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Brain 5-hydroxytryptamine metabolism: Adaptive changes after long-term administration of psychotropic drugs
Gvn. Pharmac. ~ol 9, pp 307 to 314 ¢3 Peryamotl Pres, Ltd 197g Prmt,,d tn (irt'at Brttam
0306-362] 781(X)1-0307502.00.0
MINIREVIEW BRAIN 5 - H Y D R O X Y T R Y P T A M I N E M E T A B O L I S M : A D A P T I V E C H A N G E S AFTER L O N G - T E R M ADMINISTRATION OF PSYCHOTROPIC DRUGS R. B. RASTOGI* and R. L. SINGIIAL Department of Pharmacology, Faculty of Medicine, University of Ottawa, Ottawa, Canada KIN 9A9 (Received
20
December
1977)
Abstract--l. Evidence has been presented to suggest that 5-hydroxytryptamine is a neurotransmitter important to many regulatory processes, basic life functions, and behaviour. 2. Our data demonstrate that thyroid hormone plays an important role in the control of 5-HT metabolism especially in the developing brain. 3. The possibility is raised that anxiety, hypermobility and psychoses seen in hyperthyroid subjects are associated with increased serotonergic activity in the brain. 4. Similarly, this indoleamine may bc implicated in the emotional instability seen during altered levels of adrenocortical hormones. Finally, evidence is cited to support the view that serotonergic neurons elicit adaptive changes in response to external or internal stimuli.
stores, and nialamide which causes a selective increase in the neuronal 5-HT stores by inhibiting the deaminating enzyme monoamine oxidase (MAO), the distribution of fine 5-HT nerve terminals in the septal area, anterior colliculi, preoptic area, hippocampal formation, and in the neo- and mesocortex was more easily studied (Dahlstrom & Fuxe, 1964). 6-Hydroxytryptamine is another chemical which gives a good fluorescene yield and was employed to study the distribution of 5-HT nerve endings (Jonsson & Sandier. 1969). It is interesting to note that the fluorescence is also improved by pretreatment of animals with precursor L-tryptophan, which causes a selective increase in the fluorescence intensity of 5-HT neurons.
Since the first demonstration of 5-hydroxytryptamine (5-HT) in mammalian tissues by Rapport et al. (1948), this indoleamine has become the focal point of many recent investigations in neurobiology. The gradual unfolding of the ubiquitous importance of this neurohormone had led to the concept that 5-HT is one of a dozen of the probable neurotransmitters in the central nervous system which plays an important role in behaviour and mental function. The aim of this review is to summarize recent investigations concerned with the localization and metabolism of 5-HT in the brain, the interrelationship between central 5-HT neurons and hormones, and finally, differential effects on brain 5-HT-ergic systems following acute and chronic administration of certain psychotropic drugs.
TRYPTOPHAN: A M O D U L A T O R OF 5.-HT SYNTHESIS
5-HYDROXYTRYPTAMINE IN BRAIN: CURRENT STATUS OF LOCALIZATION A N D METABOLISM
There is now ample evidence to suggest that one of the rate-limiting steps for 5-HT formation in the brain is the concentration of tryptophan (TP) at the site of synthesis. This has been explained by the relatively high K,, of the rate-limiting enzyme, tryptophan hydroxylasc (TPH). which approximates normal brain TP concentration. As a consequence, basal 5-HT synthesis is thought to be both substrate and enzyme-dependent with the basal rate approximating Vm~. Brain TP is derived from plasma and after a TP load the concentrations in brain and plasma are directly proportional to each other (GrahameSmith, 1971). Furthermore, TP is the only essential amino acid which is partially bound to albumin in the plasma (McMenamy & Oncley, 1958); the binding is pH sensitive and TP can be displaced from albumin-binding sites by agents such as free fatty acids (Curzon et al., 1973). The binding, in general, implies storage, hence it is reasonable to suggest that the quantity which reaches cerebral tissue via the blood
The histochemical fluorescence techniques have yielded important knowledge on the circuitry of 5-HT containing neuronal pathways. Evidence indicates that most of the cell bodies are localized in the raphe nuclei. However, more recent studies have demonstrated that quite a few are also localized outside the raphe region within the vcntromedial reticular formation of the pons and the mesencephalon (Fuxe & Jonsson, 1974). From these cell bodies, axons course to provide 5-HT-containing nerve terminals in most parts of the CNS and certain areas of the spinal cord. By employing pharmacological tools, such as reserpine, which depletes the catecholamine and 5-HT
* Present address: Chief Pharmacologist, Nordic Research Laboratories, Pointe Claire, Montreal. Quebec. 307
3(~
R.B. RASIO(,a and R. L. SIN(JHA[.
brain barrier is closely dcpcndent on the small proportion of the amino acid still free in plasma. Earlier studies had demonstrated the existence of saturable, neutral amino acid carrier system (Lajtha & Toth. 19611 and Oldendorf {1971) has shown that most neutral amino acids compete with TP for transport at this carrier site. A year later, Fcrnstrom and Wurtman (1972) reported that the main determinant of brain TP and in turn 5-HT concentration does not appear to be free plasma TP alone, but the ratio of this amino acid to other plasma neutral amino acids such as tyrosine, phenylalanine, leucine, isoleucin and valinc. More rccently, it was shown that at normal blood concentrations, the individual amino acids inhibit TP transport into the brain in the following order: leucin > tyrosine > threonine > phenylalanine = histidine > isoleucine = methionine > valine (Yuwiler e t al.. 1977). I..;pon reaching the brain, I P is taken tip from the external mcdium into synaptosomcs, the so-callcd pinched nerve endings. The uptake across the neuronal membrane is stereospecific, tcmperaturc dependent, inhibited by metabolic inhibitors, but probably not too sensitive to changes in extcrnal Na" or K ' . There appears to bc two systems operating for TP uptake by synaptosomcs: one is the high-affinity uptakc system (K,, = 5.5 × 10 5 M) which is energy-dependent, temperature-sensitive and drug alterable. The second is the low affinity TP uptake systcm (K,, = 3.3 × 10 3 M) which does not sccm to be drugsensitive (Knapp & Mandell. 1972a). Synaptosomcs concentrate TP to a ratio of 4:1 (int:ext){Green & Grahamc-Smith, 1975). Denizean and Sourkes (1977) observed that the rate of TP uptake was highest in slices and synaptosomes prepared from hypothalamus (rich in serotonergic nerve endings). The finding correlates well with the data of Knott and Curzon (1974) who showed a larger incrcasc of TP in the hypothalamus than in other regions after an intraperitoneal injection of this amino acid (50 mg,'kg). Intravenous infusion of TP was found to elevate brain 5-HT and 5-HIAA levels within I hr after infusion, however: a proportional rise in 5-HT release in cerebral spinal fluid (CSF) was observcd only during the 2nd hr of infusion (Tcrnaux et al.. 1976). The transport of TP into synaptosomes can bc affected directly or indirectly by drugs, lmipramine, chlorimipramine and cocaine inhibited both TP uptake and 5-HT synthesis (Hamon & Glowinski. 1974). In contrast rcscrpine (Tozer ct al.. 1966) o r lithium treatment (Knapp & Mandcll, 1973: Rastogi & Singhal, 1977) activated TP transport as well as 5-HT s',nthesis in the brain. In new born animals, TP is present almost totally in free state in the plasma (Hamon & Glowinski, 1974). This free state of amino acid is not solely related to the presence of high levels of fatty acids in the blood, but also to the differences in the binding capacit~ of albumin in new born animals. Despite high levels of brain TP (Hoff et al.. 19741, the amount of 5-HT in new born rats is very low. This lower concentration of 5-HT may, in part, be due to decreased activity of T P H in neonatal rats (Rastogi & Singhal, 1974: Schmidt & Sanders-Bush, 1971). Since the rate of 5-HT accumulation in corresponding neurons is limited by their capacity for uptake and storage into granules, the possibility also rcmains that
these mechanisms might be undcr-dcvcloped in brains of immature animals. In presence of excess substrate, an increased amount of 5-HT might be synthesizcd and in turn is rapidly metabolized by intraneuronal monoamine oxidasc (MAOL without cvcr being stored in the synaptic vesicles and taking part in neural transmission in young brain. This 5-HT can then be presumed as "nonfunctional", in contrast to the "functional pool" of 5-HT which is storcd in the synaptic vesicles and is released upon ncrvc stimulation before being finally mctabolizcd by the deaminating enzyme (Fig. 1). Thus, it might be conceived that administration of TP or 5-hydroxytryptophan does not necessarily raise the levels of "'functional" 5-HT in thc brain. Moir and Eccleston (1968) observed that acute loading doses of 5-hydroxytryptophan markedly elevated the levels of 5-hydroxyindoleacetic acid (5-HIAA) without appreciably altering the levels of brain 5-HT. A kind of "'shunt" mechanism has been hypothesized by these investigators in which 5-hydroxytryptophan was rapidly mctabolised to 5-HIAA without eliciting any effect on the level of 5-HT. However. caution must be exercised while extrapolating these findings to chronic situations. The possibility can be raised that with an acute loading dose. the rate of accumulation of 5-HT in scrotonergic neurons is limited by the capacity for uptake into the storage granules, whereas with continuous administration, the storage capacity might be augmented.
