Prog. Biophys. molec. Biol., Vol. 60, pp. 1-15, 1993. 0079-6107/93 $24.00 Printed in Great Britain. All rights reserved. © 1993 Pergamon Press Ltd

REGULATION OF SEROTONIN SYNTHESIS

MARGARET C. BOADLE-BIBER

Department of Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, U.S.A.

I. INTRODUCTION

CONTENTS

II. ORGANIZATION OF SEROTONERGIC NEURONS

IlI , SYNTHETIC PATHWAY

IV. CHARACTERISTICS OF THE ENZYMES 1. Tryptophan Hydroxylase 2. Aromatic Amino Acid Decarboxylase

V. FACTORS THAT INFLUENCE SEROTONIN SYNTHESIS 1. Tryptophan Concentration 2. Tissue Oxygen Levels 3. Tetrahydrobiopterin 4. Neuronal 1repulse Flow

(a) Electrical stimulation (b) Inhibition offiring--somatodendritic autoreceptors

5. Terminal Autoreceptors 6. Regulation of Tryptophan Hydroxylase Activity

(a) Impulse flow (b) Other factors

VI, RELATIONSHIP OF EVOKED RELEASE AND SYNTHESIS 1. Electrical Stimulation 2. Tryptophan 3. Somatodendritic Autoreceptors 4. Terminal Autoreceptors

VII. FUNCTIONAL IMPLICATIONS 10

ACKNOWLEDGEMENTS 10 REFERENCES 10

I. INTRODUCTION

This review covers the regulation of serotonin synthesis in serotonergic neurons of the central nervous system over the short term, that is minutes to hours, as opposed to days or weeks. Its purpose is to outline regulatory mechanisms that permit acute adjustments to be made in the synthesis of this transmitter in response to short term changes in physiological demand, and where possible to illustrate the functional significance of such adaptive responses. The reader is referred to earlier reviews on serotonin synthesis (Hamon et al., 1979b, 1981a,b; Boadle-Biber, 1982a; Fernstrom, 1983, 1991; Korf, 1985; Leathwood, 1987).

II. ORGANIZATION OF SEROTONERGIC NEURONS

Serotonergic neurons comprise a widely projecting and divergent system within the central nervous system with a large repetoire of actions. The neurons are largely contained within the midline raphe nuclei that extend caudally from the midbrain to the medulla (Steinbusch and Nieuwenhuys, 1983; Tork, 1985, 1990) and they are few in number compared to the totality of neurons in the brain. In the dorsal raphe nucleus of the midbrain the serotonergic cell bodies are clustered compactly together (Steinbuseh and Nieuwenhuys, 1983; Tork, 1985, 1990), a feature that permits them to be stimulated electrically or

2 M.C. BOADLE-BIBER

selectively lesioned (see Section V.4). This limited number of cell bodies sends out branching projections that reach practically all regions of the neuraxis, but have some topographical organization (Tork, 1985, 1990). Recent electron microscopic studies have also revealed that the serotonergic neurons within the dorsal raphe nucleus give off fine fibres with many terminal varicosities that form en passant synapses while those of the median raphe nucleus give rise to thicker axons with larger terminal varicosities that tend to form tight synapses on the target neurons (Descarries et al., 1990; Molliver et al., 1990). These structural differences are associated with different sensitivities to neurotoxins (Molliver et al., 1990) and suggest different physiological properties within subgroups of serotonin-containing neurons. The descending serotonergic neurons are biochemicaUy distinct from the rostrally projecting system in having peptides (e.g. substance P, thyrotropin releasing hormone) co-stored along with serotonin (Steinbusch and Nieuwenhuys, 1983; Nieuwenhuys, 1985; Chiba and Masuko, 1989). The descending serotonin neurons also exhibit other biochemical differences relevant to synthesis that will be outlined later.

III. SYNTHETIC PATHWAY

Serotonin is formed from the essential amino acid, L-tryptophan, by the 5-hydroxylation of tryptophan to form 5-hydroxytrytophan (5-HTP) followed by the rapid decarboxylation of 5-HTP to give 5-hydroxytryptamine (5-HT) also called serotonin, a name which reflects the discovery of this substance in serum and its ability to cause profound vasoconstriction (Rapport et al., 1947). The first and rate-limiting reaction in this sequence is catalyzed by tryptophan-5-monooxygenase (or hydroxylase) (EC 1.14.16.4) an enzyme which belongs to the family of aromatic amino acid hydroxylases that includes the closely related phenylalanine and tyrosine hydroxylases (Grenett et al., 1987). The 5-monooxygenase utilizes two substrates, L-tryptophan and molecular oxygen, as well as a reduced pterin cofactor, L-erythro-5,6,7,8-tetrahydrobiopterin (2-amino-4-hydroxy-6-(L-erythro-l',2- dihydroxypropyl)-5,6,7,8-tetrahydropteridine) (6R-BH4) which serves as an electron donor.

L-tryptophan + BH 4 + 02-'-} 5-HTP + quinonoid H2-biopterin + H20

In the hydroxylation reaction, 6R-BH 4 is converted to an unstable quinonoid dihydrobiopterin that is reconverted to the tetrahydro form by quinonoid dihydropteridine reductase, in the presence of NADH (or NADPH) (see Job et al., 1986 for a review). The second enzyme in the reaction pathway, aromatic L-alpha amino acid decarboxylase, (EC 4.1.1.28) is a soluble pyridoxal-5'-phosphate dependent enzyme, with a wide substrate specificity (see Bowsher and Henry, 1986 for a review). 5-HTP, which occurs in trace amounts (Long et al., 1982), is one of its preferred substrates and is rapidly decarboxylated to 5-HT in serotonergic neurons (Bowsher and Henry, 1986). As with other transmitters, serotonin is sequestered in storage vesicles where it is held prior to release (Tamir and Gershon, 1990). Serotonin that is not sequestered is rapidly metabolized to 5-hydroxyindo- leacetic acid (5-HIAA) through the actions of monoamine oxidase, which oxidatively deaminates the amine to 5-hydroxyindoleacetaldehyde, and aldehyde dehydrogenase which oxidizes the aldehyde to the acid.

IV. CHARACTERISTICS OF THE ENZYMES

1. Tryptophan Hydroxylase

Tryptophan hydroxylase is restricted to serotonergic neurons within the central nervous system (Kuhar et al., 1971). (An enzyme with somewhat different characteristics is found in rodent mast cells which store 5-HT (Hasegawa and Ichiyama, I987).) It appears to be a membrane bound enzyme, but its precise cellular location has not been identified (Joh et al., 1975). In contrast to tyrosine hydroxylase, tryptophan hydroxylase is not subject to inhibition by the end product of the reaction pathway, 5-HT (Joh et al., 1986). The pH optimum for the brain enzyme is 7.5 (Hamon et ai., 1979b). The isolated enzyme can utilize a variety of different synthetic pterin cofactors, but shows optimal activity with the naturally

Regulation of serotonin synthesis 3

occurring 6R-BH 4 (Kato et al., 1980; Bigham et al., 1987). The structure of the pterin cofactor also influences the affinity of the enzyme for its two substrates, tryptophan and oxygen, with the highest affinities seen in the presence of the naturally occurring cofactor, 6R-BH 4. In the presence of 6R-BH 4 the apparent Km of the partially purified enzyme for tryptophan and oxygen are 50/~u and 2.5% respectively; the Km for 6R-BH4 ranges from 30 to 60 #M (Friedman et al., 1972; Kato et al., 1980). In the intact neuron, the enzyme is unsaturated with both its substrates, L-tryptophan (Fernstrom and Wurtman, 1971; Carlsson and Lindquist, 1978) and oxygen (see Boadle-Biber, 1982a for a review). The question as to whether the concentration of cofactor, 6R-BH 4, is also unsaturating remains unsettled (Miwa et al., 1985; Wolf et al., 1991) (see Section V.3). Tryptophan hydroxylase has been purified from rat brain stem and is probably a tetramer of identical subunits which give a single band of 58 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (Fujisawa and Nakata, 1987). The cDNA sequence for the enzyme from rat (Kim et al., 1991) and rabbit brain (Grenett et al., 1987) have now been published, and the human (Craig et al., 1991) and murine (Stoll and Goldman, 1991) genes identified. Furthermore, studies are now being undertaken on the post-transcriptional control of gene expression in the rat brain and pineal where very different levels of message are found (Hart et al., 1992). The regulatory properties of tryptophan hydroxylase have been discussed in earlier reviews (Hamon et al., 1979b, 198 lb; Joh et al., 1986). It is a highly unstable enzyme that is inactivated by oxygen (Kuhn et al., 1980) but can be stabilized by reducing agents such as dithiothreitol (Kuhn et al., 1980; Fujisawa and Nakata, 1987). Other methods for stabilizing the purified enzyme include the use of a combination of nonionic detergents, glycerol, EDTA and the substrate L- tryptophan (Fujisawa and Nakata, 1987). The activity of the purified enzyme is enhanced by the addition of ferrous iron, but this is not neccessary with crude extracts of the enzyme (Fujisawa and Nakata, 1987). The activity of crude extracts or partially purified enzyme can be increased significantly by a variety of treatments including calcium activated proteases (Hamon and Bourgoin, 1979) polyelectrolytes (Kuhn et al., 1979), phospholipids (Hamon et al., 1978b; Imai et al., 1989) and phosphorylating conditions (for references see Hamon et al,, 198 l b; Joh et al., 1986). Recent evidence suggests that the enzyme is directly phosphorylated via the action of calmodulin-dependent protein kinase II (Ehret et al., 1989). Others have evidence that an activator protein is also required for this interaction (Ichimura et al., 1987; Isobe et al., 1991) and that in the presence of the activator, the enzyme may also be a substrate for cyclic AMP dependent protein kinase (Makita et al., 1990). Recent work done ex vivo suggests that phosphorylation may be an important physiological regulator of this enzyme (Boadle-Biber et al., 1986a), as has been found for related aromatic amino acid hydroxylases (Kaufman, 1986; Zigmond et al., 1989).

