Melatonin affects circadian rhythmicity in lizards

J. Comp. Physiol. 130, 317-323 (1979) Journal of Comparative Physiology. B �9 by Springer-Verlag 1979

Melatonin Affects Circadian Rhythmicity in Lizards

Herbert Underwood Department of Zoology, North Carolina State University, Raleigh, North Carolina 27650, USA

Accepted January 13, 1979

Summary. Subcutaneous Silastic capsules which continuously released melatonin at low rates (10 gg/day) either (i) lengthened the period of the freerunning activity rhythm in two iguanid lizard species (Scelo- porus occidentalis and S. olivaceus) exposed to either continuous illumination or continuous darkness or (ii) induced arrhythmicity in S. olivc~ceus exposed to continuous illumination. These results and previous observations that two melatonin synl:hesizing sites in lizards, the pineal organ and lateral eyes, are involved in circadian rhythmicity suggest that melatonin may be a chemical messenger between the pineal or eyes and other elements of the circadian system.

Introduction

Recent studies have shown that the pineal organ of birds and the pineal organ and lateral eyes of lizards are important components of the circadian system (Menaker and Zimmerman, 1976; Underwood and Menaker, 1976; Underwood, 1977). Removal of the pineal organ of house sparrows freerunning (showing their endogenous circadian activity rhythm) in continuous darkness renders the birds arrhythmic (continuously active) (Menaker and Zimmerman, 1976). In the iguanid lizard Scelroporus olivaceus removal of the pineal organ of lizards freerunnirLg in continuous illumination (LL) causes either: (i) a "splitting" of the circadian activity rhythm into two components which freerun with different circadian periodicities (ii) marked changes in the period of the freerunning activity rhythm or (iii) arrhythmicity (Underwood, 1977). Removal of the lateral eyes of S. olivaceus exposed to LL also causes changes in the freerunning period or arrhythmicity (Underwood and Menaker, 1976). The effects of blinding on S. olivaceus are not compatible v~ith the hypothesis that blinding has

merely reduced the effective intensity of light reaching the clock (Underwood and Menaker, 1976). These results indicate that the lizard is a "multi-oscillator" system - that is, more than one circadian oscillator is normally involved in generating the circadian activity rhythm - and are consistent with either of two hypotheses: (i) The pineal organ or lateral eyes act as coupling devices between circadian oscillators located elsewhere or (ii) the pineal organ or eyes, them- selves, contain circadian oscillators which interact with circadian oscillators located elsewhere and in- fluence their periodicities. Regardless of which of these hypotheses is correct both demand that a chan- nel of communication, either hormonal or neural, exists between the pineal and eyeg and other elements of the circadian system.

The pineal organ of vertebrates possesses abun- dant biosynthetic capabilities. Among other com- pounds the pineal organ is capable of synthesizing biogenic amines such as serotonin and melatonin (Quay, 1974; Ralph, 1976; Reiter, 1977). Historically, interest centered on melatonin as being a unique pineal hormone since melatonin was detected in the plasma of a variety of vertebrates and the terminal enzyme in the synthesis of melatonin, hydroxy-indole- O-methyltransferase (HIOMT), was believed to be present only in pineal tissue. Recently, however, other tissues have also been shown to possess melatonin synthesizing capabilities, such as the retina and the Harderian gland (Bubenik et al., 1976). The relative contribution of these areas to plasma melatonin levels in many vertebrates is unknown. A remarkable feature of pineal biochemistry in the higher vertebrates is the presence of large daily rhythms in pineal enzyme activities and biochemical concentrations (including serotonin and melatonin) (Quay, 1974; Ralph, 1976; Reiter, 1977). Studies on such rhythms within the pineal organs of lower vertebrates are scarce but a few studies show that daily rhythms are also present

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318 H. Underwood: Melatonin Affects Circadian Rhythmicity in Lizards

in the pineal organs of fish and reptiles (Vivien-Roels etal., 1971; Smith and Weber, 1976; Joss, 1977). Among the reptiles, for example, a daily rhythm in pineal serotonin content has been described in turtles (Vivien-Roels et al., 1971), and a daily rhythm in plasma melatonin levels has recently been demon- strated in a scincid lizard (Tiligua rugosa) (Kennaway et al., 1977). In addition, Quay (1965) has shown significant HIOMT activity in the pineal organ and retina of several reptiles.

