appears tha t mate rna l influences dur ing the pos tna ta l period

serve to mainta in or reinforce the coordina t ion of phase tha t has been established dur ing the prenata l period. In ra ts , the pos tna ta l materna l influence persists until the p u p develops the potent ial for direct l igh t -dark ent ra inment th rough its own eyes (via the re t inohypothalamic t ract) at the end of the first week of l i f e .

9'

4 7'

50

Maternal Entrainment of a Fetal Biological Clock 1 0 1

Potent ia l funct ions of an ent ra ined fe ta l c lock

The existence of prenata l communica t ion of circadian phase in m a m m a l s suggests that this p h e n o m e n o n is of adapt ive value. Mate rna l en t ra inment would presumably coordina te the pups to the d a m and to the environ-ment , so tha t when physiological and behavioral rhythmici ty later develops, the rhy thms are expressed in proper relat ionship to one ano ther and to the 24-hour day. This allows the young animal to more easily assume its temporal niche.

If the pups of altricial rodents were no t coordina ted to the envi ronment by the mother , they might exhibit inappropr ia te t iming of emergence from the bu r row or d isadvantageous behavioral pa t te rns when they e m e r g e d .

30

Fur the rmore , if each p u p were to develop its own rhythmici ty, there would be a period of disorganizat ion of the litter that could be detr imental to some or all members of the litter. F o r example , coord ina t ion of a dam's willingness to nurse and of all pups in the litter to suckle would clearly be of benefit. Circadian disorganizat ion at the level of the individual or litter could thus threaten survival of individual p u p s .

15

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Maternal Entrainment of a Fetal Biological Clock 1 0 3

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7

Daily Melatonin Infusion Entrains Free-Running Activity in Syrian and Siberian Hamsters R. K I R S C H , S. B E L G N A O U I , S. G O U R M E L E N a n d P. PÉVET

CNRS-URA 1332 "Neurobiologie des fonctions rythmiques et saisonnières", Université L. Pasteur, Strasbourg, France

In t roduct ion

I N RECENT years it has become clear that , in mammals , the pineal gland is essential for the regulation of photoperiodic responses and that melatonin, one of the hormones produced by this gland, is directly involved in such phenomena (for review see R e i t e r

26 and P é v e t

2 1) . Melatonin synthesis,

indeed, occurs during the dark period of the l ight/dark cycle and the durat ion of the secretion is directly dependent upon the durat ion of the dark p h a s e .

1 0'

1 6'

1 7 , 21 Moreover , subcutaneous administrat ion of melatonin in

pinealectomized animals by a method designed to produce a daily peak corresponding to either a long or a short durat ion can reproduce the effect of short or long photoper iod on e.g. their sexual a c t i v i t i e s .

6'

7'

9'

1 1'

2 0'

23 These

da ta clearly demonstra te that it is the durat ion of the nocturnal peak of melatonin which is the critical parameter in the transmission of the photoperiodic information to the central nervous system.

Another aspect of melatonin secretion, which has not to be forgotten is that its rhythm is a circadian one which, like many other circadian rhythms, is directly generated in the suprachiasmatic nuclei of the hypothalamus . This means that it persists in the absence of l ight/dark cycle (period slightly different from 24 hours) and is entrained to a 24 hour period by the light/dark cycle. Beside its role as the transducing signal for daylength, the periodic secretion of melatonin might thus be used as a circadian zeitgeber, directly or indirectly involved in the control of circadian rhythmicity.

Considerable speculation has surrounded this possible role of melatonin as a circadian zeitgeber, especially in humans for the t reatment of pathologically or socially induced disturbances of biological rhythms

107


(disturbed sleep in shift-workers or blind peoples, jet lag, e t c . . . . for review see Arend t

1) .

Although till now there is still little experimental evidences in mammals , all these informations as well as the clear da ta obtained in non-mammal ian vertebrates (for review see C a s s o n e

1 2) suggest a role of melatonin in a direct

control of circadian rhythmicity and, at least, ask for an expansion of our knowledge on this phenomenon.

In general, to date, as animal studies are concerned, there is only evidence for minor effects of the pineal on circadian timing in mammals (review in C a s s o n e

1 3) . Injections of melatonin at the same time everyday, however,

entrain the circadian locomotory pat tern of male and female rats free running in constant c o n d i t i o n s .

3 , 4 , 5'

25 Whether this observation is the

consequence of a pharmacological effect rather than a physiological one is still a debate, but it clearly established the useful properties of melatonin.

Daily melatonin injection induces, however, daily manipulat ion of the animals and this might interfere in the synchronisatory process studied. T o avoid this problem, and with the purpose to analyze the properties of melatonin, we have decided to administer the ho rmone by infusion. This technique, indeed, permit to mimic the daily nocturnal peak of plasmatic melatonin concentrat ion.

The present paper reports the results already obtained in two rodents species, the Syrian and Siberian hamsters.

Mater ia l and methods

Young adult male Syrian hamster (Mesocricetus auratus) were purchased from a commercial supplier (Centre de Product ion animale, Olivet, France) where they had been raised under a photoper iod of 14hL/10hD). Prior to experimentation the animals were adapted to the laboratory conditions for at least 2 weeks (photoperiod 14 hL/10hD, lights off at 18 hours , temperature 2 0 ± 1 ° C ) .

Male Siberian hamster (Phodopus sungorus) were raised in the laboratory (bred from animals kindly provided by D r Steinlechner in 1985). Since birth, they were raised, under constant artificial photoper iod (16 hL/8hD) with lights on at 4 hours , temperature 2 0 ± 1 ° C , until they were used for the present experiment (2 months old).

Dur ing the experiment, animals were housed individually in infusion cages, each of these cages being placed inside a light-tight and sound isolated ventilated wooden-box. Each box was photoregulated at a given condition (see below). In each infusion cage, wheel-running activity was permanently moni tored.

Pinealectomy was performed on animals anesthetized with either equithesin or pentobarbi tal . A hole centered on the l ambda was made in the

Melatonin and Circadian Locomotor Activity 1 0 9

skull. The pineal was removed from the sinus surrounding it with fine forceps. Animals were given 1 week for recovery after the operat ion before being at tached to the infusion system.

Cannulat ion of the animals for infusion was performed using a poly-ethylene tube. At 2 cm from the proximal end of the cannula a polyethylene collar was fixed and the cannula was thermoformed into a right angle. The animals were then anesthetized with halothane gas and the collar and the end of the cannula were introduced under the skin through a 3 cm incision on the neck. The collar prevented the cannula from being chewed and the extension of the cannula ensured a good efflux of the ho rmone solution. The distal end of the cannula was placed on a swivel fixed to a bar hanging on the top of the cage. The swivel was connected via polyethylene tube to a syringe. Infusions were performed using infusion pumps controlled by electronic timers. The syringes placed outside the wooden boxes were refilled every 2 or 3 days with freshly prepared solution. The cannula system was built in such a way that it did not prevent the animal to run within the wheel. The entire system enabled the animals to be infused subcutaneously during several months (e.g. 7 months in Figure 4). Evidently to obtain such da ta during a very long period of time is very difficult. Often after few weeks the cannulae were broken or obstructed, the animals were sick, refused to go within the wheel or even died. In all these situations, the partial da ta obtained, a l though often confirming our complete observations were systematically rejected. The complete da ta presented in these five illustrations were obtained from three Syrian and seven Siberian hamsters .

At the beginning of the experiment the Siberian hamsters were placed in their infusion cages and submitted to constant dim light (0.1-5 lux) (such constant conditions permit the regular cleaning of the cages). At the same time infusion of Ringer (6 or 8 hours /day depending of the animals, 33 μΐ/hours) were started. After a few weeks (3 or 4) when the free running conditions were well-established, melatonin infusion started (melatonin disolved in Ringer; 6 or 8 hours /day depending of the animals, 33 μΐ/hours; 16.5 /ig melatonin/hour) . After a few weeks (4 to 10) Ringer infusion was started again, a period which in few animals was followed by a new period of melatonin infusion.

F o r Syrian hamster , in order to test a possible effect of Ringer infusion, the animals were prepared and placed in the system a few weeks before starting the Ringer infusion. The lighting and infusion conditions were similar to those described above.

As the locomotor activity is concerned, the number of revolutions of the running wheel were sampled every 10 minutes by computer via a real time data acquisition system (built in our lab). Activity was double plotted over a 48 hour scale.

Dur ing all the experiment the animals received t ap water and food pellets ad libitum.


Results

All animals used in this study, showed in constant dim-light condit ions a classical free-run in their circadian locomotory pat tern . Mos t of the animals , either Siberian or Syrian hamsters , free-ran with a period slightly higher than 24 hours , which is classical in nocturna l species. Few animals , however, free-ran with period inferior to 24 hours and results obta ined with one of such animals are presented in Figure 5.

Infusion of Ringer, which in the Siberian hamster started immediately, did not interfere with the circadian locomotory pa t te rn , at least in the present experimental condi t ions , and a clear free run was observed (Figures 1, 2 and 3). This observat ion was confirmed in the Syrian hamsters , where the Ringer infusion started 3 weeks after the beginning of locomotor activity registration (Figures 4 and 5).

In such experimental condi t ions , in bo th species tested, daily infusions of melatonin (8 hours , Figure 1, 6 hours Figures 2, 3 ,4 and 5) were able to entrain the circadian locomotor pa t te rn to a period of 24 hours .

Some animals were entrained as soon as the infusions started (Figure 2), others only after few days (Figure 1). Sometimes considerable longer t ime, up to 59 days (Figure 4) was needed to get ent ra inment . This was due to the fact that mela tonin did not influence the activity rhy thm until the beginning of the infusion reached a certain momen t in the temporal organizat ion of the locomotor activity. Interestingly, a clear species difference was observed in this phenomenon .

In the Siberian hamster , melatonin had no effect unless the beginning of the infusion period did coincide with the animal 's activity onset (Figures 1, 2 and 3). In most of the cases the onset of activity was immediately following the beginning of mela tonin infusion (Figures 2-3) . In few situations (Figure 1), however, the onset of activity immediately preceeded the infusion period.

In the Syrian hamster , to the contrary , melatonin was able to entrain the activity rhy thm only when the beginning of the infusion period coincided with the second par t of the activity period. In this species, the onset of activity preceded always the beginning of melatonin infusion from 3 to 5 hours (Figures 4 and 5).

In bo th species, when the beginning of infusion coincided with the adequate period of the activity rhy thm, melatonin was able to entrain this rhy thm to a 24 hour period and that , even if the endogenous period of the animal was shorter than 24 hours (Figure 5).

Some animals , but not all of them (Figure 3) remained entrained as long as melatonin was administered (until 36 days in the experiments represented in Figures 1 and 2). When melatonin adminis t ra t ion was s topped, the activity rhy thm of some Siberian hamsters free ran almost immediately (Figure 1), while in others it took 4 to 14 days (Figures 2 and

Melatonin and Circadian Locomotor Activity 111

F I G . 1. D o u b l e p lo t t ed r u n n i n g wheel activity records of a Siber ian h a m s t e r . F r e e - r u n n i n g activity is unaffected by Ringer (R) infusions. After a few days of me la ton in (M) t r e a tmen t (8 h o u r s ) , an e n t r a i n m e n t of the act ivi ty r h y t h m is observed w h e n the beg inn ing of the infusion coinc ided with the onse t of act ivi ty. This e n t r a i n m e n t ceased immedia te ly when m e l a t o n i n was replaced by Ringer (R). In this a n i m a l the onse t of activity immedia te ly preceeded the m e l a t o n i n infusion pe r iod . T h e infusion pe r iod is ind ica ted by the vert ical b l ack l ines. T h e two vert ical d o t t e d lines t race onset a n d t e r m i n a t i o n of the subjective n ight

respectively.

3). In the two Syrian hamsters where the registration has been long enough to study this phenomenon , considerable time (Figure 4) elapsed from the terminat ion of mela tonin adminis t ra t ion to the re-establishement of the original free-running period.

In very few animals—all Siberian hamsters—it has been possible to study the effect of a second period of mela tonin infusion. Again, mela tonin was able to entrain the free-running activity rhy thm to a period of 24 hours and again this was possible only when the beginning of the infusion coincided with the onset of activity (Figure 2).

In Siberian hamsters , a different effect of mela tonin infusion has also


F I G . 2. Siber ian h a m s t e r activity d o u b l e plot showing the clear effect of me la ton in infusions (6 hou r s ) . In this an ima l the onset of activity is immedia te ly following the beg inn ing of infusion pe r iod . W h e n me la ton in t r e a tmen t was t e rmina ted a d i so rgan iza t ion of the activity p a t t e r n is observed before the full re -es tabl i shment of the free-run. W h e n a new sequence of me la ton in infusions s ta r ted a similar

d i so rgan iza t ion of the activity p a t t e r n is no ted before to ob t a in e n t r a i n m e n t .

Melatonin and Circadian Locomotor Activity 1

F I G . 3. In this Siber ian hams te r , infusion of m e l a t o n i n (6 h o u r s ) immedia te ly en t ra ins the f ree-running activity r h y t h m (coincidence be tween onset of act ivi ty a n d beg inn ing of the infusion pe r iod) . After 1 week, however , a d i so rgan iza t ion of the activity p a t t e r n is observed . This per iod is itself followed by new per iod (approx imate ly 2 weeks) of e n t r a i n m e n t a n d then aga in a p p e a r s a p e r t u r b a t e d p a t t e r n itself followed by a new synchron iza t ion . W h e n m e l a t o n i n t r e a t m e n t is

t e rmina t ed , after a few days , the free-running r h y t h m r e a p p e a r e d .

Light and Biological Rhythms in Man

M

ι hams te r , s la tonin (6 > days) a n d itered. T h e tided wi th


F I G . 5. D o u b l e p lo t t ed r u n n i n g wheel activity record of a Syr ian h a m s t e r f ree-running wi th a pe r iod smaller t h a n 24 h o u r s . Again an e n t r a i n m e n t of the activity was observed when the beg inn ing of m e l a t o n i n infusion coincided with the second p a r t of the act ivi ty pe r iod . Th i s effect ceased when m e l a t o n i n was

replaced by Ringer .


been observed. As shown in Figure 2 in the second period of melatonin infusion, after a few days, mela tonin clearly affected the system by disturbing the circadian pa t te rn of activity. When tha t happened , it was only after a few weeks tha t en t ra inment to a period of 24 hours was obtained. Such p h e n o m e n o n of disorganizat ion of the circadian pat tern of activity was frequently observed. F o r example, looking at Figure 3 it appears tha t mela tonin entrains the rhy thm initially for a few days and tha t afterwards this is followed by a disorganized period which itself is followed by a new ent ra inment period and so on.

Discussion

Syrian hamsters , cont rary to rats , are no t entrained by daily injections of mela tonin (for details see review of C a s s o n e ;

13 A r m s t r o n g

3) . The present

results, however, clearly demons t ra te that melatonin is able to entra in the free running rhy thm of locomotor activity of pinealectomized Syrian and Siberian hamsters when the h o r m o n e is administered in a way which duplicates its endogenous plasmatic nocturna l peak. The observed effect corresponds to a t rue ent ra inment , since terminat ion of mela tonin infusions reestablished the free-running rhy thm.

The da t a confirm the hypothesis that in m a m m a l s

3'

25 like in

non -mammal i an v e r t e b r a t e s ,

12 mela tonin is involved in the control or the

expression of circadian rhy thms . The quest ion of major interest concerning this effect of mela tonin is its

precise site and mechanism of act ion. The S C N , the circadian clock underlying rhythmicity in mammal s ,

contains mela tonin r e c e p t o r s

1 5'

3 0 , 3 3 , 34 and its metabol ism is k n o w n to be

directly affected by mela tonin (review in C a s s o n e

1 2) . Moreover , in rats

kept in constant da rk , daily injections of mela tonin entrain free-running circadian locomotor rhy thm, a result not observed in S C N lesioned a n i m a l s .

14 En t ra inment takes place when the onset of activity has just

passed injection t ime so tha t the active phase follows injection while the quiescent one precedes it. In these condit ions mela tonin induces a daily phase-advance. All these results strongly suggest tha t the circadian system is directly affected by mela tonin and, based on the phase-advancing propert ies of exogenous mela tonin , some au thors have postula ted tha t melatonin is working on the clock mechanism itself.

Apparent ly our results on the Syrian and the Siberian hamster confort such a hypothesis . A detailed analysis of our da ta , however, led us to think carefully with such an interpretat ion.

When melatonin infusion started, the en t ra inment was no t observed immediately; a period from a few days to several weeks was needed. We have to assume that , like in rat , melatonin 's entraining action was limited to a critical period of the circadian rest-activity cycle. In constant D D


condit ions the period of the free-run is slightly different from 24 hours and consequently the activity period shifted a few minutes every day. Depending on the start ing time of the infusion, a few days to several weeks are needed for the infusion period to reach the critical period of ent ra inment ability.

In Siberian hamsters , mela tonin was able to entrain the free-running activity rhy thm to a period of 24 hours only when the beginning of the infusion period coincided with the onset of activity. Again tha t agrees with the work done in the rat . The difference, however, is tha t in individual animals the onset of activity followed (Figure 2) or preceeded (Figure 1) the appar i t ion of mela tonin in the circulation. This observat ion clearly indicates tha t if mela tonin is able to entra in the circadian activity rhy thm its role in this system is no t as it has been suggested (Thomas and Armst rong , 1988) to switch from behavioral quiescence to activity.

The result obta ined in the Syrian hamster strongly suppor t this conclusion. Indeed, in this species, mela tonin was again able to entrain the rhy thm but only when the beginning of the infusion coincided with the second par t of the activity period. In these condit ions the onset of activity preceeded mela tonin for 3 to 5 hours . In fact the experimental pa rad igm used is mimicking the na tura l occurr ing covaria t ion between endogenous melatonin release and behavioral activity. Indeed, in the Siberian hamster , as in the rat , the locomotor activity starts at the beginning of the dark phase as does the mela tonin synthesis. T o the contrary , in the Syrian hamster , a l though the onset of activity is observed at the beginning of the dark phase , mela tonin synthesis is always observed 3 to 5 hours later (Skene et al, 1987). This difference in tempora l organizat ion implies a different m o d e of act ion of mela tonin in these two species.

In noc turna l animal , kept under cons tant condi t ion, the free-run period is slightly larger t han 24 hours . Consequent ly the onset of activity is delayed for a few minutes every day. In order to entrain this rhy thm to a 24 h o u r period, mela tonin thus needs to induce every day an advance in the onset of activity. Based on these results, is it possible to speak of a phase advance effect of mela tonin , a terminology which implies a direct effect of the h o r m o n e on the clock? Few of the animals used in this s tudy presented a free-running period shorter than 24 hours . In these condi t ions , perfusion of mela tonin , in the golden hamster (Figure 5) was also able to entra in the rhy thm to a 24 hou r period. T o do this, however, mela tonin has to induce a daily phase delay. Interestingly, this effect is also observed only when melatonin perfusion started 3 to 5 hours after the onset of activity, this means when the si tuation observed normal ly in Syrian hamster is mimicked. If we think in terms of a direct effect of mela tonin on the clock, we have thus to accept tha t mela tonin infused at the same circadian time is able to induce, depending of the animals , either a phase advance or a phase delay. Tha t is difficult to conceive. However , an effect of mela tonin on the


VIP-neurons of the S C N , neurons which are known to be the "gate" of the "clock" for synchronizing information, might explain our results.

Another possible mechanism of action of melatonin has to be considered. "Trigger circuits" initiating specific pat terns of h o r m o n e secretion or behaviors (e.g. locomotory activity) tha t possess a circadian componen t do not appear to be directly innervated by S C N neurons . Ma in target areas of S C N efferents are the paraventr icular nuclei of the hypo tha lamus (PVN) and of the tha lamus ( P V T ) .

1 8'

3 1'

32 As far as the

present work is concerned, this last s t ructure is of interest. The most remarkable feature of the P V T is its p rominent projection to the nucleus accumbens , olfactory tubercule, central amygdala and prefrontal c o r t e x ,

8 , 22 thus providing the S C N with an almost direct input to the

mesolimbo-cort ical complex, tradit ionally considered as the main center in the central nervous system for the initiation and maintenance of ( loco)motor behavior . Mela ton in binding sites have been observed in the P V T of the Siberian as well as of the Syrian h a m s t e r .

3 3 , 34 An effect of

melatonin at the level of the P V T might also explain the presently obtained da ta .

Are these results of physiological importance?

In our experimental condi t ions, the hamsters received 16.5 /xg/hour which corresponds to approximately a total a m o u n t of 100 /xg of melatonin infused per 6 hours and 120 ^g/8 hours . Lower doses have not yet been tested. Such dosage probably results in supraphysiological b lood levels of the ho rmone , at least at the beginning of the experiment. Wha t happens however after several mon ths of infusion (7 mon ths in Figure 4). Pi t rosky et al.,

23 indeed, have observed no significant difference in p lasma

melatonin concentra t ion when Syrian hamsters were infused for 2 mon ths with doses of mela tonin varying from 20 to 80 ng /hour and concluded that within 8 weeks, the metabol ism of mela tonin is s t imulated when melatonin doses is increased. It has no t been possible to measure mela tonin plasmatic concentra t ion in the Syrian hamster after our experiments. In the Siberian hamster , however, after 2 mon ths , a melatonin concentra t ion of 12.35 ± ng/ml has been measured in the middle of the infusion period. This value is supraphysiological bu t far from the high value found after an acute adminis t ra t ion of mela tonin (e.g. 250 ng/ml of p lasma 15 minutes after injection of 100 μg melatonin in the rat ; 100 ng/ml and 25 ng/ml, 15 minutes after injection of respectively 25 and 2.5 pg in the golden h a m s t e r ) .

1 9 , 2 4 , 27 Whether such results are pharmacological or physiologi-

cal is thus still a debate but the use of the present technique associated with lower concentra t ions of mela tonin should help answer this quest ion.

In conclusion, it appears that , in the Syrian as well as in the Siberian hamster , exogenous melatonin exerts an effect on the circadian locomotor activity. Nevertheless, at the present t ime, it is impossible to integrate such


results into any global function for the mammal i an p inea l /SCN axis. It remains to be determined if the sites of act ion for this entraining effect of melatonin is at the level of the S C N or at the level of s tructures which otherwise require the S C N (e.g. PVT) . In addi t ion, it is not yet clear whether these results are pharmacological or physiological ones.

A c k n o w l e d g e m e n t s

The au thors wish to thank Drs . M . Masson-Pévet and J. Stehle for their suggestions and critical remarks and Miss F . M u r r o for her secretarial aid.

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25 . R e d m a n J.R., A r m s t r o n g S .M. a n d N g K . T . (1983) F ree r u n n i n g activity r h y t h m s in the ra t e n t r a i n m e n t by me la ton in . Science 2 1 9 , 1089-1091 .

26. Rei ter R.J . (1987) T h e m e l a t o n i n message : d u r a t i o n versus coinc idence hypo theses . Life Sci. 4 0 , 2 1 1 9 - 2 1 3 1 .

27. Rol lag M . D . a n d S te t son M . H . (1982) M e l a t o n i n injection in to syr ian h a m s t e r s . In The Pineal and its Hormones (ed. Rei ter R.J . ) , p p . 143-152 . Alan R. Liss, N e w Y o r k .

28. Skene D.J . , Péve t P . , Vivien-Roels B. , M a s s o n - P é v e t M . a n d A r e n d t J. (1987). Effect of different p h o t o p e r i o d s o n concen t r a t i ons of 5 - m e t h o x y t r y p t o p h o l a n d m e l a t o n i n in the p ineal g land of the syr ian h a m s t e r . J. Endocrinol. 114 , 301 -309 .

29. T h o m a s E . M . W . a n d A r m s t r o n g S .M. (1988) M e l a t o n i n admin i s t r a t i on en t ra ins female ra t activi ty r h y t h m s in c o n s t a n t da rknes s bu t n o t in c o n s t a n t l ight. Am. J. Physiology 2 5 5 , R237-R242 .

30. Vanecek J., Pav l ik A. a n d I l lnerova H . (1987) H y p o t h a l a m i c m e l a t o n i n recep tor sites revealed by a u t o r a d i o g r a p h y . Brain Res. 4 3 5 , 359 -369 .

3 1 . W a t t s A . G . a n d S w a n s o n L . N . (1987) Efferent pro jec t ions of the sup rach ia sma t i c nucleus I I . Studies using r e t r o g r a d e t r a n s p o r t of fluorescent dyes a n d s imul t aneous pep t ide i m m u n o h i s t o - c h e m i s t r y in the ra t . J. Comp. Neurol. 2 5 8 , 2 3 0 - 2 5 2 .

32. W a t t s A .G . , S w a n s o n L . W . a n d S a n c h e z - W a t t s G . (1987) Efferent pro jec t ions of the sup rach i a sma t i c nucleus I. Studies us ing a n t e r o g r a d e t r a n s p o r t of Phaseolus vulgaris Leuco-agg lu t in in in the ra t . J. Comp. Neurol. 2 5 8 , 204 -229 .

33. W e a v e r D .R . , Rivkess S.A. a n d R e p p e r t S .M. (1989) Loca l iza t ion a n d cha rac te r i za t ion of m e l a t o n i n recep tors in r o d e n t b ra in by in vitro a u t o r a d i o g r a p h y . / . Neuroscience 9 , 2581-2590 .

34. Wi l l iams L . M . , M o r g a n P.J . , Has t i ngs M . H . , L a w s o n W. , D a v i d s o n G . a n d Howel l H . E . (1989) M e l a t o n i n recep to r sites in the syr ian h a m s t e r b r a in a n d p i tu i t a ry . Loca l iza t ion a n d cha rac te r i za t ion using

1 2 5I - i o d o m e l a t o n i n . / . Neuroendocrinology, 1 ,

315-320 .

8

Distribution of the Melatonin Receptor in the Central Nervous System of Vertebrates. Kinetic Parameters and Signal Transduction Pathways F R A N C O F R A S C H I N I a n d B O J I D A R S T A N K O V

Chair of Chemotherapy, Department of Pharmacology, University of Milan, Milan, Italy

Abstract

Subt le species differences a re observed in the d i s t r ibu t ion p a t t e r n of the high-affinity m e l a t o n i n recep tors in the cent ra l ne rvous sys tem. Lower ve r t eb ra tes ' b r a in s a r e enr iched , while l a b o r a t o r y m a m m a l s display m o r e restr icted d i s t r ibu t ion . T h e p a r s tubera l i s of the a d e n o p i t u i t a r y a n d the sup rach i a sma t i c h y p o t h a l a m i c nuclei have been uncovered as the ma jo r m e l a t o n i n ta rge ts in m o s t of the species s tudied , while in o the r s , neocor t ica l a reas also expressed significant r ecep tor levels. T h e m e l a t o n i n recep to r is l inked to a per tuss is toxin-sensitive G-p ro t e in , regu la t ing the adenylyl cyclase a n d possibly t he C a

++ influx, b u t

b ind ing site(s) n o t l inked to a G-p ro t e in have been descr ibed as well. Appa ren t l y m e l a t o n i n in terac ts wi th lower affinity with the G A B A a recep tor , m o d u l a t i n g the n e u r o n a l activity by po t en t i a t i on of e n d o g e n o u s G A B A effects.

In t roduct ion

AFTER THE synthesis of 2 - iodomela tonin in 1 9 8 4 , two valuable research tools for s tudying the mela tonin binding sites became available: a radioactive ligand with high specific activity and a poten t non-radioact ive mela tonin a g o n i s t .

27 Subsequently, a number of studies repor ted the

distr ibution and the kinetic parameters of the mela tonin receptor in the vertebrate brain; certain model systems were created, the signal t ransduc-tion pa thways and the mechanism of mela tonin act ion were studied. Several mela tonin analogs were synthesized and tested. This lecture will outline the recent achievements and will a t t empt to view in perspective the future t rends in this rapidly developing field.

121


Dist r ibut ion of the mela ton in receptor

Most of the animal studies have been conducted on labora tory rodents , bu t in an increasing number of reports mapp ing and character izat ion of the receptor have been performed in the brains of phylogenetically close or distant vertebrates, such as fish, amphib ians , reptiles, birds and various orders of mammal s (for review see reference 22).

In lower vertebrates ' brain, binding is widespread, and in fish, e.g. ra inbow t rout (Salmo gairdneri) and Atlantic sa lmon (Salmo salar), au to rad iography discovered highest levels in the optic tectum, nucleus semicircularis, lobus inferioris and the molecular layer of the c e r e b e l l u m .

1 , 11 The green anole lizard (Anolis carolinensis) expressed

2 - [

1 2 5I ] i o d o m e l a t o n i n binding with highest levels in the anter ior cerebral

cortex, anter ior dorsal ventricular ridge, lateral geniculate nucleus, lentiform thalamic nucleus, nucleus ro tundus , habenula , pretectum, tectum, pi tui tary and the suprachiasmat ic nuclei .

1

In the avian species studied (Gallus domesticus,

24 Coturnix coturnix, B.

Cozzi et al, unpubl ished) , the visual suprachiasmat ic nuclei, ovoid nuclei, accessory hypers t r ia tum and the neos t r ia tum were intensely labeled. Areas, such as the tectum opt icum, ec tomammilary body, dorsolateral anter ior thalamic nuclei, lateral geniculate nuclei, Edinger-Westphal nuclei, nucleus tr iangularis , nucleus ro tundus and pars magnocellularis of the nucleus isthmi expressed high receptor concentra t ions . Lower levels were recorded th roughou t the brain. The visual suprachiasmat ic nuclei are known to be the site of an endogenous pacemaker in birds and are thought to be the homologue to the mammal i an suprachiasmat ic n u c l e i

2 6. The

localization pat tern observed in the low vertebrates suggests an involve-ment of mela tonin mainly in the processing of visual information.

Mela tonin binding sites in the C N S of a primitive mammal i an species, the Bennet 's wallaby (Dendrolagus bennettianus) were also widespread th roughou t the brain , comparab le to most of the non-mammal i an spec ies .

17 The brain of the marsupials is relatively simple, however. It lacks

corpus callosum, and the neopal l ium displays absence or very simple convolut ions of the cerebral hemispheres.

In some of the higher mammal s , however, intense 2 - [

1 2 5I ] i o d o -

melatonin binding can also be found in m a n y areas of the neocortex, apar t from the paleocortex and the archicortex. In the brain of the Old Wor ld rabbi t (Oryctolagus cuniculus) 2 - [

1 2 5I ] i o d o m e l a t o n i n binding was exten-

sive, similarly to the lower vertebrates. M a n y of the phylogenetically old cortical areas, such as the h ippocampus , indusium griseum and olfactory cortex were intensely labeled. In Lagomorphs indusium griseum is well developed and has not yet undergone the part ial regression typical for the brain of the higher mammal s and Primates. Binding, however, was s t rong in the greater par t of the neocortex, as w e l l

1 9 , 20 (Figure 1).

F I G . 1. A u t o r a d i o g r a p h s from c o r o n a l (A-D) a n d pa ra sag i t t a l ( E , F ) sec t ions , showing the d i s t r ibu t ion of 2 - [ 1 2 5I ] i o d o m e l a t o n i n b ind ing in the r abb i t b ra in . A, Β a n d C are sect ions t h r o u g h the r ight hemisphe re , 300 μπι a p a r t . D : c o r o n a l sect ion t h r o u g h the cent ra l segment . C G , c ingula te gyrus ; C P , c h o r o i d p lexus of the th i rd ventr icle; D L , do r so l a t e r a l t ha l amic nuclei ; H I , h i p p o c a m p u s ; I G , i n d u s i u m gr i seum; L G , la tera l genicula te gang l ion ; O C , occipi ta l cor tex ; P C , par ie ta l cor tex ; P T , p a r s tubera l i s ; S C N , sup rach i a sma t i c nuclei ; S C , super io r col l iculus; S G , s t r a t u m g r a n u l o s u m d e n t a t u m ; S P , s t r a t u m p i r amida l e ; S U , sub icu lum; T , t a p e t u m . N o t e the diffuse b ind ing in the h y p o t h a l a m u s . Scale

b a r = 5 m m .

A c o m m o n feature in l abora tory rodent brains studied to da te is the limited distr ibut ion of 2 - [ 1 2 5I ] i o d o m e l a t o n i n binding. Three anatomical sites have been most frequently ment ioned as potent ial mela tonin targets: the pars tuberalis of the pi tui tary gland, the hypotha lamic suprachiasma-


tic and the paraventr icular thalamic n u c l e i .

5 , 2 5'

2 8'

30 Another putat ive site

of melatonin action in the rat brain is the area p o s t r e m a .

1 4'

31 Binding in

this area was not detected in any of the strains of labora tory m i c e ,

2 4'

30

Syrian (Mesocricetus auratus) and Djungar ian (Phodopus sungorus) hamster b r a i n s .

31

An apparen t exception a m o n g rodents is a New Wor ld species, the white-footed mouse (Peromyscus leucopus), where the basal forebrain and the hypo tha lamus , the amygdala , tha lamus and a number of neocortical areas were intensely l a b e l e d .

30 It is not clear why in the brain of this species

mela tonin receptor is expressed with distr ibution so different from the rest of the rodents examined until now. The difference could be due to the fact that the Connect icut and the Georgian stocks of white-footed mice have been bred in captivity for only few generat ions, compared to the labora tory albino rat , hamster and the c o m m o n labora tory strains of mice. O n the other hand , au to rad iography may have overlooked some areas because of limits in resolution. Specific 2 - [

1 2 5I ] i o d o m e l a t o n i n binding

was reported in other regions of the rat and hamster brain, such as cortex and h ippocampus , detected by in vitro l igand-receptor b i n d i n g .

1 0'

33

Binding in the olfactory bulb was repor ted in the Syrian h a m s t e r

10 and the

r a b b i t ,

1 9'

20 and au to rad iography revealed melatonin receptors in the

olfactory epithelium of the Siberian hamster (Phodopus sungorus).

5

Of the large order of Art iodactyla , two species have been a subject of study so far: the domestic sheep (Ovis aries) and the goat (Capra hircus). In the sheep, 2 - [

1 2 5I ] i o d o m e l a t o n i n binding was reportedly confined to the

pars tuberalis of the pi tui tary g land .

7 Later experiments demonst ra ted

that mela tonin receptor is more widespread in the brain of this s p e c i e s .

4 19

The suprachiasmat ic nuclei were found to express low to undetectable levels of binding, diffuse labeling being observed in the immediate vicinity. The retrosplenial, t empora l , occipital and entorhinal cortex, the subicu-lum, the molecular layers of the h ippocampus adjacent to the denta te gyrus, the medial preoptic nucleus and the preoptic area expressed high to medium melatonin receptor levels. Low levels of binding were recorded as well in the anter ior hypo tha lamus , medial basal hypo tha lamus , paraven-tricular thalamic and supramammila ry nuclei. In the midbra in the binding was restricted to the ventral raphe complex and the inferior colliculus. In the caprine brain, melatonin receptor distr ibution was similar, with the apparen t difference of higher binding levels recorded in the suprachiasma-tic nucle i .

8

Of the three recent families contained in the order Perissodactyla, only the horse (Equus caballus) has been a subject of investigation. In the brain areas examined by in vitro binding, melatonin receptor was detected with a limited distr ibution. Highest levels were recorded in the pars tuberal is / median eminence r e g i o n .

19

In the brain of the only carnivore species examined, the ferret (Mustela

Distribution of the Melatonin Receptor 1 2 5

sp.), b inding was limited to the pars tuberalis and pars distalis of the

pituitary g l a n d .

32

The first work on mela tonin receptor dis t r ibut ion in the brain of Pr imates , described high affinity 2 - [

1 2 5I ] i o d o m e l a t o n i n binding in the

suprachiasmat ic nuclei of the h u m a n hypo tha lamus . Surprisingly, binding was not repor ted in the pars tuberalis of the pi tui tary g l a n d .

18 In the

b a b o o n (Ραρίο ursinus) and the vervet monkey (Cercopithecus aethiops), melatonin receptor was expressed with high density in the pars tuberalis of the pi tui tary gland (Figure 2), and the collar of pars distalis, present in Pap io , and the suprachiasmat ic nuclei were diffusely labeled. The neurohypophysis was devoid of binding (Stankov et al, unpubl ished) . Thus , of the m a m m a l s studied so far, the h u m a n is the only species where 2 - [

1 2 5I ] i o d o m e l a t o n i n binding was no t repor ted in the pars tuberalis of

the pi tui tary gland. In general, there seems to exist a phylogenetic t rend towards a

restriction in the brain areas expressing mela tonin receptors . Apparent ly , fewer areas possess high affinity mela tonin binding sites in higher m a m m a l s . The number of compara t ive studies is still very limited and there are m a n y species that have not been the subject of investigation to date .

Kinet ic parameters and signal t ransduct ion p a t h w a y s

The first results on the mela tonin receptor kinetic parameters , ob ta ined by using 2 - [

1 2 5I ] i o d o m e l a t o n i n , were controversial . The apparen t affinity

was repor ted either in the low or medium n a n o m o l a r range in the r a t

33 and

the Syrian h a m s t e r

10 brains . At the same t ime, p icomolar affinity binding

site was described in the rat median eminence and suprachiasmat ic n u c l e i .

28 Considering that mela tonin peripheral b lood concentra t ions in

all species studied to da te are in the high p icomolar or low n a n o m o l a r range, the p icomolar K d and Ki values seemed physiologically app ro -priate and suggested tha t 2 - [

1 2 5I ] i o d o m e l a t o n i n labeled a relevant

receptor.

Later work again pointed out to the existence of two binding forms, a low and a high affinity. Thus , a hypothesis for the existence of two mela tonin receptor types (ML-1 and ML-2) was forwarded .

9 Exper iments

on t ransduct ion of the mela tonin signal at cellular level demons t ra ted tha t what has been described as high- and low affinity binding type, m a y be in fact the same receptor class, undergoing shifts in its apparen t affinity according to the process of coupling and uncoupl ing to its G - p r o t e i n ;

5'

2 0'

2 0'

30 a well documented p h e n o m e n o n for several G-prote in

coupled receptors . Moreover , pharmacological identity of the high-affinity binding site was demons t ra ted in the avian and mammal i an

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b r a i n s .

23 Inhibi t ion of forskolin-stimulated c A M P formation by physio-

logical concentra t ions of mela tonin was demons t ra ted in expiants from hamster , rat , sheep and mouse pars tuberalis , and rabbi t cortex (Figure 3, for review see reference 22). The inability of pertussis toxin to completely abolish the melatonin inhibitory effect on c A M P levels in some systems indicates part ial sensitivity to pertussis toxin. The main effect of pertussis toxin-mediated rybosilat ion is to block the interact ion between the receptor and its G-prote in . Thus , the synergistic effect of bo th pertussis toxin and G T P in inhibiting high affinity mela tonin binding in sheep pars tuberalis suggested tha t a single class of possibly two subtypes receptors is coupled to two G-proteins , pertussis toxin-sensitive and -insensitive, to regulate the adenylate cyc lase .

15 However , mela tonin binding sites tha t

are not coupled to a G-prote in have been observed in the neuronal r e t i na

6

and the basal bra in arteries (Stankov, unpubl ished da ta ) . Interestingly, melatonin receptors expressed in Xenopus oocytes following microinjec-tion of poly ( A )

+ R N A , extracted from ovine pars tuberalis , coupled to a

signal t ransduct ion mechanism with characteristics similar of an inos i to l -phospha te /Ca

+ s y s t e m .

13 In model systems that make use of pars

tuberalis cells and /o r expiants it would be of extreme interest to investigate the molecular basis of mela tonin action in the control of L H release. This exocytotic process has very complex control mechanisms, involving diverse second messenger systems and short feedback loops between L H and G n R H . Novel studies demons t ra ted that mela tonin inhibited G n R H -stimulated C a

++ elevation in neonata l rat pi tui tary cells, p resumably

acting th rough a pertussis toxin-sensitive G - p r o t e i n .

29

2 0 η 1

C F FM F M / P T

F I G . 3. Inh ib i to ry effect of m e l a t o n i n (10 nM) o n forskol in-s t imula ted (10 μΜ) a c c u m u l a t i o n of c A M P in rabb i t cor t ical exp ian t s a n d the b lock ing effect of per tussis toxin (1 μg /ml ) . C , con t ro l ; F , forskolin; F M , forskolin p lus m e l a t o n i n ; F M / P T , forskolin p lus me la ton in , p r e i n c u b a t i o n wi th per tuss is toxin . *, ρ > 0.05.

F r o m reference 2 1 .


7ΖΖΆ

(Scale = 144.8 S /Diw)

GABA \ZZ MELATONIN

β.880 hS <Sc*le = 144.

• I GABA \Z2 MELATONIN Π BICUCULLINE

F I G . 4. M e l a t o n i n (100 nM c o n c e n t r a t i o n in the e lec t rode , appl ied pressure 0.22 k g / c m

2) is ineffective per se, b u t s t rongly po ten t i a t e s the effect of G A B A in

suppress ing the s p o n t a n e o u s firing activity of the ra t re t icular t ha l amic nuclei n e u r o n s (A). T h e specific G A B A a an t agon i s t s bicucull ine coun te rac t s the effects

of ei ther G A B A or me la ton in (B). F r o m reference 2.

Recent experimental evidence also demons t ra ted that nanomola r concentrat ions of melatonin increased the inhibitory effect of GABA on the firing activity of single neurons in the rat reticular tha lamic nuclei .

2

Fur the rmore , the potent melatonin agonists 2- iodomelatonin and 2-bromomela ton in expressed benzodiazepine-like effects, similar to what was described in the rabbit c o r t e x .

20 The difference from the rabbit cortex

was that melatonin per se, in low concentrat ions was wi thout effect on the firing activity of the thalamic reticular nuclei neurons , bu t was able to increase significantly the inhibitory effect of GABA. This action could be counteracted by the G A B A a receptor antagonis t bicucul l ine

3 (Figure 4).

High affinity melatonin binding sites have not been demonst ra ted in the rat reticular thalamic nuclei. Thus , it appears that melatonin can exert

A


biological effects (benzodiazepine-like in this case), wi thout necessarily binding to its own receptor, but utilizing other complex receptor systems, i.e. acting as a modu la to r . Recently allosteric modula t ion of i - [

3 5S ] T B P S

binding by melatonin was demons t ra ted in the rat b r a i n .

16 Therefore, the

action of mela tonin and its potent agonists can apparent ly be exerted t rough a dual mechanism: (i) interact ion with its high affinity receptor in discrete brain areas, leading to an inhibit ion of the adenylyl cyclase; (ii) possibly th rough a non -GABA, non-benzodiazepine binding site mela to-nin is acting on the great GABA receptor complex, modula t ing the cellular chlorine influx and potent ia t ing the effects of endogenous GABA. These interact ions can explain the repor ted hypnot ic propert ies of mela tonin in h u m a n s and may serve as a basis for the development of clinical applications of the indole. Studies with heal thy young h u m a n volunteers demons t ra ted tha t melatonin can successfully be used as supplement t rea tment to sleep-inducing and -maintenance benzodiazepine therapy, allowing for a reduct ion in the benzodiazepine doses with significant ameliorat ion of the sleep, determined by the microst ructural changes and the subjective assessment of the sleep q u a l i t y .

12

References

1. Agge lopou los N . a n d D e m a i n e C . (1990) Q u a n t i t a t i v e recep tor a u t o r a d i o g r a p h y of me la ton in recep tors in th ree n o n - m a m m a l i a n ve r t eb ra te s : the green ano le l izard (Anolis carolinensis), the edible frog ( R a n a esculenta) a n d the r a i n b o w t r o u t (Salmo gairdneri), In Vth Colloquium of the European Pineal Study Group, Gui ld ford , U . K . , abs t r . 66.

2. Biella G. , P a n a r a C , Cozzi B. a n d Frasch in i F . (1991) M e l a t o n i n enhances G A B A -med ia t ed effects w h e n admin i s t e red by mic rop res su re ejection in single uni t n e u r o n a l record ings . In Role of Melatonin and Pineal Peptides in Neuroimmunomodulation (eds. F rasch in i F . a n d Reiter R.J . ) , p p . 191-200 . P l e n u m Press , N e w Y o r k .

3. Biella G. , P a n a r a C , S t a n k o v B., Fe r in i -S t r ambi L. a n d Frasch in i F . (1992) M e l a t o n i n -induced m o d u l a t i o n of G A B A a synapses in the cen t ra l n e r v o u s system. A m o d e l a n d a new theory . Sleep (in press) .

4. B i t t m a n E .L . a n d W e a v e r D . R . (1990) T h e d i s t r ibu t ion of m e l a t o n i n b ind ing sites in n e u r o e n d o c r i n e tissues of the ewe. Biol. Reprod. 4 3 , 9 8 6 - 9 9 3 .

5. C a r l s o n L.L. , W e a v e r D . R . a n d Reppe r t S .M. (1991) M e l a t o n i n recep tors a n d signal t r a n s d u c t i o n du r ing deve lopmen t in Siber ian h a m s t e r s (Phodopus sungorus). Devel. Brain. Res. 5 9 , 8 3 - 8 9 .

6. C h o n g N . W . S . a n d Sugden D . (1991) G u a n i n e nucleot ides regula te 2 - [

1 2 5I ] i o d o -

me la ton in b ind ing sites in the chick re t inal p igmen t ep i the l ium, bu t no t in in n e u r o n a l re t ina . J. Neurochem. 5 7 , 685 -689 .

7. DeRevie r s M - M . , Ravau l t J -P . , Tillet Y. a n d Pellet ier J. (1989) M e l a t o n i n b ind ing sites in the sheep p a r s tubera l i s . Neurosci. Lett. 100 , 8 9 - 9 3 .

8. Deveson S., H o w a r t h J., Arend t J. a n d F o r s y t h LA. (1990) In vi t ro a u t o r a d i o g r a p h i c a l local izat ion of m e l a t o n i n ( M T ) b ind ing sites in the capr ine b ra in . In Vth Colloquium of the European Pineal Study Group, Gui ld ford , U K , abs t r . 67.

9. D u b o c o v i c h M . L . (1988) P h a r m a c o l o g y a n d function of me la ton in recep tors . FASEB J. 2 , 2 7 6 5 - 2 7 7 3 .

10. D u n c a n M.J . , T a k a h a s h i J .S . a n d D u b o c o v i c h M . L . (1988) 2 - [

1 2 5I ] I o d o m e l a t o n i n

b ind ing sites in h a m s t e r b ra in m e m b r a n e s : p h a r m a c o l o g i c a l charac ter i s t ics a n d reg ional d i s t r ibu t ion . Endocrinology 1 2 2 , 1825-1833 .

11. E k s t r o m P . a n d Vanecek J. (1992) Loca l i za t ion of 2-[*

2 5I ] i o d o m e l a t o n i n b ind ing sites

in the b ra in of the At lan t ic s a l m o n , Salmo salar. Neuroendocrinology 5 5 , 529-537 .


12. Fe r in i -S t r ambi L., Z u c c o n i M. , Biella G. , S t a n k o v B. , F rasch in i F . , Z a m b o n i M . G . a n d Smirne S. (1991) Sleep l a b o r a t o r y s tudy o n me la ton in effects in hea l thy subjects . In Third Milano International Symposium on Sleep, p . 31 (Abstr . ) . Sep tember 18-19 .

13. F ra se r S.P., Bar re t t P . , D j a m g o z M.B.A. , a n d M o r g a n , P .J . (1991) M e l a t o n i n receptor m R N A express ion in Xenopus oocytes : inh ib i t ion of G-pro te in -ac t iva ted response Neurosci. Lett. 124 , 2 4 2 - 2 4 5 .

14. La i t inen J .T. , F lugge G . a n d Saaved ra J . M . (1990) Cha rac t e r i za t i on of m e l a t o n i n receptors in the ra t a rea p o s t r e m a : m o d u l a t i o n of affinity wi th ca t ions a n d guan ine nucleot ides . Neuroendocrinology 5 1 , 619 -624 .

15. M o r g a n P.J . , D a v i d s o n G. , L a w s o n W . a n d Bar re t t P . (1990) B o t h per tussis toxin-sensi t ive a n d insensit ive G-p ro t e in s l ink m e l a t o n i n recep tor to inhibi t of adeny la t e cyclase in the ovine pa r s tubera l i s . J. Neuroendocrinol. 2 , 7 7 3 - 7 7 8 .

16. Niles L . P . a n d Peace C H . , (1990) Allosteric m o d u l a t i o n of i - [

3 5S ] b u t y l b i c y c l o -

p h o s p h o r o t h i o n a t e b ind ig in r a t b r a in by me la ton in . Brain Res. Bull. 2 4 , 635 -637 . 17. P a t e r s o n A . M . , C h o n g N . , Sugden D . , Br ink low B.R. a n d L o u d o n A.S.I . (1990) Studies

of the role of me la ton in in de t e rmin ing the t rans i t ion to seasonal r eproduc t ive quiescence a n d p re l iminary obse rva t ions o n m e l a t o n i n b ind ing sites in t he b r a in of a ma r sup i a l , the Bennet t ' s wal laby . In Vth Colloquium of the European Pineal Study Group, Gui ldford , U K , abs t r . 60.

18. R e p p e r t S.M., W e a v e r D .R . , Rivkees S.A. a n d S t o p a E.G.(1988) P u t a t i v e me la ton in receptors in h u m a n biological c lock. Science 2 4 2 , 7 8 - 8 1 .

19. S t a n k o v B. , Cozzi B. , Lucini V., F u m a g a l l i P . , Scagl ione F . a n d Frasch in i F . (1991) C h a r a c t e r i z a t i o n a n d m a p p i n g of m e l a t o n i n receptors in the b ra in of th ree m a m m a l i a n species: r abb i t , ho r se a n d sheep . Neuroendocrinology 5 3 , 2 1 4 - 2 2 1 .

20. S t a n k o v B. , Cozzi B. , Lucini V., C a p s o n i S., F a u t e c k J., F u m a g a l l i P . a n d F rasch in i F . (1991) Loca l iza t ion a n d cha rac te r i za t ion of m e l a t o n i n receptors in the rabb i t b r a in by a u t o r a d i o g r a p h y a n d in vi t ro l igand- recep tor b ind ing . Neurosci. Lett. 133 , 6 8 - 7 2 .

2 1 . S t a n k o v B. , Biella G. , P a n a r a C , Lucini V., C a p s o n i S., F a u t e c k J., Cozzi B. a n d Frasch in i F . (1991) M e l a t o n i n signal t r a n s d u c t i o n a n d m e c h a n i s m of ac t ion in the Cen t r a l N e r v o u s System: Us ing the r abb i t cor tex as a mode l . Endocrinology 130 , 2152-2159 .

22. S t a n k o v B. , F rasch in i F . a n d Rei ter R.J . (1993) T h e M e l a t o n i n Recep to r : D i s t r ibu t ion , Biochemis t ry a n d P h a r m a c o l o g y . In Melatonin: Biosynthesis, Physiological Effects and Clinical Applications (eds. Rei ter R.J . a n d Yu H.S. ) , p p . 155-186. C h a p t e r 7. C R C Press , Boca R a t o n , F lo r i da .

23 . Sugden D . a n d C h o n g N . W . S . (1991) P h a r m a c o l o g i c a l ident i ty of 2 - [

1 2 5I ] i o d o -

me la ton in b ind ing sites in chicken b ra in a n d sheep pa r s tubera l i s . Brain Res. 5 3 9 , 151-158 .

24. Suiciak J.A., K r a u s e D . N . a n d D u b o c o v i c h M . L . (1991) Q u a n t i t a t i v e pha r maco log i ca l analysis of 2-\}

2 5I ] i o d o m e l a t o n i n b ind ing sites in discrete a reas of the chicken b ra in . J.

Neurosci. 1 1 , 2855 -2863 . 25 . Siuciak J. Α., F a n g J - M . a n d D u b o c o v i c h , M . L . , (1990) A u t o r a d i o g r a p h i c local iza t ion

of 2 - [

1 2 5I ] - i o d o m e l a t o n i n b ind ing sites in the b ra ins of C 3 H / H e N a n d C 5 7 B L / 6 J

s t ra ins of mice . Eur. J. Pharmacol. 180 , 387 -390 . 26. U n d e r w o o d H . (1989) T h e p ineal a n d m e l a t o n i n : r egu la to rs of c i rcadian function in

lower ver tebra tes . Experientia 4 5 , 9 1 4 - 9 2 2 . 27. V a k k u r i O . , L a m s a E. , R a h k a m a a E. , Ruo t sa l a inen H . a n d L e p p a l u o t o J. (1984)

I o d i n a t e d m e l a t o n i n : p r e p a r a t i o n a n d cha rac te r i za t ion of the molecu la r s t ruc tu re by mass a n d 1H N M R spec t roscopy . Anal. Biochem. 142 , 284 -289 .

28. Vanecek J., Pavl ik A. a n d I l lnerova H . (1987) H y p o t h a l a m i c me la ton in recep tor sites revealed by a u t o r a d i o g r a p h y . Brain Res. 4 3 5 , 359 -362 .

29. Vanecek J. a n d Kle in , D . C . (1992) M e l a t o n i n inhibi ts gonago t rop in - re l eas ing h o r m o n e - i n d u c e d e levat ion of in t racel lu lar C a

++ in n e o n a t a l ra t p i tu i t a ry cells.

Endocrinology 130 , 701-707 . 30. W e a v e r D.R. , C a r l s o n L .L . a n d Reppe r t S .M. (1990) M e l a t o n i n receptors and signal

t r a n s d u c t i o n in mela tonin-sens i t ive a n d mela tonin- insens i t ive p o p u l a t i o n s of whi te-footed mice (Peromyscus Leucopus). Brain Res. 5 0 6 , 353-357 .

3 1 . Weave r D.R. , Rivkees S.A. a n d Reppe r t S .M. (1989) Loca l iza t ion a n d cha rac te r i za t ion


of m e l a t o n i n recep tors in roden t b ra in by in vitro a u t o r a d i o g r a p h y . Neurosci. 9 , 2581-2590 .

32. W e a v e r D . R . a n d Reppe r t S .M. (1990) M e l a t o n i n recep tors are present in the ferret pa r s tubera l is and pa r s distal is , bu t no t in b ra in . Endocrinology 127 , 2607-2609 .

33. Zisapel N . , N i r I . a n d L a u d o n , M . (1988) C i r cad i an va r i a t ions in m e l a t o n i n - b i n d i n g sites in discrete a reas of the ma le rat b ra in . FEBS Lett. 2 3 2 , 172-179.

Alphabet ica l list of the abbreviat ions used in the f igures

C G , cingulate gyrus; C P , choroid plexus of the third ventricle; D L , dorsolateral thalamic nuclei; H I , h ippocampus ; I G , indusium griseum; L G , lateral geniculate ganglion; O C , occipital cortex; P C , parietal cortex; P T , pars tuberalis; S C N , suprachiasmat ic nuclei; SC, superior colliculus; SG, s t ra tum granulosum den ta tum; SP , s t ra tum piramidale; SU, subicu-lum; T, t ape tum.

9

133

Neurotransmitters and Peptides in the Pineal Gland A. Y U W I L E R

1 a n d G. L B R A M M E R

2 1 Neurobiochemistry Laboratory 7-85, West Los Angeles Veterans Alfa/rs

Medical Center Brentwood Division, Los Ange/es, CA 90073, USA 2 Β rain Research Institute and Department of Psychiatry and Biobehavioral

Sciences, UCLA School of Medicine, University of California, Los Angeles, CA 90024, USA

Abst rac t

While n o r e p i n e p h r i n e is r ega rded as the p r i m a r y regu la to r of m e l a t o n i n b iosynthes is , an increas ing n u m b e r of "classical" t r ansmi t t e r s a n d n e u r o p e p t i d e s have been impl ica ted as m o d u l a t i n g pineal funct ion. T h e a n a t o m i c a l a n d funct ional bases for these suggest ions is briefly reviewed. In add i t i on , some of the similarit ies a n d differences be tween the effects of β-adrenerg ic a n d V I P s t imula t ion on induc t ion of TV-acetyltransferase activity are discussed.

SCIENCE MOVES in successive waves of accumulat ing da ta which eventually consolidate into general principles only to swell again as new da ta accumulates . The neurosciences are in the midst of such a wave. The growing number of identified neurally active c o m p o u n d s and neuropep-tides, each with its assor tment of receptors, appear to eventually activate a series of second messenger systems. Current ly the n u m b e r of such second messenger systems is much smaller than the p roduc t of the number of t ransmit ters and their receptor subtypes. Since many cells receive multiple inputs from different t ransmit ters using the "same" second messenger systems, the quest ion arises over whether cells uniquely differentiate between different agonists s t imulat ing the same second messenger system. If so, how? If not , what is the purpose of the r edundan t incoming code?

The pineal gland has been useful in explicating noradrenergic t ransmit-ter t ransduct ion , and the discovery of multiple receptor inputs into the pineal permits its use in defining t ransmit ter interact ions as well. New information brings new complexities, and even the very well established role of norepinephr ine in mela tonin synthesis in the r at

2 5'

2 7'

67 turns out to

be at least one step more complex than originally believed. N o t only does noradrenergic s t imulat ion of the βΐ receptor drive increased N-acetyl-


T A B L E 1

Status of proposed modulators of pineal function

Presence Binding Release Act ion Inac t iva t ion

Acetylchol ine 17,44,50,57,59 14 — 15,30,56 — Adenos ine * 55 — 9,10,40,41,46,65 — D o p a m i n e — 13 — — — G A * 12 — — — G A B A 51 51 51,53 31,52 58 N o r e p i n e p h r i n e 28 24 25 25,27 — Sero ton in 29 15 1,6 61 — T a u r i n e 8 — 68 68 — E n k e p h a l i n 37 2,3 — — — N P Y 60,75 — — 42,47,64 — Subs tance Ρ 48,60 11 — — — P H I 33 62 — 38 — V I P 34,35,63 19,32 — 45,67 —

N u m b e r s refer to references in the Bib l iography . * signifies a general cell cons t i tuen t .

transferase activity but concomitant noradrenergic s t imulat ion of post-synaptic a l receptors, while having no effect of its own, augments ^-st imulated YV-acetyltransferase (NAT) synthesis. A growing list of other putat ive t ransmit ters possibly implicated in regulating melatonin synthesis has also accumulated over the years. These include a d e n o s i n e ,

9'

1 0 , 4 1 , 4 6 , 65 y-aminobutyric acid ( G A B A ) ,

5 1 , 5 2 , 53 substance

P ,

47 t a u r i n e

6 8 , 6 9 , 70 e n k e p h a l i n e s ,

2 , 3'

37 s e r o t o n i n ,

61 and more recently the

polypeptides vasoactive intestinal peptide ( V I P ) ,

2 2 , 72 peptide ^ - t e rmina l

histidine C-terminal isoleucine ( P H I )

3 3 , 3 8 , 62 and neuropept ide Y

(NPY)

3 6'

4 2'

4 7'

6 4'

7 1'

75

The actual case for these various substances as regulators of pineal function is generally incomplete , and species variat ions are great. There are four minimal requirements for a substance to be considered a regulator. First it must reach some active site. The route could be local product ion , neuronal release, or by body fluid t ranspor t . Second, it should be released in some physiologically relevant manner : if in nerves, it should be released by depolar izat ion; if in b lood or if p roduced locally, by some physiologically specific discharge. Third , it should produce some physio-logically relevant effect. Last , it mus t have some mechanism for timely removal .

H o w well the various putat ive t ransmit ters meet these requirements is summarized in Table 1. Clearly only norepinephr ine currently satisfies all requirements , and even its exact role is species dependent . F o r example, while norepinephr ine stimulates jS-adrenergic receptors on pinealocytes to elevate N A T in the rat , in the chick norepinephrine activates a postsynapt ic a2-receptor to oppose the s t imulatory action of V I P on N A T ac t iv i ty .

45

Neurotransmitters and Peptides in the Pineal Gland 135

The strength of the evidence for the other t ransmit ters varies considerably. In one way or another all of the putat ive t ransmit ters fulfill the first requirement to some degree. However , the small molecules GABA, adenosine, taur ine and serotonin do so by being m a d e in pinealocytes from which they are released dur ing noradrenergic stimula-tion. Evidence for dopaminergic innervat ion is based on detection of its synthetic enzyme dopamine β-hydroxylase ra ther than dopamine itself in some nerves, and evidence for cholinergic innervat ion rests on i m m u n o -chemical detection of choline ace ty l t ransferase .

44 High affinity binding

has been demons t ra ted for many of the candidate modula to r s , and for some, like glutamic acid. Substance P , and the enkephal ines, suppor t for modu la to ry activity is based solely on occurrence and binding. Funct ional release has only been demons t ra ted for norepinephr ine unless noradrener-gic effects on releasing GABA, adenosine, taur ine and serotonin from pinealocytes presumably dur ing noradrenergic s t imulat ion is to be regarded as "functional". O n the other hand , except for substance P , glutamic acid and the enkephal ines, all of the c o m p o u n d s in Table 1 have been reported to have some effect on the pineal. The significance of some of these effects is unclear. F o r example, taur ine weakly stimulates the β-receptor to increase N A T activity but taur ine release is dependent upon norepinephr ine which itself is a s t rong s t imulator of bo th α and β r e c e p t o r s .

68 Some of the findings are controversial . This is the case for

GABA, for example. O n e study f o u n d

54 G A B A inhibit ion of norepine-

phrine-induced increases in N A T activity, while a n o t h e r

31 and our own

results found G A B A to have no effect either by itself or in inhibiting norepinephr ine or isoproterenol induct ion on N A T . Similarly, N P Y in one study inhibited norepinephrine- induced increases in N A T activity by p inea locy tes ,

42 yet a n o t h e r

64 reported N P Y increased melatonin p roduc-

tion and enhanced norepinephr ine s t imulat ion of mela tonin , while a t h i r d

47 found that N P Y given to intact rats increased N A T activity but

nightt ime activity in blinded rats was decreased. O u r own experience with rat pineals in organ culture was that N P Y had no effect by itself nor on agonist induct ion of N A T . Some of the findings are puzzling. F o r example, in the rat , adenosine analogues given to the intact animal increased pineal N-acetylserotonin and m e l a t o n i n

10 while, in vitro, adenosine and its

analogues increased cyclic nuc l eo t i de s

41 but not N A T ac t i v i t y

40 or

mela tonin p r o d u c t i o n .

65 Also, in the chick pineal adenosine inhibits or

stimulates mela tonin product ion depending on experimental cond i t ions .

9

Some transmit ters have functions other than those involved in mela tonin product ion . This appears to be the case for acetylcholine, which has no effect on basal or induced a c t i v i t y ,

1 6'

43 but which increases synaptic

r ibbon m e m b r a n e formation in the rat p i n e a l o c y t e s

16 and stimulates

release of pineal arginine vasopres s in .

56 Finally, reuptake systems exist to

inactivate most of the small molecules which have been suggested


to modula te pineal activity, but inactivation mechanisms have not yet been demons t ra ted for any of the peptides.

Perhaps the best studied and most consistent case for a modu la to r other than norepinephrine is that for vasoactive intestinal polypeptide. Al though, as seen in Table 1, the two criteria of demons t ra ted functional release and efficient inactivation have not been met, there is now ample evidence and confirmation that in rat p i n e a l o c y t e s ,

2 2'

2 3'

72 pineals in organ

c u l t u r e

72 and pineals in situ

39,72 V I P increases cyclic neucleotides, N A T

activity and melatonin product ion (Table 2). In these activities it resembles a weak β-agonist like isoproterenol but is an order of magni tude less potent . The resemblance is further s trengthened in that just as concomi-tant s t imulat ion with an α-adrenergic agonist like phenylephrine increases

T A B L E 2

Effects of isoproterenol and VIP on pineal indole production

C o m p o u n d Loca t i on C o n t r o l I S O 10 n M V I P 1 μΜ

M e l a t o n i n pineal 4 + 0.2 13 + 2 11 + 1 m e d i u m 27 + 3 5 9 + 1 1 61 + 6

N - A c 5 H T pineal 0.1 18 + 4 18 + 1 m e d i u m 3 61 + 16 65 + 6

5 H I A A pineal 10 + 3 6 + 1 7 + 1 m e d i u m 8 6 + 1 7 73 + 12 94 + 7

5 H I O H pineal 2 + 0.4 1 + 0 . 2 1 + 0 . 2 m e d i u m 18 + 3 17 + 3 19 + 3

5 H T P pineal 0.7 + 0. 1 0.9 + 0.1 0.6 + 0.1

5 H T pineal 40 + 8 16 + 3 20 + 2

N A T pineal 0.2 + 0.09 7.7 + 1.1 6.8 + 0.3

P inea ls were cu l tu red for 48 h o u r s before cha l langed wi th agonis t as ind ica ted . P inea l indoles were assayed by H P L C using a n Axxion C I 8 R P c o l u m n a n d a mob i l phase of 0.1 M N a A c p H 4.25 with 3 5 % m e t h a n o l (for m e l a t o n i n a n d TV-acetylmelatonin) o r 1 2 % in m e t h a n o l (for o the r indoles) . Indo les were q u a n t i t a t e d by fluorescence at 345 n m u p o n ac t iva t ion at 285 n m . Values a re M E A N + S E M for 4 - 6 pineals or associa ted m e d i u m . Values for c o m p o u n d s are in ng /p inea l . Un i t s for N A T are nmole s /p inea l / hou r .

the effectiveness of i s o p r o t e r e n o l ,

66 α-agonists also increase the effective-

ness of V I P .

74 Norep inephr ine itself, of course, st imulates bo th α and β-

receptors. The purpose of a second /Mike agonist in the rat pineal is unclear. O n e possibility is tha t V I P may augment the effects of β-st imulat ion under special condi t ions. If so, the sum of maximal V I P and maximal isoproterenol s t imulat ion should be addit ive. As seen in Table 3, however, concomitant V I P and isoproterenol , V I P and norepinephrine , or V I P and the weak beta agonist 6-methoxy-2-benzoxazolinone are additive when agonist concentra t ions were low but not when concentra-tions were high. This argues against augmenta t ion and indicates that bo th receptors share the same limiting components of the N A T t ransduct ion system. N o r is tempora l additivity the answer because V I P does not

Neurotransmitters and Peptides in the Pineal Gland 1 3 7

N-acety l t ransferase Agent Conc( /*M) N o V I P + V I P (0.1 μΜ) (Expected)

N o n e 0.4 + 0.1 2.7 + 0.3 2.3 I S O 0.0001 0.9 + 0.3 3.2 + 0.6 3.2

0.001 2.5 + 0.4 5 . 1 + 0 . 3 4.8 1 9.8 + 0.4 8.0 + 0.5 12.1

N E 1 5.8 + 0.6 8.3 + 0.6 8.1 6 M B O A 1000 2.9 + 0.3 6.6 + 1.2 5.2

P inea ls were cu l tu red for 48 h o u r s before agonis t cha l lange . N-acetyl t ransferase activity was de t e rmined by the m e t h o d of Deguch i a n d Axel rod (1972). Values a re M E A N + S E M in n m o l e s / g l a n d / h o u r for N A T activity of 4 - 8 p ineals . 6 M B O A is 6 -methoxy-2-benzoxazol i -n o n e .

appreciably extend the time course of N A T activity after beta s t imulat ion nor is the t ime course of induct ion appreciably different .

72 Fur the r , the

V I P system, like the ^-adrenergic system, shows circadian sensitivity to st imulat ion of cyclic A M P

20 and N A T

72 and to agonist content in the

p i n e a l .

20

The adrenergic and V I P systems do show some differences, however, especially in response to chronic agonist challenge and to steroid exposure. Table 4 shows tha t chronic (48 hour ) s t imulat ion with high concentra t ions of V I P or isoproterenol diminishes the response of the pineal to

T A B L E 4

Diminished response after chromic stimulation

48 H o u r Cha l lenge S t imula t ion P r e - T r e a t m e n t N o n e V I P (0.1 μΜ) I S O (10 nM)

N o n e 0 . 1 + 0 . 0 2 2.8 + 0.3 3.3 + 0.4 V I P 10 μΜ 0.2 + 0.04 1 .1+0 .2* 2.8 + 0.3 I S O 10 μΜ 0.7 + 0.1 1.0 + 0 . 1 * 2.9 + 0.5

Values are M E A N + S E M in n m o l e s / g l a n d / h o u r for N A T activity of 4 - 8 p ineals .

* indica tes a significant difference from N o P r e - T r e a t m e n t cond i t ion ( p < 0 . 0 5 )

subsequent s t imulat ion by V I P but less so to subsequent challenge with isoproterenol . The requirement for more t han 24 hours of s t imulat ion before a decrement in subsequent response can be detected suggests this phenomenon results from depletion of limiting componen t s of the t ransduct ion sequence. The difference in relative sensitivities of the V I P and adrenergic systems to pr ior s t imulat ion could be due to a tighter coupling between the beta receptor and adenylate cyclase and /o r G protein and /o r protein kinase A. A tighter coupling might also explain the

T A B L E 3

Effects of concomitant agonist stimulation on Ν A Τ activity


T A B L E 5

Relative effect of corticosterone preincubation on pineal response to isoproterenol and vasoactive intestinal polypeptide

U n t r e a t e d Cor t i cos t e rone Trea t ed H o u r s I S O V I P I S O V I P

0 9 . 1 + 0 . 7 3.3 + 0.4 48 — — 4.2 + 0.6* 2.7 + 0.3 72 9.8 + 0.8 3.8 + 0.7 3.5 + 0.4* 2.7 + 0.1

P inea ls were cu l tu red in the presence or absence of cor t icoster-one (50 μΜ) for 48 or 72 h o u r s before chal lenge with 0.1 μΜ I S O or 1 μΜ V I P for 6 h o u r s . Values a re M E A N + S E M in n m o l e s / g l a n d / h o u r for N A T activity of 4-8 pineals .

* indicates a significant difference from zero- t ime con t ro l s ( p < 0 . 0 5 ) .

T A B L E 6

Effect of corticosterone on the pineal response to various agonists

N-acety l t ransferase Agonis t C o n t r o l Steroid t rea ted

I sopro te reno l 1 μΜ (4) 7.4 + 0.6 (4) 2.6 + 0.04* Cho le r a toxin 72 μg (4) 2.7 + 0.2 (4) 0.2 + 0.04* For sko l in 10 μΜ (4) 6.9 + 0.2 (4) 3.2 + 0 .3* b C A M P 1 μΜ (4) 8 . 0 + 1 . 2 ( 4 ) 2 . 0 + 0.4* V I P 1 μΜ (4) 1.4 + 0.2 (4) 1.8 + 0.2

Pineals were ma in t a ined for 48 h o u r s as descr ibed previously in the presence or absence of 100 μΜ cor t icos-te rone before a 5 h o u r chal lenge wi th the agonis t s indica ted . Values a re (N) M E A N + S E M in n m o l e s / g l a n d / h o u r for N A T activi ty.

* indicates a significant difference from C o n t r o l ( p < 0 . 0 5 ) .

T A B L E 7

Response to agonists of pineals, cultured for 48 hours, upon acute ethanol exposure

E t O H Agonis t I S O (1 nM) V I P (100 nM)

0 0 ( 8 ) 0 . 1 2 + 0.01 —

0 + (22) 6.08 + 0.58 (20) 2.15 + 0.20

25 m M + (8) 7.62 + 0.70 (12) 2.70 + 0.19

50 m M + (21) 8.78 + 0.34* (17 )2 .16 + 0.19

75 ΙΏΜ + (8) 8.94 + 0.72* (12) 2.69 + 0.27

Values a re (N) M E A N + S E M in n m o l e s / g l a n d / h o u r for N A T activity.

* indicates a significant difference from N o E t h a n o l ( p < 0 . 0 5 ) .

Neurotransmitters and Peptides in the Pineal Gland 1 3 9

greater potency, on a mola r basis, of isoproterenol in st imulat ing N A T activity and its somewhat longer dura t ion of act ion. The effects of steroid pre-exposure are shown in Tables 5 and 6. In these cases V I P st imulat ion is relatively unaffected by steroid pre t rea tment while isoproterenol st imula-t ion is markedly blocked. E thano l also reveals differences between the V I P and ^-adrenergic sys tems.

4 Concur ren t od- and βί-stimulation or a l - and

VIP-s t imulat ion increases bo th c A M P and c G M P . E thano l exposure of pinealocytes does not affect the changes produced by combined al-jSl adrenergic s t imulat ion but does block the cyclic nucleotide increases produced by combined a l - V I P st imulat ion, apparent ly by interference with C a dependent processes

5 likely linked to interact ions between Kinase

A and Kinase C. As seen in Table 7, e thanol at concentra t ions comparab le to those used in the cyclic nucleotide studies does not affect the response of pineals in o rgan culture to either isoproterenol or V I P . At higher concentra t ions , response to isoproterenol is slightly increased. This effect may be due to changes in membrane fluidity and , since V I P response was no t changed, m a y indicate tha t the s t rength of coupling between the t ransduct ion componen t s differs in these systems.

The V I P and /^-adrenergic systems respond differently to light shock. Both show increased agonist r e s p o n s e

7 , 73 and increased receptor

d e n s i t y

2 1 , 49 in light, when N A T activity is low, and decreases in these

measures in the dark when N A T activity in high. Light exposure in the da rk period produces a profound fall in N A T a c t i v i t y

1 8 , 26 and , as seen in

Table 8, an unexpected fall in sensitivity to s t imulat ion by isoproterenol as

T A B L E 8

Response to light shock

G r o u p Agonis t 4 D 4 D L

I S O (14) 1 0 1 + 2 3 ( 1 5 ) 4 4 + 8 V I P (16) 9 6 + 1 2 ( 1 5 ) 9 8 + 20

After 3 h o u r s in the d a r k p h a s e of a 14:10 L : D cycle, one g r o u p of an ima l s ( 4 D L ) were exposed to 5 m i n u t e s of light a n d then r e tu rned to d a r k n e s s while con t ro l s (4D) r e m a i n e d in d a r k . P inea l s were ob t a ined from b o t h g r o u p s 1 h o u r later , i n c u b a t e d for 1 h o u r in o r g a n cu l tu re , t ransferred to new m e d i a a n d s t imula ted wi th e i ther I S O (10 ΠΜ) o r V I P (100 nM) for 5 h o u r s . Values a re (N) M e a n - % -of-Cont ro l + SE for two poo led exper imen t s . Ac tua l m e a n values for con t ro l s (4D) in the t w o exper iments were : Exp t 1. I S O = 5.8 ± 1 . 6 ; V I P 2.8 + 1.0; E x p t 2 I S O = 7.0 + 2.7; V I P = 3 . 0 ± 0 . 4 n m o l e s / g l a n d / h o u r .


well. O n the other hand sensitivity to s t imulat ion by V I P is unaltered. This might be due to differences in receptor metabol ism or, again, to coupling between receptor and t ransduct ion . Taken together, these results would seem to suggest that the V I P and ^-adrenergic systems may differ in the coupling between componen t par ts to their t ransduct ion systems.

The physiological significance of these differences between the V I P and ^-adrenergic systems in the rat pineal gland is certainly still unclear. Whatever the differences may be, it seems more subtle than in the chick where V I P and norepinephr ine act in opposi t ion. The role, if any, of the other t ransmit ter candidates is even more obscure. As illustrated above the pineal seems an excellent system to identify actions and interactions and, ultimately, functions. Unti l these subtleties and significance are resolved, the rat pineal poses the anomaly of an organ without parasympathe t ic balance of sympathet ic act ions, which seem only augmented by and not opposed by most putat ive secondary regulators .

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62. Tsuch iya M . , K a k u K., M a t s u d a M . , K a n e k o T. a n d Y a n a i h a r a , N . (1987) D e m o n s t r a t i o n of recep tors specific for N - t e r m i n a l his t id ine a n d C- te rmina l isoleucine ( P H I ) us ing ra t P H I a n d ra t d ispersed p ineal cells. Biomed. Res. 8 , 4 5 - 5 1 .

63 . U d d m a n R., A lumets J., H a k a n s o n R., L o r e n I. a n d Sund le r F . (1980) V a s o -act ive intes t inal pep t ide (VIP) occurs in nerves of the p ineal g land . Experientia 3 6 , 1119-1120 .

64. Vacas M. I . , S a r m i e n t o M. I . , Pe r ey ra E .N . , E t chegoyen G . S . a n d Ca rd ina l i D . P . (1987) In vitro effect of n e u r o p e p t i d e Y o n m e l a t o n i n a n d n o r e p i n e p h r i n e release in r a t p inea l g land . Cell Mol. Neurobiol. 7 , 3 0 9 - 3 1 5 .

65 . Vacas M. I . , S a r m i e n t o M. I . , P e r e y r a E . N . a n d Ca rd ina l i D . P . (1989) Effect of adenos ine o n m e l a t o n i n a n d n o r e p i n e p h r i n e release in ra t p ineal exp ian t s . Acta Physiol. Et Pharmacol. Latinoamer. 3 9 , 189-195 .

66. Vanecek J., Sugden D . , Weller J. a n d Kle in D . C . (1985) Atypica l synergist ic ocl a n d β-adrenerg ic r egu la t ion of adenos ine 3 ' , 5 ' - m o n o p h o s p h a t e in r a t p inea locytes . Endocrinology 116 , 2 1 6 7 - 2 1 7 3 .

67. Weiss B. a n d C o s t a E. (1968) Selective s t imula t ion of adeny la t e cyclase of ra t p inea l g land by pha rmaco log ica l ly act ive ca t echo lamines , / . Pharm. Exp. Therap. 1 6 1 , 310-319 .

68. Whe le r G . H . , Weller J .L . a n d Klein D . C . (1979) T a u r i n e : s t imu la t ion of p ineal N-ace ty l t ransferase activity a n d m e l a t o n i n p r o d u c t i o n via a be ta -ad rene rg ic mech -an i sm. Brain Res. 166 , 6 5 - 7 4

69. Whe le r G . H . a n d Klein D . C . (1980) T a u r i n e release from the p inea l -g land is s t imula ted via a be ta -ad rene rg ic m e c h a n i s m . Brain Res. 187 , 155-164 .

70. Whe le r G . H . a n d Kle in D . C . (1979) Cyclic A M P - i n d u c e d release of [

1 4C ] t a u r i n e from

pinea locytes . Biochem. Biophys. Res. Commun. 9 0 , 22-21. 7 1 . Wi l l iams L . M . , M o r g a n P.J . , Pellet ier G. , R i d d o c h G. I . , L a w s o n W . a n d D a v i d s o n

G . R . (1989) N e u r o p e p t i d e Y ( N P Y ) inne rva t ion of the ovine p ineal g l and . J Pineal Res. 7, 3 4 5 - 3 5 3 .


72. Yuwiler A. (1983) Vasoac t ive intes t inal pep t ide s t imula t ion of p ineal s e ro ton in -N-acetyl t ransferase act ivi ty: G e n e r a l charac ter i s t ics . J. Neurochem. 4 1 , 146-153 .

73 . Yuwiler A. (1983) Light a n d agon is t s al ter p inea l N-ace ty l t ransferase i nduc t ion by vasoact ive intes t inal po lypep t ide . Science 2 3 0 , 1082-1083 .

74. Yuwiler A. (1987) Synergist ic ac t ion of pos t synap t i c a lpha -ad rene rg ic recep tor s t imula t ion o n vasoac t ive intest inal po lypep t i de—induced increases in p ineal N -acetyl t ransferase act ivi ty. J. Neurochem. 4 9 , 8 0 6 - 8 1 1 .

75 . Z h a n g E .T . , Mikke l sen J . D . a n d Mol le r M . (1991) Ty ros ine hydroxy lase - a n d n e u r o p e p t i d e Y - i m m u n o r e a c t i v e nerve fibers in the p ineal complex of u n t r e a t e d ra t s a n d ra t s following r emova l of the super io r cervical gangl ia . Cell Tissue Res. 2 6 5 , 6 3 - 7 1 .

10

The Mammalian Pineal Gland as an End Organ of the Visual System 1

R U S S E L J . REITER

Department of Cellular and Structural Biology, The University of Texas, Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7762, USA

Abstract

Visible light is the ma jo r e n v i r o n m e n t a l factor con t ro l l ing the c i rcad ian p r o d u c t i o n of m e l a t o n i n in the m a m m a l i a n pineal g land . Regardless of the activity p a t t e r n a species d isplays , i.e. d iu rna l , n o c t u r n a l o r c repuscu la r , h igh m e l a t o n i n p r o d u c t i o n is a lways associa ted wi th the d a r k p h a s e of the l ight : d a r k cycle. Light has t w o effects on the c i rcad ian m e l a t o n i n r h y t h m ; it precisely en t ra ins the r h y t h m to 24 h o u r s a n d light exposu re d u r i n g the night rapidly depresses h igh n igh t t ime m e l a t o n i n levels. T h e intensi ty of light requ i red to inhibi t n o c t u r n a l m e l a t o n i n p r o d u c t i o n varies widely a m o n g species wi th m e l a t o n i n synthesis in the p ineal g land of n o c t u r n a l an ima l s hav ing a m u c h grea ter sensitivity (lower th resho ld ) to light t h a n t h a t of d iurna l ly act ive an ima l s . Whi le op t ic enuc lea t ion p reven t s all ac t ions of l ight o n the 24 h o u r m e l a t o n i n cycle, des t ruc t ion of the major i ty , if n o t all, the re t inal p h o t o r e c e p t o r s does n o t a l ter the abil i ty of light to synchron ize the m e l a t o n i n r h y t h m a n d only slightly increases the th resho ld of the p ineal g land to acu te light exposu re . Beside l uminance or in tensi ty , the specific wave leng th of light is i m p o r t a n t in de t e rmin ing its efficacy as an inh ib i to r of pineal m e l a t o n i n synthesis . In genera l , b lue-green wave lengths seem to be m o s t inh ib i to ry to the m e l a t o n i n forming abil i ty of the p ineal . Red light ( > 6 0 0 n m ) exposure , however , a lso suppresses p ineal a n d s e r u m m e l a t o n i n levels. T h e abil i ty of red light to achieve this effect is re ta ined in ra ts wi th to ta l o r nea r to ta l des t ruc t ion of the re t inal p h o t o r e c e p t o r s . T o d a t e there is n o a d e q u a t e exp l ana t i on as to the ret inal m e c h a n i s m s involved in med ia t i ng the inh ib i to ry effect of light on the pineal g land . It is possible , however , t ha t the classical p h o t o r e c e p t o r s , i.e. the rods a n d cones , m a y no t be involved in this process .

In t roduct ion

L I G H T CONSISTS of electromagnetic radia t ion limited to a defined band of retinally-detected wavelengths referred to as the visible spectrum. Classically, the visible spectrum consists of wavelengths ranging from 400 nm to 760 nm, a l though some wavelengths shorter than 400 nm in the

1 Work by the au thor was supported by grants from the Nat ional Science Founda t ion and

from the Nat ional Institutes of Health.

145


ultraviolet (UV) range) and, perhaps under some circumstances and /o r in some species, wavelengths longer than 760 nm (in the infrared range) may be detectable by the mammal i an retina. This window of visible wavelengths is a very small por t ion of the total electromagnetic spectrum and the windowing aspect of detection is a feature c o m m o n to several sensory systems. Besides these limited wavelengths, it has recently been proposed tha t the eyes may respond to static and extremely low frequency electromagnetic fields

22 which may produce physiological effects similar

to those caused by visible l i g h t .

2 8'

30

Since light is a form of energy, it can be quantified in absolute units referred to as ergs. Physiologically, however, what is identified as the "brightness" or " luminosi ty" of light, which is of major interest to experimentalists working on the functional aspects of the visible spectrum, is not uniquely determined by the energy content of light, but ra ther it varies according to the specific wavelength. As a result, on a luminosity scale, two independent light sources have an equal intensity when they induce an equal sensation of brightness.

The effects of light on the ret ina as well as the signal t ransduct ion mechanisms in the photoreceptors have been, in par t , de f ined .

12 These

mechanisms have been studied primari ly in terms of the visual effects of light and less frequently in reference to the functional aspects of light in relat ionship to circadian and endocrine p h y s i o l o g y .

1 9'

38 Al though in

mammal s the detection of light subserving bo th systems is in the retinas, the neural pa thways mediat ing the physiological consequences are different (Figure 1). Since this repor t is concerned with the circadian and endocrine (especially pineal mela tonin) changes induced by the visible spectrum, the neural pa thways connecting the eyes with the suprachiasma-tic nuclei (SCN) and eventually with the pineal gland are of special i n t e r e s t .

24 As summarized in Figure 1, the neural pa thway from the eyes to

the pineal gland is a highly circuitous one; in terrupt ion of the connect ions anywhere between the S C N and the pineal render the gland incapable of producing its h o r m o n e melatonin and totally ineffective as an endocrine g l a n d .

23 Besides the innervat ion of the pineal gland via the peripheral

au tonomic nervous system, centrally originating fibers also enter the gland th rough its s t a l k ;

14 however, these connections seem functionally inept in

reference to the circadian mela tonin rhy thm.

Pineal me la ton in synthesis and l ight

W h a t is known of the signal t ransduct ion mechanisms related to the product ion of the key pineal h o r m o n e melatonin (7V-acetyl-5-methoxy-t ryptamine) has recently been r e v i e w e d

26 and is summarized in Figure 2.

Whereas norepinephr ine (NE) is the neurot ransmit te r primari ly respons-ible for initiating the p roduc t ion of melatonin in the pineal gland, its

Light Regulation of Pineal Gland 1 4 7

neuron

F I G . 1. Schemat ic r ep resen ta t ion of the neura l connec t ions of the re t inas with the visual cor tex a n d wi th the c i rcad ian system via the s u p r a c h i a s m a t i c nuclei ( S C N ) a n d the pineal g land . T h e visual cor tex receives pro jec t ions from the eyes by m e a n s of gangl ion cell a x o n s which synapse in the la teral genicula te nucleus ( L G N ) ; neura l fibers from the L G N then project to the occipi tal pole of the cerebra l cor tex . T h e eyes are connec ted to the p ineal g land as well wi th the p a t h w a y also involving a x o n s of ret inal gang l ion cells which synapse in the S C N ; the r ema inde r of the p a t h w a y includes synapses in the pa raven t r i cu l a r nuclei ( P V N ) , the in te rmedio la te ra l cell c o l u m n of the u p p e r tho rac ic co rd a n d the super io r cervical gangl ia . T h e pos tgang l ion ic sympa the t i c fibers t e rmina t e in the pineal g land a n d end in the p rox imi ty of the p inea locytes , the endoc r ine uni ts of

the g land .

interact ion with pinealocyte adrenergic receptors is not the only described mechanism for regulating melatonin produc t ion . Indeed, pinealocyte membranes contain m a n y other receptors which can modula te the synthesis of mela tonin to varying d e g r e e s ;

10 however, the circadian

product ion and release of mela tonin is generally considered to be exclusively related to the peripheral au tonomic innervat ion and its associated catecholaminergic neurot ransmit ter , N E .