2. Aromat ic Amino Ac id Decarboxylase

L-alpha-aromatic amino acid decarboxylase (AADC), is a soluble enzyme(s) with a very wide tissue distribution (Bowsher and Henry, 1986; Ebadi and Simmoneaux, 1991). A controversy has existed for many years as to whether the decarboxylation of L-3,4- dihydroxyphenylalanine (L-DOPA) in catecholamine containing neurons and L-5-HTP in serotonergic neurons is carried out by the same or related enzymes. Several lines of evidence argue for separate enzymes and include different pH optima for these two substrates and different ratios for their decarboxylation in different tissues and subcellular fractions (Bowsher and Henry, 1986; Ebadi and Simmoneaux, 1991). On the other hand, a monoclonal antibody prepared against the purified enzyme from rat kidney cross reacts with enzyme from a number of other tissues including striatum, pineal and adrenal medulla (Shirota and Fujisawa, 1988) and oligonucleotide probes based on rat kidney AADC eDNA hybridize with neuronal perikarya positive for tyrosine or tryptophan hydroxylase (Tison et al., 1991). Finally, a eDNA clone encoding human L-AADC from a phaeochromocytoma expressed in COS cells decarboxylated both substrates with the same pH optima (6.5 for L- DOPA and 7-8.4 for L-5-HTP) observed with enzymes from other tissue sources (Sumi et al., 1990). Nevertheless, AADC purified from the rat striatum also decarboxylates both aromatic amino acids, but has a relatively much higher activity towards L-DOPA. The variability in

4 M. C, BOADLE-BIBER

relative activity towards these two substrates suggests that different isoenzymes occur (Siow and Dakshinamurti, 1990).

AADC from a number of sources appears to be a dimer of 100-106 kDa (Shirota and Fujisawa, 1988; Siow and Dakshinamurti, 1990). It requires free sulfhydryl groups for activity and is readily inhibited by heavy metals (Siow and Dakshinamurti, 1990). In nervous tissue high concentrations of the enzyme are associated with monoamine containing neurons, although it is also found in other types of neurons as well as nonneuronal tissue (for references see Bowsher and Henry, 1986). The affinity for 5-HTP is roughly three orders of magnitude higher than that for tryptophan ensuring that 5-HT rather than tryptamine is synthesized in serotonergic neurons. Although the activity of AADC can be dramatically enhanced in vitro in the presence of certain organic solvents, no evidence currently exists for regulation of the activity of this enzyme in the brain in vivo (see Bowsher and Henry, 1986). However, the activity of the enzyme in rat retina is regulated in response to light, doubling over a 3 hr period (Hadjiconstantinou et al., 1988). In rat brain, the low endogenous concentration of 5-HTP (Long et al., 1982) is well below the K m of 10/~M. For this reason, in the intact neuron, AADC activity appears likely to be dictated by the concentration of substrate, 5-HTP.

V. FACTORS THAT INFLUENCE SEROTONIN SYNTHESIS

Serotonin synthesis is regulated by modulating the rate of conversion of L-tryptophan to 5-HTP. Several ways in which this can occur have been identified, and include alterations in the level of the subsaturating substrate, L-tryptophan, as well as changes in the kinetic properties of tryptophan hydroxylase itself. The relative importance of these regulatory mechanisms in the control of 5-HT synthesis under particular physiological conditions, however, remains unclear. Moreover, the extent to which the concentrations of other substances, such as cofactor or tissue oxygen are acutely regulated and therefore participate in the short term control of 5-HT synthesis remains to be defined. These, as well as other factors that may have a role in regulating 5-HT synthesis will be examined.

Synthesis of 5-HT can be determined in vitro or in vivo from the conversion of radiolabelled tryptophan to 5-HT (or alpha-methyltryptophan to alpha-methyl 5-HT) (steady state), or by the accumulation of endogenous 5-HTP in the presence of a decarboxylase inhibitor (preferably NSD 1015 (Johansen et al., 1991) (nonsteady state) (see (Korf, 1985) for a review). Recently 11C-alpha-methyltryptophan has been used to make repeated measure- ments of 5-HT synthesis in the same living animal under steady state conditions by positron emission tomography (Diksic et al., 1991). The present discussion will cover studies in which these approaches have been employed. It will also include a consideration of experiments in which the effects of various in vivo manipulations on tryptophan hydroxylase are examined ex vivo in crude low speed supernatant extracts of the enzyme. Finally, the significance of the acute regulation of 5-HT synthesis for the evoked release of 5-HT and its relationship to measures of turnover will be addressed.

1. Tryptophan Concentration

The concentration of tryptophan in rat brain is nonuniform and ranges from 10 to 30 #M, which is subsaturating for tryptophan hydroxylase (Young and Sourkes, 1977; Leathwood, 1987). Many studies have addressed the issue of whether 5-HT synthesis can be regulated by altering the concentration of plasma tryptophan, thereby altering the availability of tryptophan in the central nervous system for hydroxylation (Fernstrom, 1983). The transport of tryptophan occurs via the large neutral amino acid transporter at the blood brain barrier and also at the plasma membrane of the neuron or glial cell. The uptake of tryptophan at these two barriers and hence the final brain concentration of tryptophan is generally predicted by the relative proportion of plasma free tryptophan to other neutral amino acids present in the plasma that share the same transport mechanism (Leathwood, 1987; Salter et al., 1989). Administration of exogenous tryptophan leads to a rise in brain levels of tryptophan and an increase in the synthesis of 5-HT in rats (Fernstrom, 1983). Most recently this relationship has been demonstrated in the same animal in which 11C-labelled

Regulation of serotonin synthesis 5

alpha-methyltryptophan has been used to monitor 5-HT synthesis by means of positron emission tomography (Diksic et al., 1991).

A number of rather special situations can lead to increased brain tryptophan and an associated enhanced synthesis of 5-HT. Besides consumption of large quantities of L- tryptophan, these include the ingestion by previously fasted animals of carbohydrate rich (but not protein rich) meals (Fernstrom, 1991) and impaired liver metabolism of tryptophan due to drug-induced inhibition of tryptophan pyrrolase (L-tryptophan 2,3-dioxygenase) (Badawy and Morgan, 1991) or severe liver damage (Sourkes, 1978; Bartelmess et al., 1987; Bengtsson et al., 1987; Bugge et al., 1987). One important determinant of the enhanced levels of brain tryptophan under these conditions is the increase in plasma levels of tryptophan relative to the other neutral amino acids. However, other factors such as enhanced transport at the blood brain barrier have also been postulated to contribute under some pathological conditions (Fernstrom, 1983). In diabetes, diminished synthesis of brain 5-HT is associated with lowered brain tryptophan, presumably resulting from the marked rise in competing neutral amino acids in plasma (Trulson et al., 1986).

No evidence exists that the consumption of normal meals leads to significant alterations in levels of brain tryptophan or 5-HT (Leathwood, 1990; Fernstrom, 1991). However, brain tryptophan concentrations are increased in otherwise normal animals in response to a variety of stressors including immobilization (Kennett et al., 1986), footshock (Dunn and Welch, 1991) and exercise (Chaouloff et al., 1985). The mechanism for the increased brain tryptophan levels is unclear but appears to involve the sympathetic nervous system (Dunn and Welch, 1991), although changes in plasma free tryptophan levels may contribute (Chaouloff et al., 1985). Although acute stress of various types such as immobilization (Curzon et al., 1972; Morgan et al., 1975a; Joseph and Kennett, 1983), restraint (Mitchell and Thomas, 1988), footshock (Thierry et al., 1968), swim stress (Ikeda and Nagatsu, 1985), sound (Calogero and Vetrano, 1973 ), exercise (Chaouloff et al., 1985) and ether (Bourgoin et al., 1973) is generally associated with increased 5-HT turnover, enhanced synthesis as indicated by the accumulation of 5-HTP is not seen in all brain regions in which tryptophan is elevated (e.g. see Mitchell and Thomas, 1988; Chaouloffet al., 1989). Furthermore, the rise in brain tryptophan associated with another manipulation, caffeine administration, fails to change 5-HT synthesis (Fernstrom et al., 1984). Additionally, it should be noted that the concentration of tryptophan in the brain shows circadian variations which do not correlate well with 5-HT levels (Morgan et al., 1975b) or turnover (Hery et al., 1977). Finally, the formation of 5-HT is higher in females (Carlsson and Carlsson, 1988; Haleem et al., 1990) and varies with brain region, with rates being about 20-fold higher in cell bodies than in the terminals (Korf, 1985). 5-HT synthesis also shows marked regional differences in response to drugs which cannot be accounted for by changes in tryptophan concentration (Long et al., 1983). Thus, although changes in tryptophan concentration clearly influence 5-HT synthesis, their contribution to the regulation of 5-HT synthesis under normal dietary conditions remains unclear. Other factors such as neuronal firing (see Section V.4.a), and autoreceptor activation (see Section V.5) must also have a role. Indeed, neuronal firing appears to determine the extent to which a rise in neuronal tryptophan levels will enhance 5-HTP accumulation in the presence of decarboxylase inhibition. Thus, the effect of exogenously administered tryptophan on 5-HTP accumulation is less pronounced under conditions where 5-HT neuronal firing has been inhibited by somatodendritic autoreceptor activation (Fernstrom et al., 1990). Even more striking is the finding of Elks et al., 1979a on brain slices in which an increase in 5-HT synthesis with increasing concentrations of radiolabelled tryptophan was only observed in the presence of electrical field stimulation, even though tissue tryptophan levels were identically increased in stimulated and unstimulated tissues. One possible explanation is that the firing of action potentials enhances tryptophan uptake selectively into 5-HT neurons via a carrier specific to these neurons (Denizeau and Sourkes, 1978). Another contributing factor could be the enhancement of tryptophan hydroxylase activity that results from neuronal firing (Boadle-Biber et al., 1986a) (see Section V.6.a). The fact that administration of exogenous tryptophan always enhances 5-HT formation in vivo could simply reflect the fact that 5-HT neurons fire tonically in vivo (Vandermaelen, 1985).