TRYPTOPHAN HYDROXYI,ASE
Considerable attention has been devoted to studying the nature and requirements of TPH, the ratelimiting enzyme in the synthesis of 5-HT (Fig. 1). During the past few years, efforts have also been made to purify this enzyme (Lovenberg et al., 1973: Youdim e t al., 1974). Evidence indicates that TPH is a mixed function oxygenase requiring both molecular oxygen and a redudng agent for activity. Similar to other aromatic amino acids, TPH requires a tetrahydropterin as the reducing component of the system. Knapp e t al. (1975) demonstrated that calcium produced a dose-dependent activation of T P H activity in t, itro. A similar risc in activity of acetylcholincstcrase by calcium was reported by Dawson and Crone (1973). Howcver, the activation of TPH by calcium is very unlikely to be of any physiological significance as it occurs with concentrations (1.0 or 2.0 mM Ca2"), which arc much higher [10 3- 10 4 times) than those reportcd in nerve cells. Tryptophan hydroxylasc is synthesized in the cell body and undergoes axonal transport to the nerve endings, wherc 5-HT synthesis takes place (Meek & Neff, 1972). The axonal flow rate of TPH is probably 1--.2 mm/day as has been rcported for soluble proteins. It is therefore reasonable to believe that acute changes in 5-HT synthesis are mediated by regulation of enzyme activity rather than by changes in enzyme synthesis p e r se. It has been postulated that two forms of T P H may exist (lchiyama et al.. 1970; Knapp & Mandell, 1972a). The soluble form of T P H is present in cell bodies whereas the particulate enzyme is presumed to be bound to neuronal membrane of sero-
Psychotropic drugs and brain 5-hydroxytryptamine changes
I TRYPTOPHAN
[
t
-
-
309
~
I
,ID• "
e a
:
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(TPH)
• /[5 HYDROX¥ TRYPTOPHAN l I
, i ,
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FUNCTIONA LtY ~4'~--'~'~ ACTIVE
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",
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Fig. 1. The pathways of 5-hydroxytryptamine metabolism in brain. Abbreviations used: tryptophan hydroxylase (TPH); 5-hydroxytryptophan decarboxylasc I5-HTPDI: 5-hydrox',tryptamine {5-HTR monoamine oxidase (MAO): 5-hydroxyindoleacctic acid (5-HIAA}.
tonergic nerve terminals. In point of fact, an intriguing correlation has been shown between the proportion of the enzyme which is in the soluble or particulate fraction, and the relative concentration of serotoncrgic cell bodies (mid-brain) or nerve endings (septal area) (Knapp & Mandeil, 1972b). The two forms of TPH do not react in a similar way to specific inhibitors such as p-chlorophenylalanine (Knapp & Mandell, 1972a) and their affinity constants toward TP appear to be different (lchiyama et al., 1970).
M E M B R A N E R E C E P T O R SITES FOR 5-HT
The presence of a 5-HT sensitive adenylate cyclase in adult rat brain synaptosomal membrane has been reported by Pagcl et al. (1976). In addition, Haigler and Aghjanian (1974) have shown by iontophoresis that there are both postsynaptic excitatory and inhibitory binding sites for 5-HT in various brain areas as well as presynaptic uptake sites. More recently, attempt has been made to isolate and characterize the 5-HT membrane-localized receptor which appears to be a proteolipid possessing activated receptor carbonyl residue. It is interesting that fluphenazine, a very potent antipsychotic drug known to block dopamine-sensitive adenylate cyclase activity was found to produce virtually no effect on 5-HT associated C-AMP production in the brain. These findings are well correlated with the report of McClain and Christian (1975) who observed no effect of fluphenazine and other antipsychotic drugs on the binding of 5-HT to synaptosomal membranes. These data lead one to believe that antipsychotic compounds may elicit their therapeutic effects probably by affecting dopamine receptors but not 5-HT receptor sites of the brain. These suggestions are, however, speculative and future work must include a complete characterization of the receptor site.
END-PRODUCT REGULATION OF 5-HT SYNTHESIS
Our current understanding of the short term regulatory processes of catecholamine synthesis suggests that cytoplasmic dopamine or norepinephrine partially controls the rate of the conversion of tyrosine into DOPA by end product inhibition (Kehr et al., 1972). In contrast, 5-HT has no effect on the activity of soluble TPH (Jequier et al., 1969) which converts TP to 5-hydroxytryptophan. This is why the hypothesis of an in t:ivo end-product regulation of 5-HT synthesis was not retained for many years. However, on the basis of data obtained from more recent studies, this hypothesis cannot be refuted completely. It has been shown that 5-HT at high concentrations (10 -3 M)does inhibit the enzyme in a non-competitive manner (Knapp & Mandell 1973). Additionally, Macon et al. (1971) demonstrated that 5-HT synthesis was reduced both in vit'o and in t'itro when intraneuronal concentrations of the amine attained 2.5 times normal levels either following inhibition by monoamine oxidase (MAO) or exogenous 5-HT. This inhibitory process seems to occur during the first step of the indoleamine synthesis, since no reduction in [3H]5-HT formation could be noted in rats injected with MAO inhibitors when [3H]5-hydroxytryptophan was substituted for [3H]TP. Carlsson and his associates (1972) employing another pharmacological tool confirmed this hypothesis. Whether or not this mechanism really plays a part in controlling the rate of tryptophan hydroxylation under physiological stimulation or inhibition of 5-HT-ergic neuronal activity remains an important issue to debate. I N T E R R E L A T I O N S H I P BEI"WEEN CENTRAL 5-HT N E U R O N S A N D H O R M O N F ~
T h y r o i d hormone
A number of instances have been cited to support the view that thyroid hormone plays an important
3111
R.B. RASIOGI and R. L. SI,~C;IVAn
role in the structural and biochemical ontogeny of the central nervous system which are believed to bc the determinants of adult neurophysiological and behavioural processes. Excessive thyroid secretion produces psychological symptoms which include emotional lability, restlessness, irritability, overreactivcness with predominant anxiety and tension (Eayrs, 1964: Whybrow & Farrell, 1974). Psychoses of acute organic type have frequently been encountered in severe cases and also during thyroid crisis. Since 5-HT plays an important role in affcctive disorder and anxiety, we were prompted to examine the effect of neonatal hyperthyroidism on brain 5-HT metabolism (Rastogi & Singhal, 1976a). Daily administration of zAriiodothyronine (T3) for 30 days beginning from one day of age. significantly increased T P H activity and TP levels in the mid-brain (Table 1). It is suggested that T3 which also binds to albumin, displaces TP from binding site making more of it available in free form to cross blood brain barrier and reach neuronal tissue. Our more recent work demonstrated that T3-treatment significantly enhanced the rate of 5-HT synthesis in the sub-cellular fraction as well (Singhal & Rastogi. 1977). Despite this, the endogenous levels of 5-HT in various discrete brain areas remained unchanged suggesting that increased synthesis kept pace with increased utilization of this indoleamine in the brain as evidenced by increased levels of 5-HIAA (Table 1). However, hyperthyroidism failed to alter M A O activity in certain brain areas examined (Rastogi & Singhal, 1976a). Evidence has emerged to suggest a direct correlation between serotonergic activity and locomotor performance in experimental animals (Green & Kelly. 1976: Lassen et al., 1976). Clinical