Regardless of the activity pa t te rn a specific animal displays, i.e. d iurnal , nocturnal , or crepuscular, peak pineal mela tonin produc t ion and secretion is always associated with the night. Because of its unique relat ionship with the dark phase of the l igh t -dark cycle, mela tonin has been referred to as the "chemical expression of d a r k n e s s " .

27 In Scandin-

avia, melatonin could also be thought of as the "troll of the endocrine


C7 o g Postganglionic sympathetic nerve

Extracellular Space

Pinealocyte membrane β AC

Tryptophan

5 -Hydroxytryptophan

ν AAAD

7 ^ -CAMP ATP

i

\ NAT HIOMT

Other Metabolites

5 -Methoxytryptamine

F I G . 2. A simplified schemat ic r ep resen ta t ion of the signal t r ansduc t i on m e c h a n i s m s con t ro l l ing the p r o d u c t i o n of m e l a t o n i n in the m a m m a l i a n pineal g land . T h e p r i m a r y n e u r o t r a n s m i t t e r med ia t i ng the n igh t t ime rise in m e l a t o n i n is the ca t echo lamine n o r e p i n e p h r i n e ( N E ) which is released from pos tgang l ion ic sympa the t i c n e u r o n s o n c o m m a n d from the sup rach ia sma t i c nuclei in the h y p o t h a l a m u s . T h e m o s t i m p o r t a n t in te rac t ion of N E is wi th ^ -ad rene rg ic receptors in the cell m e m b r a n e of the p inea locytes ; α-adrenergic recep tors p lay a m o d u l a t o r y role in con t ro l l ing m e l a t o n i n p r o d u c t i o n . Besides the one visually depic ted here , the p inea locytes r e s p o n d to m a n y o the r n e u r o t r a n s m i t t e r s which act o n specific recep tors a n d which e i ther slightly or m a r k e d l y influence me la ton in p r o d u c t i o n . A A A D , 1-aromatic a m i n o acid deca rboxy lase ; A C , adeny la te cyclase; A T P , adenos ine t r i p h o s p h a t e ; c A M P , cyclic A M P ; D G , diacylglycerol ; G s , s t imula to ry guan ine nuc leo t ide b ind ing p ro t e in ; H I O M T , h y d r o x y i n d o l e - O -methyl t ransferase ; I P , inosi to l p h o s p h a t e ; M A O , m o n o a m i n e oxidase ; N A T , N-acetyl t ransferase; P I , phospho t idy l inos i to l ; P K C , p ro te in k inase C; P L C , p h o s p h o l i p a s e C ; T H , t r y p t o p h a n hydroxy lase ; a

1- , α-adrenergic recep tor ;

β ,β-adrenerg ic recep tor . F r o m Reiter .

system". F o r mela tonin produc t ion , as with the mythical troll, sunlight is synonymous with death .

Mela tonin produced in the pineal gland is quickly released into the blood vascular system; the release of mela tonin is so rapid that b lood levels of the h o r m o n e are generally used as an index of the a m o u n t being synthesized in the gland at virtually the same time. Remarkably , however, melatonin 's release can be further hastened by at least one maneuver , i.e. s t renuous exercise. Thus , if rats at night are forced to swim the enzymes controll ing mela tonin p roduc t ion in the pineal gland remain elevated while the mela tonin content of the gland falls to dayt ime levels. Yet, circulating concentra t ions of the indole remain high, as dur ing darkness . The best interpretat ion consistent with these findings is that the newly-synthesized melatonin is very rapidly released (more rapidly than normal ) , leading to low pineal mela tonin levels but high concentra t ions in the b l o o d .

31

Once in the blood, mela tonin has access to every other fluid in the body


and its high lipophilicity, which p romotes its ready passage th rough cell membranes , ensures that it gains admi t tance to other fluid compar tmen t s . Because of this, the circadian melatonin rhy thm, typical of the blood, is also present, albeit of somewhat lower ampl i tude , in o ther bodily fluids including saliva, ovar ian follicular fluid, male seminal fluid, cerebrospinal fluid, amniot ic fluid and fluid of the anter ior chamber of the e y e .

26

Likewise, the chief hepatic metabol i te of mela tonin , 6-hydroxymelatonin sulfate, exhibits a 24 hour cycle in the urine with high excretion of the c o m p o u n d occurring at n igh t .

6 Thus , all cells with which melatonin comes

in contact could use the mela tonin message, provided they have the cellular machinery to recognize melatonin changes over time.

Encoded in the melatonin message is bo th time-of-day (clock) and time-of-year (calendar) information. Thus , dayt ime levels of mela tonin are always low whereas night t ime levels are elevated. Providing the receptor molecules that respond to the message distinguish low (or absence) from high concentra t ions (or presence) of mela tonin , the associated metabol ic responses reflect either day or night.

The rat io of light to darkness per 24 hour period varies seasonally and is more exaggerated at higher lati tudes (Figure 3). Since the dura t ion of elevated mela tonin at night is roughly p ropor t iona l to the dura t ion of the night, the mela tonin signal thereby provides time-of-year-information; this is t rue bo th for experimental a n i m a l s

3 , 27 as well as for h u m a n s .

4 2'

43 In

animals , at least, the physiological significance of the annua l variat ion in melatonin secretion is well documented where it is known to control seasonal reproduct ive capab i l i t y .

23 Seasonal reproduct ion in m a m m a l s in

their na tura l habi ta t is an impor tan t event since it ensures the young are born at a season most conducive to their survival.

As ment ioned above, environmental light is incompat ible with high melatonin produc t ion . Sunlight as well as artificial light have two obvious effects on the 24 hou r mela tonin cycle. Thus , light entrains the circadian rhy thm of mela tonin synthesis and the in t roduct ion of light at unusual times, e.g. at night, acutely suppresses mela tonin synthesis. The entraining effect of light is readily seen in bo th experimental a n i m a l s

1 and h u m a n s

43

and is d iagrammatical ly por t rayed in Figure 4. The entraining effect of light on the circadian melatonin rhy thm is readily apparen t in h u m a n s living in Antarct ica where the 24 hour cycle of mela tonin free-runs dur ing the interval when the sun is below the h o r i z o n

13 and in blind h u m a n s who

are incapable of light p e r c e p t i o n .

16

Light intensi ty e f fec ts

The intensity of white light required to suppress pineal mela tonin product ion varies greatly a m o n g species (Table 1 ) and may be influenced by the lighting history of the a n i m a l .

33 T o illustrate the variat ion a m o n g


Vernal Summer Autumnal Winter Vernal equinox solstice equinox solstice equinox

F I G . 3. D i a g r a m m a t i c r ep resen ta t ion of seasonal va r ia t ions in day leng th in representa t ive cities a t va r ious la t i tudes . At progressively h igher la t i tudes in b o t h hemispheres , the ra t ios of dayl ight h o u r s to n igh t t ime h o u r s b e c o m e increasingly great . In the m o s t exaggera ted case, in the Arct ic a n d Anta rc t i c Circle, long

pe r iods of pers is tent l ight a n d d a r k n e s s a re exper ienced.

the mammal s investigated, pineal mela tonin product ion in the albino rat is roughly 3.7 χ 10

6 t imes more sensitive to inhibit ion by white light than is

the pineal of the Richardson 's g round s q u i r r e l .

2 43 In general, pineal

melatonin synthesis in nocturna l animals , who have rod-domina ted retinas, exhibits a greater sensitivity to light inhibit ion when compared with diurnal species who have almost exclusively cone photoreceptors . The one subter ranean m a m m a l tha t has been tested, i.e. the valley pocket gopher (Thomomys bottae), has , unexpectedly, a pineal melatonin rhy thm that is relatively insensitive to white light. It is est imated that pocket gophers spend 99 .9% of their life in underground bur rows (with closed openings to the surface) in total darkness . Their activity pat terns are seasonally dependent and range from nocturna l , to diurnal , to crepuscular depending on g round tempera ture condit ions near the surface. Despite their a lmost total lack of exposure to light in their na tura l habi ta t , the melatonin rhy thm in the pineal gland of wild-captured pocket gophers is easily synchronized by labora tory l ight :dark cycles (J.H. Sun, R.J. Reiter, K. Yaga and L.C. Manchester , unpubl ished observat ions) . Information on the photoreceptor composi t ion of the pocket gopher retina is lacking.

It is also apparen t that in a real sense the ret ina-SCN-pineal system can act as a compara to r to compare the relative intensity of light to determine what is night and what is d a y .

18 In the study in quest ion, rats were exposed

t o a L 1 2 : D 1 2 photoper iodic cycle of either darkness al ternat ing with dim light (0.1-0.3 / i W / c m

2) or the dim light al ternat ing with bright light

(45-100 juW/cm

2) . In bo th exposure si tuations the rhythmic product ion of


phase de layed

m e l a t o n i n r h y t h m

Lo J -

Night Day N ight Day N igh t

Lo —ι

Night Day Night

F I G . 4. In the case of an an ima l or a h u m a n where the me la ton in r h y t h m is phase-de layed (upper F igure ) , it can be p h a s e - a d v a n c e d (lower F igure ) by br ight light exposu re (in h u m a n s , referred to as p h o t o t h e r a p y ) in e i ther the ear ly m o r n i n g or possibly t h r o u g h o u t the day . Shifting the m e l a t o n i n r h y t h m m a y have

psychologica l a n d physiological benefits.

melatonin persisted with the maximal levels always being associated with the darkest por t ion of the photoper iodic cycle. Thus , the dim light was interpreted as either day or night depending on the i l lumination intensity dur ing the cont iguous 12 hour period. The implication is that the system exhibits some plasticity and can adap t to prevailing environmenta l condit ions. This is also suppor ted by the a l ready-ment ioned da t a showing that the lighting history of an animal may influence the sensitivity of the re t ina-SCN-pineal system to l i g h t .

33 It is presumed these adjustments are

made at the level of the so-called visual cells in the S C N .

19

C o m p a r e d to the sensitivity of the retina-visual cortical system, the re t ina-SCN-pineal system is relatively insensitive to visible light st imula-tion. F o r example, in the Syrian hamster a light i r radiance of 0.00001 μψ/ c m

2 evokes retinal neuronal activity as indicated by the firing pa t te rn of

ganglion cell axons (measured by means of the electroret inogram) in the optic n e r v e .

3 4 , 35 An examinat ion of Table 1 shows that the inhibit ion of

melatonin synthesis in this species has a threshold of approximate ly 0.1 / i W / c m

2. Therefore, the retina-visual cortical system of the hamster


T A B L E 1

Sensitivity of the melatonin rhythm to acute light exposure at night in representative mammals. Listed are the approximate intensities of light required to depress pineal

melatonin production; both the irradiance and illumination itensities are given

I r r ad iance ( / i W / c m

2 I l lumina t ion intensi ty Activity

Species cool whi te) ftc lux p a t t e r n

Alb ino ra t 0.0005 0.0002 0.0017 n o c t u r n a l (Rattus norvigecus)

C o t t o n ra t 0.01 0.003 0.034 n o c t u r n a l (Sigmodon hispidus)

Syrian h a m s t e r 0.1 0.031 0.336 n o c t u r n a l (Mesocricetus auratus)

G o a t 0.4 0.13 1.35 d iu rna l (British S a a n e n s t ra in)

Valley pocke t g o p h e r 420 131 1,412 d i u r n a l / (Thomomys bottae) n o c t u r n a l

13-Lined g r o u n d squirrel 925 289 3,109 d iu rna l (Spermophilus tridecemlineatus)

Richa rdson ' s g r o u n d squirrel 1,850 578 6,220 d iu rna l (Spermophilus richardsoni)

has a sensitivity to light which is 1,000 times greater than that for the ret ina-SCN-pineal system. Similar relationships hold for the r a t .

32 At the

other end of the spectrum, it appears that in species such as the Richardson 's g round squirrel (Table 1) the retina-visual cortical system may be millions of times more sensitive to light than is mela tonin synthesis in the pineal gland.

The suppression of high noc turna l pineal mela tonin levels by acute light exposure is very rapid with the melatonin content of the pineal gland falling to 5 0 % of the nightt ime levels in less than 10 m i n u t e s .

36 Soon after

pineal melatonin levels fall, serum levels as well as the concentra t ion of the indole in other bodily fluids are decreased (Figure 5). The dura t ion of light exposure at night required to inhibit pineal melatonin product ion is very brief. Thus , certainly a 1 sec flash of bright light at night leads to a t emporary cessation of pineal mela tonin p r o d u c t i o n .

29 The disrupt ion of

the circadian melatonin rhy thm in mammal s by very brief light periods leads to marked physiological effects

9 and probably has significance for

fossorial animals in their na tura l h a b i t a t .

25

Unques t ionably , the ability of light to inhibit pineal melatonin product ion requires the eyes of the animal to be intact. Thus , optically enucleated rats kept under a l ight :dark cycle no longer exhibit a pineal melatonin rhy thm that is either synchronized by or acutely suppressed by l i g h t

32 (Figure 6). In these animals the mela tonin rhy thm free-runs with

each animal having a mela tonin cycle with a slightly different free-running period. As a result, individual pineals of a g roup of animals killed at the


Serum melatonin Salivary melatonin

a

22 24 2 4 6 S 22 24 2 4 6 8

Time Time

F I G . 5. Se rum a n d sal ivary m e l a t o n i n response to a 1 h o u r exposu re to light be tween 02.00 a n d 03.00 h o u r s in three adu l t h u m a n males . D u r i n g the con t ro l n ight ( · ) m e l a t o n i n levels were elevated be tween 02.00 a n d 03.00 h o u r s ; w h e n the n ight was i n t e r rup t ed by light ( O ) m e l a t o n i n levels in b o t h fluids fell. S h a d e d a rea

represents d a r k n e s s .

same time dur ing the l ight:dark cycle may have high, low or intermediate levels of mela tonin p roduc t ion with the average melatonin level falling between the expected low dayt ime and high nightt ime values (Figure 6, middle panel) . This is quite different from the si tuation in rats where the photoreceptor cells are lost due to cont inual light e x p o s u r e ;

15 even in the

case of total or near total destruct ion of the retinal photoreceptors the mela tonin rhy thm is readily synchronized by the prevailing light :dark envi ronment (Figure 6, b o t t o m panel) .

As ment ioned above, in optically enucleated rats acute light exposure at night also does not impair the ability of the pineal gland to synthesize its chief ho rm ona l p roduc t m e l a t o n i n

41 (Figure 7, middle panel) . Conversely,

mela tonin synthesis in the pineal gland of rats with l ight-damaged retinas is still depressed by acute light exposure at night with the threshold of light intensity required to inhibit pineal indoleamine metabol i sm being only slightly increased over that in rats with no rma l photorecep tor e l e m e n t s .

41

Besides lighting history, ambient envi ronmenta l t empera ture may be a factor in determining the sensitivity of the mela tonin synthesis cycle to light. When Djungar ian hamsters (Phodopus sungorus) were exposed to reduced tempera ture dur ing the night the light threshold required to suppress the mela tonin forming ability of the pineal gland was increased, i.e. the system was less sensitive to i n h i b i t i o n .

37 It is assumed tha t the

threshold adjustment is m a d e at the level of the hypotha lamic S C N since this area of the bra in receives sensory projections from the tha lamus , which is an impor tan t sensory relay center to higher cortical s tructures.


3000η

2000

1000-

•Ο 0 C -£L

3° 0 0 Ο)

Ο)

2000

C Β 3 1000-φ S

S o

J

g= 3000η

Intact

Optically Enucleated

Photoreceptor Damaged

2000

1000

F I G . 6. P inea l m e l a t o n i n levels over a 24 h o u r l i gh t : da rk cycle in ra t s wi th a n in tac t re t ina ( top pane l ) , in ra t s t h a t h a d been opt ical ly enuc lea ted (middle pane l ) and in ra t s t ha t h a d their p h o t o r e c e p t o r s severely d a m a g e d by con t inua l l ight exposure ( b o t t o m pane l ) . Clear ly , the p ineal m e l a t o n i n cycle is no t synchron ized by light in the b l inded an ima l s while it is in the ra t s tha t have n o or few funct ional p h o t o r e c e p t o r s r ema in ing in their re t inas . D a r k b a r o n the ho r i zon t a l axis is the

dai ly d a r k per iod .

Light w a v e l e n g t h e f fec ts

In addi t ion to light intensity, light wavelength is impor tan t in determining the suppressive effect of visible electromagnetic radia t ion on the synthetic activity of the pineal gland. Using the activity of H I O M T , the melatonin-forming enzyme, as an index, Cardinal i et al.

1 claimed tha t a m o n g the

wavelengths tested (red, yellow, blue, green and near ultraviolet (UV-A)), green wavelengths proved mos t effective in inhibiting the biosynthetic activity of the rat pineal gland. In the Syrian hamster , green wavelengths (half-peak bandwid th = 515-550 nm) , but even to a greater extent blue wavelengths (half-peak bandwid th = 435-500 nm) , suppress noc turna l pineal melatonin levels.

4 The implication of these findings, as well as those

from a similar study in humans where a patternless monochroma t i c light (λ max 509 nm, with wavelength and irradiance adjustment with glass interference and neutral density filters) was used ,

2 is tha t scotopic

mechanisms in the ret ina are involved in mediat ing the inhibitory effect of visible light on the pineal gland. Al though no definitive claims have been


Intact Optical ly Photoreceptor enucleated damaged

0 15 30 0 15 30 0 15 30

Time after l ights on (min)

F I G . 7. Responses of the p ineal g land to acu te light exposu re a t n ight in re t inal - in tac t ra t s (left pane l ) , in opt ical ly enuc lea ted ra t s (middle pane l ) , a n d in ra t s t h a t have severely d a m a g e d re t inas d u e to p r o l o n g e d light exposu re (right pane l ) . Opt ica l ly enuc lea ted ra t s d o n o t r e spond to l ight onse t wi th m e l a t o n i n suppress ion while those wi th extensive p h o t o r e c e p t o r des t ruc t ion r e s p o n d similar

to those in ra t s wi th in tact re t inas .

m a d e that the activation of rhodops in is an impor tan t aspect of the l ight-mediated suppression of mela tonin , this is usually tacitly accepted. If t rue, it would be consistent with observat ion that very low light intensities readily suppress pineal mela tonin in animals with rod-domina ted retinas (Table 1).

In a brief report published in 1982, Vanecek and I l l n e r o v a

39 claimed

that the exposure of albino rats to red light (8 lux, from a 15 W red Tesla pho tograph ic bulb) for 30 minutes at night suppressed pineal N A T activity and , by inference, mela tonin levels (al though they were not measured) . With this par t icular red light source less than 5 % of the total emit ted light has wavelengths shorter than 600 nm. These findings are of interest because of the widespread use of red background i l lumination in animal rooms as "safe" lights and because it implies tha t rat retinal photoreceptors can respond to wavelengths greater than 600 nm; this lat ter claim could not be definitively m a d e since in fact 5 % of the emitted wavelengths were below 600 n m and , in the range of wavelengths under 600 nm, the albino rat pineal gland is very sensitive to l i g h t .

41

T o prove whether wavelengths of light greater than 600 n m do suppress the nocturna l levels of no t only N A T activity, bu t also mela tonin , rats were exposed to a red light ( > 600 n m wavelengths only; with the aid of a K o d a k 1A Wra t t en filter) of different intensities. As did Vanecek and Il lnerova, we found that at all intensities equal to or greater than 50 ^ W / c m

2, no t only

inhibited pineal N A T , but pineal and serum levels of mela tonin also were suppressed (R.J. Reiter, B. Poeggeler, K. Yaga and J .H. Sun, unpubl ished observations) as a consequence of this exposure. The findings provide


unequivocal evidence that nocturnal pineal melatonin product ion is influenced by wavelengths of 600 nm and above. Fur thermore , in the retinas of the same animals we measured the ratio of il-cis to all-frans-retinal. Normally, during darkness 8 5 - 9 0 % of the retinal aldehyde is in the 11-ds form. Whereas white light exposure at night quickly changes this r a t i o ,

12 no

alteration in the 11-ds to a\\-trans ratio could be measured in the red light exposed rats. Since the isomerization of 11-ds retinal is the initial event in the activation of rhodopsin, our findings indicate that the rod photoreceptor is not likely to be involved in the red light-mediated suppression of pineal melatonin product ion. It is possible that cone cells in the rat retina may respond to red wavelengths al though the evidence for this is weak .

8

We carried these studies one step further in an a t tempt to determine the role of the rod photoreceptors in detecting red light that was capable of suppressing pineal mela tonin produc t ion . Rats were placed under cons tant white light, a procedure known to cause the rapid destruct ion of the rod p h o t o r e c e p t o r s .

15 The animals were then returned to cyclic light

and dur ing the night they were exposed to > 6 0 0 nm light (red). Despite the severely damaged retinas with the near total loss of photoreceptors , red light readily inhibited the ability of the pineal gland to reduce melatonin . These findings suggest that rods are not involved in the detection of > 6 0 0 nm light which acts to inhibit the pineal gland, a l though it is not incontrovert ible evidence since a few cell bodies, if not the outer segments, of the rod photoreceptors m a y have survived the prolonged cont inuous light t rea tment . Also, since the relatively few cone photoreceptors that do exist in the rat retina are less readily destroyed by cont inual light t reatment , it is possible that surviving cones responded to the red wavelengths which depressed the formation of melatonin in the pineal g land .

8 These explanat ions are not the only tentable ones, however (see

Concluding remarks) .

Concluding remarks

The discrimination of light intensity or luminance in the re t ina-SCN-pineal system likely occurs at the level of the S C N .

38 W h a t are referred to

as visual cells in the S C N change their firing rate (the majority of the cells increase their discharge rate while the remainder decrease it) in response to sustained i l l umina t ion .

20 The light-activated visual cells increase their

discharge rate as light intensity increases thereby presumably coding for l u m i n a n c e .

21 Interestingly, a m o n g the species tested the light intensity

required to activate the visual cells varies with the S C N cells of the rat being most light sensitive, followed by those in the Syrian hamster , and finally by those in the g round squirrel (Spermophilus tridecemlineatus).

21

The rat and hamster are, of course, nocturnal while the g round squirrel is diurnal . Inspection of Table 1 reveals that the light intensity required to


inhibit pineal mela tonin p roduc t ion has the same rank order . Al though this certainly provides circumstantial evidence for the S C N being the site at which light intensity is discriminated in reference to its ability to inhibit the pineal gland, not all the da ta suppor t this in terpreta t ion. Thus , whereas very brief light pulses ( < 1 second) inhibit noc turna l mela tonin synthesis in the rat pineal, sustained i l lumination is required to evoke maximal s t imulat ion of S C N visual cell ac t iv i ty .

21

F o r the mos t par t , it is usually assumed tha t pineal mela tonin p roduc t ion relies on the ability of retinal photoreceptors to respond to visible electromagnetic i l lumination. However , there are observat ions that argue against this. F o r example, severely damaged retinas with total or near total destruct ion of the photoreceptors , especially the rods , are still very sensitive to light. Certainly, the possibility exists tha t very few partially surviving rods could still detect sufficient light to inhibit pineal mela tonin produc t ion . While this cannot be precluded, a substant ial a t tenuat ion of light's ability to influence the pineal gland would seemingly be expected. Likewise, it could be argued tha t ra ther than rods , surviving cones (which are more likely preserved in eyes with l ight-damaged re t inas

8) , m a y mediate the inhibi tory effects of light on pineal mela tonin

produc t ion . If, in fact, this is a function of cones then it would be expected that pineal mela tonin synthesis in diurnally active animals , which have cone-dominated ret inas, would be more sensitive to light inhibit ion than would the pineal metabol ic activity of nocturnal ly active animals which possess rod-domina ted ret inas. However , just the inverse is the case (Table 1). Also, the three proposed photop ic mechanisms in the ret ina of the rat have peaks of roughly 450 nm, 520 n m and 560 nm.

Whereas there is little doub t that wavelengths of visible light between 460 nm and 580 n m (with peak sensitivity at 495 nm) inhibit pineal N A T activity in the rat and this inhibit ion m a y be mediated by a rod pho top igmen t ,

5 there is currently no satisfactory explanat ion for the

ability of wavelengths > 6 0 0 n m (red light) to suppress the mela tonin forming ability of the pineal. We have found that the isomerizat ion of 11-ds retinal does not occur in the retinas of red light exposed rats even when the light is sufficiently intense to inhibit the ability of the pineal gland to synthesize mela tonin (J .H. Sun, R.J. Reiter and K. Yaga, unpubl ished observat ions) ; thus , photoac t iva t ion of rhodops in is clearly no t required for red light to act in an inhibi tory manne r on the pineal gland.

One implication of the information summarized in the previous pa rag raph is that there may be circumstances under which pineal melatonin synthesis is inhibited by wavelengths between 400 n m and 760 nm where the classical retinal photoreceptors m a y not be involved. Indeed, the evidence is already quite compell ing that light detected by the retinas, bu t not by rods and cones, may influence the circadian system via the SCN. According to Foster and co l l eagues ,

11 in retinally degenerate


mice (C57BL/6J mice homogenous for the rd allele: rd/rd) light can still entrain their circadian system. These workers advanced the idea that there may exist a yet unidentified g roup of photorecept ive cells in the mammal i an retina that normal ly function to entrain the circadian system. Considering the possibility of a non-classical photoreceptor in the mammal i an eye, it may be no tewor thy that the vertebrate retina contains a special class of bipolar cells which has a ciliated dentri t ic process that has extensive arbor izat ions . These structures, referred to as "Landol t ' s c lubs"

17 have been speculated to be p h o t o s e n s o r y

40 and they should be

given considerat ion as a photoreceptor which mediates the inhibi tory effect of light on pineal melatonin produc t ion in mammal s .

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43 . We t t e rbe rg L., Beck-Fri is J. a n d Kje l lman B . F . (1990) M e l a t o n i n as a m a r k e r for a s u b g r o u p of depress ion in adu l t s . In Biological Rhythms, Mood Disorders, Light Therapy, and the Pineal Gland (eds. Schafii M . a n d Sham* S.L.), p p . 7 1 - 9 5 . Amer i can Psychia t r ic Press , W a s h i n g t o n .

11

Light Entrainment of Rat and Human Circadian Melatonin Rhythms H E L E N A I L L N E R O V À , L U D M I L A S A M K O V Â a n d M I L E N A B U R E S O V A

Institute of Physiology, Czechoslovak Academy of Sciences, 14220 Prague 4, Czechoslovakia

Abst rac t

T h e r h y t h m i c p r o d u c t i o n of me la ton in in ra ts is dr iven by a c i rcad ian r h y t h m of the p ineal TV-acetyltransferase act ivi ty. T h e present s tudy c o m p a r e s light e n t r a i n m e n t of the ra t p ineal N-acetyl t ransferase r h y t h m with tha t of the h u m a n se rum or saliva m e l a t o n i n r h y t h m . E x p o s u r e to br ight light in the early night p h a s e delays the ra t N A T r h y t h m a n d the h u m a n me la ton in r h y t h m as a whole wi th in one day . E x p o s u r e to br ight light in the late n ight phase advances the m o r n i n g ra t N A T a n d h u m a n m e l a t o n i n offset wi thin one day as well; the evening ra t N A T onset is, however , no t p h a s e shifted at all, whereas the h u m a n m e l a t o n i n onset m a y or m a y no t be p h a s e a d v a n c e d . It a p p e a r s t h a t even in h u m a n s the evening m e l a t o n i n p r o d u c t i o n onset does no t necessari ly phase shift in paral le l wi th the m o r n i n g offset. Bo th the ra t N A T r h y t h m a n d the h u m a n m e l a t o n i n r h y t h m are p h o t o p e r i o d d e p e n d e n t ; ad jus tmen t of the r h y t h m s to a change from long to shor t days m a y , however , p roceed in a different way in ra t s t h a n in h u m a n s . Al toge ther , t h o u g h light e n t r a i n m e n t of the c i rcadian r h y t h m in m e l a t o n i n p r o d u c t i o n in h u m a n s is s imilar to t h a t in ra t s , s o m e differences exist as well.

In t roduct ion

T H E DAILY rhy thm of the pineal h o r m o n e melatonin produc t ion and secretion in m a m m a l s is involved in t ransduct ion of the photoper iodic signal and may play a subtle role in the circadian system e n t r a i n m e n t .

1 0 , 28

In ra ts , the rhythmic melatonin produc t ion is driven by the circadian rhy thm of the pineal N-acetyltransferase (EC 2.3.1.87) (NAT) a c t i v i t y .

1 8'

23 The N A T rhy thm is controlled by a pacemaker located in the

suprachiasmat ic nuclei of the hypo tha lamus , similarly as other rhythms a r e .

22 The N A T rhy thm may be reset by exposure of rats to light dur ing

161


their subjective n i g h t .

1 1 , 1 2'

15 Similarly, chronic and even a single bright

light exposure may reset the h u m a n melatonin r h y t h m .

4 , 5 , 2 4'

25 The

present s tudy compares the light ent ra inment of the rhythmic melatonin product ion , i.e. of the N A T rhy thm, in the rat , with the en t ra inment of the h u m a n serum or saliva mela tonin rhy thm in order to show similarities as well as eventual dissimilarities between the rat and the h u m a n melatonin rhy thm resetting.

N A T activity was determined by a modification of the me thod of Deguchi and Axelrod, as described e l s e w h e r e .

9 , 1 3 15 Mela ton in was

measured by a direct rad io imunoassay validated for use in serum and in s a l i v a .

2 7 , 31 Unless otherwise specified, rats were mainta ined under a

lighting regime with 12 hours of light and 12 hours of darkness per day (LD 12:12). Resetting of the h u m a n circadian pacemaker by artificial light was performed in winter m o n t h s in order to avoid any interference with bright ou tdoor light either in the morn ing or in the evening.

Phase delays

After a 1-minute light pulse in the early night (Figure 1 A) or after a delay of the evening darkness onset (Figure IB) , the evening N A T activity onset was phase delayed more than the morn ing offset dur ing the same n i g h t .

1 6 , 17 However , within one day, the onset and offset were phase

delayed to the same extent, eventually by as much as by 6 hours . Consequent ly, the whole N A T rhy thm was phase shifted within one cycle and its waveform did no t change.

After a 3-hour bright light pulse in the early night, the morn ing saliva mela tonin offset was phase delayed by 1-2 hours in four subjects out of four within one day; the evening melatonin onset was phase delayed by 1 hour in three subjects (Figure 2) (Samkova and Illnerova, unpubl ished results). The mechanism of phase delaying of the h u m a n melatonin rhy thm might thus be similar to the mechanism of phase delaying of the rat N A T rhy thm. The magni tude of phase delays of the rat N A T rhy thm appeared to be larger than that of the h u m a n melatonin rhy thm. Chronic bright light t rea tment of h u m a n s in the evening delays the onset of the nightt ime melatonin product ion and the whole rhy thm of 6-sulfatoxymelatonin e x c r e t i o n .

2 0 , 25

The melatonin rhy thm ampl i tude increased dur ing the night when the light pulse was applied and the next night in three subjects (Figure 2). Such a r ebound effect has been repor ted for the h u m a n serum melatonin r h y t h m ,

1 ,2 but it has never been observed in the rat N A T rhy thm. In the

h u m a n melatonin rhy thm, other factors besides the N A T activity might play a role in the rebound effect, e.g. the pineal hydroxyindole-O-methyltransferase activity or concentra t ion of s u b s t r a t e s .

11

Light Entrainment of Rat and Human Melatonin Rhythms 1 6 3

18 22 02 06 10 14 18 22 02 06 10 14

Time (h)

F I G . 1. De lay of the N A T r h y t h m . Ra t s m a i n t a i n e d in L D 12:12, wi th l ights on from 06.00 to 18.00 h o u r s , were (A) exposed to a 1 m i n u t e light pulse a t 21.00 h o u r s (dashed line) o r (B) subjected to a 4 -hou r (dashed line) o r a 6 -hour (do t ted line) delay of the evening light offset. Thereaf ter , the ra ts were released in to da rknes s a n d the N A T r h y t h m was followed du r ing the same n ight (day 0) a n d du r ing the next n ight (day 1). Ful l lines represent N A T r h y t h m s in con t ro l ra ts

und i s tu rbed by light.

Phase advances

After a 1-minute light pulse in the late night (Figure 3A) or after a 3-hour and a 5-hour advance, respectively, of the morn ing light onset (Figure 3B), the next night only the morn ing N A T offset was phase advanced whereas the evening N A T onset was not phase shifted at a l l .

1 6 , 17 Consequent ly , the

phase relat ionship between the onset and the offset became compressed. Apparent ly , the onset and the offset do not necessarily phase shift in parallel.

After an exposure of h u m a n subjects to bright light since 03.00 hours in the morn ing , the next day the serum melatonin offset was phase advanced by 1-3 hours (Figure 4 ) .

5 In cont rary to the rat N A T rhy thm, the evening

melatonin onset was eventually phase advanced as well t hough not in all


80

70

c: 60 a M 50 cx

.3 40 d ο

je 30

S 20

10

/— / ρ - α m'

/ t

\

18 20 22 24 02 04 06 08 10

Time of day (h)

F I G . 2. Effect of a 3-hour br ight light pulse in the early n ight on the saliva me la ton in r h y t h m in a representa t ive subject . Vo lun tee r s sampled saliva du r ing the first, con t ro l n ight , when only d im light of an intensi ty lower t h a n 50 lux was on (full l ine), d u r i n g the second night , when they were exposed to br ight light of intensi ty 2,500 lux from 21.00 to 24.00 h o u r s (dot ted line) a n d du r ing the th i rd n ight , when they aga in exper ienced d i m light only (dashed line). O p e n b a r u n d e r

the abcissa deno tes the light pulse .

subjects. It appears that even in h u m a n s the evening melatonin onset and the morn ing offset do not necessarily phase shift in parallel. After a 3-hour bright light pulse in the late night, the next day the morn ing saliva mela tonin offset was phase advanced in four subjects out of four by 1-3 hours whereas the evening onset was phase advanced in one subject (Figure 5) (Samkova and Il lnerova, unpubl ished results). Chronic morn-ing bright light t rea tment phase advances the onset of nightt ime melatonin product ion in winter depressive pat ients and the whole melatonin rhy thm pat tern dur ing the Antarct ic w i n t e r .

4'

25

Effect of photoper iod

In the rat , the evening light phase delays the evening N A T onset primari ly while the morn ing light phase advances the morn ing N A T offset primarily. Consequent ly , in long summer days the N A T rhy thm waveform is compressed due to light in t ruding into the late evening and early morn ing hours ; in short winter days, the waveform is d e c o m p r e s s e d .

14 Unde r all

photoper iods equal to 16 hours or shorter , the morn ing N A T offset occurs abou t 1 hou r before the light onset and is thus locked to the morn ing l i g h t .

11 It appears that the morn ing light is the entraining agent which

synchronizes the rat circadian system with the 24-hour day. The evening light has a s t rong phase delaying effect only when the morn ing light is absent . Otherwise, the evening light may serve not as a true entraining agent but ra ther as a photoper iodic signal. After t ransi t ion of rats from


18 21 24 03

Time (h)

F I G . 3. Effect of a light exposu re in the late n ight on the N A T r h y t h m . Ra t s m a i n t a i n e d in L D 12:12, wi th lights on from 06.00 to 18.00 h o u r s were (A) exposed to a 1 m i n u t e light pulse a t 03.00 h o u r s (dashed line), thereafter released in to da rknes s a n d the N A T r h y t h m was followed d u r i n g the same night (day 0) o r the next n igh t (day 1) o r (B) subjected t o a 3-hour a d v a n c e (dashed line) a n d a 5-hour a d v a n c e (dot ted line) respectively, of the m o r n i n g light onset , a n d the next day they were released in to d a r k n e s s a l ready at 14.00 h o u r s a n d the N A T r h y t h m was followed. Ful l lines represent N A T r h y t h m s in con t ro l ra ts und i s tu rbed by

light.

long to short days , the N A T rhy thm waveform extended gradually only (Figure 6 ) .

13 When the dark period was prolonged symmetricaly, the

extension proceeded mostly into the morn ing hours , so tha t the N A T decline occurred again jus t 1 hou r before the morn ing light onset (Figure 6A).

In h u m a n s , no difference in the p lasma melatonin signal dura t ion between summer and winter was observed at 50°N and 35°S lat i tude, due apparent ly to shielding of h u m a n subjects from bright ou tdoo r l i g h t .

1 9'

2 1'

29 However , at 60°N lat i tude in Sweden and at 68°S lat i tude in

Antarct ica, a slight difference between summer and winter was r e p o r t e d .

3'

26 Dur ing winter mon ths at 50°N, when na tura l daylight


(D)

18 20 22 00 02 04 06 08 16 18 20 22 00 02 04 06 08

( F )

16 18 20 22 00 02 04 06 08 16 18 20 22 00 02 04 06 08

Time of day (h)

F I G . 4. Effect of a single m o r n i n g br igh t light exposure o n the h u m a n se rum m e l a t o n i n r h y t h m . B lood was sampled from six vo lun teers , A - F , du r ing the first con t ro l n ight , when d im light only was on (full l ines). D u r i n g the second night , the subjects were exposed to b r igh t light of intensi ty 2 ,000-3 ,000 lux from 03.00 to 09.00 h o u r s in the m o r n i n g . D u r i n g the th i rd n ight , the subjects aga in exper ienced

d im light only a n d b l o o d was sampled (dashed lines). After Ref. 5.

18 20 22 24 02 04 06 08 10

Time of day (h)

F I G . 5. Effect of a 3-hour b r igh t light pulse in the late n ight on the saliva m e l a t o n i n r h y t h m in a representa t ive subject . Volun tee rs sampled saliva du r ing the first, con t ro l n igh t , w h e n only d im light was o n (full l ine), du r ing the second night , when they were exposed to br ight light from 03.00 to 06.00 h o u r s (dot ted line) a n d du r ing the th i rd n ight when they aga in exper ienced d i m light only (dashed line).

O p e n b a r u n d e r the abcissa deno tes the light pulse .


16

12 -

8 -

4 -

LD 16: 8 LD 8: 16

12

β \ * \' 16 20 24 04 08 12

Time (h)

F I G . 6 . Ex tens ion of the ra t N A T r h y t h m after symmet r ica l p r o l o n g a t i o n of the d a r k per iod (A), after p r o l o n g a t i o n of the d a r k per iod in to the evening h o u r s (B), a n d after p r o l o n g a t i o n of the d a r k pe r iod in to the m o r n i n g h o u r s ( C ) . Ra t s m a i n t a i n e d in L D 1 6 : 8 were ei ther killed in L D 1 6 : 8 (filled circles) o r were t ransferred to L D 8 : 1 6 a n d killed 3 (open circles), 6 (filled squares ) a n d 1 3 (open squares ) days la ter . Lines be low the abcissa d e n o t e pe r iods of d a r k n e s s before

( L D 1 6 : 8 ) a n d after ( L D 8 : 1 6 ) t r ans i t ion . After Ref. 1 3 .

resembled L D 8:16, the mela tonin signal dura t ion in subjects entra ined to artificial long days simulated by the bright light L D 16:8 skeleton photoper iod was shorter by 2.5 hours than the signal in subjects experiencing just short winter d a y s .

6 Hence the noc turna l mela tonin signal

dura t ion may also provide h u m a n beings with the information on day length.

Four -day exposure to the long skeleton pho toper iod dur ing winter mon ths , with bright light on from 19.00 to 22.00 hours in the evening and


again from 06.00 to 09.00 hours in the morn ing phase advanced the morn ing serum mela tonin offset in four subjects out of four (Illnerova, Buresovâ, Dvofâkovâ and Zvolsky, unpublished results). Fol lowing withdrawal of the bright light t rea tment , the phase advance of the morn ing melatonin offset persisted for three subsequent days and eventually the evening melatonin onset became phase advanced as well. In cont rary to what happens with the rat N A T rhy thm after a change from long to short d a y s ,

13 extension of the h u m a n melatonin rhy thm waveform on short days

did not proceed into the morn ing hours . The subjects were, however, woken each day at 06.00 hours in the morn ing . The forced sleep-wake schedule might thus mainta in the circadian phase advance induced by the long skeleton pho toper iod . The prevailing phase advancing effect of the long pho toper iod is in agreement with the role the morn ing light may play in the h u m a n circadian system e n t r a i n m e n t .

1 9 , 2 4'

30 At 50°N and 35°S

lat i tude, the mela tonin rhy thm in winter is phase shifted towards the morn ing hours as compared with the summer melatonin pa t te rn , due probably to the fact tha t in winter h u m a n s may experience light of sufficiently s t rong intensity later in the morn ing than in s u m m e r .

1 9 , 21

Lower ing of ampl i tude

In rats exposed to a 7-hour advance and an 8-hour advance, respectively, of the morn ing light onset (Figure 7 A) or in those exposed to a 4-hour light pulse a r o u n d the middle of the night (Figure 7B), the next day the morn ing N A T offset was phase advanced and at the same time the evening onset was phase d e l a y e d .

16 The waveform of the N A T rhy thm was compressed

considerably and the ampl i tude decreased. Apparent ly , exposure to light a round the middle of the night might have a dual effect on the N A T rhy thm, i.e. a phase delaying one on the N A T onset and a phase advancing one on the N A T offset .

12 Eventually, the onset might occur close to or at

the time of the offset and the N A T rhy thm ampli tude diminished.

In h u m a n s , a reduct ion of ampli tudes of the p lasma Cortisol and body tempera ture rhythms was observed following a repeated exposure of subjects to a long bright light pulse a round the time of the tempera ture m i n i m u m .

7'

8 O u r effort to diminish the mela tonin rhy thm ampli tude in

h u m a n volunteers by a single 3-hour or even a 7-hour exposure to bright light of intensity 2,500 lux has been unsuccessful till this t ime, due probably to inappropr ia te t iming, low intensity of light; a single exposure instead of a repeated one or to combina t ion of all the above ment ioned reasons.

Conclusions

Following exposure to bright light in the early night, the next day the evening rat N A T and h u m a n melatonin onsets are phase delayed and ,


18 20 22 24 02 04 06

Time (h)

F I G . 7. Lower ing of the N A T r h y t h m a m p l i t u d e . Ra t s m a i n t a i n e d in L D 12:12, wi th lights o n from 06.00 to 18.00 h o u r s were ( A ) subjected to a 7 -hour a d v a n c e (dashed line) o r to an 8-hour a d v a n c e (dot ted line), respectively, of the m o r n i n g light onse t a n d the next day they were released in to d a r k n e s s a l ready at 14.00 h o u r s a n d the N A T r h y t h m was followed in the subsequen t n ight , o r (B) were exposed to a 4 -hou r light pulse from 23 .00-03 .00 h o u r s , thereafter released in to d a r k n e s s a n d the N A T r h y t h m was followed the next n ight (dashed line). Ful l

lines represent N A T r h y t h m s of con t ro l r a t s und i s tu rbed by l ight.

correspondingly, the offsets are phase delayed as well. Fol lowing exposure to bright light in the late night, the next day the morn ing rat N A T offset and the h u m a n melatonin offset are phase advanced; the evening rat N A T onset is no t phase shifted whereas the h u m a n mela tonin onset may or may not be phase advanced. Dura t i on of the elevated noc turna l N A T level in rats and dura t ion of the mela tonin signal in h u m a n s depend on the pho toper iod in bo th species. Fol lowing a change from a long to a short photoper iod , the rat N A T rhy thm pat te rn extends mostly into the morn ing hours . In h u m a n s , a long pho toper iod causes primari ly a phase advance of the morn ing mela tonin offset; such an advance may persist even in short days , due probably to a fixed waking schedule. In h u m a n s , similarly as in ra ts , the evening mela tonin p roduc t ion onset does not necessarily phase shift in parallel with the morn ing offset. In summary , light en t ra inment of the h u m a n melatonin rhy thm shows striking


similarities with that of the rat pineal N A T rhythm, but some dissimilari-ties as well.


The research was part ly suppor ted by the Czechoslovak Academy of Sciences grant number 71118.

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24. Lewy A.J. (1985) Regu la t ion of m e l a t o n i n p r o d u c t i o n in h u m a n s by br igh t artificial l ight: evidence for a c lock-ga te mode l a n d a p h a s e response curve . In The Pineal Gland. Endocrine Aspects (eds. B r o w n G . M . a n d W a i n w r i g h t S.D.), Vol . 53 , p p . 2 0 3 - 2 0 8 . P e r g a m o n Press , N e w York .

25 . Lewy A.J. , Sack R.L. , Mil ler L.S. a n d H o b a n T . M . (1987) An t idep res san t a n d c i rcad ian phase-shif t ing effects of l ight. Science 3 2 5 , 352 -354 .

26. M a k k i s o n I. a n d A r e n d t J. (1991) M e l a t o n i n secret ion in h u m a n s on two different an ta rc t i c bases (68° a n d 75°S). J. Interdisciplinary Cycle Res. 2 2 , 149-150 .

27. N o w a k R., M c M i l l e n I .C. , R e d m a n J. a n d Shor t R.V. (1987) T h e cor re la t ion be tween se rum a n d sal ivary m e l a t o n i n c o n c e n t r a t i o n s a n d u r ina ry 6 -hyd roxyme la ton in su lpha te excre t ion ra tes : t w o non invas ive t echn iques for m o n i t o r i n g h u m a n c i rcad ian rhy thmic i ty . Clin. Endocrinol. 2 7 , 4 4 5 - 4 5 2 .

28. R e d m a n J., A r m s t r o n g S., N g K . T . (1983) F r e e - r u n n i n g activity r h y t h m s in the ra t : E n t r a i n m e n t by me la ton in . Science 2 1 9 , 1089-1091 .

29. R o e n n e b e r g R. a n d Aschoff J. (1990) A n n u a l r h y t h m of h u m a n r e p r o d u c t i o n . I I . E n v i r o n m e n t a l cor re la t ions . / . Biol. Rhythms 5 , 2 1 7 - 2 3 9 .

30. T e r m a n M . , Q u i t k i n F . M . , T e r m a n J .S. , S tewar t J . W . a n d M c G r a t h P .J . (1987) T h e t iming of p h o t o t h e r a p y : effects o n clinical r esponse a n d the m e l a t o n i n cycle. Psychopharmacol. Bui. 2 3 , 354—357.

3 1 . Webley G .E . , M e h l H . a n d Willey K . P . (1985) Va l ida t ion of a sensitive direct assay for m e l a t o n i n for inves t iga t ion of c i rcad ian r h y t h m s in different species. / . Endocrinol. 106 , 387-394 .

12

The Use of Melatonin as a Marker for Circadian Phase and as a Chronobiotic in Blind and Sighted Humans

A . J . L E W Y a n d R. L. S A C K

Sleep and Mood Disorders Laboratory, Departments of Psychiatry, Ophthalmology and Pharmacology, Oregon Health Sciences University, Portland, Oregon, 97201, USA

M e l a t o n i n assay

M E L A T O N I N IS produced only at night in the dark . Levels of p lasma melatonin are quite low dur ing the day (1-5 pg/ml) and increase to abou t 50-100 pg/ml at night. In order to accurately measure these low levels, we developed the gas chromatographic -nega t ive chemical ionizat ion mass spectrometric ( G C M S ) a s s a y .

17 The G C M S assay continues to be the

"gold s tandard" , because of its sensitivity, specificity and precision. It has helped improve rad io immunoassays (RIAs) over the years, directly th rough cross-validation and indirectly th rough the publ icat ion of accurate p lasma mela tonin values. The G C M S assay's future utility lies in a cont inued role for on-going validat ion of RIAs established in different laborator ies when there is a change in tissue or species, when there is a change in experimental design, or when an unexpected seemingly spurious result needs to be verified. At present, we routinely use this assay for high resolution determinat ion in p lasma samples of the endogenous mela tonin produc t ion circadian rhy thm; the G C M S assay also has great potent ia l for the rout ine determinat ion of the mela tonin rhy thm in saliva, in which melatonin levels are abou t one-third those of p l a s m a .

53

The G C M S assay has been used in the discovery of impor t an t findings, such as suppression of h u m a n melatonin produc t ion by bright l i g h t .

30 O n e

implication of this finding was that h u m a n s might have circadian and

173


seasonal rhy thms that respond to the na tura l (bright) l igh t -dark cycle and the na tura l photoper iod , respectively, and that these biological rhy thms are relatively unper turbed by the use of ordinary-intensi ty r o o m light. Pr ior to this discovery, scientists thought that social cues were the major zeitgeber (time cue) for h u m a n s .

54

M e l a t o n i n c i rcadian rhy thms in bl ind people

When we realized that light might be an impor tan t zeitgeber for h u m a n s , the quest ion natural ly arose, what were the consequences for totally blind individuals who had absolutely no light perception and thus could not perceive the l igh t -dark cycle. Initially, we identified 10 individuals who were each studied on one o c c a s i o n .

18 All of them had melatonin pat terns

with an approximately 12-hour active phase and 12-hour quiescent phase. While some of these melatonin pat terns were in a normal phase relat ionship with the l igh t -dark cycle, most were markedly phase advanced or phase delayed.

Two of the abnormal ly phased individuals were studied longitudinally on a weekly basis for 4 consecutive weeks. O n e of them appeared to be stably entrained, albeit at an abnorma l phase. The other individual was shown to be free-running, with an intrinsic period (tau) of abou t 24.7 hours .

The mela ton in onset

It is not necessary to measure the entire nightt ime profile when using melatonin to assess circadian phase . Theoretically, any par t of the curve can be used to assess phase , as long as the ampl i tude and the dura t ion (time interval between the onset and the offset) are constant . This appears to be the case in blind people. F o r most of our studies, the melatonin onset is used because it is the most clearly demarca ted par t of the melatonin curve and most accurately reflects the phase and period of the melatonin rhy thm in blind people.

We prefer the melatonin onset for bo th theoretical and practical reasons. Theoretically, the melatonin onset is tha t par t of the melatonin curve which is least affected by biochemical factors that could confound the use of mela tonin levels as a marker for circadian phase. O n e such factor is increasing subsensitivity of pineal beta-adrenergic receptors dur ing the n i g h t .

41 Fu r the rmore , melatonin levels could also decrease dur ing the

night because synthesis precursors become depleted. Consequent ly , as the night progresses, mela tonin levels are mark ing biochemical events as well as the circadian signal from the endogenous pacemaker (the suprachias-mat ic nuclei, or SCN, located in the hypotha lamus) .

Use of Melatonin as a Marker 1 7 5

The d i m l ight me la ton in onset ( D L M O )

In sighted people, the mela tonin onset is also the preferred marke r for assessment of circadian phase . T o avoid suppression of mela tonin , it is critical to d raw blood under dim light ( < 30-50 lux), beginning 1 hour before the first d raw. W e call this measure the d im light mela tonin onset, or D L M O . Blood is d r awn in the evening as frequently as resolut ion requires, usually every 30 minutes between 18.00 and 23.00, a l though sometimes we extend this interval by an hour in either direction.

F r o m our studies of blind people, we know that sleep propensi ty is phase-locked with the mela tonin onset, consistently lagging behind it by 2 -3 h o u r s .

34 We also know tha t when the mela tonin onset is within a few

hours of sleep onset, sleep tends to be phase-locked with the mela tonin onset; tha t is, they appear to be relatively c o o r d i n a t e d .

45 These findings do

not necessarily mean that the mela tonin rhy thm induces sleep, bu t ra ther that sleep is strongly influenced by a (free-running) endogenous circadian pacemaker which is marked by the mela tonin onset .

Ruling out non-phot ic e f fec ts on t h e c i rcadian system of humans

In a subsequent study of 11 blind people, 10 had taus greater t han 24 hours that ranged between 24.28 and 2 5 . 0 8 .

45 O n e person had a t au less t han

24 hours (23.88). This person happened to be the oldest person of this g roup , which is interesting in tha t some investigators believe that tau decreases with age.

If t au does change with age, it does so very slowly, because intra-individual variability in tau is small. F o r example, two free-running blind people studied on a second occasion after an interval of 1 or 2 years had taus unchanged from when they were first s tudied. An individual w h o was studied for more than 6 mon ths had a tau of 24.70 with a s t andard deviation less t han the sampling frequency of 1 hour : the regression fit was highly linear (r = 0.95, ρ < 0.001). Thus , a l though inter-individual variabi-lity is marked , intra-individual variability is small. Therefore, the study of blind people provides a means for testing non-phot ic influences on the circadian system. Incidentally, the mela tonin onset is phase-locked with the Cortisol circadian rhy thm and has the same t a u .

44

The marked inter-individual variability in the taus of free-running blind people may explain why some people are "early birds" , while others are "night owls". Sighted people are similar, in tha t D L M O intra-individual variability is small, yet a m o n g individuals D L M O s can range between 19.00 and 24.00. Whereas par t of this variability is explained by differences in light exposure and sleep times (which can super impose some structure upon the l ight-dark cycle), as with blind individuals, some of this


variability is intrinsic, in tha t a person with a greater t au is likely to have a more phase-delayed D L M O when entrained.

Al though most totally blind people appear to have free-running circadian rhy thms , a few may not be free-running. Some of these people may be entra ined to a normal phase posit ion, while others may be entrained to an abnorma l phase , and still others may have unstable circadian rhythms. An on-going s tudy in our l abora tory is to determine if there actually are blind individuals who appear to be (stably or unstably) entrained. Whereas it is straight-forward if an individual is free-running, stable en t ra inment is more difficult to demons t ra te . O n e must do studies over several weeks or mon ths to detect a change in circadian phase posit ion if the tau is close to 24 hours . Alternatively, a circadian rhy thm with a tau much greater than 24 hours would appear to be entrained every few weeks; these individuals would need to be studied at frequent intervals.

These studies are very impor tan t . If all totally blind people are free-running, it is unlikely tha t activity, food or even non-ocular light perception can affect the circadian system in h u m a n s . There is a report of a presumably entrained a lmost totally blind person; however, this person could suppress mela tonin product ion in response to 6,300 to 8,400 lux light e x p o s u r e .

32 Also, upon further evaluat ion of this person's light

response, it turned out that some response could be detected on electroret inographic testing. We know of one totally blind person who continues to have a free-running melatonin rhy thm, despite daily and vigorous use of a s ta t ionary bicycle. We also know of a totally blind shift worker whose circadian rhythms were unaffected after switching from one work shift to another .

M e l a t o n i n : f r o m a marker t o a chronobiot ic

When we started working with melatonin, we regarded it primarily as a biological marker . However , in our efforts to develop ways in which to entrain blind people, we decided to investigate the potential zeitgeber propert ies of melatonin. We were encouraged in this regard by animal studies.

In animals , the function of the pineal gland and its principle ho rmone , melatonin, was first associated with an ant igonadal effect; thus , melatonin was originally thought to be a reproduct ive h o r m o n e .

40 Then it was

discovered that melatonin regulated the seasonal rhy thm of estrus as well as other seasonal rhy thms. Tha t is, melatonin could be either ant igonadal or p rogonada l , depending on whether the animal was a spring, or a u t u m n breeder, respectively. Consequent ly , melatonin came to be associated with seasonal rhy thms , a l though there cont inues to be controversy as to whether it is the dura t ion or the phase of nightt ime melatonin secretion


that is responsible for communica t ing the time of the year to animals with seasonal r h y t h m s .

1 2'

51

A circadian function for the pineal was first shown in b i r d s .

11 In

mammal s , however, the effects of pinealectomy on circadian rhy thms was not r o b u s t .

38 Then , in 1983, R e d m a n and Armst rong showed that

melatonin could entrain the circadian activity-rest cycle in the r a t .

39

M e l a t o n i n and je t lag

H u m a n mela tonin-adminis t ra t ion studies began in the early 1980s, when melatonin was used by certain peripatetic scientists for self-treatment of jet lag; eventually, a few controlled studies were d o n e .

2'

36 However ,

melatonin 's role in helping to synchronize circadian rhythms was not clear.

In these studies, there was some concern that mela tonin was helping air travelers primarily th rough a sedative-hypnotic effect and not th rough a direct effect on circadian rhythms. Indeed, at the pharmacological doses taken (at least 2 mg po) , melatonin can have a soporific effect, and sleep loss is definitely a par t of jet lag. Moreover , mela tonin can indirectly aid in entraining circadian rhy thms, because sleep superimposes da rk on the l igh t -dark cycle. Thus , it was thought that melatonin might not have a true zeitgeber effect, at least with respect to most endogenous circadian rhythms. In one further jet lag s tudy ,

1 subjects slept better after bedt ime

melatonin adminis t ra t ion . Effects on endogenous melatonin were difficult to interpret , mainly because mela tonin da ta from subjects who did not subjectively improve were eliminated from the analysis.

Phase-advancing e f fec ts of me la ton in in s ighted people

Whether or not melatonin administered in this way was directly resynchronizing circadian rhy thms , the use of mela tonin in treat ing jet lag encouraged researchers to test the phase-shifting effects of mela tonin . This was first done by Arendt in 1 9 8 5 ,

4'

56 who gave melatonin at 17.00 hours in

order to advance the t ime when the melatonin signal occurred, yet still be cont inuous with the endogenous nightt ime bou t : the results, however, were neither robust nor consistent. Of 12 subjects given melatonin for 1 m o n t h in the spring, only two showed phase advances in the endogenous melatonin rhy thm. Exogenous melatonin was no t administered on the last (sampling) day of the study. This s tudy was repeated in the a u t u m n , in which exogenous melatonin was administered for 3 weeks, including the last (sampling) day. Five ou t of 11 subjects demons t ra ted phase advances . Par t ly because the pos t - t rea tment endogenous mela tonin levels in most of


the remaining subjects could no t be discerned from the exogenous levels, it was no t clear how many , if any, of the other subjects phase advanced.

The second g roup to investigate the phase-shifting effects of exogenous mela tonin on the endogenous mela tonin rhy thm was the Claust râ t g r o u p .

31 There was no effect immediately after administering melatonin

(8 mg po) at 22.00, a l though there was a phase advance 3 days after s topping melatonin t rea tment . However , the Cortisol rhy thm did not shift at all.

Finally, Arendt and Wever studied two individuals under free-running condit ions and failed to show any effect of mela tonin on e n t r a i n m e n t .

4'

55

In this study, mela tonin was given at bedt ime dur ing a protocol in which the l igh t -dark cycle was increasingly lengthened. Mela tonin had no effect on any of the circadian rhy thms measured, except for those of fatigue and alertness. It is no wonder , then, tha t some of these r e s e a r c h e r s

4 , 55 were

drawing quite conservative conclusions abou t the zeitgeber propert ies of melatonin from their studies. (Despite the negative findings with respect to endogenous physiological rhy thms , mela tonin did appear to have a consistent effect on s leep.)

4

Phase-advancing e f fec ts of me la ton in in bl ind people

In 1985-1986, we began to study the effects of melatonin (5 mg po) in free-running blind p e o p l e .

43 We thought tha t more robust and consistent

effects could be demons t ra ted in individuals who were not influenced by the l igh t -dark cycle. Indeed, we were eventually able to show phase-advancing effects of abou t 30 minutes per day; bo th the mela tonin onset and the Cortisol nadir were phase-shifted to the same extent following exogenous melatonin a d m i n i s t r a t i o n .

46

Mela tonin adminis t ra t ion research thus began to focus on blind people. Other groups then began to give mela tonin to blind p e o p l e ,

3'

3 5'

48 showing

at least some effect on the sleep-wake cycle. However , when a physiologi-cal marker such as mela tonin product ion was assessed, it was not clear if phase shifts actually o c c u r r e d .

3'

35 This lack of an effect was probably due

to the fact tha t ur inary melatonin product ion was measured, which is not very sensitive to small phase shifts. Consequent ly , we thought tha t par t of our ability to show robus t and consistent phase shifts in blind people was the use of a very sensitive marke r for circadian phase, the melatonin onset.

The D L M O and the phase response curve (PRC)

In sighted individuals, the D L M O is also a useful marker for circadian phase. It has been used, for example, to phase type individuals before insti tuting light t h e r a p y .

25 People with phase-advanced D L M O s require a

circadian phase delay and therefore are treated with bright light exposure


scheduled in the evening. People with phase-delayed D L M O s require a phase advance and are treated with morn ing bright light. The D L M O not only discriminates between different phase types but also indicates the phase-shifting response to light t rea tment . Interestingly, the baseline D L M O predicts phase-shifting responses; individuals who are relatively phase delayed at baseline have more of a phase-advance response to morn ing light and have less of a phase-delay response to evening light; individuals who are phase advanced have more of a phase-delay response to evening light and have less of a phase-advance response to morn ing l i g h t .

1 9 , 29 These findings suggest that the D L M O is mark ing the phase of

its phase response curve (PRC) and imply that the D L M O is mark ing the phase of its underlying circadian pacemaker .

Perhaps a comment would be appropr ia te here regarding a recent paper published by Shanahan and Cze is le r .

50 Al though we agree with these

investigators that the melatonin circadian rhy thm correlates well with the melatonin offset as well as with the melatonin m a x i m u m ,

49 we do not

agree with their s ta tement that the mela tonin onset is not as reliable as the fitted melatonin m a x i m u m for mark ing circadian phase shifts, because the high degree of noise in their mela tonin RI A appears to have confounded measurement of the low dayt ime levels needed to determine the mela tonin onset. This is not a p roblem when the melatonin onset is measured by the G C M S assay or by several other RIAs currently in use.

In 1983, we hypothesized a h u m a n P R C to bright l i g h t ,

20 based on the

c o m m o n features of P R C s observed in a variety of an ima l s .

9 37 Essentially,

there is a zone of reduced responses dur ing the day. Phase-delay responses occur in the evening and increase in magni tude dur ing the middle of the night; phase advance responses occur in the morn ing and also increase in magni tude dur ing the middle of the night. We subsequently published da ta consistent with such a P R C .

2 5 - 27 Recently, complete P R C s for light have

been described by four g r o u p s .

7 1 3'

3 3 , 55 O n e of these P R C s was

determined under free-running c o n d i t i o n s .

33

In order not to interfere with sleep, the usual times for scheduling light t rea tment are in the evening ending abou t 1 hour before bedt ime, and in the morn ing immediately upon awakening. The phase-shifting responses to bright light have been applied in the t rea tment of advanced and delayed sleep phase s y n d r o m e ,

2 0'

28 winter depression ( thought to be a phase-delay

type of d i s o r d e r ) ,

2 5'

47 jet l a g

8 and ma ladap ta t ion to shift w o r k .

6'

10 Bright

light exposure is also being investigated in the t rea tment of non-seasonal d e p r e s s i o n

15 and in a few other psychiatric disorders .

The human mela ton in PRC

Using the highly resolved D L M O (which is sensitive to small phase shifts), we were able to describe a P R C for mela tonin adminis t ra t ion in


h u m a n s .

1 6 , 2 1 - 24 We administered 0.5 mg of mela tonin for 4 days in a

placebo-control led study. F o r the first week of each trial, subjects received a (cornstarch) placebo. The second week subjects received placebo for 2 days and melatonin for 4 days. There was a D L M O determined at the end of each week.

Ten subjects part icipated in 30 trials. Subjects were asked to sleep at the same time of week two as tha t same day of week one. Only one subject failed to keep average sleep time of week two close to (within 1 hour ) week one's sleep time. This subject was eliminated from the study.

Mos t subjects received their medicat ion in two divided doses 1 hour apar t . The protocol was modified, however, when capsules were scheduled dur ing sleep for three subjects who received the entire dose in one capsule. Fu r the rmore , placebo adminis t ra t ion was eliminated for these trials in order to minimize interference with sleep.

The first D L M O was the baseline D L M O . In addi t ion to serving as a reference point for the pos t - t rea tment phase shift, the baseline D L M O was used to demarca te circadian time. F o r example, if an individual 's baseline D L M O was at 21.00 and melatonin was administered at 18.00, then the circadian time of adminis t ra t ion was C T 11. This is because, on average, the mela tonin onset is usually 14 hours after sleep offset (or "lights on") which in diurnal animals is t radit ionally designated as C T 0. If another individual 's baseline D L M O is at 19.00, then a clock time adminis t ra t ion of melatonin at 18.00 converts to a circadian time of C T 13. Between individuals, baseline D L M O s can be as early as 18.00 and as late as midnight . (Within individuals, baseline D L M O s are quite consistent.)

The results indicate a P R C similar in shape to P R C s described for various stimuli in other species. In phase , however, the mela tonin P R C is abou t 12 hours shifted from the P R C s for light. The melatonin P R C has the same phase as a dark-pulse P R C .

5 Indeed, this should not be

surprising, since melatonin is p roduced at night (in the dark) in almost all species. Interestingly, the mela tonin P R C more resembles that of the l i z a r d

52 than tha t of the r a t .

39 In the latter species, there is no delay zone

and a very limited zone of phase-advance responses (between C T 10 and 12) with no apparen t relat ionship between time of adminis t ra t ion and magni tude of phase shift.

The h u m a n melatonin P R C appears to have a "dead zone" of reduced responses between C T 14 and C T 20. Between C T 20 and C T 5, there are delay responses but no advance responses. Between C T 5 and C T 14, there are advance responses bu t no delay responses. Around C T 4 -5 there is a cross-over point .

The melatonin P R C explains why previous investigators did not find more consistent or robust results. Mela tonin was not administered at the opt imal time to induce robust phase a d v a n c e s .

4'

31 In the case where

phase-delay responses might have been effective, mela tonin was not

Use of Melatonin as a Marker 181

administered at the correct t i m e .

4'

55 It is no t surprising, then, tha t some of

these investigators were beginning to doub t that mela tonin could cause phase shifts in endogenous circadian r h y t h m s .

4'

55

At the very least, our da t a provide clear evidence for a phase-shifting effect of mela tonin . F o r phase advances , there is a clear linear relat ionship between the magni tude of the phase shift and the t ime of adminis t ra t ion . There also appears to be a delay zone, a l though because of the difficulties administer ing melatonin dur ing sleep, there were fewer trials conducted in this zone. Therefore, we cannot as yet clearly describe the precise shape of the delay zone or where the cross-over point is precisely located.

It is significant that we used a physiological dose of mela tonin in this s tudy. The 0.5 mg dose produces levels no greater than 200-300 pg/ml abou t 1-2 two hours after ingestion. Its half-life is a r o u n d 40 minutes : exogenous melatonin is removed from the circulation within a few hours . Consequent ly , exogenous melatonin did not interfere with the next day's D L M O determinat ion . F o r the modified protocol , the D L M O was determined on the evening of the same day of adminis t ra t ion, again wi thout being confounded by exogenous mela tonin .

The func t ion of endogenous me la ton in product ion in humans

The use of a physiological dose is also significant because it s t rengthens the a rgument that the mela tonin P R C suggests a function for endogenous melatonin in h u m a n s : mela tonin could act to augment en t ra inment of the endogenous circadian pacemaker by the l igh t -dark cycle. This role of endogenous melatonin is related to the suppressant effect of light. Al though it has been argued in some species tha t the appa ren t suppression of mela tonin by light may in fact be due to ins tan taneous shifts in either an onset oscillator or an offset osc i l l a to r ,

14 h u m a n s appear to have a separate

suppressant effect of light on mela tonin produc t ion . The suppressant effect of light is ins tan taneous . O n the other hand , shifts of the pacemaker , or at least of its driven rhy thms , often require t ransients; tha t is, several days may be required before steady-state phase relat ionships are resumed. (If the pacemaker shifts ins tantaneously, mela tonin p robab ly shifts the driven rhythms tha t require transients.)

The suppressant effect of light and endogenous mela tonin work together in the following way. Suppression of mela tonin at the twilight t ransi t ions prevents its s t imulat ion of specific por t ions of the mela tonin P R C . F o r example, while light is s t imulat ing the advance por t ion of the light P R C as occurs with an earlier dawn, the mela tonin offset occurs earlier tha t day, before the pacemaker tha t drives the mela tonin rhy thm has been advanced. Suppression of mela tonin in the morn ing decreases s t imulat ion


of the delay por t ion of the mela tonin P R C , thus enhancing advancement of the pacemaker .

After a few days, steady-state en t ra inment ensues. Thus , augmenta t ion of phase-shifting is p robably most impor tan t after shifts in the l igh t -dark cycle. Alterat ions in endogenous mela tonin levels may also be more impor tan t for changes in the t iming of dawn than for dusk, and there may be some differences between advances and delays.

The mela tonin P R C is the basis for t reat ing bo th phase-advance and phase-delay disorders with adminis t ra t ion of exogenous melatonin . The mela tonin P R C (and the suppressant effect of light) also provide the basis for hypothesizing a function for endogenous melatonin in h u m a n s . The mela tonin P R C makes clear tha t t ime of adminis t ra t ion must be taken into account . Whether or not knowledge of a person's baseline D L M O will be impor tan t remains to be determined. It is possible that sleep time (most likely sleep offset time) will be a fairly useful marker for circadian time.

S u m m a r y

Melatonin has had a very significant role in our unders tanding of h u m a n circadian physiology. The discovery of suppression of mela tonin by bright light changed the way in which we think abou t the regulat ion of h u m a n circadian rhy thms . Mela ton in may also be impor tan t as par t of a feed-back loop in this system.

A role for mela tonin in h u m a n s suggests tha t bright light t rea tment of circadian phase disorders might at least partially (particularly initially) shift circadian rhy thms th rough mela tonin suppression. F o r example, it is hoped tha t the study of beta-blockers in winter dépress ives

42 will be

repeated, this t ime selectively blocking endogenous melatonin product ion at a t ime predicted by the mela tonin P R C so as to cause a phase advance.

In conclusion, appropr ia te ly t imed melatonin adminis t ra t ion may be useful in the t rea tment of circadian phase disorders, alone or in conjunction with appropr ia te ly t imed bright light exposure. Determining the D L M O may be impor tan t in the opt imal scheduling of light t rea tment or mela tonin therapy. Mela tonin , therefore, continues to be impor tan t for assessing as well as t reat ing circadian phase disorders, such as advanced and delayed sleep phase syndrome, winter depression and ma ladap ta t ion to shift work and t ransmeridional air travel.

References

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Use of Melatonin as a Marker 185

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13

Overview: Unmasking Temperature Data Obtained in Field Conditions

J . W A T E R H O U S E ,

1 G . C O S T A ,

2 M . H A R M A ,

3 Ρ K N A U T H ,

4 D. M I N O R S ,

1

U. S I T K A

5 a n d D. W E I N E R T

5 1 Department of Physiological Sciences, University of Manchester, Manchester

M13 9PT, UK 2lstituto di Med ici η a del Lav or ο, Univers/ta degli Studi di Verona, 37134 Verona, Italy 3 Institute of Occupational Health, Laajaniityntie 1, S F-01620 Vanta a, Finland 4UP, Universitat Karlsruhe, Hertzstrasse 16, D-7500 Karlsruhe 21, Germany 5lnstitute for Zoologie, Universitat Halle, Domplatz 4, 0-4020 Halle, Germany

Abst rac t

U n d e r n o r m a l c i r cums tances , m a s k i n g effects d u e to a n indiv idual ' s lifestyle a n d e n v i r o n m e n t give mis lead ing in fo rma t ion w h e n the p h a s e of the e n d o g e n o u s osci l la tor(s) is inferred from over t c i rcad ian r h y t h m s . M e t h o d s to o v e r c o m e this p r o b l e m are often n o t feasible in field s tudies . H o w e v e r , we have deve loped a series of m e t h o d s t h a t can be used in such c o n d i t i o n s a n d which give a be t te r e s t ima te of the e n d o g e n o u s osci l la tor t h a n us ing raw, m a s k e d d a t a . These m e t h o d s have been assessed for val idi ty in l a b o r a t o r y - b a s e d s tudies a n d a lso used in several s tudies in the field where the a m o u n t a n d qua l i ty of d a t a collected c a n be less.

Resul ts conf i rm the view t h a t the use of m a s k e d d a t a leads to an overes t ima te of the r a t e of ad jus tmen t of the e n d o g e n o u s osci l la tor to t ime-zone t rans i t ions a n d shif twork. T h e y a lso indica te t h a t m a s k i n g factors m i g h t c o n t r i b u t e t o t he p h a s e stabi l i ty t h a t n o r m a l l y is m e a s u r e d in n y c h t h e m e r a l c i r cumstances .

T h e m e t h o d s requi re further tes t ing b u t o p e n u p the possibi l i ty of assessing m o r e accura te ly t h a n before the p rocess of ad jus tmen t of the b o d y clock t o changes in the field.

Mask ing and some w a y s of deal ing w i t h it

T H E OVERT circadian rhy thms measured in mos t variables are a mixture of endogenous and exogenous componen t s ; the endogenous componen t is due to the internal oscil lator(s)—the body c lock—and the exogenous componen t is caused by aspects of our envi ronment a n d lifestyle—and also referred to as "masking" . As a result, measured circadian rhy thms may be only poo r reflections of the internal oscillators, part icularly with regard to the phase and ampl i tude of their ou tpu t . Accurate measurements

189


of the internal oscillators—for example, of their response to a change in sleep/activity schedule, of the day-by-day variat ion in their t iming in nychthemeral circumstances, or of their development in the neonate—will all need recordings to be made in the absence of these masking influences, or at least for the record to be corrected (unmasked or purified) in some way.

Several successful approaches have been used, amongs t which are: A. The rhy thm can be measured under circumstances in which the masking influences are decreased sufficiently to become un impor tan t . F o r p lasma melatonin concentra t ion, rectal t empera ture and some other variables, this has been achieved. With melatonin, the dim light melatonin onset method of Lewy has been successful,

7 as has a version of the constant

rout ine me thod for rectal t empera ture , as refined by Czeisler's g r o u p .

1 F o r

other variables, however, including those of mental performance, of hormones influenced by plasma g l u c o s e ,

14 and of cardiovascular variables

such as heart rate and blood pressure, it is not certain that the exogenous influences have been removed fully or tha t the effects of sleep loss have not begun to exert another dis turbing effect. Clearly the constant rout ine method is unsuitable bo th for the study of several successive days and in most field condit ions. B. The rhy thm has been measured under circumstances in which masking influences are present but a t tempts have been made to separate the endogenous and exogenous effects mathematical ly . Such methods have proved to be fruitful in studies involving t ime-zone transi t ions and irregular sleep/activity s c h e d u l e s .

5'

6 12 These methods have the disadvan-

tages, however, that they can require large amoun t s of da ta and the biological interpretat ion of the mathemat ica l description of the da ta (particularly the exogenous componen t ) can be complex. C. We have developed a set of unmasking m e t h o d s

10 that , at least for

rectal tempera ture da ta , enable an est imate to be made of the phase of the endogenous componen t and yet overcome some of the difficulties discussed above. Thus :

(a) the rhy thm can be measured in nychthemeral circumstances; (b) there is good evidence that the exogenous componen t is directly

related to the sleep/activity cycle; (c) successive days can be studied; (d) the method can be used with missing da ta .

Unmasking methods: the basic concept

The basic concept is tha t a measured rhy thm consists of exogenous and endogenous components that act addit ively.

2 3 10 The endogenous

component , whose phase is sought , is assumed to be described by

Purifying Field Data 1 9 1

"normat ive endogenous da ta" . (These da t a are the mean rectal tempera-ture rhy thm from m a n y subjects studied in the supine posi t ion and corrected for any effects of sleep, when masking effects will be minimal.) The shape and t iming of the exogenous componen t depend upon several factors, including the individual 's sleep/activity cycle which can be est imated in more than one way, see below. The est imated rhy thm will be the sum of these componen t s and the me thod consists of changing the phase of the endogenous componen t until the sum of it and the exogenous componen t approximates most closely to the rhy thm tha t has been measured on the day in quest ion. This approx imat ion is assessed by finding the phase lag of the endogenous componen t which maximizes the cross-correlation coefficient between the est imated and measured circa-dian rhy thms. Alternatively, the phase lag of the endogenous componen t which minimizes the summed squared deviat ions between the est imated and measured rhy thms can be used; this me thod is required if the size of the masking effect is being assessed, see later.

In the following overview, some of the uses to which the basic concept and its var iants have been pu t will be outl ined.

Test ing th e basic m e t h o d in the laboratory env i ronment

We first tested the me thod with a simulated t ime-zone t r ans i t ion .

4 The

labora tory enables constant rout ines to be carried out also, t hough only on selected ra ther than consecutive days. Nevertheless, this will enable a compar ison to be made between shifts measured by the new and the "gold-s t andard" methods .

The me thod is applicable because the sleep/activity cycle is fairly reproducible from day to day in compar i son with the expected shift of the endogenous componen t .

The stages of the analysis would be as follows:

(a) The control days are pooled to give an average nychthemeral day. (b) The normat ive endogenous curve is subtracted to give the exogenous

profile for that subject. (c) This profile is then adjusted fully to the new local t ime after the t ime-

zone t ransi t ion. (d) Sets of test da t a are produced by combining (c) with the normat ive

endogenous da t a shifted by different a m o u n t s . In our studies we used hourly da t a points , so the shifts were in hour ly increments from 0 to 23 .

(e) The set of test da t a which correlates mos t highly with the measured rhy thm on a par t icular day is found; the shift of the normat ive endogenous da t a which contr ibute to this set of test da t a is taken as a measure of the shift of the endogenous componen t on tha t day.


Such a me thod was used for 8 successive days after a simulated t ime-zone transi t ion in seven subjects. The results indicated a progressive adjustment to the new time zone, bu t one tha t was significantly less than that measured conventionally (by cosinor analysis). Moreover , the shifts were not significantly different from those that were measured in independent experiments in which the shift of the endogenous componen t was est imated by constant rout ines. Tha t is, the me thod appears to produce an estimate of the phase of the endogenous componen t even though nychthemeral da ta are being used.

However , this me thod makes an impor tan t assumpt ion, namely it assumes that the profile of the exogenous componen t is, overall, constant from day to day. In many cases, particularly in the field, this will not be so, and a more versatile me thod is required.

The pur i f icat ion m e t h o d : deal ing w i t h var iable pat terns of sleep and act iv i ty

This, based upon the ideas of W e v e r ,

17 we have called the "purification"

method . Here the tempera ture record is corrected or "purified" in accord with categories which represent the major type of activity dur ing the previous hour . In one of our s t u d i e s ,

11 the categories used were:

"sleeping"; "lying down but awake" ; "sitting"; "active". The method then consisted of the following stages:

(a) Categorize each tempera ture on the basis of the activity in the previous hour (see above) .

(b) Raise all temperatures in the "sleeping" category by 0-1.0°C in 0.1 °C steps.

(c) Leave temperatures in the "lying down but awake" category unchanged (there is assumed to be no masking in this posture) .

(d) Lower all temperatures in the "sit t ing" category by 0-1.0°C in 0.1 °C steps.

(e) Repeat stage (d) for each of the other categories. (f ) Consider all the purified da ta sets produced by a combina t ion of the

above and compare each with normat ive endogenous da ta shifted in hourly increments from 0 - 2 3 . This enables the "best" combina t ion of purification factors and shift of normat ive endogenous da t a to be found—and hence the shift of the endogenous componen t on that day. "Best" can be assessed by finding the highest cross-correlation coefficient as before, but finding the min imum summed squared deviations between normat ive and purified da ta sets is an alternative tha t we normally use.

We have tested the purification method upon a g roup of 10 n u r s e s .

11


Each nurse was studied for at least 2 rest days (when she lived convent ional hours) before working up to 6 consecutive night shifts. Rectal tempera ture was measured th roughou t and hourly readings were used in the analysis. The nurses also kept a diary of their sleep and different categories of activity, as described above. It was established that adjustment of the endogenous componen t was progressive but incomplete (as compared with the delay in midsleep), and tha t it was slower than tha t assessed by cosinor analysis of raw (masked) da ta .

Tha t is, such a purification me thod indicates that there is an overest imate of phase adjustment when unpurified da ta are used. Whether the phase adjustment calculated by the purification me thod is "correct" is no t k n o w n since it was no t feasible for the nurses to undergo cons tant routines. However , in an earlier labora tory-based simulat ion of n igh twork ,

9 rectal t empera ture da ta were analyzed by other me thods that

also a t tempted to correct for masking effects. N o t only did these results show tha t shifts using corrected da t a were less than those using uncorrected (or masked) da ta , but also these smaller shifts were close to those est imated by constant rout ines. Tha t is, the evidence points to the results being a better assessment of the phase of the endogenous componen t when purified ra ther than raw da ta are used.

Assessing nychthemera l rhy thms using bo th methods

The aim of developing the new methods was to be able to use them in field condi t ions. Since neither of the me thods just described involves sleep loss, either can be used with da t a collected on an extended number of consecutive days to assess how reproducibly the circadian rhy thm is phased from day to day.

With subjects living in an isolation chamber , in which activity is restricted by lack of space to a mainly sedentary existence, the activity profile is very similar each day and therefore the first me thod (see the section on testing the basic me thod) can be used. We have done this with a g roup of 11 sub jec t s .

16 The analysis consisted of the following stages.

(a) All da t a were pooled to give the mean nychthemeral rhy thm. (b) The normat ive endogenous da t a were subtracted to give the mean

exogenous profile. (c) This profile was taken from each day's da t a in turn , to est imate the

endogenous componen t on that day. (d) The phase of this componen t was compared with normat ive

endogenous da ta .

As a control , the phase of the masked da ta was assessed each day by


carrying out a compar ison with normat ive da ta in the same way but wi thout having first removed the exogenous component . Results indicated that there was ent ra inment to 24 hours , but tha t there was a greater phase variat ion between days when the endogenous componen t was assessed (that is, the exogenous componen t had been removed) than when the unchanged raw da ta were assessed.

In a related e x p e r i m e n t ,

16 two subjects kept detailed activity logs for a

period of 23 consecutive days whilst they lived with their families. The purification me thod (see the section on the purification me thod) was more appropr ia te with these da ta , the normal five categories of activity being entered into the logs. Again results showed that there was ent ra inment to the 24-hour day and tha t the day-by-day phase variat ion was greater when purified ra ther than raw da ta were used. (Incidentally, if this purification me thod was used with the 11 subjects in the isolation unit then, again, the purified da ta showed more daily phase variat ion than did the raw data . )

The full implications of these results remain to be determined but they indicate that masking effects dur ing nychthemeral circumstances have the effect of stabilizing the phase of the measured circadian rhy thm in compar ison with the phase of the endogenous component . The results might also enable some est imate to be made of the phase variability of the entrained endogenous componen t . This seems an impor tan t factor when the process of adjustment and the relat ionship described by the phase response curve are considered.

Using th e models in less ideal f ie ld condi t ions

The rectal tempera ture da t a described so far have been collected from volunteers in the labora tory or, in the cases of the nurses and two subjects just described, from individuals who were able and willing to record rectal tempera ture and details of their lifestyle for extended periods of t ime. In most studies performed in the field, one or more factors prevent such "ideal" collection of da ta . Some of these will now be considered.

Changing the number of categories of activity

The nychthemeral s tudy upon two subjects living with their families made use of logs in which five types of activity (sleeping, lying but awake, sitting, s tanding, active) were used; the study using volunteers in the isolation unit used only two categories (sleeping and sitting); and the study using nurses used four categories (sleeping, lying but awake , sitting or relaxed, active or working) . The choice of categories reflects in par t what is convenient to the subjects and appropr ia te for their lifestyles, but it raises the quest ion whether more categories, which means more purification, also means that


the shift estimates are significantly improved. Closely related to this is the quest ion of whether an activity log assessed by an objective means , say a wrist accelerometer, is equally suitable.

O u r studies with the nurses doing shiftwork (above) indicated that the greater the number of categories, the better was the purification achieved. ("Better" in this context has two meanings: first it means a smaller value for the min imum summed squared deviat ions; second, it means a shift of the endogenous componen t that is less than that of raw da ta by a larger amoun t ) . However , the use of as few as two categories (sleeping and waking) produced some improvement over the use of masked da ta . (In this last case we are very close to the original process of purification as used by W e v e r .

1 7)

The record from a wrist accelerometer could also be used, the hourly counts being used as a measure of activity. Purification using categories based on these counts produced results tha t were indistinguishable from those using an activity log and the same number of categories.

Recently we have analyzed da ta from 19 nurses who wore rectal probes and whose activity record only distinguished between "sleep"; "leisure" and "work" . Results, Figure 1, compare the phase change between the control day (normal routine) and the 24 hours from the end of the first night shift to the end of the second night shift, using raw and purified da ta . The overest imate of the a m o u n t of adjustment when using raw da ta is clearly evident, as is the fact tha t adjustment of the endogenous componen t was ra ther inconsistent and , on average, very small. (Indeed it was only marginally significantly different from zero. Thus : for raw da ta , the delay was 6.47 + 0.63 hou r (mean ± SE); for purified da ta , the delay was 2.11 ± 1 . 1 0 hour , p < 0 . 1 0 . )

Estimation of rhythm changes with missing data

Assessments of circadian rhy thms in the field not only have to deal with problems caused by masking but also with those due to missing da ta . There is a general resistance to the use of a rectal p robe ( though we must reiterate our thanks to the nurses who were honourab le exceptions and whose da ta we have summarized above) . In practice, therefore, recordings dur ing sleep will be missing and even those taken dur ing the subjects' waking phase cannot be taken frequently.

A fairly typical ou tcome would be tha t the protocol would consist of measurements of oral t empera ture made every 2 or 3 hours when awake (during leisure and work time) and single readings taken on retiring and immediately after waking but before rising. We have recently investigated the adjustment of a g roup of 13 nurses dur ing two successive night shifts using the pro tocol jus t described. The process of purification needed some minor changes due to differences in the measurements . These were:


8h-i

advance

8h

delay

\ ° \

À ο

\ V

ο \

Raw 16h

16h-Purified

F I G . 1. Recta l t e m p e r a t u r e . C o m p a r i s o n of shifts of c i rcad ian r h y t h m be tween con t ro l d a y a n d night d u t y using r a w a n d purified d a t a . Ο , indiv idual nurses ; · , m e a n value + SE for g r o u p ; , line of equa l change ; d o t t e d line, m e a n delay

of mids leep

(a) Each tempera ture was assigned to the nearest clock hour . (This is required as the normat ive da t a are hourly) . When the reading was taken on the half hour , then it was assigned to bo th adjacent hours .

(b) Each tempera ture was assigned a category (in this case "sleeping"; "lying but awake" ; "sitt ing"; "s tanding"; "working") . "Sleeping" was the category assigned to the single reading obta ined immediately on waking.

(c) The purification process was unchanged except tha t : (i) the summed squared deviation was calculated only for those hours when a tempera ture reading was available; (ii) the normat ive endogenous da ta set were decreased in value slightly in order to enable better al ignment with oral temperatures which are slightly lower than those obta ined from the rectum.

As before, the average delay of the rhy thm assessed from raw da ta (4.15 + 1.01 hour ) was greater than tha t of the endogenous componen t assessed after purification (1.23 + 0.96 hour ) . An example of raw and purified tempera ture rhy thms from one subject is shown in Figure 2.