6 M.C. BOADLE-BIBER

2. Tissue Oxygen Levels

Cerebral oxygen tensions range from 1-90 mm Hg in the anesthetized cat but 70% of values are less than 30 mm Hg and 50% lie between 5 and 20 mm Hg (Smith et al., 1977). With the apparent K m of oxygen for partially purified tryptophan hydroxylase estimated at 2.5% (19 mm Hg) (Friedman et al., 1972), the oxygen concentration in the brain is subsaturating for 5-HTP formation and most likely varies along the extensive projections of 5-HT neurons. In synaptosomes, the apparent Km for oxygen has been reported to vary with the concentration of tryptophan (3--4 mm Hg at 10 #M tryptophan and 8 mm Hg at 2/~M) (Katz, 1980, 1981). The closely related enzyme, phenylalanine hydroxylase, also shows a decreased affinity for oxygen as cofactor concentration falls (Kaufman and Fisher, 1974). Thus, under hypoxic conditions it is likely that 5-HT synthesis will be more severely affected if tryptophan (or possibly cofactor concentrations as well) are also decreased. However, it is not clear that these considerations influence 5-HT synthesis under normal conditions. The great variability in the values of oxygen tension measured in different regions of cortex, makes it unlikely that changes in oxygen tension per se could be used to regulate 5-HT synthesis acutely, although clearly they influence the rate of conversion of tryptophan to 5- HTP. 5-HT synthesis is sensitive to hypoxia as was demonstrated by the linear decline in 5- HTP accumulation in whole brain of rats treated with a decarboxylase inhibitor as arterial oxygen tension fell from 100 to 30 Torr (Davis et al., 1973). Recently a 38% increase in the synthesis of 5-HT estimated by PET in the anesthetized dog was shown when the pO2 of arterial blood was raised from 76 to 106 mm Hg (Diksic et al., 1991). In other studies in which the oxygen carrying capacity of the blood was reduced by administration of 1% CO or NaNO2, brain 5-HT synthesis was reduced significantly (Freeman et al., 1986; MacMillan, 1987). No evidence has been obtained that tryptophan levels are significantly affected by acute hypoxia (Davis and Carlsson, 1973) nor overall 6R-BH 4 levels by acute ischemia (Iijima et al., 1986). Thus the critical factor here for the reduced formation of 5-HT appears to be pO 2 .

3. Tetrahydrobiopterin

6R-BH 4 is an obligatory cofactor in brain monoamine synthesis that is formed locally in nervous tissue from GTP (Duch and Smith, 1991). The requirement for 6R-BH 4 in monoamine synthesis in human brain was established with the detection of certain extremely rare forms of hyperphenylalaninemia in which the problem is a deficiency or lack, not of phenylalanine hydroxylase, but of 6R-BH 4 due to defects in its biosynthesis or regeneration from the dihydro quinonoid form (Duch and Smith, 1991). In weanling rats, a 35 % reduction in endogenous brain 6R-BH 4 levels through the inhibition of GTP cyclohydrolase activity results in a 25-35 % reduction in accumulation of 5-HTP in the presence of a decarboxylase inhibitor (Suzuki et al., 1988).

The K m of 6R-BH4 is about 30 #M (Friedman et al., 1972). Average concentrations of cofactor in rat brain are about 1/~M. However, the cofactor is undoubtedly much more concentrated in neurons that utilize 6R-BH4 as a cofactor, such as the monoamine- containing (Levine et al., 1979, 1981) as well as those that use NO as a signalling system (Duch and Smith, 1991). There is conflicting data concerning whether 6R-BH 4 concentra- tions are actually saturating for tryptophan hydroxylase. Two laboratories reported that intraventricular administration of 6R-BH 4 increases the rate of 5-HTP accumulation in vivo in the presence of a decarboxylase inhibitor (Miwa et al., 1985; Boadle-Biber et al., 1986b). Conversely, no increase in the synthesis of 5-HT was observed with added cofactor in synaptosomes or hippocampal slices (Wolf et al., 1990, 1991) whereas an increase was observed with slices of midbrain (Sawada et al., 1986). Recent studies indicate that 6R-BH 4 turnover is rapid in neuronal cultures (6.7 hr) and its synthesis can be rapidly altered under certain conditions (e.g. the presence of nerve growth factor) in cultured sympathetic ganglia or adrenal chromaffin cells as a result of increases in the levels ofGTP cyclohydrolase (Abou Donia et al., 1986; Kapatos, 1990; Duch and Smith, 1991; Hirayama and Kapatos, 1991). These studies indicate that de novo BH 4 synthesis can contribute to the regulation of

Regulation of serotonin synthesis 7

catecholamine formation. Whether similar regulation of6R-BH 4 formation can take place in serotonin-containing neurons of the CNS and influence 5-HT synthesis has not been established.

4. Neuronal Impulse Flow

(a) Electrical stimulation

A variety of studies carried out in brain slices (Elks et al., 1979a,b; Hamon et al., 1979a) or in intact animals (Shields and Eccleston, 1972; Bourgoin et al., 1980) have indicated that the formation of 5-HT is enhanced in response to depolarization or electrical stimulation of 5- HT containing neurons and that this increase in 5-HT synthesis arises as a consequence of an increase in the conversion of tryptophan to 5-HTP (Herr et al., 1975; Bourgoin et al., 1980; Boadle-Biber et al., 1983; Duda and Moore, 1985). The increase in serotonin synthesis has an absolute requirement for extracellular calcium ions (Elks et al., 1979b; Hamon et al., 1979a) and is sensitive to the frequency of electrical stimulation (Shields and Eecleston, 1972). It cannot be accounted for by increases in brain tryptophan levels (Herr et al., 1975), but rather appears to arise from an alteration in the properties of tryptophan hydroxylase (Elks et al., 1979a) (see Section V.6.a).

(b) Inhibition of firing--somatodendritic autoreceptors

Serotonergic neurons exhibit a slow steady discharge that has the characteristics of pacemaker activity (Vandermaelen, 1985). The rate of discharge appears related to state of arousal (Trulson and Jacobs, 1979), increasing with phasic auditory and visual stimuli (Heym et al., 1982). Activation of somatodendritic 5-HT1A receptors, by 5-HT or the selective 5-HTtA agonist, 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino) tetralin] inhibits 5-HT neuronal firing (Aghajanian et al., 1988) and reduces the turnover and synthesis of 5- HT in terminal fields (measured by 5-HTP accumulation) (Hjorth and Magnusson, 1988; Hamon et al., 1988; Invernizzi et al., 1991). In vitro studies on slices of midbrain which contain the cell bodies of 5-HT neurons reveal that autoreceptor activation reduces 5-HTP accumulation (Sawada and Nagatsu, 1986) possibly through inhibition of action potentials that continue to fire in slice preparations (Vandermaelen, 1985). Calmodulin antagonists also inhibit 5-HT synthesis in this preparation and the inhibition obtained is nonadditive with the autoreceptor mediated inhibition. In other studies in which the firing of 5-HT neurons is blocked by lesion or transection, 5-HT synthesis is also found to be reduced (Carlsson et al., 1973; Herr and Roth, 1976).

5. Terminal Autoreceptors

The 5-HT neuron possesses terminal autoreceptors (classified as 5HT1B in rats, 5HT m in other species) that exhibit a different pharmacological profile from those on the dendrites and soma of 5-HT neurons (see above) (Gothert, 1990) and, when activated, inhibit the evoked release of 5-HT (Gothert, 1990). RU-24969 (5-methoxy-3-(1,2,3,6-tetrahydro-4- pyrindinyl-4-yl)), a drug selective for the 5-HT1B site (rat) not only inhibits release of 5-HT in vivo, as indicated by the diminished extracellular levels of 5-HT (Brazell et al., 1985; Carli et al., 1988), but also reduces 5-HTP accumulation (Crespi et al., 1990). These actions occur in the absence of any effect on 5-HT neuronal firing (Carli et al., 1988).

6. Regulation of Tryptophan Hydroxylase Activity

(a) Impulse flow

The activity of low speed supernatant extracts of enzyme prepared from depolarized or electrically stimulated brain tissue is increased compared to that from nondepolarized or sham-stimulated tissue (Boadle-Biber, 1978; Boadle-Biber et al., 1986a). This increase in activity is also sensitive to the frequency of electrical stimulation over the range of frequencies at which 5-HT neurons are known to fire (2-20 Hz) (Boadle-Biber et al., 1986a) and has an