The observations that both the pineal and eyes are involved in circadian organization in lizards and the fact that both these organs are also active sites of melatonin synthesis led me to examine the effects of melatonin administration on circadian rhythmicity in lizards. These studies were facilitated by the introduction of a new method of melatonin administration by Turek et al. (1976). These workers have demon- strated that melatonin is released from Silastic capsules at low rates for long periods of time.

the slope of a line drawn through the successive daily onsets of activity. Empirical studies on the reliability of estimating z using eyefits have shown that eye-estimates give an accurate measure of period length (Pittendrigh and Daan, 1976a). The first 2-7 days of a freerun following a change in conditions were usually characterized by transient phase-shifting and these onsets were not used in the determination of the freerunning period. However, spontaneous changes in the periods of freerunning rhythms are a common feature in many organisms, including lizards. In the present study most lizards exhibited relatively stable freerunning periods. In those cases in which spontaneous changes were evident (as seen, for example, in the initial portion of Fig. 1 B) z estimates were more difficult. In cases where spontaneous changes were seen,

estimates were based on that portion of the record which imme- diately preceded the change in conditions for example, in the case of Fig. 1 B the estimate of z of the unimplanted lizard in LL was based on the activity onsets for 14 days preceding the melatonin implant.

The part of each cycle during which activity occurs is desig- nated c~. The value of ~ is the interval (in hours) between eye-fitted lines marking onsets and cut-offs of activity. Estimates of c~ are less precise than those of ~ because daily activity cut-offs are less well defined than daily activity onsets.

All statistical comparisons utilized the t-statistic for dependent paired data.

Materials and Methods

Experimental Conditions

The activity of individual lizards of both sexes was monitored by "tilt cages" connected to an event recorder (Esterline Angus). The tilt cages were either: (1) visually isolated from one another and exposed to constant fluorescent illumination (General Electric F48T12/cw bulbs) in a large environmental chamber held at 29 ~ (+0.5 ~ ) or were (2) individually maintained inside light-tight wooden boxes which, in turn, were kept in a large environmental chamber held at 29 ~ (+0.5~ Light sources for these boxes consisted of fluorescent bulbs (Sylvania F4TS/cw). Different inten- sities of LL were obtained by partially masking the fluorescent bulbs with aluminum foil and black type. DD lizards were maintained in constant darkness within individual boxes except for a brief light exposure during insertion or removal of a Silastic implant. Lizards in LL had food (live mealworms) and water continuously available. Lizards in DD would not feed but had water continuously available. Silastic capsules (Dow Coming catalog no. 602-235, 1.47 mm inside diameter and 1.96 mm outside diameter) 10 mm long were filled with crystalline melatonin (Sigma) and sealed at both ends with Silastic medical adhesive. Capsules were implanted under the skin on the dorsal surface. The melatonin release rate from the Silastic capsules depends upon capsule length (Turek et al., 1976). The release rate of the 10 mm capsules used in the present study was 10 +_ 1 gg/day (av. + 1 S.E.) and was determined by weighing the capsules (after cleaning and drying) both before and after the experiment. Control experiments consisted of implanting empty Silastic capsules in place of the melatonin filled capsules.

Assay

The estimate of the freerunning period (~) of the activity rhythm in constant conditions was based on the onset of activity which was a more precise "marke r " of the rhythm than the end of activity. The freerunning period was determined by calculating

Results

Subcutaneous implantation of melatonin-filled Silas- tic capsules into the iguanid lizards S. occidentalis and S. olivaceus had marked effects on the period of their activity rhythms and implicates melatonin as a chemical messenger between the pineal and eyes and other elements of the circadian system. Figure 1 shows the effect of exogenous melatonin on the freerunning circadian activity rhythm of a S. olivaceus (Fig. 1 A) and a S. occidentalis (Fig. 1 B). Note that melatonin induced a marked lengthening of v in both lizards in LL, and in the S. occidentalis shown (Fig. 1 B), a shortening in z was observed in DD after removal of the melatonin implant showing that the melatonin had induced a lengthening in z in DD as well. Exogenous melatonin induced a lengthening in r in every S. occidentalis implanted while freerunning in LL (Fig. 2A). The significance of these differences was confirmed by statistical tests. Paired ~ tests showed that melatonin caused a significant lengthening of the periods of the S. occidentalis freerunning in LL of either 10 lux (P< 0.005) or 50 lux (P<0.005). Melatonin also caused a lengthening of the period of the activity rhythm in DD in every S. occidentalis tested (Fig. 2B). These differences were significant (P < o.oos).