Whilst the results confirm previous findings, and therefore indicate that the present methods have at least some value in this type of field study, there is a caveat. As with any analysis of a circadian rhy thm, as the numbers of da t a points per cycle falls, so the est imate of phase will be influenced more by any extreme result. In the present case, the single value designated "sleeping" necessarily assumes considerable impor tance when the processes of purification and phase assessment are carried out . O n e ou tcome is that there is ra ther more inter-individual var iat ion in phase

Purifying Field Data 197

C l o c k t i m e

2 0o o 0 0o o 0 4O O 0 8O O 1 2O O 1 6O O 2 0O O QQ<

I I L

F I G . 2. R a w , O - - O , a n d purified, · — · , ora l t e m p e r a t u r e d a t a for single subject . I 1, t ime of sleep; S, s i t t ing; W , a w a k e ; A, as leep. Es t ima ted t imes of

m i n i m u m s h o w n for, Δ , r aw d a t a a n d , A , purified d a t a .

estimates than when 24 da t a points per cycle are available. Even so, it is wor th point ing out tha t the mean values for the purification factors (sleeping, sitting etc) were not different from those found when complete da t a sets were used in other studies.

A fu r the r test of t h e pur i f ica t ion method—assessing ampl i tude

In the studies so far we have always assumed that the ampl i tude of the endogenous componen t of an individual 's rhy thm was approx imated by that of normat ive endogenous da ta . The test of this a s sumpt ion—to compare normat ive endogenous da ta with those obta ined dur ing a constant rou t ine—cannot be carried out in the field condi t ions we are investigating, but it mus t be remembered that the normat ive da t a were obtained with protocols that approx imated to such condi t ions in heal thy subjects. With aged s u b j e c t s

15 and , part icularly, infants ,

8 however, there is

evidence to suggest tha t the ampl i tude of the endogenous componen t is decreased; ou r me thod would need modification if it were to be applied to such subjects, therefore.

We have performed a series of measurements upon heal thy, full-term babies dur ing the first week of extra-uterine life (day 2) and 4 weeks later. Skin and rectal t empera ture were measured hour ly for 24 hours and a record of "activity" was kept , this being divided in to: "deeply asleep"; "lightly asleep"; "awake , bu t lying passively"; "awake and moving a rms and legs"; "awake and crying". The category "awake , bu t lying passively" was assumed to be unaffected by masking, and so was no t purified. The analysis proceeded as before except for two changes.


A. The ampl i tude of the normat ive endogenous da ta could be changed. This is achieved by altering Kin the equat ion:

Yt = K(Et-ME) + ME,

where Yt is the ampli tude-corrected value for normat ive endogenous da ta at time t; Et is the uncorrected value for normat ive endogenous da ta at this t ime—this is the value used in the other me thods ; ME is the mean value for normat ive endogenous da ta .

Wi th this t ransformat ion, a value of Κ equal to zero implies a normat ive endogenous componen t with no ampl i tude and equal to the mean value, a value of unity gives values equal to those normal ly used in our other studies; other values of Κ can be tried systematically dur ing the purification process. B. The mean value for the raw da ta was set equal to ME and all individual values were adjusted appropria te ly . This is necessary to superimpose the mean values for normat ive and raw da ta , part icularly in the present case as the tempera ture of an infant might differ from tha t of an adult . Misal ignment of normat ive and raw da ta might lead to incorrect assessments of the size of masking effects in any s tudy—but it does not alter the est imate of phase of the endogenous componen t—but this could be part icularly misleading in the present case, as masking effects would be predicted to be small on account of activity being so limited in an neona te . F o r this reason also, the size of the masking effects in all categories was est imated to the nearest 0.05°C ra ther than 0.1 °C.

The results from the 11 babies showed some differences in ampl i tude and the size of masking effects between day 2 and week 4. Mask ing effects due to activity were generally small at bo th ages, but those due to sleep increased between the first and fourth week, Figure 3. The former result no doub t reflects the small a m o u n t of physical activity involved and "crying" as a category was too infrequent for a quant i ta t ive assessment of the masking effects of this to be made . The increased masking effect of sleep might indicate some consequence of the process of establishing sleep infrastructure tha t is occurring at this age (see reference 8, for example). The ampl i tude of the endogenous componen t of rectal tempera ture did not change significantly between day 2 and week 4, all ampli tudes on bo th occasions being less than values predicted for an adult , Figure 4. Such a result would cor robora te several findings on the process of development of circadian rhythmicity dur ing the weeks immediately after b i r t h ,

8'

13

though these other studies have been u p o n masked da ta . By contras t the ampl i tude of the endogenous componen t of skin tempera ture (which correlated highly with rectal tempera ture in most babies) showed a significant increase in ampl i tude between day 2 and week 4. Taken


Rectal temperature

Size of masking effect 'C

F I G . 3. F r e q u e n c y d i s t r ibu t ions of m a s k i n g effects for rectal a n d skin t e m p e r a t u r e in 11 bab ies . Closed ba r s , a t 2 days ; open ba r s , a t 4 weeks . A, deeply as leep; B,

lightly as leep; C , a w a k e a n d m o v i n g a r m s a n d legs; D , a w a k e a n d crying.

together, these results suggest that the development of the circadian rhy thm of tempera ture regulat ion is taking place by the end of the first m o n t h after bir th .

Concluding c o m m e n t s

We do not claim tha t the present me thods give estimates of the phase and ampl i tude of the endogenous componen t of the circadian rhy thm of body tempera ture tha t are as accurate as those given by cons tant routines or by sophisticated mathemat ica l analyses of da t a obta ined under nychthemeral condit ions.

We accept tha t the present me thods are based u p o n assumpt ions abou t the constancy of shape of normat ive endogenous da ta , the addit ive na ture of endogenous and exogenous componen t s , and the ability to est imate the exogenous componen t from activity records. We also accept that the methods have not been justified in all the experimental circumstances in which they have been applied. There are also the problems tha t validating a set of assumpt ions in one circumstance does not validate them in another , and tha t further difficulties might arise when combining assumpt ions . We have outl ined the evidence, bo th here and e l sewhere ,

10


1.6 Rectal

1.2

0.8

0.4

ο ο ο t ο , 6

0.0 0.0 0.4 0.8

Day 2 1.2 1.6

1.6

1.2

© 0.8 Φ

0.4

0.0 0.0

Skin

0.4 0.8 Day 2

1.2 1.6

F I G . 4 . C o m p a r i s o n of es t imated a m p l i t u d e of e n d o g e n o u s c o m p o n e n t of t e m p e r a t u r e r h y t h m at 2 days a n d 4 weeks of age in 1 1 babies . Resul ts expressed as fraction of a m p l i t u d e of n o r m a t i v e d a t a in adu l t s . O , indiv idual bab ies ; · , m e a n

value ± SE for g r o u p . , line of equa l change .

which suppor ts these assumpt ions and the results which have been obta ined by the different me thods .

Clearly, more validation is required, but we suggest that the methods offer the prospect of gaining some insight into the endogenous componen t of the circadian rhy thm of body tempera ture in field circumstances, tha t is, in circumstances where the experimental demands of the labora tory cannot be met for ethical or practical reasons. The results and prel iminary analyses reported and overviewed here confirm tha t results obta ined with


masked da t a can be misleading and tha t theoretical concepts developed in the labora tory can be applied to the field. It is hoped tha t this cross-fertilization between theory and practice, bo th in exper imentat ion and analysis, will cont inue to provide evidence relevant to an unders tanding of circadian rhy thms mechanisms.

References

1. Czeisler C.A., K r o n a u e r R . F . , Allan J .S. , Duff J .F . , Jewet t M . E . , B r o w n E . N . a n d R o n d a J . M . (1989) Br ight light i nduc t ion of s t r ong (type 0) reset t ing of the h u m a n p a c e m a k e r . Science 2 4 4 , 1328-1333 .

2. F o l k a r d S. (1988) C i r cad i an r h y t h m s a n d shif twork: Ad jus tmen t o r m a s k i n g ? In Trends in Chronobiology (eds. H e k k e n s W.Th . , K e r k h o f G .A . a n d Rietveld W.J . ) , p p . 173-182 . P e r g a m o n Press , Oxford .

3. F o l k a r d S. (1989) T h e p r a g m a t i c a p p r o a c h t o m a s k i n g . Chronobiol. Int. 6 , 5 5 - 6 4 . 4. F o l k a r d S., M i n o r s D . S . a n d W a t e r h o u s e J . M . (1991) " D e - m a s k i n g " the t e m p e r a t u r e

r h y t h m after s imula ted t ime-zone t rans i t ions . J. Biol. Rhythms 6 , 8 1 - 9 1 . 5. G a n d e r P . H . , K r o n a u e r R .E . a n d G r a e b e r R . C . (1985) P h a s e shifting t w o coup led

c i rcad ian p a c e m a k e r s : impl ica t ions for je t lag. Am. J. Physiol. 2 4 9 , R 7 0 4 - R 7 1 9 . 6. G u n d e l A. a n d Spencer M . B . (1992) A m a t h e m a t i c a l m o d e l of the h u m a n c i rcad ian

sys tem a n d its app l i ca t ion to je t lag. Chronobiol. Int. 9, 148-159 . 7. Lewy A.J. a n d Sack R .L . (1989) T h e d im light m e l a t o n i n onse t as a m a r k e r for c i rcad ian

p h a s e pos i t ion . Chronobiol. Int. 6 , 9 3 - 1 0 2 . 8. Mills J . N . (1975) D e v e l o p m e n t of c i rcad ian r h y t h m s in infancy. Chronobiologia 2 ,

3 6 3 - 3 7 1 . 9. M i n o r s D . S . a n d W a t e r h o u s e J . M . (1968) Effects u p o n c i rcad ian rhy thmic i ty of a n

a l t e ra t ion to the s leep-wake cycle: p r o b l e m s of assessment resul t ing from m e a s u r e m e n t in the presence of sleep a n d analysis in t e rms of a single shifted c o m p o n e n t . / . Biol. Rhythms 3 , 2 3 - 4 0 .

10. M i n o r s D . S . a n d W a t e r h o u s e J . M . (1992) Inves t iga t ing the e n d o g e n o u s c o m p o n e n t of h u m a n c i rcad ian r h y t h m s : a review of s o m e s imple a l te rna t ives to c o n s t a n t rou t ines . Chronobiol. Int. 9 , 5 5 - 7 8 .

11. M i n o r s D . S . a n d W a t e r h o u s e J . M . (1993) Sepa ra t i ng the e n d o g e n o u s a n d exogenous c o m p o n e n t s of the c i rcad ian r h y t h m of b o d y t e m p e r a t u r e d u r i n g n ight w o r k us ing s o m e "pur i f ica t ion" mode l s . Ergonomics 3 6 , 4 9 7 - 5 0 7 .

12. M i n o r s D .S . , N icho l son A.N. , Spencer M . B . , S tone B . M . a n d W a t e r h o u s e J . M . (1986) I r regular i ty of rest a n d act ivi ty: s tudies on c i rcad ian rhy thmic i ty in m a n . / . Physiol. 3 8 1 , 279-296 .

13. Si tka U. , Nage l F . , R u m l e r W . a n d Weine r t D . (1984) E n t w i c k l u n g der Z i r k a d i a n per iodik der K o r p e r t e m p e r a t u r bei N e u g e b o r e n e n . Dt. Gesundh. Wesen. 3 9 , 1 3 3 4 - 1 3 3 9 .

14. V a n C a u t e r E., B l a c k m a n J . D . , R o l a n d D . , Spire J -P . , Refetoff S. a n d P o l a n s k y K . S . (1991 ) M o d u l a t i o n of glucose regu la t ion a n d insulin secret ion by c i rcad ian rhy thmic i ty a n d sleep. Clin. Invest. 8 8 , 934 -942 .

15. Vitiello M.V. , S m a l l w o o d R .G. , Avery D . H . , Pa scau ly R.A. , M a r t i n D . C . a n d P r inz P . N . (1986) C i r c a d i a n t e m p e r a t u r e r h y t h m s in y o u n g adu l t a n d aged m e n . Neurobiol. Aging 7 , 97 -100 .

16. W a t e r h o u s e J . M . , M i n o r s D .S . a n d F o l k a r d S. (1993) Es t ima t ing the e n d o g e n o u s c o m p o n e n t of the c i rcad ian r h y t h m of rectal t e m p e r a t u r e in h u m a n s u n d e r g o i n g n o r m a l sleep/act ivi ty schedules . J. Interdiscipl. Cycle Res. 24 (in press) .

17. Wever R. (1985) In te rna l in te rac t ions wi th in the h u m a n c i rcad ian sys tem: the m a s k i n g effect. Experientia 4 1 , 332-342 .

14

Some Effects of Light and Melatonin on Human Rhythms

J O S E P H I N E A R E N D T

School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK

Abst rac t

In a n a t u r a l e n v i r o n m e n t , b o d y r h y t h m s are a d a p t e d to the regular a l t e rna t i on of n ight a n d day , a n d the s lower seasonal cycles. C u r r e n t social o r g a n i z a t i o n (inter al ia) leads to specific mi sma tches be tween o u r in te rna l phys io logy a n d o u r e n v i r o n m e n t . Shif t-work a n d r ap id t ravel across several t ime-zones leads to forced desynch ron i za t i on of in te rna l r h y t h m s from the externa l e n v i r o n m e n t a n d from each o the r , wi th consequen t p r o b l e m s of behav io r (e.g. sleep), phys io logy (e.g. gu t funct ion) a n d pe r fo rmance (e.g. acc ident ra te ) . Similar d i so rgan iza t ion of dai ly r h y t h m s is seen in the aged , s o m e b l ind subjects a n d in cer ta in pa tho log ica l s i tua t ions , such as de layed sleep p h a s e i n somnia , s o m e psychia t r ic d i so rders , possibly some cancers a n d o the r p a t h o l o g y .

T h e pineal h o r m o n e m e l a t o n i n conveys in fo rmat ion conce rn ing l igh t -dark cycles to b o d y phys io logy in an ima l s a n d m a y play a similar role in h u m a n s . I ts r h y t h m i c secret ion p rov ides an excellent c i rcad ian m a r k e r r h y t h m a n d has been extensively used for the inves t igat ion of the con t ro l of c i rcad ian r h y t h m s in h u m a n s , in pa r t i cu la r wi th r ega rd to the effects of l ight. I t has b e c o m e clear t h a t the h u m a n clock can be reset by su i table app l i ca t ion of br igh t l ight.

A subs tan t ia l n u m b e r of recent inves t iga t ions suggest t ha t r h y t h m d i s tu rbance can be alleviated or min imized by p h a r m a c o l o g i c a l a n d n o n - p h a r m a c o l o g i c a l m e t h o d s . L ight p rov ides a n o n - p h a r m a c o l o g i c a l a p p r o a c h to the al leviat ion of r h y t h m d i s tu rbance . Similarly, there is g o o d evidence tha t the p ineal h o r m o n e m e l a t o n i n c a n reset the h y p o t h a l a m i c clock a n d tha t in h u m a n s it h a s resynchron iz ing effects wi th r ega rd t o a n u m b e r of c i rcad ian r h y t h m s inc luding the s leep-wake cycle. Al leviat ion of r h y t h m d i s tu rbance in je t - lag , de layed sleep phase i n s o m n i a a n d selected b l ind subjects under l ines the po ten t ia l wide the rapeu t i c possibil i t ies for this n a t u r a l h o r m o n e .

In t roduct ion

M O S T IF not all circadian rhy thms in m a m m a l s are generated in the suprachiasmat ic nucleus (SCN) and entra ined (synchronized) to the 24 hour day primarily by l ight-dark cyc l e s .

1 ,2 Fac tors (zeitgebers) o ther than

l igh t -dark cycles which are involved in the en t ra inment of circadian rhythms in animals include behavioral imposi t ion, for example forced activity and rest, social and nutr i t ional (rhythmic feeding) cues, tempera-

2 0 3


ture variat ions, the pineal h o r m o n e melatonin and in h u m a n s knowledge of clock time. In a normal envi ronment these factors operate in concert . The extent to which any one of these can act as a principal zeitgeber in the absence of the others is species dependent and in general not well defined. Sufficient intensity and dura t ion of white light is, however, sufficient for ent ra inment to 24 hours in most species.

Dur ing the last decade the ability of light of suitable dura t ion and intensity to manipula te h u m a n circadian rhythms has become evident. Mela tonin provides an endogenous circadian marke r rhy thm generated in the S C N and relatively easily assessed in h u m a n plasma, urine and sal iva.

3

It has been extensively used in these investigations. It is normal ly secreted dur ing the dark phase of the day and is suppressed by sufficient intensity of light at n i g h t .

3 , 13 In addi t ion to its usefulness as an indicator of circadian

status, melatonin may well play an impor tan t role in the organizat ion of h u m a n circadian rhy thms. There is now good evidence that exogenous melatonin can manipula te some aspects of h u m a n circadian funct ion

3 and

that in vitro it is able to modify directly the phase of the (rodent) S C N .

4

This review aims to summarize some recent work.

Effects of l ight

Melatonin secretion in relation to daylength

In most species melatonin secretion is related to the length of the night: the longer the night the longer the dura t ion of secretion. This has been particularly well demons t ra ted in sheep where melatonin rises within a few minutes of lights off and in photoper iods of more than a round 14 hours of light does not decline until lights o n .

5 ,6 The dura t ion of mela tonin

secretion is a critical feature of its physiological role in photoper iodic time m e a s u r e m e n t .

5'

6

F o r many years it has proved difficult to demons t ra te a change in dura t ion of melatonin secretion with daylength in h u m a n s even in polar regions, a l though very small changes have been reported. The most consistent observat ion is that h u m a n melatonin profiles show a phase change from winter to summer , with earlier secretion in summer compared to w i n t e r .

7 ,8 If, however, h u m a n s are kept strictly in darkness for 14 hours

per day for a period of 2 mon ths , the melatonin secretion pa t te rn expands to cover almost the entire da rk period and concomitant ly , in extended periods of 14 hours of light, the rhy thm contracts to less than 10 h o u r s .

9

Thus in appropr ia te experimental condit ions h u m a n s show a dura t ion change like animals .

O u r behavior , part icularly tha t of u rban popula t ions , is such that in "no rma l " circumstances it is likely that we percieve only very small changes in daylength dur ing the year. F o r example in the U K it is fairly

Some Effects of Light and Melatonin on Human Rhythms 205

typical to rise a r o u n d 07.00 hours (well after dawn in the summer) and to be inside a dwelling from a r o u n d 19.00 hours (well before dusk in the summer) and thus no t exposed to na tura l sunlight for the entire photoper iod in summer .

Light suppression of melatonin secretion

Even a brief exposure to white or full spectrum light of sufficient intensity at night will rapidly suppress mela tonin p r o d u c t i o n .

10 The a m o u n t of light

required to suppress mela tonin secretion either dur ing the night or at the beginning and the end of the night varies considerably from species to species. A most impor tan t factor involved in this suppression is the previous exposure to light, with evidence from labora tory animals tha t prolonged main tenance in light of the intensity of na tura l light leads to decreased sensi t iv i ty .

11 It is likely tha t light sensitivity varies acutely,

increasing in the course of the night, p robably th rough an increase in retinal sensitivity.

H u m a n s require substant ial a m o u n t s of light to suppress mela tonin secretion at night. Early experiments suggested that domest ic intensity light (100-500 lux) did not suppress h u m a n mela tonin , and the inference was made that either we are resistant to such intensities or that h u m a n mela tonin could no t be suppressed by l i g h t .

12 In a classic experiment ,

however, L e w y

13 showed conclusively tha t if sufficient intensity of white

light (2,500 lux for 2 hours dur ing the night between 02.00 and 04.00 hours ) was used h u m a n mela tonin could be suppressed to basal (daytime) levels. This observat ion has been of very considerable impor tance , no t only for an unders tanding of the control of h u m a n mela tonin secretion bu t also for a general appreciat ion of the role of light in h u m a n physiology, in par t icular its impor tance in the control of h u m a n rhy thms .

In fact part ial suppression of mela tonin secretion in h u m a n s can be affected by 2-300 lux applied for 30 m i n u t e s .

14 Using this pro tocol a t ime

of day and t ime of year var iat ion in sensitivity is suggested by recent work in Antarc t ica—a region where long term exposure to domest ic intensity light is possible dur ing the winter in the absence, for prolonged periods, of na tura l sunlight. As might be expected sensitivity increases in the course of the night and was greater in winter t han in s u m m e r .

15 In heal thy subjects

there are very substant ial individual variat ions in sensitivity which may be bo th genetically and environmental ly determined.

Entrainment of the melatonin rhythm with light

Clear evidence of the impor tance of l igh t -dark for the en t ra inment of mela tonin was demons t ra ted in h u m a n s

16 where, following an inversion of

the l igh t -dark cycle, the ur inary mela tonin rhy thm adap t s to the new


photoper iod over a period of several days, finally assuming the same phase relat ionship with the new light da rk cycle as was present originally with the old light dark cycle. If h u m a n s are subjected to a forced phase shift of 24 hour time cues, such as in rapid flight across several time zones, readjustment of the phase of melatonin secretion takes on average approximate ly 1 day per hou r of shift but with substantial individual differences.

17

Light pulses are conventionally used to investigate the control of circadian rhythms according to a protocol known as the phase response curve (PRC) developed from observat ions on activity rest cyc les .

18 This

approach has been used extensively in the rat by I l l n e r o v â

19 to investigate

the circadian control of melatonin product ion with evidence for separate control of evening onset and morn ing offset of melatonin by two distinct oscil lators—Ε and M .

A single short daily pulse of light has not to the au thors knowledge been investigated in free-running h u m a n s with regard to melatonin . A week of daily exposure to bright light in the morn ing advances the onset of the evening melatonin rise (measured in dim light to prevent suppression) by up to 2.5 hours and a week of exposure to the same a m o u n t of light in the evening delays the onset by 1 hour even when the sleep wake cycle is kept c o n s t a n t .

20

Recent work designed to clarify phase shifting mechanisms in general in h u m a n s has used constant rout ine condit ions to evaluate phase and ampl i tude before and after the use of 2 to 3 daily (5-6 hours) applicat ions of bright (approximately 8,500 lux) light to induce phase sh i f t s .

21 Here

subjects are kept semi-recumbent in constant dim light and awake for 36 hours or more with identical hourly snacks. Such procedures abolish the "masking" effects of light da rk al terat ions, sleep, activity, social contacts and intermit tent meals and allow a pure assessment of circadian phase and ampli tude. Bright l ight-induced phase shifts in the core tempera ture rhy thm were accompanied by precisely equivalent shifts of the plasma melatonin r h y t h m .

21 Mela ton in is only "masked" to a major extent by

ambient light and provides probably the purest indicator of central biological clock status provided tha t light exposure is controlled.

Skeleton photoperiods

"Skeleton" light pulses at dawn and dusk, with otherwise cont inuous darkness , are read as a full pho toper iod by animals and have been used to investigate control of h u m a n melatonin rhythms. In Antarct ica, specifi-cally on bases with an imposed structure of daily activity, h u m a n melatonin rhythms show a more marked tendency to delay in winter compared to summer, than in temperate zones, presumably due to looser coupling of the circadian system to 24 hours in the dim domest ic intensity


Circadian rhythms in the blind

An u n k n o w n propor t ion of blind people have abno rma l circadian r h y t h m s .

22 Repor ts of free-running sleep-wake, Cortisol and mela tonin

rhy thms exist, however, to what extent these are representative of the total blind popula t ion is not known. In a small number of registered blind people in the U K report ing sleep dis turbance and willing to provide body fluids for the assessment of ho rmones , the majority showed free-running mela tonin with tau varying from 23.8 to 25 h o u r s .

23 O thers had a

synchronized rhy thm with a delayed phase relat ionship to the 24 hou r day. In other studies no rma l , free-running and phase delayed rhy thms have been r e p o r t e d .

22

Thus it would appear that for some individuals t ime cues other than light will main ta in synchronizat ion, albeit with the weak coupling evident from a delayed phase , whereas for others the l igh t -dark cycle is essential. Whether these differences are genetic, envi ronmenta l , related to the specific pa thology of the blindness or a combina t ion of factors remains an open quest ion. Moreover it is possible that some visually blind people still have hypotha lamic light perception via the re t ino-hypothalamic projec-t ion, and could use this information for the en t ra inment of circadian rhythms.

Social zeitgebers in dim light

Fur the r evidence for the relative impor tance of different zeitgebers for the en t ra inment of mela tonin again comes from studies in Antarct ica. O n the British base of Halley (75°S) the sun does no t rise for 3 m o n t h s in the winter and does not set for 3 m o n t h s in the summer . The m a x i m u m light intensity dur ing the winter is a r o u n d 500 lux. Sleep logs and mela tonin assessment t h roughou t the year show tha t the vast majori ty of personnel on the base of Halley remain synchronized to 24 hours at all t i m e s .

8'

24 In

view of the low intensity light in winter the predict ion was tha t rhy thms

l ight .

8 O n e hou r of light (2,500 lux) in the morn ing and in the evening for

6 weeks is sufficient to re-establish an earlier phase posi t ion comparab le to s u m m e r .

8 Pulses of 500 lux were insufficient, hence we may assume tha t in

these part icular condi t ions of dim light condi t ioning, the intensity of light experienced in the morn ing and the evening required to entra in h u m a n rhythms lies between these limits.

There is no reason to suppose tha t the intensity of light required for mela tonin suppression and the intensity of light required for en t ra inment are the same in a par t icular individual, bu t they may be related.


would desynchronize from the 24 hour day and "free-run" at their own endogenous period. In fact it is rare to observe such a p h e n o m e n o n on British bases. Rhy thms generally remain entrained at all seasons with, however, a clear delay in the t iming of sleep at weekends especially in the winter, and a delay in the t iming, and exceptionally a small increase in the dura t ion , of mela tonin in winter compared to summer , similar to changes found in temperate l a t i t u d e s .

8 , 24

In contras t , Kennaway and co l l eagues

25 have recently reported a s tudy

of four Greenpeace volunteers overwintering in Antarct ica, all of w h o m showed free-running sleep-wake and melatonin rhy thms th roughou t the period of sundown. This g roup were aware of clock t ime, main ta ined daily radio contact (a social zeitgeber) and even intermit tent personal contact with other bases, however their daily rout ine was no t otherwise s t ructured. They were free to sleep and eat as they wished, in contras t to British bases where the daily rout ine is s t ructured.

Some interesting conclusions can be d rawn compar ing these studies. Evidently in the absence of a s t rong l igh t -da rk cycle behavioral imposit ions can serve to main ta in the integrity of the circadian system. Wi thou t this stricture, daily time cues and knowledge of clock time do not suffice. The delayed, but entrained, rhy thms in winter reflect the weak na ture of the time cues. In such circumstances coupling between the pacemaker ( rhythm generat ing system) and the zeitgeber is loosened.

F o r most purposes the imposed social rout ine enforces synchronizat ion in combina t ion with the dim l igh t -dark zeitgeber and undoubted ly contr ibutes to well-being and performance. The neural and /o r biochemi-cal mechanisms whereby imposed behavior influence the circadian system remain to be determined.

Ν on-24 hour light-dark cycles

The limits of ent ra inment of mela tonin by l igh t -dark have been explored in h u m a n s kept in environmenta l isolation. Imposi t ion of a l igh t -dark cycle (domestic intensity light less than 1,000 lux) steadily increasing in length from 24 to 29 hours by 10 minutes a day leads to a phenomenon known as fractional desynchronizat ion. The sleep wake cycle remains entrained up to 29 hours but more strongly endogenous variables such as tempera ture , Cortisol and mela tonin uncouple from the zeitgeber at different imposed daylengths. Mela tonin breaks away at approximate ly 27 h o u r s

26 and free-runs with a free-running period identical to that of

temperature . If, however, bright light (3-4,000 lux) is used, all variables remain coupled up to at least 29 hours and the upper limit of en t ra inment has yet to be fully evaluated with this intensity of l i g h t .

27


Effects of me la ton in on c i rcadian rhy thms

Early work

In the late 1970s accumulated experience suggested that adminis t ra t ion of mela tonin to m a m m a l s might provide more rewarding information concerning its role in circadian rhy thms than pinealectomy. Only when the impor tance of t iming in the physiology of mela tonin was appreciated, ra ther than a pharmacological emphasis on dose, did the design of experiments p roduce coherent and interprétable results.

Two groups working simultaneously and independent ly with rats and h u m a n s , and observing the circadian effects of t imed daily mela tonin adminis t ra t ion, showed for the first t ime tha t mela tonin had entraining and phase shifting effects respectively in m a m m a l s . R e d m a n and c o w o r k e r s

31 injected mela tonin at 24 hou r intervals into free-running rats

and obta ined en t ra inment of the rest activity cycle when saline control injections were usually ineffective. Arendt and c o w o r k e r s

2 6'

3 2'

33 gave

daily mela tonin orally in the late afternoon to h u m a n volunteers and obta ined a phase advance of the endogenous melatonin rhy thm, of evening fatigue and of prolact in morn ing offset.

Timed administration of melatonin in humans

Daily feeding of low dose (2 mg) mela tonin in the late afternoon advanced the t iming of evening self ra ted fatigue, the endogenous mela tonin rhy thm and the morn ing decline of prolact in compared to placebo. The dura t ion and quali ty of sleep were not significantly altered but there was a non-significant advance of morn ing wake up t ime. The evening rise of mela tonin was advanced more than the morn ing decline.

Role of th e pineal in the cont ro l of m a m m a l i a n c ircadian rhy thms

Unti l quite recently, received opinion was that the pineal did not have a role in the m a m m a l i a n circadian system. This conclusion was essentially based on one approach to function: the lack of effect of pinealectomy on free-running rest activity cycles. However , as long ago as 1970 Q u a y

28

reported tha t when rats are subjected to forced phase shifts of the l igh t -dark cycle, pinealectomy increased the rate of reentra inment . He even suggested tha t rate of adap ta t ion to phase shift could be a diagnostic app roach to the detection of destructive lesions of the pineal in h u m a n s . Redman et al.

29 have confirmed this work and very recently Cassone and

coworkers ( 1 9 9 2 )

30 have shown that pinealectomy of hamsters in cons tant

light leads to a major disrupt ion of the circadian system.


There were no significant effects on self rated m o o d , or on L H , F S H , testosterone, Cortisol, growth h o r m o n e or T4. The effect on fatigue was not immediately apparen t bu t became significant in the g roup as a whole after 4 days. N o deleterious effects were reported by the subjects. This study, conducted twice over 4 weeks on two different occasions in spring and a u t u m n showed that in low doses mela tonin has chronobiot ic effects in h u m a n s at least with respect to sleep, its own rhy thm and p r o l a c t i n .

2 6 , 3 2'

33

It indicated tha t such a daily dose is well tolerated over 4 weeks. Impor tan t ly it showed no significant reproduct ive effects and indeed the dose is well below that needed to influence h u m a n reproduct ion as a combined contraceptive. It clearly suggested that mela tonin would be useful for phase shifting and synchronizing strategies at least for phase advances. Since this initial s tudy a substantial volume of work has served to confirm this predict ion.

Amongs t the condit ions likely to benefit from the ability to manipula te h u m a n circadian rhy thms are jet-lag, shift work , insomnia with delayed or advanced phase, blindness, old age and possibly some psychiatric condit ions.

Jet lag and shift work

Melatonin has been used in the t rea tment of jet lag in bo th real life and simulation c o n d i t i o n s .

1 7'

3 4 - 37 Field studies show that self ra ted jet lag can

be reduced on average by 5 0 % with appropr ia te ly t imed t rea tment bo th westwards and eas twards . The improvement is greater with larger numbers of t ime zones. Using advance shifts over eight t ime zones with pre-shift adminis t ra t ion of mela tonin in the late subjective day and post shift adminis t ra t ion at the desired bedt ime, the subjective impressions are reinforced by improved latency and quality of sleep, greater dayt ime alertness and more rapid resynchronizat ion of mela tonin and Cortisol rhy thms. In simulat ion studies using advanced phase shift and a similar adminis t ra t ion protocol , effects on sleep are no t significant but there is a marked increase in the rate of re-entrainment of bo th ho rmona l and electrolyte rhythms and an immediate effect of lowering body tempera ture which persists as more rapid re-entrainment .

Nei ther the dose nor the t iming of mela tonin adminis t ra t ion has been optimized for different t ime zone shifts a l though a phase response curve for mela tonin would serve to indicate preferred t rea tment times. T o date a minori ty (less than 10%) of subjects feel significantly worse after m e l a t o n i n .

35 Individual circadian status is p robably a major de terminant

of response in tha t undesirable changes in direction of ent ra inment may occur with inappropr ia te t iming of t rea tment . Moreover , unpredictable exposure to bright light can theoretically act in opposi t ion to the desired result. Bright light exposure needs to be t i trated dur ing t rea tment for jet


lag. An ambula to ry technique for ins tantaneous assessment of individual circadian phase posi t ion would be of great interest.

There is very little published work on the use of mela tonin in shift work a l though exposure to bright light dur ing the night is clearly beneficial to night shift w o r k e r s .

38 O n e study has found improved sleep and increased

dayt ime alertness in night shift workers receiving melatonin at the desired bedt ime dur ing a night shift week compared to placebo and baseline c o n d i t i o n s .

39 Here there was a suggestion that one performance task

(visual search speed) was adversely affected by mela tonin . This is a very impor tan t considerat ion if mela tonin is to be used on members of the workforce. There are no repor ted deleterious effects on performance in jet lag studies but acute studies using much higher doses (240 mg) describe a brief decline in visual reaction t ime, accompanied by an increase in fatigue ratings but with no effect on memory .

Recently, moderate ly bright light t rea tment dur ing the night has been used to force phase delays in h u m a n volunteers kept otherwise in a normal environment . Mela tonin was used in the post phase shift adap ta t ion to induce phase advances. In the course of this study no deleterious effects on performance were s e e n .

40 It will be essential to assess thoroughly

performance effects of mela tonin in the work place and on specific tasks before its use can be recommended .

Sleep disturbance in the blind

Free-running blind subjects often find this condi t ion difficult to tolerate, with intermit tent problems part icularly of sleepiness and poor perform-ance dur ing the day and poor night t ime sleep, when their pacemaker is out of phase with the environment .

Only a small number of studies have been reported, however it is clear that some blind subjects report ing sleep problems derive benefit from melatonin ingestion at desired b e d t i m e .

4 1'

23 O n e effect is a stabilization of

sleep onset, sometimes with significant improvement of some other sleep parameters such as quality and dura t ion . In some sufficiently long term experiments it is clear tha t a synchronizat ion of the sleep wake cycle has occurred under mela tonin . The successful t rea tments have included bo th free-running subjects with a period greater than 24 hours and others with abnorma l phase delay. Insufficient da ta are available to determine in which blind people melatonin is likely to be useful as yet. As melatonin may have sites of act ion within the visual system the integrity of such targets may be compromised in some blind people.

Whether synchronizat ion of sleep wake is accompanied by synchroniza-tion of other circadian variables in these circumstances is a quest ion of much interest. O n e subject with a previously free-running sleep wake cycle and whose sleep has been stabilized for a number of years showed


persistent free-run in mela tonin , Cortisol and tempera ture r h y t h m s .

42 In

spite of this his well being was restored with improvement of m o o d and dayt ime alertness and from this one case it appears tha t if the sleep problem is allayed, the behavior of other major circadian variables may be irrelevant. It also suggests tha t behavioral changes d o not necessarily entrain all of the pacemaker in the absence of any l igh t -dark cycle. There may be eno rmous individual differences however. In the work of Lewy and co l l eagues

43 and this volume, other blind subjects show clear phase shifts

and occasionally en t ra inment of the endogenous melatonin rhy thm dur ing mela tonin t rea tment .

Delayed sleep phase insomnia

Another si tuation in which phase delay forms an integral par t of a clinical problem is delayed sleep phase insomnia (DSPI) . Pat ients cannot sleep at the socially acceptable t ime of night and delay sleep onset until the early hours of the morn ing , sleeping th rough much of the day. This condi t ion is endemic in teenagers and in the s tudent popula t ion where it is usually self limiting. The obligation to earn a living provides sufficient mot ivat ion to overcome the problem. In true D S P I subjects the aber ran t phase posit ion cannot be overcome with consequent inability to work and socialize at usual times. This condi t ion has been successfully t reated with bright light in the early morn ing to induce phase advances of the clock. In others evening melatonin (5 mg) will also advance sleep time signif icant ly.

44

A PRC for melatonin

Lewy and coworkers (45 and this volume) have described a P R C for mela tonin in people. They repor t phase advances in the late subjective day and small phase delays dur ing the early morn ing . The da t a are approximate ly 12 hours out of phase with the P R C to light and are consistent with the long held view tha t mela tonin can act like darkness bo th in circadian and seasonal rhy thms.

Melatonin as an endogenous time cue

The physiological role of mela tonin in circadian organizat ion in h u m a n s can be considered as follows. Wi th a free-running period (tau) greater than 24 hours the phase advancing effects of morn ing light and the phase advancing effects of evening mela tonin may combine with other zeitgebers as yet incompletely defined to main ta in 24 hour synchrony. The reverse si tuat ion can evidently be postula ted for the rare individual with t au less than 24 hours . With this in mind it would obviously be advan tageous to combine controlled bright light (artificial or na tura l ) and mela tonin


t rea tments in the management of rhy thm disorder . Prel iminary work indicates tha t this app roach will yield a rich harvest .

If the mainta ined night t ime levels of mela tonin are impor tan t for sleep maintenance then a slow release prepara t ion given in the evening should prove of greater benefit to sleep dura t ion than a rapidly metabolized dose. G o o d slow release prepara t ions of mela tonin for h u m a n use have only been developed recently and there is a lack of information in this area.

The interest in mela tonin as an endogenous thermoregula tory factor has been reinforced by studies designed to uncover the acute effects of mela tonin and light in h u m a n s . There is an inverse relat ionship between acute suppression of melatonin and acute elevation of body tempera ture by bright l i g h t .

46 These acute and opposi te effects of bright light and

melatonin may represent the pr imary manifestation of a phase shift in the making , in terms of brain metabol ic events.

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2. M o o r e - E d e M . C . , Czeisler C A . a n d R i c h a r d s o n G . S . (1983a) C i r cad i an t imekeep ing in hea l th a n d disease. P a r t 1. Basic p roper t i e s of c i rcad ian p a c e m a k e r s . N. Eng. J. Med. 3 0 9 , 4 6 9 ^ 7 6 .

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26. A r e n d t J., Bojkowski C , F o l k a r d S., F r a n e y C , M i n o r s D .S . , W a t e r h o u s e J . M . , Wever R.A., W i l d g r u b e r C . a n d W r i g h t J. (1985) Some effects of me la ton in a n d the con t ro l of its secret ion in m a n . In Ciba Foundation Symposium 117. Photoperiodism, Melatonin and the Pineal (eds. Evered D . a n d C l a r k S.), p p . 2 6 6 - 2 8 3 . L o n d o n , P i t m a n .

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29. R e d m a n J.R., A r m s t r o n g S. a n d N g K . T . (1983) P inea l ec tomy: increased sensory inpu t or r educed h o m e o s t a t i c m e c h a n i s m ? Proc. Int. Union Physiol. Sci. 15 , 4 1 .

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3 1 . R e d m a n J., A r m s t r o n g S. a n d N g K . T . (1983) F r e e - r u n n i n g activity r h y t h m s in the ra t : e n t r a i n m e n t by me la ton in . Science 2 1 9 , 1089-1091 .

32. A r e n d t J., Borbely Α.Α., F r a n e y C . a n d Wr igh t J. (1984) T h e effect of ch ron ic , small doses of m e l a t o n i n given in the late a f te rnoon on fatigue in m a n : a p re l iminary s tudy . Neurosci. Lett. 4 5 , 3 1 7 - 3 2 1 .

33. W r i g h t J., A l d h o u s M . , F r a n e y C , Engl ish J. a n d A r e n d t J. (1986) T h e effects of exogenous m e l a t o n i n on endoc r ine funct ion in m a n . Clin. Endocrinol. 24, 375-382 .

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35. Skene D.J . , A l d h o u s M . a n d A r e n d t J. (1989) M e l a t o n i n , je t - lag a n d the s leep-wake cycle. In Proceedings of the European Sleep Congress (eds. H o m e J. a n d K a r g e r S.), Je rusa lem.

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37. Nickelsen T. , L a n g A. a n d Bergau L. (1991) T h e effect of 6-, 9- a n d 11-hour t ime shifts on c i rcad ian r h y t h m s : a d a p t a t i o n of sleep p a r a m e t e r s a n d h o r m o n a l p a t t e r n s following the in t ake of m e l a t o n i n o r p l acebo . In Advance Pineal Research (eds. A r e n d t J. a n d Pevet P . ) , Vol . 5, p p . 303-306 .

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(1990) E x p o s u r e to br ight light a n d d a r k n e s s to t rea t phys io logic m a l a d a p t a t i o n to n igh t -work . N. Engl. J. Med. 3 2 2 , 1253-1259.

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4 1 . A r e n d t J., A l d h o u s M . a n d W r i g h t J. (1988) Synch ron i sa t i on of a d i s tu rbed s leep-wake cycle in a bl ind m a n by me la ton in t r e a t m e n t . Lancet 1 , 7 7 2 - 7 7 3 .

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15

Understanding the Use of Light to Control the Circadian Pacemaker in Humans R I C H A R D E. K R O N A U E R a n d C H A R L E S A . C Z E I S L E R

Harvard University, Division of Applied Sciences, Cambridge, MA 02138 and Laboratory for Circadian and Sleep Disorders Medicine, Harvard Medical School, Β rig ham and Women's Hospital, Boston, MA 02115 USA

Abst rac t

Bright light can m a k e large changes in the a m p l i t u d e of c i rcad ian r h y t h m s as well as shift their phase . W h e n a m p l i t u d e is m u c h reduced , the p h a s e shifting abil i ty of light is great ly e n h a n c e d , in a m a n n e r which is m o s t readily c o m p r e h e n d e d by m e a n s of vec tor d i a g r a m s . T o o b t a i n large p h a s e shifts, l ight is used m o s t effectively w h e n the s t imulus reduces a m p l i t u d e a t the beg inn ing of the s t imulus ep i sode . S t imulus s t rategies which e m b o d y s t rong (Type 0) reset t ing can be very powerful , b u t to w o r k they requ i re careful adhe rence to a regimen of ex tended light exposures . A comple t e invers ion of r h y t h m p h a s e (a 12 h o u r phase shift) is possible wi th 10,000 lux episodes of 8 h o u r s d u r a t i o n o r of 6 h o u r s d u r a t i o n o n two successive days . Briefer exposures p r o d u c i n g a succession of small shifts (cumula t ive type 1) can achieve the s a m e result bu t m a y requi re several m o r e days .

Quant i fy ing pacemaker response t o l ight

O N E OF the mos t revealing demons t ra t ions of the act ion of light on the suprachiasmat ic nuclei (SCN pacemaker) was given by Inouye and K a w a m u r a .

8 Electrodes measured the circadian rhy thm of mass neurona l

discharge before a n d after 3 hours of light appl icat ion to a rat 's ret inae (Figure 1). The circadian pa t te rn of discharge is nearly sinusoidal , maximal by day and minimal by night (corresponding to metabol ic activity for all m a m m a l i a n species, whether the animal is noc turna l or d i u r n a l

2 0'

2 1) . F o r reasons which we will develop, it is mos t useful to use the

nadir of neuronal activity as a t iming reference for stimuli. Al though light was applied for 3 hours it is clear tha t the effect on the rhy thm was essentially complete in the first few minutes . This in terpreta t ion agrees with the da t a of Nelson and T a k a h a s h i

17 who showed tha t extending light

exposure to hamsters beyond abou t 5 minutes p roduced very little increase

2 1 7


ST IMULUS O N S E T

SHIFTED

MINIMUM

r MINIMUM MIN IMUM

P R O J E C T E D

MINIMUM

SHIFTED MINIMUM

F I G . 1. M a s s n e u r o n a l d i scharge in the sup rach i a sma t i c nuc leus of a ra t . A 3 h o u r light s t imulus was appl ied t o the re t ina beg inn ing 3 h o u r s before the projec ted m i n i m u m n e u r o n a l act ivi ty. In response to the s t imulus the m i n i m u m was delay-

shifted by 2 h o u r s a n d the r h y t h m a m p l i t u d e was reduced by 2 0 % .

in phase shifting. Such t ime-saturat ion of circadian responsiveness may be a protective mechanism for systems with high sensitivity (for example, 5 minutes of 250 lux light can shift the hamster rhy thm by 2 hours) . O the r less sensitive m a m m a l s such as the Polynesian r a t

6 and h u m a n s appear to

respond in a cont inuous , integrat ing way to t ime-extended stimuli. As Figure 1 shows, the effective st imulus occurred abou t 3 hours before the nadir of neuronal activity. The light produced two changes of significance: The rhy thm was phase-delayed by abou t 2 hours (30 phase degrees or 0.5 radian) and reduced in ampl i tude by abou t 2 0 % .

It is tempt ing to think that an assay of S C N electrical activity reveals directly the circadian pacemaker function. However , since the blocking of neuronal sodium channels with te t rodatoxin silences the electrical activity wi thout eliminating the rhy thm itself,

22 the neuronal firing must be

regarded as " h a n d s " of the pacemaker clock, indicative of underlying chemical rhy thms in a manne r yet to be quantified. It is reasonable to anticipate a simple relat ionship between these " h a n d s " and the chemical cycle itself. O the r commonly used h u m a n markers of pacemaker function, such as the endogenous components of core body tempera ture , p lasma Cortisol and plasma melatonin may have more complex relationships to the pacemaker .

In apprais ing S C N electrical activity we have repeatedly referred to rhy thm ampli tude in order to emphasize an impor tan t feature of circadian function which is too often ignored. The direct physiological significance of ampl i tude has yet to be worked out . Some writers have linked reduced ampl i tude to h u m a n d e p r e s s i o n

5'

24 or to a reduced circadian modula t ion

of a ler tness .

9 In insects there seems to be a connect ion between ampl i tude

and geographic l a t i t u d e .

19 Regardless of how impor tan t , physiologically,

the ampl i tude of pacemaker rhy thm is found to be, it indisputably plays a crucial role in our ability to manipula te effectively the phase of that rhy thm, as we shall e laborate below. In part icular , s t rong phase resetting of the type which W i n f r e e

28 called "Type 0", requires a stimulus capable of

reducing ampl i tude to zero!

Use of Light to Control the Human Circadian Pacemaker 2 1 9

R h y t h m assessment

The example of Figure 1 presents a pro tocol pa rad igm for all experiments which seek to quantify the effect of light on the pacemaker . The s t ructure of that experiment entails first an assessment of the state of the pacemaker (its rhythmic ampl i tude and phase) , next a quantified st imulus intervention (specified s trength and dura t ion of light) at a chosen phase of the endogenous rhy thm, and finally an assessment of the resulting state of the pacemaker . A full rhy thm cycle is needed to appraise each state.

In h u m a n experiments , assessment of initial and final rhy thm states is effected th rough subsidiary variables (e.g. t empera ture , Cortisol, mela to-nin). Tempera tu re has been most widely adop ted due to the ease with which it can be measured. However , the endogenous circadian tempera-ture pa t te rn is ordinari ly "masked" by comparab ly large tempera ture swings evoked by pos ture change, activity, sleep, showering, etc. The endogenous rhy thm is best "unmasked" by the Cons tan t Rout ine (CR) p r o c e d u r e

2 which requires moni tor ing and control of the subject with a

strictness usually achieved only in the labora tory . Repeated C R performed on control subjects mainta ined under low light levels (less than 20 lux) show ampl i tude variat ions in endogenous tempera ture of abou t 2 7 % (SD). These can be decomposed into intersubject (intrinsic) variability of abou t 2 0 % and intrasubject variability (i.e. "noise" in the C R assessment) of abou t 1 8 % . The "noise" produces errors in circadian phase assessment of abou t ± 0.9 hou r (SD). Compar i son between subjects is improved when ampli tudes are normal ized relative to each subject's intrinsic value.

Any physiological variable which can be sampled frequently (e.g. hourly) over an extended time has the potent ial to give da t a on bo th ampl i tude and phase of a circadian rhy thm. Blood p lasma Cortisol has been used to cor robora te pacemaker phase shifts inferred from tempera-ture d a t a

4 and as an alternative measure of ampl i tude in experiments

designed to reduce greatly the pacemaker a m p l i t u d e .

10 Lewy and

co-workers have used p lasma mela tonin as a phase marke r via the D i m Light Mela ton in Onset ( D L M O ) p r o c e d u r e .

15 Since Shanahan and

Cze i s l e r

23 have shown that the t iming of the peak of noc turna l mela tonin

in any subject is closely related to bo th onset and offset t iming, it is possible that mela tonin can also be used as a marke r of rhy thm ampl i tude . Hopefully, salivary or ur inary measures of Cortisol and mela tonin may be able to replace invasive b lood samples, which would greatly facilitate the use of these ho rmones as circadian markers .

Mask ing effects on ho rmona l rhy thms have been studied very little, beyond a very s t rong, acute effect of light on mela tonin p r o d u c t i o n .

1 4 , 16

Of course, b lood sampling under C R condi t ions automat ical ly controls for possible masking effects due to those variables which are held cons tant dur ing the CR.


Report ing reset t ing results

Rodent studies have been greatly simplified by the existence of a sharply defined activity onset (wheel running or drinking) which provides an easily moni to red marke r of circadian phase . Consequent ly , the more difficult task of assessing rhy thm ampl i tude in animals has largely been ignored. Unfortunately, the part ial information offered by phase alone has significantly nar rowed the perspective of much research on animal rhy thms.

When one ignores the ampl i tude of the pacemaker rhy thm, only the t iming of the cycle remains to be considered. The effect of a brief light st imulus can be described by the magni tude and direction of the phase shift which it produces , typically plot ted as a function of the phase of the cycle at which it is applied. This is the Phase Resetting Curve (PRC) in t roduced by Hast ings and Sweeney.

7 The experiment shown in Figure 1 generates one

point of a P R C . Taking the min imum of neural activity as the phase reference (phase = 0), the effective stimulus in Figure 1 was applied 21 hours after the min imum. We denote this as the initial phase , 0 i n i t, at which the st imulus was applied, with respect to the unshifted rhy thm ((/>in it = 21). The phase shift p roduced was a 2 hour delay (or —2 hours according to convent ion) . "Final phase" , 0 f i n a l, is defined as the phase at which the st imulus occurs relative to the shifted rhy thm. In this experiment, </>f i n a l= 19 hours . Phase shift is typically designated as Αφ; clearly Αφ = φ η η Ά ΐ- φ ϊ η ί ί.

When we consider rhy thm ampl i tude (designated A), bo th the experiments and their repor t ing become far more complex. We mus t select initial ampl i tude, A i n i t, as well as the stimulus timing, (/>i n i t, and we must measure A f i n al as well as 0 f i n a l. Two functions:

^final=/l(^init' init) Φϊ\η2λ

=Ϊΐ (Ainit' init)

are needed to report the results. We have found tha t a graphical presentat ion helps greatly in organizing and interpret ing them.

Consider the polar d iagram of Figure 2. The endogenous rhy thm ampl i tude* is represented by the radius from the center (designated 0), while the circadian phase (at which a stimulus is applied) is represented by the angle measured clockwise in hours from the indicated zero phase. Once a round the circle is 24 hours (i.e. 1 hour = 15°). (We are ignoring here the approximately 1-3% difference between intrinsic rhy thm period and 24 hours.) As suggested earlier, we will normalize rhy thm amplitude for each animal or h u m a n subject to the value seen under reference condi t ions. There is a long t radi t ion in rodent research to consider the free-run

T h e endogenous rhythm exists within the S C N neurons, presumably in the form of intracellular chemical oscillations.

Use of Light to Control the Human Circadian Pacemaker 221

condit ion in darkness or very dim light as the reference state. The rat repor ted in Figure 1 was in the reference state before the light s t imulus. Consequent ly , A i n it = 1.

The experiment of Figure 1 is represented by the initial and final vectors indicated in Figure 2. The initial phase is 21 hours (or —3 hours) , describing the t ime in the cycle at which the st imulus is applied. The final state vector shows the reduced ampl i tude of 0.8 and a phase of 19 hours , which describes the t ime in the newly-altered rhy thm at which the st imulus was applied. The vector d rawn from the initial state to the final state describes the change produced by the st imulus (a delay shift, —2 hours) .

+-±12

A Initial = 1.0 φ Initial = - 3hr .

F I G . 2. Schemat i c r ep resen ta t ion of the d a t a of F igu re 1. T h e filled circle represents the init ial c i rcad ian a m p l i t u d e a n d init ial c i rcad ian p h a s e a t which the s t imulus occurs . T h e o p e n circle represents the final ( reduced) c i rcad ian a m p l i t u d e a n d the final (earlier) c i rcad ian p h a s e a t which the s t imulus occu r red . Since the e n d o g e n o u s r h y t h m was delay-shifted 2 h o u r s by the s t imulus , the t iming

of the s t imulus was advanced 2 h o u r s in relat ive c i rcad ian p h a s e .

H u m a n reset t ing results

In what follows we shall describe an idealized or schematic picture of the effect of br ight light (i.e. 10,000 lux) on the h u m a n pacemaker . The representat ion is intended to be realistic and corresponds quantifiably to the results of several key experiments , which will be cited where relevant.


At the same time the schematic picture contains many details which must be extrapolated from the limited available da t a and which will require many more experiments either to be cor robora ted or modified. T o begin, we must observe that , for humans , bright light dura t ions of several hours are needed to produce measurable phase and ampl i tude changes. T o compare stimuli of different dura t ions , experiments have shown it useful to use the midpoint of the light episode for the phase reference, as did G a n d e r and Lewis .

6 By so doing, critical points of the response (i.e. st imulus

phases at which the direction of the phase shift, or the sign of Αφ, changes) are very nearly independent of st imulus dura t ion .

Since h u m a n subjects do not tolerate prot rac ted darkness , the reference condi t ion must be modified from the convent ional one for noctural rodents . We have therefore lowered ambient light exposure to the range of 10-20 lux to reduce its effect on the h u m a n pacemaker . F o r experimental convenience we find it useful to impose a 24 hour sleep/wake pat tern . This is close to free-run periods and insures bo th regular sleep dura t ion and internal synchrony. Together these const i tute our reference condi t ion.

Figure 3 shows the est imated response of a h u m a n subject to a single 5 hour episode of 10,000 lux. We assume that the subject is a typical young male placed in the reference condi t ion by being entrained to a s tandard labora tory protocol in which sleep is scheduled from 24.00 to 08.00 and waking hours are spent in a r o o m of abou t 20 lux. Given these condi t ions, the min imum of the endogenous tempera ture rhy thm would be at abou t 05.30 and A i n it = l . F o r the st imulus depicted in Figure 3 the subject is awake dur ing at least a par t of his normal sleep t ime, and bright light (10,000 lux) is viewed from 04.00 to 09.00 (centered at 06.30). Thus , φιηη=1.0 hours . The final state is shown as A f i n al = 0.56 and $ f i n ai = 2.4 hours .

The 5 hours of bright light has advanced the pacemaker rhy thm by 1.4 hours , since the t iming of the center of the light episode (06.30) now occurs 2.4 hours after t empera ture min imum (i.e. the next min imum will now be found at 04.06, which is 1.4 hours earlier than 05.30, where it would have occurred wi thout the light st imulus). The pacemaker responds to the stimulus progressively dur ing the 5 hours of light applicat ion (from 04.00 to 09.00). The stimulus interval actually encompasses the t iming of the min imum in bo th the "initial s ta te" (05.30) and "final s ta te" (04.06). Consequent ly , an experimental appraisal of the change would require compar ison of the "initial" min imum (located approximately 24 hours before the stimulus) and the "final" min imum (located approximately 24 hours after the st imulus). The vector describing the change from the initial to the final state in Figure 3 shows that the reduct ion of ampl i tude (to abou t half its reference value) is a s tronger effect of light than is the phase change.

Figure 4 shows schematically the effect of 5 hours of 10,000 lux when


18 (-6)

F I G . 3 . Schemat ic r ep resen ta t ion of the effect of a single 5 h o u r ep isode of b r igh t light o n the h u m a n c i rcad ian p a c e m a k e r . T h e filled circle represents the init ial c i rcad ian a m p l i t u d e a n d initial c i rcad ian p h a s e (referenced to the p res t imulus r h y t h m ) a t which the m i d - p o i n t of the s t imulus occurs . If the reference c i rcad ian p h a s e ( endogenous t e m p e r a t u r e m i n i m u m ) were a t 0 5 . 3 0 , as chosen , app l i ca t ion of br igh t light from 0 4 . 0 0 to 0 9 . 0 0 (centered a t 0 6 . 3 0 ) c o r r e s p o n d s to the chosen ^ i n i t i a i

= 1 h o u r . A 5 h o u r ep isode t imed in this way is p r e s u m e d to p r o d u c e a

1.4 h o u r p h a s e a d v a n c e of the e n d o g e n o u s r h y t h m a n d a r educ t ion of a m p l i t u d e to 0 . 5 6 . Relat ive to this shifted r h y t h m the s t imulus is n o w centered 2 . 4 h o u r s after the (new) cycle reference p h a s e . C o n s e q u e n t l y , # f i n al is 2 . 4 a n d Af i n a, is 0 . 5 6 . These

a re represen ted by the o p e n circle.

applied at all phases of the pacemaker cycle. The filled circles represent the initial states. They are all on the unit circle, indicating tha t the subject was in the reference condi t ion for each hypothet ical experiment, and they are located at the integer phase points . The corresponding final states are indicated by open circles and identified by the value of the corresponding initial phase . Mos t of the vector changes have been d rawn in. The dashed line connect ing the final states is the resetting contour, showing all possible final states which can be reached from the reference condi t ion ( A i n it = 1) by using the 5 hou r st imulus. The resetting con tour shown is symmetrical abou t the hor izonta l axis, indicating that advancing phase shifts and delaying shifts are equally a t ta inable . When the 5 hour bright episode is centered over the tempera ture min imum ((/>in it = 0) the phase shift is zero so ^ f i n a i = 0· However , the ampl i tude is reduced to 0.4, which is consistent with related h u m a n da t a as we shall describe below. There is an impor tan t

6 (H8)


implication for animal experiments in which only phase shifting is measured: zero phase shift may very well correspond to s t rong ampl i tude reduction.

Figure 4 shows ampl i tude to be somewhat enhanced for φ ί η ίί between 7 and —7, typically abou t 2 0 % . Whether light does enhance ampl i tude dur ing these hours (between abou t 12.00 and 22.00 for a typical subject entrained to the normal daily rout ine) must be regarded as conjectural at this t ime; the requisite experiments have yet to be performed. In fact, identifying changes in ampl i tude of this size is very difficult, since the s tandard deviation in the measured ampli tude of the endogenous tempera ture rhy thm (in the CR) is a lmost as large as the hypothesized change.

An interesting feature of Figure 4 is that the principal effect of stimuli t imed close to the reference phase (O) is to generate vector changes directed r ightward ( towards + 12). When timed exactly at reference phase , no change of phase is produced . Rightward vector changes correspond to delay shifts when they occur before reference phase and advance shifts when they occur after. T a k a h a s h i

26 has shown tha t the expression of F O S

and J U N B in the S C N of hamsters is induced by light dur ing the animal 's subjective night at very much the same levels whether the light occurs in the delay por t ion of the P R C or the advance por t ion . If we d raw an analogy between the r ightward displacement produced by light in Figure 4 and the expression of F O S or J U N B we find a possible explanat ion for how a single chemical drive would be interpreted as either a delay shift or an advance shift depending on other chemical processes in the oscillatory system.

Because of the cumulat ive effects of light, we can approximate the resetting produced by bright episodes shorter than 5 hours simply by scaling down the vector change propor t ional ly to st imulus dura t ion . F o r example, a 2 hour episode will p roduce abou t 4 0 % as much change, so the vector length would be reduced by abou t 6 0 % . However , the same principle cannot be applied to episodes much longer than 5 hours . The reason can be simply deduced from Figure 4. If we proceed a round the circadian cycle, we find that for abou t half of the cycle the rhy thm ampl i tude is reduced by light while for the o ther half-cycle it is increased. Similarly, phase is advanced for half of the cycle while for the opposi te half it is delayed. Any desired effect (e.g. phase advance) is limited to a "window of oppor tun i ty" and furthermore, near the edges of tha t window the effect is small. Thus , 12 hours of st imulus can, at best, p roduce little more effect than 9 hours , while 9 hours may produce only abou t 4 0 % more than 5 hours . A stimulus which lasts more than 12 hours can only p roduce less effect than the best 12 hours episode. The essential fact is tha t there are inescapable limits to the effects which 10,000 lux can produce within any circadian cycle. T o produce stronger changes it is necessary to wait until

Use of Light to Vontrol the Human Circadian Pacemaker 225

Reference

Phase

F I G . 4. T h e schemat ic r ep resen ta t ion of the response of the h u m a n c i rcad ian system to a single 5 h o u r ep isode of b r igh t light centered at all different phases of the e n d o g e n o u s r h y t h m . C o n v e n t i o n s a re the s a m e as in F igu re 3. Fil led circles represent the init ial c i rcad ian a m p l i t u d e (always the reference va lue of 1) a n d initial c i rcad ian p h a s e at which the mid -po in t of the s t imulus occurs . O p e n circles represent the final c i rcad ian a m p l i t u d e a n d phase a t which the s t imulus occurs . T h e n u m b e r s by the open circles identify the c o r r e s p o n d i n g initial p h a s e . T h e closed-circle a n d open-circ le pa i r identified as " 1 " show the effect examined in detai l in F i g u r e 3. T h e dashed line d r a w n t h r o u g h the open circles is the reset t ing c o n t o u r which summar i ze s the effects of the 5 h o u r b r igh t light s t imulus for any phase of app l i ca t ion . A clockwise d i sp lacemen t from a filled circle t o the c o r r e s p o n d i n g o p e n circle indicates t h a t the s t imulus has advanced the e n d o g e n o u s r h y t h m , while counterclockwise d i sp lacement indica tes delay.

A d v a n c e a n d delay shifts a re symmet r ica l in this cons t ruc t i on .

the desired "window of oppor tun i ty" comes back again on the next cycle and applies further st imulus there.

Repet i t ive s t imul i

Once we admit the opt ion of applying light on successive circadian cycles, the possible combina t ions are innumerable . F r o m the s tandpoint of experimental pro tocol it is clear that unless we are prepared to re-assay the system after the first light episode, the second episode can only be t imed relative to the pacemaker assay which preceded the first episode. In such a case, by far the simplest pro tocol is to t ime the second, third and subsequent episodes with the same relation to the original assay as the first


episode, i.e. all episodes at the same clock hour . Wi th the same objective of simplifying the protocol , all episodes should embody the same strength of light and have the same dura t ion . Five hours is a convenient dura t ion since it is readily tolerated by subjects and avoids the "diminishing re turns" of much longer episodes. These then const i tute the protocol s tandards which we will use as the basis for our further schematic representat ions.

In the 24 hours between the centers of the stimulus episodes, the pacemaker is presumed to "run free" and express its intrinsic drives. The most significant drive is that to re turn rhy thm ampl i tude to its reference value, A = 1. The simplest mathemat ica l representat ion of a self-sustaining rhy thm generator is the van der Pol oscillator, which we have used to represent the pacemaker on various o c c a s i o n s .

1 2 , 13 Only a single

non-dimensional parameter , the "stiffness" of the oscillator, is needed to characterize the ampl i tude recovery process. We have est imated this stiffness by compar ing the dura t ion of a single 10,000 lux episode which will essentially reduce A to zero (an 8 hou r episode) with the dura t ion of two equal episodes required to p roduce the same reduct ion of A (two 5.5 hour e p i s o d e s ) .

10 W h e n two episodes are used (spaced 24 hours apar t )

there is one full cycle of ampl i tude recovery intervening. The resulting est imate for stiffness is 0 . 1 3 .

12 Using this value of stiffness, it is a

s traightforward mat te r to calculate for the van der Pol oscillator the spontaneous change of ampl i tude in one cycle, ΔΑ, as a function of the ampl i tude at the start of the cycle, A i n i t. These are shown in Figure 5. When A i n it is less than 1 the ampl i tude increases (ΔΑ is positive). Ampl i tude decreases when A i n it is greater than 1. Spontaneous increases as large as 0.18 can occur in one cycle.

F I G . 5. T h e e n d o g e n o u s recovery of r h y t h m a m p l i t u d e t o w a r d its n o m i n a l value of 1. T h e change of a m p l i t u d e in a single cycle is s h o w n as a funct ion of the a m p l i t u d e at the s ta r t of the cycle. M a x i m u m a m p l i t u d e g r o w t h is 1 8 % of n o m i n a l for a n initial a m p l i t u d e of a b o u t 0.6. F o r init ial ampl i t udes larger t h a n 1, a m p l i t u d e

The second intrinsic drive of the pacemaker is embodied in its intrinsic period, τ, which is typically longer than 24 hours . In our report of the phase resetting potential of bright l ight ,

4 the da ta suggest that the pacemaker

period is longer than 24 hours by ~ 0 . 2 hour (which is considerably closer

< 50% growth per cycle Deviation of A i n it / from 1 decays 56%

1.2

<~> -0.2 L

decreases in each cycle.


to 24 hours than previous e s t i m a t e s

2 7) . Consequent ly , in the 24 hour

interval between stimuli the pacemaker will drift in a delaying sense by 0.2 hours typically. Figure 6 shows schematically the combina t ion of effects to be expected in a 2-cycle protocol where st imulus episodes are centered 24 hours apar t . Between the "final s ta te" for the first episode (open circle) and the "initial s ta te" for the second episode (filled square) there is an ampl i tude recovery and a delay phase shift of the rhy thm. Then the second episode is applied, inducing the second light effect.

6 (-18)

\ " ±

^ I

λ

18 (-6)

F I G . 6. A schemat ic r ep resen ta t ion of the e n d o g e n o u s c i rcad ian effects o p e r a t i n g be tween successive br igh t ep isodes . ( C o n v e n t i o n s a re the s a m e as in F i g u r e 3.) These inc lude a m p l i t u d e recovery a n d a p h a s e delay shift d u e to an e n d o g e n o u s pe r iod , τ , which is larger t h a n 24 h o u r s . T h e " a p p a r e n t " effect of a second ep isode

is a l tered by these processes .

Figure 7 shows the schematic resetting con tour for single 5 hour episodes of 10,000 lux (open circles, taken from Figure 4) a long with the schematic resetting con tour for two repeated episodes (open squares) . The most significant feature is the extremely low ampl i tude produced when these episodes are t imed to occur precisely over the tempera ture min imum. Related to this ampl i tude reduct ion is an extreme sensitivity of phase shift to the t iming of the stimuli. If the two episodes are centered only 30 minutes away from the tempera ture min imum the phase shift will be more than 3 hours of advance or delay, depending on the sense in which the episodes are offset. The delay shifts due to τ are quite small by compar i son , but they do make the resetting con tour slightly asymmetr ic .


+•+12

F I G . 7. A schemat ic r ep resen ta t ion of the " a p p a r e n t " a m p l i t u d e a n d p h a s e changes p r o d u c e d by a second 5 h o u r ep isode of b r igh t l ight , appl ied a t t he s a m e clock t ime as the first ep isode b u t 24 h o u r s later . As in F igures 3 a n d 4, the o p e n circles represent the e n d o g e n o u s a m p l i t u d e a n d the c i rcad ian p h a s e of the m i d p o i n t of the b r igh t l ight after t he first ep isode . T h e o p e n squa re s represen t the e n d o g e n o u s a m p l i t u d e a n d c i rcad ian p h a s e of the m i d p o i n t after the second ep isode . T h e n u m b e r s indica te the c i rcad ian p h a s e of the m i d p o i n t relat ive to the init ial (pres t imulus) r h y t h m s h o w n in F igu re 4 as filled circles. T h e open-ci rc le a n d o p e n - s q u a r e sequence labeled by the n u m b e r " 1 " c o r r e s p o n d s to the c o m b i n a t i o n of effects d i a g r a m m e d in F igu re 6. All " a p p a r e n t " changes represen ted he re c o m b i n e a m p l i t u d e recovery a n d p h a s e delay (due t o

e n d o g e n o u s τ) a long wi th the direct effect of the second br igh t light ep isode .

By waiting for the "window of oppor tun i ty" to reappear again, a third episode can be applied. Dur ing the intervening cycle ano ther recovery of ampl i tude will occur and ano ther small phase shift due to the intrinsic period, τ. Figure 8 shows the effective transi t ion for the resetting con tour from its configuration for two 5 hour episodes found in Figure 7 (open squares) to the configuration for three successive 5 hour episodes (open triangles). This t ransi t ion is very special. Whereas the resetting contours for one and two episodes each encircled the "origin" of the polar d iagram (the "phaseless" point , A = 0), for three episodes the origin lies outside the contour . The significance of this distinction is best unders tood by compar ing jus t the phase aspect of the resetting process as shown in the phase resetting curves of Figure 9.

The abscissa in Figure 9 represents the 24 hour range of initial phases (at


F I G . 8. A schemat ic r ep resen ta t ion of the " a p p a r e n t " a m p l i t u d e a n d p h a s e changes p r o d u c e d by a th i rd 5 h o u r ep isode of b r igh t light appl ied at the s ame clock t ime as the first a n d second episodes bu t o n the th i rd c i rcad ian cycle. T h e o p e n squares a re pos i t ioned as in F igu re 7. T h e o p e n t r iangles represent the e n d o g e n o u s a m p l i t u d e a n d c i rcad ian p h a s e of the ep isode m i d p o i n t after the th i rd ep i sode . As in F igu res 4 a n d 7 the n u m b e r s beside o p e n - s q u a r e a n d open- t r i ang le sequences ind ica te the c i rcad ian phase of the ep i sode m i d p o i n t relat ive to the initial (pres t imulus) r h y t h m . (To avo id c lut ter m a n y n u m b e r s a n d c h a n g e vectors have been omi t t ed . ) F o r n u m b e r 11 we observe tha t the " a p p a r e n t " c h a n g e has n o net phase c o m p o n e n t . Th i s is because the phase a d v a n c e p r o d u c e d by the br ight light exactly m a t c h e s the p h a s e delay d u e to e n d o g e n o u s τ. W e also observe t h a t the d a s h e d line j o in ing the o p e n t r iangles (the reset t ing c o n t o u r for the ent i re 3-ep isode s t imulus) does not encircle the or igin (which represents ze ro -ampl i t ude of the e n d o g e n o u s r h y t h m ) . T h e solid line jo in ing the o p e n squa res (the reset t ing c o n t o u r for a s t imulus cons is t ing of only t w o br ight light episodes) does enclose

the or igin . This t rans i t ion t hus represents T y p e 0 reset t ing.

which the first s t imulus episode is applied) while the ordina te represents the final phase , after one stimulus episode, or two episodes or three episodes as the case may be. F o r either one episode or two episodes, for which the resetting contours in Figure 6 or Figure 7 enclose the origin, the final phase advances exactly one full cycle when the initial phase advances one cycle. Tha t is, the average slope of phase resetting is unity. It is for this reason tha t W i n f r e e

28 called the process "Type 1 resetting". In contras t , for

three episodes, where the resetting con tour in Figure 8 does no t enclose the origin, all of the (/>f i n al lie in a band of abou t 12 hours width and there is no overall advance of final phase : the average slope of phase resetting is zero.


Phase resetting for 5hr. bright light episodes

φ initial

F I G . 9. A s u m m a r y of the p h a s e re la t ionships p r o d u c e d by st imuli consis t ing of one episode of br ight l ight , two episodes o r three episodes , t a k e n from Figures 4 , 7 a n d 8. T h e abscissae c o r r e s p o n d to the c i rcad ian p h a s e of the m i d p o i n t of the 5 h o u r episodes relat ive to the p res t imulus c i rcad ian r h y t h m . Second a n d th i rd episodes a re given a t the same clock t ime as first ep isodes . T h e o rd ina te s c o r r e s p o n d to the c i rcadian p h a s e of these episodes after the des igna ted n u m b e r of episodes have been given. T h e response curves c o r r e s p o n d i n g to one a n d t w o episodes a re " T y p e 1" since </>f i n al increases by one cycle for a n increase in </>init of one cycle. T h r e e ep isodes p r o d u c e " T y p e 0" response since there is zero overal l increase in 0 f i n al for a n increase in </>init of one cycle. All response curves intersect a t 0 i n it = 1 1 . This special cond i t i on , des igna ted φϊηϊί, is where φ{inal = </>init + δ (where δ is j u s t the a d v a n c e requ i red to offset t he delay p r o d u c e d by e n d o g e n o u s τ in each cycle). Th is is the s table e n t r a i n m e n t cond i t i on for which 0 f i n al = ^

i n i t i a l. T h e o the r

special ^ cond i t i on

des igna ted </>init c o r r e s p o n d s to a t iming of the 3-episode s t imulus which will b r ing the c i rcad ian r h y t h m to s table e n t r a i n m e n t in precisely those three episodes (by

induc ing a p h a s e shift of a b o u t 11 hou r s ) .

Winfree called this "Type 0 resetting". In Figure 9, if we look at the range 2 < 0 i n i t< 2 2 we find the resetting curves for one, two and three episodes progress in an orderly sequence of stronger phase shifts, a l though three episodes do no t quite p roduce three times the shift of one episode. However , if we look at φ close to zero (or close to 24, which is the same thing) we see tha t by progressing from two to three episodes the slope of the resetting curve changes from steeply positive (about + 1 0 ) to steeply negative (about —10). It is for these 0 i n it tha t Type 0 resetting is essentially


different from Type 1 resetting. If we consider the st imulus to be centered very soon after the reference phase so tha t φ ι ηη is, say, 0.25 hour we find that after the first light episode the phase is advanced abou t 1 hour , after the second episode (at the same clock time as the first episode) the phase is advanced (overall) abou t 3 hours , but after the third episode it is advanced (overall) a lmost 9 hours . The extraordinari ly s t rong phase-shifting effect of the third episode is due to a great reduct ion of ampl i tude produced by the first two episodes.

Type 0 resetting can occur only if the cumulat ive effect of individual light episodes is able to reduce the ampl i tude to zero. Figure 5 shows that the max imum growth of ampl i tude (for van der Pol "stiffness", μ = 0.13) in a single cycle is approximate ly 0.18. Thus , any light st imulus which can reduce ampl i tude by more than 0.18 in a single episode is capable of producing Type 0 resetting (al though the number of repetit ions required may be very large). Weaker stimuli can only give Type 1 resetting. An impor tan t corollary to these observat ions is that if the single episode has sufficient s trength to reduce ampl i tude to zero by Ν repeti t ions, then the resetting will certainly be Type 0 for all protocols in which the st imulus is repeated more than Ν times, and will certainly be Type 1 when the repetit ions are fewer than N.

The distinction is shown qualitatively in Figure 10. F o r simplicity, endogenous τ is assumed to be exactly 24 hours , and also only half of the phase resetting curve is shown. Figure 10a por t rays the weak stimulus si tuation for which the endogenous recovery of ampl i tude is s t rong enough to prevent the st imulus from driving ampl i tude to zero. N o mat te r how many times the st imulus episode is repeated there will always be a band of 0 i n it close to zero for which phase shifts are small. Figure 10b shows the si tuation in which Ν repeti t ions can produce zero ampl i tude . The resetting pa t te rn bifurcates at TV repeti t ions. When the repeti t ions, n, are fewer than N, the resetting curves all pass th rough the point 0 i n it = 0, </>f inal = 0 (much as they do for Figure 10a) but when n>N the resetting curves all pass th rough the point 0 i n it = O, 0 f i n al = 12.

When under tak ing to classify experimental resetting da t a as Type 1 or Type 0 the protocol must be designed to provide m a x i m u m discriminabi-lity in the critical region near 0 i n it = O. The da t a points must be dense enough so tha t the sense in which the phase resetting curve is cyclically closed is unambiguous (despite "noise" in the assessment of each point) . This means also that the total st imulus strength must be weak enough so that the band near 0 i n it = O, for which phase shifts would be small if the resetting were Type 1, is clearly defined. F o r the h u m a n resetting we have r epo r t ed ,

4 the da t a points were principally clustered in the critical region

while the choice of a three-episode st imulus was the fewest repeats of 5 hour episodes which could produce Type 0.

The transi t ion between Type 1 and Type 0 resetting is tradit ionally


0 ώ

i2 0 ώ

12

ι initial r initial

(a) (b) F I G . 10. Schemat ic r ep resen ta t ion of p h a s e reset t ing curves for increas ing n u m b e r of repea ted s t imulus episodes , (a) T h e ind iv idua l s t imulus ep isode is weake r t h a n the m a x i m u m e n d o g e n o u s recovery of a m p l i t u d e be tween episodes . T h e reset t ing is T y p e 1 howeve r m a n y the repet i t ions , (b) T h e ind iv idua l s t imulus ep isode is s t rong e n o u g h to reduce the a m p l i t u d e to zero in TV repe t i t ions . T h e reset t ing is

T y p e 1 for η < Ν, bifurcates w h e n η = Ν a n d is T y p e 0 for η > Ν.

discussed in terms of a single (brief) st imulus of increasing strength in which the strength increase may come abou t th rough brighter light or a longer e p i s o d e .

1 8'

28 Here our st imulus consists of a pa t terned repetit ion of

separated episodes; increasing overall st imulus strength is obta ined by increasing the number of episodes. Wi thou t ampl i tude recovery, Type 0 resetting will ult imately be found for any repeated stimulus episode which reduces ampl i tude , however weak tha t reduct ion may be for each repeti t ion.

S t r o g a t z

25 reviewed the mathemat ica l a rguments which show how

repeated applicat ions of Type 1 resetting can never produce Type 0 resett ing—a result which may superficially seem to contradict the presentat ion here. However , Strogatz also observed that if a sequence of resettings gives Type 0 as the final result then one member of the sequence must be Type 0. In Figures 4 and 7 the resettings are each Type 1 since bo th the "initial" and "final" contours in each figure encircle the "phaseless po in t" (the origin). It is in Figure 8 tha t the resetting (due to the third bright light episode) is Type 0, and this establishes Type 0 for the overall sequence of three episodes. Strogatz discussed ampl i tude reduct ion and subsequent endogenous recovery in terms of two limiting cases: rapid recovery and slow recovery. The h u m a n response to 5 hou r bright light epsiodes which we have depicted is intermediate between these. Significant ampl i tude recovery does occur between the first and second episodes, but the total reduct ion achieved by these episodes is s t rong enough to win the competi t ion. Once the ampl i tude is close to zero, the recovery per cycle is presumed to be small (Figure 5).


Ent ra inment and r e - e n t r a i n m e n t

All of the resetting curves of Figure 9 intersect at φίηι1 = 1 1 hours implying that neither the second nor third st imulus produces any net phase shift. In fact, they each p roduce a phase advance of 0.2 h o u r which jus t offsets the daily phase delay drift due to intrinsic τ = 24.2 hours . The pacemaker is seen to be stably entrained by daily 5 hours br ight episodes when they are centered 11 hours after t empera ture min imum. However , a person who has τ = 24.4 hours requires a daily phase advance of 0.4 hou r for ent ra inment to an imposed 24 hour rhy thm. A daily 5 h o u r bright light episode will provide this if centered at 10 hours after the circadian reference phase . In daily life the pat terning of the light s t imulus is dictated primari ly by the social constraints of employment and recreat ion which are tied to solar t ime. Thus st imulus t iming is primari ly independent of a person's individual τ. In tha t case, persons with longer τ will have their reference circadian phase at a later clock hour . If the total daily st imulus approximately matches the strength of the 5 hours of br ight light discussed here, the clock time of the reference phase would be \ hour later for each 0.1 hou r increase of τ. F o r weaker stimuli the effect of τ on en t ra inment phase would be even greater.

En t ra inment is the condi t ion for which 4>init = φ{inal, after the drift due to intrinsic τ has been taken into account . The hor izonta l line in Figure 9 at </>f i n a l= 11 hours is a representat ion of the entra ined condi t ion equivalent to 0 i n i t= 11 hours . This hor izonta l line has a second intersection with the three-episode resetting curve at 0 i n it = 24.2 hours (or equivalently (/>init = 0.2 hours) . The significance of this second intersection is tha t three episodes critically t imed in this precise way, relative to the initial state of the pacemaker , will theoretically advance the pacemaker by 10.8 hours exactly so tha t no further phase adjustment is required for stable ent ra inment .

A basic app roach to the re-entra inment needed to cope with t ransmeri-dional jet travel is to separate the phase shifting process from tha t of ent ra inment . O n e or more bright light episodes are t imed to develop the required phase change rapidly and conveniently. Once the new phase has been approximate ly achieved, the na tura l stimuli in the new envi ronment are relied upon to complete and mainta in en t ra inment . The bright light episodes chosen for this scenario are often much different from either 0 i n it or <Lit-

In the preindustr ial ages artificial light was a scarce commodi ty and so the ent ra inment of the circadian pacemaker was necessarily governed by natura l light which is centered abou t noon . This would have placed the pacemaker reference phase shortly after midnight , say 01.30. We have found that present-day young adul t men (18-31 years) have their reference phase close to 06.45 + 1.3 hours S D while persons of age 60-85 show


reference phase of 04.55 + 2.0 hour S D .

3 These present-day ent ra inment

phases appear to have two principal causes. The first is a reduced light exposure dur ing dayt ime hours , compared to that in an agrar ian setting. Kr ipke and coworkers have f o u n d

11 surprisingly low daily exposures,

even in the favorable climate of San Diego, California. The second is the extension of i l luminated hours well beyond sundown. Indoo r light, though more than 50 times lower in physical intensity than typical ou tdoor light appears to have a significant cumulat ive effect.

There appears to be a strongly "compressive" nonl inear relat ionship between the physical measure of light (e.g. lux) and the effect it exerts on the pacemaker . We have proposed that the effect is p ropor t iona l to the cube-root of the lux m e a s u r e .

12 In labora tory studies where h u m a n

subjects are allowed to select their own sleep/wake schedule (human "free-run" protocol) they tradit ionally have been allowed to turn on r o o m lights dur ing waking hours (150-200 lux). The free-run period could often be abou t 25.5 hours with the pacemaker remaining entrained to the sleep/wake rhy thm. Tha t is, the pacemaker could be drawn-off from its na tura l period by more than an h o u r .

13 O u r current da t a indicate that

ent ra inment of the pacemaker to such a different period was effected solely by the self-selected dark/ l ight cycle which was coincident with the sleep/wake cycle. Thus it appears that abou t 17 hours of 100-200 lux can shift the pacemaker phase by as much as 1 hour per day.

Conclusions

When multiple episodes of light are used on successive days , analysis of experimental da ta is m a d e more difficult by the endogenous rhythmic processes which intervene between episodes. Before spontaneous internal desynchrony was reported by Aschoff in 1965

1 it was thought that the

free-run period which characterized internally synchronized records (approximately 25.5 hours) represented the intrinsic pacemaker period. F r o m a study of free-run records in which internal desynchrony was seen, it was recognized that with synchrony the t rue pacemaker period was being compromised by the endogenous sleeep/wake rhy thm. W e v e r

27

ascribed to the pacemaker the period 24.85 + 0.3 (SD) hours . K r o n a u e r et α / .

13 pointed out that pacemaker periods observed even in desynchrony

could be somewhat high due to a residual effect of the sleep/wake cycle. The phase shifting results of Czeisler et al.

4 on which many features of the

present discussion are based, indicated tha t the daily phase delay a t t r ibutable to pacemaker period were in fact considerably smaller than Wever's est imate, being only abou t 0.2 hours per cycle ra ther than 0.85 hours .

In the vector presentat ion used in Figure 6, the 0.2 hours delay drift is seen to be small compared to the recovery of ampli tude which occurs in a


single cycle. Since Type 0 resetting is a powerful me thod of achieving rapid phase change, and since Type 0 resetting requires large ampl i tude reduct ion, endogenous ampl i tude recovery has emerged as having as s t rong an impact on phase resetting as phase drift due to endogenous τ. We conclude that to utilize the Type 0 strategy the effect of individual bright light episodes must be s t rong: ampl i tude recovery makes Type 0 inaccessible if light exposure is casual or weak.

Finally, we wish to emphasize that the resetting contours of Figures 4, 7 and 8 are idealizations based upon limited da ta . They are the result of a simplified m o d e l

12 with parameters adjusted to ma tch the da t a reasonably

well. As experiments are refined and repeated the details of the resetting contours may change but we expect the essential features to remain.


We thank M e g a n E. Jewett for her thoughtful criticism of this manuscr ip t . This research analyzed herein was suppor ted in par t by N I A 1-R01-AG06072, N I A 1-P01-AG-09975, N I M H 5-R01-MH45130, N A S A N A G 9-524 and N I H D R R G C R C 5 - M 0 1 - R R 0 0 8 8 8 . Richard E. K r o n a u e r is a G o r d o n M c K a y Profesor of Mechanica l Engineering at H a r v a r d University. Charles A. Czeisler and Richard E. K r o n a u e r serve as consul tants to Light Sciences, Inc., Brookline, Massachuset ts .

References

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W . O . (1985). A clinical m e t h o d to assess the e n d o g e n o u s c i rcad ian phase ( E C P ) of the deep c i rcad ian osci l la tor in m a n . Sleep Res. 14 , 295.

3. Czeisler C.A., D u m o n t M . , Duffy J . F . , S te inberg J . D . , R i c h a r d s o n G . S , B r o w n E . N . , Sanchez R., Rios C D . a n d R o n d a J . M . (1992). Assoc ia t ion of s leep-wake hab i t s in o lder people wi th changes in o u t p u t of c i rcad ian p a c e m a k e r , The Lancet, 3 4 0 , 9 3 3 - 9 3 6 .

4. Czeisler C.A., K r o n a u e r R.E. , Al lan J .S. , Duffy J .F . , Jewet t M . E . , B r o w n E . N . a n d R o n d a J . M . (1989). Br ight light i nduc t ion of s t r ong (type 0) reset t ing of the h u m a n c i rcadian p a c e m a k e r . Science 2 4 4 , 1328-1333 .

5. Czeisler C.A., K r o n a u e r R.E. , M o o n e y J.J. , A n d e r s o n J .L . a n d Allan J .S . "Biologic r h y t h m d i sorders , depress ion , a n d p h o t o t h e r a p y . A new hypo thes i s " , Psychiatric Clinics of North America, Vol . 10, N o . 4, "Sleep D i so rde r s " , p p . 6 8 7 - 7 0 9 . W . B . S a u n d e r s C o , Ph i l ade lph ia , D e c . 1987.