8 M.C. BOADLE-BIBER

absolute requirement for calcium ions (Boadle-Biber, 1978). Indeed, manipulations that are known to raise the free intracellular concentration of calcium ions will enhance 5-HT synthesis (Elks et al., 1979b; Hamon et al., 1979a) and also increase enzyme activity (Boadle- Biber, 1979, 1982b; Boadle-Biber and Phan, 1987). The increase in enzyme activity is reversible in situ within 1 hr after cessation of electrical stimulation or depolarization (Boadle-Biber, 1982b; Boadle-Biber et al., 1986a) and can also be reversed in vitro by incubation of the enzyme extract with alkaline phosphatase (Boadle-Biber et al., 1986a; Boadle-Biber and Phan, 1987), an enzyme that removes phosphate groups from protein. Finally, the increase in enzyme activity obtained with depolarization is nonadditive with the increase obtained when enzyme extract is incubated under phosphorylating conditions (Boadle-Biber et al., 1986a; Boadle-Biber and Phan, 1987). These findings strongly suggest that the activity of tryptophan hydroxylase, like that of the other aromatic amino acid hydroxylases (Kaufman, 1986; Zigmond et al., 1989) may be regulated by phosphorylation. As noted earlier, direct phosphorylation of the enzyme has been demonstrated with a purified calcium-calmodulin kinase II (Ehret et al., 1989). The observation that calmodulin antagonists block the increase in enzyme activity obtained in response to treatments that raise intracellular levels of free calcium is consistant with participation of calmodulin kinase II in the regulation of this enzyme in the 5-HT neuron (Boadle-Biber, 1982b; Boadle- Biber and Phan, 1987). However, it remains to be demonstrated that the enzyme is directly phosphorylated as a result of depolarization of intact 5-HT neurons. Changes in the kinetic properties of the enzyme have been detected when it is incubated under phosphorylating conditions, including increases in the affinity for its cofactor (the synthetic analogue, 6- methyl-5,6,7,8-tetrahydropterin) (Hamon et al., 1978a; Lysz and Sze, 1978; Kuhn et al., 1978; Yamauchi and Fujisawa, 1979a,b) substrate (Hamon et al., 1978a) and an increase in Vma x (Yamauchi and Fujisawa, 1979a,b). Electrical stimulation produces an increase in Vm~ ~ but does not change the affinity for either cofactor or substrate (Boadle-Biber et al., 1986a). Similar increases in enzyme activity have been obtained in response to exposure of awake rats to an acute (1-2 hr) sound (Boadle-Biber et al., 1989), a stressor which enhances 5-HTP accumulation in the presence of a decarboxylase inhibitor (Dilts, Phan and Boadle-Biber, unpublished data). The effect of sound stress on enzyme activity appears to involve enhanced firing of 5-HT neurons since it is blocked by local infusion into the dorsal raphe nuclei of the 5HT1A agonist, gepirone (Corley et al., 1990, 1992), a compound that inhibits the firing of 5-HT neurons through its action on the somatodendritic autoreceptors (see Section V.4.b).

It is noteworthy that these regulatory properties of tryptophan hydroxylase are only detected in the enzyme from rostrally projecting 5-HT neurons. Electrical stimulation of the descending tracts, while increasing 5-HT synthesis (Bourgoin et al., 1980) does not result in any detectable increase in enzyme activity assayed ex vivo (Bourgoin et al., 1980; Boadle- Biber et al., 1986a). Apparently there are differences in the regulatory properties of the enzyme from these 5-HT neuronal systems.

(b) Other fac tors

Tryptophan hydroxylase is found to be activated following treatments in vivo that lower brain tryptophan levels. Once again, the increase in enzyme activity which was reflected by an increase in Vmax was obtained in extracts of forebrain, but not of spinal cord (Neckers et al., 1977). The physiolgical significance of this finding is unclear.

VI. RELATIONSHIP OF EVOKED RELEASE AND SYNTHESIS

1. Electr ical S t imulat ion

Microdialysis has revealed that electrical stimulation of the dorsal raphe over the range of frequencies at which the 5-HT neurons are known to fire (2-20 Hz) increases the level of 5- HT in the dialysate by a mechanism that is attenuated, although not completely abolished, by tetrodotoxin and calcium-free perfusate (Sharp et al., 1989, 1990). Like the evoked release

Regulation of serotonin synthesis 9

of 5-HT, both the depolarization-induced increase in 5-HT synthesis (Elks et al., 1979b; Hamon et al., 1979a) and the activation of tryptophan hydroxylase (Boadle-Biber, 1978, 1979) require extraceUular calcium ions. Thus, a common dependence on calcium appears to link increased transmitter utilization and increased transmitter synthesis.

The 5-HT released by nerve activity is the newly synthesized fraction (Hery et al., 1979, 1983). However, only a small proportion of the 5-HT in the terminal is apparently destined for release. Indeed, about 70% of intraneuronal 5-HT is metabolized to 5-HIAA without being released (Lookingland et al., 1986; Wolf and Kuhn, 1986). Tissue levels of 5-HIAA are a good index of overall 5-HT turnover (Shannon et al., 1986), but do not reveal whether turnover is related to functional release of transmitter. Extracellular levels of 5-HIAA are increased by electrical stimulation (De Simoni et al., 1987; Crespi et al., 1988), but are not a reliable indicator of 5-HT release under a variety of conditions such as autoreceptor activation (Crespi et al., 1990).

2. Tryptophan

A number of studies utilizing the technique of in vivo microdialysis have now examined the question of whether the increase in synthesis of 5-HT, that results when exogenous tryptophan is given to rats, actually leads to an increase in the amount of transmitter in the extracellular fluid. The level of 5-HT in the dialysate from frontal cortex and striatum was found to be increased about two-fold by tryptophan administration (100 mg/kg i.p.) (Carboni et al., 1989; During et al., 1989) and, in the study on frontal cortex (Carboni et al., 1989), the level of dialysate 5-HT was depressed by perfusiuon with tetrodotoxin or calcium- free Ringer's medium, treatments known to block evoked release of 5-HT (Sharp et al., 1989, 1990). Thus the enhanced release of 5-HT appears to be linked to neuronal firing. In another study, a dose of tryptophan (100 mg/kg i.p.) that caused a 300% increase in dialysate 5-HTP, an index of 5-HT synthesis, also increased 5-HT release by 50% in separate experiments (Westerink and De Vries, 1991). The conclusion is that enhanced availability of tryptophan, in vivo, not only leads to increased formation of 5-HT but also to increased release. In view of the fact that tryptophan administration actually reduces the discharge rate of 5-HT neurons acutely (Aghajanian and Wang, 1978), the findings of increased dialysate 5-HT after a tryptophan load suggest that the output of the 5-HT per impulse must be increased. Conceivably the 5-HT content of the storage vesicles in 5-HT terminals is increased by tryptophan loading. In vitro studies on slices have also shown that an increase in the concentration of tryptophan leads to an increase in both spontaneous and evoked release of transmitter (Schaechter and Wurtman, 1989, 1990).

3. Somatodendritic Autoreceptors

Local infusion of the 5-HTIA, agonist, 8-OH-DPAT, into the dorsal raphe nucleus of anesthetized rats depresses both 5-HT neuronal firing and synthesis (see Section V.4.b), and also reduces the levels of 5-HT in the dialysate from ventral hippocampus (Sharp et al., 1989) a dorsal raphe projection area (Azmitia and Segal, 1978). The decline in dialysate 5-HT presumably results from the inhibitory effect of 8-OH-DPAT on 5-HT neuronal firing via its local action on the somatodendritic autoreceptors.

4. Terminal Autoreceptors

Local in vivo infusion of RU24969, which is selective for the 5-HT 1B terminal autoreceptor, reduces the release of 5-HT as indicated by the level of 5-HT in the dialysate from frontal cortex (Brazell et al., 1985; Crespi et al., 1990). This drug has no effect on firing of 5-HT neurons in the dorsal raphe (Carli et al., 1988) but does reduce 5-HT synthesis as well as release (see Section V.5 above). Inhibition of the evoked release of 3H-5-HT by 5-HT and related agonists has also been demonstrated in vitro (Gothert, 1990). One common mechanism that would link these inhibitory effects on synthesis and release involves autoreceptor-mediated inhibition of calcium entry into the nerve terminal during depolarization, as described for the norepinephrine autoreceptor (Lipscombe et al., 1989).

10 M.C. BOADLE-BIBER

VII. F U N C T I O N A L I M P L I C A T I O N S

The importance of 5-HT synthesis for function is underscored by two observations, the preferential release-of newly synthesized amine in response to depolarization, and the marked behavioral consequences seen when synthesis is blocked, such as sensory overreactivity, hyperalgesia and insomnia (Vogt, 1982). There appears to be a coupling between evoked release of transmitter and the increase in synthesis that may arise through a common signal, the elevation in intracellular concentration of free calcium that occurs in nerve terminals in response to electrical activity. Such a rise in calcium is required for transmitter release and also activates tryptophan hydroxylase, at least in the rostrally projecting 5-HT neurons. The enhanced synthesis of 5-HT seen when brain tryptophan levels rise also appears to be linked to neuronal firing and leads to enhanced amine output possibly via elevated vesicle content of amine. In this case increased synthesis of 5-HT may reflect the altered kinetic properties of tryptophan hydroxylase and/or enhanced precursor uptake into the 5-HT terminal in response to impulse flow. The inhibitory effect of somatodendritic autoreceptor activation on 5-HT synthesis and release is presumably also linked through the inhibition of 5-HT neuronal firing. Finally, the ability of the terminal autoreceptor to reduce synthesis and release of 5-HT, in the absence of any change in 5-HT impulse flow could be accounted for by inhibition of calcium flux into the terminal during depolarization as has been described for presynaptic autoreceptors sensitive to norepinephrine. Thus one can make the case that 5-HT synthesis and neuronal firing appear to be coupled. On the other hand it would be misleading to propose that all the factors that can potentially influence synthesis such as brain tryptophan concentration and its regulation by stress, and tissue levels ofpO 2 or BH 4 are well understood. The fact that most of the amine that is synthesized is apparently destined for intraneuronal metabolism rather than release remains puzzling. Such profligacy suggests that the role of this transmitter in the central nervous system does not require that its levels be tightly regulated.

Serotonin's actions in the central nervous system appear to be those of a modulator or gain setting device, altering for example the level of sensory responsiveness or motor activity, but not actually mediating the responses themselves (Vogt, 1982). This modulatory role is reflected in the fact that, with one known exception (the 5-HT 3 receptor) the plethora of 5-HT receptors identified and characterized so far by ligand binding (Sanders-Bush, 1988), cloning (Hartig, 1989), second messenger production (Saunders-Bush, 1988) and electrophysiology (Vandermaelen, 1985; Aghajanian et al., 1988) are G-protein coupled and predominently metabotropic (Sanders-Bush, 1988; Julius, 1991). Serotonin clearly must have a large repertoire of actions in the central nervous system. But metabotropic signalling may not require that transmitter output be regulated with the same degree of precision as the ionotropic signalling that conveys information rapidly through the nervous system. The challenge for the future, then, lies in understanding the relationship between regulation of transmitter stores, 5-HT neuronal firing, release of transmitter and function.