Similar results were obtained with S. olivaceus. Of 11 S. oIivaceus tested in LL, a 10 mm melatonin capsule induced lengthening of the freerunning period in 9 lizards (Figs. 1 A, 3) and induced arrhythmicity in two (Fig. 4). The freerunning periods of the S.

H. Underwood: Melatonin Affects Circadian Rhythmicity in Lizards 319

Fig. 1 A and B. Cont inuous records of the locomotor activity of two lizards in constant conditions in which 10 m m Silastic capsules containing melatonin were implanted. The records were pho- tographically duplicated and double plotted on a 48-h time basis to aid in interpretation. Record (A) shows a S. olivaeeus implanted (a) while freerunning in LL (10 lux). Note the marked lengthening in r after implantation. Record (B) shows a lengthening in r also occurs in a S. occidentalis implanted (a) while freerunning in LL (10 lux). When the lizard bearing the implant was placed into continuous darkness (b) ~ shortened and, after removal of the implant in DD (c), z shortened even further. The shortening which occurred when the lizard was placed into DD is consistent with the observation that the diurnal S. oecidenmlis obey Aschof?s Rule for nocturnal auimals (see text)

olivaceus tested in LL of 50 lux significantly (P < 0.005) lengthened in the presence of melatonin. In LL of t0 lux the sample size was too small (2 cases) for statistical comparison but both lizards clearly lengthened ~ in the presence of exogenous melatonin (Fig. 3). Although a systematic investigation of the effects of exogenous melatonin on S. olivaceus in DD was not undertaken, in the only two S. olivaceus tested, removal of the melatonin capsules in DD elicited a shortening in ~ (not shown) showing that S. olivaceus responds similarly to S. occidentalis in DD.

Control experiments consisted of removing the melatonin-filled capsules in 10 lizards (4 S. olivaceus and 6 S. occidentalis) in LL and replacing them with empty capsules (Fig. 5). In these 10 lizards a clear shortening of freerunning periods was observed after implantation of the empty capsules showing that the effect of the melatonin-filled capsules was due to melatonin and not to some other aspect of the experimental manipulation.

A dose-response relationship between melatonin and r was not systematically investigated. However 2 lizards (1 S. occidentalis and 1 S. olivaceus) were tested for a dose-response effect of melatonin. One of these is shown in Fig. 6. In both lizards a 10 mm melatonin-filled capsule caused a lengthening in v and replacement of the 10 mm capsule with a 20 mm capsule induced a further lengthening.

The activity records shown in Fig. 1 B, 5 and 6 strongly suggest that melatonin may affect activity time (~) as well as ~ in S. occidentalis. This relationship was confirmed by statistical tests. In S. occidentalis melatonin caused a significant (P < 0.001) decrease (an average of 3.6 h) of ~ in LL and a significant (P<0.05) decrease (an average of 2.2 h) of c~ in DD as well. However, no significant effects of melatonin on ~. in S. olivaceus were noted.

Aschoff's Rule

Aschoff (1960) formulated an empirical generalization about the relationship between the diurnal or nocturnal habits of animals and the length of their circadian periods in different constant conditions. This generalization, which has become known as "Aschoff 's Rule" postulates that (1) light-active (diurnal) animals show a shorter circadian period in LL than in DD while the opposite holds for dark-active (nocturnal) animals, and (2) in LL the period decreases with increasing intensity of illumination in diurnal animals, and increases in nocturnal animals. Although most animals examined to date obey the rules, exceptions are known (Hoffmann, 1965). An additional general-