6. G a n d e r P . H . a n d Lewis R . D . (1983). P h a s e reset t ing ac t ion of l ight o n the c i rcad ian r h y t h m of Rattus Exulans. Am. J. Physiol. 2 4 5 , R 1 0 - R 1 7 .

7. Has t ings J . M . a n d Sweeney B.W. (1958). Biol. Bull. 115 , 4 4 0 - 4 5 8 . 8. I n o u y e S.T. a n d K a w a m u r a H . (1982). Charac te r i s t i cs of a c i rcad ian p a c e m a k e r in

sup rach i a sma t i c nuc leus . J. Comp. Physiol. 1 4 6 , 153-160 . 9. Jewet t M . E . a n d Czeisler C.A. (1992). T h e role of e n d o g e n o u s c i rcad ian a m p l i t u d e in

the m o d u l a t i o n of a ler tness a n d pe r fo rmance : An inves t iga t ion of the s ingular region. Sleep Res. 2 1 , 378.

10. Jewet t M . E . , K r o n a u e r R .E . a n d Czeisler C.A. (1991). A l ight - induced suppress ion of e n d o g e n o u s c i rcad ian a m p l i t u d e in h u m a n s . Nature 3 5 0 , 5 9 - 6 2 .

11. K r i p k e D . F . a n d G r e g g L . W . (1990). C i r c a d i a n effects of va ry ing e n v i r o n m e n t a l l ight .


In Medical Monitoring in the Home and Work Environment (eds. Miles L .E . a n d B r o u g h t o n R.J . ) , p p . 187-195 . Raven Press , N e w York .

12. K r o n a u e r R .E . (1990). A quan t i t a t i ve m o d e l for the effects of l ight o n the a m p l i t u d e a n d phase of the deep c i rcad ian p a c e m a k e r , based o n h u m a n d a t a . Sleep '90 (Proceedings of the Tenth European Congress on Sleep Research, Strasbourg), (ed. H o r n J .A.) , B o c h u m : P o n t e n a g e l Press , 306 -309 .

13. K r o n a u e r R.E. , Czeisler C.A., P i l a to S., M o o r e - E d e M . C . a n d W e i t z m a n , E . D . (1982). M a t h e m a t i c a l m o d e l of the h u m a n c i rcad ian sys tem wi th two in te rac t ing osci l la tors . Am. J. Physiol. 2 4 2 , R 3 - R 1 7 .

14. Lewy A.J. , W e h r T.A. , G o o d w i n F .K . , N e w s o m e D.A. a n d M a r k e y S.P. (1980). L ight suppresses m e l a t o n i n secret ion in h u m a n s . Science 2 1 0 , 1267-1269 .

15. Lewy A.J. , Sack R .L . a n d Singer C M . (1985). I m m e d i a t e a n d de layed effects of br ight light o n h u m a n m e l a t o n i n p r o d u c t i o n : Shifting " d a w n " a n d " d u s k " shifts the d i m light m e l a t o n i n onset ( D L M O ) . Ann. NY Acad. Sci. 4 5 3 , 253 -259 .

16. M c l n t y r e I .M. , N o r m a n T.R. , B u r r o w s G . D . a n d A r m s t r o n g S .M. (1989). H u m a n me la ton in suppress ion by light is intensi ty d e p e n d e n t . / . Pineal Res. 6 , 149-156 .

17. Ne l son D . E . a n d T a k a h a s h i J .S . (1991). Sensitivity a n d in teg ra t ion in a visual p a t h w a y for c i rcad ian e n t r a i n m e n t in the h a m s t e r (Mesocricetus Auratus). J. Physiol. 4 3 9 , 115-145 .

18. P e t e r s o n E .L . (1980). Phase - rese t t ing a m o s q u i t o c i rcadian osci l la tor . / . Comp. Phyisol. 138 , 2 0 1 - 2 1 1 .

19. P i t t end r igh C.S. , K y n e r W . T . a n d T a k a m u r a T . (1991). T h e a m p l i t u d e of c i rcad ian osci l la t ions: T e m p e r a t u r e d e p e n d e n c e , l a t i tud ina l clines, a n d the p h o t o p e r i o d i c t ime m e a s u r e m e n t . / . Biol. Rhythms 6(4), 2 9 9 - 3 1 3 .

20. Reppe r t S. a n d Schwar t z W . (1983). M a t e r n a l c o o r d i n a t i o n of the fetal biological clock in u t e ro . Science 2 2 0 , 9 6 9 - 9 7 1 .

2 1 . Schwar t z W.J . , Dav idsen L .C . a n d Smi th C .B . (1980) In vivo me tabo l i c act ivi ty of a pu ta t ive c i rcad ian osci l la tor , the ra t sup rach i a sma t i c nuc leus . J. Comp. Neurol. 189 , 157-167 .

22. Schwar tz W.J . , G r o s s R . H . a n d M o r t o n , M . T . (1989) T h e sup rach i a sma t i c nuclei con ta in a t e t r o d o t o x i n res is tant c i rcad ian p a c e m a k e r . Proc. Natl. Acad. Sci. 8 4 , 1694-1698.

23 . S h a n a h a n T .L . a n d Czeisler C A . (1991). Light exposu re induces equiva len t p h a s e shifts of the e n d o g e n o u s c i rcadian r h y t h m s in c i rcula t ing p l a s m a m e l a t o n i n a n d core b o d y t e m p e r a t u r e in m e n . J. Clin. Endocrinol. Metab. 73 (2) , 2 2 7 - 2 3 5 .

24. Soue t re E. , Salvat i E. , Be lugou J . -L. , P r inguey D . et al. (1989) C i r cad i an r h y t h m s in depress ion a n d recovery: Evidence for b lun ted a m p l i t u d e as the m a i n ch ronob io log ica l a b n o r m a l i t y . Psychiatry Res. 28 (3 ) , 2 6 3 - 2 7 8 .

25 . S t roga tz S.H. (1990). C o m m e n t a r y : In t e rp re t ing the h u m a n p h a s e response curve to mul t ip le br ight- l ight exposures . / . Biol. Rhythms 5(2) , 169-174 .

26. T a k a h a s h i J .S . Biological R h y t h m s : F r o m G e n e Express ion to Behavior . Th i s vo lume , p p . 3 -20 .

27. Wever R.A. (1979). The Circadian System of Man. Spr inger , N e w Y o r k . 28. Winfree A .T . (1979). The Geometry of Biological Time. Spr inger , N e w Y o r k .

16

237

Sleep/Wake Regulation T O R B J Ô R N Â K E R S T E D T

1 a n d S I M O N F O L K A R D

2 1IPM & Stress Research, Karolinska Institute, Stockholm, Sweden 2MRC Social and Applied Psychology Unit, Sheffield, UK

A MAJOR reason for having the present symposium is tha t misal ignment frequently occurs between behavior and the circadian system and tha t this seriously affects the individual. In part icular , one observes dis turbed alertness pa t te rns tha t lead to excessive sleepiness, d is turbed sleep, and a loss of capacity to function properly. This is seen in clinical popula t ions as demons t ra ted in other contr ibut ions to the present meeting. It also occurs, however, in healthy popula t ions , and then as perfectly "normal" , non-pathological phenomena . In part icular , we can observe the effects in relation to shift work and t ransmeridian jet travel. This presentat ion will give an overview of such effects and their causes, and will p ropose a practical mathemat ica l model for the predict ion of sleepiness/alertness.

Shi f t w o r k

A number of studies have repor ted tha t shift workers feel sleepy dur ing part icularly the night sh i f t .

41 Recently, this has been verified with

ambula to ry E E G methods . Figure 1 shows the h y p n o g r a m of a control r o o m worker falling asleep three times dur ing a night sh i f t .

31 This type of

behavior occurred in 2 5 % of the subjects and was no t condoned by managemen t or unions . In fact, it was a reason for dismissal. Interestingly, the sleep incidents were in most cases no t recognized by the subjects themselves—a circumstance tha t consti tutes an addi t ional problem. Similar pa t te rns of sleep intrusions into the waking E E G have been demons t ra ted for train d r i v e r s

29 as well as for t ruck d r i v e r s .

19 In bo th

cases spectral power density in the a lpha (8-12 Hz) and theta (4-7 Hz) bands reached high levels towards the early morn ing and were closely correlated with self-rated sleepiness. High levels of E E G a lpha and theta


I Work J I Sleep

06

Time of day (h)

18 06

F I G . 1. 2 4 h o u r h y p n o g r a m involving a n ight shift for a process o p e r a t o r . Sleep stages 0 - 4 a n d R E M are indica ted .

activity are essentially incompat ible with purposeful interact ion with the external w o r l d .

2 5'

3 0'

33

If sleepiness is excessive dur ing work one would expect an accompany-ing performance degradat ion and an increased accident risk at work. This appears to be the case, indeed. Thus , gauge reading errors of process o p e r a t o r s

2 and response latency of swi tchboard opera tors increase dur ing

the night shift.

5 M o r e impor tant ly , the risk of single vehicle road accidents

reaches a p ronounced peak in the morn ing h o u r s

17 and night shift

sleepiness has been identified as a major cont r ibutor to many of the recent nuclear reactor accidents and i nc iden t s .

22 M u c h research remains to be

done in this area, however. The effects of shift work on sleep have been studied in a number of E E G

studies. Essentially, the results show that sleep is shortened by 2 -4 hours in connect ion with night shift and morn ing shift w o r k .

1 1'

2 8'

3 1'

32 Most ly the

sleep loss affects the total amoun t s of stage 2 and R E M sleep. Slow wave sleep (SWS = stages 3 and 4) does not seem to be much affected. Interestingly, sleep after the night shift is often spontaneously terminated, leaving a certain feeling of r e c u p e r a t i o n .

45 The sleep before a morn ing

shift, however, is terminated by the a larm clock (or similar ar rangements) under considerable resistance. The latter leaves the sleeper with a feeling not being refreshed by sleep. The afternoon shift, in contrast , is usually perceived as very comfortable with respect to sleep or wakefulness.

Jet lag

Jet lag is a problem similar to that of shift work. It usually involves out-of-phase feelings of excessive sleepiness and sleep disturbances after t ransmeridian flights across three or more time z o n e s .

36

After a westward flight across, for example, eight time zones, alertness and performance will be reduced in the afternoon and evening of the new time Zo n e .

6'

2 3'

2 5'

3 4'

37 After an eastward flight alertness and performance

Sleep/ Wake Regulation 239

The mechanism of d isturbed sleep

The reason for the sleep dis turbances in shift work and jet travel seem rather straightforward. Control led l abora tory studies (under opt imal sleep condit ions) show that sleep that is displaced towards later times of night gradually decreases in length with increasing displacement (see Figure 2 ) .

42 But only up to a point—from noon , when sleep length is

between 4 and 5 hours , a lengthening occurs, reaching a m a x i m u m of 11 hours after a bedt ime a round 19.00 hours . This then, clearly shows tha t the short post-night shift sleep of the shift worker , or the post-flight sleep of the t ransmeridian jet traveller, is a par t of a general pa t te rn of greatly reduced dayt ime sleep propensi ty, i.e. a time of day pa t te rn . External influences, such as noise, need not be invoked to explain shor tened day time sleep, a l though such influences may exacerbate the problems. Interestingly, also R E M sleep shows a morn ing peak in d u r a t i o n .

7'

1 8'

3 8'

42

will be reduced from the early morn ing to the beginning of the night. Interestingly, the performance effects correspond to those seen in connect ion with b lood alcohol levels of 0.05 % }

With respect to sleep, a westward flight involves a rapid sleep onset but an early (local t ime) awakening. Both progressively app roach normal i ty across a w e e k .

8'

2 4'

2 6'

35 Eas tward flights mainly involve problems of

initiating sleep and of difficulties a w a k e n i n g .

8 , 26 Frequent ly , the first sleep

after arrival is quite normal , whereas the second sleep is dis turbed, followed by subsequent , gradual recovery.


M e c h a n i s m of excessive sleepiness

Inspired by the sleep model we have constructed a practical model for sleepiness regulation. Using published da ta from experiments with life on a 22.0 hour d a y ,

9 sleep d e p r i v a t i o n

1 0 - 14 and other studies, we have found

that subjective sleepiness (or alertness) is predictable using three processes: S, C and W. Space does not permit here a discussion of the derivat ion of the

Isolat ion studies have demons t ra ted that the time of day pa t te rn is highly correlated with the phase of the rectal tempera ture rhy thm, such that high or rising por t ions are associated with a low sleep propensi ty as well as with a low R E M p r o p e n s i t y .

6'

39 Thus , the phase of rising body

tempera ture is associated with s t rong sleep offset tendencies (not necessarily causal). Also sleep latency is influenced by circadian factors such tha t it reaches a min imum in the early m o r n i n g .

2 0'

2 3 , 27 Even a very

gradual phase advance of bedt ime will increase sleep l a t e n c y .

44

It should be emphasized that the circadian influence on sleep and wakefulness is dependent on the rate of adjustment of circadian rhythmicity to phase shifts of the sleep/wake pat tern . Wi th respect to shift work the adjustment is only m a r g i n a l .

40 Even in pe rmanent night workers

the circadian phase may only be delayed by 1 or 2 hours . The adjustment to time zone shifts is approximate ly 1 hou r per day after eas tward flights and slightly more after westward flights.

37 N o t e , however, tha t masking

effects may cause an impression of faster a d j u s t m e n t .

21 As is discussed in

several other contr ibut ions to the present symposium, light plays a major role in the adjustment process.

The circadian influence on sleep may be sizeable, but it should be strongly emphasized that sleep is equally strongly regulated by homeo-static influences. Thus , for example, a dayt ime sleep started at 11.00 hours will be differentially long depending on the a m o u n t of sleep dur ing the prior n i g h t .

43 The resulting T S T may vary from 4.5 hours , after no prior

night sleep, via 3.5 hours after 2 hours of night sleep and 2.8 hours after 4 hours of night sleep to 2 hours after a full night 's sleep.

The homeosta t ic influence is also clearly evident in the a m o u n t of deep sleep—stage 3 and 4 sleep (SWS). Both the sleep stage measure (SWS) and the computer derived measure (slow wave act ivi ty—SWA) are almost entirely dependent on the a m o u n t of pr ior w a k i n g

4'

1 8'

34 or the a m o u n t of

prior s l e e p .

43 If SWS is suppressed (by e.g. noise), sleep is spontaneously

extended until the SWA needed has been r ecove red .

15

Borbély and others have combined the two main regulatory influences into a "two-process model of sleep regula t ion" .

3 Together (approximately

5 0 % each), homeosta t ic and ci rdadian regulat ion account for most of the effects of bo th shift work and t ransmeridian travel on sleep.


0 06 12 18 24

Time

F I G . 3. T h e three c o m p o n e n t s S, C a n d W of the aler tness regu la t ion m o d e l (plot ted in hour ly intervals) .

T A B L E 1

Mathematical functions of the model of alertness regulation

C=Mcos{t-p)n/\2 o r C = 2 . 5 C O S ( M 6 . 8 ) T U / 1 2

S=(Sa-L)e~

00353t + L or 5 = ( 1 4 - 2 . 4 ) < ? - ° ·

0 3 5 3ί + 2.4

S'=U-(U-Sr)e-°

38U or S'= 1 4 . 3 - ( 1 4 . 3 - 7 . 9 6 ) é > - ° -

3 8 1i

W=5J2e-

U5U

A = C + S (or S')-W S' + C=U

Process S

This is an exponential function of the time since awakening. M a x i m u m alertness is reached upon awakening and alertness initially falls rapidly but levels out and gradual ly approaches an asympto te .The rate of decrease of the exponential function corresponds to 3 .5% of the previous value per

model bu t the reader is referred to Fo lka rd and Â k e r s t e d t .

10 Here will be

described the main traits of the model .

Process C

This represents sleepiness due to circadian influences and has the general sinusoidal form indicated below (Figure 3 and Table 1), where M = ampli-tude (in arbi t rary units on a scale from 1 to 21), p = acrophase (in decimal hours) , and i = time of day (in decimal hours) . T o the right, model default parameters have been inserted. O u r a t tempts to model empirical da ta suggest that the phase est imate may have to be delayed to somewhat later hours in s tudents , shift workers or other groups in which evening type preferences domina te .


hour . Sa = value of S at awakening, L = lower asymptote , i = time since awakening. At sleep onset process S is reversed and called S' and recovery occurs as an exponential function tha t initially increases at a very rapid rate but subsequently levels off towards an upper asymptote . Sr = S value at retiring; U= upper asymptote . The rate of increase of the exponential function is 28 .7% per hour , i.e. much steeper than the decay dur ing the day.

The final componen t is the wakeup process W, or sleep inertia, after forced awakenings. This function is also exponential but with an even steeper initial decrease—the rate of decrease is 62 .7% per hour , i.e. already after the first hou r most of the inertia has dissipated. W is subtracted (in effect) from the S+ C level. Spontaneous awakenings const i tute a special problem since the exact t ime of awakening often is not availabe, and since the time between the first formal awakening and the time of finally rising might be considerable. Tentatively, we assume that such a process might take approximately 1 hour .

The est imated alertness (or sleepiness) is then expressed as the ar i thmetic sum of the three functions above. Using the model we have found it possible to predict with accurracy the variat ion of subjective sleepiness under condit ions of sleep deprivat ion and various experimental or natural ly occurring al ternat ions of sleep/wake b e h a v i o r .

10 Figure 4

shows an example of predict ion of latency to stage 1 sleep dur ing a 30 minute vigilance test performed every 2 hours in a study of 64 hours of cont inuous activity. The study involved 12 subjects under condit ions of isolation from external synchron ize r s .

16 The circadian acrophase was set

at 20.48 hours . The covariat ion between the curves is very close, reaching an r

2 of 0.70. N o t e the line at 7 units , denot ing the "critical level". This level

was determined after l abora tory experiments and represents the level at which EEG-signs of sleep will start appear ing within 5 minutes in a waking subject with open eyes focusing on a spot on the wall.

Figure 4 also shows the predict ion of (mean) sleepiness in 20 workers in connect ion with night shift w o r k .

31 Again the variance accounted for is

very high. EEG-defined sleep incidents start to occur a round and below level 7.

Sleep and t h e alertness model

In order to be comprehensive the sleepiness model also needs to be able to predict sleep. In a previous study we have demons t ra ted that SWA dur ing sleep is predicted by componen t 5 " ,

41 tha t is SWA follows the same

pa t te rn as does S". We have also recently started to apply the model to sleep termination

(unpublished). This applicat ion is based on the assumpt ion that sleep terminat ion occurs when S+C reach a certain level of alertness (see

Sleep/Wake Regulation 243

OS

16

14

« 12 ΧΛ

G

10

6 Η

r2 Predicted

= 0.96 -

\ //—Rated \ Crit level \ y

- Sleep · " * \ * incidents \J 1 1

f Sleep

1 1 16 04 16 04

Time (h)

O

Ο-

Ι* O

14 h

12

10

8

6

1

oc

Prediction r2 = 0.70 -

- V,. -

- \ \ Latency

— Critical level *. \

* * \ ·*/

1 1

•λ y · I 1 1

40

30

20

10

12 24 36

Time awake (h)

48 60

o

O

c

F I G . 4 . C o m p a r i s o n of mode l p red ic t ion a n d empir ica l d a t a . A b o v e : ra ted aler tness a n d sleep incidents in n ight shift w o r k e r s . Below: sleep la tency d a t a

(g roup m e a n ) du r ing a 6 4 h o u r sleep dep r iva t ion exper iment .

Figure 5). Fu r the rmore , we assume that the value in quest ion is the upper asymptote , since it may be argued tha t 5" would reach close to its asymptote if circadian factors would not interfere. Hence, we propose that sleep terminat ion occurs when S' + C=U (the latter is the upper asymptote of 5").

Figure 5 illustrates the applicat ion of the model . We assume 16 hours of waking preceeding each of the bedtimes indicated. Alertness is regained during sleep according to process S"—up to the point where circadian influences s top the process. In the afternoon this interference will occur very early and in the early morn ing it will occur much later. We also assume that the influence (ampli tude) of C is less dur ing sleep. We therefore sought the o p t i m u m ampl i tude of C by modell ing empirical sleep length da ta from two earlier studies of displaced s l e e p .

4 2 , 43 This was done


Prediction of sleep offset

Sleep offset

Sleep onset

0 6 12 18 24 06 36

Time of day (h)

F I G . 5 . Use of the a ler tness regu la t ion m o d e l for p red ic t ion of sleep length . See text for further in fo rmat ion .

by varying the ampl i tude of C in the function S' + C=U. The results showed that op t imum (96%) predict ion could be obta ined by using an ampl i tude of 1. This yielded a regression coefficient of 1.01 ( through the origin) for the predict ion of sleep length by the model . The r

2 reached 0.98

for g roup mean sleep lengths. This new, sleep terminat ing, version of C h a s been named C

Final c o m m e n t s

This presentat ion has wanted to emphasize tha t alertness and sleep are severely affected by phase shifts of sleep wake behavior but that appreciat ion of the mechanism and provisions for countermeasures make it necessary to consider not only circadian rhythmicity, but also homeostasis . The latter has a tendency to be left out in connect ion with work on rhy thms and light.

References

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20. Lavie P . (1986) U l t r a s h o r t s leep-waking schedule . I I I . " G a t e s " a n d " forb idden z o n e s " for sleep. Electroenceph. Clin. Neurophysiol. 6 3 , 4 1 4 - 4 2 5 .

2 1 . M i n o r s D . S . a n d W a t e r h o u s e J . M . (1989) M a s k i n g in h u m a n s : T h e p r o b l e m s a n d some a t t e m p t s to solve it. Chronohiol. Intern. 6 , 2 9 - 5 3 .

22. Mit ler M . M . , C a r s k a d o n M.A. , Czeisler C.A., D e m e n t W . C . , Dinges D . F . a n d G r a e b e r R .C . (1988) C a t a s t r o p h e s , sleep a n d publ ic policy. C o n c e n s u s Repo r t . Sleep 1 1 , 100-109 .

23 . M o n k T . H . a n d M o l i n e M . L . (1989) T h e t iming of bed t ime a n d wake t ime decis ions in f ree-running subjects . Psychophysiology 2 6 , 304 -310 .

24. N icho l son A.N. , P a s c o e P.A. , Spencer M . B . , S tone B . M . a n d G r e e n R .L . (1986). N o c t u r n a l sleep a n d d a y t i m e aler tness of a i rcrew after t r an smer id i an flights. Aviat. Space Environ. Med. 5 7 , B 4 2 - 5 2 .

25 . O ' H a n l o n J . F . a n d Beat ty J. (1977) C o n c u r r e n c e of e l ec t roencepha lograph ic a n d pe r fo rmance changes d u r i n g a s imula ted r a d a r wa tch a n d s o m e impl ica t ions for the a rousa l t heo ry of vigilance. In Vigilance (ed. M a c k i e R.R.) , p p . 189-202 . P l e n u m Press , N e w York .

26. Sasaki M . , K u r o s a k i Y., M o r i A. a n d E n d o S. (1986) P a t t e r n s of sleep-wakefulness before a n d after t r ansmer id i an flight in commerc i a l air l ine p i lo ts . Aviat. Space Environ. Med. 5 7 , B 2 9 - 4 2 .

27. S t roga tz S.H., K r o n a u e r R .E . a n d Czeisler C A . (1987) C i r cad ian p a c e m a k e r interferes with sleep onset a t specific t imes each day : role in i n somnia . Am. J. Physiol. 2 5 3 , 172-178 .

28. Tilley A.J. , Wi lk inson R.T . a n d D r u d M . (1981 ) N igh t a n d day shifts c o m p a r e d in t e rms of the qua l i ty a n d q u a n t i t y of sleep recorded in the h o m e a n d pe r fo rmance m e a s u r e d at w o r k : a pi lot s tudy . In Night and Shift Work. Biological and Social Aspects (eds. Re inberg Α., Vieux Ν . a n d A n d l a u e r P . ) , p p . 187-196. P e r g a m o n Press , Oxford .


29. Torsva l l L. a n d Âkers ted t T . (1987) Sleepiness o n the j o b : con t inuous ly m e a s u r e d E E G changes in t ra in dr ivers . Electroenceph. Clin. Neurophysiol. 6 6 , 5 0 2 - 5 1 1 .

30. Torsva l l L. a n d Âkers t ed t T . (1988) E x t r e m e sleepiness: quant i f ica t ion of E O G a n d spectra l E E G p a r a m e t e r s . Int. J. Neurosci. 3 8 , 4 3 5 - 4 4 1 .

3 1 . Torsva l l L. , Âkers t ed t T. , G i l l ande r Κ . a n d K n u t s s o n A. (1989) Sleep on the n ight shift: 24 -hour E E G m o n i t o r i n g of s p o n t a n e o u s s leep /wake behav io r . Psychophysiology 2 6 , 352-358 .

32. Torsva l l L. , Âkers ted t T . a n d Gi l lberg M . (1981) Age, sleep a n d i r regular w o r k h o u r s : a field s tudy wi th E E G record ing , ca t echo lamine excre t ion , a n d self-ratings. Scand. J. Work Environ. Health 7 , 196-203 .

33 . Valley V. a n d B r o u g h t o n R. (1983) T h e physiological ( E E G ) n a t u r e of d rowsiness a n d its re la t ion to pe r fo rmance deficits in narco lep t ics . Electroenceph. Clin. Neurophysiol. 5 5 , 2 4 3 - 2 5 1 .

34. W e b b W . B . a n d Agnew J . H . W . (1971) Stage 4 sleep: influence of t ime course var iables . Science 174 , 1354-1356.

35. W e g m a n n H . M . , G u n d e l Α., N a u m a n n M . , Samel Α., Schwar tz E. a n d Vejvoda M . (1986) Sleep, sleepiness a n d c i rcad ian rhy thmic i ty in a i rcrews o p e r a t i n g o n t r ansa t l an t i c rou tes . Aviat. Space Environ. Med. 5 7 , 5 3 - 6 4 .

36. W e g m a n n H . M . a n d Kle in K . E . (1985) Jet - lag a n d a i rcrew schedul ing . In Hours of Work (eds. F o l k a r d S. a n d M o n k T .H . ) , p p . 539-552 . J o h n Wiley, Chiches te r .

37. W e g m a n n H . M . , Kle in K . E . , C o n r a d B. a n d Esser P . (1983) A m o d e l for p red ic t ion of r e synchron iza t ion after t ime-zone flights. Aviat. Space Environ. Med. 5 4 , 524—527.

38. Zul ley J. (1980) D i s t r i bu t i on of R E M sleep in en t r a ined 24 h o u r a n d free r u n n i n g s l eep—wake cycles. Sleep 2 , 377 -389 .

39. Zul ley J., Wever R. a n d AschoffJ . (1981) T h e dependence of onset a n d d u r a t i o n of sleep on the c i rcadian r h y t h m of rectal t e m p e r a t u r e . Pflugers Arch. 3 9 1 , 314 -318 .

40. Âkers t ed t T . (1985) Ad jus tmen t of physiological c i rcad ian r h y t h m s a n d the s leep-wake cycle to shift w o r k . In Hours of Work (eds. M o n k T . H . a n d F o l k a r d S.), p p . 185-198 . J o h n Wiley, Chiches te r .

4 1 . Âkers t ed t T . (1988) Sleepiness as a consequence of shift w o r k . Sleep 1 1 , 17-34 . 42. Âkers t ed t T . a n d Gi l lberg , M . (1981). T h e c i rcad ian va r i a t ion of exper imenta l ly

d isplaced sleep. Sleep 4 , 159-169 . 43 . Âkers t ed t T . a n d Gi l lberg , M . (1986) A dose- response s tudy of sleep loss a n d

s p o n t a n e o u s sleep t e rmina t i on . Psychophysiology 2 3 , 293 -297 . 44. Âkers t ed t T. , H u m e K. I . , M i n o r s D .S . , W a t e r h o u s e J . M . a n d F o l k a r d S. (1992) Sleep

on a sho r t en ing day /n igh t schedule . Electroencephalogr. Clin. Neurophysiol. 8 2 , 1 0 2 - 1 1 1 .

45 . Âkers t ed t T. , K e c k l u n d G . a n d K n u t s s o n A. (1991) Spectra l analysis of sleep e l ec t roencepha log raphy in ro t a t i ng three-shift w o r k . Scand. Work Environ. Health 17 , 330-336 .

17

Hormone Rhythms and Sleep J . S A I N I , G. B R A N D E N B E R G E R , C. S I M O N a n d M . F O L L E N I U S

Laboratoire de Physiologie et de Psychologie Environnementales, INRS/CNRS, 21, rue Becquerel, 67087 Strasbourg Cedex, France

Abstract

Recent studies suggest tha t the t empora l relat ionships between h o r m o n e rhy thms a n d sleep are m o r e complex t h a n previously believed. T o clarify the s i tuat ion, an acute shift in the sleep per iod was used to dissociate sleep from circadian influences.

T h e results d e m o n s t r a t e d tha t h o r m o n e s from a wide range of endocr ine systems, namely the anter ior p i tu i tary , hydrominera l regulat ing a n d pancrea t ic systems all show t empora l associat ions with sleep. F o r certain h o r m o n e s the occurrence of sleep was the major factor de termining the 24-hour profile, ei ther being related to sleep as a whole or to the internal sleep s t ructure . Pro lac t in was influenced by the presence of sleep ra ther t han sleep qual i ty , wi th an immedia te shift in the noc tu rna l rise as soon as sleep was displaced. A significant pulse was observed at the t ime of hab i tua l sleep suggesting a possible weak circadian c o m p o n e n t to the profile which is d o m i n a t e d by the s leep-wake cycle. T h e profiles of G H a n d P R A were also de te rmined by sleep, bu t m o r e precisely by the internal sleep s t ruc ture . G H showed some associat ion with S W S , par t icular ly with the first S W S episode of b o t h night a n d day sleep. As for prolact in an "anamnes t i c " pulse was observed dur ing the night of sleep depr iva t ion , possibly reflecting intrinsic rhythmici ty . In con t ras t to a specific sleep stage, P R A oscillations were associated with sleep stage a l te rnance . T h e acute shift in sleep confirmed tha t the increase in noc tu rna l P R A is sleep related a n d no t c i rcadian in na tu re . Glucose a n d insulin oscil lations were also amplified du r ing sleep bu t were no t related to the sleep cycles. F o r o ther h o r m o n e s , such as T S H a n d Cortisol, t empora l associat ions with sleep stages were super imposed on a s t rong circadian r h y t h m . Sleep onset h a d an inhibi t ing effect o n T S H a n d declining levels of T S H and Cortisol pulses coincided with deep sleep.

These t empora l associat ions p robab ly reflect the close proximi ty of the central control l ing mechan i sms involved in sleep a n d h o r m o n e secretion. In oppos i t ion A N P proved to be indépendan t of sleep and ci rcadian rhythmici ty confirming per iphera l mechanica l regulat ion.

In s u m m a r y , these results h ighl ight the complex i ty of h o r m o n e a n d sleep in te rac t ions in t ha t n o t w o h o r m o n e s relate to sleep in exactly the s a m e m a n n e r a n d p rov ide a basis for further research inc lud ing m a n i p u l a t i o n s of the l i g h t - d a r k cycle.

In t roduct ion

O V E R THE last 20 years a large body of experimental evidence has accumulated concerning the description of temporal associations between hormones and sleep in man . These relationships involve endocrine systems with very different functions, including hydromineral regulation, glucose

2 4 7


metabolism, lactation and growth. Mullen in 1 9 8 3 ,

21 classified hormones

into three categories; (1) those dependent on sleep; (2) those not influenced by sleep; (3) those related to a specific sleep stage. The current use of precise and sensitive radioimmunoassays, frequent blood sampling methods and deconvolution models to determine hormone secretion rates from plasma concentrations is providing evidence that these relationships are more complex than originally believed.

M a n y hormones show a 24 hour rhythm, which may be driven by an internal clock and termed "circadian" or synchronized to the sleep-wake cycle or derived from a combinat ion of the two. Superimposed on the 24 hour profile, ultradian oscillations may be associated with the internal sleep structure so that any given hormone can exhibit more than one relationship with sleep and recent research suggests that this is frequently the case rather than the exception. Hormones such as adrenocort icotropic hormone (ACTH) and Cortisol considered for a long time to be circadian in nature may have a sleep related c o m p o n e n t .

1'

16 Others , for example growth hormone

(GH) and prolactin thought to be solely sleep dependent appear to possess an inherent rhythmic c o m p o n e n t .

34 F o r renin, a key enzyme in the angiotensin-

aldosterone hydromineral regulatory system, a close association exists between the ultradian oscillations of plasma renin activity (PRA) and the rapid-eye movement ( R E M ) — n o n R E M sleep cycles. P R A levels are in the increasing phase during N R E M sleep and decrease as sleep becomes lighter(3). These investigations have also established the consequential relationship between a l d o s t e r o n e

20 and sleep, but as yet the absence of a

circadian rhythm in P R A has not been confirmed.

5 Controversy persists as to

the presence or absence of a circadian rhythm for the recently identified hormone , atrial natriuretic peptide (ANP), bu t recent da ta suggests that A N P shows no associations with internal sleep s t ruc tu re .

15

A variety of methods have been used to investigate s leep-hormone interactions, including: complete or partial sleep deprivation; acute or chronic shifts in sleep time or complete sleep-wake reversal as occurs with transmeridian travel or shift work; administrat ion of pharmacological agents which per turb either sleep or hormone secretion and finally the study of specific pathologies which manifest either endocrine or sleep disturb-ances. This paper presents current knowledge of the relationships between hormones of different endocrine systems, namely the anterior pituitary, hydromineral and pancreatic systems, and presents findings using one approach, an acute shift in sleep time, to dissociate circadian and sleep related influences.

Experimental procedures

Six healthy male subjects with regular sleep wake pat terns underwent two 24-hour studies (23.00-23.00 hours) , following a night of habi tuat ion. O n e

Hormones and Sleep 249

study was considered as a reference period in which the subjects slept at night (23.00-07.00 hours) and the other an experimental session in which the sleep period was shifted by 8 hours so the subjects slept during the day (07.00-15.00 hours) .

F r o m 8 hours before and throughout the study the subjects received continuous enteral nutri t ion, to avoid the acute effects of meals on hormone profiles. Blood sampling began at 23.00 hours and was pumped continu-ously from an indwelling intravenous catheter until the end of the experiment. Samples were collected at 10 minute intervals for subsequent plasma hormone analysis. Sleep was recorded during sleep times and examined in parallel with the ho rmone profiles.

Analysis of hormone profi les

Plasma ho rmone concentrat ions were measured using radioimmunoassay methods as previously d e t a i l e d .

3'

1 5'

1 6'

26 T o evaluate ho rmone pulsatility an

objective pulse detection program (ULTRA) was employed. The general principle of the U L T R A algori thm is to eliminate all pulses of plasma concentrat ion for which either the increment or the decrement does not exceed a pre-defined threshold, based on measurement e r r o r .

29 As plasma

hormone concentrat ions reflect not only secretion rates but also rates of clearance and redistribution, a mathematical deconvolution model , des-cribed by Ea ton et al.

12 was used to estimate ho rmone secretory rates from

plasma concentrat ions.

Results

Pi tui tary hormones

Prolactin

The nocturnal prolactin increase was displaced by the acute shift in sleep time (Figure 1 ). Neither the sleep induced rise nor the levels observed during the waking periods differed significantly in the two conditions, suggesting that the prolactin rhythm is solely determined by the sleep-wake pat tern. In addit ion no differences were found in the number or ampli tude of the sleep associated oscillations. However, a significant prolactin pulse was identified in all subjects, during the night in which sleep was deprived, at the time that habitual sleep occurred (Figure 1). The mean maximal value of this peak was at 3.15 hours ± 1 9 minutes and is perhaps indicative of some innate circadian rhythmicity.

Analysis of ascending and descending phases of prolactin plasma and secretory pulses in relation to the different sleep stages, confirmed that neither slow-wave sleep (SWS) nor nocturnal awakenings were associated with any trend in prolactin. F o r R E M sleep, a temporal association was


23 1 3 5 7 9 11 13 15 17 19 21 23 T I M E ( h o u r s )

F I G . 1. Ind iv idua l 2 4 - h o u r p l a s m a pro lac t in profiles wi th n igh t t ime sleep ( top) a n d day t ime sleep ( b o t t o m ) . S h a d e d a reas represent R E M sleep.

apparent in that 9 1 % of plasma and 7 7 % of secretory pulses were in a descending phase at the onset of R E M sleep (p< 0.001, /?<0.02 respec-tively). Prolactin secretion was then seen to increase following 20 (p<0 .05) and 30 (p<0 .02) minutes of R E M sleep.

Growth Hormone

Plasma and secretory G H profiles consisted of low basal levels interrupted by episodic pulses. During nighttime and daytime sleep a large pulse occurred soon after sleep onset. The ampli tude of this pulse varied widely between individuals but was not altered by the time of sleep, giving a mean maximum concentrat ion of 20.04 + 4.76 n g - m l

-1 at 01.10 hours + 16

minutes for night sleep and 19.52 + 5.59 n g - m l

-1 at 09.00 hours + 15

minutes for day sleep. As for prolactin a significant pulse was detected in four out of the six subjects during the night of sleep deprivation at the habitual time of the nocturnal pulse (Figure 2). The number of plasma pulses


I 1 I ! I 1 I 1 1 X 1 I 1 1 i 1 1 1 1 1 1 1 1—ι—ι 23 1 3 5 7 9 11 13 15 17 19 21 23

T IME ( h o u r s )

F I G . 2. Ind iv idua l 24 -hour p l a s m a G H profiles wi th n igh t t ime sleep ( top) a n d d a y t i m e sleep ( b o t t o m ) . S h a d e d a reas represent S W S .

for the six subjects during night and day sleep was 21 and 25 pulses respectively corresponding to 35 and 41 secretory pulses. In relation to sleep stages, 5 9 % and 6 8 % of plasma and 4 6 % and 6 3 % of secretory pulses of the night and day time sleep periods were associated with SWS episodes and for SWS, 7 4 % of night and 9 2 % of day time episodes were associated with a significant G H secretory pulse.

Thyrotropin (TSH)

Under basal condit ions, plasma T S H concentrat ions showed a circadian rhythm with a slow rise during the evening, a peak a round the time of sleep onset and a subsequent decrease during the night hours and low values during the day (Figure 3). Sleep deprivation during the night preceding day sleep was associated with significantly higher levels, and a decline as soon as sleep onset occurred. In addit ion to a sleep onset influence on the T S H


T I M E ( h o u r s )

F I G . 3. Ind iv idua l 24 -hou r p l a s m a T S H profiles with n igh t t ime sleep ( top) a n d day t ime sleep ( b o t t o m ) . Shaded a reas represent S W S .

rhythm the temporal association which has recently been described between SWS episodes and declining plasma T S H levels was conf i rmed.

18

ACTH and Cortisol

The shift in sleep time had very little influence on the Cortisol rhythm (Figure 4), reflecting the dominant circadian rhythmicity of the controlling pituitary hormone A C T H . A C T H is rarely in the ascending phase at the time of R E M sleep o n s e t

16 and as Cortisol pulses lag behind those of A C T H the number of

pulses in the descending phase at R E M sleep onset is reduced. Dur ing day sleep the major SWS episodes coincided with a period of enhanced Cortisol secretion, allowing the analysis of Cortisol secretory episodes concomitantly with a greater number of SWS episodes than for night sleep. In confirmation of a prior exper iment ,

13 using partial sleep deprivation, SWS episodes

tended to occur when adrenal cortical activity was diminishing (Figure 4).

Hydrominera l hormones

Renin-angiotensin-aldosterone system

The profiles of PRA, a key enzyme in the angiotensin-aldosterone system, in a subject who had regular R E M - N R E M sleep cycles during night and daytime sleep are presented in Figure 5. Pulse analysis revealed no significant difference in the number , durat ion or ampli tude of the oscillations under the


I 1 1 1 1 1 I I -J U I I ι - ι i~ ι ι ι ' ι ι ι ι ι

23 1 3 5 7 9 11 13 15 17 19 21 23 T IME ( h o u r s )

F I G . 4 . Ind iv idua l 2 4 - h o u r plasma Cortisol profiles wi th nighttime sleep (top) and daytime sleep (bottom). S h a d e d areas represent S W S .

two conditions, but in each case their absolute ampli tude was 1.5 times greater during sleep compared to waking periods. The previously described relationship of increasing P R A levels with the occurrence of N R E M sleep and decreasing levels coinciding with R E M sleep persisted during daytime sleep. Throughout the waking periods, P R A oscillations existed but were damped and irregular. The P R A rhythm appeared to be determined by sleep and independent of intrinsic circadian rhythmicity.

ANP

A N P fluctuated throughout the 24 hours showing between two and five transient increases, which was not altered by a shift in sleep time (Figure 6).


I 1 I I t 1 I I I I I I I 1 1 1 1 1 1 1 1 1 — ι — ι — ι

0 -I ι ι ι I I I I I t_ I I JL I I 1 1 1 I I I I I I I

23 1 3 5 7 9 11 13 15 17 19 21 23 T IME ( h o u r s )

F I G . 5. Ind iv idua l 24 -hour P R A profiles wi th n igh t t ime sleep ( top) a n d day t ime sleep ( b o t t o m ) . Shaded a reas represent R E M sleep.

N o differences were found between either the mean level, 38 .0+ 3.7 p g - m l

- 1, for the normal sleep wake cycle and the shifted sleep cycle,

39.9 ± 3.2 pg · m l "

1 or the concentrations during the sleep or wake periods.

Under standardized conditions, the profile of A N P was unaffected by sleep and seemed to possess no circadian rhy thmic i ty .

14

Pancreatic hormones

Glucose and insulin oscillate regularly throughout the 24 hours with a periodicity of about 80 minutes with a greater ampli tude following m e a l s .

26

Under the steady state condition obtained by administrat ion of continous enteral nutri t ion, plasma insulin and glucose demonstrated larger oscil-lations during both night and day time sleep. The mean amplitude of plasma glucose was 0.26 ± 0.03 and 0.28 ± 0.02 g · 1 "

1 for the respective 8 hour sleep

periods. These values were significantly higher (p<0 .01) than those obtained for the corresponding waking periods (mean values: 0.14 + 0.02 and 0.19 + 0.02 g l "

1) . Fo r plasma insulin the pulse ampli tude was

16 .6+1.7 u U - m l "

1 and 22.4 + 2.7 u U - m l "

1 for night and day sleep also

differing significantly (/?<0.01) from corresponding periods during which the subjects were awake (11.5 + 1 . 2 u U - m l "

1 and 12 .6+1.2 u U - m l

-1

respectively). It appeared that sleep itself amplified the pulses, a l though


20 h E cn a.

Q-z <

5 0 1

30 L

2θ\-

23 5 9 ) Π 13 TIME ( h o u r s )

15 17 19 21 23

F I G . 6. Ind iv idua l 24 -hour p l a s m a A N P profiles with n igh t t ime sleep ( top) a n d d a y t i m e sleep ( b o t t o m ) . S h a d e d a reas represent R E M sleep.

despite their similar periodicity, could not be related to R E M - N R E M sleep cycles (Figure 7).

Discussion

An acute shift in sleep time is one of a variety of methods that can be used to investigate relationships between hormone rhythms and sleep. The findings from this study along side current da ta highlight the complexity of these inter-relationships, with hormones from different endocrine systems, with different controlling mechanisms exhibiting temporal relationships with sleep.

Temporal associations were present between basal rhythms of all the hypothalamo-pi tui tary hormones studied and sleep, which is a reflection of their central control , a l though each ho rmone related to sleep in a unique manner . Hormones such as prolactin and G H , which have a certain amoun t of structural homology, are secreted by adjacent pituitary acidophil cells and show increased plasma concentrat ions during exercise and other conditions of s tress ,

9 have very different relationships with sleep.

Prolactin is classically known to be influenced by sleep as a whole with sleep deprivation abolishing the nocturnal prolactin rise and a shift in sleep immediately displacing the nocturnal secretory e p i s o d e .

24 Findings of Desir

et α / .

10 have challenged the concept that the prolactin rise is solely sleep

dependent, firstly because the sleep associated acrophase was advanced after a westward and delayed after an eastward flight and secondly, as we observed, following an acute shift in sleep, an "anamnest ic" pulse was present in 9 out of 10 profiles at the time of pre-travel sleep.


I I L I I i I J J I I I I I I I 1 1 1 1 1 1 1 1 1

10 L I 1 1 1 1 1 1 l 1 ι ι ι J ι ι ι ι ι ι ι ι ι ι

23 1 3 5 7 9 11 13 15 17 19 21 23 T IME ( h o u r s )

F I G . 7. Ind iv idua l 24 -hour p l a s m a insulin profiles wi th n igh t t ime sleep ( top) a n d day t ime sleep ( b o t t o m ) . Shaded areas represent R E M sleep.

Reports have been contradictory concerning the relationship between nocturnal prolactin oscillations and sleep s t r u c t u r e .

2 3'

32 Analysis of the

prolactin pulses, as previously described, were rarely in the ascending phase at R E M sleep o n s e t ,

16 but also demonstrated that pituitary prolactin

secretion is reduced at this time and then subsequently increases particularly if the R E M sleep episode is of long durat ion.

In contrast to prolactin, where sleep as a whole is the major determinant of the 24-hour profile, many studies have described a temporal relationship between G H and SWS, especially between the major secretory pulse occurring soon after sleep onset and the first SWS episode. Discordance in this temporal relationship has, however, been repor ted .

2 P lasma G H levels

may begin to increase even before sleep onset or be displaced after the SWS ep i sode .

28 Deconvolut ion methods which permit an accurate definition of

the onset and offset of G H secretion reveal an increased number of secretory pulses, with a single plasma pulse often reflecting more than one secretory episode. The number of G H secretory pulses observed to coincide with a SWS episode was lower than that reported by Van Cauter et al.

33 but when

the percentage of SWS episodes was considered the degree of concordance was similar. This was true for both night and day sleep and in both cases G H secretion increased in all subjects during the first SWS episode. Recent findings suggest that SWS facilitates G H secretion but SWS is not


obligatory for its occur rence .

31 As for prolactin, a significant G H secretory

pulse was observed in most subjects during the night of sleep deprivation at the time of the habitual sleep associated pulse, again possibly representing an innate circadian component of the hormone r h y t h m .

34

Other anterior pituitary hormones , such as A C T H and T S H , show strong circadian rhythmicity. Fo r T S H an inhibitory effect of sleep onset is superimposed on the intrinsic rhythm as when sleep is deprived elevated T S H levels are obse rved .

22 Decreasing levels of T S H and Cortisol pulses

were associated with S W S ,

1 3'

18 suggesting that some mechanisms regulat-

ing SWS may modula te their secretion or conversely increased hormone concentrations prevent SWS occurrence. The decline in plasma Cortisol appears to slightly precede SWS onset indicating that it may be enhanced Cortisol activity that prevents sleep deepening rather than an effect of sleep on Cortisol secre t ion .

13

The temporal associations between hormones under hypotha lamo-pituitary regulation and sleep are probably a reflection of the intimacy of sleep generating processes and the central control of endocrine secretion. A number of neuroendocrine peptides and transmitters, including noradrena-line, dopamine and 5-hydroxytryptamine have all been shown to affect s l eep .

11

PRA, reflecting angiotensin-aldsoterone activity demonstrates a clear relationship with the internal sleep structure. The acute shift in sleep confirmed that increased nocturnal P R A levels is a sleep related phenom-enon and not circadian in nature. The ampli tude as well as the frequency of the oscillations was dependent on the regularity of the R E M - N R E M sleep cycles, with increasing P R A levels coinciding with N R E M and decreasing levels with R E M sleep. Modula t ing renin release, either using a jS-blocker to suppress or a low sodium diet to stimulate renin release, only alters the magni tude of the pulses and does not disturb the relationship with the sleep cycles.

4 The importance of the internal sleep structure in maintaining

rhythmicity of P R A is supported by the nocturnal profiles of patients with various sleep disturbances such as obstructive sleep apnea and narcolepsy. In these cases, P R A profiles reflect all the irregularities of the sleep pat tern, but as soon as sleep quality is restored by treatment and regular R E M -N R E M sleep pat terns are re-established, P R A rhythmicity returns and the amplitude of the oscillations increases .

17 A "renin-releasing factor" located

in the hypotha lamus has been identified which under serotoninergic innervation possibly acts as a central control of renin re lease .

35 At present, it

is not known whether this partially characterized peptide is a candidate for the common factor involved in P R A and sleep stage alternance.

Aldosterone, the active ho rmone of this hydromineral regulating system, is also under the influence of A C T H and, as for A C T H , levels are low early on in the night and increase towards the morning. Stimulating and suppressing P R A reveal the relative influence of the two hormone systems


on aldosterone secretion. When P R A is stimulated aldosterone oscillations reflect those of P R A with a delay of 20 minutes and when P R A is depressed the oscillations follow those of A C T H by 10 minutes. Thus the relationship of the aldosterone rhythm to sleep is dependent on that of P R A and A C T H .

20

The diurnal variations of A N P have often been compared to the renin-angiotensin-aldosterone system activity, bo th being sodium and volume dependent. The acute shift in sleep established that , contrarily to PRA, sleep regulatory processes seem not to influence its re lease ,

15 neither did A N P

profile show any circadian rhy thmic i ty .

14 This da ta substantiates the

argument that , unlike P R A in which central mechanisms are clearly involved, A N P is released in response to peripheral mechanical factors.

A number of studies indicate that the time of day influences the regulation of blood glucose levels, with higher levels obtained in the afternoon or evening compared with the morning, following either oral or intravenous glucose administrat ion, as well as in response to m e a l s .

19 In normal subjects

glucose tolerance reaches a minimum around mid-sleep, suggesting circadian rhy thmic i ty .

25 A recent study, monitor ing glucose and insulin

levels during a constant glucose infusion suggest that both circadian and sleep modula te the regulation of g lucose .

30

Both glucose and insulin oscillate regularly throughout the 24 hours and in normal subjects show a concomitancy of about 9 0 % , suggesting interdependence. However, results from noninsulin dependent diabetic patients question the hypothesis of an intrinsic insulin-glucose regulatory loop as compared to control subjects these patients have regular glucose oscillations, but less regular, less frequent and smaller ampli tude insulin pulses showing a poor association with those of g lucose .

27 Oscillations of

glucose and insulin secretion were amplified in this study, both during nighttime and daytime sleep, reflecting either central mechanisms or changes in glucose metabolism during sleep.

In conclusion, these results demonstra te the diverse relationships between hormones and sleep. Sleep may be the major factor determining the basal 24-hour rhythm, either by being temporally associated with sleep as a whole, for example prolactin, or linked to the internal sleep structure, such as P R A which reflects sleep stage alternance, and G H which is specifically associated with SWS. F o r other hormones , the relationship with sleep is superimposed on a strong circadian rhythm, as for A C T H , Cortisol and T S H . Melatonin, the pineal hormone is also dominated by a circadian rhythm but recent reports do not favor the presence of any direct relationship between melatonin secretion and sleep.

7 Both melatonin and Cortisol as well as the

circadian rhythm of body temperature can be entrained by light e x p o s u r e ,

6 ,8

however, the influence of the l ight-dark cycle on other ho rmone rhythms has been little studied. The present da ta provides a basis for further investigations into this area.


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18

Biological Rhythms and Sleep Disorders in Man: The Delayed Sleep Phase Syndrome J . V I G N A U , M . D A H L I T Z , J . A R E N D T , J . E N G L I S H a n d J . D . P A R K E S

University Department of Neurology, King's College Hospital and Institute of Psychiatry, London SE5 9RS, UK School of Biological Sciences, University of Surrey, Guildford, UK University of Lille, France

Abst rac t

T h e delayed sleep phase s y n d r o m e is a form of i n somnia with inabil i ty to fall as leep at conven t iona l t imes b u t wi th n o r m a l sleep at a de layed clock t ime in respect of l i g h t - d a r k cycles, social , e c o n o m i c a n d family d e m a n d s . T h e s y n d r o m e is the m o s t c o m m o n intr insic d i sorder of h u m a n s leep-wake rhy thmic i ty wi th a r epo r t ed p o p u l a t i o n preva lence ra te of be tween 0.1 a n d 0 . 4 % . In some clinics it a c c o u n t s for 2 - 5 % of all subjects wi th a p r i m a r y compla in t of evening i n somnia . T h e s y n d r o m e is familiar in a b o u t one q u a r t e r of all subjects , m o r e c o m m o n in males t h a n females a n d usual ly presen t a r o u n d p u b e r t y . It needs to be d is t inguished from mo t iva t ed s leep-phase delay assoc ia ted wi th psychia t r ic p r o b l e m s , psychologica l factors o r school avo idance behav io r in adolescence . T h e delayed sleep phase s y n d r o m e is life-long a n d m a y involve several biological r h y t h m s coup led to the s leep-wake cycle inc luding core t e m p e r a t u r e , a ler tness a n d m e l a t o n i n r h y t h m s .

T h e p l a s m a m e l a t o n i n pulse d u r a t i o n a n d a m p l i t u d e is n o r m a l , wi th a slight de lay in low-light m e l a t o n i n onset as c o m p a r e d wi th subjects wi th a n o r m a l s leep-wake schedule in a similar e n v i r o n m e n t . Light suppress ion ( m o d e r a t e in tensi ty : 600 lux a t 00 .01-00 .30) of me la ton in secret ion is of n o r m a l a m p l i t u d e . M e l a t o n i n 5 m g p o will a d v a n c e s leep-phase bu t the response is n o t m a i n t a i n e d o n s topp ing t r e a t m e n t a n d d e p e n d s o n the exact t iming of me la ton in a d m i n i s t r a t i o n in re la t ion to the e n d o g e n o u s m e l a t o n i n cycle. T h e phase -response curves of s leep-wake cycles, b o d y t e m p e r a t u r e a n d sal ivary m e l a t o n i n profile after ora l m e l a t o n i n are descr ibed .

T r e a t m e n t s t rategies inc lude the use of c h r o n o t h e r a p y wi th a round- the -c lock successive 2 h o u r delay in scheduled sleep onset t ime, p h o t o t h e r a p y (10,000 lux for 30 minu te s a t 08.00 for 14 days ) , a n d m e l a t o n i n 5 m g p o at 22.00. Also, v i t amin Β12 t r e a t m e n t has been r epor t ed effective in s o m e b u t n o t all subjects .

T h e cause of the de layed sleep p h a s e s y n d r o m e is unce r t a in , bu t b o t h genet ic a n d e n v i r o n m e n t a l factors a re involved. T h e r e is n o evidence of in t ra -u te r ine or pe r ina ta l invo lvement a n d the r epo r t ed incidence of b reas t feeding is n o r m a l . C h i l d h o o d d e v e l o p m e n t is a lso n o r m a l . B o t h p a t e r n a l a n d m a t e r n a l inher i t ance have been r epo r t ed . O n e m e c h a n i s m , as s h o w n by the occas iona l progress ive delay in sleep p h a s e m a y involve the nea r 25 -hour pe r iod of the e n d o g e n o u s p a c e m a k e r . Also , there m a y be a failure of n o r m a l e n t r a i n m e n t m e c h a n i s m s in the a d v a n c e p h a s e of the s leep-wake cycle.

261


In t roduct ion

I N THE London Spectator of 26 April 1711, there is a report of a group of philosophers at Cambridge and Oxford Universities called "the Loungers" .

1 Also, W.B. Yeats in "The Trembling of the Veil" refers to the

long literary tradition of a delayed sleep phase syndrome

1

"When I had first gone to see him in 1889 at the Charlot te Street house I had called at about 5 in the afternoon but the manservant told me he was not yet up , adding with effusion: 'He is always up for dinner at Τ This habit of breakfasting when others dined had been started by insomnia, but he came to defend it for its own sake ."

1

Clinical features

The delayed sleep phase syndrome was first described by Weitzman et al. in 1981 in a group of insomniac patients who complained of inability to fall asleep at a socially acceptable time but were able to sleep normally at a clock-time independent of social, economic and family demands .

2 The

subjects comprised about 7 % of the total populat ion presenting at a Sleep Disorders Clinic with insomnia. Delayed sleep phase often began in childhood and on the owl-lark, "morning-evening" scale derived by H o m e and Ôstberg, all the subjects were extreme evening types .

3 Thirteen of 30

subjects had a psychiatric disorder but the nature and distribution of this was no different from other insomniacs. Almost every subject had taken many different sedatives without success.

In the last 10 years about 75 subjects with the delayed sleep phase syndrome have been r e p o r t e d .

4"

15 The age of onset varies from infancy to

the 6th decade and is most often at adolescence. The sex incidence was equal in the 30 subjects originally reported by

Weitzman et al.

2 but most reports have stressed male predominance. The

syndrome is similar in children and adults, characterized by habitual sleep onset and wake times several hours later than desired. Children and adolescents have a poor school record and often truancy whilst adults often have many short periods of employment, marital and financial problems and increasing social i so la t ion .

10

A recent Norwegian epidemiological study suggests a fairly high populat ion prevalence rate for the delayed sleep phase syndrome of between 0.1 and 0 . 4 % .

16 Possible latitude variation was not recorded in

this study. The delayed sleep phase syndrome has been distinguished from a

motivated sleep phase syndrome in adolescents, where school avoidance, psychological and psychiatric factors are usually very prominent with more normal sleep patterns during school hol idays .

5

Delayed Sleep Phase Syndrome 263

Diagnostic cr i ter ia

We have adopted the following diagnostic criteria for the delayed sleep phase syndrome from those of Weitzman et al, Thorpy and the American Sleep Disorders Association C lass i f i ca t ion .

2 10

(1) Complaint of inability to fall asleep and wake spontaneously at the desired clock-time.

(2) A phase delay of the major sleep episode in relation to the desired time for sleep.

(3) Symptoms present for at least 12 months . (4) N o evidence of any medical, psychological, environmental or

psychiatric factor sufficient to explain the above symptoms. (5) Absence of any anatomical lesion on head C T scan; and normal

polysomnographic findings apar t from an atypical time of sleep onset and waking.

Technological problems in the study of the delayed sleep phase syndrome

In our investigation of subjects with the delayed sleep phase syndrome we have met a number of problems with technology. These have included:

(1) Difficulties with accurate core temperature continuous measurement . Available systems need to be refined. Few patients can tolerate an indwelling rectal probe for long periods and these are often displaced during sleep periods. This technology is not very suitable for use in the home environment. There have been frequent problems in our experience with the CorTemp gut capsule broadcast ing system—in particular with the signal reception via flimsy antenna devices.

(2) Cont inuous melatonin sampling from plasma or saliva raises formid-able problems in the home or ward environment over the long term. Salivary samples interrupt sleep; a t tempts to use a dental sucker without arousing the subject have not been successful in our experience. Intravenous cannula require constant monitoring, the use of heparin to keep patent and may need re-inserting into different veins. In the long term, urinary melatonin metabolite sampling may be the only practical technique but will not allow a detailed analysis of melatonin rhythms.

Details of subjects with the delayed sleep phase syndrome seen in a 5-year period at the King's College Hospi ta l /Maudsley Hospital Sleep Disorders Clinic are shown in Table 1.

TA

BL

E

1

Clin

ical

fe

atur

es

of 1

4 ca

ses

of d

elay

ed

slee

p ph

ase

synd

rom

e

Sle

ep-w

ake

Pa

ram

ete

rs

HL

A

Ty

pe

Dec

imal

ti

me

IQ

Sle

ep

Ag

e at

F

amil

y D

evel

op

men

tal/

o

nse

t W

ak

e A

lert

nes

s C

ase

Ag

e S

ex

pu

ber

ty

his

tory

en

vir

on

men

tal

fact

ors

D

R

DQ

m

ea

n m

ea

n ac

rop

has

e P

erfo

rman

ce

Ver

bal

1 14

M

8

14

a P

rolo

ng

ed

lab

ou

r l,

x l,

x 0

1.4

8 11

.90

19.2

2 8

0 7

5 2

39

M

33

14

EB

V1

2,6

l,x

07

.95

11.8

1 0

3.4

3 12

5 10

3 3

18

M

16

13

b P

rem

atu

re

bir

th

EB

V1

3,x

* 0

8.1

0 17

.15

20

.19

103

74

4 6

1 M

4

5 13

4

,13

1,3

04

.05

15.8

7 0

4.6

0 12

6 9

2 5

22

M

12

15

Bir

th

hy

po

xia

1,

2 1,

3 0

1.1

7 0

8.1

2 0

1.9

3 11

9 7

6 6

20

M

8 15

2

,11

1,3

02

.68

11.0

0 15

.75

101

70

7 42

M

2

7 16

c

2,x

l,x

03

.25

10.8

0 2

0.7

2 10

8 9

3 8

58

M

53

14

Infl

uen

za2

1,7

1,2

05

.30

17.3

0 2

3.5

0 12

6 11

2 9

22

M

16

12

1/1

0,4

1,

3 0

0.2

2 1

2.0

0 10

.18

NT

N

T

10

14

M

10

11

c 8,

4 3,

x 0

3.0

0 12

.28

20

.11

NT

N

T

11

17

M

25

15

Pro

lon

ged

la

bo

ur

4,6

1,3

01

.48

12.0

4 13

.59

111

99

12

39

M

8 16

d

2,3

1,2

01

.55

08

.01

04

.46

126

86

13

71

M

18

12

d 2,

6 l,

x 0

2.0

0 0

9.0

0 2

3.0

5 N

T

NT

14

28

F

18

13

ado

pte

d T

on

ic-c

lon

ic

seiz

ure

s, a

ge

12

* *

05

.00

16.1

0 N

T

NT

86

a F

amil

y h

isto

ry

of d

ayti

me

slee

pin

ess.

b

Fam

ily

his

tory

of

nar

cole

psy

an

d ca

tap

lex

y,

c F

ath

er

an

d so

n.

d F

ath

er

an

d so

n.

1 O

nse

t of

del

ayed

sl

eep

ph

ase

syn

dro

me

foll

ow

ing

Ep

ste

in-B

arr

v

iral

in

fect

ion

. 2

On

set

afte

r in

flu

enza

-lik

e il

lnes

s.

* F

aile

d H

LA

ty

pin

g.

NT

N

ot

test

ed.

Sle

ep-w

ake

par

amet

ers

giv

en

in

dec

imal

ti

me,

i.

e.

01

.30

= 0

1.5

0.

Sle

ep-w

ake

da

ta

der

ived

fr

om

m

ea

n 2

8-d

ay

slee

p lo

gs.

A

cro

ph

ase:

ti

me

of

sub

ject

ive

hig

hes

t al

ertn

ess

self

rat

ing

(co

mp

uti

ng

da

ta

by

cosi

no

r an

aly

sis:

fro

m

me

an

28

-day

se

lf

reco

rdin

g).


(3) M o t o r activity recording with act imetry is a simple technique but will not separate for example sleep from quiet immobil i ty.

(4) There are considerable difficulties in determining tempera ture phase shifts over a short t ime span if these are minor . F o r example, it is very difficult to evaluate the possible effect of a single small oral dose of mela tonin on the tempera ture rhy thm over a single 24-hour cycle.

(5) Pract ical pat ient considerat ions . Few subjects will tolerate detailed prolonged circadian studies in their no rmal envi ronment and the use of a chronobiology labora tory , with, e.g. a 40-hour protocol , is a lmost certainly necessary for analytical studies.

Delayed, advanced and non-24 hour s l e e p - w a k e phase syndromes

As well as the delayed sleep phase syndrome, a number of less c o m m o n rhythmic and ar ry thmic sleep-wake pat terns have been described including the rare advanced sleep phase syndrome. Non-24-hour sleep-wake pat terns are sometimes found in blind or mentally re tarded i n d i v i d u a l s .

1 7'

18 In these condi t ions , the exact sleep-wake pa t te rn may be

determined largely by life-style, social and other time cues and different pa t terns of sleep phase may occur in the same individual. In the delayed sleep phase syndrome prevalence study in N o r w a y referred to above , no subject with advanced sleep phase was identified.

Pathophysio logy of the delayed sleep phase syndrome

The clinical presentat ion of the delayed sleep phase syndrome, with normal sleep architecture a l though at an unusual t ime as well as the normal quality of wakefulness suggest a chronobiological disorder with preserved mean levels and ampli tudes of sleep-wake and sleep-wake coupled biological rhy thms including those of the tempera ture and melatonin cycle: in contras t to an expected delay in tempera ture and melatonin cycles accompanying the delay in sleep onset and wake times, alertness and m o t o r activity rhy thms . It should be noted tha t in some blind subjects, t empera ture and melatonin rhy thms are not always closely coupled to the sleep-wake cycle.

It has been shown that oral mela tonin causes a minor sleep phase advance in the delayed sleep phase syndrome a l though the effect of melatonin on sleep-coupled rhy thms has no t been determined. The actions of melatonin would be expected to depend on the exact t iming of melatonin adminis t ra t ion in relation to the endogenous mela tonin cycle and to show a definite phase-response curve. Present da t a suggest that the response to mela tonin is greatest in the 6 hou r period prior to the


endogenous melatonin p e a k .

19 However , in the delayed sleep phase

syndrome it has been suggested there may be a diminshed phase advance capabi l i ty .

7

The delayed sleep phase syndrome and mela ton in

(a) Endogenous melatonin rhythmicity

Detailed information on p lasma melatonin and ur inary excretion of the melatonin metabol i te a M T 6 s as clock markers in the delayed sleep phase syndrome in the free-running condit ion is not available. In a fixed 24 hour l ight .dark wake.bed schedule, Dahl i tz et al showed tha t p lasma melatonin urinary 6-sulphatoxy melatonin profiles, pulse dura t ion and peak concentrat ions were within the normal range for men aged 20-60 living under normal l ight:dark environmental condi t ions. However there was a minor delay in plasma melatonin acrophase as compared with normal subjects (mean 04.43 + 0.67 ν 03.15 ± 1.00: decimal times + 1 S D : groups not identical in number and a g e ) .

15

(b) Effect of exogenous melatonin

Exogenous melatonin 5 mg at 22.00 given 5 hours pr ior to the est imated mean time of sleep onset in the delayed sleep phase syndrome results in a mean advance in sleep onset time of a little over 60 minutes , and also results in an advance in wake time without pro longat ion of the total sleep time. It has not been established if this results from a specific phase-advancing ra ther than minor hypnot ic effect of melatonin on sleep—or indeed whether these two actions are separate . However , in most normal subjects, melatonin 5 mg does not have a reliable hypnot ic effect. In the delayed sleep phase syndrome, the advance is not mainta ined when melatonin is d i s con t inued .

15 The magni tude of the phase shift in sleep

onset time in the delayed sleep phase syndrome is closely comparable to that produced by the same dose of melatonin (5 mg) in normal subjects after a simulated time shift. (A single individual has been described with a delay, not advance, in sleep phase with m e l a t o n i n .

2 0) A sleep phase shift of

similar magni tude occurs in blind sub jec t s .

19

The magni tude of phase shift in endogenous melatonin rhythmicity induced by exogenous melatonin 5 mg may be of slightly greater magni tude than the advance in sleep phase produced by the same dosage of melatonin at the same phase of the endogenous melatonin cycle. Arendt et al. reported a 1-3 hour phase advance in the rhy thm of endogenous melatonin in 5 of 11 normal subjects given melatonin 2 m g .

21


(c) Light suppression of melatonin

Although h u m a n light sensitivity is less than tha t repor ted in m a n y other animal species, a short low intensity light exposure will result in part ial suppression of h u m a n mela tonin p roduc t ion or r e l ea se .

22 T h e degree of

mela tonin suppression by light (600 lux—this m a y be above the threshold to detect individual var iat ion in sensitivity) found in the delayed sleep phase syndrome falls within the range repor ted in normal subjects at 00.00-00.30, and there is no evidence from this da t a of ana tomica l involvement of ret inal-pineal-melatonin mechanisms in the delayed sleep phase syndrome a l though the phase-response curve to light has no t been d e t e r m i n e d .

23 Bojkowski et al. (1987) repor ted that 300 lux induced

differential suppression (from 0 - 1 0 0 % ) in no rma l s u b j e c t s

24 (see Table 2).

Phase-response curve t o me la ton in in delayed sleep phase syndrome

Subjective response

Mela tonin 5 mg was given single blind as to d rug time in four subjects (subjects no 2, 8, 14, Table 1 and one addi t ional female subject) with the delayed sleep phase syndrome at 06.00, 12.00, 18.00 and 00.00 under a fixed l ight-dark wake-bed schedule repeated at 1 week intervals on four separate occasions. Two subjects, bo th female, described a subjective 1-2 hou r advance in sleep phase following mela tonin adminis t ra t ion, one at 06.00, one at 18.00 but not at o ther t imes. The two male subjects gave no preference for t ime of mela tonin adminis t ra t ion . In a s tudy of blind and sighted subjects, Lewy et al. found a mean sleep phase advance of 38 minutes in the sighted g roup following mela tonin 5 mg, the m a x i m u m advance occurring when mela tonin adminis t ra t ion preceded endogenous melatonin onset by abou t 6 hours , less phase advance when the interval was greater than 6 h o u r s .

19

Temperature rhythm in delayed sleep phase syndrome

The original description of the delayed sleep phase syndrome made no ment ion of the characteristics of other rhy thms a l though Wagner et al. subsequently described a late phase of tempera ture nadir in a single subject with the delayed sleep phase s y n d r o m e .

7

In a s tudy in tempora l isolation, six subjects with the delayed sleep phase syndrome had the nadir of tempera ture in the last 2 hours of sleep compared with one of eight no rmal subjects.

The circadian rhy thm of body tempera ture is coupled to the t iming of R E M sleep as well as mela tonin excretion in m a n , the body tempera ture

TA

BL

E

2

Lig

ht

supp

ress

ion

of m

elat

onin

in

the

de

laye

d sl

eep

phas

e sy

ndro

me

Tim

e

Co

nst

ant

dar

k 2

3.0

0-0

8.0

0

60

0 lu

x 0

0.0

0-0

0.3

0

20

.00

21

.00

5 +

5 1

1+

6

2 +

2 9

+ 3

22

.00

16 +

7

23

.00

18 +

8

00

.00

28 +

10

0

0.3

0 2

9 +

10

01

.00

29

+1

2 0

1.3

0 3

4+

10

02

.00

03

.00

37 +

13

39 +

13

10 +

4

14 +

5

18 +

7

25

+ 10

16

+ 6

22

+ 9

31

+ 1

2 38

+

15

LIG

HT

M

ea

n P

lasm

a m

elat

on

in

(+1

SE

M:p

g/m

l)

η =

9

Del

ayed

sl

eep

ph

ase

syn

dro

me

Co

nd

itio

ns

of c

on

stan

t d

ark

, 2

3.0

0-0

8.0

0;

co

mp

are

d w

ith

60

0 lu

x li

gh

t ad

min

istr

atio

n at

0

.00

-00

.30

.


low corresponding to the mela tonin a c r o p h a s e .

2 5 - 27 A slight delay in the

phase of core body tempera ture from that of no rma l subjects in the na tura l environment would be expected to accompany delayed sleep phase periods in the delayed sleep phase syndrome. This may be the case despite problems of masking as well as of possible differences in correlat ions between spontaneous , and p lanned, sleep times.

T r e a t m e n t strategies in the delayed sleep phase syndrome

F o r successful t rea tment , the delayed sleep phase syndrome should be distinguished from the mot ivated sleep delay syndrome. However , despite the diagnostic criteria outl ined, it can be difficult to separate the two condit ions.

Whatever app roach to managemen t is used, good subject compliance is essential and an exact t rea tment pro tocol is necessary. Wi thou t bo th factors, success is u n c o m m o n . Trea tment is rarely practical in the h o m e environment ; in part icular , phase advance schedules are no t practical at home in young children. See Table 3 for var ious t rea tment strategies.

T A B L E 3

Treatment strategies

Stra tegy

Chronotherapy

A r o u n d the clock p h a s e delay wi th successive 2 h o u r delay in bed t ime unti l the new desired sleep phase is reached

Phototherapy 10,000 lux (Wolff L ight ing Systems) for 30 minu te s a t 08.00 for 14 days . This requi res enforced a rousa l a t 08.00.

Vitamin Β12 100—1000 m i c r o g r a m s dai ly: o ra l o r sc: for 4 weeks a t 08.00 (requires enforced a rousa l a t 08.00)

Melatonin 5 m g p o for 2 - 4 0 weeks at 22.00

Morning CNS stimulants ( a m p h e t a m i n e s ) with Evening hypnotics (benzodiazepines)

Overa l l results in o u r exper ience

In ado lescen t s , success ra te a b o u t 5 0 % . N e w sleep p h a s e is re ta ined for 3-8 m o n t h s .

Var iab le r e sponse in ado lescen ts ; ? be t te r resul ts in adu l t s

N o effect

Pa r t i a l a d v a n c e in sleep p h a s e m a i n t a i n e d d u r i n g t r e a t m e n t pe r iod

Successful in 1 adu l t subject ; unsuccessful in o the r s


Conclusion

The delayed sleep phase syndrome has a fairly characteristic phenotype. It has been recognized with increasing frequency in the last decade. In our experience it is sometimes difficult to disentangle the role of social, environmental , psychological, psychiatric factors. There is increasing evidence for phase delay in a number of biological rhythms coupled to the sleep-wake cycle including melatonin and temperature al though the phase of alertness and moto r activity rhythms during wakefulness may depend largely on environmental factors.

Although the syndrome is likely to be multi-factorial, about one quarter of cases have an affected first degree relative and genetic factors may be involved. The anatomical integrity of the retinal-pineal pathways appears to be intact as indicated by normal light suppression of the melatonin rhythm al though the physiological function of this pa thway has not yet been established. However, there may be a low response in the advance port ion of the phase-response curve or subjects may have an unusually long endogenous cycle. N o treatment is universally successful. Chrono-therapy with high motivat ion and exact schedule may be the best initial option, followed by a trial of light or melatonin if the first approach is unsuccessful. Perhaps the combinat ion of light with melatonin may prove to be the most effective remedy.

Acknowledgments

J.V. was supported by a grant from the British Council, M . D . by the King's College Hospital Joint Research Commit tee. We gratefully acknowledge the helpful review of D . Minors and J. Waterhouse and the assistance of J. Gr imshaw.

References

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2. Wei t zman E .D. , Czeisler C.A., C o l e m a n R .M. , Spielman A.J., Z i m m e r m a n J . C , D e m e n t W., R ichardson G . a n d Pol lak C P . (1981) Delayed sleep phase syndrome . A chronobiologica l d isorder with sleep-onset insomnia . Arch. Gen. Psychiatry 38(7) , 737-46 .

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5. Ferber R. and Boyle M . P . (1983) Delayed sleep phase synd rome versus mot iva ted sleep phase delay in adolescents . Sleep Res. 2 1 , 239.

6. Ferber R. and Boyle M . P . (1983) P h a s e shift dyssomnia in early ch i ldhood. Sleep Res. 12, 242 (abstract) .

7. W a g n e r D.R. , M o t i r a M . C , Pol lak C P . a n d Czeisler C.A. (1986) En t ra ined sleep and t empera tu re rhy thms in delayed sleep phase syndrome . Sleep Res. 15 , 179 (abstract) .


8. Pe layo R .P . , T h o r p y M J . a n d Glovinsky P . (1988) Prevalence of delayed sleep phase synd rome a m o n g adolescents . Sleep Res. 17, 392.

9. K a m g a r - P a r s i B. , W e h r T.A. and Gillin J .C . (1983) Successful t r ea tmen t of h u m a n non -24-hour s leep-wake synd rome . Sleep 6(3), 257-264 .

10. T h o r p y M.J . , K o r m a n E., Spei lman A.J. and Glovinsky P . B . (1988) Delayed sleep phase s y n d r o m e in adolescents . J. Adolesc. Health Care 9 , 22 -27 .

11. Ozak i N . , Iwa ta T., I t oh Α., O h t a T., O k a d a T. a n d K a s a h a r a Y. (1989) A t r ea tmen t trial of delayed sleep phase s y n d r o m e with t r i azo lam. J. Neurol. Neurosurg. Psychiat. 43 , 51 -55 .

12. Ozak i N . , Iwa ta T., I toh Α., K o g a w a S., O h t a T. , O k a d a T. a n d K a s a h a r a Y. (1988) Body t empera tu re mon i to r ing in subjects with delayed sleep phase synd rome . Neuropsychobiology 20, 174-177.

13. O k a w a M . , Mi sh ima K., N a n a m i T., Shimizu T. , Iijima S., H i sh ikawa Y. a n d T a k a h a s h i K. (1990) Vi tamin Β12 t r ea tmen t for s leep-wake r h y t h m disorders . Sleep 13, 15-23 .

14. deBeck T .W . (1990) Delayed sleep phase syndrome—cr imina l offence in the mil i tary? Milit. Med. 155, 14-15.

15. Dahl i tz M.J . , Alvarez B., Vignau J., English J., Arend t J. a n d Pa rkes J . D . (1991) Delayed sleep phase synd rome response to mela ton in . Lancet 337, 1121-1124.

16. Schrader H. , Bovin G . a n d Sand T. (1992) Prevalence of delayed a n d advanced sleep sleep phase synd rome . J. Sleep Res. 1 Suppl . 1 415, 208 (abstract) .

17. K a m e i R., H u g h e s L., Miles L. and D e m e n t W . (1979) Advanced sleep-wake s y n d r o m e studied in a t ime-isolat ion facility. Chronohiology 6 , 115.

18. Weber A.L. , Ca ry M.S . , C o n n o r N . et al. (1980) H u m a n non-24-hour s leep-wake cycles in an everyday env i ronment . Sleep 2, 347-354 .

19. Lewy A.J., Sack R.L. a n d L a t h a m K . L . (1990) Exogenous mela ton in phase shifts c i rcadian r h y t h m s accord ing to a phase response curve. Proceedings of E u r o p e a n Pineal S tudy G r o u p Universi ty of Gui ldford , 2 -7 September , Abs t rac t 0 2 1 .

20. Samel Α., W e g m a n n H . M . a n d Vejvoda M . (1992) Ci rcad ian a d a p t a t i o n of the s imulated t ime shifting effect of mela ton in . J. Sleep Res. 1 Suppl . 1 205 (abstract 410).

2 1 . Arend t J., Bojkowski C , F o l k a r d S. et al. (1985) Some effects of me la ton in and the cont ro l of its secretions in h u m a n s . In Photoperiodism, Melatonin and the Pineal Ciba Foundation Symposium 117, p p . 266-283 . P i t m a n , L o n d o n .

22. M c l n t y r e I .M. , N o r m a n T.R., Bur rows E . G . D . and A r m s t r o n g S.M. (1989) Q u a n t a l mela ton in suppress ion by exposure to low intensity light in m a n . Life Sci. 45 , 327-332 .

23 . Vignau J., Dahl i t z M. , P a r k e s J .D . , Alvarez B., Arend t J. a n d English J. (1992) T h e delayed sleep phase s y n d r o m e : light suppress ion of mela ton in . / . Sleep Res. 1 Suppl . 1 243 (abstract 485).

24. Bojkowski C , A ldhous M. , English L., F r a n e y C. et al. (1987) Suppress ion of noc tu rna l p lasma mela ton in a n d 6-sulphatory mela ton in by br ight and d im light in m a n . Hormone Metabol. Res. 18, 4 3 7 ^ 4 0 .

25 . Czeisler C.A., Z i m m e r m a n J . C , R o n d a J . M . a n d M o o r e - E d e M . C . (1980) T iming of R E M sleep is coupled to the circadian r h y t h m of b o d y t empera tu re in m a n . Sleep 2, 329-346.

26. Lewy A.J. a n d Sack R.L. (1989) T h e d im light me la ton in onset is a m a r k e r for c i rcadian phase posi t ion. Chronohiol. Int. 6 , 93 -102 .

27. Wever R.A. (1986) Character is t ics of c i rcadian r h y t h m s in h u m a n functions. / . Neural. Transmis. 2 1 , 323-374.

19

The Influence of Age, Sex, Height, Weight, Urine Volume and Latitude on Melatonin Concentrations in Urine from Normal Subjects: A Multinational Study L E N N A R T W E T T E R B E R G ,

1 G O R A N E B E R H A R D ,

2 L A R S V O N K N O R R I N G ,

3

Μ . Α . Κ Ο Η Α Ν ,

4 EVA Y L I P Â À ,

5 W O L F G A N G R U T Z ,

6 T R O N D B R A T L I D ,

7 V E R E N A

L A C O S T E ,

8 C H R I S T O P H E R T H O M P S O N ,

9 H I R O S H I Y O N E D A ,

10 N O R M A N E.

R O S E N T H A L ,

11 M I C H A E L M c G U I R E ,

12 A L E S S A N D R O P O L L E R I ,

13 M A R G A

F R E E D M A N ,

14 D. J . M O R T O N ,

15 J E N N Y R E D M A N ,

16 J O A N N E D. B E R I G A N N A K I ,

17

C O L I N S H A P R I O ,

18 H E L E N D R I V E R

18 a n d A R T H U R Y U W I L E R

19 1Karolinska Institute, Department of Psychiatry, St Goran's Hospital, Stockholm, S-11281

Sweden;

2University of Lund, Department of Psychiatry, Ma/mo Hospital, Sweden;

3University of Uppsala, Department of Psychiatry, Uppsala;

4Mare del Plata, Republica

Argentina;

5Department of Psychiatry, St. Goran's Hospital, Stockholm, Sweden;

6Psykiatriska kliniken, Visby lasarett, Visby, Sweden;

7Àsgàrd sykehus, Ν-9010 Àsgàrd,

Norway;

8Psychiatrische Universitàts Klinik Basel, 4025 Basel, Wilhelm Klein-Strasse 27,

Switzerland;

9Department of Psychiatry, 1q Royal South Hants., Hospital, Graham Road,

Southampton, Hants S09 4EE, England;

10Department of Neuropsychiatry, Osaka Medical

College, Daigakumachi 2-7, Takatsuki City, Osaka 569, Japan;

11 Outpatient Services,

Clinical Psychobiology Branch, NIMH, Room 4S-239 Building 10, 9000 Rockville Pike, Bethesda, MD 20892, USA;

12NPI, UCLA, 760 Westwood Plaza, Los Angeles, CA 90024,

USA;

13University of Genoa, Department of Endocrinology and Metabolic Sciences, Chair of

Internal Medicine, Via le Benedetto XV, 6, 16132 G e nova, Italy;

14Departemento. de

Fisiologia, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 7ο Piso, CP. 1121 Buenos Aires, Argentina;

15Harare University, Faculty of Medicine, Ρ Ο Box MP

167, Mt Pleasant, Harare, Zimbabwe;

16Department of Psychology, Monash University,

Victoria, Australia;

17Athens University Medical School, Department of Psychiatry, Eginition

Hospital, Athens, Greece;

18Department of Psychiatry, Toronto Western Hospital, 399

Bathurst Street, Toronto, M5T Canada and Department of Physiology, University of Witwatersrand, Johannesburg, South Africa;

19Department of Psychiatry and Biobehavioral

Sciences, University of California at Los Angeles, Los Angeles, CA 90024, USA and VA Medical Center, West Los Angeles (Brentwood Division) Los Angeles, CA 90073, USA

In t roduct ion

T H E DAILY rhythmic rise and fall of mela tonin formation is driven by the nightly release of norepinephr ine from sympathet ic terminals innervat ing

2 7 5


the pineal g l a n d .

20 This rhy thm reflects the free-running activity of a

central pacemaker in the suprachiasmat ic nucleus which is synchronized to the daily 24 hou r cycle by light information passed to it by the r e t i n a .

24

Because melatonin formation involves the st imulated metabol ism of one m o n o a m i n e by the t ransmit ter actions of ano ther in accordance with the coupling between an endogenous , free-running pacemaker and light, it has been studied in a number of clinical condit ions as a possible marke r for endogenous rhythmicity and for noradrenergic activity. Syndromes associated with possible metabol ic dysynchrony such as d e p r e s s i o n ,

5'

9'

1 0'

1 8'

2 1'

2 2'

2 3'

2 5'

3 4'

3 6'

4 0'

42 seasonal affective

d i s o r d e r ,

11 '

1 9'

29 premenst rual s y n d r o m e ?

1 6'

3 7'

3 8>

42

a n (j degenerative diseases of the e l d e r l y ,

3 1'

3 2'

33 have been given part icular a t tent ion. Age

and sex distr ibutions in these popula t ions often differ from tha t in the general popula t ion and defining the influences of sex and age on melatonin metabol ism and distr ibut ion is of obvious impor tance to s tudy design and da ta interpretat ion.

The association between mela tonin and age has been studied in bo th animals and m a n with the general consensus that mela tonin product ion shows a post -puber ta l decline with a g e

7'

1 4'

1 7'

2 6'

2 7>

2 8'

3 0>

3 2>

35 a l though this

relat ionship has no t been seen in all s t u d i e s .

2'

5'

1 2'

43 There is less

information on gender differences in mela tonin product ion in m a n a l though Tou i tou et α / .

35 repor ted slightly higher p lasma melatonin in

women and Wetterberg et al*

1 found a similar relat ionship in one

popula t ion of control bu t no t another . These differences may be due to the statistical vagaries associated with small popula t ions or to environmental or seasonal influences a l though seasonal effects in m a n may be m o d e s t .

1 5'

30

A recent worldwide mul t inat ional study on seasonal and geographic influences on h u m a n mela tonin produc t ion provided multiple urine samples from a large popula t ion of geographically dispersed normal subjects. In tha t study, month ly measures of nightt ime ur inary melatonin concentra t ion were obta ined th roughou t the year on 370 normal subjects at 19 centers in 14 countries distr ibuted on 5 cont inents at lati tudes from 3 Γ 0 Γ South to 69°40' N o r t h . Comple te demographic and related da ta was also available on 321 of the 370 subjects. A value for "yearly" average nightt ime ur inary mela tonin concentra t ion could therefore be calculated for each individual which would be more representative than one obtained on a r a n d o m sample at some unspecified t ime of the year. Fur ther , because sampled popula t ion consisted of heal thy subjects of varying ethnicity living in widely different climatic, dietary, social and geographic circumstances, the g roup da ta should be widely generalizable.

The present paper reports on the relat ionship between yearly nightt ime melatonin concentra t ion and sex, age, height, weight and urine volume on the 321 subjects for w h o m da ta was complete on these measures . Finally,

Age, Sex and Urinary Melatonin 277

Analytical procedure

Urine was analyzed for mela tonin using a specific r ad io immunoassay developed for use with urine and blood samples (Wetterberg et α/., 1978).

the relat ionship between geographic location and mela tonin was exam-

ined.

M e t h o d s

Subjects

Subjects were 321 heal thy volunteer university s tudents and faculty (165 men and 156 women) , aged 18-62 at medical centers t h roughou t the world for w h o m information on sex, age, height, weight, urine volume and urinary melatonin concentra t ion was available. Wri t ten informed consent was obta ined from all subjects. Volunteers were medically and psychiatri-cally screened to exclude subjects with somatic or mental disease and subjects taking medicat ions. Use of oral contraceptives was permit ted provided they were employed consistently th roughou t the s tudy period.