A C K N O W L E D G E M E N T S

Work from the author's laboratory was supported by NIH grant NS 14090 from NINDS. Thanks are due to Dr Roger P. Dilts, Jr for helpful discussions and to Thomas U. L. Biber for the 486 computer used to prepare the manuscript.

REFERENCES Aaot3 DONIA, M. M., WILSON, S. P., ZIMMERMAN, T. P., N~CnOL, C. A. and VIWROS, O. H. (1986) Regulation of

guanosine triphosphate cyclohydrolase and tetrahydrobiopterin levels and the role of the cofactor in tyrosine hydroxylation in primary cultures of adrenomedullary chromatfin cells. J. Neurochem. 46, 1190-1199.

AGHAJANIAN, G. K., SPROUSE, J. S. and RASMUSSEN, K. (1988) Elcctrophysiology of central scrotonin receptor subtypes. In The Serotonin Receptors, pp. 225-252, (ed. E. SANDERS-Bus'n) The Humana Press, Clifton, NJ.

AGRAJANIAN, G. K. and WANG, R. Y. (1978) Physiology and pharmacology of central serotonergic neurons. In Psychopharmacology: A Generation of Progress, pp. 171-183, (eds. M. A. LIPTON, A. DIMASOO and K. F. KILLAM) Raven Press, NY.

AZblITIA, E. C. and SEGAL, M. (1978) An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. coml~. Neurol. 179, 641~67.

Regulation of serotonin synthesis 11

BADAWY, A. A. and MORGAN, C. J. (1991) Effects of acute paroxetine administration on tryptophan metabolism and disposition in the rat. Br. J. Pharmacol. 102, 429-433.

BAR~LM~SS, P., BENGTSSON, F., NOmN, A., JEPPSSON, B., HERL1N, P. and BUOOE, M. (1987) The effect of blood ingestion on brain serotonin synthesis in portacaval-shunted rats. Res. exp. Med. (Bert.) 187, 353-358.

BENGTSSON, F., BUOOE, M., VAOIANOS, C., JEPPSSON, B. and NomN, A. (1987) Brain serotonin metabolism and behavior in rats with carbon tetrachloride-induced liver cirrhosis. Res. exp. Med. (Berl.) 187, 429-438.

BIGHAM, E. C., SMITH, G. K., REINHARD, J. F. JR., MALLORY, W. R., NICHOL, C. A. and MORRISON, R. W. JR. (1987) Synthetic analogues of tetrahydrobiopterin with cofactor activity for aromatic amino acid hydroxylases. J. med. Chem. 30, 40-45.

BOAOLE-BIBER, i . C., JOHANNESSEN, J. N., NARASIMHACHARI, N. and PHAN, T. H. (1983) Activation of tryptophan hydroxylase by stimulation of central serotonergic neurons. Biochem. Pharmacol. 32, 185-188.

BOADLE-B1BER, i . C. (1978) Activation of tryptophan hydroxylase from central serotonergic neurons by calcium and depolarization. Biochem. Pharmacol. 27, 1069-1079.

BOADLE-BIBER, M. C. (1979) Activation of tryptophan hydroxylase from slices of rat brain stem incubated with agents which promote calcium uptake or intraneuronal release. Biochem. Pharmacol. 28, 2129-2138.

BOADLE-BIBER, i . C. (1982a) Biosynthesis of serotonin. In Biology of Serotonergic Transmission, pp. 63-94, (ed. N. N. OSBORNE) John Wiley.

BOADLE-BmER, M. C. (1982b) Further studies on the role of calcium in the depolarization-induced activation of tryptophan hydroxylase: Effect of verapamil, tetracaine, haloperidol and fluphenazine. Biochem. Pharmacol. 31, 2495-2503.

BOADLE-BIBER, M. C., JOHANNESSEN, J. N., NARAS1MHACHARI, N. and PHAN, T.-H. (1986a) Tryptophan hydroxylase: Increase in activity by electrical stimulation of serotonergic neurons. Neurochem. Int. 8, 83-92.

BOADLE-BIBER, M. C., JOHANNESSEN, J. N., REINHARD, J. F. JR. and NICHOL, C. A. (1986b) Effect of tetrahydrobiopterin on brain serotonin synthesis in dorsal raphe nucleus stimulated rats. Fedn Proe. 45, 437.

BOADLE-BIBER, i . C., CORLEY, K. C., GRAVES, L., PHAN, T.-H. and ROSECRANS, J. (1989) Increase in the activity of tryptophan hydroxylase from cortex and midbrain of Fischer 344 rats in response to acute or repeated sound stress, Brain Res. 482, 306-316.

BOAOLE-BIBER, M. C. and PHAN, T.-H. (1987) Involvement of calmodulin-dependent phosphorylation in the activation of tryptophan hydroxylase induced by depolarization of slices or other treatments that raise intracellular free calcium. Biochem. Pharmacol. 36, 1174-1176.

BOURGOIN, S., MOROT-GAUDRY, Y., GLOWINSKI, J. and HAMON, i . (1973) Stimulating effect of short term ether anaesthesia on central 5-HT synthesis and utilization in the mouse brain. Eur. d. Pharmacol. 22, 209-211.

BOURGOIN, S., OLIVERAS, J. L., BRUXELLE, J., HAMON, M. and BESSON, J. M. (1980) Electrical stimulation of the nucleus raphe magnus in the rat Effects on 5-HT metabolism in the spinal cord. Brain Res. 194, 377-398.

BOWSHER, R. R. and HENRY, D. P. (1986) Aromatic L-amino acid decarboxylase: Biochemistry and Functional Significance. In Neuromethods Series I: Neurochemistry. Neurotransmitter Enzymes, pp. 33-78, (eds. A. A. BOULTON, G. B. BAKER and P. H. Yu) Humana Press, Clifton, NJ.

BRAZELL, i . P., MARSDEN, C. A., NISBET, A. P. and ROUTLEDGE, C. (1985) The 5-HT1 receptor agonist RU-24969 decreases 5-hydroxytryptamine (5-HT) release and metabolism in the rat frontal cortex in vitro and in vivo. Br. J. Pharmacol. 86, 209-216.

BUGGE, M., BENGTSSON, F., NOBIN, A., JEPPSSON, B. and HERLIN, P. (1987) The effect of liver ischaemia on brain monoamine synthesis in the rat. Res. exp. Med. (Berl.) 187, 119-130.

CALOGERO, B. and METRANO, G. (1973) Variations of the biogenic amines content in some different areas of the brain of rats subjected to physiological auditory stimulation and acoustic trauma. Rev. Laryng. V94, 429-443.

CARBONI, E., CADONI, C., TANDA, G. L. and DI CHIARA, G. (1989) Calcium-dependent, tetrodotoxin-sensitive stimulation of cortical serotonin release after a tryptophan load. J. Neurochem. 53, 976-978.

CARLI, M., INVERNlZZl, R., CERVO, L. and SAMANIN, R. (1988) Neurochemical and behavioural studies with RU- 24969 in the rat. Psychopharmacology (Berl.) 94, 359-364.

CARLSSON, A., LINDQUIST, M., MAGNUSSEN, T. and ATACK, C. (1973) Effect of acute transection on the synthesis and turnover of 5-HT in the rat spinal cord. Naunyn Schmiedeber#'s Arch. Pharmacol. 277, 1-12.

CARLSSON, A. and LINDQUIST, i . (1978) Dependence of 5-HT and catecholamine synthesis on concentrations of precursor amino acids in rat brain. Naunyn Schmiedeberg's Arch. Pharmacol. 303, 157-164.

CARLSSON, i . and CARLSSON, A. (1988) In vivo evidence for a greater brain tryptophan hydroxylase capacity in female than in male rats. Naunyn Schmiedeberg's Arch. Pharmacol. 338, 345-349.

CHAOULOFF, F., ELGHOZI, J. L., GUEZENNEC, Y. and LAUOE, D. (1985) Effects of conditioned running on plasma, liver and brain tryptophan and on brain 5-hydroxytryptamine metabolism of the rat. Br. J. Pharmacol. 86, 33-41.

CHAOULOFF, F., LAUOE, D. and ELGHOZI, J. L. (1989) Physical exercise: evidence for differential consequences of tryptophan on 5-HT synthesis and metabolism in central serotonergic cell bodies and terminals. J. neural Transm. 78, 121-130.

CHINA, T. and MASUKO, S. (1989) Coexistence of varying combinations of the neuropeptides with 5- hydroxytryptamine in neurons of the raphe pallidus and obscurus projecting to the spinal cord. Neurosci. Res. 7, 13-23.

CORLEY, K. C., SINGH, V. B., PHAN, T.-H. and BOADLE-B1BER, i . C. (1990) Gepirone infused into dorsal raphe nucleus blocks sound stress induced increases in tryptophan hydroxylase in rat. FASEB J. 4, A809--3146.

CORLEY, K. C., SINGH, V. B., PHAN, T.-H. and BOADLE-BIBER, i . C. (1992) Effect of gepirone on increases in tryptophan hydroxylase in response to sound stress. Fur. J. Pharmacol. 213, 417-425.

CRAIG, S. P., BOULARANO, S., DARMON, i . C., MALLET, J. and CRAIG, I. W. (1991) Localization of human tryptophan hydroxylase (TPH) to chromosome l l p 1 5 . 3 - - - p14 by in situ hybridization. Cytogenet. Cell Genet. 56, 157-159.

CRESPI, F., MARTIN, K. F. and MARSDEN, C. A. (1988) Simultaneous in vivo voltammetric measurement of striatal extracellular DOPAC and 5-HIAA levels: effect of electrical stimulation of DA and 5-HT neuronal pathways. Neurosci. Lett. 90, 285-291.