studies demonstrated enhanced levels of 5-H1AA in ccrebrospinal fluid of man subjected to increased psychomotor activity (Post et al., 1973). Hence it is likely that hyperactivity seen in hyperthyroid animals (Rastogi & Singhal. 1976a) might, in part, be associated with increased scrotonergic activity, in addition to enhanced catccholaminergic activity in the brain (Rastogi & Singhal, 1976b; Singhal et al., 1977). In contrast to hyperthyroidism, thyroid deficiency was accompanied by decreased activity of T P H (Rastogi & Singhal, 1974: Rastogi et al.. 1976) as well as decreased levels of 5-1-|T in several brain areas of developing rats [Table 1). Toth and C ~ b a 11966) had earlier reported significant reduction in 5-HT levels of brain stem and blood in thyroidcctomized rabbits. Clinical studies reported a decline in blood 5-HT in "'early myxedema" with return to normal following thyroid therapy (L. T. Giron-Personal Communication). One aspect of 5-HT metabolism after neonatal thyroidectomy which warrants consideration is the fact that in contrast to the observed lowering in 5-HT levels, the concentration of 5-HIAA was significantly increased in whole (Rastogi & Singhal, 1974) as well as discrete areas of the brain (Table 1}. Whereas radiothyroidcctomy at birth decreased the activity of dcaminating enzyme M A O in the hypothalamus (by 141~,,), it was significantly increased in mid-brain (by 14°o) and remained unaltered in cortex, brain stem and striatum. It is likely that brains of thyroid-deftcient rats may have an impaired efflux mechanism for 5-HIAA (Singhal et al., 1975). Clinical studies (Dewhurst et al., 1969) have demonstrated low levels of thyrotropic hormone in the blood of patients suffering from endogenous depression, an affective dis-
Table 1. Changes in 5-hydroxytryptaminc metabolism during altered thyroidal states and corticosterone treatment Mid-brain TP TPH (Pg.:g) (nmoles/g/hr) Control Hypothyroid Control Hyperthyroid
5.71 _+0.31 5.37 _+0.29 6.4 _+0.41 9.02 _+0.63*
9.13 _+1).71) 6.94 _+0.36* 11).78 -+0.96 13.78 _+ 1.10"
Brian stem Sham operated (Control) Adrenalectomized 115 days) Adrenalectomizcd + Corticosteronc (7 days)
2.99 _+0.18 2.45 -+1).14" 2.92 _+0.161
7.34 _+0.82 5.73 _+0.41" 7.11 _+0.46t
5-HT (pg/g) Midbrain
5-HIAA (,ug/g)
Striatum
Hypothalamus
Midbrain
Striatum
Hypothalamus
1.69 _+0.05 1.20 _+0.04* 1.76 _+0.09 1.71 _+0.11
1.44 _+0.08 0.99 _+0.1)6" 1.52 _+0.18 1.16 _+0.17
2.12 _+0.15 1.84 +0.13 2.49 _+0.17 1.97 -+1).18"
1.53 _+0.08 1.94 +0.12" 1.64 .+_0.07 1.98 -+0.09"
1.32 _+0.09 1.80 -+0.14" 1.37 _+0.08 2.32 -4-0.11"
2.04 _+0.22 2.37 _+0.26 1.95 _+0.19 2.96 -+0.24*
Brain stem
Striatum
Hypothalamus
Brain stem
Striatum
Hypothalamus
0.68 _+0.04 0.50 _+0.02* 0.61 _+0.02t"
1.32 _+0.09 0.92 _+0.03* 1.14 _+0.10"1
1.83 _+0.11 1.61 _+0.15 1.92 _+1).25
0.62 _+0.03 0.76 _+0.04* 0.63 _+0.02~
0.98 -+0.06 1.23 _+0.05* 0.86 _+0.08t
1.73 +0.07 2.02 _+0.15 1.88 -+0.07
Each value represents the mean + S.E.M. of 6 animals in the group. Neonatal hypothyroidism was induced by a single injection of 200 ,uCi of t'~Xl at one-day of age and animals were sacrificed 30 days later. Neonatal hyperthyroidism was induced by daily injections of T 3 111)pg/100 g) for 30 days beginning from the one-day of age. Rats were sacrificed at 30 days of age. A group of adrenalectomized rats received corticosterone (10 mg/kg/day) for 7 days beginning from 8 days after adrcnalectomy. * Statistically significant difference when compared with the control values (P < 0.051. + Statistically significant difference when compared with the value of adrenalectomizcd rats (P < 0.05).
Psychotropic drugs and brain 5-hydroxytryptamine changes order which shows certain biochemical and psychological features common with hypothyroidism. Evidence also indicates that a number of depressed patients with latent hypothyroidism respond well to thyroid medication in addition to tricyclic antidepressant drugs (Hatotani et al., 1977). It is therefore reasonable to believe that hypomobility and suppressed behaviour seen during "cretinoid" syndrome might, at least in part, be associated with decreased functioning of 5-HT-ergic neurons in addition to decreased dopaminergic (Singhal et al., 1976) and enhanced cholinergic activity in brain (Hrdina et al., 1975). Adrenocortical hormone
The pituitary-adrenal activity is known to be particularly sensitive to fluctuations in the external environmental milieu with which the internal homeostasis of an organism must equilibrate. There is emerging evidence to suggest that serotonergic nerve fibers containing structures like cortex, hippocampus and amygdala constitute a functional unit which modulates the regulation of ACTH (Scapagnini et al., 1971) and the pharmacological agents which affect brain 5-HT metabolism, abolish the daily rise in plasma 17-hydrocorticosteroids (Krieger & Rizzo, 1969). On the other hand, manipulation of adrenocortical levels has been found to decrease the biosynthesis of 5-HT in the brain (Rastogi & Singhal, 1978). Sze et al. (1976) reported that adrenalectomy interfered with the developmental as well as reserpine-induced rise of TPH activity in neonatal as well as adult brain. We found that even though 5-HT levels were lowered, the concentration of 5-HIAA was increased following adrenalectomy (Table 1). It is likely that the rise in brain 5-HIAA levels might, in part, be due to enhanced intraneuronal deamination of 5-HT by MAO, whose activity was significantly increased by 20~o following adrenalectomy. Our finding that the replacement corticosterone therapy restored 5-HT metabolism to virtually normal limits suggests that the observed neurochemical changes might be specific to adreno-corticoid hormones. Daily administration of corticosterone for 6 days not only increased TP and TPH activity, but also 5-HT levels in brains of adrenalectomized rats. Corticosterone-induced rise in TPH enzyme has been found to be cycloheximide-sensitive, suggesting that the rise in TPH activity represents de novo synthesis (Azmitia & McEwen, 1969). Glucocorticoids also stimulate the uptake of TP by nerve terminals and thereby accelerate the rate of 5-HT synthesis in the brain (Neckers & Sze, 1975). These authors have postulated that glucocorticoids in its "'immediate" action, rapidly accelerate 5-HT synthesis through an increased uptake of TP by nerve terminals. In the "slow" action, the hormone probably plays a permissive role in the induction of TPH by some unknown mechanisms. D I F F E R E N T I A L EFFECTS ON BRAIN 5-HT-ERGIC SYSTEM F O L L O W I N G ACUTE AND C H R O N I C ADMINISTRATION OF PSYCHOTROPIC DRUGS
Earlier, it was considered that alterations in neuro-
311
transmitter dynamics following chronic treatment were merely temporal extensions of those seen after acute treatment, requiring the continued presence of the agent inducing the change. However, a number of findings reported by Mandell (1975) and his associates suggest that changes in central amines, particularly NE and 5-HT metabolism, may not necessarily be in the same direction after long-term administration of a psychotropic drug, as those seen after a single injection. The so-called "secondary adaptive change" which has achieved common usage in discussions of intraneuronal regulation of transmitter synthesis, involves "product-feed back inhibition". In other words, the increased outward flow of intraneuronal transmitter stores results in a lowering of endogenous amine levels which in turn minimizes the inhibitory influence on the synthesizing enzyme. In contrast, decreased release results in decreased rate of synthesis (Macon et al., 1971). This section will attempt to examine the recent work on the influence of short and long-term administration of certain psychotropic drugs on brain 5-HT metabolism. Lithium