Sample collection

Urine samples were collected on the first Wednesday of each m o n t h ( + one day) for a period of 12-16 mon ths . Subjects were given gradua ted plastic beakers to measure urine volume and plastic vials for s torage. They were instructed to empty the bladder at bedt ime (usually 10-11 p.m.) on the night of the collection period, discard the urine, and record the specific time of voiding. Thereafter the total urine produced dur ing the night, including the first morn ing ur inat ion (at approximate ly 7 a.m.), was collected into the g radua ted plastic beaker, the total volume and time of any nocturna l voiding recorded, and a por t ion of the total urine poured into the plastic bott le . The bott le was then taken in the morn ing to a collection point where bottles were frozen and stored at — 20°C. Samples were not taken from subjects traveling more than 300 miles (500 km) nor th or south of their residence dur ing the m o n t h preceding sampling. At the end of the study, all urine samples were t ranspor ted to Stockholm, Sweden on dry ice, where they arrived in frozen condi t ion, and were retransferred to — 20°C freezers until assayed. Previous studies have shown tha t ur inary melatonin is stable under these condi t ions of s torage and t r ans fe r .

37

Previous studies have also shown that ur inary melatonin concentra t ion in samples collected in this way correlates highly with the 2 a.m. peak value of serum melatonin (N=64; r = 0.8; p < 0 . 0 0 1 ) .

1


The assay had a sensitivity of 0.01 nmoles/1. Interassay variability was 4 . 8 % for melatonin levels above 0.15 nmol/1 ( N = 6 0 ) . Melatonin values were expressed as concentrat ion (nM).

Statistics

Correlations, scatter plots and estimates of statistical significance of correlations were computed using the B M D P programs 6D, 1R and 2R, while 2V was used for the analysis of gender difference covaried for age. For each subject the da ta used was the mean of all available monthly values for that individual. The log of urinary melatonin concentrat ion was used for computat ions of statistical significance because the log, but not the concentration itself, was normally distributed, ρ < 0.05 was considered significant.

Results

Demographic da ta on the subjects at the 19 centers are presented in Table 1. Mean ages ranged from 30 (Genoa) to 44 (Umea). Subjects at the most southerly site, M a r De Pla ta Argentina, consisting of one male and five females, were the shortest (mean height = 1 6 0 cm) and lightest (61 kg) while those at Visby Sweden, consisting of four males and six females, were the tallest (177 cm) and heaviest (77 kg). The correlations between age and melatonin at each center is shown in Table 2 and in Figure 1. Overall, the correlation coefficient was 0.19 for males, females, and both groups combined (p< 0.001 in each case). Correlation coefficients between age and melatonin at individual sites were negative in 15 and positive in 4 of the cases. The positive correlations were generally small (0.02, 0.04, 0.08 and 0.23) while negative correlations were generally more robust . Fou r of the negative correlations were statistically significant. There did not appear to be a relationship between latitude and the magnitude or sign of the correlation coefficient. However, as seen in Table 3, there was a statistically significant relationship between latitude and melatonin for the whole sample (R = 0.202, ρ < 0.001), for subjects in the nor thern hemi-sphere alone ( iV=241, i? = 0.157, p = 0.015) and there was a similar, but nonsignificant, correlation for subjects in the Southern hemisphere (7V = 80, 7? = 0.197, ρ = 0.81). There was no significant relationship between melatonin and longitude (R = —0.07; NS) .

Table 3 also presents correlations between melatonin and height, weight and urine volume. Height was not significantly correlated with melatonin (r= —0.004, 0.018 respectively) for males or females, but was significantly correlated for the whole group ( — 0.114; ρ < 0.04). Male body weight (R= - 0 . 1 7 ; p = 0.03), but not female body weight ( - 0 . 1 4 ; NS) , correlated

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F I G . 1. Scatter d i ag ram of ur inary mela ton in concen t ra t ion for males (closed circles) and females (open circles).

T A B L E 3

Correlations between melatonin concentration and height, weight, urine volume, latitude and longitude

Male F e m a l e To ta l

Me la ton in vs: R (P) R (P) R (P) Height - 0 . 0 0 4 (NS) - 0 . 0 1 8 (NS) - 0 . 1 1 4 (0.04) Weight - 0 . 1 6 6 (0.03) - 0 . 1 4 3 (NS) - 0 . 2 3 0 ( < 0.001) Ur ine V - 0 . 2 3 4 (0.002) - 0 . 2 3 9 (0.003) - 0 . 2 4 6 ( < 0.001) La t i tude 0.202 ( < 0.001) Long i tude - 0 . 0 6 8 (NS)

R — corre la t ion coefficient, ρ = probabi l i ty , N S = no t significant.

with melatonin while weight and melatonin were correlated for the entire group ( r = - 0 . 2 3 ; p < 0 . 0 0 1 ) .

Correlations between age and the other variables are shown in Table 4. The correlation between log melatonin and age was even stronger than that between melatonin and age. Normalizing melatonin to body weight did not materially alter the relationship between melatonin concentrat ion and age or between log melatonin and age. Melatonin content in the urine of males correlated with age but not in the urine of females or the melatonin content for both groups combined. As expected, urine volume was strongly negatively correlated with melatonin concentrat ion for males ( Λ = - 0 . 2 3 , p = 0.002), females {R=-024, p < 0 . 0 0 3 ) and bo th groups together (R= - 0 . 2 4 6 , ρ<0.001) (Table 3).

Gender differences are shown in Table 5. As might be expected, the sexes differ in height and weight. They also differ in melatonin concentrat ion. For this reason an analysis of variance with sex as covariate was carried


Male Female To ta l

Age vs: R (P) R (P) R (P) Mela ton in ( n M ) - 0 . 1 9 3 (0.01) - 0 . 1 9 0 (0.02) - 0 . 1 8 8 ( < 0.001) Log mela ton in - 0 . 2 4 5 ( < 0.001) - 0 . 2 3 7 (0.001) - 0 . 2 4 5 ( < 0.001)

(nM) Mela ton in (nM) /kg

wri - 0 . 2 4 0 (0.002) - 0 . 2 1 2 (0.008) - 0 . 2 0 4 ( < 0.001)

WI Log mela ton in - 0 . 1 7 7 (0.023) - 0 . 1 8 6 (0.02) - 0 . 1 7 8 ( < 0 . 0 0 1 )

(nM) /kg wt. Mela ton in (pmoles) - 0 . 1 4 9 (0.06) + 0.040 (NS) - 0 . 5 5 (NS) Height - 0 . 0 7 5 (NS) - 0 . 0 9 7 (NS) - 0 . 0 6 1 (NS) Weight - 0 . 2 4 3 ( < 0 . 0 1 ) - 0 . 1 4 7 (NS) - 0 . 1 4 7 (0.01) Ur ine V - 0 . 1 4 5 (NS) - 0 . 3 3 5 ( < 0.001) - 0 . 2 3 7 ( < 0.001)

R = Corre la t ion coefficient, ρ = probabi l i ty , N S = no t statistically significant.

T A B L E 5

Gender differences in melatonin, body height and weight, and in urine volume

Male Female

M e a n S D M e a n SE F Df Ρ

Height 178 7 165 7 298 1,319 < 0.0001 Weight 77 10 60 9 257 1,319 < 0.0001 Mela ton in 0.250 0.129 0.300 0.151 10 1,319 0.0016 U vo lume 409 139 386 145 2 1,319 0.2 N S log Mela ton in - 0 . 6 6 5 0.250 - 0 . 5 8 0 0.018 10 1,319 0.0017

out confirming the effect of age on melatonin (Df=l, 1, 318, F=12, ρ = 0.0006) with age adjusted mean melatonin concentrations of 0.250 + 0.129 for males and 0.300 + 0.151 for females.

Discussion

Our finding of a negative correlation between age and nighttime urinary melatonin concentrat ion is consistent with other reports in the l i t e r a t u r e

5 , 1 4 1 7'

2 6'

2 7 , 2 8 , 3 0 , 3 2 , 35 and seems generalizable because of the

diverse populat ion sampled. The magni tude of the correlation in this study, however, is considerably smaller than reported elsewhere (e.g. 0.47 by Sack et al.

30 measuring urinary 6-hydroxymelatonin and 0.38 by

Sharma et al.

32 measuring the acrophase of the blood melatonin rhythm),

perhaps due to environmental influences on the measures or to the differences in methodology. The unexpected correlation between latitude and melatonin could be interpreted as support ing an environmental influence on melatonin al though a genetic influence on melatonin from

T A B L E 4

Correlations between Age and melatonin concentration, height, weight and urine volume


geographically separated genetically different populat ions could also explain the results.

As seen in Table 1 the magni tude of the age-melatonin correlation varied considerably between centers and correlations between age and melatonin concentrat ion in these small populat ions only reached statisti-cal significance in 4 of the 19 participating centers (Stockholm, London , Genoa and Harare) . It approached significance in two others (Los Angeles and Osaka) .

The negative correlation between body height and urinary melatonin found in this study corresponds to the correlation found by Beck-Friis et ai

5 between height and plasma melatonin and by Sack et al.

30 between

height and urinary 6-hydroxymelatonin. However, it was much weaker and was only seen for the whole populat ion but not for males or females alone.

The inverse relationship between melatonin concentrat ion and over-night urine volume was expected as a natural consequence of the free passage of melatonin from blood into the renal tubule. Since blood melatonin peaks a round 2 a.m., urine passing into the bladder at this time is also high and concentrat ion is diluted by urine collecting before and after that period in accordance with the lowered blood levels at those times.

In agreement with Ferrier et al.

12 (who did not , however, find an

age-melatonin correlation) and Arend t ,

3 we found a significant correlation

between melatonin and weight. However, normalizing melatonin to body weight did not materially affect the correlation of melatonin with age indicating that age correlate is not a simple reflection of age-related weight changes.

Our finding of a gender difference in urinary melatonin product ion which persisted after age correction, supports and extends the observation by Toui tou et al

35 of higher blood melatonin in aged women than in aged

men. This does not appear to be accounted for by gender differences in body mass.

The mechanism accounting for the relationship between age and melatonin is not clear. Age-linked losses of noradrenergic neurones ,

8

decreases in catecholamine m e t a b o l i s m

13 or a fall in the pineal enzyme,

hydroxy-O-methyltransferase, which converts 7V-acetylserotonin to me la ton in

4 could each contribute to the relationship. A more practical

issue is that of evaluating the contributions of sex, age, height and weight to the results of studies on the clinical significance of melatonin product ion. While we find a consistent and statistically significant relationship between melatonin and age, the magni tude of the relationship is small accounting for less than 5 % of the variance. This may account for the the lack of agreement between studies on the linkage between age and melatonin. Therefore, these results with normals suggest that age would have a significant but small effect in studies compar ing populat ions


provided only that the quantitative relationship between age and melatonin is not altered by clinical state. Whether this proviso is true, however, needs to be established.

Similar considerations apply to assessing the influence of gender on studies of melatonin. Females do have significantly higher urinary melatonin concentrations than males after age correction but again the differences are relatively small so that incomplete sex matching of experimental and control populat ions is unlikely to result in significant populat ion differences when TV's are small but may lead to statistically significant, but physiologically trivial differences when TV's are large. Obviously good experimental design requires close matching for bo th age and sex.

References

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14. Grinevich Y.A. and Lebune tz I .F . (1986) Mela ton in , thymic serum factor and Cortisol levels in heal thy subjects of different age and pat ients with skin m e l a n o m a . J. Pineal Res. 3, 263-275 .

15. Griffiths P.A., F o l k a r d S., Bojkowski C , English J. and Arendt J. (1986) Persis tent 24-hr var ia t ions of ur inary 6-hydroxymela tonin sulfate and Cortisol in Antarc t ica . Experentia 15, 430-432 .


16. H a r i h a r a s u b r a m a n i a n N . , Na i r N . P . V . a n d Pilapil C. (1984) Ci rcad ian r h y t h m of p lasma mela ton in and Cortisol dur ing the mens t rua l cycle. In The Pineal Gland: Endocrine Aspects (eds. B r o w n G . M . a n d Wainwr igh t , S.D.), p p . 31 -36 . P e r g a m o n Press , Oxford.

17. Iguchi H. , K a t o K.J . a n d Ibayashi , H . (1982) Age-dependent reduct ion se rum mela ton in concen t ra t ion in heal thy h u m a n subjects. J. Clin. Endocrinol. Metab. 55 , 27-29 .

18. J imerson D . C , Lynch H.J. , Pos t R .M. , W u r t m a n R.J. a n d Bunney W . E . (1977) Ur ina ry mela ton in rhy thms dur ing sleep depr iva t ion in depressed pat ients a n d no rma l s . Life Sci. 2 0 , 1 5 0 1 - 1 5 0 8 .

19. K e v a n S. (1980) Perspectives on season of suicide: A review. Soc. Sci. Med. 14 ,3 6 9 - 3 7 8 . 20. Klein D . C , Weller J .L. a n d M o o r e R.Y. (1971) Me la ton in metabo l i sm: neura l

regulat ion of pineal sero tonin N-acetyl transferase activity. Proc. Natl. Acad. Sci. U.S.A. 6 8 , 2 1 0 7 - 3 1 1 0 .

2 1 . M c l n t y r e L, J u d d F . , N o r m a n T. a n d Bur rows G . (1986) P l a s m a mela ton in concent ra t ions in depression. Aust. NZ Psychiat. 2 0 , 381-383 .

22. Mendlewics J., Branchey L., Weinberg U. , Branchey M. , L inkowski P . and W e i t z m a n n E . D . (1980). T h e 24 h o u r pa t t e rn of p lasma mela ton in in depressed pat ients before and after t r ea tment . Commun. Psychopharmacol. 4 , 4 9 - 5 5

23 . Miles A. a n d Phi lbr ick D .R .S . (1988) Me la ton in a n d Psychia t ry . Biol. Psychiat. 2 3 , 4 0 5 ^ 2 5 .

24. M o o r e R.Y., Heller Α., W u r t m a n R.J. a n d Axelrod J. (1967) Visual p a t h w a y s media t ing pineal response to env i ronmenta l light. Science 155, 220-223

25. N a i r N .P .V . , H a r i h a r a s u b r a m a n i a n N . and Pilapil C. (1984) Ci rcad ian R h y t h m of p lasma mela ton in in endogenous depress ion. Prog. Neuropsychopharmacol. Biol. Psychiat. 8 , 715-718 .

26. Na i r N .P .V . , H a r i h a r a s u b r a m a n i a n N . , Pilapil C , Isaac I. a n d Thivundayi l J .X. (1986) P l a s m a mela ton in , an index of b ra in aging in h u m a n s ? Biol. Psychiat. 2 1 , 141-150.

27. P a n g S.F., T s a n g C.W. , H o n g G.X., Yip P .C . , T a n g P .L . a n d Brown G . M . (1990) F luc tua t ion of b lood mela ton in concen t ra t ions with age: result of changes in pineal mela ton in secretion, b o d y g rowth , and aging. / . Pineal Res. 8 , 179-92.

28. Reiter R.J., Craft C M . , J o h n s o n L.Y., K i n g T.S. , R icha rdson B.A., V a u g h a n G . M . a n d V a u g h a n M . K . (1981) Age -associated reduct ion in noc tu rna l pineal mela ton in levels in female ra ts . Endocrinol. 109 , 1295-1297.

29. Rosen tha l N . E . , Sack D.A. and Jacobsen F . M . (1986) Me la ton in in seasonal affective d isorder a n d p h o t o t h e r a p y . J. Neural Trans. (Suppl.) 2 1 , 257-267 .

30. Sack R.L. , Lewy A.L. , H e r b D.L. , Vol lmer W . M . a n d Singer C M . (1986) H u m a n mela ton in p roduc t i on decreases with age. «/. Pineal Res. 3 , 379-388 .

31 . Sandyk R., Ann inos P .A. and Tsagas N . (1991) Age related d is rupt ion of c i rcadian rhy thms : possible re la t ionship to m e m o r y impa i rmen t a n d impl icat ion for the rapy with magnet ic fields. Int. J. Neurosci. 5 9 , 259-62 .

32. S h a r m a M. , Palacios-Bois J., Schwar tz G. , I skanda r H. , T h a k u r M. , Qu i r ion R. a n d N a i r N . P . (1989) Ci rcad ian rhy thms of mela ton in a n d Cortisol in aging. Biol. Psychiatry. 2 5 , 305-19 .

33. Skene D.J. , Vivien-Roels B., Sparks D.L . , H u n s a k e r J . C , Pevet P . , Ravid D . a n d S w a a b D . F . (1991) Daily var ia t ion in the concen t ra t ion of me la ton in a n d 5-methoxyt rypto-phol in the h u m a n pineal g land: Effect of age a n d Alzheimer 's disease. Brain Res. 5 2 8 , 170-174.

34. Stewart J .H . a n d Halbre ich U . (1989) P l a s m a mela ton in levels in depressed pat ients before a n d after an t idepressant medica t ion . Biol. Psychiat. 2 5 , 33 -38 .

35. T o u i t o u Y., F e v r e - M o n t a n g e M. , P r o u s t J., Kl inger E. a n d N a k a c h e T . P . (1985) Age-and sex- associated modif icat ion of p l a sma mela ton in concen t ra t ions in m a n . Rela t ionship to pa tho logy , mal ignan t or no t , and au topsy findings. Acta Endocrinol. 108, 135-144.

36. T h o m p s o n C , F r a n e y C , Arendt J. a n d Checkley S.A. (1988) A compar i son of mela ton in secretion in depressed pat ients a n d n o r m a l subjects. Br. J. Psychiat. 152, 260-265

37. Webley G . E . a n d Leidenberger , F . (1986) T h e circadian pa t t e rn of me la ton in a n d its


positive relat ionship with proges te rone in w o m e n . J. Clin. Endocrinol. Metab. 63 , 323-328.

38. Wet te rberg L., Arendt J., Paun ie r L., S izonenko P . , van Donse laa r W. and Heyden T. (1976) H u m a n serum mela ton in changes dur ing the mens t rua l cycle. J. Clin. Endocrinol. Metab. 42, 185-199.

39. Wet te rberg L., Er iksson O. , Fr iberg Y. and V a n g b o B. (1978) A simplified r ad io immunoassay for mela ton in and its appl ica t ion to biological fluids. Pre l iminary observat ions on the half-life of p lasma mela tonin in m a n . Clin. Chim. Acta. 8 6 , 1 6 9 - 1 7 7 .

40. Wet te rberg L., Beck-Friis J., Aperia B. a n d Pe t te r son U. (1979) Mela tonin /cor t i so l ra t io in depression. Lancet 2, 1361

4 1 . Wet te rberg L., Aperia B., Gorel ick D.A., G w i r t z m a n H.E . , M c G u i r e M .T., Serafeti-nides E.A. and Yuwiler A. (1992) Age, a lcohol ism, and depression are associated with low levels of ur inary mela ton in . J. of Psychiat. Neurosci. 17, 215-224 .

42. Wirz-Justice A. and Arendt J (1979) Diurna l , mens t rua l cycle a n d seasonal indole rhy thms in m a n and their modif icat ion. In Biological Psychiatry Today (eds. Obio l s J., Ballus C , Gonza le s -Monc lus E. and Pujol E.), p p . 294-302 . Elsevier /Nor th Hol l and , Ams te rdam.

43 . Wirz-Just ice A. a n d Richter R. (1979) Seasonali ty in biochemical de te rmina t ions : A source of var iance and a clue to the t empora l incidence of affective illness. Psychiat. Res. 1, 53-60 .

20

Individual Variations of Rhythms in Morning and Evening Types wi th Special Emphasis on Seasonal Differences

V E R E N A L A C O S T E

1 a n d L E N N A R T W E T T E R B E R G

2 1 Psychiatric University Clinic, W il helm Klein-Str. 27, C H-4025 Basel,

Switzerland 2Department of Psychiatry, St. Goran's Hospital, S-11281 Stockholm, Sweden

T H E PRESENT communica t ion focuses on h u m a n rhy thm pat terns and their modification by the individual "diurnal type" (evening-/morning-) , and thus relates to a respectable Scandinavian t radi t ion: Swedish scientists such as Ôquis t , Ôstberg , F rôberg , Pa tka i , Torsvall and Akerstedt a m o n g others , have essentially contr ibuted to the progress in various areas of diurnal typology including the development of self-assessment i n s t r u m e n t s

1 1 , 3 2 , 3 4'

41 and shift-work c o n c e r n s .

7 , 3 3 , 3 4 , 41

Since recognit ion of the existence of so called "morn ing" and "evening" active persons by Kle i tman 1 9 3 9 ,

18 var ious synonymous terms have

appeared in chronobiological l i terature, ranging from the more abstract "subjective circadian phase posi t ion" to the poetical terms like larks and owls (Table 1). C o m m o n l y morn ing (M-) and evening (E-) types are regarded as two extremes of a con t inuum on which the intermediate (I-) ones represent the largest c a t e g o r y .

17 But, for example according to

W e n d t ,

45 morningness-eveningness self-reports are more complex and

would be more appropr ia te ly described within a mult idimensional model .

In search for factors which might influence the t ime structure of a h u m a n individual, measurable in biological rhy thm characteristics such as phase posit ion, ampl i tude and waveform, up to now individual , social and environmenta l sources have been proposed (Table 2). Based on circadian rhy thm s t u d i e s ,

17 Kerkhof concluded that the morningness-eveningness

dimension is the most impor tan t factor associated with reliable interindivi-dual differences.

287


1. D i u r n a l type 2. Subjective c i rcadian phase pos i t ion 3. Time-of-day preference 4. C i r cad i an activity type 5. C i r cad ian c h r o n o t y p e 6. M o r n i n g / e v e n i n g type 7. Ear ly / la te sleeper 8 . L a r k / o w l

T A B L E 2

Factors which contribute to the variability of rhythm patterns

Ind iv idua l • Age and gender • Height and weight • Ethnic i ty • Persona l i ty t ra i t s • D iu rna l type (morningness-eveningness) • Activity load a n d sleep • Illness related features

Social • W o r k cond i t ions and vaca t ion • Altered s leep /wake schedules (e.g. shift work ,

t r ansmer id i an flights) • Die ta ry cons t i t uan t s a n d meal t iming • Psychosoc ia l " s t ressors"

E n v i r o n m e n t a l • C l ima te • W e a t h e r cond i t ions • Light a n d t e m p e r a t u r e • G e o g r a p h i c a l la t i tude • Al t i tude

Here we will briefly summarize li terature dealing with morningness-eveningness as related to the circadian system and circadian rhy thms, and then in a second par t present new da ta that indicate an effect of the morningness-eveningness factor on seasonal rhy thms.

Morningness-eveningness and the human circadian system

Diurnal typology is mainly based on the individual circadian phase posit ion of spontaneous sleep-wake rhythms and subjective alert-n e s s

1 2 , 1 7'

3 4 , 42 and , as an external validation criterion, body

t e m p e r a t u r e .

12 M-types generally retire and arise earlier and show less

T A B L E 1

Terms used in literature to describe the morningness-eveningness dimension

Rhythm Patterns in Morning and Evening Types 289

variable sleep length and awakening time as compared to Ε-types and oral tempera ture peaks approximate ly 2 hours earlier.

It was suggested that interindividual differences in subjective circadian phase posit ion could be related to the a u t o n o m o u s intrinsic frequency of the circadian rhy thm of an individual, which generally deviates from the 24-hour- rhythm of external z e i t g e b e r s .

1 , 2 , 1 2 , 17 Thus , M - and E-types

could mir ror differences whith regard to the t iming of their biological oscillators, whereby according to Fore t and Benoi t ,

6 the morningness-

eveningness index seems to reflect the sleep/wake oscillator ra ther than the tempera ture oscillator of an individual. In favor of such a view is the observat ion that circadian sleep/wake cycles in h u m a n temporal isolation paradigms exhibit individual differences with a range of 16- to 5 6 -hou r -da ys .

43 Fu r the rmore , in animal studies the dependency of phase

posit ion upon the free-running circadian activity period length has clearly been d e m o n s t r a t e d .

1 , 2 10 Alternatively differences in circadian phase

posit ions between M - and Ε-types could be a consequence of their habi ts (e.g. sleep times), a view suppor ted by results from F r ô b e r g .

7 Whether

morningness-eveningness is of endogenous or exogenous origin still remains to be clarified. Al though the two explanat ions no t mutual ly exclude one ano ther and interactive effects of bo th are most plausible.

Morningness-eveningness and c i rcadian rhy thms

Even in the 19th century interindividual differences in sleep pat terns , namely depth of sleep, have been a t t r ibuted to individual diurnal type related features (see the waking experiments of Michelson 1891 ,

2 9

Figure 1). But morningness-eveningness related l i terature expanded only in the seventies of the actual century, in parallel to the increasing interest in circadian rhy thms . Differences between M - and Ε-types with regard to circadian parameters have been observed in various behavioral , psycho-physiological and biological measures and are reviewed in detail by Ke rkho f .

17 The majority of studies and the most replicable M - E

differences concern the phase or the t iming of the rhy thms. All studies agree that the m a x i m u m value for the M-types precedes that for E-types. Regarding the magni tude of the difference, a metaanalysis showed that for body tempera ture the average M - E difference is 121 minutes (11 studies), for the alertness rhy thm 171 minutes (7 studies) and for sleep-time 80 minutes (4 studies). The scarce results on ampl i tude and shape lack consistency.

Morningness-eveningness has also been connected with personali ty and coping s t y l e s ,

2 7 3 , 2 8 , 31 adaptabi l i ty to night- and s h i f t - w o r k

4 , 7 , 9 , 3 3 , 41 and

j e t - l a g .

46 There is general agreement that E-types show greater tolerance


Waking experiments

E. Michelson 1891

H.D.I. H.D.II.

0 1 2 3 4 5 6 7

B.I. B.II.

M-type I-type E-type

F I G . 1. Ind iv idua l sleep p a t t e r n s ( "dep th" of sleep) eva lua ted by a rousa l s t imuli presented r a n d o m l y across the n ight , as a t t r i bu ted to the m o r n i n g , in t e rmed ia te a n d evening types , respectively. O r d i n a t e : intensi ty of acous t ic a rousa l s t imuli Abscissa: h o u r s after sleep onset . D a s h e d line: modif ica t ion of the indiv idual

p a t t e r n by externa l events . F r o m M i c h e l s o n .

29

to shift- and night-work as compared to M-types . M-types appear to have more difficulty to adjust to westward, Ε-types to eastward t ransmeridian flights.

A further condi t ion associated with changing external t ime cue schedules is season, raising the quest ion abou t a possible link between morningness-eveningness and seasonal adap ta t ion , an issue however not considered so far.

Morningness-eveningness as re lated t o seasonal rhy thms and subject ive seasonal i ty

Part ic ipat ion in the "Season Around the Wor ld" mul t inat ional project (Wetterberg et aL, 1993, Chap te r 19, this volume) rendered it possible to address the quest ion whether seasonal rhy thm pat terns and /o r "seasona-lity" are modified by the individual diurnal type. In this purpose the local Basel s tudy g roup was investigated in more detail, applying in addi t ion to the s tandard protocol a Morningness-Eveningness-Quest ionnaire across the year.


Study

Subjects and methods

According to a G e r m a n version of the Horne-Ôs tbe rg Morningness-Even ingness -Ques t ionna i re

11 administered to 20 (11m, 9f) healthy

volunteers aged between 23 and 52 years (mean: 32.0 + 7.4) and living in Basel (47.3°N; 7.4°E) under normal daily rout ine lighting condi t ions , M- , E- and I-type groups of individuals were formed and compared with regard to the following measures assessed over 14 mon ths :

(1) Night t ime melatonin and cort isol/urine (RIA method) and overnight urine volume.

(2) Self-ratings (100 m m Visual Analogue Scales, VAS) of m o o d and sleep including: sadness, elation, fatiguability, b roken and dis turbed sleep, sleep need and difficulties falling asleep.

(3) The degree of diurnali ty (morningness-eveningness) using the Horne -Ôstberg index, corresponding to the summary score of the 19 item Horne-Ôs tbe rg Q u e s t i o n n a i r e .

11

(4) In addi t ion, at the end of the investigation period the Seasonal Pa t te rn Assessment Ques t ionnai re ( S P A Q ) ,

36 designed to assess the extent to

which each subject had previously been affected by seasonal changes, was administered. F o r analysis the seasonality change score was used.

As to the statistical analysis, considering the small N ' s , part icularly in the extreme groups (M- and E-) priori ty was given to an explorative approach and a descriptive presentat ion. Complementa ry statistical analysis comprises: 1-way A N O V A for repeated measures , 1- and 2-way A N O V A for g roup compar ison and g roup χ t ime interact ion, as well as Pearson p roduc t -momen t correlat ions.

Results

Test-retest-reliability of the Horne-Ôs tbe rg index (H-O index) ranged depending upon the test-retest interval (1-13 months ) between 0.67 and 0.96, mean : 0.84 ± 0 . 0 8 ) and thus can be considered as adequate . However , since it has been shown that the H - O Scale undergoes seasonal v a r i a t i o n

20

(Figure 2), we considered the average value of the 14 investigations obta ined from each individual for typology. Accordingly, the diurnal types were normal ly distr ibuted, 12 subjects falling in the I-, 4 in the M - and 4 in the Ε-categories, respectively.

As shown in Table 3, the subgroups are comparab le with regard to their mean age, height, weight and body surface area but differ in their sex distr ibution and as a t rend in the seasonality score. A subsequent separate analysis for male and female subjects revealed no further sex difference,


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F I G . 2. M e a n m o n t h l y changes in the degree of morn ingness -even ingness in eight hea l thy m e n and w o m e n ( M a r b u r g / G e r m a n y , 50°N) from three different ques t ionna i re s . C o m p a r i s o n showed t h a t only the Ho rne -Ôs tbe rg -Sca l e a n d tha t

of W e n d t shifted significantly across the year . F r o m K l ô p p e l .

19

apar t from the H - O index ( p < 0 . 0 1 ) and with exception of urine volume being higher in males (p = 0.04).

Yearly means of psychobiological measures in Μ-, Ι-Ε-types

and

Significant g roup differences concern mainly fatiguability and sleep items, but also sadness and Cortisol, as can be seen in Table 4. Sleep measures as well as fatiguability and sadness generally scored higher in the extreme groups (M- and E-) part icularly in the E-types. Cortisol was highest in the M-group , mela tonin in the Ε-group, the latter however wi thout reaching

J A S O N D J F M A M J J A

I I I I I I I I I I I I I I


T A B L E 3

Subject characteristics by diurnal type subgroups

M - t y p e E- type I- type N = 4 N = 4 N=12 p-level

Sex ra t io (m/f ) 3/1 1/3 7/5 Age (years) 35.0 (12.0) 32.5 (7.1) 30.8 (4.6) 0.58 He ig th (cm) 175.3 (10.2) 165.0 (6.0) 172.8 (10.3) 0.29 Weight (kg) 65.3 (11.1) 60.5 (10.5) 66.0 (10.3) 0.66 Body surface 1.79 (0.21) 1.64 (0.15) 1.78 (0.19) 0.39

a rea ( m

2)

H - O index 62.7 (2.7) 36.2 (4.9) 51.9 (3.2) 0.00 (p ts )

a 51.9 (3.2)

Seasona l i ty— 5.50 (3.7) 8.75 (3.3) 5.92 (1.1) 0.09 score ( p t s )

b m e a n S D a

S u m m a r y score from the H o r n e - Ô s t b e r g 19-item Morn ingness -Even ingness Q u e s t i o n n a i r e ( M E Q ) ,

11 reflecting the subjective c i rcad ian phase pos i t ion a n d

serving as the basis for d iu rna l typology . b As der ived from the Seasona l P a t t e r n Assessment Q u e s t i o n n a i r e ( S P A Q )

from R o s e n t h a l et al.,

36 r epresen t ing the s u m of six i tems ( a m o u n t of sleep per

day , social act ivi ty, m o o d , weight , appe t i t e a n d energy level) for which the individuals ra ted the degree they cons idered themselves to be affected t h r o u g h o u t the year .

significance, provided winter values are considered (December: ρ < 0.01). Tha t g roup differences were greater in winter as compared to summer applies to all measures investigated, except elation.

Seasonal rhythm patterns of psychobiological measures in M-, I- and Ε-types

Figure 3 demonst ra tes that the subjective circadian phase posi t ion (H-O index) varies th roughou t the year in the Ε-group and to a lesser extent in the M-group , while it remains stable in the I -group.

Wi th regard to m o o d and sleep impai rment , in addi t ion to the higher levels (yearly means) , M - and Ε-types also exhibit larger seasonal variat ions than the I-types, as illustrated in Figure 4. The items difficulties falling asleep and broken and dis turbed sleep show statistical t rends ( p < 0 . 1 0 ) for a g roup χ t ime of year interact ion.

Regarding mela tonin (Figure 5), it is the only variable studied with a statistically significant seasonal rhy thm (p< 0.0001). The seasonal pa t te rn is similar in all d iurnal type subgroups , with low values dur ing the summer half and high levels dur ing the winter half of the year, a l though the E-type shows a more p ronounced winter increase resulting in an almost significant g roup χ t ime of year interact ion (July/December: ρ = 0.05).

With regard to Cortisol (Figure 6), it is notable that peak values are reached in M a r c h (M-type) and Oc tober (E-type), respectively.

A differential seasonal pa t te rn was also observed in urine volume: while


Var iab le M - t y p e Ε- type I- type

N=4 N=4 N=\2 p-level

M o o d Sadness (mm) 33.0 (10.8) 51.5 (4.5) 25.5 (16.9) 0.049 E la t ion (mm) 47.9 (5.4) 36.2 (1.6) 50.7 (16.5) 0.701 Fa t iguab i l i ty (mm) 43.3 (20.4) 66.1 (11.4) 31.8 (17.5) 0.009

Sleep Sleep need (mm) 45.6 (18.3) 56.3 (7.1) 28.7 (17.3) 0.019 Broken a n d 33.4 (10.8) 58.8 (4.5) 20.3 (16.9) 0.002

d i s tu rbed sleep (mm)

Difficulties falling 27.3 (8.9) 55.9 (32.8) 14.5 (3.9) 0.006 asleep (mm)

Physio logica l M e l a t o n i n (nmol/1) 0.33 (0.15) 0.41 (0.11) 0.32 (0.10) 0.360 Cor t i so l (nmol/1) 201.0 (200) 138.6 (44) 126.2 (60.1) 0.034 Ur ine vo lume 336.0 (95.3) 236.4 (101.9) 463.9 (186.8) 0.107

(8 h o u r s overn igh t )

ο

30

40

Ο

ε ο

Subjective circadian phase posit ion

+ Morning (Ν = 4) • Intermediate (Ν = 12) * Evening (Ν = 4)

50 h

60

70 I I I I I

>

M J J A S O N D J F M A

Month

F I G . 3. Seasona l p a t t e r n (mon th ly m e a n s ) of the H o r n e - Ô s t b e r g index separa te ly for the d iu rna l type s u b g r o u p s . T h e ho r i zon ta l lines represent the cut-off po in t s o n

which classification is based . L o w e r values indica te increas ing eveningness .

T A B L E 4

Yearly means and standard deviations of self rated mood and sleep (100 mm Visual Analogue Scale) and physiological measures by diurnal type subgroups

Rhythm Patterns in Morning and Evening Types

Difficulties falling asleep Fatigability

295

>

100

V A A A ' A A A A t t /

MJ J A S O N D J F M A

Month

Broken and disturbed sleep

M J J A S O N D J F M A

Month

Sadness

[J J A S O N D J F M A

Month

. I I I I I I . . . . MJ J A S O N D J F M A

Month F I G . 4. Seasona l pa t t e rn s (mon th ly m e a n s ) of self-rated m o o d a n d sleep (100 m m Visual A n a l o g u e Scale) by d iu rna l type s u b g r o u p s . U p p e r curve : E- type , b o t t o m :

I- type, in be tween : M - t y p e .

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0.6

0.5

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0.1

0

• Intermediate (N = 12)

* Evening (N = 4)

+ Morning (N = 4)

I I I I I I I J_J L M J J A S O N D J F M A M J

Month

F I G . 5. Seasona l p a t t e r n (mon th ly m e a n s ) of n igh t t ime u r ina ry me la ton in by d iu rna l type s u b g r o u p s .


I-types showed high levels in winter, M - and E-types reached minimal values at this time of the year, where g roup differences become significant on the 1% level (data not shown).

Correlations

As indicated by the coefficients of correlat ions listed in Table 5, the H - O index correlated negatively with fatiguability and sleep complaints . A low negative correlat ion was obta ined between the H - O index and the seasonality score (Figure 7a), becoming significant when winter values were considered (December: r= —0.58, /?<0.01) .

The seasonality score itself showed more substantial relations, being highly positively correlated with fatiguability, sleep complaints , part icu-larly difficulties falling asleep (Figure 7b) but also with sadness and wintert ime melatonin (December: r = 0.64, ρ < 0.01). Fu r the rmore , the degree of seasonality appears to increase with age.

G r o u p differences were found in the tempora l relat ionship between melatonin and Cortisol, the correlat ions being positive in the M - (r = 0.71, p < 0 . 0 1 ) , negative ( r = — 0.41, p = 0.10) in the Ε-type, as described e l sewhere .

23

Discussion

According to our results morningness-eveningness appears to be a relatively stable individual characteristic tha t nevertheless can vary in degree across the seasons. This is in agreement with reports from literature demonst ra t ing a high reliability independent of the popula t ion studied and the quest ionnaire u s e d .

1 4'

1 6'

4 0 , 41

In a longitudinal study applying concomitant ly three different morning-ness-eveningness quest ionnaires , two out of them—tha t of H o m e and Ostberg and that of Wend t—have shown to undergo seasonal variat ions with shifts towards an eveningness phase posit ion in w i n t e r

19 (Figure 2).

In the present study a similar seasonal pa t te rn has been observed in the E-type g roup (Figure 3). Parallel to the within subject studies, in a previous transverse popula t ion study a likewise varying distr ibution of self-est imated M - and E-types in function of season has been n o t i c e d .

21

A winter phase delay in circadian rhythms of various vegetative func t ions ,

19 but also of ho rmones such as m e l a t o n i n

5 , 13 has been

described. Thus , seasonal var iat ion in subjective circadian phase posit ion may have a physiological basis in the annual ly changing phase posit ion of circadian rhy thms.

As concerns age, we did not find an association between morningness and increasing age, as has systematically been r e p o r t e d .

2 8'

3 2'

41 But in

agreement with epidemiological findings issued from a survey in the general


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• Intermediate (Ν = 12)

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F I G . 6. Seasonal pattern (monthly means) of nighttime urinary Cortisol by diurnal type subgroups.

T A B L E 5

Pearson s product-moment correlations of diurnality and seasonality with psychobiological measures (N = 20)

H - O i n d e x

a Seasonal i ty score

r Γ

Age (years) 0.19 — 0.45 Heigh t (cm) 0.20 — 0.12 H - O i n d e x

3 - 0 . 3 7 ( * )

Seasonal i ty score (pts) - 0 . 3 7 ( * ) Sadness (mm) - 0 . 3 6 0.53* Ela t ion (mm) 0.27 0.06 Fat iguab i l i ty (mm) - 0 . 4 3 * 0.61** Sleep need (mm) - 0 . 2 1 0.54** Broken a n d d i s tu rbed - 0 . 4 6 * 0.54**

sleep (mm) Difficulty falling

asleep - 0 . 4 3 * 0 γι*** M e l a t o n i n (nmol/1) - 0 . 3 7 ( * ) 0.39(*) Cor t i so l (nmol/1) 0.34 0.13 Ur ine vo lume

( 8 h o u r s , overn igh t ) 0.20 - 0 . 1 7 a A high H - O index is associa ted with m o r n i n g n e s s . ( * ) p < 0 . 1 0 . * / ? < 0 . 0 5 . ** p<0.0l. *** p < 0 . 0 0 1 .

350 ι


2 h

r = -0.373 Ρ < 0.10

Ο

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12

10

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- Eveningness Morningness

r = 0.706 Ρ < 0.001

He · · · t o o

I- · ο

30 40 50 60

Horne-Ostberg scale

70 J -

20 40 60 80 100

Difficulties falling asleep (mm)

F I G . 7. Morn ingness -even ingness ( H o r n e - Ô s t b e r g index) a n d "difficulties falling as leep" p lo t ted in function of the degree of subjective seasonal i ty as der ived from the S P A Q in 20 hea l thy subjects . P e a r s o n cor re la t ion coefficient = r. O p e n circles:

m e n , closed circles: w o m e n

popula t ion in Mon tgomery C o u n t r y / M a r y l a n d ,

15 we could establish a

negative correlat ion between subjective seasonality and age. O u r results, a l though prel iminary considering the small sample studied,

are in suppor t of a view assuming that morningness-eveningness contr ibutes to seasonal variability. M- , I- and Ε-types have shown to differ in objective (seasonal rhythms) and subjective (seasonality score) measures and indirectly, in seasonal sensitivity and /o r seasonal adap -tat ion. Tha t there is a link between diurnali ty and various aspects of seasonality is not surprising, since the circadian system is closely related to the seasonal system by the interdiurnal changes in daylight over the year. Also the lacking or reduced rhythmicity observed in the I-type corres-ponds somewhat to expectat ion. The comparab ly large seasonal varia-tions seen in the E-type are in agreement with one of the rare studies available, report ing a greater number of significant seasonal rhy thms in E-types as compared to M - t y p e s

19 (Figure 8). In the same investigation it

was shown that , when only weekends were considered, the number of significant rhy thms substantially increased in bo th M - and E-types, suggesting that social constraints may partially be responsible for "a-seasonali ty" in m a n .

In the present study M - and Ε-types were ra ther close together deviating both in the same direction from the I-types, at least as far as psychometr ic measures (levels) are concerned. In most work dealing with diurnal

Rhythm Patterns in Morning and Evening Types 2 9 9

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ALL SUBJECTS ( n - 7 ) ANALYZED FUNCTIONS : 84 SIGNIFICANT ANNUAL RHYTHMS ( p < 1 % ) : 40

MORNING TYPES (n = 4) ANALYZED FUNCTIONS : 48 SIGNIFICANT ANNUAL RHYTHMS ( p < 1 % ) : 18

EVENING TYPES (n = 3) ANALYZED FUNCTIONS : 36 SIGNIFICANT ANNUAL RHYTHMS ( p < 1 % ) : 22

• WEEKEND

• WORKDAY

HARMONIC ANALYSIS OF THE DAILY BEHAVIOR-PARAMETERS

F I G . 8. F r e q u e n c y of significant ( 1 % level) a n n u a l r h y t h m s in dai ly behav io r -p a r a m e t e r s separa te ly for M - a n d E - t y p e s . F r o m K l ô p p e l .

19

typology however, M - and E-types were opposed one to another . Given the period length of a free-running intrinsic rhy thm is shorter with regard to the 24-hour envi ronment in M-types , (tending to phase advance) and longer in E-types (tending to phase delay) it is conceivable that M - and E-types have more difficulties to remain synchronized with the geophysical environment and become easier dis turbed in their equil ibrium as reflected by the higher levels of sadness, fatiguability and sleep complaints . Thus , including the I-type in g roup compar i son appears to be essential and may provide complementary information.

Another impor tan t finding is that diurnal type related differences in self-rated m o o d and sleep as well as in physiological measures were greatest in winter. Likewise, the individual values within the whole g roup scattered wider in winter as compared to summer . A similar dis t r ibut ion across the year has previously been described for total m e t a n e p h r i n e s

47 and can

tentatively be interpreted as a consequence of the weak or lacking zeitgeber effect of winter light condit ions in tempera te zones.


Among the three diurnal type subgroups, the E-type appears to be particularly susceptible to seasonal changes in the exogenous light/dark cycle, with prevailing difficulty to adapt to winter conditions, as suggested by findings summarized in Table 6. It is noteworthy that the E-type not only scored highest in subjective seasonality and exhibited largest seasonal amplitudes in self-rated m o o d and sleep, but also showed highest levels of nighttime melatonin in comparison to M - and I-types. This is in accordance with clinical findings from a depressed popula t ion ,

3 relating low nocturnal

melatonin to diminished rhythmicity in depressive symptomatology. While there is some support from literature for an association between eveningness and day-time f a t i gue ,

7'

38 an association between eveningness and increased

seasonality however, has not yet been described.

T A B L E 6

Synopsis of characteristics associated with an eveningness-typical subjective circadian phase position

M o o d

Sleep

Phys io logy

Seasonal i ty

G e n d e r d i s t r ibu t ion

• S a d n e s s j

• F a t i g u a b i l i t y j

• Sleep c o m p l a i n t s ! (especially difficulties falling asleep)

• M e l a t o n i n / u r i n e (overn igh t ) î

• Subjective Seasonal i ty score ( S P A Q )

• Object ive A m p l i t u d e ( annua l ) P a t t e r n : u n i m o d a l with winter p e a k s

• P r e d o m i n a n t l y w o m e n

this s tudy

Age · H igh p r o p o r t i o n of y o u n g subjects 41

P s y c h o p a t h o l o g y · Neu ro t i c i sm 3 1 , 4 1 • Anx ious a n d neuro t i c persona l i ty 29 • Cyc lo thymic persona l i ty 32

Catel l ' s 16 P F • H igh p r o p o r t i o n of E- types 30

in b ipo la r depress ion (free in terval )

Clinical issues

Two chronobiological disorders affected by season are of par t icular interest with regard to eveningness: the so-called midwinter insomnia (MI) occurring frequently in the far no r th (e.g. in 23 .7% of the general adul t popula t ion in T r o m s ô

2 6) and winter depression, a form of seasonal

affective disorder (SAD) described by Rosenthal and c o w o r k e r s ,

37 with a


varying prevalence rate in function of geographical l a t i t u d e .

35 M I as well

as S A D manifest increased day-t ime drowsiness and sleepiness dur ing the dark period in winter, and M I is characterized by a predominant ly initial type of insomnia , ranging in degree from modera t e difficulty in getting asleep, to an almost total inability to sleep dur ing the whole n i g h t .

27 In

addi t ion, bo th are illness condi t ions , where a tendency to circadian phase delay due to the lack of regulatory influence of normal day light is presumed, and bo th respond favorably to therapy with artificial bright l i g h t .

2 5'

27 As summarized in Table 6, the Ε-type appears to share some

c o m m o n characteristics with M I and S A D , such as the high degree of subjective seasonality, the un imodal seasonal pa t te rn with regard to sadness, fatiguability and sleep complaints (initial insomnia)—all peaking a round winter—as well as the predominance of female subjects. Tha t in the Ε-type the latest subjective circadian phase posi t ion (maximum evening-ness) occurred in late au tumn/win te r and coincided with the max ima of sadness, fatiguability and difficulties falling asleep add indirect suppor t to the hypothesis that SAD and M I may reflect underlying circadian phase delay of some or all circadian rhy thms . In accordance with our findings, in a recent review abou t seasonal rhy thms in heal thy subjects, it was shown that changes in metabol ic processes, increased sleep and circadian phase delay occurs somewhat physiologically in winter, and that winter impai rment of m o o d , sleep/-wake- and vegetative functions is not restricted to M I and SAD, but occurs in an a t tenuated form in normals as w e l l .

22

In opposi te to the phase delay hypothesis of SAD, a phase advance hypothesis , based a.o. on earlier peaking of the 24-hour body tempera ture and early morn ing awakening, has previously been formulated for major affective d i sorder ,

8 being of interest with regard to a morningness phase

posi t ion, where an ana logous phase advance in circadian rhy thms is p r e s u m e d .

17 According to our results, morningness appears to be related

to high noc turna l Cortisol, corresponding to elevated Cortisol levels found in major affective d i s o r d e r ,

24 but not in S A D .

39 This fact, together with the

observat ion tha t extreme M-types are strikingly often seen in melancholic depressed pat ients dur ing their symptom free i n t e r v a l ,

30 brings the M-type

near to this clinical category of affective disorder. Whether an extreme phase posi t ion, part icularly eveningness, would

predispose to seasonal variat ions and sleep and m o o d pa thology mus t be cor robora ted by further studies. Such future research might include the evaluat ion of dis tr ibut ion pat terns of M- , I- and E-types with regard to M I and SAD as well as to the Delayed Sleep Phase Syndrome ( D S P S ) — another chronobiological sleep disorder but with a chronical c o u r s e

4 4— a n d also with regard to male and female subjects and in

function of geographical lat i tude. In conclusion, the "diurnal type" appears to modify aspects of


seasonality in healthy volunteers. Whether this is due to differences in the s tructure of the circadian system of M- , I - and Ε-types, or to a differential response to exogenous zeitgebers th rough an association between personali ty traits and cognitive and social factors affecting ent ra inment of circadian rhythms to the sleep/wake cycle, remains to be clarified. Nevertheless, our findings suggest, tha t including diurnal typology in chronobiological studies, especially those focusing on seasonal concerns, is a promising area of research, with impor tan t implications for sleep and m o o d disorders and providing new insights into mechanisms relating the circadian and the seasonal system in man .

References

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2. Aschoff J. (1965) T h e phase -ang le difference in c i rcadian per iodici ty . In Circadian Clock (ed. Aschoff J .) , p p . 262 -276 . N o r t h - H o l l a n d , A m s t e r d a m .

3. Beck-Fri is J., Kjel lman B.F . , Aper ia B. , U n d e n F . , L jungren J . G . a n d Wet t e rbe rg L. (1985) Se rum me la ton in in re la t ion to clinical var iables in pa t iens with major depressive d i sorder a n d a hypothes i s of a low me la ton in s y n d r o m e . Acta Psychiat. Scand. 7 1 , 319-330 .

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5. B r o a d w a y J .W. , Arend t J. a n d F o l k l a n d S. (1987) Bright light phase-shifts the h u m a n me la ton in r h y t h m d u r i n g the an ta rc t i c winter . Neurosci. Lett. 79, 185-189 .

6. F o r e t J. a n d Benoit Ο . (1981) Ind iv idua l factors , sleep character is t ics a n d c i rcadian evolu t ion of b o d y t e m p e r a t u r e . In Sleep 1980. 5th Eur . C o n g r . Sleep Res. A m s t e r d a m , p p . 195-197 . Ka rge r , Basel.

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3 1 . M u r a L E . L . a n d Levi A. (1986) Re la t ionsh ip be tween neuro t i c i sm a n d c i rcad ian r h y t h m s . Psychol Reports 5 8 , 298.

32. Ô q u i s t O . (1970) K a r t l â g g n i n g av individuel la dygns ry tmer . Thesis at the D e p t . of Psycho logy . Univers i ty of G ô t e b o r g .

33. Ô s t b e r g O . (1973) In te r ind iv idua l differences in c i rcad ian fatigue p a t t e r n s of shift worke r s . British Journal of Industrial Medicine 3 0 , 341-351

34. P a t k a i S.G. (1971) T h e d iu rna l r h y t h m of ad rena l ine secret ion in subjects wi th different w o r k i n g hab i t s . Acta Physiol. Scand. 8 1 , 3 0 - 3 4 .

35. P o t k i n S.G., Ze tk in M . a n d S t a m e n k o v i c V. (1986) Seasona l affective d i so rde r : prevalence varies wi th l a t i tude a n d c l imate . Clin. Neuropharmacol. 9 (Suppl . ) 4, 181-183 .

36. R o s e n t h a l N . E . , B rad t G . H . a n d W e h r T.A. (1984) Seasonal Pattern Assessment Questionnaire. N a t i o n a l ins t i tu te of M e n t a l H e a l t h , Be thesda M L .

37. Rosen tha l N . E . , Sack D.A. , Gil l in C , Lewy A.J. , G o o d w i n K. , M tiller D.A. , N e w s o m e D.A. a n d W e h r T . (1984) Seasona l affective d i sorder . A descr ip t ion of the s y n d r o m e a n d pre l iminary findings. Arch. Gen. Psychiatry 4 1 , 7 2 - 8 0 .

38. S a n o K . (1987) C i r cad i an r h y t h m of C F F in m o r n i n g n e s s a n d eveningness chi ldren . Neurosciences 13 , 298 -300 .

39. Skwever R .G . , J a c o b s o n F . M . , D u n c a n C .C . , Kelly K.A. , Sack D .A . a n d T a m a r k i n L. (1989) N e u r o b i o l o g y of seasonal affective d i so rde r a n d p h o t o t h e r a p y . In Seasonal


Affective Disorders and Phototherapy (eds. Rosen tha l N . E . a n d Blehar M . ) , p p . 311-332 . Gui l ford Press , L o n d o n / N e w York .

40. Sverko B. a n d Fabu l i c L. (1985) Stabil i ty of morn ingnes s - eveningness : retest changes after seven years (engl. abs t r ac t ) . Revij za psihologiju 15, 7 1 - 7 8 .

4 1 . Torsva l l L. a n d Akers t ed t (1980) A d iu rna l type scale: cons t ruc t ion , consis tency a n d va l ida t ion . Scand. Environ. Health 6 , 283 -290 .

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43 . W e h r T.A. a n d G o o d w i n F . K . (1981) Biological r h y t h m s a n d psychia t ry . In American Handbook of Psychiatry. Vol . 7 Advances and Directions (eds. Arieti S. a n d Brod io M . K . ) , p p . 4 6 - 7 4 . Basic B o o k s Inc . Pub l i shers , N e w York .

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46. Winge t C M . , D e R o s h i a C . W . a n d Hol ley D . C . (1985) C i r cad ian r h y t h m s a n d athlet ic pe r fo rmance . Med. Sci. Sports Exerc. 17, 4 9 8 - 5 1 6 .

47. Y a m a m o t o T. , T a k e u c h i Y., B a b a M . a n d T a n a k a M . (1976) Seasona l va r i a t ion of u r ina ry excret ion of to ta l m e t a n e p h r i n e s . Clinica Chemica Acta 6 8 , 241 -244 .

21

Abstract

T h e 1967 r epo r t of E d m u n d D e w a n tha t a beds ide 100 wa t t b u l b cou ld stabil ize i r regular mens t rua l cycles was initially greeted with skept ic ism. Recent s tudies of light t r e a t m e n t s of h u m a n s have ind ica ted tha t b o t h br ight a n d d im light have real biological effects. T o examine the validity of D e w a n ' s r epor t , o u r g r o u p has t rea ted two samples of w o m e n with i r regular m e n s t r u a t i o n wi th r andomly-as s igned 100 wa t t beds ide n ight l ights o r d im red light p l acebos . In b o t h g r o u p s , the 100 wa t t t r e a t m e n t s significantly decreased the d u r a t i o n of m e n s t r u a l cycles a n d reduced their var iabi l i ty . Effective were t r e a t m e n t s b o t h from days 10-14 a n d from days 13-17 of the m e n s t r u a l cycle (count ing the first b leeding as day 1 ). T h e night light effect on long cycles in o l igomenor rhe ic w o m e n seems to be a genu ine a n d r a the r powerful biological effect a n d no t a result of sugges t ion .

Use of light to regula te m e n s t r u a l cyclicity m a y have clinical value b o t h for r h y t h m con t racep t ive m e t h o d s a n d for t r ea tmen t s of infertility. F u r t h e r s tudy of night light effects on the m e n s t r u a l cycle m a y e x p a n d o u r u n d e r s t a n d i n g of r ep roduc t ive endoc r ino logy . Extensive clinical tes t ing is needed to de t e rmine the op t ima l intensi t ies , t iming , a n d d u r a t i o n s of n ight light t r e a t m e n t s for p r o d u c i n g endoc r ine benefits.

Light regulat ion of the menstrua l cycle

I N 1967, E d m u n d Dewan repor ted that by simulating moonl ight with nocturnal light exposures, the menstrual cycles of women could be b rought nearer to the lunar cycle of 29.5 d a y s .

9 Dewan argued tha t such

dim light might be used to augment na tura l contracept ive methods . Dewan published several follow-on r e p o r t s ,

1 0'

11 and a feminist popular i -

zat ion called "Lunacep t ion" a p p e a r e d ,

16 but for 20 years, Dewan ' s work

3 0 5

Light Regulation of the Menstrual Cycle D A N I E L F. K R I P K E *

Department of Psychiatry, UCSD and the Sam and Rose Stein Institute for

Research on Aging, La Jolla, CA 92093-0667, USA

' S u p p o r t e d b y M H 0 0 1 1 7 , by H L 4 0 9 3 0 , by t h e D e p a r t m e n t o f Ve te rans A f f a i r s , a n d by a g i f t f r o m A p o l l o L i g h t S y s t e m s , Inc .


was virtually ignored by the scientific communi ty . Perhaps the idea that mere moonl ight had biological effects on h u m a n s strained the credulity of the t ime.

In the intervening years, more evidence has accumulated that circadian systems of plants , nocturna l r o d e n t s ,

22 and other organisms are indeed

sensitive to light exposures in the range of moonl ight . Fu r the rmore , th rough effects on the circadian t iming system, in nocturnal rodents , dim light exposures are able to trigger a variety of reproduct ive endocrine responses including seasonal reproduct ion .

Whereas it once seemed accepted that h u m a n beings were immune to the phot ic effects which regulate lower organisms, pioneering work of Wetterberg, Lewy, and colleagues demons t ra ted that very bright light does indeed influence h u m a n circadian and melatonin s y s t e m s .

1 9 , 26 In the

1980s, we have witnessed an expanding science of pho to the rapy , inspired by the concept that i l lumination of at least 1,500-2,500 lux is required for melatonin suppression a n d related effects in h u m a n s . Meanwhi le , the field has tended toward experiments with very bright i l lumination levels. Te rman et al.

25 found that photo therapeut ic effects of 10,000 lux for 30

minutes exceeded effects of 3,000 lux for 30 minutes and approx imated effects of 2,500 lux for 2 hours . Meanwhi le , Wever reported tha t s t rong circadian synchronizing effects required exposures up to 4,000 lux for as much as 8 h o u r s .

27 Czeisler and c o l l e a g u e s

8 , 13 obta ined surprisingly

s trong Type-0 circadian phase-shifting effects from 7-12,000 lux exposures for 5 hours on 3 consecutive days. Such da ta have encouraged some investigators to work with 5,000-10,000 lux i l luminations, even though there is some da ta in monkeys suggesting a possibility of eye damage with prolonged exposures to 10,000 l u x .

24

In retrospect, the not ion of a threshold of suppression of 1,500-2,500 lux was based on a small s tudy of only a few c a s e s .

19 Subsequent studies

showed that melatonin suppression in h u m a n s is a graded effect produced by graded intensities and dura t ions of i l l u m i n a t i o n .

6'

21 Indeed part ial

h u m a n melatonin suppression can be produced by 500 l u x ,

18 300 lux ,

4 or

even 17 l u x

5 in part icular circumstances. Nevertheless, little study has

been done of effects on h u m a n melatonin of relatively dim i l lumination. The intensity/response relat ionship for h u m a n circadian phase shifting

has likewise been examined only vaguely. Wi thou t s t rong empirical evidence, K r o n a u e r

15 suggested that circadian phase shifting might be

related to the cube root of i l lumination intensity. If a cube root rule is correct, 8,000 lux should be only twice as powerful as 1,000 lux. Fur ther , 8,000 lux might be only five times as powerful as 64 lux and only 20 times as powerful as 1 lux. A logari thmic rule might produce relationships of similar magni tude , e.g. 10,000 lux would be four times as powerful as 10 lux. Whether longer dura t ions compensate for d immer i l luminations by cube, logari thmic or more linear rules is essentially unknown . F r o m such

Light Regulation of the Menstrual Cycle 307

considerat ions, the possibility that Dewan 's report could be correct

became more plausible.

First San Diego replication: experimental procedures

At U C S D , Lin et al.

20 decided to replicate Dewan 's reports with

placebo-control similar to designs we had used for studies of pho to the rapy of depression. W o m e n were recruited who reported long (or irregular) menstrual cycles averaging 46-54 days. To focus on women who were likely to have o l igomenorrhea of functional hypotha lamic origin, we excluded women with grossly abnorma l body weights, those with any appearance of androgenizing syndromes, and those with any history of major psychiatric or endocrine disorders . Each w o m a n was followed prospectively for one untreated menst rual cycle. In the following cycle, randomized women were treated with a 100 watt light bulb placed by the side of the bed, approximate ly 1 m from the head, on the nights following the 13th—17th days after the start of mens t rua t ion . T o assure that the women had some exposure to this t rea tment , which measured 235 lux, we asked these women to read for 0.5 hour in bed. Randomized controls used a dim red pho tograph ic safe light which produced only 1.7 lux by photop ic measurement . A red light placebo was chosen, because in previous pho to the rapy studies, we had found that subjects commonly believed that dim red light was the active t rea tment , thus balancing expectat ions.

First San Diego replication: results

In this study, we were able to study seven subjects who received the 100 watt light and nine who received the dim red placebo. All of the women exposed to the 100 watt bulb reduced their menst rual cycle dura t ions : from an average of 45.7 days at baseline to 33.1 days dur ing the treated cycle. Placebo had no significant effect, thus , differences between the 100 watt t rea tment and placebo were robust ly significant. O n e problem with this study was that eight subjects assigned to the 100 watt bulb had d ropped out , apparent ly either because the light disturbed their sleep or because of negative expectat ions. Another problem was failure to formally balance pre-sleep behaviors and expectat ions. Thus , further replication was needed.

Second San Diego replication: experimental procedures

In our next study, D r e n n a n et al.

12 sought to determine if the effect of night

lights was limited to women with long and irregular menst rual cycles, or


whether there was also an effect on the ovulat ion of women with normal cycles. We did not know Dewan 's empirical basis for originally recommending nights 14-17 of the menst rual cycle for bedside lights and for stating that such st imulat ion was ineffective a few days ea r l i e r .

10

Because m a n y normal women might already have ovulated by days 13-17 of the menst rual cycle, we decided to test light effects somewhat earlier in the follicular phase on days 10-14 of their cycles.

Two groups of women were studied. The first g roup reported regular 28-32 day menstrual cycles. The second g roup reported long and irregular cycles averaging 41-45 days. As in our first s tudy, women were selected whose physical examinat ion and medical histories tended to rule our anorexia, androgenizing syndromes, or discrete psychiatric and endocrine disorders, however, the subjects with reported o l igomenorrhea were not evaluated sufficiently to definitively diagnose functional hypotha lamic ol igomenorrhea . As in the previous study, subjects from each g roup were randomly assigned either to 100 wat t beside lights or to dim red photograph ic safe light placebos.

Second San Diego replication: results

With the 100 watt bulbs, the women with long cycles again showed significant shortening from a baseline of 44.9 days to 33.2 days dur ing the t rea tment cycle. Again the women treated with the dim red placebo showed no effect, so tha t the g roup responses were significantly different. Unl ike the women in the first replication who had been treated from days 13-17 of their cycles and who reverted to their prior long cycles in the follow-up m o n t h , these second-replication women treated from days 10-14 had cycle dura t ions of 35.4 days on follow-up, which was almost significantly shorter than baseline. This suggested some possibility of a carry-over effect from 100 watt t rea tments from days 10-14 of the cycle to the next cycle. The women with normal 28-32 day cycles showed no significant effects on cycle length either from 100 watt or from dim red lights. Endocr ine studies of these women will be reported elsewhere.

Perhaps because these subjects were being paid to contr ibute b lood specimens, in the second replication, d rop-ou t rates were not as t roublesome. Also, bo th subjects randomized to 100 wat t and dim red t rea tments had spent 0.5 hou r in bed reading, so this was balanced. Finally, positive expectat ions were found to actually be somewhat higher a m o n g the subjects who were randomized to placebo, so the likelihood of suggestion as confounder was remote . This replication added s t rong assurance that effects of 100 watt bulbs on long and irregular menstrual cycles are real biological effects.


Discussion

F r o m the results of these two studies, we are quite confident that as Dewan had reported, 100 watt bedside lights can indeed shorten and regularize the menstrual cycles of women whose cycles were long and irregular. We agree with Dewan that these effects may have potent ial usefulness bo th for c o n t r a c e p t i o n

9'

11 (by facilitating rhy thm methods) and perhaps for

t reatments of infertility as well. If night lights influence reproduct ive endocrinology, they may have more extensive applicat ions in endocr ino-logy than we can currently envision. Unfortunately, e laborate clinical trials will be needed before we can develop more than speculations as to the mechanisms by which 100 watt lights achieve their effects.

We would quest ion Dewan ' s theory that night lights are simulating effects of moonl ight . O u r placebo dim red lights, a l though only abou t 1.7 lux, are nevertheless of greater pho top ic brightness than the full m o o n . Red lights are much brighter than the m o o n in the red por t ion of the visible spectrum but perhaps d immer in some blue and green regions, neverthe-less, there is currently no basis for supposing that moonl ight would have stronger effects than 1.7 lux red light. It would certainly be valuable to obtain dose/response da t a for night lights across the range of roughly 1-500 lux. Unfortunately, dose/response da t a curves will require labor ious clinical studies.

O u r da ta do not confirm Dewan 's s ta tements that days 14-17 of the menst rual cycle are the opt imal times for bedside light t rea tment . We achieved comparab le effects using days 13-17 and days 10-14, though bo th protocols included day 14, which D e w a n had suggested might be the critical day. N o t e also that most women did not achieve a shortening of their cycles to 29-30 days, which would be expected if the effect s imulated lunar synchronizat ion.

The issue of brightness is complicated by uncertainty as to what i l lumination exposures bedside lights p roduce dur ing sleep. If one places a pho tomete r by the subject's head aimed toward a 100 wat t bulb at 1 m, then the brightness is 235-250 lux, however, brightness is certainly much less if the pho tomete r is turned away from the light, as the subject's head may be. Fol lowing Dewan ' s model , we have permit ted our subjects to close their eyes and roll over, so tha t the i l lumination actually reaching the eyes must be extremely variable. I l luminat ion reaching the cornea must be much less than 235 lux when subjects' eyes are closed, when they face away from the night lights, and when they may possibly obscure their eyes with pillows, l imbs, or bedclothes. In future experiments , we hope to study women using Dr . Cole's lighted sleep m a s k ,

7 which should be very helpful

in defining and s tandardizing the i l lumination exposures of sleeping subjects. Unti l these studies can be done , the t ime/dura t ion curves of i l lumination actually reaching our subjects' eyes can only be guessed, and


indeed, it is surprising that the results seem so consistent when the i l lumination exposures were p robably erratic.

We believe tha t light masks may prove the best me thod for administer-ing night lights for several reasons besides s tandardiza t ion of light exposure. If they use light masks , women desiring to regulate their menst rual cycles need not dis turb bed par tners or roommates . Night lights must have real potential to dis turb other occupants of the bed room, if we admit that night lights are biologically active. In rustic and third-world settings, 100 watt bedside lights m a y be impractical , but light masks could probably be powered by low-cost solar batteries, since they require much less power than 100 wat t bulbs .

Another variable is the t iming of night light exposures. In our first two experiments, we had asked subjects to give themselves 30 minutes exposure to 235 lux before going to sleep, in order to assure the strongest effects possible. So far as we know, Dewan 's subjects did not receive this prel iminary exposure, but results were quite similar. O u r current ongoing study exposes subjects to 235 lux only from 30 minutes after bedt ime to 30 minutes before wake-up time, nevertheless, the first subject has already shown a typical shortening of the cycle. T o systematically explore different dura t ions of night light exposure given at varying times of night will require large-scale clinical exper imentat ion, multiplying the complexity of intensity/response studies.

The m o d e of act ion of Dewan ' s effect is currently purely a mat te r of speculation. Nei ther Dewan ' s subjects nor ours have been well-character-ized from the viewpoint of reproduct ive endocrinology. Having a t tempted to exclude volunteers with a variety of psychiatric and endocrine disorders , anorexia, and androgenizing syndromes, we believe that most of our subjects were experiencing forms of functional hypotha lamic o l igomenor rhea ,

2 a syndrome resulting primari ly from inadequate

pulsatile secretion of gonado t rop in releasing ho rmone , with resultant inadequate ovulat ion and decreased corpus- luteum product ion of proges-terone. The mechanisms producing such hypotha lamic failures are poorly characterized. In a series of pat ients with hypotha lamic amenor rhea (a more extreme disorder, possibly with similar etiology), Berga et al.

3 found

that nocturna l mela tonin secretion was elevated. In pat ients with amenor rhea related to s t renuous exercise, similar elevations of melatonin have been desc r ibed .

17 Thus , it is tempt ing to speculate that our subjects

may have been suffering from excessive nocturnal mela tonin secretion, and that night lights blunt this excessive secretion, nevertheless, currently we have no empirical confirmation of this speculation.

Par ry et al. have described a g roup of women with premenstrual depressions who appeared to have an early offset of noc turna l mela tonin secretion, associated with decreased total area under the nocturna l melatonin c u r v e .

23 Pe rhaps premenstrual depressions resemble the "low


References

1. Beck-Fri is J., Kje l lman B.F . , Aper ia B., U n d e n F . , von Rosen D . , L junggren J . -G . a n d Wet t e rbe rg L. (1985) Se rum me la ton in in re la t ion to clinical var iables in pa t i en t s with major depress ive d i sorder a n d a hypo thes i s of a low me la ton in s y n d r o m e . Acta Psychiatr. Scand. 7 1 , 319 -330 .

2. Berga S.L. a n d Danie ls T .L . (1991) Use of the l a b o r a t o r y in d i sorders of r ep roduc t ive n e u r o e n d o c r i n o l o g y . Journal of Clinical Immunoassay 14(1), 2 3 - 2 8 .

3. Berga S.L., M o r t o l a J . F . a n d Yen S.S.C. (1988) Amplif icat ion of n o c t u r n a l me la ton in secret ion in w o m e n with funct ional a m e n o r r h e a . J. Clin. Endocrinol. Metab. 6 6 , 242-244 .

4. Bojkowski C.J., A l d h o u s M . E . , Engl ish J., F r a n e y C , P o u l t o n A.L. , Skene D.J . a n d Arend t J. (1987) Suppress ion of n o c t u r n a l p l a s m a me la ton in a n d 6 - su lpha toxyme la to -nin by br ight a n d d im light in m a n . Horm. Metab. Res. 19 , 4 3 7 - 4 4 0 .

5. B ra ina rd G . C , Lewy A.J. , M e n a k e r M. , F r e d r i c k s o n R .H . , Miller L.S. , Weleber R .G . , C a s s o n e V. a n d H u d s o n D . (1988) Dose - re sponse re la t ionsh ip be tween light i r r ad iance a n d the suppress ion of p l a s m a me la ton in in h u m a n vo lun teers . Brain Res. 4 5 4 , 2 1 2 - 2 1 8 .

6. Byerley W . F . , Risch S . C , Gil l in J . C , J a n o w s k y D.S . , P a r k e r D . , R o s s m a n L . G . a n d K r i p k e D . F . (1989) Biological effect of br ight light. Prog. Neuropsychopharm. and Biol. Psychiatry 3 , 683 -686 .

7. Cole R.J. a n d K r i p k e D . F . (1991) Light exposu re du r ing sleep wi th a br ight light m a s k : effects on h u m a n b o d y t e m p e r a t u r e r h y t h m . Sleep Res. 2 0 , 450 (Abs t rac t ) .

8. Czeisler C.A., K r o n a u e r R.E. , Allan J .S. , Duffy J .F . , Jewet t M . E . , B r o w n E . N . a n d R o n d a J . M . (1989) Bright light i nduc t ion of s t rong (type 0) reset t ing of the h u m a n c i rcadian p a c e m a k e r . Science 2 4 4 , 1328-1332.

9. D e w a n E . M (1967) O n the possibil i ty of a perfect r h y t h m m e t h o d of b i r th con t ro l by per iodic light s t imula t ion . Am. J. Obstet. Gynecol. 9 9 , 1016-1018 .

10. D e w a n E . M . (1969) R h y t h m s . Science and Technology J a n u a r y , 2 0 - 2 8 . 11. D e w a n E . M . , M e n k i n M . F . a n d Rock J. (1978) Effect of pho t i c s t imula t ion on the

h u m a n m e n s t r u a l cycle. Photochem. Photobiol. 2 7 , 581 -585 . 12. D r e n n a n M . D . , K r i p k e D . F . a n d Berga S.L. (1991) N igh t light shor t ens long a n d

i r regular , bu t no t regular , m e n s t r u a l cycles. Society for Light Treatment and Biological Rhythms. Abstracts 3 , 20 (Abst rac t ) .

13. Jewet t M . E . , K r o n a u e r R.E . a n d Czeisler C.A. (1991) L igh t - induced suppress ion of e n d o g e n o u s c i rcadian a m p l i t u d e in h u m a n s [ l e t t e r ] . Nature 3 5 0 , 5 9 - 6 2 .

14. K r i p k e D . F . , M u l l a n e y D.J . , K l a u b e r M.R. , Risch S.C. a n d Gil l in J .C . (1992) Con t ro l l ed tr ial of b r igh t light for n o n s e a s o n a l ma jo r depress ive d i so rde r s . Biol. Psychiatry 3 1 , 119-134 .

melatonin syndrome" described a m o n g major depressive pat ients by Beck-Friis and col leagues.

1 Al though light is generally conceived as

melatonin-suppressing, pat ients with major depressions do respond to bright light t r e a t m e n t .

14 Thus , a l though our subjects had no overt major

depressions, the alternative hypothesis that night lights somehow combat a low-melatonin condi t ion cannot presently be excluded.

Unfortunately, a barrier of disbelief delayed exploitat ion of Dewan ' s discovery for two decades. We hope that others will add to the replications from our labora tory , and tha t night light effects on the menstrual cycle will become an accepted reality. This apparent ly powerful—yet simple and seemingly safe—methodology should lead to exciting progress in our unders tanding of h u m a n reproduct ive endocrinology and probably to very useful clinical approaches .


15. K r o n a u e r R .E . (1987) A m o d e l for the effect of light o n the h u m a n " d e e p " c i rcad ian p a c e m a k e r . Sleep Res. 16 , 6 2 1 .

16. Lacey L. (1975) Lunaception: A Feminine Odyssey into Fertility and Contraception, C o w a r d , M c C a n n & G e o g h e g a n , N e w York .

17. Laugh l in G.A. , L o u c k s A.B. a n d Yen S.s. (1991) M a r k e d a u g m e n t a t i o n of n o c t u r n a l m e l a t o n i n secret ion in a m e n o r r h e i c a th le tes , b u t n o t in cycling a th le tes : una l te red by opio idergic o r d o p a m i n e r g i c b l o c k a d e . / . Clin. Endocrinol. Metab. 73 (6 ) , 1321-1326.

18. Lewy A.J. , N u r n b e r g e r J .L , W e h r T.A., P a c k D . , Becker L.E. , Powel l R. a n d N e w s o m e D.A. (1985) Supersensi t ivi ty t o l ight: possible t ra i t m a r k e r for manic-depress ive illness. Am. J. Psychiatry 142 , 725 -728 .

19. Lewy A.J. , W e h r T.A. , G o o d w i n F .K . , N e w s o m e D.A. a n d M a r k e y S.P. (1980) Light suppresses m e l a t o n i n secret ion in h u m a n s . Science 2 1 0 , 1267-1269.

20. Lin M . C . , K r i p k e D . F . , P a r r y B.L. a n d Berga S.L. (1990) N igh t l ight a l ters m e n s t r u a l cycles. Psychiatry Res. 3 3 , 135-138 .

2 1 . M c l n t y r e I .M. , N o r m a n T.R. , B u r r o w s G . D . a n d A r m s t r o n g S .M. (1989) H u m a n m e l a t o n i n suppress ion by light is in tensi ty dependen t . / . Pineal Res. 6 , 149-156 .

22. N e l s o n D . E . a n d T a k a h a s h i J .S . (1991 ) C o m p a r i s o n of visual sensitivity for suppress ion of p ineal m e l a t o n i n a n d c i rcad ian phase-shif t ing in the go lden hams te r . Brain Res. 5 5 4 , 272-277 .

23 . P a r r y B.L. , Berga S.L., Laugh l in G.A. , K l a u b e r M.R . , Yen S.S.C., K r i p k e D . F . a n d Gil l in J .C . (1990) Al tered waveform of p l a s m a n o c t u r n a l m e l a t o n i n secret ion in p r e m e n s t r u a l depress ion . Arch. Gen. Psychiatry 4 7 , 1139-1146.

24. Sykes S.M., R o b i s o n W . G . J r . , Wax ie r M . a n d K u w a b a r a T . (1981) D a m a g e to the m o n k e y re t ina by b r o a d - s p e c t r u m fluorescent l ight. Investigative Ophthalmology & Visual Science 20 (4) , 4 2 5 ^ 3 4 .

25 . T e r m a n J .S. , T e r m a n M . , Schalger D . , Rafferty B. , Rosofsky M . , L ink M.J . , Ga l l in P . F . a n d Q u i t k i n F . M . (1990) Efficacy of brief, in tense light exposu re for t r e a t m e n t of winter depress ion . Psychopharmacol. Bull. 26 (1) , 3 - 1 1 .

26. We t t e rbe rg L. (1978) M e l a t o n i n in h u m a n s : physiological a n d clinical s tudies . J. Neural Transm. S u p p l . 13 , 289 -310 .

27. Wever R.A., Po la sek J. a n d Wi ldg rube r C M . (1983) Bright light affects h u m a n c i rcadian r h y t h m s . Pflugers Arch. 3 9 6 , 8 5 - 8 7 .

22

Circannual Variation of some Endocrine and Neuroendocrine Functions in Humans

W A N T O U I T O U

1 a n d E R H A R D H A U S

2 1 Department of Biochemistry, Faculté de Médecine Pitié-Salpétrière, 91, bd de

l'Hôpital, 75634 Paris Cedex 13, France 2Department of Anatomic and Clinical Pathology, Ramsey Clinic, Saint Paul Ramsey Medical Center, 640 Jackson Street, Saint Paul, Minn, 55101-2595, USA

In t roduct ion

T H E MAMMALIAN organism is characterized by a complex time structure of biologic rhy thms which are present at all levels of organizat ion ranging from popula t ion and g roup rhy thm to individuals, o rgan systems, organs and tissue, cells and subcellular s tructures. These rhy thms cover a large frequency range (infradian, circadian, u l t radian) providing multiple and highly complex interact ions. Mos t au thors suppor t the view tha t the na tura l apparent ly genetically fixed period of biologic rhy thms will cont inue in the absence of environmenta l forces e.g. l ight /dark, magnet -ism, cosmic radia t ions . . . a l though certain endogenous or exogenous factors can exert a considerable influence upon t h e m .

60

Seasonal variat ions of various body functions may be induced environmental ly and may represent the organism's response to variat ions in the envi ronment such as changes in tempera ture , in the length of the daily light and dark spans (photoper iod) , and in seasonal availability of food for, e.g. free-living animals . However , such seasonal rhy thms persist in many functions even in a control led envi ronment of the labora tory , i.e. under constant condi t ions of tempera ture , lighting regimen and food u p t a k e .

19 It appears likely that m a n y of the apparen t seasonal variat ions

of body functions are endogenous circannual rhy thms which may be synchronized (determined in their t iming) by environmenta l factors but are not directly induced by them.

313


Circannual rhythms are defined as rhythms that persist for more than one cycle under condit ions that provide no information abou t the dura t ion of a year. The period of these rhy thms may deviate under constant environmental condit ions from 12 mon ths ( ± 2 mon ths as a rule) thus attesting to their endogenous n a t u r e .

5 In h u m a n s it is not

possible, for obvious reasons, to provide a constant environmental condi t ion over 2 or more years and it is therefore no t possible to ascertain if such a rhy thm is totally endogenous or not . However such changes persist in some variables studied under as far as feasible constant condit ions of tempera ture , lighting regimen, and food u p t a k e .

1 9'

68 In

addi t ion, a circannual free-running from the calendar year has been shown for 17-ke tos te ro ids

17 and for h u m a n blood p r e s s u r e .

1 2'

21 It is also

interesting to note that a circannual rhy thm established in one hemi-sphere was found to persist with the same t iming several years after the subject changed from the southern to the nor thern h e m i s p h e r e .

41

Therefore, it appears that some, if not many , h u m a n seasonal rhythms are endogenous and probably generated by a circannual oscillator. The term seasonal variations should be used whenever environmental factors like light, t empera ture , etc . . . seem to determine the changes observed. The term circannual rhythm should be reserved for variat ions with a period of abou t 1 year for which an endogenous rhy thm componen t has been shown, irrespective of whether it may be modula ted and /o r synchronized by the seasonal changes in the environment . The question of potential Zeitgebers for circannual rhythms is of great interest and experimental work suggests that the same kind of cues (photoper iod, tempera ture , social cues . . .) in the environment act upon bo th circadian and circannual rhy thms. The photoper iod is one of the major cues with ample evidence for the photoper iodic regulation of seasonal reproduc-tion. Tempera ture is ano ther major Zeitgeber, e.g. transfer of g round squirrels from w a r m to cold can phase shift the circannual rhy thm of hibernat ion. Finally, social cues have also to be taken into account , especially in humans .

The physiological basis of circannual rhythmicity is poorly under-s tood. Organisms frequently restrict energetically expensive activities to a specific time of the year, e.g. reduction of activity or animal migrat ion when food availability is low. Reproduct ion , p repara t ion for migrat ion and other various energy demanding activities also coincide with a b u n d a n t local food resources and environmenta l condit ions tha t p romote survival. Seasonally breeding animals frequently detect and respond to environmental cues that accurately signal well in advance, the arrival or depar ture of seasons favoring reproductive success. However , one of the most impor tan t factors is the day length (photoper iod) . Seasonal changes in behavior are also observed in tropical animals despite relatively constant pho toper iod and tempera ture condi t ions.

Circannual Variation of Functions in Humans 315

They could be due , e.g. to regular t iming of the onset of rain in these lat i tudes.

The neural pa thways involved in the generat ion of c ircannual rhy thms are unkown . F o r instance the integrity of the suprachiasmat ic nuclei (SCN) which is essential for the normal expression of a number of circadian rhy thms is not required for the generat ion of c ircannual rhy thms in g round squ i r r e l s .

72

M é t h o d o l o g i e considerat ions

Sampling

F o r a given physiologic function bioperiodicity can be expressed in several frequency domains whatever the species. When sampling is limited to once or twice daily at month ly intervals over the period of a year, collected da ta may reflect a change of the circadian phase ra ther than the existence of a c i rcannual rhythmici ty. A seasonal change of a morn ing or an evening value in b lood could reflect a modification of one (or more) of the parameters characterizing a biological rhy thm, e.g. the phase , the ampl i tude or the mesor , bu t wi thout indicating which one is affected. Circannual rhy thms can be shown in longitudinal studies of the same subjects by repeatedly sampling over one or several years or by the study of different subjects only once or a few times dur ing different seasons.

The interactions of rhythms of different frequencies have also to be taken into account . Single sampling at a fixed clock hour , t h roughou t the year may be misleading since circannual changes in circadian ampl i tude or acrophase would not be recognized which could lead to misinterpre-tat ion of the results.

Ethnic-geographic differences

Ethnic-geographic differences (climate, diet, social cus toms . . .) have been repor ted in the rhy thm parameters of several frequencies, including circannual rhy thms .

M e l a t o n i n

Melatonin is mainly secreted by the pineal gland, the secretion being larger at night bo th in diurnal and noc turna l a n i m a l s .

33 The pho toper iod

(changes in day length in non-equator ia l regions) controls seasonal cycles in m a n y species, e.g. the reproduct ive cycles, behavior , coat growth and color, etc. . . In a large number of species the pineal gland plays a major role in the transmission of the information on pho toper iod to the body. As the onset darkness is followed by an increase in the synthesis of


m e l a t o n i n ,

33 the greatest activity of the gland can be expected to occur

dur ing the shortest days, i.e. in winter. This dogma , commonly accepted for animals in their na tura l s t a t e

48 has no t been verified in healthy h u m a n

sub jec t s .

58 Whatever the length of na tura l light, longer in summer and

shorter in winter, the circadian acrophases were located a round 03.00 hours all year long (Figure 1). This stability suggests that the p lasma level

A Β

F I G . 1. C i r cad ian p a t t e r n s of p l a s m a m e l a t o n i n in y o u n g m e n ( A ) , elderly m e n ( • ) , elderly w o m e n ( · ) a n d elderly d e m e n t e d subjects ( O ) d o c u m e n t e d in

J a n u a r y (A), M a r c h (B), J u n e (C) a n d O c t o b e r (D) . F r o m T o u i t o u et α / .

58

of melatonin is relatively independent of the onset of darkness o u t d o o r s .

58 H u m a n s are not considered to be a photoper iodic species.

However the control of indoor light (i.e. artificial photoper iod) , as well as indoor tempera ture by h u m a n s part iculary in u rban si tuations, might cue

Circannual Variation of Functions in Humans 3 1 7

the circadian (and seasonal) rhy thm of mela tonin a l though it has been shown tha t exposure to artifical light dur ing the da rk phase does no t reduce p lasma mela tonin levels in h u m a n s u b j e c t s

2 8'

4 0 , 6 4 , 65 except when

very bright artificial light is u s e d .

3 7'

38 In nor the rn tempera te zones and

Antarct ica a phase advance of the mela tonin circadian rhy thm occurs in summer compared to winter. A similar phase advance can be achieved when submit t ing h u m a n subjects to bright light ( > 2,500 lux) t rea tment dur ing the Antarct ica win ter .

7 In seasonally breeding species the

circadian mela tonin rhy thm may be the h o r m o n a l signal t ransducing environmental light information to the reproduct ive s y s t e m .

4 8 - 50 H o w -

ever in nonseasonal breeders the physiologic role of this molecule remains unclear. The circadian rhy thm of mela tonin in h u m a n body fluids has been demons t ra ted by several groups with a decrease of mela tonin secretion in the elderly (review in 59). Evaluat ion of seasonal differences in the circadian 24-hour mean of p lasma mela tonin concent ra t ion showed apparen t peaks in J a n u a r y / M a r c h in the elderly and in June in young men bo th with significant peak- t rough differences (Figure 2). These da t a extend and clarify those obta ined by o t h e r s

2'

3'

69 with one or two

samplings per day at month ly intervals. Indeed in such designs a shift in the acrophase or a change in the circadian ampl i tude would also result in yearly fluctuating concentra t ions of the h o r m o n e documented at a single sampling time wi thout overall modification of the circadian mean of mela tonin secretion.

A study was performed by T a m a r k i n et al.