12 M. C, BOADLE-BIBER

CRESPI, F., GARRATT, J. C., SLEIGHT, A. J. and MARSDEN, C. A. (1990) In vivo evidence that 5-hydroxytryptamine (5-HT) neuronal firing and release are not necessarily correlated with 5-HT metabolism. Neuroscience 35, 139-144.

CuRZON, G., JOESPH, M. H. and KNOTT, P. J. (1972) Effects of immobilization and food deprivation on rat brain tryptophan metabolism. J. Neurochem. 19, 196%1974.

DAVIS, J. N., CARLSSON, A., MACMILLAN, V. and SIESJO, B. K. (1973) Brain tryptophan hydroxylation: Dependence on arterial oxygen tension. Science 182, 72-74.

DAvis, J. N. and CARLSSON, A. (1973 ) The effect of h ypoxia on monoamine synthesis, levels and metabolism in rat brain. J. Neurochem. 21, 783-790.

DE SIMONI, M. G., SOKOLA, A., FODRITTO, F., DAL TOSO, G. and ALGERI, S. (1987) Functional meaning of tryptophan-induced increase of 5-HT metabolism as clarified by in vivo voltammetry. Brain Res. 411, 89-94.

DENIZEAU, F. and SOURKES, T. L. (1978) Regional transport of tryptophan in rat brain. J. Neurochem. 28, 951-959. DESCARRIES, L., AUDET, M. A., DOUCET, G., GARCIA, S., OLESKEVICH, S., SEGUELA, P., SOGHOMONIAN, J.-J. and

WATKINS, K. C. (1990) Morphology of central serotonin neurons: Brief review of quantified aspects of their distribution and ultrastructural relationships. Ann. N.Y. Acad. Sci. 600, 81-92.

DIKSIC, M., NAGAHIRO, S., CHALY, T., SOURKES, T. L., YAMAMOTO, Y. L. and FEINDEL, W. (1991) Serotonin synthesis rate measured in living dog brain by positron emission tomography. J. Neurochem. 56, 153-162.

DUCH, D. S. and SMITH, G. K. (1991) Biosynthesis and function of tetrahydrobiopterin. J. nutr. Biochem. 2, 411-423. DUDA, N. J. and MOORE, K. E. (1985) Simultaneous determination of 5-hydroxytryptophan and 3,4-

dihydroxyphenylalanine in rat brain by HPLC with electrochemical detection following electrical stimulation of the dorsal raphe nucleus. J. Neurochem. 44, 128-144.

DUNN, A. J. and WELCH, J. (1991) Stress- and endotoxin-induced increases in brain tryptophan and serotonin metabolism depend on sympathetic nervous system activity. J. Neurochem. 57, 1615-1622.

DURING, M. J., FREESE, A., HEYES, M. P., SWARTZ, K. J., MARKEY, S. P., ROTH, R. H. and MARTIN, J. B. (1989) Neuroactive metabolites of L-tryptophan, serotonin and quinolinic acid, in striatal extraceilular fluid. Effect of tryptophan loading. FEnS Lett. 247, 438-444.

EBADI, M. and SIMMONEAUX, V. (1991) Ambivalence on the multiplicity of mammalian aromatic L-amino acid decarboxylase. In Kynurenine and Sertonin Pathways, pp. 115-126, (eds. R. SCHWARCZ, S. N. YOUNG and R. R. BROWN) Plenum Press.

EHRET, M., CASH, C. D., HAMON, M. and MAITRE, M. (1989) Formal demonstration of the phosphorylation of rat brain tryptophan hydroxylase by calcium/calmodulin-dependent protein kinase. J. Neurochem. 52, 1886-1891.

ELKS, M. L., YOUNGBLOOD, W. W. and KIZER, J. S. (1979a) Serotonin synthesis and release in brain slices: Independence of tryptophan. Brain Res. 172, 471-486.

ELKS, M. L., YOUNGBLOOD, W. W. and KIZER, J. S. (1979b) Synthesis and release of serotonin by brain slices: Effect of ionic manipulations and cationic ionophores. Brain Res. 172, 461-469.

FERNSTROM, J. D. (1983) Role of precursor availability in control of monoamine biosynthesis in brain. Physiol. Roy. 63, 484-546.

FERNSTROM, J. D. (1991) Effects of the diet and other metabolic phenomena on brain tryptophan uptake and serotonin synthesis. In Kynureninc and Serotonin Pathways, pp. 369-376, (Adv. Exp. Biol. Med., Vol. 294) (eds. R. SCHWARCZ, S. N. YOUNG and R. R. BROWN) Plenum Press.

FERNSTROM, J. D. and WURTMAN, R. J. (1971) Brain serotonin content: physiological dependence on plasma tryptophan levels. Science 173, 149-152.

FERNSTROM, M. H., BAZIL, C. W. and FERNSTROM, J. D. (1984) Caffeine injection raises brain tryptophan level, but does not stimulate the rate of serotonin synthesis in rat brain. Life Sci. 35, 1241-1247.

FERNSTROM, M. H., MASSOUD1, M. S. and FERNSTROM, J. D. (1990) Effect of 8-hydroxy-2-(di-n-propylamino)- tetralin on the tryptophan-induced increase in 5-hydroxytryptophan accumulation in rat brain. Life Sci. 47, 283-289.

FREEMAN, G. B., NIELSEN, P. and GIBSON, G. E. (1986) Monoamine neurotransmitter metabolism and locomotor activity during chemical hypoxia. J. Neurochem. 46, 733-738.

FRIEDMAN, P. A., KAPPELMAN, A. H. and KAUFMAN, S. (1972) Partial purification and characterization of tryptophan hydroxylase from rabbit hind brain. J. biol. Chem. 247, 4165-4173.

FUJISAWA, H. and NAKATA, H. (1987) Tryptophan 5-monooxygenase from rat brain stem. Methods Enzymol. 142, 83-87.

GOTHERT, M. (1990) Presynaptic serotonin receptors in the central nervous system. Ann. N. Y. Acad. Sci. 604, 102-112.

GRENETT, H. E., LEDLEV, F. D., REED, L. L. and WOO, S. L. (1987) Full-length eDNA for rabbit tryptophan hydroxylase: functional domains and evolution of aromatic amino acid hydroxylases. Proc. hath. Acad. Sci. U.S,A. 84, 5530-5534.

HADJICONSTANTINOU, M., ROSSETTI, Z., SILVIA, C., KRAJNC, D. and NEFF, N. H. (1988) Aromatic L-amino acid decarboxylase activity of the rat retina is modulated in vioo by environmental light. J. Neurochem. 51, 1560-1564.

HALEEM, D. J., KENNETT, G. A. and CURZON, G. (1990) Hippocampal 5-hydroxytryptamine synthesis is greater in females rats than in males and more decreased by the 5-HT1A agonist 8-OH-DPAT. J. neural Transm. Genet. Sect. 79, 93-101.

HAMON, M., BOURGOIN, S., HENRY, F. and S1MMONET, G. (1978a) Activation of tryptophan hydroxylase by adenosine triphosphate, magnesium and calcium. Molec. Pharmacol. 14, 99-110.

H~ON, M., BOURGOIN, S., HERY, F. and SIMMONET, G. (1978b) Phospholipid-induced activation of tryptophan hydroxylase from the rat brainstem. Biochem. Pharmacol. 27, 915-922.

HAMON, M., BOURGOIN, S., ARTAUD, F. and GLOWINSKI, J. (1979a) The role of intraneuronal 5-HT and of tryptophan hydroxylase activation in the control of 5-HT synthesis in rat brain slices incubated in K +-enriched medium. J. Neurochem. 33, 1031-1042.

HAMON, M., BOURGOIN, S. and YOUDIM, M. B. H. (1979b) Tryptophan hydroxylation in the central nervous system

Regulation of serotonin synthesis 13

and other tissues. In Aromatic Amino Acid Hydroxylases and Mental Disease, pp. 233-297, (ed. M. B. H. YOUDIM) John Wiley and Sons, New York.

HAMON, M., BOURC, OIN, S., ARTAUD, F. and EL MESTmAWV, S. (1981a) The respective roles of tryptophan uptake and tryptophan hydroxylase in the regulation of serotonin synthesis in the central nervous system. J. Physiol. (Paris) 77, 269-280.

HAMON, M., BOURGOIN, S., ARTAUD, F. and NELSON, D. (1981b) Regulatory properties of neuronal tryptophan hydroxylase. Adv. exp. Med. Biol. 133, 231-251.

HAMON, M., FATTACCINI, C. M., ADRmN, J., GALLISSOT, M. C., MART1N, P. and GOZLAN, H. (1988) Alterations of central serotonin and dopamine turnover in rats treated with ipsapirone and other 5-hydroxytryptaminelA agonists with potential anxiolytic properties. J. Pharmacol. exp. Ther. 246, 745-752.

HAMON, M. and BOURGOIN, S. (1979) Characterization of the Ca 2 +-induced proteolytic activation of tryptophan hydroxylase from the rat brain stem. J. Neurochem. 32, 1837-1844.

HART, R. P., YANG, R., RILEY, L. A. and GREEN, T. L. (1992) Post-transcriptional control oftryptophan hydroxylase gene expression in rat brain stem and pineal gland. J. molec. Cell Neurosci. 2, 71-77.

HARTIG, P. R. (1989) Molecular biology of 5-HT receptors. Trends pharmacol. Sci. 10, 64-69. HASEGAWA, H. and ICmVAMA, A. (1987) Tryptophan 5-monooxygenase from mouse mastocytoma: high-

performance liquid chromatography assay. Methods Enzymol. 142, 88-92. HERR, B. E., GALLAGER, D. W. and ROTH, R. H. (1975) Tryptophan hydroxylase: Activation in vivo following

stimulation of central serotonergic neurons. Biochem. Pharmacol. 24, 2019-2023. HERR, B. E. and ROTH, R. H. (1976) The effect of acute raphe lesion on serotonin synthesis and metabolism in the rat

forebrain and hippocampus. Brain Res. 110, 189-193. HERY, F., CHOUVET, G., KAN, J. P., PUJOL, J.-F. and GLOWlNSKL J. (1977) Daily variations of various parameters of

serotonin metabolism in the rat brain. II Circadian variations in serum and cerebral tryptophan levels: Lack of correlation with 5-HT turnover. Brain Res. 123, 137-145.