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H. Underwood: Melatonin Affects Circadian Rhythmicity in Lizards

i I I 1 L L L L+ Melatonin DD D D + Melatonin

Fig. 2A and B, Lengthening in the period of the activity rhythm of S. occidental& in response to 10 mm melatonin capsules. In (A) the lizards were freerunning in LL of either 10 lux (circles) or 50 lux (triangles). B shows the results of two kinds of DD experiments: either melatonin capsules were implanted in lizards while they were freerunning in DD (squares) or melatonin capsules were removed from lizards while they were freerunning in DD (closed circles) (refer to Fig. 1B)

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Fig. 3. Lengthening in the period of the activity rhythm of S. olivaceus in response to 10 mm melatonin capsules. The lizards were freerunning in LL of either 10 lux (circles) or 50 lux (triangles)

Fig. 4. Arrhythmicity in a S. olivaceus in response to a 10 mm melatonin capsule (a). The lizard was maintained in LL (50 lux)


Fig. 5. A control S. olivaceus in which a 10 mm melatonin capsule [implanted at (a)] was replaced by an empty 10 mm capsule (b). The lizard was maintained in LL (50 lux)

Fig. 7. A record of a (diurnal) S. occidentalis obeying Aschoff's Rule for nocturnal animals. The lizard was initially freerunning in DD and was then placed in LL (10 lux) at (a). Note that rLL is much longer than ZDD

Fig. 6. A dose-response effect ofmelatonin on ~. The S. occidentalis, freerunning in LL (50 lux), was implanted with a 10 mm melatonin capsule (a) ; this implant was removed and replaced with a 20 mm melatonin capsule (b)

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Fig. 8. The effect of different constant conditions on the period of the activity rhythm of the diurnal S, occidentalis. Normal (unimplanted) lizards exhibit a shorter ~ in DD than they do in LL of either 10 lux (circles) or 50 lux (triangles) showing that they obey Aschoff's Rule for nocturnal animals

322 H. Underwood: Melatonin Affects Circadian Rhythmicity in Lizards

ization which has never been formally incorporated into the rule is that at sufficiently high levels of LL most animals will become arrhythmic. The intensity of LL which produces arrhythmicity in lizards (as in other animals) shows both inter- and intraspecific variation. The arrhythmicity thresholds for S. olivaceus are usually above 450 lux (Underwood and Menaker, 1976) whereas thresholds in individual S. occidentalis range from 5 to 90 lux. We have previously shown that the diurnal S. olivaceus tend to obey Aschoff's Rule for diurnal animals (Underwood and Menaker, 1976). However in the present study, almost without exception, the diurnal S. occidentalis obeyed Aschoff's Rule for nocturnal animals. In 11 of 13 cases examined the lizards exhibited a shorter z in DD than in LL (Fig. 7, 8). These differences were highly significant (P < 0.001).

Discussion

Melatonin has a marked and consistent effect on the circadian oscillator(s) driving the activity rhythm of lizards it induces a lengthening of the period of these oscillators or arrhythmicity. These effects of melatonin are particularly remarkable in view of the marked resistance of circadian oscillators to chemical perturbations (Pittendrigh and Caldarola, 1973). Re- sistance of circadian oscillators to perturbations of any kind is of obvious adaptive value since the circadian clock must be homeostatically conserved against any change it is likely to encounter - otherwise it could not function very effectively as a true clock (Pitten- drigh and Caldarola, 1973). Only a few substances have been shown to affect the period of circadian oscillators [e.g., deuterium oxide, ethanol (Enright, 1971a, b)] but most of these substances are not ordi- narily found in the body. Therefore, the evidence that melatonin normally plays a significant role in circadian organization is twofold: (1) exogenous melatonin affects circadian oscillators and (2) melatonin is endo- genously synthesized in the body. Significantly, removal of two melatonin synthesizing organs - the pineal and the eyes - also causes major effects on circadian organization in lizards (Underwood and Menaker, 1976; Underwood, 1977). To my knowledge the only other naturally occurring substances which have been shown to affect circadian rhythms in vertebrates are testosterone and estradiol. Testoster- one implants (or castration) has been shown to affect circadian rhythmicity in starlings and mice (Gwinner, 1974, 1975; Daan et al., 1975). For example, exogenous testosterone can cause the circadian activity rhythm of the starling to "spl i t" into two components which can temporarily run with different circadian

frequencies whereas, in mice, exogenous testosterone can cause a lengthening of the period of the circadian activity rhythm (Gwinner, 1974, 1975; Daan et al., 1975). Estradiol implants shortened the freerunning activity rhythm of hamsters (Morin et al., 1977a). Interestingly, in both birds and mammals testosterone and estradiol can have a feed-back effect on the synthesis of indoleamines by the pineal organ (Nagle etal., 1974; Cardinali etal., 1975; Preslock, 1976) and it is possible that testosterone and estradiol are affecting circadian rhythmicity via their effect on melatonin synthesis.