The promise offered by lithium in combatting mania or hypo-mania has added considerable impetus to investigations of the mechanisms by which this alkali metal elicits its beneficial effects. Since 5-HT was implicated in the pathogenesis of mania, many investigators have directed their attention to studying the effects of lithium on serotonergic neurons in the brain. These reports have been somewhat contradictory. For example, whereas some workers reported an increase in 5-HT turnover following lithium (Perez-Cruet et al., 1971; Rastogi & Singhal, 1977al others found either an opposite (Ho et al., 1970) or no effect (Kuriyama & Speken, 1970). Of course, some of these discrepancies may be related to differences in the dose and duration of lithium treatment and the regions of brain examined. Knapp and Mandell (1973) demonstrated that after 5 days of lithium treatment, there was stimulation of the high-attinity TP uptake system into synaptosomes. This resulted in increased synthesis of 5-HT in striatum, a region rich in 5-HT nerve terminals (Ternaux et al., 1977). Lithium is also known to suppress the impulse-induced release of 5-HT from serotonergic neurons (Schubert, 1973), resulting in an elevation of intraneuronal stores of 5-HT. The high level of 5-HT will presumably inhibit the rate of synthesis by lowering the formation of TPH within the cell bodies localized in mid-brain. Indeed, an increased TPH activity in striatum and a decreased TPH activity in mid-brain has been reported after short-term (5 days) treatment with lithium (Knapp & Mandell, 1973). However, after 21 days of treatment, these authors found a decrease in TPH activity of synaptosomes, when the values were compared with those seen after 5 days of lithium treatment. The apparent delay in decrease of TPH activity in serotonergic nerve endings is probably related to slow axoplasmic flow of the enzyme as stated elsewhere in this review. Morphine
A similar type of adaptive change in TPH activity following short and long-term administration of mor-
312
R.B. r a s t ( ~ l and R. L. Sl~(.atat, Table 2. Effects of short- and long-term DZP treatment on 5-HT synthesis m midbrain and P2 pellet Tissue examined Mid-brain
Parameter
Acute Control Treated
TP (,ug gl
10.29 4-0.73
7.82 _+0.52*
T P I I Inmol g hr)
8.21 _-0.N)
6.01 F_t).40*
5-HT lt~g.gl TP I/~g."gl
5-HT 0~g,gl
7.86
7.56 _+0.34* 10.71
2.16
_+0.74* 2.80
+_0.12"
± 0.(?9
_+0.19"
16.49 -:- 1.03 0.92
3.42 _+o.25 15.26 _+1.31 1.12
3.62 z0.12 1791 ..+ 1.12 t).7g
7.~6 _+0.69* 22.92 _+ 1.46" I. 17
r0.06
+0.07*
_+().(X~
4-0.1 I*
1.65 3.98 _+0.28
5-HT Synthesis "
5.73 4-0.28 _+0.45 1.61
~(1.08
P2 pellet
('hronic ('ontro] Treated
Values represent the means -4- S.E.M. of 6 rats in the group. DZP (10 mg.kg) was administered s.c. either acutel,, or chronically for 22 days. In acute study, animals were sacrificed 2 hr after the single injection whereas in chronic experiments, rats were killed 6 hr after the 22nd injection of DZP. Corresponding controls received an equal volume of the vehiclc. "pmoles ~'~CO2..mg protein25 min. * Statistically significant difference when compared with thc values of control rats (P < 0.051. phine has also been observed (Knapp & Mandell. 1972b). Whereas single injection of morphine decreased the activity of T P H in 5-HT nerve terminals, it failed to change T P H in mid-brain area. In contrast, chronic exposure to morphine by pellet implantation for 5 days resulted in a significant increase in the activity of nerve ending enzyme but not in cell body preparation. Morphine treatment at no time produced any significant change in the activity of 5-hydroxytryptophan decarboxylase, an intermediate enzyme involved in 5-HT synthesis. Cocaine
Much of the work on the effects of cocaine on the central nervous system has been concerned with central catecholaminergic systems. However, recent studies have demonstrated that cocaine manifests its behavioural effects partly by stimulating 5-HT receptors, which by a negative feedback mechanism affects the synthesis of this neurotransmitter. Taylor and Ho (1976) reported that short-term (5 days) treatment with cocaine preferentially inhibited the soluble form of TPH, presumed to be localized in the cytoplasm of cell bodies. When the treatment was extended to 45 days, the activity of soluble TPH returned to normal, and that of the particulate enzyme (localized in the nerve terminals) was significantly increased. It would appear that following short-term administration of cocaine, there is a marked stimulation of 5-HT receptors which by a negative feedback mechanism reduces the activity of T P H in cell bodies. The increase in particulate fraction of TPH after chronic cocaine treatment might represent a compensatory mechanism to control the stimulation of rcceptor.
demonstrated that over-functioning of serotoncrgic neurons is associated with anxiety and that 1.4-benzodiazepines elicit their anxiolytic effect by reducing the turnover (release) of 5-HT in brain. The view gains support from our findings in which a single injection of D Z P (10 mg/kgl increased the endogenous levels of 5-HT in several brain areas (Rastogi et al., 19771 as well as in synaptosomal preparation (P_, pellet) without producing any significant effect on the rate of 5-HT synthesis and TP level in the P2 pellet (Tablc 2). Acute D Z P treatment produced no effect on M A O activity suggesting that the elevated level of the amine was not associated with its reduced deamination. Furthermore, short-term treatment with D Z P was found to produce a significant rise in the absolute concentrations of labelled 5-HT in brains of rats pretreated with [t4C]5-HT by thc intraventricular route (Chase et al.. 1970). Since brain 5-HT also has been implicated to play a role in ,~izures (Kilian & Frey, 1973). it is postulated that D Z P produces its anticonvulsant effect by diminishing 5-HT turnover. When D Z P or a relatively new benzodiazepine, bromazepam, was injectcd repeatedly over a period of 22 days. TPH activity in mid-brain was significantly enhanced (Agarwal et al., 1977). We believe that diminished outward flow of 5-HT, after short-term treatment. results in lowering of thc amine level at the vicinity of reccptor sites. This by a feedback mechanism, results in increased T P H activity in mid-brain of rats (Agarwal et al., 1977). The compensatory rise in TPH formation in serotonergic cell bodies culminates in an increased rate of 5-HT synthesis in synaptosomal fraction of brain of rats exposed to DZP over a long period (22 days) of time (Singhal & Rastogi. 1977).
Diazepam
More recently, we have found differential effects on brain T P H activity following single and repeated injections of diazepam (DZP) in rats (Singhal & Rastogi, 1977). Using the "conflict test", Wise et al. (1972)
c O n c l . I 'SIONS The investigations briefly s u m m a r i z e d herein can
be viewed as emerging research trend in identifying
Psychotropic drugs and brain 5-hydroxytryptamine changes the role o f 5 - h y d r o x y t r y p t a m i n e r g i c n e u r o n s in the regulation o f various b e h a v i o u r a l processes as well as the m e c h a n i s m s of p s y c h o t r o p i c drug effects. O u r data d e m o n s t r a t e that thyroid a n d a d r e n o c o r t i c a l h o r m o n e s affect the m e t a b o l i s m of this i m p o r t a n t n e u r o t r a n s m i t t e r in the brain. Also, it seems i m p o r tant to investigate the influence of b o t h short- a n d long-term t r e a t m e n t of p s y c h o t r o p i c drugs on the 5 - h y d r o x y t r y p t a m i n e r g i c system. Indeed, it would be quite deceptive to p r e s u m e the c h r o n i c effects of a drug on a given n e u r o t r a n s m i t t e r system, on the basis of acute findings.