HERY, F., SIMMONET, G., BOURGOIN, S., SOUBRIE, P., ARTAUD, F. and HAMON, M. (1979) Effect of nerve activity on the in vivo release of I-3H]-serotonin continuously formed from [3H]-tryptophan in the caudate nucleus of the cat. Brain Res. 169, 317-334.

HERY, i . , FAUOON, M. and HERY, F. (1983) In vitro release of newly synthesized serotonin from superfused rat suprachiasmatic area--ionic dependency. Eur. J. Pharmacol. 89, 9-18.

HEYM, J., TRULSON, M. E. and JACOBS, B. L. (1982) Raphe unit activity in freely moving cats: effects of phasic auditory and visual stimuli. Brain Res. 232, 29-39.

HIRAYAMA, K. and KAPATOS, G. (1991) Dissociation of changes in GTP cyclohydrolase, tetrahydrohiopterin and tyrosine hydroxylase induced by NGF in superior cervical ganglia. Soc. Neurosci. Abstr. 17, 1118.

HJORTH, S. and MAGNUSSON, T. (1988) The 5-HT 1A receptor agonist, 8-OH-DPAT, preferentially activates cell body 5-HT autoreceptors in rat brain in vivo. Naunyn Schmiedeberg's Arch. Pharmacol. 338, 463-471.

ICHIMURA, T., ISOBE, T., OKUYAMA, T., YAMAUCH1, T. and FUJISAWA, H. (1987) Brain 14-3-3 protein is an activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca 2 +, calmodulin-dependent protein kinase II. FEBS Lett. 219, 79-82.

I1JIMA, S., HARA, K., SUGA, H., NAKAMURA, S. and KAMEYAMA, i . (1986) Effect ofischemia on hydroxylase cofactor (tetrahydrobiopterin) and monoamine neurotransmitters in rat brain. Stroke 17, 529-533.

IKEOA, M. and NAGATSU, T. (1985) Effect of short term swimming stress and diazepam on 3,4-dihydroxyphenyl- acetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) levels in the caudate nucleus: an in vivo voltammetric study. Naunyn Schmiedeberg's Arch. Pharmacol. 331, 23-26.

IMAL Y., YAMAUCm, T. and FUJISAWA, H. (1989) Modulation of tryptophan hydroxylase activity by phospholipids: stimulation followed by inactivation. J. Neurochem. 53, 1293-1299.

INVERNIZZL R., CARLL M., Dl CLEMENTE, A. and SAMANIN, R. (1991) Administration of 8-hydroxy-2-(Di-n- propylamino)tetralin in raphe nuclei dorsalis and medianus reduces serotonin synthesis in the rat brain: differences in potency and regional sensitivity. J. Neurochem. 56, 243-247.

ISOBE, T., ICHIMURA, T., SUNAYA, T., OKUYAMA, T., TAKAHASHI, N., KUWANO, R. and TAKAHASHI, Y. (1991) Distinct forms of the protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. J. molec. Biol. 217, 125-132.

JOH, T. H., SHIKIML T., PICKEL, V. M. and REIS, O. J. (1975) Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain. Proc. natl Acad. Sci. U.S.A. 72, 3575-3579.

JOH, T. H,, HWANG, O. and ABATE, C. (1986) Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. In Neuromethods Series 1: Neurochemistry. Neurotrasmitter Enzymes, pp. 1-32, (eds. A. A. BOULTON, G. B. BAKER and P. H. Yu) Humana Press, Clifton, NJ.

JOHANSEN, P. A., WOLF, W. A. and KUHN, D. M. (1991) Inhibition of tryptophan hydroxylase by benserazide and other catechols: Biochem. Pharmacol. 41, 625-628.

JOSEPH, M. H. and KENNE'rT, G. A. (1983) Stress-induced release of 5-HT in hippocampus and its dependence on increased tryptophan availability: An in vivo electrochemical study. Brain Res. 270, 251-257.

JuHus, D. (1991) Molecular biology of serotonin receptors. Ann. Rev. Neurosci. 14, 335-360. KAPATOS, G. (1990) Tetrahydrobiopterin synthesis rate and turnover time in neuronal cultures from embryonic rat

mesencephalon and hypothalamus. J. Neurochem. 55, 129-136. KATO, T., YAMAGUCH1, T:, NAGATSU, T., SUGIMOTO, T. and MATSUURA, S. (1980) Effects of structures of

tetrahydropterin cofactors on rat brain tryptophan hydroxylase. Biochim. biophys. Acta 611, 241-250. KATZ, I. R. (1980) Oxygen affinity of tyrosine and tryptophan hydroxylases in synaptosomes. J. Neurochem. 35,

760-763. KATZ, I. R. (1981) Interaction between the oxygen and tryptophan dependence of synaptosomal tryptophan

hydroxylase. J. Neurochem. 37, 447-451. KAUFMAN, S. (1986) Regulation of the activity of hepatic phenylalanine hydroxylase. In Advances in Enzyme

Regulation, Vol. 5, pp. 37-63, (ed. G. WEBER) Pergamon Press, New York.

14 M.C. BOADLE-BIBER

KAUFMAN, S. and FISHER, D. B. (1974) Pterin-requiring aromatic amino acid hyroxylases. In Molecular Mechanisms of Oxygen Activation, pp. 285-369, (ed. O. HAYAISHI) Academic Press, New York.

KENNETT, G. A., CURZON, G., HUNT, A. and PATEL, A. J. (1986) Immobilization decreases amino acid concentrations in plasma but maintains or increases them in brain. J. Neurochem. 46, 208-212.

KIM, K. S., WESSEL, T. C., STONE, D. M., CARVER, C. H., JOH, T. H. and PARK, D. H. (1991) Molecular cloning and characterization of eDNA encoding tryptophan hydroxylase from rat central serotonergic neurons. Brain Res. molec. Brain Res. 9, 277-283.

KORF, J. (1985) Turnover rate assessments of cerebral neurotransmitter amines and acetylcholine. In Neuromethods, Vol 2: Amines and Their Metabolites, pp. 407-456, (eds. A. A. BOULTON, G. B. BAKER and J. M. BAKER) Humana Press, Clifton, NJ.

KUHAR, M. J., ROTH, R. H. and AGHAJANIAN, G. K. (1971) Selective reduction of tryptophan hydroxylase activity in rat forebrain after midbrain raphe lesions. Brain Res. 35, 167-176.

KUHN, D. M., VOGEL, R. L. and LOVENaERG, W. (1978) Calcium-dependent activation of tryptophan hydroxylase by ATP and magnesium. Biochem. biophys. Res. Commun. 82, 759-763.

KUHN, D. M., MEYER, M. A. and LOVE~nERG, W. (1979) Activation of rat brain tryptophan hydroxylase by polyelectrolytes. Biochem. Pharmacol. 28, 3255-3260.

KUHN, D. M., RUSKIN, B. and LOVENBERG, W. (1980) Tryptophan hydroxylase. The role of oxygen, iron, and sulfhydryl groups as determinants of stability and catalytic activity. J. biol. Chem. 255, 4137-4143.

LEATHWOOD, P. D. (1987) Tryptophan availability and serotonin synthesis. Proc. Nutr. Soc. 46, 143-156. LEATHWOOD, P. D. (1990) Tryptophan availability and serotonin synthesis. In Serotonin: From Cell Biology to

Pharmacology and Therapeutics, pp. 221-225, (eds. R. PAOLETT1, P. M. VANHOUTTE, N. BRUNELLO and F. M. MAGGI) Kluwer Academic Publishers.

LEVlNE, R. A., KUHN, D. M. and LOVENBERG, W. (1979) The regional distribution of hydroxylase cofactor in rat brain. J. Neurochem. 32, 1575-1578.

LEV1NE, R. A., MILLER, L. P. and LOVENBERG, W. (1981) Tetrahydrobiopterin in striatum. Science 214, 919-921. LIPSCOMBE, O., KONGSAMUT, S. and TSIEN, R. W. (1989) Alpha-adrenergic inhibition of sympathetic

neurotransmitter release mediated by modulation of N-type calcium-channel gating. Nature 340, 639--642. LONG, J. B., YOUNGaLOOD, W. W. and KIZER, J. S. (1982) A microassay for simultaneous measurement of in vivo

rates of tryptophan hydroxylation and levels of sertonin in discrete brain nuclei. J. Neurosci. Meth. 6, 45-58. LONG, J. B., YOUNGBLOOD, W. Y. and KIZER, J. S. (1983) Regional differences in the response of serotonergic

neurons in rat CNS to drugs. Fur. J. Pharmacol. 118, 89-97. LOOKINGLAND, K. J., SHANNON, N. J., CHAr'IN, D. S. and MOORE, K. E. (1986) Exogenous tryptophan increases

synthesis, storage, and intraneuronal metabolism of 5-hydroxytryptamine in the rat hypothalamus. J. Neurochem. 47, 205-212.

Lvsz, T. W. and SZE, P. Y. (1978) Activation of brain tryptophan hydroxylase by phosphorylating system. J. Neurosci. Res. 3, 411-416.

MACMILLAN, V. (1987) A comparison of the effects of carbon monoxide intoxication and low-oxygen gas mixtures on cerebral biogenic amine metabolism. Brain Res. 408, 40-46.

MAKITA, Y., OKUNO, S. and FUJISAWA, n. (1990) Involvement of activator protein in the activation of tryptophan hydroxylase by cAMP-dependent protein kinase. FEBS Lett. 268, 185-188.

MITCHELL, S. N. and THOMAS, P. J. (1988) Effect of restraint stress and anxiolytics on 5-HT turnover in rat brain. Pharmacology 37, 105-113.