The exact role of melatonin in the lizard's circadian system will still require elucidation. Although it seems likely that melatonin, itself, is released from the pineal and/or eyes and affects other components of the circadian system, the present data do not ex- clude the possibility that melatonin exerts its major effects within these organs and controls the release of some other substance(s). A knowledge of target sites for melatonin within the central nervous system is obviously of great importance. In this regard the observations of Bubenik et al. (1976) are probably very pertinent. Using immunohistochemical tech- niques Bubenik et al. (1976) have identified melatonin in the suprachiasmatic nuclei (SCN) of the hypothal- amus of rats. Destruction of the SCN has been shown by a number of investigators to disrupt circadian rhythmicity in mammals (Stephan and Zucker, 1972; Moore and Eichler, 1976; Stetson and Whatson- Whitmyre, 1976; Morin etal., 1977b). In view of these observations it would obviously be worthwhile to investigate the role of the SCN in circadian rhythmicity in lizards.

Among other vertebrates a recent report has shown that melatonin affects circadian rhythmicity in birds as well; implantation of melatonin filled Silas- tic capsules into house sparrows freerunning in DD caused a shortening in r or arrhythmicity (Turek et al., 1976). The diurnal house sparrow obeys Aschoff's Rule for diurnal animals and Turek et al. (1976) have suggested that exogenous melatonin may mimic the effects of constant dim light (shortening of ~) or pro- duce an effect that resembles, but may not be identical with, the effect of bright constant light (arrhythmicity). In lizards, however, the effects of melatonin cannot be equated with the effects of light; light has opposite effects in the two diurnal species examined (light shortens ~ in S. olivaceus and lengthens z in S. occidentalis) yet melatonin consistently lengthens r in both species regardless of lighting conditions. The reason for the two different effects of melatonin between birds (melatonin shortens r in DD) and lizards (melatonin lengthens ~ in DD and LL) is unknown. A possible explanation involves a potential


ro le for m e l a t o n i n in a m u l t i o s c i l l a t o r sys tem. P i t ten-

d r igh (1974) a n d P i t t e n d r i g h a n d D a a n (1976b) h a v e p r o p o s e d tha t the ac t iv i ty r h y t h m s o f at least s o m e

a n i m a l s ref lec t the c o u p l e d o u t p u t o f two (or m o r e )

i nd iv idua l osc i l la tors . Th i s h y p o t h e s i s is ce r t a in ly ten- ab le in l izards since, a f t e r pinealec '~omy, the ac t iv i ty

r h y t h m s o f s o m e l izards spl i t in to t w o c i r c a d i a n c o m -

p o n e n t s w h i c h f r ee run w i t h d i f fe ren t f r equenc i e s ( U n -

d e r w o o d , 1977). P i t t e n d r i g h a n d D a a n fu r t he r p ro -

pose tha t the p e r i o d o f the ac t iv i ty r h y t h m is a func-

t ion o f the i nna t e ~'s o f the i nd iv idua l osc i l l a to r s

c o m p r i s i n g the c o u p l e d m u l t i o s c i l l a t o r sys tem as wel l

as a f u n c t i o n o f the s t r eng th o f the c o u p l i n g a m o n g

the i n d i v i d u a l osc i l la tors . T h e d i f fe ren t effects o f mel -

a t o n i n on bi rds a n d l izards may , the re fo re , ref lec t a d i f fe ren t i a l e f fec t o f m e l a t o n i n o n e i the r the i n n a t e

r ' s o f the i n d i v i d u a l osc i l l a to r s or d i f fe rences in the

effects o f m e l a t o n i n on the c o u p l i n g a m o n g these osc i l la tors .

This study was supported by NSF grant PCM76-06842.

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Melatonin affects circadian rhythmicity in lizards

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