REFERENCES AGARW^L R., LAPIERR~, Y. D., RASTOGX R. B. & StNGHAL R. L. (1977) Alterations in brain 5-hydroxytryptaminc metabolism during the "withdrawal" phase after chronic treatment with diazepam and bromazepam. Br. J. Pharmac. 60, 3-9. AZMrnA E. C. & McEwxN B. S. (1969) Costicosterone regulation of tryptophan hydroxylase in mid-brain of the rat. Science 166, 1274-1276. C^RLSSON A., DArts J. N., KEHR W., LtNt)QUIST M. & ATACK C. (1972) Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in brain in vivo using an inhibitor of aromatic amino acid decarboxylase. Naunyn-Schmiedeherg"s Arch. exp. Path. Pharmak. 275, 153-168. CHASE T. N., KATZ R. I. & KOPIN I. J. (1970) Effect of diazepam on fate of intracisternally injected serotonin-14C. Neuropharmacolo~ty 9, 103-110. Ct;RZON G., FmEDEL J. & KNOTT P. J. (1973) The effect of fatty acids on the binding of tryptophan to plasma protein. Nature. Lond. 242, 198-200. DAHLSTROM A. & Ft:xr K. (1964) Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta. physiol, scand. 62, Suppl. 232, 1 55. DAWSON R. M. & CRONE H. D. (1973) Inorganic ion effects on the kinetic parameters of acetylcholinesterase. J. Neurochem. 21, 247-249. DENIZE^N F.. & SOURKES T. L. (1977) Regional transport of tryptophan in rat brain. J. Neurochem. 28, 951--959. DEWHURST K. E., ELKAmR D. J. & HARRlS G. W. (1969) Observation on the blood concentration of thyrotropic hormone (TSH) in schizophrenia and affective states. Br. J. Psychiat. 115, 1003-1011. EXYRS J. T. (1964) Endocrine influence on cerebral development. Archs Biol., Paris. 75, 529-565. FVRNSTROMJ. D. & WURTMAN R. J. (1972) Brain serotonin content: Physiological regulation by plasma neutral amino acids. Science 178, 414-416. FUXE K. & JONSSON G. (1974) Further mapping of central 5-hydroxytryptamine neurons: studies with the neurotoxic dihydroxytryptamines. In Serotonin--New Vistas: Histochemistry and Pharmacology,-Advances in Biochemical Psychopharmacolooy. Vol. 10, pp. 1-12. Raven Press, New York. GRAHAME-SMITH D. G. (1971) Studies in vivo on the relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L-tryptophan. J. Neurochem. 18, 1053-1066. GREEN A. R. & GRAHAME-SMITH D. G. (1975) 5-hydroxytryptamine and other indoles in the central nervous system. In Handbook of Psychopharmacology, (edited by SNVDER S. H. & IVERSEN L. L.) pp. 169--245. Pergamon Press, N.Y.
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the 5-HT uptake inhibitors chlorimipramine and FG 4963. Neuropharnmcolo,qy, 15, 665-668. LOVENBERG W.. BESSEt.AARG. H., BENSINGFR R. [". & JACKSOY R. L. (1973) Physiological and drug induced regulation of sertonin synthesis. In Serotonin and Behaviour, (edited by BARCHASJ. & L/St)tN E.). pp. 49 54. Academic Prcss, N.Y. MACON J. B., SOKOLO[[. L. & (h,owIYSKI J. 11971l Fecdback control of rat brain 5-hydroxytryptamine synthesis. J. Neuroehem. 18. 323 331. MANDE-LL A. J. (19751 Neurobiological mcchanism of prcsynaptic mctabolic adaptation and their organization: implications for a pathophysiology of the affcctivc disorders. In Neurobioloyieal Mechanisms o1" Adaptation and Behariour {edited by MANDII.I.A.J.I. pp. I 32. Raven Press, N.Y. M('CLAIN L. D. & CHRISIIAN S. T. 119751 5-hydrox~tryptamine and u-Lysergic acid diethylamine binding to rat brain synaptosomal membranes. Fedn Proc. Fedn ,tin. Sots exp. Biol. 34, 801. McMLNANY R. H. & ONCLI:'," J. I.. 11958) The specific binding of t.-tryptophan to serum a l b u m i n J. biol. ('hem. 233, 1436 1447. MHiK J. L. & NtH- N. H. (19721 Tryptophan 5-hydroxylase: Approximation of half-life and rate of axonal transport. J. Neurochem. 19, 1519 1525. MOtR A. T. B. & F.CCI.ESION D. (1968) The effects of precursor loading in the cerebral metabolism of 5-hydroxyindoles. J. Neurochem. 15, 1093 1108. Nt!CKt!RS L. & SzE P. Y. (1975) Regulation of 5-hydroxytryptamine metabolism in mouse brain by adrenal gh,cocorticoids. Brain. Res. 93, 123--135. OLDliNDORr W. H. 0971), Brain uptake of radiolabeled a m i n o acids, amines and hexoses after arterial injection Am. J. Physiol. 221, 1629-1639. PAGEL J.. CHRISTIAN S. T., Qt:AYLIi E. S. & MONTI J. A. (1976) A serotonin sensitive adenylatc cyclase in mature rat brain synaptic membranes. L!~, Sci. 19, 819.829. PEREZ-CRt:E'I J.. TA(;I.IAMONTI; A., TAGI,IAMONTIi P. & (hiSSA G. L. (lt-~71) Stimulation of scrotonin s.~nthcsis b) lithium J. Pharmac. exp. Ther. 178, 325 331). Pos3 R. M., KOTtN J.. GCX)DWlN F. K. & GORtXON E. K. (1973) Psychomotor activity and cerebrospinal fluid amine metabolites in affective illness. Am. a. Psychiat. 130. 67-72. RAPPORT M, M., Gat¢t-:Y A. A. & PAGr I. H. (1948) Crystalline serotonin. Science 10g, 329 330. RASI(X;I R. B. & SINGIIAL R. L. (1974) Thyroid hormone control of 5-hydroxy tryptaminc metabolism in dcveloping rat brain. ,/. Pharmac. exp. Ther. 191, 72 81. RAs'r~X,3t R. B. & Sty~;ttAt. R. L. (1976a) Influence of neonatal lind adult hyperthyroidism on behaviour and biosynthetic capacity for norepinephrine, dopamine and 5-hydroxytryptaminc in rat brain. J. Pharmac. exp. Th&'. 198, 609618. RAST(mt R. B. & St~GHAL R. L. (1976b) Neonatal hyperth.vroidism, vlterations in behavioural activity and the metabolism of brain norcpinephrine and dopaminc. Lift" Sci. 18. 851- 858. rAsIO(il R. B. & ,~IN(;HAL R. L. (1977) I,ilhium: Modilication of bchavioural activity lind brain biogcnie amines in developing h)perthyroid flits. J. Pharmac. exp. Ther. 201, 92.102. RASTO(;I R. B. & SINGHAt R. L. (1978) Adrcnocorticoids control 5-hydroxytryptaminc metabolism in rat brain. J. Neural. Trans. In press. RASTOG~ R. B.. LAPH!Rm: Y. D. & S~Y{;ttAL R. L. (19761 Evidence for the role of brain biogenic amines in depresscd motor activity scen in chemically thyroidectomized rats. J. Neurochem. 26, 443 449. RASTOGI R. B., I.APURRf{ Y. I). & Stx{;HAt R. I.. (1977)
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0306-362] 781(X)1-0307502.00.0
MINIREVIEW BRAIN 5 - H Y D R O X Y T R Y P T A M I N E M E T A B O L I S M : A D A P T I V E C H A N G E S AFTER L O N G - T E R M ADMINISTRATION OF PSYCHOTROPIC DRUGS R. B. RASTOGI* and R. L. SINGIIAL Department of Pharmacology, Faculty of Medicine, University of Ottawa, Ottawa, Canada KIN 9A9 (Received
20
December
1977)
Abstract--l. Evidence has been presented to suggest that 5-hydroxytryptamine is a neurotransmitter important to many regulatory processes, basic life functions, and behaviour. 2. Our data demonstrate that thyroid hormone plays an important role in the control of 5-HT metabolism especially in the developing brain. 3. The possibility is raised that anxiety, hypermobility and psychoses seen in hyperthyroid subjects are associated with increased serotonergic activity in the brain. 4. Similarly, this indoleamine may bc implicated in the emotional instability seen during altered levels of adrenocortical hormones. Finally, evidence is cited to support the view that serotonergic neurons elicit adaptive changes in response to external or internal stimuli.
stores, and nialamide which causes a selective increase in the neuronal 5-HT stores by inhibiting the deaminating enzyme monoamine oxidase (MAO), the distribution of fine 5-HT nerve terminals in the septal area, anterior colliculi, preoptic area, hippocampal formation, and in the neo- and mesocortex was more easily studied (Dahlstrom & Fuxe, 1964). 6-Hydroxytryptamine is another chemical which gives a good fluorescene yield and was employed to study the distribution of 5-HT nerve endings (Jonsson & Sandier. 1969). It is interesting to note that the fluorescence is also improved by pretreatment of animals with precursor L-tryptophan, which causes a selective increase in the fluorescence intensity of 5-HT neurons.