54 in 20 women with stage I

F I G . 2. C i r c a n n u a l p a t t e r n s of 24 h o u r - m e a n levels of p l a s m a m e l a t o n i n in y o u n g m e n ( A ) , elderly m e n ( • ) , elderly w o m e n ( · ) a n d elderly d e m e n t e d subjects

( O ) . F r o m T o u i t o u et ai

58


or II breast cancer and eight no rmal control women. Samples were obtained every 3 hours 1 day before or 5 days after surgery. A section of the mal ignant breast tissue resected was analyzed for estrogen receptors (ER) The circadian melatonin rhythms of some patients from breast cancer showed differences with those of normal subjects. Al though all subjects had a nocturna l peak of p lasma melatonin a round 02.00 it was much smaller in ER positive pat ients . The au thors were able to put in evidence a significant inverse relation between ER concentra t ion and peak melatonin concentra t ion. Unfortunately no ment ion was made of the stage of the menst rual cycle at the time of sampling which could have in t roduced a bias since large variat ions of mela tonin concentra t ions have been reported by Wetterberg et al.

69 at the time of mens t rua t ion . The

question remains unanswered whether the decline in the nocturna l peak of melatonin represents a long-term pineal failure or a change occurring at the time of breast cancer development . T o know whether the al terat ion of the melatonin rhy thm precedes the presence of mal ignant tissue will require longitudinal and familial studies.

Nevertheless these da ta suggest that the lower the nocturna l peak of plasma melatonin the greater the probabil i ty that the tumor may be hormonal ly dependent . Thus , the modification of the mela tonin rhy thm may serve as a marker for increased risk of ER positive breast cancer development . Seasonal fluctuations of ER concentra t ions in h u m a n breast cancer have been d e m o n s t r a t e d .

2 5'

26 The possible relations

between these variat ions and the seasonal variat ions of p lasma melatonin should be investigated as they might be consistent with the seasonal incidence of breast cancer m o r t a l i t y

3 5a n d seasonal occurrence of the

disease.

9

Prolact in

Prolact in (PRL) , a polypeptide ho rmone secreted by the pitui tary, is released episodically over the 24 hour span and the frequency and magni tude of the episodes are , in par t , under the control of central nervous system rhythms (time of day, sleep-wake cycle). The circadian pat tern is characterized by a min imum a round noon and a nightt ime increase with a max imum a round midsleep (Figure 3). Whereas the par t played by P R L in condit ions such as lactat ion and amenor rhea is well known, its role in the genesis of m a m m a r y cancer in h u m a n beings is still c o n t r o v e r s i a l .

2 0'

2 2'

2 7'

67

A number of hormones are known to affect the secretion of prolact in in humans , e.g. adminis t ra t ion of e s t r o g e n s

1 4'

71 or m e l a t o n i n

31 which

increase p lasma prolact in levels in young adult and adult men and women. Al though the secretion of bo th m e l a t o n i n

59 and e s t r o g e n s

66 is reduced in

Circannual Variation of Functions in Humans 3 1 9

15

10

~ 5 ε c

15

10

J L

Elderly men

_ _ J I L

ο u

15

10

Elderly demented subjects

J I I L

0 7

4 51 1

45 1 5

4 51 9

45 2 3

45 0 3

45

Elderly women

J 1 I L

0 74 5 n4 5 1 54 S 1 94 5 2 34 5 ^ 4 5

F I G . 3. C i r cad i an pa t t e rn s of p l a s m a pro lac t in in y o u n g m e n ( A ) , elderly m e n ( • ) , elderly w o m e n ( · ) a n d elderly d e m e n t e d subjects ( • ) . F r o m T o u i t o u

et a l

51

the elderly, no apparen t effect could be seen on prolact in levels of elderly subjects when compared to young m e n .

59 A number of studies agree with

the absence of detectable circannual rhy thm of prolact in in young or elderly men (Figure 4 )

1 1»

1 5'

47 whereas c i rcannual rhythmici ty was vali-

dated in heal thy young and elderly w o m e n .

55

Prolact in acts on the estrogen-primed breast to st imulate and main ta in lactat ion. It is associated with the functioning of differentiated cells ra ther than with cell g rowth and thus would be expected to decrease breast cancer risk. The l i terature reports conflicting results on p lasma prolact in


18 h

10±r Τ _ j ι ι i _

January March June October

F I G . 4. C i r c a n n u a l pa t t e rn s of 24 h o u r - m e a n level of p l a s m a pro lac t in in y o u n g m e n ( • ), elderly men ( • ), elderly w o m e n ( · ) and elderly demen ted subjects ( Ο ).

F r o m T o u i t o u et ai

51

concentrat ions in h u m a n breast c a n c er .

6'

1 0'

1 3'

1 4'

1 6'

2 4'

3 4'

51 This may be

due to the rhythmic characteristics of prolact in secretion as well as to geographic l o c a t i o n

52 and ethnic differences

20 a l though this latter point

might in fact merely represent environmental and nutr i t ional f a c t o r s .

4'

56

M a n y of the controversies in the study of the potential role of prolactin in the etiology and /o r pathogenesis of breast cancer are due to a lack of regard paid to the multifrequency time structure in prolactin secretion and serum concentra t ions . Studies at a single clock hour only or at different clock hours in different studies and /o r l imitation to a single, e.g. the circadian frequency, may often be the source of nonreproducible , controversial and even misleading results.

Ha us et al.

20 studied the ethnic-geographic differences in prolactin

serum concentrat ions between Japanese women in Kyushu , J apan and in American women in Minnesota , USA. The Japanese and half the Minnesota popula t ion were selected for absence of epidemiologically determined risk factors to develop breast cancer, while in the American women, half of the par t ic ipants in the study were selected to present such risk factors (especially a history of first degree female relatives with breast cancer). This study extended over three age ranges, namely adolescent, young adult , and menopausa l women. Sampling at 20 minute intervals was carried out over a 24-hour span four time in each woman , once dur ing each of the four seasons. The rhythmometr ic analysis of the da ta indicated statistically highly significant differences between Japanese and American women with higher values of serum prolact in concentra t ion in the Japanese only dur ing the night hours in winter and spring. Dur ing the day time (when most investigators choose to sample), there was usually no difference in plasma prolact in concentrat ions between the two groups of


women. These differences disappeared dur ing summer and began to reappear in fall. The rhy thmometr ic analysis revealed that this difference was due to a larger circadian ampl i tude and a circannual rhy thm (or seasonal variat ion) in the Japanese women with acrophase dur ing the second par t of November , which was not statistically significant in the Americans. A repeat study dur ing M a r c h confirmed the nocturnal differences in p lasma prolact in concentra t ions dur ing spring. Depending on the clock hour of sampling along the circadian and circannual scale, the Japanese had either higher, comparab le or lower plasma prolact in concentra t ions than the Americans.

The double ampl i tude of the circannual variat ion (a measure of the extent of predictable change) amoun ted in the Japanese women to abou t 4 0 % of the mesor .

The circadian rhy thm characteristics between the low and high risk groups of Minneso ta women showed a difference in the circadian ampl i tude of the two risk groups in Minneso ta dur ing fall only (p = 0.04).

In menstrual ly cycling subjects, there was a difference in prolact in circadian mesor between Japanese and Americans at the end of the luteal phase. A menstrual cycle related rhy thm in p lasma prolact in concentra-tions was described only in the USA high risk g roup with an acrophase in the luteal stage.

An age t rend was found with lower circadian prolact in mesor in pos tmenopausa l women in bo th countr ies . An age related decrease of the circadian ampl i tude , however, was found in the Japanese only.

Since dietary and climatic condi t ions were not controlled, it remains to be clarified whether or not these facts reflect a true ethnic-geographic difference between American and Japanese women (the latter of which are epidemiologically classified as low-risk).

The observat ion of a higher circannual ampl i tude associated with a lower risk to develop breast cancer may correspond to the results of a s tudy performed in I t a l y

55 involving women with a related yet different

risk, namely with fibrocystic mas topa thy . As compared with healthy controls , the subjects with fibrocystic mas topa thy revealed a lack of circannual variat ion of p lasma prolact in as g r o u p phenomenon . All subjects in this s tudy were followed at the same geographic location with comparab le diet and climate exposure. In bo th studies involving I tal ian, Japanese and American women, a negative correlat ion was found between breast cancer risk and ampl i tude of the p lasma prolact in circannual variat ion.

It would be desirable to follow a larger g roup of women of different risk to develop breast cancer over their entire reproduct ive life using chronobiologic techniques and rhy thmomet r ic analyses. This has thus far not been feasible. In the meant ime, chronobiologic methodology will have to be followed to obta in meaningful results. The accumulat ing da t a will


have to be carefully evaluated and any conclusions to be d rawn will have to keep the multifrequency time structure of prolact in secretion in mind.

V i t a m i n D, parathyro id hormone , ca lc i tonin and e lec t ro ly te - re la ted metabo l ism

Before vitamin D can exert its biologic effects, two hydroxylat ion reactions are required. It is first metabolized to 25-hydroxyvitamin D (25-OH-D) in the liver and subsequently in the kidney to Ια-25-dihydroxyvitamin D ( l , 2 5 - ( O H ) 2D ) , which is the biologically active metabol i te . l , 2 5 - ( O H ) 2D plays a major role in the regulat ion of the mineral metabol ism by st imulat ing the active intestinal absorp t ion of calcium (Ca) and phos-phorus (P) and regulating their flux into and out of bone . The renal synthesis and the circulating concentra t ions of l , 2 5 - ( O H ) 2D are, in turn , controlled by the serum levels of Ca and inorganic phospha te (Pi) either directly or, in the case of Ca, th rough para thyro id h o r m o n e (PTH) .

The serum concentra t ion of l , 2 5 - ( O H ) 2D does not undergo large variat ions a long the 24-hour scale but is mainta ined within relatively na r row limits t h roughou t the d a y

1'

46 though some young men were found

to exhibit a circadian rhy thm with a small ampl i tude of abou t 10% or l e s s .

18 Circulat ing levels of calcitonin are also circadian periodic in normal

subjects, with a peak a round m i d d a y .

23

The vi tamin D 3 metabol i te concentra t ion would be expected to vary according to climate and sunlight exposure. A seasonal variat ion (with higher levels dur ing the summer) has been described for 2 5 - O H - D ,

3 6'

53 for

2 4 , 2 5 - ( O H ) 2D32

and for l , 2 5 - ( O H ) 2D30

in young and elderly subjects though seasonal variat ions were no t found by some investigators for this latter m e t a b o l i t e .

8'

39

In recent years, osteomalacia has been named as a possible cont r ibutory factor in femoral neck fractures, and lower values of vitamin D metaboli tes are often encountered in the e lde r ly .

36 Meller et α / .

44 have observed in

geriatric patients with long bone fractures that the serum concentra t ions of vi tamin D metaboli tes correlate positively while tha t of para thyro id ho rmone correlates negatively with day length and global solar radiat ion. The high prevalence of fractures in young and elderly people during the winter mon ths can be related, at least in par t , to these biologic variat ions.

Seasonal variat ions in serum concentra t ions of P T H were described in elderly subjects with higher levels in winter when concentrat ions of 25-O H - D and 2 4 , 2 5 - ( O H )2D were lowest, which suggests a possible functional relat ionship between these h o r m o n e s .

3 6'

44

Plasma total Ca and M g undergo circadian and seasonal variat ions in the elderly with a small rhy thm a m p l i t u d e .

4 5 , 6 0 , 61 The circadian

variat ions of bo th cations (Figure 5) have been thought to reflect predominant ly the diurnal changes in p lasma p r o t e i n s .

2 9 , 42 However , this


Young men serum

Na CI Ca

Creatinine Mg

Urate Urea

Κ 3 _ L Ρ

Elderly women serum

M I I I I I 0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24

Elderly men serum

Creatinine Urea

Ca Urate

Ρ Na CI κ

2 4 6 8 10 12 14 16 18 20 22 24

Peak-trough differences (%)

Cl<

Young men urine

I I I I 1 I I 1 I I I I I O J 2 4 6 8 10 12 14 16 18 20 22 24

Peak-trough differences (%)

F I G . 5. C i r cad i an var ia t ions of se rum cons t i tuen t s . F r o m T o u i t o u et αϊ}

does not fit well with the large differences in the circadian ampli tudes of the p lasma total proteins as compared to the much smaller ampli tudes of Ca and M g as found by Tou i tou et al.

62,63 and the phase difference between

plasma Ca and plasma proteins as reported by Nicolau et al.

45 Markowi tz

et α\

43 have described the tempora l relat ionship between the circadian

rhythms of serum P T H and Ca concentra t ions and have found, at least in healthy young men, tha t the changes in ionized calcium (Cai) concentra-tions precede inverse changes in P T H levels by 2 hours whereas changes in P T H precede similar directional al terat ions in Cai by abou t 4 hours .

The circadian variat ions in p lasma inorganic phosphorus (Pi) are large with a peak located at night in bo th young and elderly s u b j e c t s .

2 9 , 63 In

bo th diurnal and noc turna l animals the peak values are observed at the end of the resting period.

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59. T o u i t o u Y., Fèvre M. , Lagoguey M. , C a r a y o n Α., B o g d a n Α., Re inberg Α., Beck Η. , Cesselin F . a n d T o u i t o u C . (1981) Age a n d m e n t a l hea l thre la ted c i rcadian r h y t h m of p l a s m a levels of m e l a t o n i n , p ro lac t in , luteinizing h o r m o n e a n d foll icule-st imulating h o r m o n e in m a n . J. Endocrinol. 9 1 , 4 6 7 - 4 7 5 .

60. T o u i t o u Y. a n d H a u s E. (eds.) (1992) Biologic Rhythms in Clinical and Laboratory Medicine. Spr inger , Berlin, N e w Y o r k , L o n d o n , Pa r i s .

6 1 . T o u i t o u Y., T o u i t o u C , B o g d a n Α., Beck H . a n d Reinberg A. (1978) Se rum m a g n e s i u m c i rcadian r h y t h m in h u m a n adu l t s wi th respect to age, sex a n d m e n t a l s ta tus . Clin. Chim. Acta 8 7 , 3 5 - 4 1 .

62. T o u i t o u Y., T o u i t o u C , B o g d a n Α., Re inbe rg Α., Auzéby Α., Beck Η . a n d Guil le t P . (1986) Differences be tween y o u n g a n d elderly subjects in seasonal a n d c i rcadian var ia t ions of to ta l p l a s m a pro te ins a n d b lood vo lume as reflected by h e m o g l o b i n , hema toc r i t a n d e ry th rocy te c o u n t s . Clin. Chem. 3 2 , 801-804 .

63 . T o u i t o u Y., T o u i t o u C , B o g d a n Α., Re inberg Α., M o t o h a s h i Y., Auzéby A. a n d Beck H . (1989) C i r cad i an a n d seasonal va r ia t ions of electrolytes in aging h u m a n s . Clin. Chim. Acta 180 , 245 -254 .

64. V a u g h a n G . M . , Bell R. a n d de la P e n a A. (1979) N o c t u r n a l p l a s m a me la ton in in h u m a n s : episodic p a t t e r n a n d influence of l ight. Neurosci. Lett. 14 , 81 -84 .

65. V a u g h a n G . M . , P e l h a m R.W. , P a n g S.W., Lough l in L.L. , Wi l son K . M . , S a n d o c k K .L . , V a u g h a n M . K . , K o s l o w S.H. a n d Reiter R.J . (1976) N o c t u r n a l e levat ion of p l a s m a me la ton in a n d u r ina ry 5-hydroxyindoleace t ic acid in y o u n g m e n : a t t e m p t s a t modif ica t ion by brief changes in e n v i r o n m e n t a l l ighting a n d sleep a n d by a u t o n o m i c d rugs . / . Clin. Endocrinol. Metab. 4 2 , 752 -764 .

66. Vermeulen A. (1976) T h e h o r m o n a l activity of the p o s t m e n o p a u s a l ova ry . J. Clin. Endocrinol. Metab. 4 2 , 2 4 7 - 2 5 3 .

67. Vôlker W. , Leiper t K . P . , Gehr l ings H . , S tauch G. , von zur M u h l e n A. a n d Schneider J. (1986) M a s t o p a t h i e u n d M a m m a c a r c i n o m : Besteht ein typisches H o r m o n Profil? Geburtsch u. Frauenheilk. 4 6 , 284—289.


68. Wal lace A . L . C . (1979) Var ia t ions in p l a s m a thyrox in c o n c e n t r a t i o n s t h r o u g h o u t one year in penned sheep on a uniform food in t ake . Aust. J. Biol. Sci. 3 2 , 371-374 .

69. We t t e rbe rg L., A r e n d t J., P a u n i e r L., S i zonenko P .C . , van D o n s e l a a r W . and H e y d e n T . (1976) H u m a n me la ton in changes du r ing the m e n s t r u a l cycle. J. Clin. Endocrinol. Metab. 42 , 185-188 .

70. Wirz-Jus t ice A. a n d Richter R. (1979) Seasonal i ty in b iochemica l d e t e r m i n a t i o n s : a source of va r iance a n d a clue to the t e m p o r a l incidence of affective illness. Psychiatry Res. 3 1 , 5 3 - 6 0 .

7 1 . Yen S.S.C., E h a r a Y. a n d Silver T . M . (1974) A u g m e n t a t i o n of p ro lac t in secret ion by es t rogen in h y p o g o n a d a l w o m e n . / . Clin, invest. 5 3 , 6 5 2 - 6 5 5 .

72. Z u c k e r I., Boshes M . a n d D a r k J. (1983) S u p r a c h i a s m a t i c nuclei influence c i r cannua l a n d c i rcadian r h y t h m s of g r o u n d squirre ls . Am. J. Physiol. 244 {Regulatory integrative Comp. Physiol. 13), R 4 7 2 - R 4 8 0 .

23

Peptic Ulcer and Melatonin F. I. K O M A R O V , S. I. R A P O P O R T , N. K. M A L I N O V S K A Y A , M . V. N E V E R O V A a n d V. A . B O R I L K O

Center of Gastroenterology, Department of Propedeutical Therapy, I.M. Setchenov Mockow Medical Academy, Pogodinskaya str.-5, Moscow 119435, Russia

S u m m a r y

T h e epiphysis e x t r a c t — e p i t h a l a m i n e — w a s used in t r e a t m e n t of d u o d e n a l ulcer. Ep i tha l a -mine was admin i s t e red i.m. in a dose of 10 m g a day . A rap id response to the t r e a t m e n t was ob ta ined . D u o d e n a l ulcer remiss ion was n o t e d at the 10th day of t r e a t m e n t in four pa t i en t s a n d on the 20th d a y in five pa t i en t s . Th is effect of ep i tha l amine can be expla ined by its a d a p t o g e n e activity a n d synch ron iza t ion of h u m a n biological r h y t h m s . T h e role of ep i tha l amine in t r ea t ing of desynchronos i s is discussed.

In t roduct ion

I N THIS repor t the first results of the work on this subject are presented. The problem of h u m a n adap t ion to the envi ronment and the changes in

physiological rhy thms at their d is turbance a t t ract the a t tent ion of all investigators. The most impor tan t feature of this condi t ion for the clinicians is desynchronosis—divergence of the na tura l physiological rhythms of the organism between themselves, and with t ime. The degree of the desynchronosis can determine the severity of the disease and in some cases it can act as a separate disease: after an ab rup t change in climate, dur ing "sleep-awakening" dis turbances, after changes in social rhy thm sensors, and dur ing chronic stress and emot ional t ens ion .

4

The phase dyssynchrony hypothesis includes a central , integrative and controll ing mechanism that provides a framework for the m o d e in which the pharmacological p repara t ion works in rhy thm disorders . In the present study we test the hypothesis tha t t rea tment with pineal gland extract forms a role in the en t ra inment of dis turbed biological rhy thms in patients with duodena l ulcer.

It is obvious, tha t the epiphysis is no t a rud imentary formation, bu t a special neuroendocr inal gland, having an unusual evolution and combin-

3 2 9


ing morphological features of the neural and endocrinal tissues and producing a b road number of biological active substances and h o r m o n e s .

3'

6 These very features are the cause of the specialty of this

neuroendocr inal gland and its b road part ic ipat ion in the vegetative functions and homeostasis system regulat ion, which realize its act ion th rough direct and indirect connect ions with hypo tha lamus , hypophysis , endocrinal glands, immune system, central and peripheral sympathie nervous system.

The study of the epiphysal hormonopoes i s of serotonin, melatonin , and enzymes part icipat ing in their synthesis, demons t ra ted the clear circadian, ovarian, menstrual , and seasonal rhy thms of this p r o c e s s .

2'

13 In

morphological studies on some species of pr imary vertebrates and m a m m a l s was ment ioned, that neuroendocr inal cells of the epiphysis (pinealocytes) are the rudimentary photoreceptor c e l l s .

1 1'

12 This fact,

unique evolutional epiphysis t ransformation from the photoreceptive organ to the neuroendocr inal gland and its extreme biochemical and functional rhy thms are the basis of the special studies on the relation of this gland to the h u m a n and animal "biological watch" system. The fundamental studies on the gland innervat ion in epiphysis and hypophysis tissues and experimental works on the gland denervat ion detected, that epiphysis is one of the leaders of the "biological watch" system and it realizes the photoper iodical synchronizat ion of the physiological functions.

5 This circumstance allowed us to use the extract of epiphysis in

correction of desynchronosis .

Earlier in our works , fulfilled in our labora tory under the leadership of the academics F . I. K o m a r o v and Prof. S. I. Rapopor t , it was demonst ra ted , that any duodena l ulcer relapse is accompanied by the divergence of the organism physiological functions and the degree with which it occurs correlates with the severity of the disease. Dur ing the remission the degree of the desynchronosis decreased and it was modera te dur ing the recovering.

7

The role of substances, secreted by epiphysis, in the pathogenesis and clinic of peptic ulcer was no t studied earlier. At the same time the periodicity of this disease, the seasonal character of its relapses, and clear rhythms in pain and dyspepsia indicate the possible role of this substance in the pathogenesis and formation of the clinical features of this disease. Taking into account a cyclical melatonin secretion dur ing the day and its seasonal changes one can explain the periodicity of peptic ulcer.

All these facts allowed the study to be carried out , in which the acetic cattle extract of bovine epiphysis—epithalamine—was used as a medicine. The subs t ra tum was obta ined in Leningrad Mili tary Medicine Insti tute under the leadership of Prof. V. Ch. Havinsone . Chemically it is a polypeptide substance, consisting at least of five homogenous active peptides wi thout indol subs tances .

6 According to the l i terature to da te

Peptic Ulcer and Melatonin 3 3 1

epi thalamine was successfully used in oncology and gerontology (it was confirmed in clinical and experimental studies). In some papers its high effectiveness as an adap togene is n o t e d .

8

Mater ia ls

M o n o t h e r a p y of epi thalamine was carried out in 10 pat ients with uncomplicated duodena l ulcer and wi thout accompanying pa thology (all men 2CM5 years old); the pat ients had frequent relapses, equal b io rhy thms ("evening type"), hyperacidity, and dura t ion of anamnesis over 2 years. Pat ient 's examinat ion and t rea tment was carried out in the hospital . Examina t ion was done twice and included gastroscopy, b lood examin-at ion, p lasma secretory IgA concentra t ion by the Manchin i test, urine examinat ion, examinat ion of feces, E C G findings, sonography , chest R-ray, intragastr ic pH-met ry with his tamine st imulat ion. Special bio-rhythmological examinat ions were adminis t ra ted before and after ther-apy: 3-hours body tempera ture and pulse measur ing (at 8 a.m., 11 a.m., 2 p.m., 5 p.m., 8 p.m., and 11 p.m.) ; as psychophysiology was used, a "line-test" was carried out at the same t ime: the pat ient was ordered to d raw on a sheet of paper the maximal number of a vertical lines 10 m m long. It is known, that its number correlates well with the neuropsychical condi t ion of a pat ient and with the speed (r= + 0 . 8 5 ) of a simple sensor imotor reaction. This test is used for the est imat ion of the circadian "sleep-awakening" r h y t h m .

10

Daily 3-hour por t ions of urine before and after t rea tment were obta ined (eight por t ions a day) for the measurement of mela tonin metabol i tes excretion.

Epi tha lamine was injected at 10 a.m. at a dose of 10 mg i.m. (this scheme was recommended by the producers of epi thalamine) after general and biorhythmological examinat ion of a pat ient . The calculation was done using cosinor and correlat ion tests.

Results

The da ta , describing rapid healing of ulcer after the epi thalamin adminis t ra t ion, are presented in Table 1; the pain and dyspepsia relieved on the 3 rd -5 th day of t rea tment . Duodena l ulcer remission was noted at the 10th day of the t rea tment in four pat ients and on the 20th day in five pat ients . In one pat ient the ulcer was indolent . The healing of duodena l ulcer was accompanied by the remaining inf lammation of duodena l mucosa . O n e m o n t h after healing there was no inf lammation in the duode num. The statistically unreliable increase of p lasma secretory IgA was noted at the m o m e n t of ulcer relapse (0.86 + 0.09 g/1 before and 0.93 + 0.09 g/1 after t rea tment ) .

TA

BL

E

1

Clin

ical

ch

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teri

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s of

the

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ep

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rese

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n D

ysp

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nd

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e

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pic

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size

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er

0.5

0.5

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1

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n re

lief

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ysp

epsy

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lief

U

lcer

h

eali

ng

ero

sio

n an

d b

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itis

af

ter

trea

tmen

t >

5 5

>5

10

20

>2

0

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er

a m

on

th

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eatm

ent

pat

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ere

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No

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1 cm

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ays

day

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ays

day

s d

ays

day

s d

ays

day

s d

ays

Yes

N

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es

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1 +

+ +

+ +

+ 2

+ +

+ +

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3 +

+ +

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+ +

+ 4

+ +

+ +

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+ +

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+ +

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+ +

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+ +

7 8 9 +

+ + + + +

+

+ + +

+ +

+ +

+ +

+ + +

+ + + 10

+

+ +

+ +

+ +

Peptic Ulcer and Melatonin 333

37.0

36,2 ^ \ -

3 6 , 8ρ · · * · *

β\ , · — \ / - - '

%"

#

L 2fei I I 36 .31 1V IV I

1 12 18 12 18

90

64 Γ / ν · . . / W s / ^ - -

9 0F ^ n / " " - > / X

L£ Ty * ι · ι 7 41 IV y % 6 12

Β

ι

r 0

ρ, In

18

Before

P.*

12

_ _ _ _ _ _ _

1 0.25 0.43 0.12

1 1 1 t, In

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r 0

-1

After

0.61

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0.43

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0.49

I p, In t, In

F I G . 1. Pa t i en t C . D u o d e n a l ulcer, (a) T e m p e r a t u r e , pulse a n d " l ine" test before a n d after t r ea tmen t , (b) Ca lcu la t ion coefficient before a n d after t r e a t m e n t .

There was no changes in b lood and urine analysis dur ing epi thalamin t reatment , and no allergic reactions.

Discussion

The rapid healing indicates high effectiveness of epi thalamine. The c o m m o n dura t ion of duodena l ulcer epithelisation is greater than 30 days.

Δ

Before After

Ά 9T a 1 1 3i>Aa 49 r / ι —J ! ι 7 61 ^ V i v ^ Ν»

κ J

1 6 12 18 1 6 12 18


Before After

1 6 12 18 1 6 12 18

1 6 12 18 1 6 12 18

Β

- ι

r 0

-1

- Before

. -0.03

0.60

1 1

0.03

1 p, In t, In

- After

-0.04 m -0.73 u

1

• 0.26

1

- i -1

P» In P.* t, In

F I G . 2. Pa t i en t K. D u o d e n a l ulcer, (a) T e m p e r a t u r e , pulse a n d " l ine" test before and after t r ea tmen t , (b) Ca lcu la t ion coefficient before a n d after t r e a tmen t .

H2-h i s t amine receptor blocking drugs heal duodenal ulcer dur ing 3 weeks t rea tment and epi thalamine does it within the same period with very rapid reduction of pain and dyspepsia. The clinical effect of epi thalamine can be explained by its adap togen activity, hypothetically working th rough mechanisms which synchonize the h u m a n biological rhy thms. It is well demons t ra ted on the following clinical examples: pat ient C (Figure 1) had intensive divergence of pulse, tempera ture and "line-test" rhy thms and

Peptic Ulcer and Melatonin 335

positive correlat ion analysis da t a after ulcer healing (after 10 days t rea tment) . The opposi te observat ion is demons t ra ted in pat ient Κ (Figure 2): the duodena l ulcer did no t heal in 20 days, and after t rea tment all b iorhythmological da t a worsened; negative correlat ion appeared between pulse and "line-test" and between tempera ture and "line-test", the circadian rhy thm did no t appear before, but after the t rea tment . It indicates the increasing degree of desynchronosis . We must note tha t some patients did no t demons t ra te a severe decrease in degree of desynchronosis after the ulcer healing. It is connected with the presence of duodena l erosions and bulbitis. Earlier in our works on the gastric mucosal protein synthesis rhy thms , the absence of rhy thm res torat ion to the no rma l level when inf lammation in duodena l mucosa is present was demons t r a t ed .

1 It

means the absence of desynchronosis decreases.

The absence of statistically reliable changes in p lasma secretory IgA level means tha t immunological mechanisms are not dominan t in the epi thalamine effect. The decrease in the pat ients ' desynchronosis after the epi thalamine t rea tment confirms the connect ion of its therapeutical effect with its synchronizing activity on the pat ients ' b io rhy thms . W e mus t note , tha t the me thod of au tomeasur ing , used in our work , was to some extent imperfect due to pat ients ' incompetence.

Undoub ted ly the moni tor ing of physiological da t a is opt imal . In conclusion it mus t be noted tha t ou r results of the t rea tment of

desynchronosis are very perspective for the future study and for the therapeutical adminis t ra t ion of epi thalamine as a medicine. These results are the first step of the work on this topic. In future we intend to enlarge our investigations a n d to s tudy the dynamics of mela tonin metabol i tes ' excretion.

References

1. Brodsk i V.L . a n d F a t e e v a V.I . (1984). T h e presence of a b o u t h o u r r h y t h m s of the p ro te in synthesis in h u m a n gas t r ic m u c o s a l b iops ies . Bull. Exper. Biol. 5 , 612 -614 .

2. Ca rd ina l i D . P . (1981). M e l a t o n i n . A m a m m a l i a n p inea le h o r m o n e . Endocr. Rev. 2 , 2 3 7 - 2 5 8 .

3. C h a s o v E.I . a n d I s a c h e n k o v V.A. (1974). Epiphysis: the Role in the Neuroendocrine Regulation. Med ic ina , M o s c o w .

4. Chronobiology and chronomedicine (1989). Med ic ina , M o s c o w . 5. D i l m a n V . M . (1986) The Large Biological Watch. Z n a n i e , M o s c o w . 6. E b a d i M . (1984) Regu la t ion of the syntesis of m e l a t o n i n a n d its significance to

n e u r o e n d o c r i n o l o g y . In The Pineal Gland (ed. Rei ter R J . ) , p p . 1-38. R a v e n Press , N e w York .

7. E r e m i n a L.V. (1988) The Circadian Rhythms of Psychophysiological Functions in Patients with Peptic Ulcer. M o s c o w .

8. K a r p o v R.S. , S lepushk in V . D . , M o r d o v k i n V . F . et al. (1985) The Using of Epiphysis Extracts in Clinic. T o m s k i Univers i ty , T o m s k .

9. M a e s t o n i G. I . , C o n t i A. a n d P ie rpao l i W . (1987) T h e pineal a n d the c i rcad ian op ia te rg ic , i m m u n o r e g u l a t o r y role of m e l a t o n i n . Ann. N.Y. Acad. Sci. 496 , 61-11.

10. M a k a r o v V.I . (1986) B io r i thmologyca l test . Nauka ijizn. 1 , 79.


11. Rei ter R.J. (1980) T h e pineal a n d its h o r m o n e s in the con t ro l of r e p r o d u c t i o n in m a m m a l s . Endocr. 1, 109 -131 .

12. Rei ter R.J . (1981) T h e m a m m a l i a n pineal g land : s t ruc ture a n d function. Anat. 162, 2 8 7 - 3 1 3 .

13. Vaskovsk i B.V., Micha leva 1.1., I v a n o v V.T. et al. (1990) T h e excret ion of the biological active pep t ides from bovin epiphysis ext rac t . In Vsesousni Symposium on the Peptide Chemistry. Riga. p p . 16.

24

The Relationship between the Pineal Gland and Cancer: Seasonal Aspects H. B A R T S C H ,

1 C. B A R T S C H ,

1 D. M E C K E

2 a n d T. H. L I P P E R T

1 1 Division of Clinical Pharmacology, Department of Gynaecology and

Obstetrics, University of Tubingen, Tubingen, Germany 2Institute of Physiological Chemistry, University of Tubingen, Tubingen, Germany

DESPITE THE invention of electric light and central heat ing as well as cons tant food availability t h roughou t the year m o d e r n m a n has , to a certain extent, main ta ined seasonal rhy thms in many body f u n c t i o n s

13

including r e p r o d u c t i o n .

23 It is therefore no t surprising that also numerous

diseases can show very s t rong seasonal rhythmici ty in their occurrence, one of the most p rominent examples being seasonal affective disorder (see elsewhere in this volume). There are also m a n y reports on the seasonality of the occurrence of h u m a n cancer of which only some examples are given in the following chapter .

Seasonal i ty in t h e occurrence of h u m a n cancer

Cohen et α\}

2 repor ted more cases of breast cancer being diagnosed in

spring and O w n b y et al

21 observed that breast tumors at the time of

opera t ion were bigger in spring. Increased appearance of breast cancer in spring and of pros ta te and testicular cancer in winter are described by Jacobson et α / .

15 When drawing conclusions from such da t a one has to be

very careful since the observed manifestations of the tumor , like e.g. first symptoms , size, appearance of metastases , depend on the type of tumor , its localization, and growth kinetics. Fu r the rmore , the me thods of detection vary widely for different types of cancer. Therefore, the rhy thm parameters can differ bu t the occurrence of seasonality may still be due to the same biological factors.

3 3 7


Relat ionship b e t w e e n the pineal gland and cancer

The pineal gland is affected by growth of malignant tumors , particularly of breast and prostate, and the pineal ho rmone melatonin as well as other yet unidentified pineal substances are capable of inhibiting a number of experimental tumors in vivo and in vitro (for review see Ref. 8). The contribution to this book on the subject deals with the seasonal rhythmicity of both aspects of this relationship: in humans and experimental animals seasonal rhythms have been observed in melatonin product ion by the pineal gland and the unidentified tumor-inhibiting substance(s) could be found in h u m a n urine and in rat pineal glands mainly in summer.

Seasonal rhy thmic i ty of serum mela ton in is deple ted in human cancer pat ients

H u m a n pat ients suffering from breast and pros ta te cancer show a tumor-s tage-dependent depression of circadian serum melatonin rhythms when compared to age-matched pat ients with benign t u m o r s .

2 , 4'

8 The

rhy thm parameters obta ined by a circadian cosinor a n a l y s i s

19 were

subjected to a subsequent circannual analysis giving possible evidence for seasonal rhythmicity. The results are shown in Tables 1-4.

Among the female patients only the g roup with benign breast diseases showed significant seasonal rhy thms of the circadian M E S O R (rhythm adjusted mean of all time points over the 24-hour cycle analyzed) and

T A B L E 1

Circannual cosinor analysis of the circadian MESOR of melatonin in patients with breast cancer and their controls

R h y t h m Ampl i t ude A c r o p h a s e detect . m e a n % of m e a n

H o r m o n e G r o u p N o . ( P < ) M E S O R ± S E M range M E S O R range

M e l a t o n i n A 28 0.005 0.146 + 0.029 0.082 56 2/1 [ p m o l / m l ] 0 .030-0 .134 1/XII-2/ I I

Β 23 N S (0.90) 0.078 + 0.030 0.009 12 1/XII A 13 0.01 0.142 + 0.031 0.107 75 1/XII

(19-41 0 .031-0 .183 2 / X I I - 2 / I I years)

A 15 N S (0.25) 0.148 + 0.053 0.069 47 i / i ( > 41 -72

i / i

years) A(Pre - 19 0.005 0.146 + 0.025 0.078 53 2/1

M P ) 0 .024-0.132 1/XII-2/ I I A 9 N S (0.50) 0.144 + 0.086 0.094 65 2/1

( P M P )

Abbrev ia t ions : A: con t ro l s , pa t i en t s wi th ben ign breas t disease (n = 28 , average age: 43 + 3 years) , i.e. fibrocystic m a s t o p a t h y (n = 14), fibroadenoma (n = 10) a n d o the r b reas t diseases (n = 4); B : pa t i en t s wi th p r i m a r y u n o p e r a t e d breas t cancer (n = 23, average age : 58 + 2 years) . P r e - M P : p r e - m e n o p a u s a l ; P M P : p o s t - m e n o p a u s a l . 1 = first, 2 = second half of a given m o n t h ; m o n t h s a re ind ica ted by R o m a n n u m b e r s .

Relationship between Pineal Gland and Cancer 339

T A B L E 2

Circannual cosinor analysis of the circadian amplitude of melatonin in patients with breast cancer and their controls

R h y t h m A m p l i t u d e A c r o p h a s e detect . m e a n % of m e a n

H o r m o n e G r o u p N o . (P<) M E S O R ± S E M range M E S O R range

M e l a t o n i n A 28 0.01 0.162 + 0.035 0.087 54 2/1 [ p m o l / m l ] 0 .022-0 .152 1/XII- l / I I I

Β 23 NS(0 .95) 0 . 0 8 1 + 0 . 0 3 5 0.009 11 2/II A 13 0.025 0 . 1 6 7 ± 0 . 0 3 8 0.119 l / I I

(19-41 0 .021-0 .217 71 2 / X I I - l / I I I years)

A 15 N S (0.25) 0.153 + 0.063 0.074 48 i / i ( > 4 1 - 7 2

i / i

years) A (Pre- 19 0.025 0.168 + 0.029 0.087 52 l / I I

M P ) 0 .039-0 .135 2 / X I I - l / I I I A 9 N S (0.50) 0.148 + 0.102 0.107 72 2/1

( P M P )

Abbrev ia t i ons : see T a b l e 1.

T A B L E 3

Circannual cosinor analysis of the circadian MESOR of hormones in patients with benign and malignant prostate tumors


H o r m o n e G r o u p N o . (P<) M E S O R + S E M range M E S O R range

M e l a t o n i n B P H 13 0.01 0.065 + 0.019 0.048 74 2/II [ p m o l / m l ] 0 .013-0 .083 1/I-l /IV

P C 9 NS(0.50) 0.033 + 0.023 0.015 46 1/XI P ro lac t in B P H 12 NS(0.25) 226 + 66 79 35 1/V

[μΐυ/ml] P C 9 NS(0.975) 229 + 74 12 5 2/IV T S H B P H 13 NS(0 .75) 1.14 + 0.65 0.40 35 2/III

[μΐυ/ml] P C 8 NS(0 .75) 0.33 + 0.40 0.29 88 l / I I h G H B P H 12 NS(0 .75) 2 . 7 7 + 1 . 0 0 0.62 22 1/IV [ n g / m l ] P C 9 NS(0 .50) 2 . 5 5 + 1 . 2 4 1.71 67 2/II L H B P H 13 NS(0 .50) 16.10 + 7.60 7.60 47 2/XI [ m I U / m l ] P C 9 NS(0.50) 13.90 + 9.30 10.00 72 2 /VII I F S H B P H 12 NS(0 .50) 7.59 + 4.02 3.09 40 2/XI [ m I U / m l ] P C 9 NS(0.50) 11.82 + 9.78 11.19 95 1/IX T h y r o x i n e B P H 13 NS(0.25) 6.97 + 0.91 1.21 17 1/X [>g /d l ] P C 9 NS(0.95) 7 . 8 9 + 1 . 5 1 0.46 6 2/VII Tes tos t e rone B P H 13 NS(0.50) 20.50 + 5.30 4.30 21 2/1 [ p m o l / m l ] P C 9 NS(0 .50) 18.70 + 6.70 9.90 53 1/VIII Cor t i so l B P H 12 NS(0 .50) 293 + 107 108 37 2/VI [ p m o l / m l ] P C 8 NS(0 .10) 263 + 53 118 45 2/II Se ro ton in B P H 13 NS(0.90) 5 0 + 1 7 8 16 1/VIII [ n g / m l ] P C 8 NS(0 .75) 63 + 36 22 35 2/IX

Abbrev ia t ions : B P H = pa t i en t s with benign p ros ta t i c hyperp las ia ; P C = pa t ien t s with p ro s t a t e c a r c i n o m a . 1 = first, 2 = second half of a given m o n t h ; m o n t h s a re indica ted by R o m a n n u m b e r s .


T A B L E 4

Circannual cosinor analysis of the circadian amplitude of hormones in patients with benign and malignant prostate tumors


H o r m o n e G r o u p N o . ( P < ) M E S O R ± S E M range M E S O R range

M e l a t o n i n B P H 13 0.05 0.068 + 0.024 0.047 69 l / I I [ p m o l / m l ] 0 .005-0.089 2 /XI-2 / IV

P C 9 N S (0.25) 0.025 + 0.020 0.018 72 1/XI P ro l ac t in B P H 12 N S (0.50) 72 + 30 29 40 1/V

[μΐυ/ml] P C 9 N S (0.75) 63 + 28 17 27 2 /VII Cor t i so l B P H 12 N S (0.25) 140 + 31 40 29 2/V [ p m o l / m l ] P C 8 N S (0.50) 1 3 1 + 6 2 43 33 2/IV

Abbrev ia t ions : see T a b l e 3.

ampli tude (half of the rhythmic variability per 24 hours) with acrophases (peak time of the cosine function used to approximate the rhy thm) in Janua ry while there was no indicat ion of seasonal rhythmicity in pat ients with mal ignant breast tumors . When the g roup of pat ients with benign breast diseases was sub-divided between individuals below and above 40 years of age or between pre- and pos tmenopausa l women seasonal rhythmicity was detectable only in the younger ( < 4 0 years) and the premenopausa l women. Therefore, the lack of a seasonal mela tonin rhy thm in cancer pat ients can be a t t r ibuted to age and not to the presence of a mal ignant as opposed to a benign t u m o r whereas the observed decrease in circannual M E S O R s and ampli tudes is specific to the cancer patients and is independent of age (see Tables 1 and 2). Interestingly, seasonality in the detection of breast cancer as repor ted by Cohen et al.

12

was most p ronounced in pat ients younger than 55 years.

Elderly men suffering from benign prostat ic hyperplasia show signifi-cant seasonal rhy thms of circadian melatonin M E S O R and ampl i tude with acrophases in Februa ry bu t age-matched pat ients with pros ta te carc inoma lack such rhythmicity. A number of other hormones were also analyzed in these patients bu t none of them showed any significant seasonal rhythmicity in bo th groups (see Tables 3 and 4).

These da ta indicate tha t in h u m a n s not only the acrophase undergoes a seasonal s h i f t

1 0'

14 but that also M E S O R and ampl i tude of the circadian

melatonin rhy thm show seasonal rhythmicity. In women, this rhy thm gets lost after menopause while it is mainta ined in elderly men free of mal ignant tumors .

Seasonal rhy thmic i ty of pineal me la ton in product ion in f e m a l e rats is d isturbed by mal ignant but not benign t u m o r s

Melatonin product ion by the pineal gland can be estimated by determina-tion of its metabolic end-product , 6-sulphatoxymelatonin (aMT6s), in


u r i n e .

11 The nocturnal urinary excretion (23.00-07.00 hours) of 6-

sulphatoxymelatonin was determined by rad io immunoassay

1 in 21 inbred

female Fischer F344 rats over one year. Four teen animals had received a single intragastral dose of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA, 10 mg/100 g body weight in 1 ml of peanut oil) on their 50th day of life to induce m a m m a r y tumors . Seven animals developed malignant m a m m a r y tumors (mostly adenocarcinomas) and seven animals showed benign m a m m a r y tumors (mostly fibroadenomas). In the control group which had received only vehicle nocturnal a M T 6 s showed clear changes over the year with higher values in summer than in winter despite constant environmental conditions (photoperiod, temperature , air humidity). The presence of seasonal rhythmicity was tested by the cosinor analysis and a highly significant rhythm (p<0.005) with an acrophase in mid-August was detected in controls (Table 5). Animals with benign tumors also showed a significant rhythm (p<0 .025) with an acrophase in mid-July. Animals with malignant tumors , however, had no significant seasonal rhythm (p<0 .25) , showing a less marked depression of a M T 6 s in winter as compared to the controls (see Figure 1). At this time, the tumor development began which

T A B L E 5

Results of the cosinor analysis of nocturnal 6-sulphatoxymelatonin excretion (ng/hour) in female Fischer rats

R h y t h m A m p l i t u d e A c r o p h a s e * detect . m e a n % of m e a n

G r o u p η ( P < ) M E S O R ± S E M range M E S O R range

C o n t r o l s 210 0.005 39.50 ± 1 . 5 2 3.95 10.0 15/VIII 1.70-6.20 7 /VII -17/ IX

Benign t u m o r s 221 0.025 41.99 + 1.65 3.33 7.9 15/VII 0 .87-5 .79 19/V-31/VIII

M a l i g n a n t 223 0.25 4 0 . 7 2 + 1 . 4 7 1.84 4.5 4 /VI I I t u m o r s

* D a y of m o n t h is given in Arab i c , m o n t h in R o m a n n u m b e r s .

may be the cause for the observed elevated melatonin product ion. Using another t umor model , i.e. spontaneous carcinoma of the endometr ium in female rats, a similar observation was made : a g roup of animals developing malignant tumors had no seasonal rhythm in its 6-sulphatoxymelatonin excretion while another g roup of rats which did not develop tumors due to hormonal t reatment showed a highly significant rhythm with a peak in M a y (Deerberg and Bartsch, unpublished observations).

Unident i f i ed pineal substances w i t h t u m o r - i n h i b i t i n g act iv i ty

It is k n o w n tha t the s t imulatory effect of pinealectomy on the development and growth of experimental m a m m a r y tumors induced by D M B A cannot


1 9 9 0 1 9 9 1 Week of t h e Y e a r 29 39 49 7 17 27 27 47

Benign Tumors

Summer Autumn * Î I

lnt«r 3prui( ! ï.

S u a" ~ \ i

50 120 190 260 330 400 Age [ d a y s ]

1 9 9 0 1 9 9 1 Week of t h e Y e ar 29 39 49 7 17 27 37 47

Ζ 50

Malignant Tumors

50 120 190 260 330 400 Age ( d a y s )

F I G . 1. N o c t u r n a l ( 2 3 . 0 0 - 0 7 . 0 0 h o u r s ) 6 - su lpha toxymela ton in excre t ion over a comple te year of hea l thy female ra t s (cont ro ls , t o p ) , seven ra t s deve lop ing ben ign m a m m a r y t u m o r s (centre) , a n d seven ra t s wi th m a l i g n a n t m a m m a r y t u m o r s ( b o t t o m ) . E a c h value represents the m o n t h l y average of 2 - 4 d e t e r m i n a t i o n s per

m o n t h . a M T 6 s = 6 - su lpha toxymela ton in .


be completely reversed by the applicat ion of m e l a t o n i n .

25 This observa-

tion leads to the assumpt ion that the pineal gland may produce other substances which can inhibit t u m o r growth . Evidence has existed for quite some time for the presence of cytostatic factors in pineal extracts free of me la ton in .

3 Such substances have been found in urine as wel l .

3

A n t i - t u m o r act iv i ty in human urine w a s de tec tab le only in s u m m e r

In an a t tempt to purify tumor-inhibit ing substance(s) with possible pineal origin from h u m a n urine a number of experiments were carried out using three different experimental tumor systems.

9 The aim was to find a suitable

tumor system for the detection of the ant i - tumor activity during the course of its purification. The experiments were carried out over 4 years. Strong tumor-inhibit ion as well as complete lack of effect was observed when rats and mice were treated with h u m a n urine administered in drinking water. Only after analyzing the da ta on a seasonal basis was a clear result obtained: the activity was present only in summer. The cosinor analysis revealed a significant rhythm (p< 0.005) with an acrophase in August (see Figure 2).

D J F M A M J J A S O N D

Month

F I G . 2. Seasona l r h y t h m of t umor - inh ib i t i ng activity in h u m a n ur ine as assessed in three t u m o r mode l s (open circles: a d e n o c a r c i n o m a of the b reas t in female H o l t z m a n ra t s ; filled circles: solid Ehr l ich t u m o r s in Swiss mice ; o p e n t r iangles :

fibrosarcoma ascites in Swiss mice) [ f rom 9 ] .

A n t i - t u m o r act iv i ty in t h e rat pineal g land peaks in summer

The tumor- inhibi t ing activity in individual rat pineal glands was analyzed over a complete year in two separate e x p e r i m e n t s .

6'

7'

9 The first one was


6 0

0 - I—ι—\—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—\-D J F M A M J J A S O N D

M o n t h

F I G . 3. Seasona l r h y t h m of a n t i - t u m o r activity in ra t p ineal g lands as de t e rmined in t w o different exper imen t s (1986/87: filled circles; 1989/90: o p e n circles). Ant i -t u m o r activity was defined as the rec iprocal I C 5 0- v a l u e , i.e. the c o n c e n t r a t i o n necessary to ob t a in 5 0 % inhib i t ion of cell g r o w t h as c o m p a r e d to u n t r e a t e d

con t ro l s , a n d expressed as (pineals /10 μΐ test v o l u m e )

-1 (from Ref. 9).

carried out in 1986/87 with 167 pineal glands from rats of bo th sexes, aged between 20 and 120 days, killed at different times of the day. E thano l extracts were prepared and applied to h u m a n erythroleukemia cells K562. Ant i - tumor activity was defined as the reciprocal I C 5 0- v a l u e (i.e. the concentra t ion necessary to obta in 5 0 % inhibit ion of cell g rowth as compared to untreated controls) and expressed as (pineals/10 μΐ test v o l u m e )

- 1. A significant seasonal rhy thm was observed with all animals

(p< 0.0001, acrophase in July, the absolute peak occurring in August , see Figure 3, filled circles) as well as with a sub-group consisting of only female rats , aged 100-120 days, killed at 9 a.m. (p < 0.001, acrophase in May) . The second experiment was carried out in 1989/90 with 52 270 day old male rats killed at 11 a.m. Again, a significant seasonal rhy thm was obta ined ( p < 0 . 0 0 1 , acrophase in July, absolute peak in August , see Figure 3, open circles). The decreased peak values and lower ampl i tude and M E S O R may be due to the advanced age of these animals as compared to the first experiment.

Conclusions

The data presented above suggest that pineal function in m a n as well as in laboratory animals shows seasonal rhythmicity. As far as h u m a n beings are concerned this observation is not surprising since it has been shown that al though the modern way of life screens them from their natural surround-ings to a great degree they have still mainta ined seasonality in m a n y body functions. However , l abora tory animals kept under cons tant environ-mental condit ions of light, t empera ture and air humidi ty t h roughou t


the year for m a n y generat ions are expected to be "s tandardized" also with respect to lack of seasonality. Nevertheless, there is an increasing number of repor ts abou t the main tenance of seasonal rhy thms even under cons tant environmenta l condi t ions . In this cont r ibut ion we will confine ourselves to those related to pineal activity: ra t pineal content of immunoreact ive a rg in ine -vaso toc in

22 and the related nonapept ides vasopressin and

o x y t o c i n

17 were found to have p ronounced peaks in August ; in an electron

microscopic s tudy over 2 years synaptic r ibbons and spherules were found to display distinct seasonal rhy thms in the rat pineal g l a n d ;

16 noc turna l

p lasma mela tonin in rats was approximate ly twice as high in July as compared to M a r c h ,

5 an observat ion which agrees with the elevated

6-sulphatoxymelatonin excretion in summer described above. Thus , it appears tha t pineal activity of l abora to ry animals is generally higher in summer than in winter, in its p roduc t ion of mela tonin , its content of nonapept ides , and in the presence of yet unidentified tumor- inhibi t ing substance(s). In h u m a n s , the existence of an t i - tumor activity in urine shows its peak also in summer a l though it still has to be verified whether this substance really is of pineal origin. H u m a n mela tonin , however, showed its m a x i m u m in winter—as expected due to its dependence on the length of the pho toper iod .

The quest ion however arises, what may be the cause of the observed seasonal rhythmici ty in l abora tory animals? It is of course possible and likely tha t the rhy thm is of an endogenous na ture . In this case, however, it would be expected to free-run if there is no external zeitgeber as observed with circadian rhy thms . The described observat ions suggest a ra ther stable acrophase over various years, part icularly in the case of the nonapept ides and the an t i - tumor activity in the rat pineal gland but the number of experiments may not be enough to actually prove it. If the peaks are really as stable as they appear to be then an u n k n o w n exogenous zeitgeber must exist—even under so-called s tandardized l abora to ry condi t ions . It is easily conceivable tha t olfactory stimuli reaching the animals via the air-condi t ioning m a y be such factors. Another possibility is an influence of the geomagnet ic field on the activity of the pineal gland. It is becoming more and more evident that the pineal is par t of a neural system in the brain that perceives and responds to changes in the hor izonta l componen t of the geomagnet ic field,

20'

24'

26 which shows distinct seasonal r h y t h m s .

18 T o

prove the involvement of olfactory or geomagnet ic factors as zeitgebers for seasonal rhy thms experiments will have to be performed under complete shielding of such influences which require highly sophist icated experi-menta l setups.

Irrespective of the factors acting as zeitgebers for the synchronizat ion of endogenous rhy thms under control led l abora to ry condi t ions the fact remains that such rhy thms canno t be easily extinguished, neither in h u m a n s nor in experimental animals . Thus the quest ion arises: what may


be the physiological significance for these highly stable rhythms? It has to be assumed that the organism requires regular seasonal "ups and downs" in diverse body functions in order to mainta in health. This should be considered if we try to eliminate the na tura l "downs" for the sake of higher professional performance since they may have considerable impor tance as a reminescent of h ibernat ion or as a resting period serving regenerat ion of the organism which may be as indispensible as is our daily sleep.

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25

Light Treatment of Depressive States: Swedish Experiences at Latitude 59° North B E N G T F. K J E L L M A N , B J Ô R N - E R I K T H A L È N a n d L E N N A R T W E T T E R B E R G

Karolinska Institute, Department of Psychiatry, St. Goran's Hospital, Stockholm, Sweden

Abst rac t

T o explore the poss ible m e c h a n i s m s med ia t i ng the clinical effect of light t r e a t m e n t , the covar i a t ion be tween b iochemica l r h y t h m m a r k e r s a n d the clinical o u t c o m e of light t r e a t m e n t was eva lua ted . A n o t h e r a im of the s tudy was to test for a b iochemica l p red ic to r of the clinical o u t c o m e of l ight t r e a t m e n t . Resul ts from 109 pa t i en t s wi th ma jo r depress ion s tudied a u t u m n 1988 to spr ing 1991 were inc luded . O n D a y 1 b lood s ampl ing was d o n e 20 .00-08 .00 hours m e a s u r i n g se rum m e l a t o n i n , Cortisol, prolactin a n d T S H . O n D a y 3 clinical r a t ing wi th the use of the C o m p r e h e n s i v e P s y c h o p a t h o l o g i c a l Ra t i ng Scale ( C P R S ) a n d the H a m i l t o n Depres s ion R a t i n g Scale ( H A M - D ) was per formed . T h e pa t i en t s were exposed to b r igh t l ight ( luminance : 350 c a n d e l a / m

2) for 1 h o u r (22.00-23.00 h o u r s ) a n d

b l o o d sampl ing was d o n e . T h e light t r e a t m e n t was given for 10 consecut ive days in a r o o m with indirect l ight of 350 c a n d e l a / m

2 l uminance . T h e pa t i en t s were t r ea ted wi th light

06 .00-08 .00 h o u r s (morn ing l ight) o r 18.00-20.00 h o u r s (evening l ight) . Of the 109 depress ive pa t i en t s 94 comple t ed the t r e a t m e n t p r o c e d u r e s a n d of the pa t i en t s wi th win ter depress ion t r ea ted wi th m o r n i n g light 7 5 % were i m p r o v e d . W h e n the s e rum m e l a t o n i n level du r ing the l ight test was lower at 23.00 h o u r s t h a n at 22.00 h o u r s , clinical i m p r o v e m e n t in the pa t i en t s wi th win ter depress ion t rea ted wi th m o r n i n g light was 8 8 % . T h e a rea u n d e r the profile t ime curve ( A U C ) of m e l a t o n i n decreased significantly m o r e after l ight t r e a t m e n t in the i m p r o v e d . T h e t ime center of gravi ty (TC) of m e l a t o n i n was phase a d v a n c e d by light t r e a t m e n t in the who le g r o u p of depress ive pa t i en t s ; 06 .00-08 .00 h o u r s light i nduced a p h a s e a d v a n c e a n d 18.00-20.00 h o u r s l ight a p h a s e delay of T C . T h e A U C of Cortisol was increased by m o r n i n g light a n d decreased by evening light in the whole g r o u p of depress ive pa t i en t s . In the i m p r o v e d pa t i en t s a p h a s e a d v a n c e of Cortisol T C was seen in o p p o s i t i o n to a p h a s e delay in the n o n i m p r o v e d pa t i en t s . In the pa t i en t s wi th win ter depress ion a p h a s e a d v a n c e of T C was also seen in i m p r o v e d pa t i en t s . M o r n i n g a n d evening light h a d differential effects o n the A U C a n d T C of T S H in the whole g r o u p of depress ive pa t i en t s wi th a decrease of A U C a n d a phase a d v a n c e of T C wi th m o r n i n g light. T h e clinical effects of light t h e r a p y seem to be re la ted to a l t e ra t ion of biological r h y t h m m a r k e r s .

S U N L I G H T IS of great impor tance for living beings. O n e major effect is the entraining and synchronizing effect on circadian rhy thms . Thus the nonvisual effects of light entering the eye influence a rhy thm generat ing

351


system in which the suprachiast ic nucleii (the circadian clock mechanism) and the pineal gland with its main h o r m o n e melatonin form the major par ts .

The first controlled study of light t rea tment in depression was performed by Kr ipke and coworkers in San Diego, USA treat ing male patients with nonseasonal depression with bright white light or red (placebo) l ight .

8 Lewy and coworkers in 1982 reported the successful

t rea tment with bright light in a pat ient with seasonal dep re s s ion .

11 Winter

depression occurs dur ing the winter with seasonal pa t te rn according to Diagnost ic and Statistical M a n u a l of Menta l Disorders (DSM-I I I -R) .

1

Some of the pat ients with winter depression report atypical symptoms as increased appeti te , weight gain and hypersomnia . Impa i rment of m o o d , cognitive function, and fatiguability are other main f ea tu res .

15 Light

t rea tment has been used dur ing the last decade mainly in patients with Seasonal Affective Disorder (SAD) of the winter type (winter dep re s s ion ) .

19

Light t rea tment of depressive states hypothetically works th rough effects on the rhy thm generat ing system. Lewy and coworkers had proposed that an advancement of the evening rise of mela tonin dur ing dim light indicates a phase advancement of the circadian r h y t h m .

1 2'

13 They

have published da t a relating this phase advancement to the clinical effect of light t rea tment in the m o r n i n g .

12 Rosenthal and coworkers suppor t

other hypotheses claiming the rhy thm changing effects of light to be of less impor tance and emphasize the p robable effect of light t rea tment on the function of t ransmit ter substances in the b r a i n .

7'

1 5'

22

This presentat ion will comprise results of light t rea tment from a 3 year period from a u t u m n 1988 to the spring 1991. T o explore the response to light t rea tment in different diagnostic categories we included pat ients meeting the D S M - I I I - R criteria of major depressive episode with either seasonal or nonseasonal pa t terns .

T o explore the possible mechanisms mediat ing the clinical effects of light t rea tment we tested the hypothesis that there were biological rhy thm marker changes covarying with the clinical ou tcome of light t rea tment . Another aim was to evaluate if there were biochemical markers that could help to predict the therapeut ic ou tcome of light t rea tment .

Mater ia ls and methods

Altogether 109 patients with a major depressive episode were included in the study, 83 were female. The mean age was 47 years with a range of 18-74 years. The patients were referred by physicians or contacted us directly. Pat ients with nonseasonal depression and pat ients with seasonal pa t te rn according to the D S M - I I I - R were included. The latter were of three different types: spring, fall, and winter depression. The definition of the

Light Treatment, Swedish Experiences 353

T A B L E 1

DSM-III-R criteria for major affective disorder according to seasonal pattern at latitude 59°N

Seasona l p a t t e r n T i m e of onse t D u r a t i o n

Spr ing After 1st F e b r u a r y before 1st J u n e

Longe r t h a n 2 weeks

A u t u m n After 1st Augus t before 1st N o v e m b e r

Longe r t h a n 2 weeks Shor t e r t h a n 3 m o n t h s

Win te r After 1st Sep tember before 1st F e b r u a r y

L o n g e r t h a n 3 m o n t h s

N o n s e a s o n a l N o n s e a s o n a l Longe r t h a n 2 weeks

four categories is shown in Table 1. O n lat i tude 59°N fall and spring depressions are recognized types of seasonal affective disorders .

The number of pat ients in each diagnostic g roup is shown in Table 2. The majority of the pat ients were of the winter depression type. The patients with spring and fall depression who had documented episodes in bo th seasons were diagnosed according to the season when they were treated.

T A B L E 2

Number of patients in different diagnostic groups (total n = 109)*

Seasona l type of depress ion F e m a l e M a l e T o t a l

Win te r 47 17 64 Spr ing 14 2 16 Fal l 5 3 8 N o n s e a s o n a l 17 4 21

T o t a l 83 26 109

* 15 of t h e m did no t comple t e light t r e a t m e n t .

Light treatment and light test

The light t rea tment at St. Goran ' s Hospi ta l was applied in a light t rea tment r o o m with white colored ceiling, walls, a n d floor. The reflection of 24 fluorescent tubes in the ceiling gave indirect light with a luminance of 350 c a n d e l a / m

2 (1,500 lux, 0.8 m above the floor). The pat ients were also

clad in white clothes to minimize light absorpt ion . The light source was a full spectrum light with two different light

temperatures , 4,000 Kelvin (n = 29) and 6,500 Kelvin (n = 65). The time of light t rea tment was either 06.00-08.00 hours (morning light) or 18.00-20.00 hours (evening light) for 10 consecutive days . The decision of the time of light t rea tment was not r a n d o m , bu t to some degree influenced


by the clinical symptoms, predominant ly the sleep-wake rhy thm. Late sleepers were treated with morn ing light and pat ients with early morn ing awakening with evening light.

The light test was performed between 22.00 hours and 23.00 hours using the light tubes of 6,500 Kelvin with a luminance of 350 c a n d e l a / m

2 as the

light source. The light test was performed in the light r o o m and for the pat ients t reated with 4,000 Kelvin in another r o o m.

Design

The design of the s tudy is shown in Table 3. O n D a y 1 a clinical interview rat ing according to D S M - I I I - R took place and the pat ients also performed a self-rating using the Beck's Depression Inventory ( B D I ) .

4

T A B L E 3

Design (overview) of the investigation schedule

D a y 1 D a y 3 D a y 15 D a y 17

Clinical B D I C P R S B D I C P R S ra t ings H A M - D H A M - D Biochemical 20 .00-08 .00 20 .00-08 .00 20 .00-08 .00 20 .00-08 .00 r h y t h m h o u r s h o u r s h o u r s h o u r s m a r k e r s B l Light Test 1 B2 Light Test 2

M T M T M T M T C O R T C O R T C O R T C O R T

T S H T S H T S H T S H P R L P R L P R L P R L

D a y 4 - 1 4 Light t r e a t m e n t 06 .00-08 .00 h o u r s (M)

or 18.00-20.00 h o u r s (E) for 10 days

F o r further in fo rmat ion see text .

Dur ing the night at 20.00, 22.00,22.30, 23.00, 24 .00,02.00,04.00,06.00 and 08.00 hours b lood samples were d rawn from an indwelling catheter measur ing mela tonin , Cortisol, prolact in, and T S H (Basal 1 (Bl)) . The pat ients slept in darkness between 23.00 and 06.00 hours . T w o days later clinical rat ings were made by two doctors independently, using the Comprehensive Psychopathological Rat ing Scale (CPRS) items measur-ing depression and the first 18 items of the Hami l ton Depression Rat ing Scale ( H A M - D ) .

3'

5 In the night the b lood samples were taken at the same

time points as on D a y 1, bu t the pat ient underwent the light test between 22.00 and 23.00 hours . After 10 days of light t rea tment the same protocol was applied as before t rea tment . These results are named Basal 2 (B2) and Test 2, respectively.

Light Treatment Swedish Experiences 355

Laboratory measurements

The serum samples of mela tonin were analyzed by the me thod of Wet terberg and c o w o r k e r s

23 (1978). The lower limit of detection was 0.01

nmol/1, intra-assay coefficient of var iat ion (CV) 7 .4% and the inter-assay CV for samples above 0.015 nmol/1 was 4 . 8 % . The serum samples for Cortisol and prolact in were analyzed by commercial RIA kits (Fa rmos Diagnost ica , Tu rku , F in land) . In t ra- and inter-assay CV were for Cortisol 2 . 9 % and 5 .8%, and for prolact in 2 . 9 % and 5 . 1 % , respectively. T S H was analyzed with two commercial kits (Spectra T S H I R M A , F a r m o s , Tu rku , F in land and from 910301 Birilux h T S H , Behringewerke, M a r b u r g , Germany) . Sensitivity was 0.05 mU/1 and 0.02 mU/1 , respectively and inter-assay CV in the normal range (0.2—4.0 mU/1) was 5 % and 3 % , respectively.

Stat is t ica l methods

In this design different measures before t rea tment were compared to measures after t rea tment to evaluate the t rea tment ou tcome. Several measures describing the status of the pat ients and of their ho rmona l levels were defined. The nightly h o r m o n e results before and after t rea tment are called B l and B2, respectively, and the nightly h o r m o n e curves where the light test are included at 22.00-23.00 hours before and after t rea tment are called Test 1 and Test 2, respectively.

A set of repeated measurement of serum h o r m o n e levels dur ing one night from 8.00 p .m. to 8.00 a.m. is called the h o r m o n e profile. Single lacking da t a points in the profiles were subst i tuted by linearly interpolated points (or extrapola ted points at the ends).

Pat ients with two adjacent lacking da t a points were excluded from the mater ial . F o r each h o r m o n e and each individual the area under the profile t ime curve (AUC) was calculated by means of the t rapezoidal rule. Since the time points are not equidistant , A U C is a better est imate of the average h o r m o n e level than the ar i thmetic mean value.

As an estimate of the "mean appearance t ime" the first-order time momen t , T C = "t ime center of gravity", of the profile t ime curve was calculated, also using an t rapezoidal rule (linear interpolat ion between time points) .

A negative difference (TC B 2 - T C B l ) is defined as a phase advance and a positive difference as a phase delay. When the differences between B l and B2 in A U C and T C were normal ly distr ibuted, a two sided ί-test was used to test the difference between B l and B2. When the difference was non-normal ly distr ibuted a logari thmic t ransformat ion was performed to obta in a no rma l dis t r ibut ion or the nonparamet r i c Wilcoxon signed rank test was used. A two-factor A N O V A was used to examine the significance


of the clinical response (nonresponse vs. response) and light t iming condi t ion (morning vs. evening). P-values < 0 . 0 5 were considered statistically significant.

Results

Some of the clinical features of the 64 pat ients with winter depression are shown in Table 4. The majority of the pat ients had been on sick-leave and h a d been treated for depressive states. Ca rbohydra te craving was repor ted by 6 0 % but only 3 0 % had increased appeti te . Fat iguabil i ty was c o m m o n (88%), bu t only 3 3 % were hypersomnic and as much as 5 3 % were hyposomnic .

T A B L E 4

Patients with winter depression (n = 64, F =47, M =17): selected clinical history, symptoms, and diagnosis

Percen t Clinical history:

Earl ier hosp i ta l i za t ion (depress ion) 36 Sick leave for depress ion 75 C o n s u l t e d phys ic ian for depress ion 89 Ear l ier t r e a t m e n t for depress ion 83 D r u g t r e a t m e n t for depress ion 79 P s y c h o t h e r a p y 38

Reported symptoms: Increased appe t i t e 30 Decreased appe t i t e 48 C a r b o h y d r a t e c rav ing 60 Increased b o d y weight 38 Decreased b o d y weight 31 Fa t iguab i l i ty 88 H y p e r s o m n i a 33 H y p o s o m n i a 53

Diagnosis: U n i p o l a r 73 Bipo la r I 5 Bipola r II 22

The therapeut ic ou tcome in the different diagnostic groups is shown in Table 5. Fifteen pat ients of the initial 109 did no t complete the research protocol most ly due to technical problems. A pat ient was considered as improved (responder) , if a 5 0 % or more reduct ion of depression scores in H A M - D and /o r C P R S rat ings was found after 10 days of light t rea tment .

Of the pat ients with winter depression 37 (63%) improved and 22 (27%) were non improved . In the pat ients with nonseasonal depression 4 (21%) improved and 15 (79%) were nonimproved .

In the total g roup of pat ients and in the pat ients with winter depression


T A B L E 5

Outcome of 10 days of light treatment in the morning 06.00-08.00 hours (M) or in the evening 18.00-20.00 hours (E) in 94 patients

Diagnos i s F e m a l e M a l e T o t a l D iagnos i s I N I I N I I N l

Win te r M 21 7 9 3 30 ( 7 5 % ) 10 ( 2 5 % ) Win te r Ε 6 8 1 3 7 ( 3 9 % ) 11 ( 6 1 % )

Spr ing 4-Fal l M 2 2 0 3 2 ( 2 9 % ) 5 ( 7 1 % ) Spr ing 4-Fal l Ε 3 6 1 0 4 ( 4 0 % ) 6 ( 6 0 % )

N o n s e a s o n a l M 1 6 1 1 2 ( 2 2 % ) 7 ( 7 8 % ) N o n s e a s o n a l Ε 1 7 1 1 2 ( 2 0 % ) 8 ( 7 8 % )

T o t a l M 24 15 10 7 34 ( 6 1 % ) 22 ( 3 9 % ) T o t a l Ε 10 21 3 4 13 ( 3 4 % ) 25 ( 6 6 % )

I m p r o v e d (I) = a t least 5 0 % reduc t ion of ra t ings in H A M - D a n d / o r C P R S ( responders ) .

N o n i m p r o v e d (NI ) = o the rs ( non re sponde r s ) . In the pa t i en t s wi th winter depress ion , a n d in the to ta l g r o u p , a stat ist ical ly significant

difference (2p = 0.02) be tween m o r n i n g a n d evening light was found (Fischer exact test) .

the morn ing light t rea tment was significantly bet ter than light t rea tment in the evening ( p < 0 . 0 5 ) . N o statistically difference in improvement rate was found between the two light sources (4,000 Kelvin vs. 6,500 Kelvin).

In a g roup of 33 pat ients with winter depression t reated with morn ing light the results of the light test are given in Table 6. (Patients with zero levels of mela tonin at 22.00, 22.30 and 23.00 hours at B l were excluded in this calculation.)

T A B L E 6

Light test as a predictor of treatment outcome in 33 patients with winter depression treated with light 06.00-08.00 hours

M e l a t o n i n ( M T ) changes I N I

M T levels h igher 23.00 h o u r s 3 ( 3 7 % ) 5 ( 6 3 % )

t h a n 22.00 h o u r s M T levels lower 23.00 h o u r s 22 ( 8 8 % ) 3 ( 1 2 % )

t h a n 22.00 h o u r s o r u n c h a n g e d

(2p = 0.0099) (Fischer exact tes t) . Of the seven pa t i en t s wi th zero m e l a t o n i n levels 22 .00-23 .00

h o u r s , five (71 % ) i m p r o v e d a n d t w o were r a t ed as n o n i m p r o v e d .

O u t of 8 pat ients with higher serum mela tonin levels at 23.00 hours than 22.00 hours , 3 (37%) improved and 5 (63%) did not improve . O u t of 25 pat ients with lower or the same levels of mela tonin at 23.00 hours compared to 22.00 hours , 22 (88%) were improved and 3 (12%) were non improved . The difference was statistically significant ( p < 0 . 0 5 ) . Thus , when the mela tonin level after the light test was lower or the same at 23.00 hours than at 22.00 hours , the chance of improvement with morn ing light in winter depression was increased from 3 7 % to 8 8 % .


Biological r h y t h m markers

Melatonin

The serum melatonin levels before (Bl ) and after (B2) 10 days of t rea tment with morn ing light and evening light in the whole g roup of depressive pat ients are shown in Figures 1A and I B . Figures 2A and 2B show B l and B2 of the improved and non improved pat ients . There was a significant decrease of A U C between B l and B2 in the whole g roup of depressive patients (Table 7).

Morning light (N=51)

α ο

1 ο s α

Diff. B2-B1

Time (h)

F I G . 1. M e a n se rum m e l a t o n i n levels (nmol/1) of B l a n d B2 a n d the difference (B2-B1) (mean ± S E M ) in all depress ive pa t ien t s t rea ted wi th m o r n i n g light (1A)

a n d evening light ( IB) .

The difference between B2 and B l of A U C was significantly smaller in the non improved g roup compared to the improved g roup . N o significant difference was found in the A N O V A between morn ing and evening light t rea tment (Table 8). The T C for the whole pat ient g roup was significantly earlier for B2 than for B l (Table 7).

There was a significant difference in the A N O V A between the difference (B2-B1 ) in T C between morn ing light t rea tment ( — 0.99 ± 0.14 hours) and evening light t rea tment (0.405 + 0.14 hours) , but not between the improved and the non improved pat ients (Table 8).

The serum melatonin results for B l and B2 in the patients with winter depression treated with morn ing light are shown in Figure 3. The mean ± S E M for log A U C of B l was 0.30 ± 0.04 (log h χ nmol/1) and for B2


0.30 ρ

0.25 -

0.20 -

0.15 -

0.10 -

0.05 V*

b Responders (N«41)

α -005 -I -o.io -* -0.15

L

0

Diff. B2-B1 ^

Time (h)

Non-re s ponder s (N=42)

0.15 μ

0.10

0.05 04 06 Β 2 ^

-0.05 -0.10 -0.15

Diff. B2-B1 Time (h)

F I G . 2. M e a n se rum me la ton in levels (nmol/1) of B l a n d B2 a n d the difference (B2-B1 ) (mean ± S E M ) in all depress ive pa t i en t s , i m p r o v e d pa t i en t s ( responders )

(2A) a n d n o n i m p r o v e d (non re sponde r s ) (2B).

0.24 + 0.04 (log h χ nmol/1), a statistically significant difference (t = —2.88, 2p = 0.006).

The mean + S E M of the T C was 2.73 ± 0 . 1 5 hours before light t rea tment (Bl) and 1.74 + 0.14 hours after (B2) ( i = —5.88, 2p 0.0001). In the A N O V A no significant differences due to sex or improvement and non improvement were found in A U C and T C .

Cortisol

The serum Cortisol levels of B l and B2 in all depressive pat ients t reated with morn ing and evening light are shown in Figures 4A and 4B, and the improved and non improved pat ients in Figures 5A and 5B.

In the A N O V A the difference of A U C increased with morn ing light (275 + 103) and decreased with evening light t rea tment ( — 555 + 112) (Table 8). The difference was significant. N o significant difference in A U C was found between improved and non improved pat ients in the A N O V A , and no significant difference in A U C was found in B l and B2 in the whole g roup (Table 7).

Table 7 also shows that there was no statistically significant change of the T C for the whole g roup between B l and B2. In the A N O V A a significant difference was found between the improved and the non-

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T A B L E 8

The statistically significant différencies of melatonin (MT), Cortisol (CORT), prolactin (PRL), and TSH between nonresponders and

responders and between morning and evening light in the ANOVA

M T Diff. C O R T Diff. T S H Diff.

A U C : (h χ nmol/1) (h χ nmol/1) (h χ mU/1) M o r n i n g 275 + 103 - 3 . 8 9 + 1.86 Even ing - 5 5 5 + 1 1 2 + 0 . 1 2 + 1 . 0 6 Even ing

ρ < 0.0001 ρ = 0.0360 N o n r e s p o n d e r s - 0 . 0 8 5 + 0.108 Responde r s - 0 . 3 7 4 + 0.098

ρ = 0.04

T C : M o r n i n g - 0 . 9 9 + 0.141 - 0 . 2 4 5 + 0.045 Even ing 0.405 + 0.144 0.082 + 0.054

ρ < 0.0001 ρ < 0.0001 N o n r e s p o n d e r s 0 . 3 2 3 + 0 . 1 1 4 Responde r s - 0 . 2 2 5 + 0.117

p = 0.0024

T C is s ta ted as h u n d r e d t h of a n h o u r ins tead of minu t e s .

Winter dcpr., morning light (N=36)

F I G . 3. M e a n m e l a t o n i n s e rum levels (nmol/1) of B l a n d B2 a n d the difference (B2-B1) (mean + S E M ) in the win ter depress ive pa t i en t s t r ea ted with m o r n i n g

l ight.

improved pat ients in T C (improved —0.225 + 0.12 hours , non improved 0.323 + 0.11 hours) (Table 8).

T h e B l a n d B2 levels of s e r u m Cor t i so l in t h e p a t i e n t s w i t h w i n t e r

depression treated with morn ing light are shown in Figure 6. The mean + S E M of log A U C was 3.40 + 0.02 (log h χ nmol/1) for B l

and 3.44 + 0.02 (log h χ nmol/1) for B2. The increase of log A U C was statistically significant (i = 2.42, 2/7 = 0.02). The mean + S E M of the T C was for B l 3.62 + 0.10 hours and for B2 3.56 + 0.09 hours . The difference was not statistically significant (i = 0 .31, 2p = 0.76).

In the A N O V A no significant influence due to sex or improvement and non improvement was found on A U C . A significant effect of improvement



F I G . 4. M e a n serum Cortisol levels (nmol/1) of B l a n d B2 a n d the difference (B2-B1 ) (mean + S E M ) in all depressive patients t r ea ted with m o r n i n g light (4A)

and evening light (4B)

600

500

400

300

200

100

0

-100

-200

Β Non-responders (Ν=42)^

B l .

02

Time (h)

F I G . 5. M e a n serum Cortisol levels (nmol/1) of B l a n d B2 and the difference (B2-B1) (mean + S E M ) in all depressive patients, improved (responders) (5A)

and nonimproved (nonresponders) (5B).


Winter dcpr., morning light (N»36) BL

F I G . 6. M e a n serum Cortisol levels (nmol/1) of B l a n d B2 a n d the difference (B2-B1) ( m e a n ± S E M ) in the winter depressive patients treated wi th morning

light.

was found for T C (2p = 0.05) with a phase advance of T C in the improved g roup but no t in the non improved g roup .

Prolactin

Figures 7A and 7B show the serum prolact in levels of B l and B2 in all depressive pat ients t reated with morn ing or evening light. Figures 8A and 8B show B l and B2 in the same g roup divided in improved and non improved pat ients . Table 7 shows a significantly larger A U C for B2 than for Β1 . There were no significant differences in the A N O V A (Table 8). Table 7 shows tha t the T C in the whole g roup for B l was 2.69 ± 0 . 0 6 hours and for B2 2.59 ± 0.05 hours {t = 1.74,2p = 0.09). N o significant differences were found in the A N O V A (Table 7).

The prolact in levels of winter depressive pat ients t reated with morn ing light are shown in Figure 9. The mean + S E M for log A U C was for B l 1.94 + 0.03 ( log/zx/zg/ l ) and for B2 1.96 + 0.04 ( l o g / i x ^ g / l ) (i = 0.6, 2p = 0.55). The mean + S E M for T C was for B l 2.77 + 0.09 hours and for B2 2.53 + 0.09 hours . The difference was statistically significant (t= - 2 . 7 8 , 2p = 0.0084). In the A N O V A n o significant influences of sex and improvement or non improvement could be found.

TSH

The nightly serum T S H levels of the whole g roup of depressive pat ients divided into those t reated with morn ing , and respectively evening light are shown in Figures 10A and 10B, while the T S H levels of the improved and nonimproved pat ients are displayed in Figures 11A and 1 IB . In the whole g roup there was no significant change of A U C between B l and B2, while the T C was significantly phase advanced (Table 7).

Evening and morn ing light t rea tment had differential effects bo th on mean T S H , A U C , and T C in the whole g roup of depressive pat ients with a



Time (h)

F I G . 7. M e a n se rum pro lac t in levels ( / ig / l )o fBl a n d B 2 a n d the difference (B2-B1) ( m e a n ± S E M ) in all depressive pa t i en t s t rea ted wi th m o r n i n g light (7A) a n d

evening light (7B).

decrease of A U C and a phase advance of T C with morn ing light and the opposi te with evening light t rea tment .

The Bl and B2 levels of T S H in the pat ients with winter depression treated with morn ing light are displayed in Figure 12. There was no significant difference in A U C between B2 and Bl. But T C in B2 was significantly earlier t han Bl (Bl 1.94 + 0.06, B2 1.74 + 0.06, t = —2.71, 2p = 0.0102). N o significant effects of clinical ou tcome and sex were found.

Discussion

A compar ison with other materials of pat ients with winter depression sampled in a similar manne r as ours in Washing ton , U S , Basel, Switzerland, and L o n d o n , U K shows that hypersomnia was less c o m m o n in our sample (33%) than in the o ther three (78-97%) . Increased appeti te was most c o m m o n in the U S and the U K samples ( 6 0 - 7 4 % ) .

1 5'

2 1'

25 The

percentage of the Swiss sample (45%) was intermediate and our sample had the lowest frequency (30%) . The difference in frequency of carbo-hydra te craving was less p ronounced . In our sample 6 0 % compared to 7 7 - 8 2 % in the other three. The lower percentage of atypical symptoms in our sample is in accordance with the findings of S a k a m o t o and coworkers in J apan reported in pat ients , like ours , not recruited via the m e d i a .

17


Time (h)

F I G . 8. M e a n s e r u m pro lac t in levels (μ%/\) of B l a n d B2 a n d the di f ference (B2-B1) (mean + S E M ) in all depress ive pa t i en t s , i m p r o v e d ( responders ) (8A)

a n d n o n i m p r o v e d (non re sponde r s ) (8B).

α

Cl,

Time (h)

F I G . 9. M e a n se rum pro lac t in levels ^ g / l ) of Β1 a n d B2 a n d the difference (B2-B1 ) (mean + S E M ) in the win ter depressive pa t i en t s t r ea ted with m o r n i n g l ight.

These differences show that the symptom picture could vary in different par ts of the world possibly due to lat i tude, genetic and cultural factors, and contradicts the creat ion of an in ternat ional diagnose system based on symptom profiles.

The diagnosis and symptomalogy of winter depression are discussed in Chapte r 30 of this volume by J o h a n Beck-Friis.

The therapeut ic ou tcome in winter depression is in agreement with the findings of other groups . The differential effect of morn ing and evening


ΧΛ

H

Β

A 3.0 r~ 2, 2, 1.5 Κ-Ι.0 0.5

0 -0.5 -0.1 -1 .5

Evening light (N«=51) B l '

B2 • o •

22 24 02 04 06 08

Diff. B2-B1 Time (h)

Β 3.0 2-5 h 2.0 1.5 1.0 0.5 h

0 -0.5 -1.0 -1.5

Evening light (N=32)

B l

Diff. B2-B1 06

f : : : : : : A ^ : ^ : : ^ : : : " j r > « . . . . A . ^

Time (h)

F I G . 10. M e a n se rum T S H levels (mU/1) of B l a n d B2 a n d the difference (B2-B1) ( m e a n ± S E M ) in all depress ive pa t i en t s t rea ted with m o r n i n g light (10A) a n d

evening light (10B).

X 00 Η

3.0 2.5 2.0 1.5 1.0 0.5

0 -0.5 -1.0 -1.5

3.0 2.5 2.0 1.5 1.0 0.5

0

-0.5 -1.0 -1.5

Responders (N=41)

— Bl

B2

22 24 02 04 06 08

Diff. B2-B1

Time (h)

Β Non-responders (Ν=42) _ Β1 Λ

ϊ J Ό • - °

Β2

22 24 02 04 06 08

I I I • • • • Δ < Λ · . . .

~Α.///...£..:τ Diff. Β2-Β1

Time (h)

F I G . 11. M e a n se rum T S H levels (mU/1) of B l a n d B2 a n d the difference (B2-B1) ( m e a n ± S E M ) in all depressive pa t i en t s improved ( responders ) (11 A ) a n d

n o n i m p r o v e d (nonresponde r s ) (1 IB) .


Winter depr., morning light (N=36)

F I G . 12. M e a n se rum T S H levels (mU/1) of B l a n d B2 a n d the difference (B2-B1) ( m e a n ± S E M ) in the win ter depressive pa t i en t s t rea ted wi th m o r n i n g l ight .

light in our sample of pat ients with winter depression is in agreement with the results of the me ta analysis performed by T e r m a n and c o w o r k e r s

19

(1989). They repor ted on 322 pat ients from 14 research centers and found that the remission rates were 6 2 % under morn ing light exposure compared to 2 8 % under evening light. The difference was statistically significant in the pat ients with low severity on the baseline H A M - D ratings.

Mos t of these studies used a crossover design. Later addi t ional suppor t to the hypothesis tha t morn ing light is more effective than evening light has been published by Avery and coworkers and Sack and c o w o r k e r s .

2'

16 In a

recent s tudy Wirtz-Justice and coworkers found no statistical difference between morn ing (06.00-08.00 hours ) and evening light (21.30-23.00 h o u r s ) .

26

The differential clinical effect of morn ing and evening light in winter depression and between winter and nonseasonal depression suppor t s the hypothesis tha t light t rea tment has more than a placebo effect.

The clinical effects on nonseasonal pat ients were small, bu t in accordance with the results of Kr ipke and c o w o r k e r s .

9'

10

Like in our earlier report the light test also in these larger samples had a predictive v a l u e .

20 The chances of improvement were reduced by 5 8 %

when the melatonin level was higher at 23.00 hours than 22.00 hours after the light test. Thus the non improved pat ients seem to be less sensitive to light induced suppression of mela tonin levels at 22.00-23.00 hours .

In the whole g roup of depressive pat ients light t rea tment caused a lowered secretion of mela tonin measured as A U C more in the improved patients than in the non improved pat ients . Evening light t rea tment caused a phase delay of the T C of mela tonin while morn ing light t rea tment led to a phase advance of the T C . There was no significant difference in the phase advancing effect between the improved and the non improved pat ients .

M o r n i n g light t rea tment caused an increased A U C of Cortisol and evening light a decreased A U C in the whole g roup of depressive pat ients .


In the improved pat ients the T C was phase advanced and in the nonimproved it was phase delayed.

In the pat ients with winter depression treated with morn ing light a significant decrease of A U C of melatonin and a significant phase advancement of the T C was seen, while no significant differences due to therapeut ic ou tcome could be recorded. Regarding Cortisol a significant increase of A U C was seen and the T C was significantly more phase advanced in the improved than in the nonimproved g roup .