MIWA, S., WATANABE, Y. and HAYAISHI, O. (1985) 6R-L-erythro-5,6,7,8-tetrahydrobiopterin as a regulator of dopamine and serotonin biosynthesis in the rat brain. Arch. biochem. Biophys. 239, 234-241.

MOLLIVER, M. E., BERGER, U. V., MAMOUNAS, L. A., MOLLIVER, D. C., O'HEARN, E. and WILSON, M. A. (1990) Neurotoxicity of MDMA: Anatomical studies: In The Neuropharmacology of Serotonin, (eds. P. M. WHITAKER-AZMITIA and S. J. PEROUTKA) (Ann. N. Y. Acad. Sci. 600, 640-661).

MORGAN, W. W., RUDEEN, P. K. and PFEIL, K. A. (1975a) Effects of immobilization stress on serotonin content and turnover in regions of the rat brain. Life Sci. 17, 143-150.

MORGAN, W. W., SALDANA, J. J., CATHERINE, A. Y. and MORGAN, J. F. (1975b) Correlation between circadian changes in serum amino acids or brain tryptophan and the contents of serotonin and 5-HIAA in regions of the rat brain. Brain Res. 84, 75-86.

NECKERS, L. M., BIGGIO, G., MOJA, E. and Mm:K, J. L. (1977) Modulation of brain tryptophan hydroxylase activity by brain tryptophan content. J. Pharmacol. exp. Ther. 201, 110-116.

Nn"UWENHUYS, R. (1985) Chemoarchitecture of the Brain, pp. 1-246, Springer Verlag. RAPPORT, M. M., GREEN, A. A. and PAGE, I. H. (1947) Purification of the substance which is responsible for the

vasoconstrictor activity of serum. Fedn Proc. 6, 184. SALTER, M., KNOWLES, R. G. and POGSON, C. I. (1989) How does displacement of albumin-bound tryptophan cause

sustained increases in the free tryptophan concentration in plasma and 5-hydroxytryptamine synthesis in brain? Biochem. J. 262, 365-368.

SANDERS-BusH, E. ED. (1988) The Serotonin Receptors, pp. 1-388, The Humana Press, Clifton, NJ. SAWADA, M., SUGIMOTO, T., MATSUURA, S. and NAGATSU, T. (1986) (6R)-tetrahydrobiopterin increases the activity

of tryptophan hydroxylase in rat raphe slices. J. Neurochem. 47, 1544-1547. SAWADA, M. and NAGATSU, T. (1986) Stimulation of the serotonin autoreceptor prevents the calcium-calmodulin-

dependent increase of serotonin biosynthesis in rat raphe slices. J. Neurochem. 46, 963-967. SCHAECHTER, J. D, and WURTMAN, R. J. (1989) Tryptophan availability modulates serotonin release from rat

hypothalamic slices. J. neurochem. 53, 1925-1933. SCHAECHTER, J. D. and WURTMAN, R. J. (1990) Serotonin release varies with brain tryptophan levels. Brain Res. 532,

203-210. SHANNON, N. J., GUNNET, J. W. and MOORE, K. E. (1986) A comparison of biochemical indices of

5-hydroxytryptaminergic neuronal activity following electrical stimulation of the dorsal raphe nucleus. J. Neurochem. 47, 958-965.

SHARP, T., BRAMWELL, S. R., CLARK, D. and GRAHAME-SMITH, D. G. (1989) In vivo measurement of extracellular 5-

Regulation of serotonin synthesis 15

hydroxytryptamine in hippocampus of the anaesthetized rat using microdialysis: Changes in relations to 5-hydroxytryptaminergic neuronal activity. J. Neurochem. 53, 234-240.

SHARP, T., BRAMWELL, S. R. and GRAHAm'E-SMITH, D. G. (1990) Release of endogenous 5-hydroxytryptamine in rat ventral hippocampus evoked by electrical stimulation of the dorsal raphe nucleus as detected by microdialysis: Sensitivity to tetrodotoxin, calcium and calcium antagonists. Neuroscience 39, 629-637.

SHIELDS, P. J. and ECCLESTON, D. (1972) Effects of electrical stimulation of rat midbrain on 5-hydroxytryptamine synthesis as determined by a sensitive radioisotope method. J. Neurochem. 19, 265-272.

SmROTA, K. and FUJISAWA, H. (1998) Purification and characterization of aromatic L-amino acid decarboxylase from rat kidney and monoclonal antibody to the enzyme. J. Neurochem. 51,426-434.

Slow, Y. L. and DAKSHINAMURTI, K. (1990) Purification of dopa decarboxylase from bovine striatum. Molec. Cell Biochem. 94, 121 131.

SMITI~, R. H., GUILBEAU, E. J. and RENEAU, D. D. (1977) The oxygen tension field within a discrete volume of cerebral cortex. Microvasc. Res. 13, 233-240.

SOURKES, T. L. (1978) Tryptophan in hepatic coma. J. neural Transm. Suppl. 14, 79-86. STEINaUSCH, H. W. M. and NIEUWENHUVS, R. (1983) The raphe nuclei of the rat brain stem: A cytoarchitectonic and

immunohistochemical study. In Chemical Neuroanatomy, pp. 131-207, (ed. P. C. EMSON) Raven Press, New York.

STOLE, J. and GOLDMAN, O. (1991 ) Isolation and structural characterization of the murine tryptophan hydroxylase gene. J. Neurosci. Res. 28, 457-465.

SUMI, C., ICmNOSE, H. and NAGATSU, T. (1990) Characterization of recombinant human aromatic L-amino acid decarboxylase expressed in COS cells. J. Neurochem. 55, 1075-1078.

SUZUKI, S., WATANABE, Y., TSUBOKURA, S., KAGAMIYAMA, H. and HAYAISHI, O. (1988) Decrease in tetrahydrobiopterin content and neurotransmitter amine biosynthesis in rat brain by an inhibitor ofguanosine triphosphate cyclohydrolase. Brain Res. 446, 1-10.

TAMIR, H. and GERSHON, M. D. (1990) Serotonin-storing secretory vesicles. Ann. N. Y. Acad. Sci. 600, 53-66. THIERRY, A.-M., FEKETE, M. and GLOWINSKI, J. (1968) Effects of stress on the metabolism of noradrenaline,

dopamine and serotonin (5-HT) in the central nervous system of the rat: II Modifications of serotonin metabolism. Eur. J. Pharmacol. 4, 384-389.

TISON, F., NORMAND, E., JAaER, M., AUBERT, I. and BLOCH, B. (1991) Aromatic L-amino-acid decarboxylase (DOPA decarboxylase) gene expression in dopaminergic and serotoninergic cells of the rat brainstem. Neurosci. Lett. 127, 203-206.

TORK, I. (1985) Raphe nuclei and serotonin containing systems. In The Rat Nervous System. Vol. 2. Hindbrain and Spinal Cord, pp. 43-78, (ed. G. PAXlNOS) Academic Press.

TORK, I. (1990) Anatomy of the serotonergic system. In The Neuropharmacology ofSerotonin (eds. P. M. WmTAKER- AZMIT1A and S. J. PEROUTKA) (Ann. N. Y. Acad. Sci. 600, 9-35).

TRULSON, M. E., JACOBV, J. H. and MACKENZIE, R. G. (1986) Streptozotocin-induced diabetes reduces brain serotonin synthesis in rats. J. Neurochem. 46, 1068-1072.

TRULSON, M. E. and JACOBS, B. L. (1979) Raphe unit activity in freely moving cats: Correlation with level of behavioral arousal. Brain Res. 163, 135-150.

VANDERMAELEN, C. P. (1985) Serotonin. In Neurotransmitter Actions in the Vertebrate Nervous System, pp. 201-240, (eds. M. A. ROGAWSKI and J. L. BARKER) Plenum Press, New York.

VOGT, M. (1982) Some functional aspects of central serotonergic neurones. In Biology of Serotonergic Transmission, pp. 299-315, (ed. N. N. OSBORNE) John Wiley.

WESTERINK, B. H. and DE VRIES, J. B. (1991) Effect of precursor loading on the synthesis rate and release of dopamine and serotonin in the striatum: a microdialysis study in conscious rats. J. Neurochem. 56, 228-233.

WOLF, W. A., ANASTASIADIS, P. Z., KUHN, D. M. and LEVINE, R. A. (1990) Influence of tetrahydrobiopterin on serotonin synthesis, metabolism and release in synaptosomes. Neurochem. Int. 16, 335-340.

WOLF, W. A., Z1AJA, E., ARTHUR, R. A. Ja., ANASTASIADIS, P. Z., LEVZNE, R. A. and KUHN, D. M. (t991) Effect of tetrahydrobiopterin on serotonin synthesis, release, and metabolism in superfused hippocampal slices. J. Neurochem. 57, 1191-1197.

WOLF, W. A. and KUHN, D. M. (1986) Uptake and release oftryptophan and serotonin: an HPLC method to study the flux of endogenous 5-hydroxyindoles through synaptosomes. J. Neurochem. 46, 61-67.

YAMAUCHI, T. and FUJISAWA, H. (1979a) Regulation of rat brainstem tryptophan 5-monooxygenase. Calcium- dependent reversible activation by ATP and magnesium. Arch. biochem. Biophys. 198, 219-226.

YAMAUCHI, T. and FUJISAWA, H. (1979b) Activation of tryptophan 5-monooxygenase by calcium-dependent regulator protein. Biochem. biophys. Res. Commun. 90, 28-35.

YOUNG, S. N. and SOURKES, T. L. (1977) Tryptophan in the central nervous system: Regulation and significance. Adv. Neurochem. 2, 133-191.

ZIGMOND, R. E., SCHWARZSCmLD, M. A. and RITrENHOUSE, A. R. (1989) Acute regulation of tyrosine hydroxylase by nerve activity and by neurotransmitters via phosphorylation. Ann. Rev. Neurosci. 12, 415-461.

JPIB 60:I-B