Since the first demonstration of 5-hydroxytryptamine (5-HT) in mammalian tissues by Rapport et al. (1948), this indoleamine has become the focal point of many recent investigations in neurobiology. The gradual unfolding of the ubiquitous importance of this neurohormone had led to the concept that 5-HT is one of a dozen of the probable neurotransmitters in the central nervous system which plays an important role in behaviour and mental function. The aim of this review is to summarize recent investigations concerned with the localization and metabolism of 5-HT in the brain, the interrelationship between central 5-HT neurons and hormones, and finally, differential effects on brain 5-HT-ergic systems following acute and chronic administration of certain psychotropic drugs.
TRYPTOPHAN: A M O D U L A T O R OF 5.-HT SYNTHESIS
5-HYDROXYTRYPTAMINE IN BRAIN: CURRENT STATUS OF LOCALIZATION A N D METABOLISM
There is now ample evidence to suggest that one of the rate-limiting steps for 5-HT formation in the brain is the concentration of tryptophan (TP) at the site of synthesis. This has been explained by the relatively high K,, of the rate-limiting enzyme, tryptophan hydroxylasc (TPH). which approximates normal brain TP concentration. As a consequence, basal 5-HT synthesis is thought to be both substrate and enzyme-dependent with the basal rate approximating Vm~. Brain TP is derived from plasma and after a TP load the concentrations in brain and plasma are directly proportional to each other (GrahameSmith, 1971). Furthermore, TP is the only essential amino acid which is partially bound to albumin in the plasma (McMenamy & Oncley, 1958); the binding is pH sensitive and TP can be displaced from albumin-binding sites by agents such as free fatty acids (Curzon et al., 1973). The binding, in general, implies storage, hence it is reasonable to suggest that the quantity which reaches cerebral tissue via the blood
The histochemical fluorescence techniques have yielded important knowledge on the circuitry of 5-HT containing neuronal pathways. Evidence indicates that most of the cell bodies are localized in the raphe nuclei. However, more recent studies have demonstrated that quite a few are also localized outside the raphe region within the vcntromedial reticular formation of the pons and the mesencephalon (Fuxe & Jonsson, 1974). From these cell bodies, axons course to provide 5-HT-containing nerve terminals in most parts of the CNS and certain areas of the spinal cord. By employing pharmacological tools, such as reserpine, which depletes the catecholamine and 5-HT
* Present address: Chief Pharmacologist, Nordic Research Laboratories, Pointe Claire, Montreal. Quebec. 307
3(~
R.B. RASIO(,a and R. L. SIN(JHA[.
brain barrier is closely dcpcndent on the small proportion of the amino acid still free in plasma. Earlier studies had demonstrated the existence of saturable, neutral amino acid carrier system (Lajtha & Toth. 19611 and Oldendorf {1971) has shown that most neutral amino acids compete with TP for transport at this carrier site. A year later, Fcrnstrom and Wurtman (1972) reported that the main determinant of brain TP and in turn 5-HT concentration does not appear to be free plasma TP alone, but the ratio of this amino acid to other plasma neutral amino acids such as tyrosine, phenylalanine, leucine, isoleucin and valinc. More rccently, it was shown that at normal blood concentrations, the individual amino acids inhibit TP transport into the brain in the following order: leucin > tyrosine > threonine > phenylalanine = histidine > isoleucine = methionine > valine (Yuwiler e t al.. 1977). I..;pon reaching the brain, I P is taken tip from the external mcdium into synaptosomcs, the so-callcd pinched nerve endings. The uptake across the neuronal membrane is stereospecific, tcmperaturc dependent, inhibited by metabolic inhibitors, but probably not too sensitive to changes in extcrnal Na" or K ' . There appears to bc two systems operating for TP uptake by synaptosomcs: one is the high-affinity uptakc system (K,, = 5.5 × 10 5 M) which is energy-dependent, temperature-sensitive and drug alterable. The second is the low affinity TP uptake systcm (K,, = 3.3 × 10 3 M) which does not sccm to be drugsensitive (Knapp & Mandell. 1972a). Synaptosomcs concentrate TP to a ratio of 4:1 (int:ext){Green & Grahamc-Smith, 1975). Denizean and Sourkes (1977) observed that the rate of TP uptake was highest in slices and synaptosomes prepared from hypothalamus (rich in serotonergic nerve endings). The finding correlates well with the data of Knott and Curzon (1974) who showed a larger incrcasc of TP in the hypothalamus than in other regions after an intraperitoneal injection of this amino acid (50 mg,'kg). Intravenous infusion of TP was found to elevate brain 5-HT and 5-HIAA levels within I hr after infusion, however: a proportional rise in 5-HT release in cerebral spinal fluid (CSF) was observcd only during the 2nd hr of infusion (Tcrnaux et al.. 1976). The transport of TP into synaptosomes can bc affected directly or indirectly by drugs, lmipramine, chlorimipramine and cocaine inhibited both TP uptake and 5-HT synthesis (Hamon & Glowinski. 1974). In contrast rcscrpine (Tozer ct al.. 1966) o r lithium treatment (Knapp & Mandcll, 1973: Rastogi & Singhal, 1977) activated TP transport as well as 5-HT s',nthesis in the brain. In new born animals, TP is present almost totally in free state in the plasma (Hamon & Glowinski, 1974). This free state of amino acid is not solely related to the presence of high levels of fatty acids in the blood, but also to the differences in the binding capacit~ of albumin in new born animals. Despite high levels of brain TP (Hoff et al.. 19741, the amount of 5-HT in new born rats is very low. This lower concentration of 5-HT may, in part, be due to decreased activity of T P H in neonatal rats (Rastogi & Singhal, 1974: Schmidt & Sanders-Bush, 1971). Since the rate of 5-HT accumulation in corresponding neurons is limited by their capacity for uptake and storage into granules, the possibility also rcmains that
these mechanisms might be undcr-dcvcloped in brains of immature animals. In presence of excess substrate, an increased amount of 5-HT might be synthesizcd and in turn is rapidly metabolized by intraneuronal monoamine oxidasc (MAOL without cvcr being stored in the synaptic vesicles and taking part in neural transmission in young brain. This 5-HT can then be presumed as "nonfunctional", in contrast to the "functional pool" of 5-HT which is storcd in the synaptic vesicles and is released upon ncrvc stimulation before being finally mctabolizcd by the deaminating enzyme (Fig. 1). Thus, it might be conceived that administration of TP or 5-hydroxytryptophan does not necessarily raise the levels of "'functional" 5-HT in thc brain. Moir and Eccleston (1968) observed that acute loading doses of 5-hydroxytryptophan markedly elevated the levels of 5-hydroxyindoleacetic acid (5-HIAA) without appreciably altering the levels of brain 5-HT. A kind of "'shunt" mechanism has been hypothesized by these investigators in which 5-hydroxytryptophan was rapidly mctabolised to 5-HIAA without eliciting any effect on the level of 5-HT. However. caution must be exercised while extrapolating these findings to chronic situations. The possibility can be raised that with an acute loading dose. the rate of accumulation of 5-HT in scrotonergic neurons is limited by the capacity for uptake into the storage granules, whereas with continuous administration, the storage capacity might be augmented.