Regarding prolact in an overall increase of A U C was seen after light t rea tment , but no differential effects were found with evening or morn ing light t rea tment or between improved and non improved pat ients .

The T S H rhy thm was differentially affected by morn ing and evening light with a decrease of A U C , and a phase advancement of T C with morn ing light and the opposi te with evening light. N o significant differences were found between improved and nonimproved pat ients . In the interpretat ion of these findings the s t rong influence of the patients with winter depression in the results of the pat ients in the whole g roup should be taken into considerat ion.

The effects of light on melatonin have been studied by Te rman and coworkers and Lewy and coworkers , bo th groups report ing phase advancing effects of morn ing light in pat ients with winter d e p r e s s i o n .

1 2 , 1 3 , 18 Lewy and coworkers also show these changes of

melatonin phase to be of therapeut ic impor tance , relating a phase advancement of the mela tonin rise in the evening after t rea tment with a clinical effect.

13 Rao and coworkers on the other hand found no significant

effects of morn ing light t rea tment on melatonin rhythms in nonseasonal depressive p a t i e n t s .

14 Win ton and coworkers in a small pat ient g roup

(n = 7) found that the clinical effect of light t rea tment was dissociated from the effect on the nightly mela tonin r h y t h m .

24

Plasma Cortisol and the dexamethasone suppression test had not been found to be abnorma l . In a recent report , Joseph-Vanderpool and coworkers report of a delayed and blunted A C T H response to C R H st imulat ion in nine patients with winter depression, suggesting hypofunc-tional C R H n e u r o n s .

7 After 9 days of light t rea tment a significant increase

of the A C T H and Cortisol responses to C R H was observed. T o our knowledge, this report is the first on Cortisol, prolactin, and T S H rhythms in a larger g roup of pat ients with winter depression.

The increased prolact in levels in the patients after light t rea tment could be interpreted as effects of lowered dopaminergic and /o r increased noradrenergic and /o r serotonergic function.

Perhaps the most interesting result of this prel iminary report on an ongoing study is the differential effect regarding the clinical ou tcome of light treatment on the changes of Cortisol T C , both in the whole depressive group and in the g roup of pat ients with winter depression.


Thus a relation between therapeut ic beneficial effects of light and a phase advancement of the Cortisol rhy thm seem possible, but the findings have to be evaluated further in a larger sample and in other pat ient groups .

Whether or no t clinical da t a as diagnosis and repor ted symptoms could predict the clinical ou tcome of light t rea tment is still unsolved. We are examining this quest ion in a larger sample of pat ients .


The au thors acknowledge the grants from KI fonder, Svenska lâkaresâlls-kapet , Medicinska forskningsrâdet (3371) och Gadeliusstiftelsen. We also thank Ylva Friberg, Klas Levin, Lars Môrk r id and M a r i a n n Zet tergren for excellent help.

References

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6. J a m e s S., W e h r T.A. , Sack D . a n d P a r r y B. (1988) T h e d e x a m e t h a s o n e suppress ion test in seasonal affective d i sorder . Compr. Psychiatry. 27, 224 -226 .

7. J o s e p h - V a n d e r p o o l J .R., Rosen tha l N . E . , C h r o u s o s G . P . , W e h r T.A., Skwerer R., K a s p e r S. a n d G o l d P . W . (1991) A b n o r m a l p i tu i t a ry -ad rena l responses to co r t i co t ro -pin-re leas ing h o r m o n e in pa t i en t s wi th seasona l affective d i so rde r : Clinical a n d pa thophys io log ica l impl ica t ions . J. Clin. Endocrinol. Metab. 72(6), 1382-1387.

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9. K r i p k e D . F . , M u l l a n e y D.J . , Savides T.J . a n d Gil l in J .C . (1989) P h o t o t h e r a p y for nonsea sona l ma jo r depress ive d i so rders . In Seasonal Affective Disorders and Photother-apy (eds. R o s e n t h a l N . E . a n d Blehar M . C . ) , p p . 342-356 . Gui l ford , N e w Y o r k .

10. K r i p k e D . F . , M u l l a n e y D.J . , K l a u b e r M.R. , Risch S.G. a n d Gil l in J .C . (1992) Con t ro l l ed trial of br ight light for n o n s e a s o n a l ma jo r depress ive d i so rders . Biol. Psychiatry 3 1 , 119-134 .

11. Lewy A.J. , H e r b e r t K.A. , Rosen tha l N . E . a n d W e h r T.A. (1982) Bright artificial l ight t r e a tmen t of a m a n i c depressive pa t i en t wi th a seasona l m o o d cycle. Am. J. Psychiat. 1 3 9 , 1 4 9 6 - 1 4 9 8 .

12. Lewy A.J. , Sack R.L. , Mil ler L .S . a n d H o b a n T . M . (1987) An t idep res san t a n d c i rcad ian phase-shif t ing effects of l ight. Science 235 , 352 -354 .

13. Lewy A.J. , Sack R.L. , Singer C M . (1985) Bright l ight, m e l a t o n i n a n d biological r h y t h m s : Impl i ca t ions for the affective d i so rders . Psychopharmacol. Bull. 2, 368 -372 .

14. R a o M . L . , M ù l l e r - O e r l i n g h a u s e n B. , M a c k e r t Α., Strebel B. , Stieglitz R . D . a n d Vol tz H . P . (1992) B lood se ro ton in , s e rum m e l a t o n i n a n d light t h e r a p y in hea l thy subjects a n d in pa t i en t s wi th n o n s e a s o n a l depress ion . Acta Psychiatr. Scand. 86 , 127-132 .

15. R o s e n t h a l N . E . , Sack D.A. , Gil l in J . - C , Lewy A.J. , G o o d w i n F .K . , D a v e n p o r t Y.,


Muel le r P .S . , N e w s o m e D.A. a n d W e h r T.A. (1984) Seasona l affective d i so rder : A descr ip t ion of the s y n d r o m e a n d p re l iminary findings wi th l ight t he rapy . Arch. Gen. Psychiatry. 4 1 , 7 2 - 8 0 .

16. Sack R.L. , Lewy A.J. , W h i t e D . M . , Singer C M . , F i r e m a n M.J . a n d Vand ive r R. (1990) M o r n i n g vs. evening light t r e a t m e n t for win ter depress ion . Evidence t h a t the the rapeu t i c effects of l ight a re m e d i a t e d by c i rcad ian phase shifts. Arch. Gen. Psychiatry. 4 7 , 3 4 3 - 3 5 1 .

17. S a k a m o t o K. , K a m o T. , N a k a d a i r a S., T a m u r a A. a n d T a k a h a s h i K . (1993) A n a t i o n w i d e survey of seasonal affective d i so rde r a t 53 o u t p a t i e n t universi ty clinics in J a p a n . Acta. Psychiatr. Scand. 8 7 , 2 5 8 - 2 6 5 .

18. T e r m a n M . , T e r m a n J .S . , Q u i t k i n F . M . , C o o p e r T .B. , L o E.S. , G o r m a n J . M . , S tewar t J . N . a n d M c G r a t h P . J . (1988) Response of m e l a t o n i n cycle to p h o t o t h e r a p y for seasonal affective d i sorder . / . Neural. Transm. 7 2 , 147-165 .

19. T e r m a n M . , T e r m a n J .S . a n d Q u i t k i n F . M . (1989) Light t he r apy for seasonal afffective d i sorder : A review of efficacy. Neuropsychopharmacology 2 , 1-22.

20. T h a l é n B-E. , Kje l lman B. a n d W e t t e r b e r g L. (1993) P h o t o t h e r a p y a n d me la ton in in re la t ion to seasonal affective d i so rde r a n d depress ion . In Melatonin, Biosynthesis, Physiological Effects, and Clinical Application (eds. Yu H . -S . a n d Reiter R.J . ) , p p . 4 9 5 - 5 1 1 . C R C Press , Inc . , B o t a R a t o n .

2 1 . T h o m p s o n C. a n d Isaacs G . (1988) Seasona l affective d i s o r d e r — A Brit ish sample . S y m p t o m a t o l o g y in re la t ion to m o d e of referral a n d d iagnos t ic sub type . / . Affective Disord. 14 , 1 -11 .

22. W e h r T.A. , J a c o b s e n F . M . , Sack D.A. , A r e n d t J., T a m a r k i n L. a n d Rosen tha l N . E . (1986) P h o t o t h e r a p y of seasona l affective d i sorder . T i m e of d a y a n d suppress ion of m e l a t o n i n a re n o t critical for an t idep res san t effects. Arch. Gen. Psychiatry. 4 3 , 8 7 0 - 8 7 5 .

23 . W e t t e r b e r g L. , E r iks son O . , F r i b e r g Y. a n d V a n g b o B. (1978) A simplified r a d i o i m m u n o a s s a y for m e l a t o n i n a n d its app l i ca t ion to biological fluids: P re l imina ry obse rva t ion o n the half-life of p l a s m a m e l a t o n i n in m a n . Clin. Chem. Acta. 8 6 , 1 6 9 - 1 7 7 .

24. W i n t o n F . , C o r n T. , H u s o n L .W. , F r a n e y C , A r e n d t J. a n d Checkley S. (1989) Effects of light t r e a tmen t u p o n m o o d a n d m e l a t o n i n in pa t i en t s with seasonal affective d i sorders . Psychol. Med. 19 , 585 -590 .

25 . Wir tz -Jus t ice Α., Bucheli C , G r a w P . , Kie lholz P . , F i sh H . - U . a n d W o g g o n B. (1986) Light t r e a tmen t of seasona l affective d i so rde r in Swi tzer land. Acta Psychiatr. Scand. 7 4 , 193-204.

26. Wir tz -Jus t ice Α., G r a w P . , K r â u c h i K. , Gis in B. , J o c h u m Α., A r e n d t J., F i sh H . -U . , B u d d e b e r g C. a n d Pô ld inge r W . (1993) Light t he r apy in seasona l affective d i sorder is i ndependen t of t ime of d a y of c i rcadian p h a s e . Arch. Gen. Psychiatry (in press) .

26

A Comparison of Two Different Placebo-Controlled SAD Light Treatment Studies C. I. E A S T M A N ,

1 M . A . Y O U N G

2 a n d L F. F O G G

2 1 Biological Rhythms Research Laboratory, Psychology Department,

Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL, USA 2Psychiatry Department and Psychology Department, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL, USA

Abst rac t

High intensi ty light is widely used as a n an t idep res san t for the t r e a t m e n t of winter depress ion . T h e r e is n o d o u b t t ha t t r e a tmen t wi th light can subs tan t ia l ly reduce a n d even e l iminate all s y m p t o m s . H o w e v e r , es tabl ish ing tha t this effect is g rea te r t h a n w h a t cou ld be p r o d u c e d by a p l acebo -con t ro l t r e a t m e n t is h a m p e r e d by the difficulty in finding an a p p r o p r i a t e p l acebo for light t r e a t m e n t . Th is p a p e r c o m p a r e s t w o light t r e a t m e n t s tudies wi th g o o d p l acebo con t ro l s . T h e first s tudy employed a c rossover design be tween m o r n i n g light a n d m o r n i n g p l acebo . T h e second, which is still in p rogress , emp loys a paral le l design with m o r n i n g light, m o r n i n g p lacebo a n d evening light g r o u p s . T h e first s tudy did no t show a statist ically significant difference be tween light a n d p l acebo . A l t h o u g h the response ra tes in the second s tudy a re m u c h grea ter t h a n in the first s tudy , there is still n o significant difference be tween light a n d p l acebo . T h e response to light t r e a t m e n t is m u c h grea te r t h a n in the first s tudy , b u t the r e sponse to p l acebo t r e a t m e n t is a lso m u c h grea ter . Several possible reasons for the g rea te r an t i dep re s san t effects in the second s tudy a re d iscussed, such as a larger p l acebo c o m p o n e n t in all three t r e a t m e n t s d u e to a m o r e opt imis t ic staff. A l t h o u g h it wou ld be p r e m a t u r e to d r a w final conc lus ions a b o u t efficacy, we h a v e s h o w n tha t for light t r e a tmen t , like an t idep res san t d r u g t r e a t m e n t , m o s t of the an t idep res san t effect can be a t t r i bu ted to p l acebo effects.

M A N Y RESEARCHERS and clinicians assert tha t bright light t rea tment is an effective ant idepressant for winter depression or seasonal affective disorder (SAD). However , even a placebo can be "effective" in tha t it can produce large reduct ions in d e p r e s s i o n .

6 18 The difficulty in finding an appropr ia te

placebo control for light t rea tment makes conclusions abou t efficacy beyond placebo effects p r o b l e m a t i c .

3'

4 A major goal of our studies is to

371


establish the efficacy of high intensity light t rea tment for winter depression. In this paper we will repor t results from two studies which compare high intensity light t rea tment to a placebo control t rea tment . The first was a crossover design between light and placebo, conducted dur ing the two consecutive winters from 1988 to 1990.

5 The second study, a

parallel design, began the next winter and is currently in progress. Since we can only report the results from the first two winters of this study, conclusions must be tentat ive. These studies are repeated for more than one winter in order to obta in a larger sample of subjects.

We decided to test light t rea tment in the morn ing , because of the evidence that t rea tment at this t ime of day produces the greatest ant idepressant effect.

21 Since the quest ion of efficacy is our major focus, it

was impor t an t to use an appropr ia te placebo control t rea tment for morn ing light t rea tment . We did not want to use dim light as a control , because we did not think our pat ients would believe it to be a plausible t rea tment (especially in a crossover design). The media in our city, and other nor the rn cities in the USA, has focused so much a t tent ion on "br ight" light as a t rea tment , tha t we think that our pat ients would expect bright light to work better than dim. Even in the first mult i-pat ient s tudy of bright light t r e a t m e n t ,

14 pat ients predicted tha t the bright light would

work better than the dim. If pat ients expect one t rea tment to work better, then the placebo effect may be greater for that t rea tment . Fu r the rmore , several studies have not shown the expected differences between bright and dim l i g h t ,

9 , 1 9'

24 leading to suggestions that dim light might be an active

t r e a t m e n t .

12 24 O u r goal was to find a placebo control t rea tment tha t is

inert, plausible to pat ients , and that produces similar expectat ions for success as light t rea tment . Some investigators have asserted tha t evening light is a good control t rea tment for morn ing l i g h t .

1 0 , 11 Al though evening

light may be a plausible t rea tment to pat ients , m a n y researchers claim that evening light is an effective a n t i d e p r e s s a n t ,

1 5 , 25 i.e. tha t it is no t inert.

Morn ing light t rea tment can produce profound changes in the sleep schedule. Pat ients usually have to wake u p earlier in order to have time for the morn ing t rea tment . They may go to bed earlier and /o r they may become partially sleep deprived. M o r n i n g t rea tment may also serve to regularize the sleep schedule, if pat ients are asked to awaken at the same time every day. We wanted a placebo control tha t would produce the same changes in sleep as morn ing light t rea tment . Light t rea tment also necessitates a regular period of inactivity. This may be an enjoyable, restful t ime for some people, or alternatively, an aversive bore or inconvenience. In any case, we wanted a placebo t rea tment tha t would also require the same period of inactivity.

There is a large scientific and popula r l i terature abou t how negative air ions can improve m o o d , energy and affect serotonin transmission. There is even evidence that our exposure to negative ions varies seasonally. A

Two Placebo-Controlled SAD Light Treatment Studies 373

M e t h o d — S t u d y No . 1

Subjects

Subjects were diagnosed by the usual criteria for seasonal affective d i s o r d e r ,

14 but in addi t ion we required the "atypical" symptoms of

increased appeti te or weight, and increased sleep. Mos t SAD pat ients have these "atypical" symptoms, and this gave us a more homogenous and more typical sample of S A D subjects. Subjects also had to score 21 or more on the Hami l ton Depression Scale composed of the 17 original i t e m s

8 and

7 "atypical" i t e m s

13 ( H A M 17 Ο + 7 A). All items were administered with

the S I G H - S A D semi-structured in t e rv iew.

22 All subjects were free of

psychotropic medicat ions for several m o n t h s , and none had previously received bright light t rea tment .

Experimental protocol

The design was a 5-week counterbalanced crossover, with a baseline (B) week, 2 consecutive weeks of morn ing light (ML) t rea tment and 2 consecutive weeks of morn ing placebo ( M P ) t rea tment . Both t rea tments were 1 hou r in dura t ion beginning immediately upon waking in the morn ing .

The light box was 65 cm wide and 43.5 cm tall (Apollo Light Systems, Orem, U tah ) . It contained six horizontal ly m o u n t e d cool-white fluores-cent lamps . Subjects sat at a table or desk with the light box directly in front of them at a distance of 12 inches (30.5 cm). Subjects were provided with 12 inch rulers. They usually read dur ing the t rea tment t ime, which produced an i l luminance of abou t 7,000 lux.

The negative ion generator was a commercial ly available air cleaner (28 χ 13 χ 18 cm). It was placed on the same table or desk as the light box, posit ioned 61 cm away from the subject, so that the negative ions were blown across the table in front of the subject. Some of the generators were deactivated by disconnecting a circuit within the generator . However , this

negative ion generator is similar to a light box, in tha t it is an electrical device that produces a na tura l environmenta l factor which travels th rough the air to impinge upon the subject. We thought that negative ion t rea tment could be plausible to our pat ients , and could generate good expectat ions for improvement . However , in order to have a control t rea tment that was inert, some of the negative ion generators were altered so that they did not actually produce negative ions. This repor t only includes da t a from the patients who received "deact ivated" generators , i.e. the placebo t rea tment .


procedure did not disable the indicator light or fan. Subjects were informed tha t they might receive an active or a deactivated generator , but that they would not be able to tell which type they had received.

D r Lahmeyer directed the staff, at the Psychiatry Depa r tmen t at the University of Illinois Medical Center , tha t conducted the diagnoses and weekly interviews for depression ratings (S IGH-SAD) . This staff was "bl ind" to t rea tment condi t ion. After the weekly interviews the subjects walked a few blocks to the Biological Rhy thms Research Labora to ry at Rush-Presbyter ian-St Luke's Medical Center where they received or exchanged their equipment and delivered their quest ionnaires and logs. This division of functions between the two medical centers helped to main ta in the blind. Fu r the rmore , pat ients were coached to use the word "equipment" ra ther than "lights" or "genera tor" in order to help mainta in the blind. Subjects completed the Beck Depression Inven to ry

1 at the end

of each week.

Before subjects agreed to par t ic ipate , D r Eas tman , at Rush Medical Center , showed them a light box and negative ion generator and explained the rat ionale for these t rea tments . They were given a packet of information from the scientific and lay l i terature abou t negative ions and light t rea tment . They were told that some people responded better to ions than light, some responded better to lights than ions, some responded to both , and some to neither. The at t i tude tha t the research staff at bo th medical centers displayed to subjects with regard to bo th t rea tments was generally neutral and caut ious ra ther than enthusiastic. However , bo th staffs were frequently coached to use the word "equipment" ra ther than "lights" or "genera tors" as much as possible, and to show equal interest in bo th t rea tments .

Together with D r Eas tman each subject was required to select a fixed wake-up time for the entire 5-week study, including weekends. Subjects were told that an early and regular wake-up time was par t of the t rea tment . Subjects were required to go to bed at least 7 hours before their scheduled wake-up t ime. They were permit ted to go to bed earlier, but not later than this t ime. N a p s were permit ted as long as they started at least 2 hours after the scheduled wake-up t ime. Dur ing the study, compliance was encour-aged and moni to red by periodic morn ing phone calls to the subjects and by reviewing the daily sleep logs once or twice a week.

Subjects were informed of their order g roup assignment at the beginning of the baseline week. At the end of the baseline week subjects completed an Expectat ion Quest ionnaire containing four 100 m m visual analog scales, one for each of the upcoming four t rea tment weeks. Thus , all ratings were performed before t rea tment began. The 100 m m lines were anchored with "will feel much bet ter" and "will feel much worse." The t rea tment p lanned for each of the 4 weeks, "l ights" or " ions ," was writ ten on to each individual 's Expectat ion Quest ionnai re .


Data analysis

Statistical analyses were performed on the composi te Hami l ton Depres-sion Scale ( H A M 17 Ο + 7 A), the original 17-item Hami l ton scale and the Beck scale. Since these scales are intercorrelated measures of depression, we conducted a mult ivariate analysis of variance ( M A N O V A ) using a repeated measures d e s i g n .

23 The between subject factor was order g roup

(light first, light last) and the within-subjects factor was t rea tment (baseline, end of light t rea tment , end of placebo t rea tment) . In all statistical tests we used a significance level of 0.05, two-tailed, except where noted.

M e t h o d — S t u d y No. 2

The methods are the same as for the first s tudy with the following changes.

Experimental protocol

Trea tment dura t ions were increased from 1 to 1.5 hours . We are using a parallel design with three t rea tment groups : morn ing light (1.5 hours upon awakening) , morn ing placebo (1.5 hours upon awakening) , and evening light (1.5 hours before bed, ending 1 hour before the latest permit ted bedt ime).

The same model light box is used, but it is placed 15 inches (38 cm) in front of the pat ient , or 3 inches (7.6 cm) further away than in the first s tudy. This reduces the average i l lumination while reading to abou t 6,000 lux, but gives the pat ients a larger work area in front of the lights. Pat ients are provided with 15 inch rulers.

New negative ion generators were built for this study. They are enclosed in shiny black cylinders, 32 cm tall, d iameter 15 cm. Three small lights on the front change rapidly between red and green. Since the ion generator makes a soft hissing sound, a white noise generator was added to all the units . This makes it impossible to tell from listening whether the generator is active or not . The ion generators used in the previous study conta ined a fan which masked the noise. T w o generators are set u p on a desk or table in front of the pat ient , 15 inches (38 cm) apar t , with 15 inches between the pat ient and each generator . Pat ients are provided with rulers.

There is a different staff who performs diagnoses and depression rat ings, directed by D r Young in the Psychiatry Depa r tmen t at Rush Medical Center . After the weekly S I G H - S A D ratings pat ients only have to walk abou t one block to go to the Biological Rhy thms Research Lab . However , there is the same division of functions between the two groups which helps to mainta in the blind. In the meeting with potent ial pat ients , D r Eas tman acts more enthusiast ic and confident abou t the success of the t rea tments


than in the previous study. She coaches the staff in bo th depar tments to be optimistic in general, as well as to be equally enthusiastic abou t light and ion t rea tment . We try to refer to subjects as pat ients , ra ther than subjects, and to emphasize that they are receiving t rea tment , ra ther than tha t this is a research study.

Together with D r Eas tman each pat ient is required to select a wake-up time tha t is earlier than usual , so tha t they will have time in their schedule for t rea tment if they are assigned to a morn ing t rea tment g roup . They are required to go to bed between 7 and 8 hours before their scheduled wake-up t ime. N a p s are permit ted within a 6-hour window in the middle of their waking period. Thus , naps are no t permit ted near the time of morn ing or evening t rea tment . Pat ients are told that an early and regular sleep schedule is par t of the t rea tment . Compl iance is encouraged and moni tored by requiring tha t pat ients call a t ime-s tamp answering machine three times each morn ing , and by reviewing the daily sleep logs.

As in the first s tudy, pat ients are told their g roup assignment at the beginning of the baseline week. At the end of the baseline week they complete an expectat ion quest ionnaire which asks them to ra te , on a 7-point scale, how they expect to feel at the end of the 4-week t rea tment , from "will feel as good as summer" (1) to "no change" (7).

Data analysis

Although we are theoretically opposed to da ta presentat ion and publicat ion before a s tudy is completed, prel iminary analyses were performed for the purpose of compar ing these two studies for this paper . A repeated measure A N O V A was performed on the H A M 17 Ο + 7 A scores. The between-subjects factor was t rea tment g roup (morning light, evening light, morn ing placebo), and the within-subjects factor was time (treatment weeks 1, 2, 3 and 4). All scores were input as difference from baseline scores.

Resul ts—Study No. 1

Depression ratings

Figure 1 shows tha t the Hami l ton depression scores declined over the course of the study in bo th order groups (light first and light last). The Beck scale produced a similar pa t te rn of results. The M A N O V A followed by post-hoc tests indicated tha t depression was significantly reduced with bo th morn ing light and morn ing placebo t rea tments , but that there was no significant difference between them in their ant idepressant effects.

A responder to t rea tment was defined as a subject whose H a m 17 Ο + 7 A score was reduced to at least 5 0 % of baseline. By this criterion, 18 .8% (6/32) of the subjects responded to morn ing light but no t morn ing placebo,


35 r

30 -25 -

+ ο 20 -t \ 15 -

10 -< X 5 -

0 -

LIGHT LAST ( N = 1 4 )

PRE-B Β MP MP ML ML

LIGHT FIRST ( N = 1 8 )

PRE-B Β ML ML MP MP

F I G . 1. Average H a m i l t o n depress ion scores over the course of s tudy N o . 1. T h e b o t t o m of each b a r represents the or iginal 17-item scale, the t o p represen ts the 7-

i tem a typica l scale.

6 . 3 % (2/32) responded to morn ing placebo but no t morn ing light, 15 .6% (5/32) responded to bo th t rea tments , and 5 9 . 3 % (19/32) responded to neither. Thus , 34 .4% (11/32) were morn ing light responders and 21 .9% (7/32) were morn ing placebo responders . Using a 1-tailed exact p rob -ability tes t ,

2 this difference in p ropor t ions was no t significant.

Expectations

Subjects had good expectat ions for bo th t rea tments , but they were slightly bet ter for light t rea tment t han for ion t rea tment . The mean + S D expectat ion scores were 18.8 ± 10.9 m m for the end of light t rea tment and 25.8 + 13.4 m m for the end of deact ivated ion generator t rea tment ( p < 0 . 0 1 , by a paired i-test).

Sleep

Sleep parameters were averaged for the week before the study (pre-baseline) and each of the 5 weeks of the pro tocol . The only significant changes occurred between the pre-baseline and baseline weeks. W a k e - u p time advanced, sleep onset did not change significantly, and n a p time was small, so tha t total sleep t ime was reduced. Sleep parameters remained stable dur ing the 5-week protocol .


The changes from pre-baseline to baseline were: Wake-up time advanced 1.5 hours from 7.8 + 1.4 hours after midnight to 6.3 + 0.9 (p< 0.001, paired ί-test). Tota l sleep time (including naps) decreased 1.4 hours , from 8.9 + 1.1 hours to 7.5 + 0.7 hours ( p < 0 . 0 0 1 , paired i-test). Thus , the experimental protocol caused an advance in wake-up time and some sleep loss, but these changes occurred prior to bo th light and placebo t rea tment . Sleep parameters did not differ between the light and placebo weeks. Thus , in terms of sleep, the placebo t rea tment was a good control for light t rea tment .

Resul ts—Study No. 2

Depression ratings

Figure 2 shows that the Hami l ton depression scores declined over the course of the study in all three groups (morning light, evening light and morn ing placebo). The Beck scale produced a similar pa t te rn of results. The A N O V A showed a significant t ime effect. The t rea tment g roup factor was no t significant, nor was the t rea tment g roup by time interact ion. Thus , patients improved over time regardless of t rea tment g roup . The results so far do not reveal any significant differences between light and placebo t reatments .

A responder to t rea tment was defined the same way as for experiment N o . 1, as a subject whose Hami l ton score was reduced by at least 5 0 % . By this criterion, 84 .6% (11/13) of the subjects responded to morn ing light, 75 .0% (9/12) responded to evening light, and 66 .7% (8/12) responded to morn ing placebo. This difference was not significant by a Pearson 's Chi-square test.

Expectations

Subjects had good expectat ions for all t reatments , and there were no significant differences in expectat ions between the three groups . The mean + SD expectat ion scores were 3.3 + 1.5 for morn ing light, 3.3 + 1.1 for evening light, and 3.8 + 1.3 for morn ing placebo.

Sleep

As in study N o . 1, the only significant change in sleep occurred between the pre-baseline and baseline weeks. Wake -up time advanced 1.7 hours , from 7.5 to 5.8, for the morn ing light g roup ( p < 0 . 0 0 1 , paired i-test), 0.9 hours , from 7.3 to 6.4, for the evening light g roup (p< 0.001), and 1.8 hours , from 8.1 to 6.3, for the morn ing placebo g roup (p<0.001). Tota l sleep time (including naps) decreased 1.2 hours , from 8.2 to 7.0 hours , for the morn ing light g roup (p<0.001, paired i-test), 0.6 hours , from 7.7 to 7.1

Two Placebo-Controlled SAD Light Treatment Studies 3 7 9

MORNING LIGHT ( N = 1 3 )

PRE-B Β ML ML ML ML

EVENING LIGHT (N = 1 2 )

PRE-B Β EL EL EL EL

MORNING PLACEBO (N=

PRE-B Β MP MP MP MP

F I G . 2. Average H a m i l t o n depress ion scores for the d a t a collected so far in s tudy N o . 2.

hours , for the evening light g roup ( p < 0 . 0 1 ) , and 1.1 hours , from 8.1 to 7.0 hours , for the morn ing placebo g roup (p < 0.001). As for s tudy N o . 1, these sleep changes occurred pr ior to all t rea tments , and sleep parameters dur ing the 5-week protocol did no t differ a m o n g the t rea tments . However , the changes from pre-baseline to baseline were more similar between the morn ing light and morn ing placebo groups . Thus , the placebo g roup was a better control for the morn ing light g roup than for the evening light g roup regarding potent ial ant idepressant effects caused by sleep changes.

Discussion

The first s tudy did no t demons t ra te efficacy for morn ing light t rea tment ; morn ing light did not p roduce a significantly greater ant idepressant effect than the placebo t rea tment . This result is even more striking given the fact


tha t pat ients expected the light t rea tment to work slightly better than the placebo t rea tment . Expectat ions are thought to account for at least par t of the placebo effect. If there had been a larger difference between light t rea tment and placebo, it might have been explained by this difference in expectat ions.

F o r the next s tudy, several design changes were made in an a t tempt to demons t ra te a superiority for light t rea tment over placebo. The placebo l i terature shows that the physician's a t t i tude toward a t reatment , his or her belief, enthusiasm, conviction, commitment , opt imism, etc. may be necessary for the success of that t rea tment . N o t only are positive at t i tudes necessary to enhance the placebo effect, which is an impor tan t componen t of all t rea tments , but positive at t i tudes may work synergistically with a t rea tment and be necessary to release the therapeut ic effect of a t r e a t m e n t .

4 , 7'

16 Since the a t t i tude of the research staff was neutral and

caut ious in the first s tudy, we changed it to be more enthusiast ic and confident in the second study.

Another change in the study design was to increase the dura t ion of light t rea tment from 1 to 1.5 hours , in case the 1 hour t reatment did not provide a large enough "dose." We also changed the length of light t rea tment from 2 to 4 weeks, in case a significant difference between light and placebo could only be detected after several weeks, as in ant idepressant drug s t u d i e s .

17

Two other design changes were made in an a t tempt to make subsequent interpretat ion of results more straightforward. We changed to a parallel groups design, to avoid the potent ial complicat ions of order and carryover effects. We switched to a more impressive negative ion generator , and gave each patients two generators instead of one, in order to generate equal expectations for light and ion t rea tment . Another design change was the addi t ion of an evening light t rea tment g roup , to include a test of evening light against placebo.

So far, the results of the second study do not show a statistically significant difference in ant idepressant effect between light and placebo. We do not know whether the difference will become significant after more subjects are added to the sample. We can, at this point , est imate the magni tude of the benefit of light t reatment over placebo, and the p ropor t ion of light t rea tment effect that can be a t t r ibuted to placebo effects, if we assume that the total ant idepressant effect of light t reatment is a simple addi t ion of the placebo effect and an active light effect. If we use Hami l ton (17 Ο + 7 A) difference scores (the difference between baseline and the last week of t rea tment) , then morn ing light t reatment reduced the average score 20.0 points , and morn ing placebo reduced it 13.9 points . Therefore, the benefit of using morn ing light over placebo was 6.1 points . Fu r the rmore , 7 0 % (13.9/20) of the reduction in score produced by morn ing light can be a t t r ibuted to placebo effects. If we base estimates on


the response rates previously calculated, then the benefit of morn ing light t rea tment is 17 .9% (84.6-66.7). In other words , we can assume tha t 17 .9% of the pat ients who responded to morn ing light would not have responded to placebo. Also, 7 9 % (66.7/84.6) of the pat ients who responded to morn ing light t rea tment would have responded to the placebo t rea tment .

Al though these differences between light and placebo m a y seem small, they are actually similar to the differences between m a n y commonly used ant idepressant drugs and placebo p i l l s .

4'

6 Studies tha t show statistically

significant benefits for ant idepressant drugs over placebo often include hundreds of p a t i e n t s .

17 It is more difficult to obta in large sample sizes for

SAD light t rea tment studies, because da t a can only be collected dur ing a few mon ths of the year, and because pat ients are less likely to agree to the more demanding t rea tment procedures and protocols . Those who believe that the benefit of ant idepressant drugs over placebo is clinically impor tan t , p robably feel the same way abou t the benefit of light t rea tment . Others may consider the active drug and light effects to be relatively un impor tan t compared to the placebo effects.

It is most interesting to compare the results of the two studies. All three t rea tment groups in the second study improved more than the groups in the first s tudy. This is no t due to the fact tha t the t rea tments were increased from 2 to 4 weeks, because the differences between the studies were already evident dur ing the first two t rea tment weeks. N o r can this be ascribed to different styles of Hami l ton rat ings between the two research teams, because the Beck self-ratings showed the same differences between the studies.

O n e possibility is tha t the pat ients in the second study did bet ter because their wake-up times were earlier. The median scheduled wake-up time was 6:30 a.m. in the first s tudy and 6:00 a.m. in the second study. However , there was no correlat ion between clinical improvement and wake-up time in either s tudy, no r was greater clinical improvement associated with any part icular range of wake-up times.

Another possible reason for the improved ant idepressant response in the second study could be the increase in t rea tment dura t ions from 1 to 1.5 hours . This increased investment of t ime on the par t of the pat ients could increase the placebo effect of all three t rea tments . It is also possible tha t the increased light exposure could have a greater physiological effect, account ing for pa r t of the improved morn ing light response. The change from one to two negative ion generators , and to a more impressive generator , could be responsible for par t of the improved placebo response. Another factor which probably increased the placebo effect in all three t reatments was the more optimistic a t t i tude of the research staff.

This discussion illustrates that the magni tude of the ant idepressant response to light depends on many factors besides intensity, dura t ion , and other parameters that might relate to the physiological dose. Similarly,


ant idepressant response rates to placebo pills vary widely a m o n g studies—from 0 to 9 1 % according to one review.

6 Therefore, concepts

such as "a placebo response rate for S A D " or "a response rate to 1 hou r morn ing light at 6 a.m." are er roneous . It is usually meaningless to compare response rates between studies. F o r example, a new light delivery device, such as a visor, cannot be considered effective jus t because it produces a response rate similar to a more conventional light box, or even because it produces a high response ra te . Each s tudy must have its own control t rea tment . Reviews of response rates from many studies, such as the study by Te rman et al.

21 can be of heuristic value. However , as noted

by T e r m a n ,

20 this type of review cannot be used to draw final conclusions

or take the place of well-controlled studies.

It may take m a n y years to establish whether light t rea tment is more effective than a placebo, and this work is impor tan t in order to help unders tand the mechanisms and etiology of winter depression. Mean-while, there is no doub t that t rea tment with light can greatly reduce the suffering of those with SAD. Given the low risk and minimal side effects of this t reatment , at least one of us (CIE) believes that it is ethical to prescribe its use. As long as the research, clinical and medical professionals cont inue to be enthusiastic abou t light t rea tment , and pat ients do not read papers like this one, we can cont inue to improve the lives of pat ients with winter depression.


We thank Eva Cherin, Mari lyn Per lman and K a y Peterson for diagnoses and S I G H - S A D ratings, and Linda Gal lo , Suk-fong Tang , Michael M a h o n e y and Charles Splete for technical assistance. Suppor ted by Nat iona l Insti tutes of Menta l Heal th grant MH42768 .

References

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3. B r o w n W.A. (1990) Is light t r e a tmen t a p l acebo? Psychopharmacol. Bull. 2 6 , 527-530 . 4. E a s t m a n C.I . (1990) W h a t the p l acebo l i te ra ture can tell us a b o u t light t h e r a p y for S A D .

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10. Lewy A.J. , Sack R.L. , Mil ler L .S . a n d H o b a n T . M . (1987) An t idepressan t a n d c i rcad ian phase-shif t ing effects of light. Science 2 3 5 , 352 -354 .

11 . Lewy A.J. , Sack R.L. , Singer C M . , W h i t e D . M . a n d H o b a n T . M . (1989) W i n t e r depress ion : the p h a s e angle be tween sleep a n d o the r c i rcad ian r h y t h m s m a y be cri t ical . In Seasonal Affective Disorder (eds. T h o m p s o n C. a n d Si lvers tone T.) , p p . 2 0 5 - 2 2 1 . C N S Clinical Neurosc ience , L o n d o n .

12. M o u l D . E . , He l lekson C.J. , O r e n D.A. , F r a n k Α., B r a i n a r d G. , M u r r a y M . G . , W e h r T.A. a n d R o s e n t h a l N . E . (1990) T rea t i ng S A D with a light visor: A mul t i cen te r s tudy . Soc. Light Treatment Biol. Rhythms Abst. 2 , 15.

13. R o s e n t h a l N . E . a n d HefTernan M . M . (1986) Bul imia , c a r b o h y d r a t e c rav ing , a n d depress ion : A cent ra l connec t i on? In Nutrition and the Brain (eds. W u r t m a n R.J . a n d W u r t m a n J .J . ) , Vol . 7, p p . 139-166 . R a v e n Press , N e w Y o r k .

14. R o s e n t h a l N . E . , Sack D.A. , Gil l in J . C , Lewy A.J. , G o o d w i n F . K . , D a v e n p o r t Y., Muel le r P .S . , N e w s o m e D.A. a n d W e h r T.A. (1984) Seasona l Affective D i s o r d e r : A descr ip t ion of the s y n d r o m e a n d p re l iminary findings wi th l ight t he rapy . Arch. Gen. Psychiatry 4 1 , 7 2 - 8 0 .

15. R o s e n t h a l N . E . , Sack D.A. , Skwerer R .G . , J a c o b s e n F . M . a n d W e h r T.A. (1988) P h o t o t h e r a p y for seasonal affective d i sorder . J. Biol. Rhythms 3 , 101-120 .

16. Shap i ro A .K . (1971 ) P l a c e b o effects in medic ine , p s y c h o t h e r a p y , a n d psychoana lys i s . In Handbook of Psychotherapy and Behavior Change: An Empirical Analysis (eds. Bergin A.E . a n d Garfield S.L.), p p . 4 3 9 - 4 7 3 . J o h n Wiley & Sons , Inc . , N e w Y o r k .

17. S ta rk P . a n d H a r d i s o n C D . (1985) A review of mul t i cen te r con t ro l l ed s tudies of fluoxetine vs. i m i p r a m i n e a n d p l acebo in o u t p a t i e n t s wi th ma jo r depress ive d i sorder . J. Clin. Psychiatry 4 6 , 5 3 - 5 8 .

18. S tewar t J. (1990) P l acebos in eva lua t ing light t h e r a p y for seasonal affective d i sorder . Psychopharmacol. Bull. 2 6 , 525 -526 .

19. Te icher M . H . , G l o d C.A., O r e n D.A. , L u e t k e C , Schwar t z P . , B r o w n C. a n d R o s e n t h a l N . E . (1992) T h e p h o t o t h e r a p y light visor: there is m o r e to it t h a n meets the eye. .Soc. Light Treatment Biol. Rhythms Abst. 4 , 20.

20. T e r m a n M . (1989) Ed i to r i a l : Deve lop ing the case for efficacy. Soc. Light Treatment Biol. Rhythms Newsletter 1 , 4.

2 1 . T e r m a n M . , T e r m a n J .S. , Q u i t k i n F . M . , M c G r a t h P.J . , S tewar t J .W. a n d Rafferty B . (1989) Light t h e r a p y for seasonal affective d i so rder : A review of efficacy. Neuropsycho-pharmacology 2 , 1-22.

22. Wi l l i ams J .B .W. , L i n k M.J . , R o s e n t h a l N . E . a n d T e r m a n M . (1988) Structured Interview Guide for the Hamilton Depression Rating Scale—Seasonal Affective Disorder Version ( S I G H - S A D ) . N Y Sta te Psych ia t r i c Ins t i tu te , N e w Y o r k .

23 . W i n e r B.J. (1971) Statistical Principles in Experimental Design. M c G r a w - H i l l , N e w Y o r k .

24. Wirz-Jus t ice Α., Bucheli C , G r a w P . , Kie lholz P . , F isch H . U . a n d W o g g o n B. (1986) Light t r e a t m e n t of seasona l affective d i so rde r in Swi tzer land . Acta Psychiatr. Scand. 74 , 193-204.

25 . Wirz-Jus t ice Α., G r a w P . , K r a u c h i K. , A r e n d t J., A l d h o u s M . , Gis in B. , J o c h u m A. a n d Po ld inge r W . (1989) M o s t S A D pa t i en t s a re p h a s e delayed in win ter b u t r e s p o n d equal ly t o m o r n i n g o r evening l ight. Soc. Light Treatment Biol. Rhythms Abst. 1 , 19.

27

Effect of Light on Unmasked Circadian Rhythms in Winter Depression

A N N A W I R Z - J U S T I C E , PETER G R A W , K U R T K R À U C H I , H A N S - J O A C H I M H A U G , G E O R G L E O N H A R D T a n d D A N I E L P. B R U N N E R

Psychiatric University Clinic, CH-4025 Basel, Switzerland

Abstract

N i n e w o m e n wi th win ter depress ion carr ied ou t a " c o n s t a n t r o u t i n e " p r o t o c o l of 40 h o u r s sus ta ined wakefulness to m e a s u r e e n d o g e n o u s c i rcad ian r h y t h m s (rectal t e m p e r a t u r e , m o o d , a ler tness , pe r fo rmance) . Bright light given in the midd le of the d a y reduced depressive s y m p t o m s a n d i m p r o v e d m o o d , as did the sleep dep r iva t i on of the c o n s t a n t rou t ine p r o t o c o l itself. M o o d a n d aler tness showed a b i m o d a l r h y t h m u n d e r u n m a s k e d cond i t i ons , wi th b o t h a n o c t u r n a l a n d af te rnoon t r o u g h . Light did no t modify the e n d o g e n o u s r h y t h m of a ler tness b u t d id i m p r o v e a s imple pe r fo rmance task a t all c i rcad ian phases b o t h in winter a n d in s u m m e r .

In t roduct ion

SEASONAL AFFECTIVE D I S O R D E R (SAD), winter pa t te rn , is a syndrome of

recurrent depressive phases in a u t u m n and winter often associated with increased appeti te , ca rbohydra te craving, weight gain, and hypersomnia , which improves spontaneously in spring and s u m m e r .

2'

24 Al though a

consensus has been reached within the field tha t bright light is the therapy of choice for S A D ,

25 there is little consensus as to mechanisms underlying

the rapid ant idepressant effect.

6

Two major hypotheses for the pathophysiology of S A D involve the circadian system: tha t SAD arises as a consequence of abnormal ly phase-delayed circadian r h y t h m s

22 or of diminished circadian a m p l i t u d e .

14

Light is therefore ant idepressant because of its phase-advancing propert ies (when given in the morning) or its ability to enhance ampl i tude (when given in the d a y t i m e ) .

21 Testing these circadian hypotheses requires more

stringent experimental me thods than usual in clinical trials, since under

3 8 5


nychthemeral condit ions circadian rhy thms are masked by sleep, mo to r activity, meals , t empera ture and variable light e x p o s u r e .

1 1'

13 Thus

measurement of phase and ampl i tude , for example of core body tempera ture in SAD, have yielded equivocal results. Only one study has yet investigated endogenous circadian rhy thms using a 27-hour "constant rout ine" unmasking protocol . SAD patients were found to have phase-delayed circadian rhy thms in core body tempera ture , melatonin, T S H and Cortisol, which were advanced by therapeutic morn ing light.

3'

4

In an ongoing study, we are investigating the effect of light on ampl i tude and phase of circadian rhy thms in S A D in more detail. Pi lot studies were set up in a u t u m n 1989 and regular investigation began in a u t u m n 1990. W o m e n diagnosed as SAD were studied in winter, at the beginning of their depressive phase , and in summer when euthymic, as were age-matched controls . When possible, four separate 40-hour constant rout ine (CR) protocols were carried out : in winter and in summer , before and after 5 days t rea tment with 6,000 lux light from 10-14 hours , a time predicted to augment circadian a m p l i t u d e .

21 As of summer 1992, nine SAD patients

have been studied in winter; six SAD patients and four controls have completed the seasonally paired protocol .

There is as yet insufficient da ta to test the main hypothesis of whether circadian rhy thms in S A D pat ients are phase delayed and /o r diminished in ampl i tude, or whether midday light t rea tment can modify phase or ampli tude of the core body tempera ture circadian rhy thm. However , some of the effects of light appear not to be related to winter depression per se. We present three pa rame te r s—mood , alertness, and performance—in order to illustrate the effects of light t rea tment on unmasked circadian rhythms in SAD. This is a first, prel iminary presentat ion of our CR data , and is intended as a descriptive ra ther than analytic or exhaustive summary .

Exper imenta l procedure

SAD patients (27-66 years) fulfilled the criteria for winter d e p r e s s i o n .

24

They were studied in the follicular phase of the menstrual cycle, if applicable. The constant rout ine protocol as developed by Czeisler (details i n

1 3) was carried out in a r o o m controlled for tempera ture (22°C),

humidi ty (67%) and light ( < 8 0 lux). Subjects remained awake in semi-recumbent pos ture for 40 hours , with isocaloric meals every 2 hours . Rectal tempera ture , peripheral skin tempera ture (10 posit ions) and heart ra te were measured on line. Self-rating of m o o d , alertness, tension and hunger were made at half-hourly intervals with linear nonnumer ic 100-m m bipolar Visual Analogue Scales (VAS),

1 together with collection of

saliva for assay of melatonin

16 and Cortisol. Performance was assessed at

2-hour intervals with a shortened form of a validated letter-cancellation

Effect of Light on Unmasked Circadian Rhythms in Winter Depression 387

test for concent ra t ion/a t ten t ion (d2), which measures the speed and precision of discr iminatory perception of similar visual s t imul i .

7

Results

Light administered in the middle of the day was an effective t rea tment for SAD patients in winter (N=9). The observer rat ing of depression (Hamil ton Rat ing S c a l e

1 8) decreased from 14.0 ± 5.5 [ S D ] to 2.9 ± 1.4, the

Atypical I tems s c o r e

23 from 7.7 ± 3.4 to 1.1 ± 1 . 3 , depression se l f - ra t ings

26

from 16.0 + 6.2 to 8.8 ± 3 . 3 . A pa radox of the CR protocol is that in order to unmask circadian rhy thms, subjects must undergo a complete sleep deprivat ion. Sleep deprivat ion itself can be an ant idepressant intervention, and indeed, all SAD patients improved on day 2 of the protocol , as can be seen in the m o o d self-ratings. This "unwan ted" effect in terms of changed clinical state may thus have int roduced a new source of masking.

Mood

O u r SAD pat ient collective has previously been characterized by diurnal variat ion of m o o d dur ing depression that persists, albeit dampened , when successfully treated with light, and is present also in summer (measured with VAS in the morn ing upon awakening and in the evening before r e t i r i n g .

1 7 , 19 The C R provides an oppor tun i ty to follow the endogenous

rhy thm of m o o d with great precision.

The time course of half-hourly m o o d ratings is presented in Figure 1. Depressed S A D pat ients in winter began the CR with low morn ing m o o d which improved in the afternoon and declined again at night. The sleep deprivat ion response could be seen after 6 a.m. Around midday, m o o d was higher on day 2 than day 1, but was followed by an afternoon s lump, with m o o d improvement again in the evening. Thus , under the unmask ing condit ions of the CR, diurnal variat ion of m o o d reveals itself to be a complex phenomenon . N o t only is there a circadian componen t of low mood , but sleep deprivat ion also "unmasks" a second 12-hour componen t .

After light t rea tment a b imodal rhy thm of m o o d was also found. The t iming and extent of the afternoon dip was identical on days 1 and 2. SAD patients dur ing depression (day 1) appeared to have an inverse diurnal rhy thm of m o o d . O n day 2 after response to sleep deprivat ion, the afternoon dip was identical in t iming to that after light t rea tment . This indicates that sleep deprivat ion induced a normal , non-depressed diurnal m o o d pa t te rn . The nocturna l t rough of m o o d lasted from ca. 9 p .m. to 6 a.m. before and from ca. 1 a.m. to 10 a.m. after light t rea tment .

When the smaller g roup of SAD patients studied bo th winter and summer were compared (7V=6, da t a not shown) , m o o d was increased by light t rea tment in winter to the same level as found in bo th CRs in summer


60

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ο

CO

< >

After light 25

F I G . 1. E n d o g e n o u s c i rcad ian r h y t h m of half-hourly self-ratings of m o o d ( V A S : 0 m m = extremely depressed m o o d ; 100 m m = extremely g o o d m o o d ) in S A D pa t ien t s in winter (N=9). 2-way A N O V A for repea ted measu re s using 2 -hour m e a n s revealed a significant effect of l ight ( F ( 1 > 8) = 5.6, ρ = 0.05), a significant t ime

course ( F ( 17 1 3 6) = 3.2, ρ<0.001), a n d in te rac t ion n.s .

(three-way A N O V A [season, before and after light, t ime course] interact ion F(1 5) = 9.0, ρ = 0.03). Light t rea tment in summer had no significant m o o d augment ing effect. VAS m o o d ratings were averaged across the CR (N=T4 scores/person) and then across individuals (N=6). This mean m o o d score (in m m + SD) illustrated the mood-normal iz ing effect of light t rea tment in winter: winter before light: 38 ± 7 ; after light: 54 + 14; summer before light: 53 + 18; after light: 53 + 2 1 .

Alertness

The circadian rhy thm of alertness in depressed SAD pat ients in winter also showed a b imodal pa t te rn : a slight midday dip in alertness was present on day 1 and accentuated on day 2, with a clear circadian t rough from ca. 8 p .m. to 6 a.m. In contras t to m o o d , which improved after sleep deprivat ion, alertness declined (values on day 2 were lower than the equivalent values on day 1). Light t rea tment had no effect on any aspect of the alertness rhy thm (Figure 2).

When the smaller g roup of SAD patients studied bo th winter and summer were compared (N=6, d a t a no t shown) , the circadian rhy thm of alertness in all four CRs was similar. Thus surprisingly, light t rea tment did not modify the endogenous circadian rhy thm of alertness in winter no r in summer , no r was there a difference between winter or summer . VAS alertness ratings were averaged across the C R (N=14 scores/person) and then across individuals (N=6). M e a n alertness scores (in m m + SD) were in winter before light: 33 + 15; after light: 32 + 7; summer before light: 38 + 7; after light: 35 ± 9 .


55

F I G . 2. E n d o g e n o u s c i rcad ian r h y t h m of half -hourly self-ratings of a ler tness (VAS: 0 m m = extremely t i red; 100 m m = ext remely aler t ) in S A D pa t ien t s in winter (N=9). A N O V A for r epea ted measu re s us ing 2 -hour m e a n s revealed a significant t ime course (F{11 1 3 6) = 9.2, ρ < 0.001), b u t n o significant effect of light

o r in te rac t ion t e rm.

Performance

A learning effect (decline in number of errors) was found in the first four tests of the first CR. The n u m b e r of errors thereafter remained cons tant . The quant i ta t ive performance (total number of letters cancelled minus errors) followed a significant circadian rhy thm. The rhy thm of perform-ance in S A D pat ients in winter was low in ampl i tude , with a wide t rough from ca. 2 a .m.-8 a.m. O n the afternoon following sleep deprivat ion, performance rose to similar values as on day 1, unlike alertness, which declined. After light t rea tment , performance was markedly increased at all times of day, as was the ampl i tude of the rhy thm. The wave form also changed, with only a na r row t rough of low performance a r o u n d 6 a.m. Fol lowing sleep deprivat ion, performance rose later, after 4 p.m., to similar values as on day 1. The 2-hourly interval of testing did no t reveal any mid-afternoon dip in performance (Figure 3).

When the smaller g roup of SAD patients studied bo th winter and summer were compared {N=5, d a t a no t shown) , there was n o seasonal difference in performance levels. However the dura t ion of the noc turna l t rough in summer was shorter than tha t in winter (interaction term F ( 1 8 7 2) = 2 . 1 , p < 0 . 0 1 ) . Light t rea tment increased performance to the same extent in winter and summer . The dura t ion of the t rough after light was decreased compared with before light. Performance was averaged across the C R (N=19 tests/person) and then across individuals (N=5). This mean score ( ± S D ) illustrates the performance-enhancing effect of light t rea tment independent of season: winter before light: 176 + 38; after light: 200 + 42; summer before light: 168 + 28; after light: 196 + 31 .


22a

F I G . 3. E n d o g e n o u s c i rcad ian r h y t h m of pe r fo rmance in a s imple letter cancel la t ion test (d2) in S A D pa t i en t s in winter (N=9). A N O V A for r epea ted measu res revealed a significant effect of l ight (F{1 8) = 61 .5 , ρ < 0.001), a significant t ime course ( F ( i 81 4 4) = 6.6, ρ<0.001), a n d a significant in te rac t ion (F(i8144) =

1 . 7 , p < 0 . 0 5 ) .

Temperature

The rectal t empera ture da t a have not yet been subjected to the appropr ia te wave-form fitting and statistic analyses. A g roup mean of the unfitted raw da ta (not shown) showed min imum tempera ture to be a r o u n d 5 a.m. Analysis by two-way A N O V A (using 2-hour mean values as for the VAS) revealed a highly significant t ime course C F ( i7, i 3 6 )

= 33.9, p < 0 . 0 0 0 1 ) but

no effect of midday light t rea tment (interaction NS) .

Conclusions

These prel iminary da ta exemplify the complex interactions between clinical state, season, and the biological effects of light on unmasked circadian rhy thms in SAD pat ients . In the nine SAD patients studied in winter, midday light exposit ion for 5 days effectively reduced depressive symptoms. However , the circadian rhy thms of core body tempera ture , alertness, and salivary mela tonin (data no t shown, collaborat ive study with J. Arendt and J. English, University of Surrey) were no t significantly modified by light. Whether the t iming of the tempera ture min imum a round 5 a.m. is phase delayed, or the ampl i tude diminished, requires future compar ison with age-matched control women. However the clinical improvement tha t occurred was no t associated with any apparen t phase-shift or augmenta t ion of ampl i tude of three major circadian rhythms driven by the endogenous pacemaker .

M o o d was clearly modified by sleep deprivat ion, light and season. O u r da t a suppor t o ther unpubl ished observat ions (T. A. Wehr , D . Avery) that


SAD patients , as other sub-groups of depressed pat ients , respond to sleep deprivat ion and relapse after recovery sleep. The mood-elevat ing effect of sleep deprivat ion was, however, less profound and less long-lasting than after light.

Diurna l var ia t ion of m o o d dur ing depression could be clearly followed in the cons tant rout ine pro tocol and revealed that the endogenous m o o d rhy thm was b imodal . The extent of this diurnal variat ion was enhanced dur ing the day following sleep depr ivat ion, bu t reduced after light t rea tment and in summer . This replicates our previous finding of a reduced morning-evening m o o d difference after light t rea tment and in s u m m e r .

19

The t iming of the afternoon dip in m o o d dur ing euthymia was parallel to the afternoon dip in alertness. This latter was small bu t increased after sleep deprivat ion. There was no significant afternoon dip in alertness in summer . N o afternoon dip in alertness before or after the sleep deprivat ion of the constant rout ine has been found in heal thy young m e n .

8 , 15

An impor t an t finding was that light could increase performance independent of clinical state or season. Light and summer also modified the wave form of the circadian rhy thm of performance, reducing the dura t ion of the t rough of low performance. Previous studies have shown that light can improve performance in bo th heal thy c o n t r o l s ,

5 , 9 , 1 0'

1 2,

Grunberger et al, unpubl ished da ta ) and severely depressed non-seasonal pat ients , independent of clinical s t a t e .

20 We now show tha t this

augmenta t ion occurs at all circadian phases , is not associated with a phase shift, and is independent of season. It is of par t icular interest tha t an increase of performance th roughou t the entire 24 hours could be at ta ined by increasing light intensity only dur ing the dayt ime. A practical consequence of this would be daylight augmenta t ion of light intensity to improve noc turna l performance wi thout necessarily inducing phase shifts.

It is difficult to m a k e compar isons of these findings with previous studies in SAD pat ients , since most have administered light in the morn ing and /o r evening, and no t over midday , as well as having been carried out under the usual masked condi t ions . Whether S A D is a circadian rhy thm disorder , as hypothesized, is still unclear, until complet ion of the study and compar i son with age-matched controls . However , these prel iminary results suggest tha t the ant idepressant effect of midday light is not necessarily media ted by a phase-advance nor by ampl i tude enhancement .


O u r studies were designed and initiated in col laborat ion with J. Anderson (supported by an N S F exchange fellowship S N F #83NI-028030) . We thank J. Duffy and C A . Czeisler, also from the Labora to ry for Circadian and Sleep Disorders Medicine, H a r v a r d Medical School, for advice in setting up the C R protocol ; G. Mol l , C. Hetsch, P . van der Velde,


A. Sarrafzadeh, J. Schulman, T. Bruhl, E. Weber and E. Holsboer-Trachsler for collaborat ive suppor t ; the s tudents who worked the shifts and the subjects who volunteered. This study was suppor ted by S N F G r a n t #32-28741.90, the Hor t en Founda t i on , and the Roche Research F o u n d a t i o n (DB).

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13. Czeisler C.A., K r o n a u e r R.E. , Al lan J .S . , Duffy J . F . , Jewet t M . E . , B r o w n E . N . a n d R o n d a J . M . (1989) Br ight light i nduc t ion of s t rong (type 0) reset t ing of the h u m a n c i rcadian p a c e m a k e r . Science 2 4 4 , 1328-1333 .

14. Czeisler C.A., K r o n a u e r R.E. , M o o n e y J.J. , A n d e r s o n J .L . a n d Allan J .S . (1987) Biologic r h y t h m d i so rde r s , depress ion , a n d p h o t o t h e r a p y : a new hypo thes i s . Psy. Clin. N. Amer. 10 , 687 -709 .

15. Dijk D.-J . , Duffy J . F . a n d Czeisler C.A. (1992) C i r cad i an a n d s leep /wake d e p e n d e n t aspects of subjective a ler tness a n d cogni t ive pe r fo rmance . / . Sleep Res. 1 , 112-117 .

16. Engl i sh J., M i d d l e t o n B.A., A r e n d t J. a n d Wirz-Jus t ice A. (1993) R a p i d direct m e a s u r e m e n t of m e l a t o n i n in saliva us ing a n iod ina ted t racer a n d solid phase second a n t i b o d y . Ann. Clin. Biochem. 3 0 , 4 1 5 - 4 1 6 .

17. G r a w P . , K r â u c h i Κ. , Wirz-Jus t ice A. a n d Pô ld inge r W . (1991) D i u r n a l va r ia t ion of s y m p t o m s in seasona l affective d i sorder . Psychiatry Res. 3 7 , 1 0 5 - 1 1 1 .

18. H a m i l t o n M . (1967) D e v e l o p m e n t of a ra t ing scale for p r i m a r y depress ive illness. Br. J. Soc. Clin. Psychol 6 , 2 7 8 - 2 9 6 .

19. H a u g H.-J . , G r a w P . , H a u g M . a n d Wirz-Jus t ice A. (1991) C o u r s e of d iu rna l va r i a t ion of m o o d in pa t i en t s wi th " seasona l affective d i so rde r " (SAD) . Abs t r ac t , VII Annual Meeting of the European Society for Chronobiology, M a r b u r g .

20. Ho l sboe r -Trachs l e r E. , S tohler R., Mùl le r J., G e r h a r d U . a n d H o b i V. (1991) Cogni t ive


a n d p s y c h o m o t o r pe r fo rmance d u r i n g sleep dep r iva t ion a n d light t he r apy . Biol. Psychiatry. 29 , 194S.

2 1 . Jewet t M . E . , K r o n a u e r R .E . a n d Czeisler C A . (1991) L igh t - induced suppress ion of e n d o g e n o u s c i rcad ian a m p l i t u d e in h u m a n s . Nature 350, 5 9 - 6 2 .

22. Lewy Α., Sack R., Singer C . a n d W h i t e D . (1987) T h e p h a s e shift hypo thes i s for b r igh t l ight 's t he rapeu t i c m e c h a n i s m of ac t ion : theore t ica l cons ide ra t ions a n d exper imen ta l evidence. Psychopharmacol. Bull. 23 , 3 4 9 - 3 5 3 .

23 . R o s e n t h a l N . E . a n d Heffernan M . M . (1986) Bul imia , c a r b o h y d r a t e c rav ing a n d depress ion : a cent ra l c o n n e c t i o n ? In Nutrition and the Brain (eds. W u r t m a n R.J . a n d W u r t m a n J .J . ) , Vol . 7, p p . 139-166 . R a v e n Press , N e w Y o r k .

24. R o s e n t h a l N . E . , Sack D.A. , Gil l in J . C , Lewy A.J. , G o o d w i n F . K . , D a v e n p o r t Y., Muel le r P .S . , N e w s o m e D .A . a n d W e h r T.A. (1984) Seasona l affective d i so rder : a descr ip t ion of the s y n d r o m e a n d p re l iminary findings wi th l ight t he r apy . Arch. Gen. Psychiatry. 4 1 , 7 2 - 8 0 .

25 . Society of L ight T r e a t m e n t a n d Biological R h y t h m s . (1990) C o n s e n s u s s t a t e m e n t o n the efficacy of light t r e a t m e n t for S A D . LTBR Bulletin 3 , 5 -9 .

26. Zerssen D.v . a n d Koe l le r D . M . (1976) Pa r ano id -Depre s s iv i t â t s - ska l a . Beltz Testgesel ls-chaft, W e i n h e i m .

28

Light Treatment versus Drug Treatment in Winter Depression O. L I N G J / E R D E , T. R E I C H B O R N - K J E N N E R U D , A . H A G G A G , Ε. M . B E R G , K. N A R U D a n d I. G A R T N E R

Gaustad Hospital, Oslo, Norway

N U M E R O U S STUDIES seem to prove beyond reasonable doub t that light is an effective t rea tment for winter depression. This has been demons t ra ted also in controlled studies, bu t no t in truly double blind studies. In fact, since it is literally impossible to study the effect of light t rea tment under double blind condi t ions , it is still uncertain how much of the observed improvement after light t rea tment must be ascribed to the pat ient 's expectat ion of effect, and how much to a true therapeut ic , physiological effect of light—if indeed these two aspects can be clearly separated!

It is easier to perform a true double blind t rea tment study with drugs , a l though marked side effects of a d rug sometimes can m a k e the blindness illusory in at least some pat ients . Therefore, besides being of interest in their own right since drugs are an al ternative to light t rea tment , d rug t rea tment studies in winter depression can possibly contr ibute to an elucidation of placebo responsiveness in persons suffering from Seasonal Affective Disorder . It may therefore be of interest at this meet ing t o present some relevant findings from a study we carried out in Oslo last winter, compar ing light t rea tment , the selective M A O - A inhibi tor moclobemide (Aurorix "Roche") , and placebo. First , however , we will give a short survey of other d rug studies in winter depression.

In general, there have been surprisingly few such studies, and most of them have been uncontrol led.

As to convent ional tricyclic ant idepressants (TCA), we are no t aware of any systematic study in winter depression, but anecdota l repor ts seem to indicate that such drugs often have an unsatisfactory effect, or give too many side effects.

Since T C A seems to be less effective in winter depression than in

3 9 5


endogenous depression—which is in itself an interesting observat ion— several newer and unconvent ional drugs have been tried. The beta-adrenergic blocker atenolol was found to be without effect in a small cross-over study by Rosenthal and coworker s .

9 But other drugs like

L - t r y p t o p h a n ,

6 fenfluramine,

8 a l p r a z o l a m ,

10 t r any lcypromine ,

2 and

b u p r o p i o n

3 have all been repor ted to have more or less effect, a l though

most often in uncontrol led studies. In a small, likewise uncontrol led study, Lingjaerde and H a g g a g

4 observed a rapid effect of moclobemide in five

women with winter depression. This p rompted us to perform the following study from November 1991 to April 1992.

5 Details of the study will be

published elsewhere.

A total of 79 outpat ients with winter-SAD or subsyndromal winter-SAD, recruited mainly by newspaper advertising, were included. O u r plan was to allocate the pat ients r andomly to light t rea tment and coded drugs . However , there was a surprisingly high number of patients who would only accept light t rea tment . We chose to place these pat ients non-randomly in the light g roup , instead of excluding them from the study. This, besides the fact that the compar i son between drug and light could not be m a d e double blind, means that compar ison between the results in the light t rea tment g roup and the d rug groups has to be m a d e with due reservations. However , the compar i son of the effects of moclobemide (400 mg daily) and placebo is unbiased for the first 3 weeks (the more so since moclobemide gave no more side effects than did placebo). F o r ethical reasons, nonresponders after 3 weeks on coded drugs (less than 2 0 % reduct ion in total score on the extended Montgomery-Asberg Depression Rat ing Scale, M A D R S

7) were changed to open moclobemide. Therefore,

in the following we will ony present results from the first 3 weeks after the t rea tment started.

F o r light t rea tment , we used the only type of light equipment that was easily obta inable and in some use in N o r w a y at that t ime, with fluorescent white light (Truelite) giving a light intensity of 1,500 lux at eye level. This is less than usually recommended, but we considered it of great interest, bo th practically and theoretically, to see whether this light intensity—which, as far as we know, has never been tested before as t rea tment for SAD—is also effective in winter depression. Light t rea tment was administered for 2 hours in the morn ing—but not earlier than from 7 a.m.—for 6 consecutive days.

O u r main ins t rument for assessment of therapeut ic ou tcome was the M A D R S , extended with ano ther nine symptoms often seen in SAD, most of them taken from the Comprehensive Psychopathological Rat ing Scale ( C P R S

1) (Increased sleep, Increased appeti te , Ca rbohydra te craving,

Reduced sexual feelings, Hypochondr ia , Get t ing easily tired, Reduced speech, M o t o r re tardat ion and M o t o r agi tat ion).

Assessments with extended M A D R S , Clinical Global Impression, and a

Light Treatment versus Drug Treatment 397

side effect checklist were performed at baseline, and after 1, 3, 6, 10 and possibly 14 and 18 weeks.

Five pat ients d ropped out after less than 1 week of t rea tment , leaving 74 pat ients for analysis. Some of their main characteristics are shown in Table 1.

Table 2 shows the extended M A D R S scores in the three t rea tment groups dur ing the first 3 weeks of the study.

T A B L E 1

Main characteristics of treatment groups

Light P l a c e b o M o c l o b e m i d e

Ν 31 18 16 Male / female 4/27 5/13 4/12 Age, m e a n 42.9 43.2 43.0 Age, r ange 2 4 - 6 3 2 0 - 6 2 2 0 - 6 5 M a j o r depress ion , p resen t ep isode 18 13 13 M a j o r depress ion , wors t ep isode 28 15 12 Age 1st W D , m e a n 23.2 26.1 23.1 Age 1st W D , r ange 5 -50 12-45 11-38 N o . W D , m e a n 16.9 16.5 15.9 N o . W D , r ange 4^59 + 4 5 3 -30

T A B L E 2

Mean total score on extended MADRS at baseline and after 1 and 3 weeks. SD in parenthesis

Light M o c l o b e m i d e P l a c e b o

Basel ine: Ν 31 16 18 Ex t . M A D R S 31.5 (10.1) 38.1 (9.3) 32.3 (7.9)

O n e week: Ν 31 16 18 Ext . M A D R S 16.7 (11.8) 31.3 (13.2) 26.8 (7.7)

T h r e e weeks : Ν 27 16 16 Ext . M A D R S 12.9 (11.5) 23.9 (13.7) 21.4 (11.3)

The main findings to be read from this table are the following: (1) Light t rea tment is reasonably effective, with marked improvement after 1 week (immediately after the end of t rea tment) , a n d — w h a t is perhaps more no tewor thy—the improvement is even more marked 2 weeks afterwards. (In fact, mos t pat ients main ta ined the improvement for the rest of the season.) (2) Improvement in the drug groups is inferior to the improve-ment on light, and with little difference between moclobemide and placebo. In fact, to our d i sappoin tment there were abou t 5 0 % non-responders in bo th groups after 3 weeks. However , after having changed from coded tablets to open moclobemide , most of these pat ients also showed a rapid improvement (not shown) .


Since abou t half of the pat ients on placebo showed marked improve-ment after 3 weeks, we tried to identify possible predictors of placebo response, and we found one factor explaining a surprisingly high percentage of the response variance, namely the pat ients ' age.

T A B L E 3

Improvement after 3 weeks (per cent reduction of total extended MADRS score) in relation to age. r = linear

regression coefficient

Light M o c l o b e m i d e P l acebo

Ν 26 16 15 r - 0 . 0 5 - 0 . 4 8 - 0 . 7 6 Ρ 0.80 0.06 0.001

As seen from Table 3, regression of per cent improvement after 3 weeks on age shows a highly significant negative correlat ion, with a linear regression coefficient as high as —0.76 and ρ = 0.001, explaining more than half of the variance. There is a non-significant tendency in the same direction in the moclobemide group , bu t not in the light t rea tment g roup .

The negative correlat ion between per cent improvement and age might be due to the older pat ients being more severely ill, but this was not the case. Nei ther was there a significant correlat ion between improvement and total illness dura t ion , or number of winter depression periods.

Due to the part icularly marked placebo effect in younger pat ients , we compared the effect of moclobemide and placebo separately for pat ients below and above the median age of 45 years. N o difference was found below 45 years, whereas the difference was barely significant (p < 0.05, one-sided test) in pat ients above 45 years.

Since to our knowledge such a negative correlat ion between age and placebo improvement has never been found in non-seasonal depression, the quest ion arises whether this is characteristic of patients with SAD. Is it possible that the pathophysiology of SAD, including whatever mech-anisms are mediat ing the placebo response, is different for young and somewhat older SAD patients?

As to placebo response on light t rea tment , the lack of age effect would seem to argue against the improvement being a pure placebo effect—at least in patients of middle or higher age. At any rate, our findings should p romp t anyone doing t rea tment studies in SAD to look for possible effects of age.

References

1. Âsberg M. , Per r i s C , Schal l ing D . a n d Sedvall G . (1978) T h e C P R S — D e v e l o p m e n t a n d app l ica t ion of a psychia t r ic ra t ing scale. Acta Psychiatr. Scand. S u p p l . 2 7 1 .

Light Treatment versus Drug Treatment 399

2. Di lsaver S . C , De l M e d i c o V.J. , Q u a d r i A. a n d Jaeckle R .S . (1990) P h a r m a c o l o g i c a l responsiveness of win ter depress ion . Psychopharmacol. Bull. 2 6 , 303 -309 .

3. Di l saver S . C , Q u a m a r A .B . a n d Del M e d i c o V.J . (1992) T h e efficacy of b u p r o p i o n in winter depress ion : resul ts of a n o p e n tr ial . / . Clin. Psychiatry 5 3 , 2 5 2 - 2 5 5 .

4. Lingjaerde O . a n d H a g g a g A. (1992) M o c l o b e m i d e in win ter depress ion : s o m e resul ts from a n o p e n tr ia l . Nord. J. Psychatry 4 6 , 2 0 1 - 2 0 3 .

5. Lingjaerde O . , H a g g a g Α., R e i c h b o r n - K j e n n e r u d T. , Berg E . M . , N a r u d K . a n d G a r t n e r I. (1992) T r e a t m e n t of winter depress ion : a c o m p a r a t i v e tr ial wi th b r igh t l ight , m o c l o b e m i d e , a n d p l acebo . Clin. Neuropharmacol. 15 , Supp l . 1, P a r t Β , 183B.

6. M c G r a t h R.E. , Buckwa ld B. a n d Resnick E.V. (1990) T h e effect of / - t r y p t o p h a n o n seasonal affective d i sorder . / . Clin. Psychiatry 5 1 , 162 -163 .

7. M o n t g o m e r y S.A. a n d Âsbe rg M . (1979) A new depress ion r a t ing scale des igned to be sensitive to change . Brit. J. Psychiatry 134 , 382 -389 .

8. O ' R o u r k e D . , W u r t m a n J.J. , W u r t m a n R.J . , Cheb l i R. a n d G l e a s o n R. (1989) T r e a t m e n t of seasonal depress ion wi th d-fenfluramine. J. Clin. Psychiatry 5 0 , 3 4 3 - 3 4 7 .

9. R o s e n t h a l N . E . , J a c o b s e n F . M . , Sack D.A. , A r e n d t J., J a m e s S.P., P a r r y B.L. a n d W e h r T.A. (1988) Ateno lo l in seasonal affective d i so rder : A test of the m e l a t o n i n hypo thes i s . Amer. J. Pschiatry 145 , 5 2 - 5 6 .

10. Teicher M . H . a n d G l o d C.A. (1990) Seasona l affective d i so rde r : r ap id reso lu t ion by low-dose a l p r a z o l a m . Psychopharmacol. Bull. 2 6 , 197-202 .

29

Light Therapy of Premenstrual Depression BARBARA L P A R R Y

Department of Psychiatry, UCSD, 0804, La Jolla, CA 92093, USA

Abst rac t

T h e p u r p o s e of this s tudy was to test the efficacy of b r igh t l ight t r e a t m e n t in pa t i en t s wi th p r e m e n s t r u a l depress ion . In a 3 m o n t h c rossover des ign, we admin i s t e red br igh t ( > 2,500 lux) whi te m o r n i n g , br igh t whi te evening, a n d p lacebo d im ( < 10 lux) red evening l ight, dai ly for 1 week of the p r e m e n s t r u a l p h a s e to 19 pa t i en t s wi th la te luteal phase d y s p h o r i c d i so rde r ( L L P D D ) a n d to 11 n o r m a l con t ro l s . In con t r a s t to o u r p rev ious pi lot s tudy in this a rea , all l ight t r e a t m e n t s significantly reduced depress ive ra t ings from basel ine . N o n e of the light t r e a t m e n t s differed significantly from each o the r . T h u s , ne i ther m o r n i n g or evening br igh t light were s h o w n to have inc rementa l benefits over p l acebo in this s tudy of pa t i en t s wi th p r emens t rua l m o o d d i so rders .

B R I G H T LIGHT has been used to treat seasonal affective d isorder ,

1 some

patients with major depressive d i sorders ,

2 seasonal premenst rua l

s y n d r o m e ,

3 and most recently nonseasonal premenst rual s y n d r o m e .

4 In

our pilot s t udy ,

4 we repor ted greater effectiveness of evening, compared

with morn ing bright light in the t rea tment of six pat ients with prospectively documented premenst rua l depression. T o extend our prel iminary investigation, in this s tudy we repor t on effects of light t rea tment in an addi t ional 19 pat ients with late luteal phase dysphor ic disorder and in 11 normal controls . In our initial pilot s tudy, morn ing bright light t rea tment , hypothesized to exacerbate symptoms by phase-advancing already pathologically advanced circadian rhy thms in pat ients with premenst rual depression, was intended to serve as an internal control . In that study, depression rat ings, a l though not statistically different from ratings obta ined at baseline (before t rea tment) , did decrease after morn ing light t rea tment . Thus , morn ing bright light did not serve as a sufficient control condi t ion. In the present study, we added an addi t ional placebo control condi t ion: dim red evening light. In this s tudy, we repor t the effects on m o o d of morn ing bright white, evening bright white, and evening dim

401


red light administered in a crossover design to pat ients with L L P D D depression and to normal control subjects.

Subjects and methods

The pat ients were referred by local professionals or were recruited by advert isement. Screening procedures , inclusions and exclusion criteria have been described previously (4). Subject characteristics are described in Table 1.

T A B L E 1

Reported mood, behavior or physical symptoms for PMS (N=19) and normal controls (N=ll)

M o o d , behav io r o r % Modera t e - seve re physical change P M S N C

Appe t i t e 66.7 0 Anx ious or j i t te ry 66.7 0 Increase in acc idents 27.8 0 T h o u g h t s of h u r t i n g self 16.7 0 Difficult to enjoy self 64.7 0 I r r i tab le 83.3 0 Difficulty c o n c e n t r a t i n g / m a k i n g 68.4 9.1

decis ions Feel sad /c ry 77.8 0 Feel guil ty 38.9 0 Decrease in efficiency 55.6 0 C r a v e specific foods 72.2 9.1 Excessive sleeping 27.8 0 Decrease in sexual dr ive 38.9 0 Loss of energy o r fatigue 77.8 0 Seasona l change in energy level 21.1 0 Seasona l change in m o o d 26.3 0 Exper ienced p o s t p a r t u m depress ion 57.1 0

ρ < 0.05 for all var iables l isted.

Subjects selected for the study underwent , in r a n d o m order , a crossover trial of bright ( > 2 , 5 0 0 lux) white morn ing light (6.30 a .m.-8.30 a.m.), bright white evening light (7.00 p .m.-9 .00 p.m.) , or placebo dim ( < 10 lux) red evening light t rea tment . Each t rea tment was administered for 7 consecutive days dur ing the luteal phase of a separate menst rual cycle (7-10 days before the onset of menses). We administered the dim red light placebo in the evening to contras t with the evening bright light, which our prel iminary study had indicated was more effective than morn ing light in alleviating P M S symptoms .

Mens t rua l cycle phase was determined by using a colorimetric ur inary immunoassay (Ovustick C o m p a n y , Irvine, CA) for the midcycle luteiniz-ing h o r m o n e surge. Pat ients were asked to sit 3 feet from the light source. The bright light was a por table i l lumination box from Apollo Light which

Light Therapy of Premenstrual Depression 403

produced 2,500 lux at 3 feet by cool white fluorescent light bulbs . The dim light was one fluorescent bu lb behind a red filter encased in the same size box producing 10 lux at 3 feet. Subjects were asked to keep their eyes open and to gaze every few minutes at the light. Dur ing each m o n t h of s tudy, pat ients were asked to mainta in their normal sleep pat terns , to take no naps , and to document their sleep times on daily sleep logs. They also were asked to wear da rk sunglasses or goggles to block out light when ou tdoors between 6:30 a.m. and 8:30 a.m. when they were being treated with evening light, or between 7:00 p.m. and 9:00 p .m. when they were being treated with morn ing light, in order to balance the a m o u n t of light exposure on bo th t rea tment condi t ions. Subjects also completed daily light logs, expectat ion forms (100 m m visual analog scales from "much better than usual" to "much worse than usual" for each light t rea tment condi t ion administered at the beginning of the study), and Horne-Os tbe rg r a t i n g s

10

for morningness and eveningness.

At the end of 7 days of light t rea tment in the luteal phase , m o o d over the past several days was assessed by two raters , who were blind to the t rea tment condi t ion, using the Hami l ton depression scale, using an a d d e n d u m to the Hami l ton scale that assessed atypical depressive symptoms such as fatigue, social wi thdrawal , ca rbohydra te craving, weight gain, and hypersomnia (maximum of 23 points) , and using a man ia rat ing s c a l e .

11 Criteria of Te rman et ai

12 were used to determine

responders to light t rea tment : a 5 0 % reduct ion in scores on the 21-item Hami l ton Scale to a score of less than 8. In addi t ion, after each t rea tment , subjects completed the Beck Inventory for Depression, and they were asked to cont inue filling out the daily ratings forms th roughou t the light s tudy.

Subjects had been off psychoactive medicat ion for at least 2 mon ths before the init iation of the study. All gave writ ten informed consent after the procedures had been fully explained.

Follow up study

After complet ion of the study, P M S subjects who wished to cont inue with the light t rea tments were invited to do so. Mens t rua l phase cont inued to be moni tored by ur inary L H assay, and that light t rea tment (bright white a.m., bright white p .m. or d im red p.m.) , which was found to be most effective in the initial s tudy, was administered for a subsequent 6-18 m o n t h period in the premenst rual phase . Each m o n t h Hami l ton ratings were done following a week of the specified light t rea tment . Pat ients cont inued to complete daily m o o d rat ings, sleep and light logs and Beck inventories for depression dur ing each light t rea tment .


Analyses

Baseline measures for each ra t ing scale (Hamil ton, Beck, atypical, mania , or daily ratings) were determined by taking the mean of the ratings dur ing the premenst rual weeks of the 2 -3 m o n t h evaluat ion phase before t rea tment . There was n o spontaneous improvement in symptoms over a 2-m o n t h interval before the t rea tment pro tocol was begun. F o r each of the rat ing scales normal i ty was tested by the Shapiro-Wilk approach . There were no statistically significant effects by order of t rea tment ; hence, t rea tment order was not taken into account in the statistical model . The nonparamet r i c tests used were those of F r i edman and Wilcoxon. L L P D D and normal control subjects were analysed separately. Since this was a small-sample explora tory s tudy, no adjustment for mult iple compar isons was used. The 5 % level was used for statistical significance.

Results

Clinical rating scales (see Figures 1-4)

Hami l ton , Beck, and atypical rat ings all showed statistically significant {p< 0.005) effects of condi t ion (baseline, a.m. bright white, p .m. bright white, p .m. d im red light) in L L P D D subjects. Baseline rat ings (before t rea tment) were significantly ( p < 0 . 0 1 , Wilcoxin) greater t han those obta ined after t rea tment with each light t rea tment (see Figure 1) bu t none of the light t rea tments differed significantly from each other .

«5

T3

α ο

Effect* of light treatment 30 r~ I—I

PMS

20

10

- J L

Control

ι I

Baseline AM light PM light Red light

F I G . 1. M e d i a n , first a n d th i rd quar t i les of H a m i l t o n depress ion ra t ings in P M S (N= 19) a n d n o r m a l con t ro l (n= 11) subjects , by basel ine a n d t r e a t m e n t va lues . E a c h of the light t r e a t m e n t s (br ight whi te a.m., b r igh t whi te p .m. , d i m red p .m. ) were significantly ( p < 0 . 0 1 ) different from basel ine , b u t n o n e of the l ight

t r e a t m e n t s differed significantly from each o ther .


Effects of light treatment

-

• τ

PMS ^ } Control

-

T Γ Ί τ

τ Τ


F I G . 2. M e d i a n , first a n d th i rd quar t i l es of Beck depress ion ra t ings in P M S (N— 19) a n d n o r m a l con t ro l (n = 11) subjects , by basel ine a n d t r e a t m e n t values . E a c h of the light t r e a t m e n t s (br ight whi te a .m. , b r igh t whi te p .m. , d im red p .m. ) were significantly ( p < 0 . 0 1 ) different from basel ine , b u t n o n e of the light

t r e a t m e n t s differed significantly from each o the r .

Effects of light treatment 20 r- . .

PMS

•3 10

CL,

• I

Control

I 1 y


F I G . 3. M e d i a n , first a n d th i rd quar t i l es of a typica l depress ive ra t ings in P M S (n= 19) a n d n o r m a l con t ro l (n = 11) subjects , by basel ine a n d t r e a t m e n t values . E a c h of the light t r e a t m e n t s (br ight whi te a.m., b r igh t whi te p .m. , d i m red p .m. ) were significantly ( p < 0 . 0 1 ) different from basel ine , b u t n o n e of the l ight

t r e a t m e n t s differed significantly from each o the r .

Using the criteria of T e r m a n et al.,

12 in which a response to light therapy

is determined by a 5 0 % reduct ion in Hami l ton ratings to a value less than 8,8 (39%) of pat ients responded to a.m. bright light, 4 (22%) responded to p .m. bright light and 5 (28%) responded to p .m. d im light.

In normal controls , Hami l ton , Beck and atypical rat ings showed no statistically significant effects of light t rea tment .

M a n i a rat ings showed significant ( p < 0 . 0 2 , Fr iedmans) effects of condi t ion for no rma l controls (see Figure 4 results of Wilcoxin tests), but


•I 3

S 2

Effects of l ight treatment

Mania ratings

I 1 PMS

LXXI Control

I Baseline AM light PM light

I Red light

F I G . 4. M e d i a n , first a n d th i rd quar t i les of m a n i a ra t ings in P M S (n = l9) a n d n o r m a l con t ro l (n=il) subjects , by basel ine a n d t r e a t m e n t values . In n o r m a l con t ro l s , there was a significant difference in m a n i a ra t ings of c o m p a r i n g basel ine vs. d i m red p . m . light ( p < 0 . 0 5 ) a n d in b r igh t whi te p . m . vs. d im red p . m . light

( p < 0 . 0 5 ) .

not for L L P D D pat ients . N o n e of the elevation of man ia score ratings were considered clinically impor tan t .

Daily ratings

Only daily rat ings after 4 days of light t rea tment were used in the analysis, because lights generally take 3-4 days to exert their therapeut ic effects.

1

The daily visual analog rat ings of depression (p<0 .04 ) , irritability (p < 0.001 ), and physical symptoms (p < 0.002) showed significant effects of light t rea tment (Fr iedman's test). Improvement in each of these symptoms was observed between baseline vs. a.m., baseline vs. p.m., and baseline vs. red light t rea tments ( p < 0 . 0 5 , Wilcoxin). Anxiety, appeti te , fatigue, and withdrawal symptoms did no t show significant effects of light t rea tment .

Subject expectations

F o r L L P D D pat ients , the only statistically significant correlat ions between expectat ion of improvement with a.m., p.m., or red light and clinical response by observed ratings were seen in Beck rat ings for bright white evening light (p = 0.015) and in Hami l ton ratings for d im red evening light ( p<0 .03 ) .

Horne-Ostberg ratings

11

There were no statistically significant correlat ions of morning—evening-ness with response to a.m., p .m. , or red light t rea tment based on Hami l ton , Beck, atypical, or man ia rat ings.


Sleep logs

In P M S pat ients , there was a statistically significant difference (Fried-man ' s test, ρ < 0.02) in a m o u n t of sleep repor ted by condi t ion (baseline premenst rual m o n t h s before t rea tment vs. t rea tment m o n t h s after either a.m., p .m. , or red light). By Wilcoxon tests, there was a statistically significant ( p < 0 . 0 1 ) reduct ion of sleep repor ted dur ing a.m. compared with red light t rea tment in P M S pat ients . There were no impor t an t differences in sleep dura t ion dur ing different condi t ions in no rma l control subjects.