TRYPTOPHAN HYDROXYI,ASE
Considerable attention has been devoted to studying the nature and requirements of TPH, the ratelimiting enzyme in the synthesis of 5-HT (Fig. 1). During the past few years, efforts have also been made to purify this enzyme (Lovenberg et al., 1973: Youdim e t al., 1974). Evidence indicates that TPH is a mixed function oxygenase requiring both molecular oxygen and a redudng agent for activity. Similar to other aromatic amino acids, TPH requires a tetrahydropterin as the reducing component of the system. Knapp e t al. (1975) demonstrated that calcium produced a dose-dependent activation of T P H activity in t, itro. A similar risc in activity of acetylcholincstcrase by calcium was reported by Dawson and Crone (1973). Howcver, the activation of TPH by calcium is very unlikely to be of any physiological significance as it occurs with concentrations (1.0 or 2.0 mM Ca2"), which arc much higher [10 3- 10 4 times) than those reportcd in nerve cells. Tryptophan hydroxylasc is synthesized in the cell body and undergoes axonal transport to the nerve endings, wherc 5-HT synthesis takes place (Meek & Neff, 1972). The axonal flow rate of TPH is probably 1--.2 mm/day as has been rcported for soluble proteins. It is therefore reasonable to believe that acute changes in 5-HT synthesis are mediated by regulation of enzyme activity rather than by changes in enzyme synthesis p e r se. It has been postulated that two forms of T P H may exist (lchiyama et al.. 1970; Knapp & Mandell, 1972a). The soluble form of T P H is present in cell bodies whereas the particulate enzyme is presumed to be bound to neuronal membrane of sero-
Psychotropic drugs and brain 5-hydroxytryptamine changes
I TRYPTOPHAN
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-
-
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Fig. 1. The pathways of 5-hydroxytryptamine metabolism in brain. Abbreviations used: tryptophan hydroxylase (TPH); 5-hydroxytryptophan decarboxylasc I5-HTPDI: 5-hydrox',tryptamine {5-HTR monoamine oxidase (MAO): 5-hydroxyindoleacctic acid (5-HIAA}.
tonergic nerve terminals. In point of fact, an intriguing correlation has been shown between the proportion of the enzyme which is in the soluble or particulate fraction, and the relative concentration of serotoncrgic cell bodies (mid-brain) or nerve endings (septal area) (Knapp & Mandeil, 1972b). The two forms of TPH do not react in a similar way to specific inhibitors such as p-chlorophenylalanine (Knapp & Mandell, 1972a) and their affinity constants toward TP appear to be different (lchiyama et al., 1970).
M E M B R A N E R E C E P T O R SITES FOR 5-HT
The presence of a 5-HT sensitive adenylate cyclase in adult rat brain synaptosomal membrane has been reported by Pagcl et al. (1976). In addition, Haigler and Aghjanian (1974) have shown by iontophoresis that there are both postsynaptic excitatory and inhibitory binding sites for 5-HT in various brain areas as well as presynaptic uptake sites. More recently, attempt has been made to isolate and characterize the 5-HT membrane-localized receptor which appears to be a proteolipid possessing activated receptor carbonyl residue. It is interesting that fluphenazine, a very potent antipsychotic drug known to block dopamine-sensitive adenylate cyclase activity was found to produce virtually no effect on 5-HT associated C-AMP production in the brain. These findings are well correlated with the report of McClain and Christian (1975) who observed no effect of fluphenazine and other antipsychotic drugs on the binding of 5-HT to synaptosomal membranes. These data lead one to believe that antipsychotic compounds may elicit their therapeutic effects probably by affecting dopamine receptors but not 5-HT receptor sites of the brain. These suggestions are, however, speculative and future work must include a complete characterization of the receptor site.
END-PRODUCT REGULATION OF 5-HT SYNTHESIS
Our current understanding of the short term regulatory processes of catecholamine synthesis suggests that cytoplasmic dopamine or norepinephrine partially controls the rate of the conversion of tyrosine into DOPA by end product inhibition (Kehr et al., 1972). In contrast, 5-HT has no effect on the activity of soluble TPH (Jequier et al., 1969) which converts TP to 5-hydroxytryptophan. This is why the hypothesis of an in t:ivo end-product regulation of 5-HT synthesis was not retained for many years. However, on the basis of data obtained from more recent studies, this hypothesis cannot be refuted completely. It has been shown that 5-HT at high concentrations (10 -3 M)does inhibit the enzyme in a non-competitive manner (Knapp & Mandell 1973). Additionally, Macon et al. (1971) demonstrated that 5-HT synthesis was reduced both in vit'o and in t'itro when intraneuronal concentrations of the amine attained 2.5 times normal levels either following inhibition by monoamine oxidase (MAO) or exogenous 5-HT. This inhibitory process seems to occur during the first step of the indoleamine synthesis, since no reduction in [3H]5-HT formation could be noted in rats injected with MAO inhibitors when [3H]5-hydroxytryptophan was substituted for [3H]TP. Carlsson and his associates (1972) employing another pharmacological tool confirmed this hypothesis. Whether or not this mechanism really plays a part in controlling the rate of tryptophan hydroxylation under physiological stimulation or inhibition of 5-HT-ergic neuronal activity remains an important issue to debate. I N T E R R E L A T I O N S H I P BEI"WEEN CENTRAL 5-HT N E U R O N S A N D H O R M O N F ~
T h y r o i d hormone
A number of instances have been cited to support the view that thyroid hormone plays an important
3111
R.B. RASIOGI and R. L. SI,~C;IVAn
role in the structural and biochemical ontogeny of the central nervous system which are believed to bc the determinants of adult neurophysiological and behavioural processes. Excessive thyroid secretion produces psychological symptoms which include emotional lability, restlessness, irritability, overreactivcness with predominant anxiety and tension (Eayrs, 1964: Whybrow & Farrell, 1974). Psychoses of acute organic type have frequently been encountered in severe cases and also during thyroid crisis. Since 5-HT plays an important role in affcctive disorder and anxiety, we were prompted to examine the effect of neonatal hyperthyroidism on brain 5-HT metabolism (Rastogi & Singhal, 1976a). Daily administration of zAriiodothyronine (T3) for 30 days beginning from one day of age. significantly increased T P H activity and TP levels in the mid-brain (Table 1). It is suggested that T3 which also binds to albumin, displaces TP from binding site making more of it available in free form to cross blood brain barrier and reach neuronal tissue. Our more recent work demonstrated that T3-treatment significantly enhanced the rate of 5-HT synthesis in the sub-cellular fraction as well (Singhal & Rastogi. 1977). Despite this, the endogenous levels of 5-HT in various discrete brain areas remained unchanged suggesting that increased synthesis kept pace with increased utilization of this indoleamine in the brain as evidenced by increased levels of 5-HIAA (Table 1). However, hyperthyroidism failed to alter M A O activity in certain brain areas examined (Rastogi & Singhal, 1976a). Evidence has emerged to suggest a direct correlation between serotonergic activity and locomotor performance in experimental animals (Green & Kelly. 1976: Lassen et al., 1976). Clinical
studies demonstrated enhanced levels of 5-H1AA in ccrebrospinal fluid of man subjected to increased psychomotor activity (Post et al., 1973). Hence it is likely that hyperactivity seen in hyperthyroid animals (Rastogi & Singhal. 1976a) might, in part, be associated with increased scrotonergic activity, in addition to enhanced catccholaminergic activity in the brain (Rastogi & Singhal, 1976b; Singhal et al., 1977). In contrast to hyperthyroidism, thyroid deficiency was accompanied by decreased activity of T P H (Rastogi & Singhal, 1974: Rastogi et al.. 1976) as well as decreased levels of 5-1-|T in several brain areas of developing rats [Table 1). Toth and C ~ b a 11966) had earlier reported significant reduction in 5-HT levels of brain stem and blood in thyroidcctomized rabbits. Clinical studies reported a decline in blood 5-HT in "'early myxedema" with return to normal following thyroid therapy (L. T. Giron-Personal Communication). One aspect of 5-HT metabolism after neonatal thyroidectomy which warrants consideration is the fact that in contrast to the observed lowering in 5-HT levels, the concentration of 5-HIAA was significantly increased in whole (Rastogi & Singhal, 1974) as well as discrete areas of the brain (Table 1}. Whereas radiothyroidcctomy at birth decreased the activity of dcaminating enzyme M A O in the hypothalamus (by 141~,,), it was significantly increased in mid-brain (by 14°o) and remained unaltered in cortex, brain stem and striatum. It is likely that brains of thyroid-deftcient rats may have an impaired efflux mechanism for 5-HIAA (Singhal et al., 1975). Clinical studies (Dewhurst et al., 1969) have demonstrated low levels of thyrotropic hormone in the blood of patients suffering from endogenous depression, an affective dis-