Side effects checklist

Only P M S pat ients repor ted significant side effects from the light t rea tments . The mos t frequently repor ted side effect of a.m. light was increased activity (agitat ion) repor ted in 6 of 10 pat ients (3 repor ted mild symptoms , 3 modera te ) . Eye strain was the mos t frequently repor ted symptom from p.m. light repor ted in 5 of 10 pat ients (3 repor ted mild, 1 modera te , and 1 marked symptoms) . Drowsiness was the most frequently repor ted symptom under red light, repor ted in 7 of 10 pat ients (1 mild, 5 modera te , 1 marked) .

Follow-up study (see Figure 5)

N o pat ient w h o par t ic ipated in the follow-up study did the best on red light t rea tment dur ing the initial s tudy and , therefore, red light t rea tment was not used in the follow-up study. F o u r pat ients (two who chose to cont inue on a.m. light and two who chose to cont inue on p .m. light) completed at least 12 m o n t h s and three pat ients 18 m o n t h s of follow-up bright light t rea tment . M e a n rat ings for Hami l ton (p< 0.008), Beck (p< 0.005), and atypical scales ( p < 0 . 0 4 ) cont inued to show statistically significant reduct ions in depressive symptoms compared with baseline at 18 m o n t h s after br ight light t rea tment . M a n i a rat ings did no t differ dur ing the m o n t h s of light t rea tment . There were no statistically significant differences between a.m. vs. p .m. light t rea tment on any of the ra t ing scales (Hamil ton , Beck, atypical , mania) . F u r t h e r m o r e , there were no statistic-ally significant differences in Hami l ton , Beck, atypical , or man ia rat ings when m o n t h 1-3 vs. m o n t h 4 -6 were compared or when m o n t h s 1-6 were compared with m o n t h s 7-12, or m o n t h s 13-18 in the follow-up study.

Daily ratings

The daily rat ings, which after follow-up br ight light t rea tment showed statistically significant improvement compared with baseline rat ings,


Follow-up study mood ratings

25 r-

20

c 2 is

42 10

Hamilton ratings

BSL 6 MO 12 MO 18 MO

20

15 -

10

5

0

Beck ratings

rh fin ΓΪ1 BSL 6 MO 12 MO 18 MO

20 r

15 h

cu 10

Atypical ratings


4

* 3

S 2

ι H

Mania ratings


F I G . 5. Resul ts of an 18 m o n t h fol low-up s tudy of light t r e a t m e n t in P M S subjects c o m p a r i n g basel ine (before t r e a t m e n t ) p r e m e n s t r u a l H a m i l t o n Beck, a typical a n d m a n i a ra t ings wi th the s ame p r e m e n s t r u a l ra t ings ob t a ined after br igh t whi te m o r n i n g , o r b r igh t whi te evening light t r e a t m e n t . C o m p a r e d with basel ine ra t ings , m e a n H a m i l t o n , Beck a n d a typical ra t ings were significantly ( p < 0 . 0 5 ) reduced d u r i n g follow u p m o n t h s 1-6, 7—12 a n d 13-18 . Ra t ings o b t a i n e d du r ing m o n t h 1-6, vs. m o n t h s 7 -12 vs. m o n t h s 13-18 did no t differ from each o ther . M a n i a ra t ings did no t significantly c h a n g e d u r i n g the m o n t h s of light t r ea tmen t

c o m p a r e d with basel ine.

included withdrawal ( p < 0 . 0 2 ) , irritability (/?<0.04) and physical discom-fort ( p<0 .04 ) .

Discussion

In summary , light t rea tments had no significant effects on m o o d ratings in normal control subjects. In L L P D D , depression ratings fell significantly from baseline after bright white morn ing , bright white evening, and dim red evening light t rea tments . The lack of discernable order of t rea tment effects is consistent with the observat ion that the three t rea tments were nearly equally effective. Because the active t rea tments did not show incremental benefits over placebo t reatments , we cannot claim efficacy for light t rea tment in L L P D D based on the present study.

Thus , a placebo effect of light therapy cannot be ruled out , as E a s t m a n

13

described in her discussion of the placebo response of pho to the rapy in


seasonal affective disorder. Fu r the r controlled follow-up trials are indicated in a larger number of subjects. Such a trial should include a completely untreated , i.e. negative control g roup , especially to evaluate any long term effects. It is possible that we have not yet developed an appropr ia te placebo control and that the different light interventions may be working th rough different mechanisms, i.e. th rough altering phase or ampl i tude of circadian rhythms or serving as a da rk pulse.


This work was suppor ted in par t by N I M H grant # R 2 9 M H 4 2 8 3 1 , C R C grant #M01RR00827 , N I M H C R C grant # M H 3 0 9 1 4 - 1 4 . D . F . Kr ipke , M . D . , served as a consul tant .

References

1. Rosen tha l N . E . , Sack D.A. , C a r p e n t e r C.J. , P a r r y B.L., M e n d e l s o n W . B . a n d W e h r T.A. (1985) An t idep res san t effects of light in seasonal affective d i sorder . Am. J. Psychiatry 142 , 163-170.

2. K r i p k e D . F . , K l a u b e r M.R. , Risch S.C. a n d Gill in J .C . (1992) Con t ro l l ed trial of br ight light for n o n s e a s o n a l ma jo r depress ive d i so rders . Biol. Psychiatry 3 1 , 119-134.

3. P a r r y B.L., Rosen tha l N . E . , T a m a r k i n L. a n d W e h r T.A. (1987) T r e a t m e n t of a pa t i en t wi th seasonal p r e m e n s t r u a l s y n d r o m e . Am. J. Psychiatry 144 , 762-766 .

4. P a r r y B.L., Berga S.L., Mostof i N . , Sependa P .A. , K r i p k e D . F . a n d Gil l in J .C . (1989) M o r n i n g versus evening br ight light t r e a t m e n t of late luteal phase d y s p h o r i c d i so rder . Am. J. Psychiatry 146 , 1215-1217.

5. Roy-Byrne P . P . , R u b i n o w D.R. , H o b a n C M . , P a r r y B.L. , R o s e n t h a l N . E . , N u r n b e r g e r J . I . a n d Byrnes S. (1986) P r e m e n s t r u a l C h a n g e s : A c o m p a r i s o n of five p o p u l a t i o n s . Psychiatry Research 17 , 7 7 - 8 5 .

6. Spitzer R.L. , Wil l iams J .B . W., G i b b o n M . a n d Fi rs t M B . (1990) Structured Clinical Interview for DSM-III-R Patient Edition ( S C I D - P , Vers ion 1.0). Amer i can Psych ia t r i c Press , W a s h i n g t o n D C , 1990.

7. H a m i l t o n M . (1967) D e v e l o p m e n t of a ra t ing scale for p r i m a r y depress ive illness. Br. J. Soc. Clin. Psychol. 6 , 278 -296 .

8. Beck A.T. , W a r d C H . , M e n d e l s o n M. , M o c k J .E . a n d E r b a u c h J. (1961) Inven to ry for m e a s u r i n g depress ion . Arc. Gen. Psychiatry 4 , 561 -569 .

9. Amer i can Psych ia t r i c Assoc ia t ion , C o m m i t t e e o n N o m e n c l a t u r e a n d Stat is t ics . Diagnostic and Statistical Manual of Mental Disorders. Revised 4 th Ed. , W a s h i n g t o n D C , Amer i can Psych ia t r i c Assoc ia t ion , 1987.

10. H o m e J.A. a n d O s t b e r g O . (1976) A self-assessment ques t ionna i r e to de t e rmine mor ingness -eveningness in h u m a n c i rcad ian r h y t h m s . International Journal of Chrono-hiology 4 , 97 -110 .

11. Rosen tha l N . E . a n d Heffernan M . M . (1986) Bul imia , c a r b o h y d r a t e c rav ing a n d depress ion : A cent ra l connec t i on? In Nutrition and the Brain (eds. W u r t m a n R.J. a n d W u r t m a n J . J ) , Vol . 7, p p . 139-166 . Raven Press , N e w Y o r k .

12. T e r m a n M . , T e r m a n J .S. , Q u i t k i n F . M . , M c G r a t h D.J . , S tewar t J . W . a n d Rafferty B. (1989) Light t he r apy for seasonal affective d i sorder . Neuropsychopharmacology 2 , 1 - 2 2 .

13. E a s t m a n C.I . (1990) W h a t the p l acebo l i te ra ture can tell us a b o u t p h o t o t h e r a p y for S A D . Psychopharmacol. Bull. 26 (4) , 4 9 5 - 5 0 4 .

30

Seasonality and Seasonal Affective Disorders: Diagnostic Implications for the Outcome of Phototherapy J O H A N B E C K - F R I I S

Karolinska Institute, Department of Psychiatry, St Gorans Hospital, S-11281 Stockholm

SEASONS ARE the na tura l consequences in na tu re due to the earth 's ro ta t ion a round the sun. Pho tope r iod and tempera ture are examples of basic elements forming the seasons. By seasonality we refer to a seasonal rhy thm, which may impact any physiological or behavioral variable. Seasonal rhy thms are c i rcarhythms like circadian rhy thms . W e talk abou t c i rcannual rhy thms because seasons vary from year to year. There is also a direct correlat ion between circadian and seasonal /circannual rhy thms , insofar as the circadian rhy thm relates to the year according to the photoper iod . This means tha t seasons do vary from lat i tude to la t i tude.

These aspects of seasonality also have impor t an t implicat ions for the diagnosis of seasonal affective disorders (SAD) and its subdromal form i.e. subdromal-seasonal affective disorder (S-SAD). In this paper the d iagno-sis of SAD, S-SAD as well as m o o d disorders in general will be discussed. The art of diagnosing always holds at least two aspects: reliability and validity. An ideal system of classification would be one with highest possible reliability as well as validity. This is the aim we as researchers may all aspire to .

However , in a commenta ry F a b r e g a

7 discussed social and cultural

factors influencing systems of psychiatric classification and diagnosis . H e concluded tha t the process of developing psychiatric knowledge and diagnosing ins t ruments must be recognized as interminable , since psychiatric disorders are const i tuted by a dialectical interplay of biology, history, society and culture.

411


Aspects of rel iabi l i ty

The reliability of the SAD diagnosis is one example of this complexity referring to Fabrega ' s conclusion. As seasons vary from year to year due to the weather , from lat i tude to lat i tude due to the pho toper iod and tempera ture and as cultural aspects of seasons vary between societies (e.g. Chr is tmas celebration varies between societies from the N o r t h e r n and Southern hemispheres according to different seasons) problems in reaching high reliability are evident. Thus , reliability pr imarly has to be established on the different lat i tudes.

In the ongoing work on developing the D S M - I V the efforts to improve the reliability have been focused on the dura t ion aspects of the S A D diagnosis, leaving the problems of symptomato logy besides. There is an agreement that the D S M - I I I - R c r i t é r ium

2 concerning a 60 day period of

symptom on/offset is too na r row and therefore is under eva lua t ion .

3 In

this way a broadening of the diagnosis is proposed , p robably resulting in a higher reliability.

However , regarding the diagnoses of S A D (an episode of Bipolar or Bipolar II Disorder or Major Depressive Disorder , Recurrent) , the "depressive atypical" s y m p t o m s

24 such as hypersomnia , weight gain and

carbohydra te craving have been shown to be of impor tance in the symptomato logy of S A D but d o vary acccording to the seasonal type: seen in the winter-type bu t not in the spring-type or summer- type . Fur ther -more , also these "atypical" winter symptoms seem to vary with lati tudes. K j e l l m a n

15 has repor ted that the frequency of "atypical" symptoms at the

lat i tude of Sweden differ from the corresponding symptoms at the lat i tude of, e.g. Wash ing ton D C . This seems also to be true for seasonal symptoms in heal thy subjects. In their review Lacoste and Wirz-Justice showed a seasonal variat ion in several variables related to , e.g. climatologie f ac to r s .

17

Lat i tude differences are also obvious for meteorological condi t ions. An example is the fact that in springtime at the lat i tude of Stockholm (59°N) the air is less cloudy than in the a u t u m n . This implies that there is a disconformity in the springtime between radiancy and pho toper iod at this lat i tude, which differs from other seasons and might differ from condit ions at o ther lat i tudes.

Also differences in psychological aspects are obvious in this respect. Ann ive r sa r i e s

22 have been proposed to trigger some of the m o o d

reactions, which might cor respond to episodes of SAD al though they by definition should be separated. Precipi tat ing factors for an anniversary reaction have mainly been described as memories of losses by different causes, with reappearance in almost a predictable/compulsive manner . Such a memory m a y act disturbingly by its own but might also be charged with social factors as, e.g. seasonal unemployment , resulting in a m o o d

Seasonality and Seasonal Affective Disorders 413

dysfunction appear ing in a seasonal way. The quest ion whether such an episode should be indicated as an episode of S A D or no t is relevant in this discussion.

These aspects are examples of factors being relevant to consider in discussing the reliability of the SAD-diagnosis and in evaluat ing the relat ionship between reliability and the validity of S A D .

Aspects of va l id i ty

The validity of the diagnosis of depression and S A D seems to be even more intriguing.

It is implicit tha t seasonality pr imarly refers to heal th and not to disease. But as we all know, diseases may also be closely linked to seasons. The quest ion is, wha t is the difference between heal th and disease according to the rhythmic function? O r is there no difference? Regarding S A D : is there a difference in rhythmici ty between no rma l seasonal m o o d swings, S-SAD and SAD?

I assume seasonal diseases, like S A D and peptic ulcers, to be stress reactions to different seasonal factors. They have to be caused either by the patient 's maladapt ive response to elements or functions of the season itself, like for example high or low tempera ture , short or long pho toper iod or even psychological/social factors as, e.g. unemployment or it could be caused by the pat ient 's difficulty in adap t ing to the changes from one season to another . T o unders tand whether it may be the elements of the season or the seasonal change which are mos t likely to be the pa thogenic factor, s tudying the quali ty of the disease symptoms seems to be relevant.

We may ask: which is the core feature of the major depression? In a lmost all diagnostic manua l s including the D S M - I I I - R and the D S M - I V the depressed m o o d of distinct quali ty is the core characterist ic in depressions, and the elevated m o o d corresponding in manic states. However , van P r a a g and c o w o r k e r s

4 23 have discussed anxiety and aggression to be the

core characterist ic in some subgroups of depressions, the m o o d lowering being merely a derivative p h e n o m e n o n from aggression, in which they hypothesize serotonin dis turbances to play an essential pa thogenic role. They point to something crucial, namely the role of aggression in the pathogenesis of m o o d disorders , an hypothesis m a d e by A b r a h a m

1 and

discussed by m a n y au thors (e.g. Ref. 8). In this context it is of interest tha t the m ode rn serotonin reup take inhibit ing agents seem to be effective no t only in m o o d disorders bu t also in e.g. panic disorders , social phobia , Premens t rua l S y n d r o m e

27 and Late Luteal Phase Dysphor ic D i s o r d e r ,

26

suggesting serotonin to be involved in different clinical states involving anxiety and irritability as impor tan t features.

Before discussing the aggressive drive in depression in more detail I first


would like to contr ibute to the discussion of the distinct quality of the depressed m o o d . The impor tan t quest ion is: what characterizes the distinct quality of depressed m o o d ? Available diagnostic systems includ-ing the D S M - I I I - R and D S M - I V seem to lack the answer. However , the quest ion is relevant and has to be addressed.

The de l im i ta t ion b e t w e e n mourn ing and depression

Clinically, two states seem often to be mis taken for a p roper depressive state: mourn ing and the asthenic reaction. F r e u d

9 discussed the impor tan t

similarity and distinction between mourn ing and melancholia and made the classical formulat ion: "in mourn ing it is the world outside which has been poor and empty, in melancholia it is the ego itself." (Melancholia is here used in the historical sense, corresponding to major depressive episode in the mode rn D S M sense). W h a t has been lost within the ego making it poor and empty in melancholic pat ients has by m a n y a u t h o r s

13

been translated into the loss of the self-regard and has by them been described as the main feature in all kinds of depressions. The self-regard is defined as a concept involving the libidinal as well as the aggressive components of the self-preservation drive and including concepts such as self-esteem, self-confidence, self-evaluation and self-respect. The struggle for mainta in ing the self-confidence is central in personalities apt for depressive react ions; the loss of self-regard being the line of demarca t ion for a depressive episode proper .

Self-regard is closely connected to the concept of narcissism. A normal and healthy development of a person's narcissism is the g round for a healthy self-regard in adult life. A loss of the self-regard could be initiated in disposed individuals for different r e a s o n s .

12 It could be a loss of an

impor tan t and introjected object or it could be a loss of an ideal. A loss of self-regard could also be initiated by the loss of energy, giving rise in disposed persons to an impai rment of the self-preservation defence mechanisms.

F reud together with A b r a h a m

1 also discussed the impor tance of the

inward direction of aggressions in melancholic pat ients . They described the inward direction of the aggressive drive directed to a beloved but dependency evoking object, internalized into the self. W h a t is impor tan t abou t this aggressive drive towards introjected objects is its oral character , a finding which may be of interest in unders tanding the repor ted relation between m o o d and food in winter-depressive S A D p a t i e n t s .

16

With these characteristics of the self-regard including the direction of aggressive drive the border between mourn ing and depression should be distinct.


The de l im i ta t ion b e t w e e n t h e asthenic react ion and depression

This demarca t ion is in clinical practice a work of the same impor tance and frequency, bu t less well described or documented . The impor tance of this task is exemplified by the fact tha t a genuine asthenic react ion, cont rary to a major depressive episode, does not respond to , e.g. tricyclic ant idepres-sants . The asthenic reaction, which seems to be one of the most c o m m o n syndromes in psychiatry, is a syndrome distinct from the major depression. In the D S M - I I I - R it is no t specifically described but seems closest to be related to the diagnosis of Dys thymia or an Organ ic M o o d Syndrome.

According to the Swedish textbook in psychiatry by O t t o s s o n

19 the

reaction is characterized by its main symptoms , which are fatigue and irritability. The loss of energy seems to be the core in this syndrome and could be triggered by various wearing stress factors: (1) longstanding psychic stress; (2) physical stress after surgery, infections etc; déficiences of minerals , vi tamins etc.; or secondary to endocrine diseases, cancer etc.

In m a n y ways this state resembles a depressive one and like the state of dysthymia the asthenic reaction may transi t into a major depressive episode. But wha t from a clinical point of view is of impor tance is the fact tha t in its genuine form the asthenic reaction has no depressive quali ty, insofar as it does no t present a depressed m o o d of distinct quali ty. Thus , a major depressive state is separated from bo th the state of mourn ing and tha t of as thenia by the regulat ion of the self-regard and the direction of the aggressive drive. This is i l lustrated in Table 1, for compar i son together with the equivalences for mania .

T A B L E 1

Differentiation between depression and some related affective states according to the maintenance of self-regard

and the main direction of the aggressive drive

Self-regard M a i n d i rec t ion of aggress ions

M o u r n i n g In t ac t N e u t r a l Depres s ion I m p a i r e d or lost I n w a r d As then ia In t ac t O u t w a r d M a n i a Elevated O u t w a r d

The de l im i ta t ion b e t w e e n S A D and S - S A D

The diagnosis of S-SAD has been proposed to be characterized by the same symptoms as in the SAD-diagnosis , excluding the symptom of depressed m o o d .

14 At the same time it was repor ted tha t the subsyndromal


states of winter -SAD equally benefit from p h o t o t h e r a p y .

14 Therefore, it

seems likely tha t in the SAD-pat ient the core symptom sensitive to light t rea tment might not be the depressed m o o d but something else. T e r m a n

25

has repor ted a s tudy abou t Depression Scale Predictors in SAD patients according to the balance of atypical and typical symptoms . The "atypical" symptoms are the increased eating, the carbohydra te craving, weight gain, hypersomnia , p ronounced afternoon s lump and the fatigue. H e found the atypical balance to be the strongest predictor for responsiveness to light-therapy in SAD pat ients . He also found fatigue to be the most universal symptom. In the Stockholm s t u d y ,

15 fatigue was seen in 8 8 % of the

pat ients .

In many ways a l though not overall these "atypical" symptoms resemble those of the genuine asthenic reaction described by Ot tosson . They are seen bo th in the SAD and in the S-SAD pat ients , suggesting the possibility of a complex of neurovegetat ive symptoms being the core symptoms of SAD and S-SAD. In the same context , it is no doub t that the depression of winter-type (true S A D winter-type) fulfills the criteria for a major depressive episode including the above ment ioned criterion of a loss of self-regard and the inward direction of the aggressive drive, seen in the SAD but no t in the S-SAD winter type.

I therefore suggest the S-SAD of winter-type to correspond to a classical form of asthenic reaction, which is very similar to "the seasonal anergy syndrome", repor ted by Lacoste and Wi rz - Jus t i ce

17 and the SAD to

correspond to a major depressive winter episode, which has been superimposed in pat ients especially disposed. Such a disposit ion could be the narcissistic vulnerability for a loss of the self-regard.

Discussion

The aim to reach a high degree of validity in diagnosing mental dysfunctions will always be of significance. M o d e r n diagnostic systems as, e.g. D S M , which use symptomato logy as the main diagnostic ins t rument , however seem to obtain diagnoses with high reliability at the expense of a lower validity. Any trial to rise the extent of validity wi thout losing the sharpness of reliability then must be impor tan t . The definition abou t the loss of self-regard as the core symptom in major depression and the turning of the aggressive drive has here been proposed for improvement of the validity of the depression diagnosis by adding the classical psycho-analytical concept of depression to the modern way of diagnosing.

In this context a valid diagnosis of SAD must contr ibute to a proper discrimination between SAD and S-SAD. This discrimination focuses bo th on what separates the condit ions but also on what they share in c o m m o n . The "atypical" symptoms of fatigue etc. seem to be what link the condit ions of winter-type and therefore is here proposed to be the main


target for the effect by pho to the rapy . If this holds t rue the classification as affective disorder may be misleading. According to the winter-type therefore a m o r e relevant nomencla ture is here suggested by the use of "seasonal anergy syndrome" , as described by Wark , Wirz-Justice and G r a w (see Ref. 17). The effect by pho to the rapy on the affective componen t in the syndrome—i.e . the depressed m o o d of distinct qual i ty—then should be interpretated as secondary to the pr imary effect on the energy componen t . This is in line with the considerat ions by Mueller and D a v i e s ,

18 who proposed the classification as "seasonal energy syndrome" .

They discussed the symptoms of anhedon ia and low energy level in the fall-winter and hyperphor ia , racing thoughts , and agi ta t ion in the sp r ing-summer per iod as the ha l lmarks of this syndrome. However , they did no t discuss in detail the discrimination between the energy-anhedonia symptoms and the t rue affective compla in ts ; i.e. the del iminat ion between the symptoms of SAD and S-SAD respectively nor the distinct quali ty of the depressed m o o d .

This differentation between SAS and S-SAD might be relevant also to the diagnosis of Premens t rua l Syndrome (PMS) , corresponding to a differentation between P M S and Late Luteal Phase Dysphor ic D i s o r d e r

3

with reference to a distinct quali ty of depressed m o o d . The effect by pho to the rapy on these condit ions described by Pa r ry et α / .

20 in ana logue

might then be interpretated as secondary to the effect on an energy/neuro-vegetative componen t .

Fu r the rmore , the val idat ion of the distinct quali ty of depressed m o o d could be of interest in unders tanding the mechanisms of placebo in pho to the rapy .

This possible placebo effect in the bright light t rea tment of S A D repor ted by E a s t m a n at this s y m p o s i u m

6 might be menacing to those who

regard the placebo effect in any way as contradic tory to a biologic effect, seeing the placebo effect as an unspecific psychological background effect.

The issue of placebo as a non-specific, active or intent ional p h e n o m e n o n has been discussed by e.g. G r i i n b a u m .

1 0'

11 In placebo effects unconscious

thoughts , emot ions and memories as well as magical and unrealistic expectat ions, claims, yearnings etc. m a y be involved. The role of faith in therapy and t rea tment has been d i s cus sed

21 as well as the role of

expectancy in behavior change .

5 It seems likely tha t these factors might be

of a specific na tu re . Concerning the placebo effect in pho to the rapy these factors might be specific to the pat ients ' experiences of light and da rk as well as colors, t empera ture , social contact etc.

The psychological experiences of light and da rk per se certainly then are factors of impor tance to s tudy in this context . F r o m the large a n t h r o p o -logical l i terature we also can learn abou t the impor tance of sun-worship in old cultures, as e.g. in Babylonia and old Egypt . Also in the nor th as a m o n g the sames/ laps worship of the sun and "re turning of the sun" has


been of central impor tance . The sames regard themselves as " the sons and daughters of the sun".

H u m a n beings experiences of seasons also involve the process of mourn ing . At higher lat i tudes seasons are more distinct and definite and therefore also remind people more clearly abou t the passage of t ime. This implies in a certain way the necessity of being able to m o u r n . You have to m o u r n the loss of the old season before you may enter the new one. M a y b e the unmis takeable sound of tender sadness and sweet melancholy, which is so typical in Nord ic folklore music, reflects this need of mourn ing .

However , the close similarity and the distinct del imitat ion between mourn ing and a depressive state again should be kept in mind . The validat ion of the distinct quali ty of depressed m o o d by the use of the concept "loss of self-regard" could be one step forward in this ongoing work of differential diagnosing and its implications for S A D and the ou tcome of pho to the rapy .

Conclusion

I discuss the impor tance of a valid diagnosis of depression by focusing on the distinct quality of the depressed m o o d and by propos ing a definition of this quali ty. This definition I p ropose by in t roducing the concept of self-regard and I stress the loss of the manifestation of self-regard as the core symptom of any depression. This core symptom is suggested to demarca te SAD from its subdromal form S-SAD.

In the further work of reaching reliable and valid diagnoses of SAD and S-SAD the use of psychological models seem to be of value and therefore are suggested to be added a m o n g the scientific tools . The use of " the loss of self-regard" in diagnosing a major depressive disorder might be one possible tool in this cont inued work.

References

1. A b r a h a m K . (1911) N o t e s o n the psycho-ana ly t ica l inves t iga t ion a n d t r e a t m e n t of manic-depress ive insani ty a n d allied cond i t ions . In ( A b r a h a m ) Selected Papers, p p . 137-156.

2. Amer i can Psych ia t r i c Assoc ia t ion (1987) Diagnostic and Statistical Manual of Mental Disorders. 3rd ed. , rev. Amer i can Psych ia t r i c Associa t ion , W a s h i n g t o n , D C .

3. Amer i can Psychia t r i c Assoc ia t ion (1993) DSM-IV Draft Criteria, 1 M a r c h 1993, Amer i can Psych ia t r i c Assoca t ion , W a s h i n g t o n , D C .

4. Ap te r Α., van P r a a g H . M . , P lu t ch ik R., Sevy S., K o r n M . a n d B r o w n S.L. (1990) In te r re la t ionsh ips a m o n g anxie ty , aggress ion , impuls ivi ty , a n d m o o d : a se ro ton in -ergically l inked c lus ter? Psychiatry Res. 32 (2), 191-199 .

5. Boo tz in R . C . (1985) T h e role of expec tancy in behav io r change . In Placebo. Theory, Research, and Mechanisms (eds. W h i t e L. , T u r s k y B. a n d Schwar t z G .E . ) , p p . 196-210 . T h e Gui l ford Press , N e w Y o r k .

6. E a s t m a n C. See this v o l u m e . 7. F a b r e g a H . Jr . (1992) C o m m e n t a r y : Diagnos i s i n t e rminab le : t o w a r d a cul tural ly

sensitive D S M - I V . The Journal of Nervous and Mental Disease 180, 1, 5 -7 .


8. F a r m e r R. (1987) Host i l i ty a n d de l ibera te self-poisoning: the role of depress ion . Br. J. Psychiatry 150 , 609 -614 .

9. F r e u d S. (1917) M o u r n i n g a n d me lancho l i a . In The Standard Edition of the Complete Psychological Works of Sigmund Freud (ed. S t rachey J.) , Vol . XIV, p p . 239 -260 . T h e H o g a r t h Press , L o n d o n .

10. G r u n b a u m A. (1985) Expl ica t ion a n d impl ica t ions of the p l acebo concep t . In Placebo. Theory, Research, and Mechanisms (eds. W h i t e L., T u r s k y B. a n d Schwar t z G .E . ) , p p . 9 - 3 6 . T h e Gui l ford Press , N e w Y o r k .

11. G r u n b a u m A. (1986) T h e p l acebo concep t in medic ine a n d psych ia t ry . Psychological Medicine 16 , 19 -38 .

12. G r u n b e r g e r B. (1979) Narcissism. Psychoanalytic Essays. I n t e r n a t i o n a l Univers i t ies Press , Inc . N e w Y o r k .

13. J a c k s o n S.W. (1986) C h a p t e r N i n e : M e l a n c h o l i a a n d depress ion in the twent ie th cen tu ry . In ( Jackson) Melancholia and Depression—From Hippocratic Times to Modern Times, p p . 188-246 . Yale Univers i ty Press , N e w H a v e n a n d L o n d o n .

14. K a s p e r S., Roger s S.L.B., Yancey Α., Schulz P . M . , Skwerer R . G . a n d Rosen tha l N . E . (1989) P h o t o t h e r a p y in ind iv idua ls with a n d w i t h o u t s u b s y n d r o m a l seasona l affective d i sorder . Arch. Gen. Psychiatry 4 6 , 837 -844 .

15. Kje l lman B.K. See this vo lume . 16. K r â u c h i Κ. , Wirz-Jus t ice A. a n d G r a w P . (1990) T h e re la t ionsh ip of affective s ta te to

d ie tary preference: win ter depress ion a n d light t h e r a p y as a m o d e l . J. Aff. Disorders 2 0 , 4 3 - 5 3 .

17. Lacos t e V. a n d Wirz-Jus t ice A. (1989) Seasona l va r i a t ion in n o r m a l subjects : a n u p d a t e of var iables cu r ren t in depress ion research . In Seasonal Affective Disorders and Phototherapy (eds. R o s e n t h a l N . E . a n d Blehar M . C . ) , p p . 167-229 . T h e Gui l ford Press , N e w Y o r k .

18. Muel le r P .S . a n d Davies R . K . (1986) Seasona l affective d i so rde r s : Seasona l energy s y n d r o m e ? Let te r t o Ed . (with reply by R o s e n t h a l N . E . ) , Arch. Gen. Psychiatry 4 3 , 188-189 .

19. O t t o s s o n J . O . (1983) Psykiatri. Almqvis t & Wiksel l , S t o c k h o l m . 20. P a r r y B. See this vo lume . 2 1 . P l o t k i n W . B . (1985) A psychologica l a p p r o a c h to p l a c e b o : the role of faith in t h e r a p y

a n d t r e a t m e n t . In Placebo. Theory, Research, and Mechanisms (eds. W h i t e L., T u r s k y B. a n d Schwar t z G .E . ) , p p . 237 -254 . T h e Gui l ford Press , N e w Y o r k .

22. Po l lock G . H . ( 1970) Annive r sa ry reac t ions , t r a u m a a n d m o u r n i n g . Psychoanal. Q. 3 9 , 3 4 7 - 3 7 1 .

23 . van P r a a g H . M . , K a h n R.S. , Asnis G . M . , Wetz le r S., B r o w n S.L., Bleich A. a n d K o r n M . L . (1987) Review. D e n o s o l o g i z a t i o n of biological psych ia t ry or the specificity of 5-H T d i s tu rbances in psychia t r ic d i so rders . J. Aff. Disorders 13 , 1-8.

24. R o s e n t h a l N . E . , Sack D.A. , Gil l in J . C , Lewy A.J. , G o o d w i n F . K . , D a v e n p o r t Y., Muel le r P .S . , N e w s o m e D.A. , a n d W e h r T.A. (1984) Seasona l affective d i so rder . A descr ip t ion of the s y n d r o m e a n d p re l iminary findings wi th l ight t he rapy . Arch. Gen. Psychiatry 4 1 , 7 2 - 8 0 .

25 . T e r m a n M . See this vo lume . 26. S tone A.B. , Pear l s te in T .B . a n d B r o w n W.A. (1991) F luoxe t ine in the t r e a t m e n t of late

luteal p h a s e d y s p h o r i c d i sorder . J. Clin. Psychiatry 5 2 7 , 2 9 0 - 2 9 3 . 27. W o o d S.H., M o r t o l a J .F . , C h a n Y .F . , M o o s s a z a d e h F . a n d Yen S.S.C. (1992)

T r e a t m e n t of p r e m e n s t r u a l s y n d r o m e wi th fluoxetine: a doub le -b l ind , p l acebo -cont ro l led , c rossover s tudy . Obstetrics and Gynecology 8 0 , 3 , 1, 339 -344 .

31

Problems and Prospects for use of Bright Light as a Therapeutic Intervention M I C H A E L T E R M A N

Department of Psychiatry, Columbia University and New York State Psychiatric Institute, New York, USA

T H I S W E N N E R - G R E N symposium has b rought together specialists in light presentat ion, light reception, and light therapeutics , and has served in delineating certain ambiguit ies in current research and point ing towards solutions. At issue are definition of the dosing dimensions of light presentat ion (above and beyond listings of obvious physical parameters) , and whether light therapy p romotes ant idepressant response (specifically, in winter seasonal affective disorder, SAD) exceeding placebo rates. If the latter issue were resolved in the negative, then the dosing issue either becomes m o o t or degenerates into a quest ion of how lighting manipula-tions affect the rate of placebo response (Eas tman et ai, this volume).

St imulus speci f icat ion

Radiometr ic measures of light intensity (e.g. i r radiance in juw/cm

2) specify

the st imulus in units of physical power wi thout account ing for wavelength-specific variat ions in retinal sensitivity. A physiological t ransform of the proximal s t imulus would m o r e closely describe the central nervous system-activating event. Pho tomet r i c measures of light intensity make such a t ransform for a " s tandard pho top ic observer", though the result for any individual may be inaccurate . As W i b o m (this volume) and others (for review, see Ref. 7; for p roposed s tandards , see Ref. 18) explain, the stimulus can be specified in terms of the propert ies of the i l luminating source (e.g. luminance in c d / m

2) or of the a m o u n t of light reaching the target surface

(e.g. i l luminance in lux, as has been conventional ly repor ted in the light therapy l i terature). W i b o m argues for the luminance measure because, for

421


a uniform diffuse light source, the visual sensation of brightness is invariant with the observer 's spatial posi t ion.

O n the other hand , the observer can actively influence proximal stimulus intensity by head and body movements , and direction of gaze relative to the light source. Some researchers (e.g. Kjellman et al, this volume) have opted for whole- room il lumination systems, in which case the observer 's momen ta ry posi t ion introduces less variability in retinal s t imulat ion. Others have opted for head-mounted "visor" systems, in which the light source moves with the subject, obviat ing the effect of head m o v e m e n t s .

2 0 , 25 Unde r s tandard light therapy, the pat ient sits before a

box of lamps covered with a diffusion s c r e e n .

22 Al though the pat ient is not

ambula to ry , even small changes in posture , head posi t ion and gaze can lead to large variat ions in proximal st imulus intensity (see, e.g. Ref. 3).

It has been generally assumed—but never demons t ra ted—tha t consis-tency of bright light exposure enhances the t rea tment effect. Trea tment protocols have often instructed patients to look directly into the light at frequent intervals, to maximize st imulat ion. In studies in my labora tory , patients have been instructed not to look into the light, but to concentra te on the i l luminated table surface (e.g. Ref. 22), resulting in indirect i l lumination of the central visual field with some direct i l lumination in the periphery. Despite the potent ial p roblem of variable i l luminance, clinical improvement in light box studies is not perforce inferior to that in whole-room and visor studies, though direct compar isons have yet to be made . If such clinical improvement were mainly a placebo response, the issue of i l luminance variability might be moo t . O n the other hand , stimulus variability can serve to potentiate retinal responses .

1

In therapeut ic si tuations where i l luminance varies dynamically, it can be approximately est imated by affixing a photosensor to the observer 's head and t ime-sampling the received signal. This me thod does no t account for variat ions in gaze at a given head posit ion, eye and eyelid movements , pupil size, and retinal adap ta t ion state. But it is sensitive to the major behavioral "gate", head posi t ion, and the measure serves to demons t ra te tha t proximal var iat ion in the stimulus does not necessarily reduce t rea tment effectiveness. O n the other hand , it has yet to be determined whether part ial responders or non-responders to light therapy tend to receive less s t imulat ion dur ing t rea tment sessions than d o responders .

We at tached an Actillume™ (Ambulatory Moni tor ing, Inc., Ardsley, New York) photosensor on a cap, centered on the forehead, and sampled maximum illuminance within successive 1-minute intervals during 10,000 lux treatment sessions at home. Subjects had previously shown remission of winter depressive symptoms under similar t reatment conditions (for criteria, see Ref. 28), and had relapsed upon withdrawal from the lights. Figure l a shows illustrative results across days for a depressed patient treated with evening light in 45-minute sessions beginning at 22.00 hours . Minute- to-

Therapeutic Light 423

minute variability in light exposure within sessions was large, as was variability across sessions. In a retrospective review of these data , the patient was unaware that his average head position varied substantially from day to day. (Illuminance of 10,000 lux would be achieved with the head facing the light box screen at about 30 cm distance.) O n some days, 10,000 lux was never achieved even briefly; on other days, there was substantial time in which illuminance far exceeded 10,000 lux. Despite the daily and local variations, the depression remitted during the treatment period.

Figure l b reduces these da ta into a frequency histogram, collapsing across t reatment sessions. Moda l illuminance was between 6,000-8,000 lux. There was an orderly decrease to either side of the mode , with a strong skew toward high intensities. The largest fall-off in exposure occurred between 8,000-10,000 lux and 10,000-12,000 lux. In this sense, the nominal value of 10,000 lux is a valid descriptor of the dose. Average illuminance, however, was 8,905 + 4,823 lux ( m e a n ± S D ) , which would more accurately describe the "effective" dose. Similar measurements have been performed with four SAD patients, and the shape (skewness, kurtosis) and mean of the frequency distributions differ individually. In three of the cases, however, modal illuminance was at 8,000-10,000 lux, and in all cases, the major fall-off in illuminance occurred just above 10,000 lux.

M y associates and I

27 and o t h e r s

33 have hypothesized that therapeutic

dose is determined by an interaction of light intensity and exposure durat ion. Patients have shown similar positive response to (nominal) 2,500 lux exposure at 2 hours , and 10,000 lux at 30 m i n u t e s .

22 The putative tradeoff

relation between intensity and time {Ixt = c) has not yet been systematically tested within subjects (but see Ref. 27, Figure 1), and the shape of the I χ t function as well as its limits are unknown. If integrated momenta ry illuminance, in conjunction with exposure durat ion, comprised a basic metric, we might want to specify light therapy dose as "lux · min". Under this assumption, a dosimeter recently has been developed (Photodose™, P h o t o n Therapeutics, Mission Viejo, California) for on-line moni tor ing of integrated light exposure, using a head-mounted photosensor . The lights can be automatically extinguished when a specified dose, in lux min, has been attained. The patient can be alerted when momenta ry light levels fall above or below a specified intensity range, guiding adjustments of head position. However, limit parameters based on therapeutic and safety considerations have yet to be determined. The dosimeter can also serve as a compliance monitor . Whether the integrated lux · min metric improves upon simpler formulations of therapeutic dose is now a feasible question for research.

The placebo s w o r d over our heads

As has been the case with antidepressant drugs, placebo response to light therapy for SAD and other disorders (e.g. Parry , this volume) may be


Β 10 000

5 000

Sess ion t ime ( 1 - m i n int)

Β

30

J.L.,cf47 yr, SAD Medic-light mini-1 OK system Actillume forehead placement 10 days / 450 minutes mean ± SD = 8905 ± 4823 lux

2-4 6-8 10-12 14-16 18-20 22-24

F I G .

I l l u m i n a n c e (kLux)

1. (a) M o m e n t a r y i l luminance m e a s u r e d at the head of a pa t i en t d u r i n g 10 successive daily light t h e r a p y sessions. E a c h session is s epa ra t ed by a space . With in-sess ion pa t t e rn s were m e a s u r e d as the m a x i m u m i l luminance (in lux) recorded in successive 1-minute in tervals , (b) F r e q u e n c y h i s t o g r a m of i l luminance

received across sessions.

substant ia l .

4 The problem becomes pivotal in experiments that show no

advantage of light beyond the placebo control . Inconsistent results, including several major trials with apparent light-placebo equivalence (e.g. Refs 6, 20), will keep this problem alive for years to come. As in psychopharmacology research, the apparent efficacy of placebo controls has


varied widely. However, if there were at least a few compelling demonstra-tions of light-specific effectiveness, it would quell the nagging doubt that we have been chasing an illusory goal. There are indeed some promising signs.

Intensity and duration

The earliest experiments used, as controls, reduced light intensity or exposure durat ion, and a cross-center pooled da ta analysis clearly showed an advantage of higher intensity, longer durat ion e x p o s u r e .

26 However,

some of the underlying experiments could be faulted for revealing the ineffective t reatment to patients, and reducing their expectations for the "inactive" controls, leading to a questionable conclusion of specific efficacy. Other experiments showed no significant difference between the putative placebo and active conditions, but the clinical response to bo th was poor (e.g. Ref. 8).

Time of day

A second type of control has been manipu la t ion of time of day of t rea tment , s temming from the hypothesis tha t morn ing light is specifically ant idepressant , and evening light is inactive or merely "ac t iva t ing" .

11

Evening light would be ineffective because of its failure to elicit a circadian phase advance , and such light could exacerbate the depression if it elicited a phase delay. Phase advances and delays are measured in clock t ime, a l though in one e labora t ion of the hypothesis they should be measured against the sleep interval as a phase-angle difference.

13 Sleep phase ,

however, never has been directly measured in these experiments , though subjects are instructed to restrict sleep between 10 p .m. and 6 a.m. (which is a very early wake-up time for a depressed over-sleeper). The general hypothesis , relating differential phase shifts to clinical efficacy, has been sustained in several individual experiments (e.g. Ref. 12). The claim for a morning-evening light clinical differential—without actually measur ing circadian phase—was s t rengthened by a cross-center a n a l y s i s .

26 Because

pat ients in these early studies did no t expect the t ime of day of t rea tment to determine efficacy, evening light could be said to have succeeded as a placebo control .

However , when period 1 of a morning-evening crossover design was separately analyzed (e.g. Ref. 28), or parallel g roups were studied wi thout crossover (e.g. Ref. 34), the effect of t ime of day of t rea tment vanished. (For an unresolved exception to this rule, in which morn ing light was superior to evening light in parallel g roups , see Kjellman et al, this volume. In this study, subjects were no t r andomly assigned to morn ing or evening


t rea tment groups , but ra ther scheduled according to convenience with respect to their habi tual sleep schedule.)

Nondifferential morning-evening results raise a problem bo th for the phase-shift hypothesis and the claim of light-specific efficacy. It mus t be emphasized that for an experiment to refute the phase-shift hypothesis it is insufficient to show that morn ing and evening light are similar in their clinical effect. Rather , one must directly show that l ight-induced phase shifts are not correlated with clinical effect.

The clinical advantage of morn ing over evening light appears to be a byproduct of the crossover d e s i g n .

28 The phenomenon becomes obvious

under the "balanced", expanded crossover des ign

9 in which the two

t rea tment times are al ternated or are repeated within groups (Figure 2). Improvement scores do not differ for morn ing and evening light when (a) either t rea tment is given first, (b) either t rea tment follows itself, or (c) morn ing t rea tment follows evening t rea tment . However , clinical improve-ment is reduced when evening treatment follows morning treatment. Morn ing light appears to "prevent" evening light effect.

17 This is also t rue for

cross-center pooled da t a analysis of morning-evening/evening-morning crossover studies using 2,500 lux light in 2-hour sessions (Figure 3). Thus , t ime of day is an impor tan t factor, t hough its influence is buried within a sequential dependency.

10,000 lux / 30 min / morning vs. evening light 100 r

AM AM AM PM PM PM first post post first post post

AM PM PM AM

F I G . 2. M e a n percent r educ t ion ( ± S E M ) of S I G H - S A D depress ion scale scores relative to basel ine , in six cells of a " b a l a n c e d " c rossover design. Efficacy of evening light is r educed in re la t ion to the o the r cond i t ions only w h e n it follows m o r n i n g light in crossover . Stat ist ical significance is i l lus t ra ted in a n i n d e p e n d e n t ί-test for g r o u p s receiving evening light as t r e a t m e n t 2, p receded ei ther by

m o r n i n g light o r evening light as t r e a t m e n t 1.


A M - P M c r o s s o v e r s Four Centers Pooled, N=70

2500 lux, 2 h

F a i r b a n k s , AK (N = 5)

New York , NY (N = 19)

P o r t l a n d , OR (N = 2 0 )

S e a t t l e , WA (N = 2 6 )

AM P M P M AM

f i r s t p o s t f i r s t p o s t

AM P M

F I G . 3. P o o l e d cross-center analysis of morn ing -even ing c rossover s tudies . T h e o u t c o m e var iab le is the p r o p o r t i o n of pa t i en t s ( ± 9 5 % confidence in terval ) showing p o s t - t r e a t m e n t r educ t ions of a t least 5 0 % in H a m i l t o n depress ion scores , relat ive to basel ine . Efficacy of evening light is r educed in re la t ion to the o the r cond i t ions only when it follows m o r n i n g light in c rossover . Stat is t ical significance is i l lus t ra ted in a hypo thes i s test of p r o p o r t i o n s for i n d e p e n d e n t g r o u p s receiving evening light as t r e a t m e n t 1, o r evening light as t r e a t m e n t 2 following m o r n i n g

light as t r e a t m e n t 1.

The crossover experiments suppor t a phase-shift hypothesis , t hough altered from Lewy and Sack's original f o r m u l a t i o n :

11 light therapy is

effective at any time of day as long as it does not induce a circadian phase delay. Circadian phase advances to morn ing light do no t mediate the ant idepressant effect. Rather , phase delays to evening light weaken the t rea tment effect, or render it ineffective or even depressogenic. W h e n evening light is given as initial t rea tment , it may not cause a phase delay, because it falls on or near the dead zone of the phase responsive c u r v e .

16

M o r n i n g light, however, elicits a phase advance, mak ing the pat ient vulnerable to phase delays when evening light follows morn ing light in a crossover.

This altered hypothesis is a current focus of research by my group , using the dim light meletonin onset ( D L M O )

10 as the marke r for circadian

phase . However , it mus t be admi t ted tha t the experiment has already been done , if no t analyzed in these terms. In their recent expansion on earlier


findings, Sack and colleagues cont inue to report the relative superiority of morn ing l ight—accompanied by circadian phase advances—based on da ta pooled across the morning-evening c ros sove r .

21 The D L M O was

first assessed at "pre-baseline", pr ior to experimental interventions. Before administering light, subjects adjusted their sleep interval to fall between 10 p .m. and 6 a.m. ("baseline"). F o r the t rea tment periods, subjects were given morn ing or evening light t rea tment in a counterbalanced crossover.

In a reanalysis of Lewy and Sack's da ta for 24 pat ients , J. S. Te rman , L. Amira and I teased apar t H A M - D and D L M O effects for the two periods of crossover (Figure 4), with the following results and conclusions.

Ο

Q

F I G . 4. Crossove r analysis of d i m light m e l a t o n i n onse t ( D M L O ) p h a s e a n d H a m i l t o n ( H A M - D ) score (mean ± S E M ) for two g r o u p s of subjects w h o received 2,500 lux m o r n i n g light t r e a t m e n t (6 -8 a .m.) before evening light t r e a t m e n t (7 -9 p .m . or 8-10 p .m. ) , a n d vice versa. S ta r t ing wi th basel ine , sleep was pe rmi t t ed only be tween 10 p . m . a n d 6 a .m. D a t a were poo led from Ref. 12 (N=\6) a n d Ref. 21

(N=S).

1 . Pre-basel ine t o baseline change

A 2-way analysis of variance for the D L M O in pre-baseline and baseline condit ions indicated a significant effect of t ime (F= 91.0, ρ = 0.0002), with an overall mean phase advance from pre-baseline to baseline of 0.75 hour . There was no significant g roup difference (morning or evening light first) or g roup χ time interact ion. A corresponding analysis of H A M - D scores showed a significant g roup difference (F= 13.7, ρ = 0.034), with morning-first subjects slightly more depressed than evening-first subjects at pre-baseline (14.8 vs. 12.4, ρ = 0.015 by 2-tailed ί-test; multiple-test correction, ρ = 0.03), but not after sleep adjustment at baseline. Pointedly, H A M - D scores did not improve in parallel to D L M O phase advances from pre-baseline to baseline. Thus , the sleep adjustment served to advance a circadian rhy thm, perhaps by exposing over-sleepers to dim ambient r o o m

Therapeutic Ligh

f 429

light earlier in the morn ing . Sleep might even have acted as a zeitgeber in its own right (cf., Ref. 5). However , this phase advance was not therapeut ic . If phase shifts of the D L M O and sleep interval were equal , there would be no net change in phase-angle difference. T o know for sure, the sleep interval would have to be measured.

2. Changes in cl inical s ta te and c i rcadian phase, w i t h l ight t r e a t m e n t dur ing morn ing -even ing crossover

D L M O and H A M - D da ta were then compared for the two periods of the crossover. Because the hypothesis predicts tha t failure of response to light is caused by a circadian phase delay, D L M O da t a were analyzed as phase shifts from baseline to t rea tment period 1, or t rea tment period l - to-2, ra ther than absolute values of the D L M O . H A M - D da ta were analyzed as absolute values. The D L M O showed significant effects of order of t rea tment ( Ρ = 1 0 . 5 , p = 0.01), t ime of day of t rea tment ( F = 6 9 . 0 , p = 0.0001), and the interact ion of order χ t ime of day (F= 6.3, ρ = 0.033). The D L M O showed a large mean phase advance to morn ing light whether in t rea tment period 1 or 2 (1.77 hours vs 1.57 hours , NS). There was a small phase delay to evening light in t rea tment 1, but a large delay in t rea tment 2, following morn ing light ( — 0.69 hour vs. — 2.12 hour , ρ = 0.0001 ; multiple-test correct ion, ρ = 0.0002). H A M - D scores showed significant effects of t ime of day of t rea tment (F= 16.9, ρ = 0.003) and the interact ion of order of t rea tment χ t ime of day (F= 5.6, ρ = 0.042), bu t not order of t rea tment per se (F= 0.577, NS). The g roup receiving evening followed by morn ing light (E1M2) showed no significant difference in mean H A M - D score (8.64 vs. 6.86, NS); by contras t , the g roup receiving morn ing followed by evening light (M1E2) showed a s t rong disadvantage of evening light (3.8 vs. 13.4, Ρ = 0.0001; multiple-test correct ion, p = 0.0003). Thus, within-group evi-dence for α morning-evening light differential stems only from subjects receiving the M1E2 sequence. This g roup is also the only one to show a major D L M O phase delay to evening light.

3. Sources of per iod 1 change

Nonetheless , subjects receiving evening light as t rea tment 1 appeared to show higher H A M - D pos t - t rea tment scores (poorer clinical response) than those receiving morn ing light, in an independent -groups compar ison (8.64 vs. 3.8, ρ = 0.02; multiple-test correct ion, ρ = 0.06). Even if this t rend were found to be reliable it would be impor t an t to recognize tha t the response to evening light was still an improvement relative to baseline (12.1 vs. 8.64, p = 0.003). The D L M O phase shift to baseline sleep adjustments may have been responsible for the morning-evening t rend


differences of t rea tment 1. The H A M - D score is correlated with the D L M O phase shift (period 1, r= - 0 . 4 2 5 ; period 2, r= - 0 . 4 6 4 ; bo th ρ < 0.05). Since the baseline sleep adjustment produced a mean D L M O phase advance (0.75 hours) , one would expect D L M O phase delays to evening light to exceed those which might be measured wi thout p repara to ry sleep adjustment . M y own studies, which have not imposed a 6 a.m. wake-up time, have shown no statistical difference in clinical response to morn ing and evening light in period 1.

A corollary of the original phase-shift hypothesis still seems viable: as fall and winter set in, S A D patients show drift toward later circadian phase, perhaps t racking the delay in ou tdoor dawn i l lumination (cf. I l lnerova et al.9 this volume). Fol lowing this spon taneous , seasonal depressogenic phase delay, artificial evening light loses its phase-delaying capacity because the delaying l imb of the phase response curve has also drifted toward a later nightt ime hour . Unde r this delayed condi t ion, artificial morn ing and evening light are bo th ant idepressant . M o r n i n g light, however, exerts a phase-advancing effect tha t reverses the natural ly-occurring, seasonally delayed phase. Fol lowing morn ing light t rea tment , phase delays to evening light are enabled, and evening light loses its ant idepressant quality. Given a si tuation under which evening light fails therapeutically, the crossover experiment provides a type of placebo control—albei t indirect—support ing the hypothesis that light, appropr i -ately delivered, is a specific ant idepressant .

Dummy treatment with a "behavioral placebo"

Eas tman et al. (this volume) have used a deactivated negative ion generator as a placebo control . The pat ient sits either at a light box or an ion generator for equivalent t rea tment dura t ions , thereby equat ing the daily behavioral " investment" in the procedure , which might affect expectations and placebo rate . In contras t , the placebo pill—in studies showing poorer response than to light (e.g. Lingjaerde et al, this volume)—requires less behavioral investment in the therapy, and thus can be faulted for diminishing expectat ions. Eas tman ' s s tudy, which is still in progress, has thus far failed to find a differential benefit of light therapy over placebo in a 4-week t rea tment pro tocol . However , as early as the first week of t reatment , morn ing light showed a substantial size of effect relative to placebo (d = 0.9; Cohen , 1988). As the weeks progressed, pat ients tended to improve regardless of t rea tment mode . It seems quite possible that larger sample size will eventually reveal a statistically significant overall advantage of light (whether for morn ing , or bo th morn ing and evening), with a size of effect similar to that of many large-sample placebo-controlled ant idepressant d rug trials.


Perhaps "brief t rea tment p robes" (e.g. 7-10 days) would be likely to reveal such differences more expeditiously than longer dura t ion t rea tment . O n the one hand , light therapy studies have been criticized for reliance on brief t r e a t m e n t .

26 O n the o ther hand , given the episodic na tu re of S A D ,

spontaneous remissions predictably increase as a function of t rea tment dura t ion . In one sample of 28 SAD pat ients , spon taneous remissions, repor ted retrospectively, began shortly after the winter solstice, in J anua ry (4%) . Twenty- two per cent of pat ients repor ted remission in February , 5 9 % in M a r c h , and 1 3 % in April (Ref. 27, Table 1). Thus , a placebo-controlled s tudy showing cont inuous g roup improvement over the winter mon ths—such as Eas tman ' s—might be expected to show improvement in bo th experimental and control groups .

The end-of-season confound could be eliminated by wi thdrawing pat ients from successful t rea tment and retaining only those showing relapse for g roup compar i sons . Fair ly few patients might be retained, however, after a full m o n t h of t rea tment which terminates in Februa ry or M a r c h . A further complicat ion is the probabi l i ty of relapse even earlier in the season. Some centers have routinely observed relapse within a week or two of wi thdrawal (e.g. B e t h e s d a ,

19 New York C i t y

2 2) while others (e.g.

B a s e l ,

32 G r o n i n g e n ,

14 Oslo [Lingjaerde et al, this vo lume] , R o c h e s t e r

3 5)

have observed little or no relapse. Indeed, the Gron ingen g roup has p roposed a model in which 5 days of light therapy serves to "prevent" recurrence of symptoms across the entire winter s e a s o n .

1 4'

15 In an

a t tempted replication of their pa rad igm in New York, however, only abou t 2 0 % of pat ients resisted relapse across 8 weeks of w i t h d r a w a l .

23

Whether the propensi ty for relapse is a function of par t icular t rea tment parameters , subjects' expectat ions, popula t ion differences, or o ther factors, is a quest ion of increasing urgency.

Selective predictors of clinical response as prima facie evidence against a placebo explanation

If there are aspects of symptomato logy tha t correlate with t rea tment ou tcome in the absence of differential expectat ions, a placebo explanat ion would be unlikely even in the absence of p lacebo control . We examined the two componen t depression scales of the S I G H - S A D

3 1— t h e 21 items of the

H A M - D scale and eight supplementary items characterist ic of atypical depression (ATYP)—in a large set of pat ients while depressed, before they received br ight light t h e r a p y .

30 F o r inclusion, the S I G H - S A D total score

was at least 20, with at least 10 points on the H A M - D and 5 points on the A T Y P subscales. Seventy-one pat ients were judged to be " responders" by the following strict criteria: S I G H - S A D score reduced by at least 5 0 % from baseline, H A M - D and A T Y P scores b o t h 7 or b e l o w ,

28 and Clinical


Global Impress ions—Improvement (CGI-I) scores of 1 ("very much improved") or 2 ("much improved") . Fifteen pat ients were judged to be non-responders by the following criteria: S I G H - S A D scores increased, unchanged, or reduced by less than 2 5 % , meeting original baseline inclusion criteria, with CGI - I score of 4 (unchanged) or higher (worsened). Additionally, a l though the analysis was restricted to initial t rea tment response, patients were not classified as non-responders if they responded even partially in any subsequent t rea tment period.

Figure 5 presents a scat tergram of H A M - D vs. A T Y P scores at baseline, with responders distinguished from non-responders . Positive response was found for every case with a H A M - D baseline score less than 15, regardless of A T Y P score. Fu r the rmore , there were no responders with atypical balance scores [ ( A T Y P / S I G H - S A D ) χ 100] less than 2 9 % . The higher the atypical balance at baseline, the greater the percent improve-ment in the pos t - t rea tment S I G H - S A D score (r = 0.26, /?<0.05) . Expec-tat ion rat ings, however, were not correlated with t rea tment ou tcome for either g roup . Thus , details of the symptom profile—above and beyond the global diagnosis of major depressive disorder with seasonal pa t t e rn—may prove useful in predicting t rea tment ou tcome. The definition of S A D might now be refined, as a light-responsive syndrome characterized by the presence of atypical symptoms (see also, Beck-Friis, this volume).

We examined the frequency and severity of all symptoms on the S I G H -SAD, and the derived scale scores, in an ordered ar ray of effect size (d) between responders and non-responders at baseline (Figure 6). Positive effect size can be considered predictive of response to light therapy, with the mean item score of responders exceeding that of non-responders . In contrast , negative effect size can be considered predictive of non-response.

The strongest positive predictor is the atypical balance score while the strongest negative predictor is overall H A M - D severi ty—both show more than 1 S D unit difference between responders and non-responders , which is a very large size of effect. By compar ison , total symptom severity, as measured by the overall S I G H - S A D score, is a weak negative predictor (d= —0.42). So is depressed m o o d itself (d= —0.24)—which, by defini-t ion, is seen in all pat ients at baseline, responders and non-responders alike. The strong positive predictors of light response (d>0.5) are all atypical symptoms, with hypersomnia , afternoon or evening s lump, and reverse diurnal variat ion ("pre-sleep worse") being most prominent . The appeti te increase and weight gain symptoms follow, with decreasing effect size. The severity of fatigability at baseline (a symptom seen in 9 8 % of patients) , does not predict response to light therapy (d = 0.03) even though responders to light t rea tment always show increased energy. Social wi thdrawal—which was added to the supplementary A T Y P scale even though it is not a neurovegetative symptom per se—falls away from the atypical cluster and is a weakly negative predictor (d= —0.39); this item


Symptom severity/depressed

30

25

s

20

Ο υ M

3

15

"Ε.

< ίο

5

0 5 10 15 20 25 30

Hamilton score

F I G . 5. C o r r e l a t i o n of H A M - D a n d s u p p l e m e n t a r y a typical ( A T Y P ) scale scores at basel ine , for 86 pa t i en t s w h o me t en t ry cr i ter ia for o u r light t r e a t m e n t s tudies : c o m b i n e d scale to ta l ( S I G H - S A D score) of at least 20, H A M - D score of a t least 10, a n d A T Y P score of at least 5. O p e n circles d e n o t e pa t i en t s w h o met remiss ion cri ter ia following light t he r apy . Closed circles a re for n o n - r e s p o n d e r s . Pa t i en t s w h o showed in t e rmed ia t e response were omi t t ed from this analys is . T w o dis t inct clusters of d a t a a re d r a w n , for s u b g r o u p s of pa t i en t s w h o showed : (a) H A M - D scores less t h a n 15, in which case the response ra te was 1 0 0 % ; a n d (b) a typica l

ba lance scores less t h a n 2 9 % , in which case the response ra te was 0 % .

might well be excised from the A T Y P scale. The strongest negative predictors of response to light t rea tment form a distinctly "melanchol ic" g roup , including re tarda t ion , suicidality, typical diurnal var iat ion ("post awakening worse") , somatic anxiety and late insomnia ("early awaken-ing"). Depersonal izat ion/dereal izat ion—which s tands apar t from the core depressive symptoms of the H A M - D scale—also falls squarely in the middle of the negative predictor g roup (d= —0.76).

With such a distinction between positive and negative predictors at baseline, the clinician has a da ta-based tool to begin answering the inevitable quest ion from pat ients , " H o w likely is it tha t light therapy will work for me?" , and perhaps to counsel a l ternate t rea tment modes when the atypical balance is very low. We could no t make such assertions if light were "merely" a p lacebo, unless there were an obscure mechanism linking clinical ou tcome to high expectat ions given an atypical profile, but no such correlat ion given a melancholic profile. The da ta show no such differences, and expectat ion rat ings were not significantly correlated with S I G H - S A D , H A M - D , A T Y P or C G I severity scores at baseline.

Responders (N - 71) Nonresponders (N • 15)

Ο Ο

ATYP total score <29%

1


ATYPICAL BALANCE

* Hypersomnia (73**) *PM Slump (69)

*Pre-Sleep Worse (34) ATYPICAL SCORE

•Carbohydrate Craving (85) -

* Increased Eating (69) -* Weight Gain (59) -

* Appetite Increase (71) -•Fatigability (98) -

Somatic Symptoms - Fatigue (91) -Libido (79) -

Hypochondriasis (37) -Insomnia - Middle (53) -Depressed Mood (100)

•Social Withdrawal (94) Activity (100)

SIGH-SAD SCORE

Guilt (80) Appetite Loss (24)

Psychic Anxiety (86) - | Insomnia - Early (24) Insomnia - Late (33)

Somatic Anxiety (64) Post-Awakening Worse (48)

CGI GLOBAL SEVERITY

Depersonalization (20) Suicidality (39)

Retardation (21) HAMILTON SCORE

-1.5

K W W N

S 3 S3 1

ΕΣ Ε

E S

E S S

WSSSNSM

71 responders 15 non-responders 86 total subjects

* atypical symptoms ** item frequency

*** responders minus non-responders

-1.0 -0.5

Effect Size (d)***

F I G . 6. R e s p o n d e r s a n d n o n - r e s p o n d e r s to light t r e a t m e n t a re c o m p a r e d in t e rms of effect sizes of scale a n d i tem-within-scale scores a t basel ine . Resul ts a r e r a n k -o rde red from the grea tes t posi t ive effect size to the greates t negat ive effect size. T h e frequency of occur rence of each s y m p t o m is s h o w n in paren thes i s , wi th s y m p t o m s showing less t h a n 2 0 % frequency omi t t ed from the analys is . S y m p t o m frequency is n o t cor re la ted wi th effect size—if near ly all pa t i en t s show a s y m p t o m (e.g. fatigabili ty), it c a n n o t be a p red ic to r of t r e a t m e n t response . H o w e v e r , these s ame s y m p t o m s show ma jo r i m p r o v e m e n t u n d e r successful l ight t he r apy , wi th little o r

n o change a m o n g the n o n - r e s p o n d e r s .


This work was sponsored in par t by G r a n t MH43219 from the U S Nat iona l Inst i tute of Menta l Heal th . I thank J iuan Su Terman , P h . D . , for col laborat ion in all its aspects; Leora Amira , P h . D . , and D o n a l d C. Ross, P h . D . , for contr ibut ions to da t a analysis; C h a r m a n e Eas tman , P h . D . , Alfred J. Lewy, P h . D . , M . D . , and Rober t L. Sack, M . D . , for sharing da ta ; and William Gruen , M.S. , of Ambula to ry Moni to r ing , Inc. , for loan of the Acti l lume™ appara tus . (The au tho r is co-inventor of the 10,000 lux light therapy appara tus , with rights assigned to the Research F o u n d a t i o n for Menta l Hygiene, Inc. , New York Psychiatric Insti tute Division.)


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30. T e r m a n M . , T e r m a n J .S . a n d A m i r a L. (1992) Light-refractory a n d l ight- responsive S A D pa t i en t s : depress ion scale p red ic to r s . Soc. Light Treatment Biol Rhythms Abst. 4, 18.

3 1 . Wil l iams J .B.W. , L ink M.J . , Rosen tha l N . E . , A m i r a L. a n d T e r m a n M . (1992) Structured Interview Guide for the Hamilton Depression Rating Scale—Seasonal Affective Disorder Version (SIGH-SAD). Rev. ed. N e w Y o r k Sta te Psych ia t r i c Ins t i tu te , N e w York .

32. Wirz-Jus t ice Α., Bucheli C , G r a w P . , Kie lholz P . , F isch H . - U . a n d W o g g o n B. (1986) Light t r e a tmen t of seasonsal affective d i so rde r in Swi tzer land. Acta Psychiatrica Scand. 74, 193-204.

33. Wirz-Jus t ice Α., Schmid A.C . , G r a w P . , K r â u c h i Κ. , Kie lholz P . , Pô ld inge r W. , F isch H . - U . a n d B u d d e b e r g C . (1987) D o s e re la t ionships of m o r n i n g br ight whi te light in seasonal affective d i so rde r (SAD) . Experientia 4 3 , 574-576 .

34. Wirz-Jus t ice Α., G r a w P . , K r â u c h i Κ. , Gis in Β. , J o c h u m Α., Arend t J., F isch H . -U . , B u d d e b e r g C . a n d Pô ld inge r W . (1993) Light t h e r a p y in seasonal affective d i sorder is i ndependen t of t ime of d a y or c i rcad ian phase . Arch. Gen. Psychiatry, in press .

35. Yerevan ian B.I. , A n d e r s o n J .L. , G r o t a L.J. a n d Bray M . (1986) Effects of br ight incandescen t l ight o n seasona l a n d n o n s e a s o n a l ma jo r depress ive d i sorder . Psychiatr. Res. 18, 355-364 .

32

Melatonin: Historical Aspects* A A R O N B. L E R N E R

Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510, USA

O N L Y A handful of people were involved in the isolation of melatonin from the pineal gland. D r Yoshiyata Takahash i and I initiated the work at Yale in 1955, and it was completed by D r James Case and me in 1959. Assays of the pineal extracts for activity were carried out by D r Takahash i , D r Mor i or D r M . Ru th Wright . Biologic studies were done with D r W a t a r u Mor i and Jack Barchas . Fresh frozen bovine pineal glands were supplied to us by the thousands th rough D r Joseph Fisher of the A r m o u r Labora tor ies in K a n k a k e e , Illinois. Assistance with the chemistry came from D r Richard V. Heinzelman of the Upjohn C o m p a n y in K a l a m a z o o , Michigan, and the determinat ion of the s t ructure of the methyl-ester of 5-methoxyindole acetic acid was done with D r Klaus Biemann at the Massachuset ts Inst i tute of Technology.

In our research on normal and mal ignant pigment cells, we were studying the act ion of ho rmona l and neural factors on the dispersion and aggregation of melanosomes in frog melanocytes . It was 1955 and we had just come to Yale from the University of Oregon Medical School. At Oregon we had isolated a melanocyte st imulat ing h o r m o n e (MSH)—the dispersing factor—from the pi tui tary gland. Even though the M S H work was going to be cont inued at Yale, we wanted to know whether there were any neural aggregating factors in addi t ion to adrenal ine, noradrenal ine , and acetylcholine. D r Takahash i found a single reference to a paper by M c C o r d and Allen in The Journal of Experimental Zoology, in 1917, in which the au thors described the l ightening—that is, aggregating ac t ion— of extracts from the pineal gland. In the pi tui tary work, there were numerous papers going back to 1914. But for the pineal gland there was only a single paper . Could the agent in the pineal gland be adrenal ine,

* This paper is modified in parts from an essay written by the au thor for the European Pineal Study G r o u p Newsletter, number 4, September 1980.

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noradrenal ine , or acetylcholine? It was easy for us to show tha t the chemical propert ies of the pineal factor did no t match any of those three compounds . W a s it a k n o w n factor or something not yet described? D r Takahash i and I decided to m a k e a determined effort to find out .

T o test for the aggregat ion factor, which we later called mela tonin , D r Takahash i modified the assay tha t Dr . K a z u o Shizume and I had developed for M S H . In tha t in vitro assay, we measured by reflectance the darkening produced by M S H on isolated pieces of frog skin. The new assay for mela tonin turned out to be more tricky because we had to first use M S H to darken the skin in order to later detect the presence of a l ightening substance. Takahash i performed the bioassays, and I did all the extract ions. We did no t know tha t it would take 4 years of ha rd work and 250,000 pineal glands to finish the project.

At the end of 2 years, we had a reliable assay, and we knew many of the properties of melatonin. We could follow the activity through silicic acid column chromatography, and we were able to measure the agent's fluorescence spectrum. At that time, D r Takahashi had to return to Japan . He was afraid that the people in Japan would forget about him and that he would not be able to find a j o b . The assays were taken over by Drs Wata ru Mor i and M . Ruth Wright. F o r help with the isolation, D r James D . Case, whom I had known as a student at the University of Oregon Medical School, came to learn clinical dermatology and, at the same time, carry out biochemical investigations. He brought with him tremendous ability and a great capacity for work. His untimely death a year after he was made head of the section of dermatology at the University of Washington Medical School in Seattle was an enormous loss from which none of us will ever recover.

In abou t a year's t ime, J im Case and I were able to show that melatonin was an indole derivative and that on electrophoresis it had no charge. We were also able to isolate, in approximate ly 10 times greater quant i ty than melatonin , a substance tha t had the same fluorescence and ultraviolet light absorp t ion spectra and also gave the same color tests as melatonin . Yet, the c o m p o u n d was inactive biologically and behaved as an organic acid on electrophoresis. Since we could never get as much as 1 mg of melatonin to work with, it seemed tha t we should hold off working on its s t ructure and concentrate on the biologically inactive acid.

I would take the 5 a.m. train from New Haven to Boston, carrying samples to D r Klaus Biemann at M.I .T . D r Biemann was a then relatively u n k n o w n young m a n doing research on the s tructure of organic compounds by using mass spectrometry to determine molecular weights of fragments obta ined from subjecting the compounds to an electric discharge. N o w D r Biemann is world famous for his research. The biologically inactive organic acid turned out to be non-volati le. F o r this reason, we made the methyl-ester. Wi th only a milligram or so of the methyl-ester, we got an accurate molecular weight determinat ion of the

Melatonin: Historical Aspects 439

entire molecule as well as the fragments. It was obvious that the s t ructure of the molecule had to be 5-methoxy-indole acetic acid. There was no alternative. This c o m p o u n d was then m a d e by the people at Up john , and they confirmed the s t ructure tha t we h a d derived from mass spectro-metry.

We still were unable to obta in enough pure mela tonin to carry out experiments on s tructure. We could not even get an elementary analysis for carbon, hydrogen, ni t rogen, and oxygen. The material was not volatile enough to use in the mass spectrometer . W e realized tha t well over a million pineal glands would have to be collected and worked up in order to isolate as much as 1 mg of mela tonin . At this point , because of our inability to get any sizeable quanti t ies of mela tonin , the work became very discouraging. J im Case and I decided tha t if we could no t find a good lead within a mon th ' s t ime, it would be best to d r o p the isolation project, go on to something else, and wait until one of the pharmaceut ica l houses would carry out the extract ions of the millions of glands required to solve the problem. Abou t a week before our deadline, which represented the t ime I was to go with my family to Cape Cod , I was discussing the metabol ism of serotonin with ano ther associate, D r Joseph McGui r e . We were reviewing the work done at the Cleveland Clinic which showed that the major p roduc t of serotonin metabol ism was N-acetylserotonin. As the discussions progressed, I in terrupted Joe McGui r e and said, "I know what the s t ructure of mela tonin is. It undoubted ly is the methoxy derivative of JV-acetylserotonin." Within an h o u r J im Case and I looked over the numerous indole derivatives we had collected for reference and found tha t we had 5-ethoxytryptamine. W e acetylated the c o m p o u n d and gave it to Ru th Wright to assay the next morn ing . A couple of hours later, Ru th came in and said, "This is it!" Never had she tested anything as poten t as the new c o m p o u n d . We k n o w we were on the right t rack and now had to m a k e the methoxy instead of the ethoxy derivative. J im Case did this a week later when I was at C a p e C o d and everything checked. Even though we were no t able to determine the s t ructure of mela tonin by a direct a t tack, perserverance, and a lucky guess paid off. The problem was solved.

Before we found the s t ructure of mela tonin , everyone said tha t biologic amines, such as adrenal ine, noradrenal ine , his tamine, all the antihista-mines and m a n y of the tranquil izers, had to have a ni t rogen carrying a positive charge. When acetylated, the charge was gone and biologic activity lost. But in p igmenta t ion , removing the charge on the ni t rogen greatly enhances the potency of the substance. Mela ton in was 100,000 times more po ten t t han noradrena l ine or acetylcholine in l ightening frog skin. Also, mela tonin and 5-methoxy-indole acetic acid turned out to be the first methoxy-indoles isolated from m a m m a l i a n tissue.

An impor tan t reason for the success of the mela tonin project was the


good fortune I had to work with people who were unusually capable . Drs Takahash i , Mor i , Case, Barchas , and Wright were exceptional indi-viduals. There were no technicians other than our excellent dishwasher, Mrs K a t e Lehmann .

Biographical sketches

YOSHIYATA TAKAHASHI , M . D . After he was gradua ted from the University of Tokyo Medical School, D r Takahash i did a residency in internal medicine before coming to the Uni ted States. W e worked together for 1 year at Oregon on the mechanism of pigment granule movement and 2 years at Yale on the mela tonin project. He became Cha i rman of the Depa r tmen t of Internal Medicine at Gifu University and then President of the hospital in that city. He was the leading hepatologist in J apan . W A T A R U M O R I , M . D . D r M o r i was gradua ted from the University of Tokyo Medical School and took a residency in pathology. We worked together at Yale on the biology of mela tonin and the assay for almost 3 years. He re turned to J a p a n as a member of the faculty in the Depa r tmen t of Pa thology . He later became Cha i rman of that Depa r tmen t and then Dean of the Medical School. He recently completed a 4-year term as President of T o k y o University. He is a foreign associate of the Insti tute of Medicine of the Na t iona l Academy of Sciences. JAMES CASE, M . D . James Case received his medical degree from the University of Oregon Medical School. As a medical s tudent he was familiar with our research on pigment cells. He came to Yale to do a residency in dermatology in our Depa r tmen t and to take par t in the isolation of mela tonin . J im Case was a brilliant s tudent . He left Yale to become head of the Dermato logy Division at the University of Wash-ington in Seattle. His dea th at an early age was a blow to all of us. M. R U T H W R I G H T , M . D . Dur ing the Second World War , D r Wright was a medical s tudent at the Universi ty of Manches te r in England. After 2 years, she was sent to Vanderbi l t Medical School to complete her medical training. She and D r Mor i did the assays for melatonin after D r Takahash i left. J A C K B A R C H A S , M . D . As a medical s tudent at Yale dur ing the t ime that the mela tonin project was going on, D r Barchas did his work on a thesis with me. He was interested in neurochemistry and carried out research on the presence of mela tonin in peripheral nerves. D r Barchas became Co-cha i rman of the Depa r tmen t of Psychiatry at Stanford Medical School where he studied the process of sleep. He went on to become Dean of Neurosciences at U C L A and now is Professor and C h a i r m a n of the Depa r tmen t of Psychiatry at Cornell Medical School.

Melatonin: Historical Aspects 441

A reun ion wi th A a r o n Lerne r in 1969 of p rev ious fellows a n d their wives. S t a n d i n g (left to r ight ) : M r s M o r i ; D r N o b u H a t t o r i , Professor of O n c o l o g y ; D r W a t a r u M o r i , Professor of P a t h o l o g y ; D r Yosh iya t a T a k a h a s h i , Professor of Medic ine ; M r s T a k a h a s h i ; D r K a z u o Sh izume, Professor of Medic ine . Seated (left to r ight ) : D r T a r o K a w a m u r a , Professor of D e r m a t o l o g y ; D r M a r g u e r i t e Lerner ; D r

A a r o n Lerner ; M r s K a w a m u r a ; a n d M r s Sh izume .


K A T E L E H M A N N came from a well-to-do family in Ge rmany . She was told tha t women did not need an educat ion because they would have suppor t from their husbands . She was an exceptionally able and alert person who would have received professional t raining under other circumstances. Her dedicat ion to keeping our glassware uncontamina ted so that the assays could be carried out was a critical and difficult task.

Afterword The Wenner-Gren Center Foundation for Scientific Research

B y D A V I D O T T O S O N

A VISITOR to S tockholm coming from the a i rpor t and entering the city from the N o r t h canno t escape from seeing a high building of mode rn architecture su r rounded by a lower semicircular apa r tmen t house . This is the Wenner -Gren Center . It was created in 1955 th rough a dona t ion by a wealthy Swedish industrialist Axel Wenner -Gren . H e was a self-made m a n w h o star ted from no th ing to become one of the richest men in Sweden a n d in Eu rope at this t ime. He star ted by selling vacuum-cleaners and was so successful in doing this tha t he after a few years established the now well-k n o w n company Elektrolux. He expanded his activities to a n u m b e r of countr ies and by skilled investments soon created a world-wide industrial empire . At the same t ime, being a smar t business m a n he was also an idealist and something of a dreamer . O n e of his d reams was to create an in ternat ional science center where scientists could come together to exchange ideas a n d feel at home . H e desired this center to also provide accommoda t ion for their families so as no t to be only a meeting point bu t also a place where scientists could stay for longer periods of t ime. He d reamed of such centers being established in various countries bu t he only saw his d ream realized only in Sweden with the creat ion of the Wenner-Gren Center .

The center consists of three buildings named the Pylon, the Helicon and the Te t ragon . The Pylon, 24 storeys high, provides space for adminis t ra-tive depar tments of m a n y of Sweden's research organizat ions , the Helicon living quar ters and depar tments for visiting scientists and their families and the Te t ragon , an aud i to r ium for conferences.

With in the framework of its scientific activities the Wenne r -Gren Center F o u n d a t i o n is annual ly organizing 6-8 in ternat ional and nat ional conferences covering a variety of themes, bu t mainly in the field of

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444 Afterword

neuroscience. The proceedings of these conferences are published in the Wenner -Gren In terna t ional Series. The present volume is the 63rd in this series. In addi t ion to the symposium p r o g r a m m e the F o u n d a t i o n has developed an extensive fellowship p r o g r a m m e for foreign scientists doing research in Sweden and for Swedish scientists going a b r o a d for research work.

In 1992, 82 foreign scientists from 30 countries received fellowships for work in Sweden and 26 Swedish scientists for work ab road . The F o u n d a t i o n is also providing travel fellowships for young scientists to a t tend conferences ab road .

The F o u n d a t i o n has th rough these various p rogrammes established itself as an impor t an t body for suppor t of science in Sweden and the Wenner -Gren Center has been recognized as a truly internat ional science center, thereby fulfilling the d ream of its creator , Axel Wenner -Gren .

SUBJECT INDEX

Adenosine, melatonin synthesis regulation 134-140

Adrenocorticotrophic hormone 251 Age, influence on urine melatonin

concentration 275-284 Asthenic reaction, differences from

depression 415 Atrial natriuretic peptide 252

Beck's Depression Inventory 354, 405 Blind humans,

circadian rhythms 207 melatonin rhythms 174 sleep disturbance 211

Cfos 8-14 gene expression 98

Cancer, and pineal gland 338 seasonality 337

Circadian, melatonin rhythm 145-158 oscillators, definition 2 pacemaker, effect of light 217-235 pacemakers, definition 3 photoreception 73-88 photosensitivity, assay method 82 rhythms,

in blind humans 207 morningness-eveningness

287-304 signals, maternal communication to

fetus 94 systems,

components 2 physiological organisation 2

Circannual rhythms, definition 314 Cocaine 98 Comprehensive Psychopathological

Rating Scale 354,396 Cortisol 251

Delayed sleep phase insomnia (DSPI), 212 syndrome 261-270

Depression, light treatment 351-369 Diagnosis, seasonal affective disorders

411-418 Diagnostic systems, for mental

dysfunction 416 Diagnostic & Statistical Manual of

Mental Disorders (DSM-III-R) 352,411

Dim light melatonin onset (DLMO) 175

Dopamine DI receptors 98 system, in fetal SCN 98

Drug treatment, winter depression 395-398

Electrical activity, SCN 218 Endocrine function, circannual

variation 313-323 Endogenous components, of circadian

rhythm measurements 189 Enkephalins, melatonin synthesis

regulation 134-140 Entrainment 233

in circadian function 73 melatonin infusion 107-119 melatonin rhythm with light 205 of fetal SCN 93-101 postnatal maternal 100 social factors 209

Epithalamine 329-335 Evening active persons 287-304 Evolution, melatonin rhythm

generating system 65 Exogenous components, of circadian

rhythm measurements 189 Extraretinal photoreceptors 74 Eye, in melatonin rhythm generating

system 58

445

446 Subject index

GABA-a receptor, melatonin interaction 128

GABA, melatonin synthesis regulation 134-140

Gas chromatographic-negative chemical ionization mass spectrometric assay, 173 gaze direction, influence on light effects 33

Gene expression, circadian rhythms 1-15

Growth hormone 249

Hamilton Depression Rating Scale 354, 373, 405

Hamilton Depression Rating Scale 43 Height, influence on urine melatonin

concentration 275-284 Hormone rhythms, relationship with

sleep 247-258

Insulin 253

Jet lag, effect of melatonin 177, 210 sleep disturbance 240

Jun Β 8-14

Late luteal phase dysphoric disorder 401,415

Latitude, influence on urine melatonin concentration 275-284

Lenticular transmission 35 Light,

as therapeutic intervention 421-434

control of human circadian pacemaker 217-235

effects on circadian rhythm in depression 385-391

effects on human rhythms 203-213 efficacy as melatonin synthesis

inhibitor 145-158 intensity, suppression of pineal

melatonin 149 physical aspects 23-28 quantitative measures 23-28

treatment, depressive states 351-369 premenstrual depression

401-409 seasonal affective disorder 371

wavelength, suppression of pineal melatonin 149

Luminous flux 25

Macromolecular synthesis, role in circadian rhythms 6

Masking, circadian rhythm measurements 189-201

Maternal entraining signals 96 Maternal-fetal communication,

circadian phase 96 Mathematical modelling,

sleepiness/alertness 237-246 Melatonin 55-66

as endogenous time cue 212 assay 173 biosynthesis, norepinephrine

133-140 daily infusion, hamsters 107-119 delayed sleep phase syndrome 266 effects on human circadian rhythms

203-213 history 437-442 jet lag, 177 light-induced suppression 30 maternal-fetal communication 96 onset, marker for circadian phase

174 production, endogenous, function

181 receptors, brain distribution

121-129 rhythm

generating system, mammalian 56

human saliva 161-170 seasonal

rhythmicity in cancer 338 variations 315-318

secretion, and daylength 204 light suppression 204

urine concentration, influencing factors 275-284

use in jet lag 210 shift workers 211

Fetal biological clock 93-101

Subject index 447

Melatonin-suprachiasmatic nucleus feedback loop 63

Menstrual cycle, and light 305-311 Moclobemide 395-398 Montgomery-Asberg Depression

Rating Scale 396 Morning active persons 287-304 Mourning, differences from

depression 414

Neuroendocrine function, circannual variation 313-323

Neuropeptide Y, melatonin synthesis regulation 134-140

Norepinephrine 60,146 regulation of melatonin synthesis

133-140 Nychthemeral rhythms 193

Ocular media transmission, influence on light effects 34

Pancreatic hormones 253 Paraventricular

nuclei 118 nucleus of hypothalamus 60

Pars tuberalis, melatonin target 121-129

Peptic ulcer 329-335 Phase

advances, melatonin rhythms 163 delays, melatonin rhythms 162 response curve, melatonin 180

Photic entrainment, functional properties

4 regulation, of early genes in SCN 8

Photometric quantities, definitions 25 Photoperiodic signals 164 Photoreceptor proteins 81 Photoreceptors,

identification in mammals 80 identification in non-mammalian

vertebrates 78 Pineal gland 107,133-140,145-158

and cancer 338 in melatonin rhythm generating

system 59 role in circadian rhythm control,

mammals 211 melatonin synthesis, and light 146

N-acetyltransferase activity, rat 161-170

signal transduction 60 substances, and tumour inhibition

341 Placebo

control, for light treatment 371, 425

controlled light treatment studies 371-382

Postnatal maternal entrainment 100 Premenstrual depression 401-409 Prolactin 249 Protein synthesis inhibitors 7 Proto-oncogenes 8-14 Pupillary diameter, mediation of

biological light effects 38 Purification methods, for field rhythm

data 189-201

Rectal temperature rhythm 191 Reentrainment 233 Renin-angiotensin-aldosterone system

252

Seasonal affective disorder (SAD) 43, 352,

385,411-418 light treatment 371-382 reliability of diagnosis 412 subdromal 411-418

breeding, non-mammalian vertebrates 76

differences, morningness-eveningness 287-304

variations, definition 314 Seasonality, pineal turn our-inhibiting

substances 343 Serotonin 55

melatonin synthesis regulation 134-140

Sex, influence on urine melatonin concentration 275-284

Shift work, effect of melatonin 211 workers, sleep disturbance 237

Siberian hamsters 107-119 Signal transduction,

melatonin receptors 125-129 pineal gland 60

Skeleton photoperiods 208

448 Subject index

Sleep activity cycle 192 disturbance,

blind humans 211 mechanism 239

relationship with hormone rhythms 247-258

wake regulation 237-246 Substance P, melatonin synthesis

regulation 134-140 Superior cervical ganglia 59 Suprachiasmatic nuclei 217

electrical activity 218 endogenous pacemaker 174 melatonin target 121-129

Suprachiasmatic nucleus 81, 93-101, 203

mammalian 3,30,41,59 Syrian hamsters 107-119

Taurine, melatonin synthesis regulation 134-140

Thyrotropin 250 Tricyclic antidepressants 395

Ultraviolet radiation 25 Unmasking methods, circadian rhythm

measurements 189-201 Urine volume, influence on urine

melatonin concentration 275-284

Vasoactive intestinal peptide (VIP), melatonin synthesis regulation 134-140

Visible spectrum 24

Wavelength sensitivity 42 Weight, influence on urine melatonin

concentration 275-284 Winter depression 43, 385-391,

395-398 light treatment 371-382,395-398

Light and Biological Rhythms in Man

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