Table 1. Changes in 5-hydroxytryptaminc metabolism during altered thyroidal states and corticosterone treatment Mid-brain TP TPH (Pg.:g) (nmoles/g/hr) Control Hypothyroid Control Hyperthyroid
5.71 _+0.31 5.37 _+0.29 6.4 _+0.41 9.02 _+0.63*
9.13 _+1).71) 6.94 _+0.36* 11).78 -+0.96 13.78 _+ 1.10"
Brian stem Sham operated (Control) Adrenalectomized 115 days) Adrenalectomizcd + Corticosteronc (7 days)
2.99 _+0.18 2.45 -+1).14" 2.92 _+0.161
7.34 _+0.82 5.73 _+0.41" 7.11 _+0.46t
5-HT (pg/g) Midbrain
5-HIAA (,ug/g)
Striatum
Hypothalamus
Midbrain
Striatum
Hypothalamus
1.69 _+0.05 1.20 _+0.04* 1.76 _+0.09 1.71 _+0.11
1.44 _+0.08 0.99 _+0.1)6" 1.52 _+0.18 1.16 _+0.17
2.12 _+0.15 1.84 +0.13 2.49 _+0.17 1.97 -+1).18"
1.53 _+0.08 1.94 +0.12" 1.64 .+_0.07 1.98 -+0.09"
1.32 _+0.09 1.80 -+0.14" 1.37 _+0.08 2.32 -4-0.11"
2.04 _+0.22 2.37 _+0.26 1.95 _+0.19 2.96 -+0.24*
Brain stem
Striatum
Hypothalamus
Brain stem
Striatum
Hypothalamus
0.68 _+0.04 0.50 _+0.02* 0.61 _+0.02t"
1.32 _+0.09 0.92 _+0.03* 1.14 _+0.10"1
1.83 _+0.11 1.61 _+0.15 1.92 _+1).25
0.62 _+0.03 0.76 _+0.04* 0.63 _+0.02~
0.98 -+0.06 1.23 _+0.05* 0.86 _+0.08t
1.73 +0.07 2.02 _+0.15 1.88 -+0.07
Each value represents the mean + S.E.M. of 6 animals in the group. Neonatal hypothyroidism was induced by a single injection of 200 ,uCi of t'~Xl at one-day of age and animals were sacrificed 30 days later. Neonatal hyperthyroidism was induced by daily injections of T 3 111)pg/100 g) for 30 days beginning from the one-day of age. Rats were sacrificed at 30 days of age. A group of adrenalectomized rats received corticosterone (10 mg/kg/day) for 7 days beginning from 8 days after adrcnalectomy. * Statistically significant difference when compared with the control values (P < 0.051. + Statistically significant difference when compared with the value of adrenalectomizcd rats (P < 0.05).
Psychotropic drugs and brain 5-hydroxytryptamine changes order which shows certain biochemical and psychological features common with hypothyroidism. Evidence also indicates that a number of depressed patients with latent hypothyroidism respond well to thyroid medication in addition to tricyclic antidepressant drugs (Hatotani et al., 1977). It is therefore reasonable to believe that hypomobility and suppressed behaviour seen during "cretinoid" syndrome might, at least in part, be associated with decreased functioning of 5-HT-ergic neurons in addition to decreased dopaminergic (Singhal et al., 1976) and enhanced cholinergic activity in brain (Hrdina et al., 1975). Adrenocortical hormone
The pituitary-adrenal activity is known to be particularly sensitive to fluctuations in the external environmental milieu with which the internal homeostasis of an organism must equilibrate. There is emerging evidence to suggest that serotonergic nerve fibers containing structures like cortex, hippocampus and amygdala constitute a functional unit which modulates the regulation of ACTH (Scapagnini et al., 1971) and the pharmacological agents which affect brain 5-HT metabolism, abolish the daily rise in plasma 17-hydrocorticosteroids (Krieger & Rizzo, 1969). On the other hand, manipulation of adrenocortical levels has been found to decrease the biosynthesis of 5-HT in the brain (Rastogi & Singhal, 1978). Sze et al. (1976) reported that adrenalectomy interfered with the developmental as well as reserpine-induced rise of TPH activity in neonatal as well as adult brain. We found that even though 5-HT levels were lowered, the concentration of 5-HIAA was increased following adrenalectomy (Table 1). It is likely that the rise in brain 5-HIAA levels might, in part, be due to enhanced intraneuronal deamination of 5-HT by MAO, whose activity was significantly increased by 20~o following adrenalectomy. Our finding that the replacement corticosterone therapy restored 5-HT metabolism to virtually normal limits suggests that the observed neurochemical changes might be specific to adreno-corticoid hormones. Daily administration of corticosterone for 6 days not only increased TP and TPH activity, but also 5-HT levels in brains of adrenalectomized rats. Corticosterone-induced rise in TPH enzyme has been found to be cycloheximide-sensitive, suggesting that the rise in TPH activity represents de novo synthesis (Azmitia & McEwen, 1969). Glucocorticoids also stimulate the uptake of TP by nerve terminals and thereby accelerate the rate of 5-HT synthesis in the brain (Neckers & Sze, 1975). These authors have postulated that glucocorticoids in its "'immediate" action, rapidly accelerate 5-HT synthesis through an increased uptake of TP by nerve terminals. In the "slow" action, the hormone probably plays a permissive role in the induction of TPH by some unknown mechanisms. D I F F E R E N T I A L EFFECTS ON BRAIN 5-HT-ERGIC SYSTEM F O L L O W I N G ACUTE AND C H R O N I C ADMINISTRATION OF PSYCHOTROPIC DRUGS
Earlier, it was considered that alterations in neuro-
311
transmitter dynamics following chronic treatment were merely temporal extensions of those seen after acute treatment, requiring the continued presence of the agent inducing the change. However, a number of findings reported by Mandell (1975) and his associates suggest that changes in central amines, particularly NE and 5-HT metabolism, may not necessarily be in the same direction after long-term administration of a psychotropic drug, as those seen after a single injection. The so-called "secondary adaptive change" which has achieved common usage in discussions of intraneuronal regulation of transmitter synthesis, involves "product-feed back inhibition". In other words, the increased outward flow of intraneuronal transmitter stores results in a lowering of endogenous amine levels which in turn minimizes the inhibitory influence on the synthesizing enzyme. In contrast, decreased release results in decreased rate of synthesis (Macon et al., 1971). This section will attempt to examine the recent work on the influence of short and long-term administration of certain psychotropic drugs on brain 5-HT metabolism. Lithium
The promise offered by lithium in combatting mania or hypo-mania has added considerable impetus to investigations of the mechanisms by which this alkali metal elicits its beneficial effects. Since 5-HT was implicated in the pathogenesis of mania, many investigators have directed their attention to studying the effects of lithium on serotonergic neurons in the brain. These reports have been somewhat contradictory. For example, whereas some workers reported an increase in 5-HT turnover following lithium (Perez-Cruet et al., 1971; Rastogi & Singhal, 1977al others found either an opposite (Ho et al., 1970) or no effect (Kuriyama & Speken, 1970). Of course, some of these discrepancies may be related to differences in the dose and duration of lithium treatment and the regions of brain examined. Knapp and Mandell (1973) demonstrated that after 5 days of lithium treatment, there was stimulation of the high-attinity TP uptake system into synaptosomes. This resulted in increased synthesis of 5-HT in striatum, a region rich in 5-HT nerve terminals (Ternaux et al., 1977). Lithium is also known to suppress the impulse-induced release of 5-HT from serotonergic neurons (Schubert, 1973), resulting in an elevation of intraneuronal stores of 5-HT. The high level of 5-HT will presumably inhibit the rate of synthesis by lowering the formation of TPH within the cell bodies localized in mid-brain. Indeed, an increased TPH activity in striatum and a decreased TPH activity in mid-brain has been reported after short-term (5 days) treatment with lithium (Knapp & Mandell, 1973). However, after 21 days of treatment, these authors found a decrease in TPH activity of synaptosomes, when the values were compared with those seen after 5 days of lithium treatment. The apparent delay in decrease of TPH activity in serotonergic nerve endings is probably related to slow axoplasmic flow of the enzyme as stated elsewhere in this review. Morphine
A similar type of adaptive change in TPH activity following short and long-term administration of mor-
312
R.B. r a s t ( ~ l and R. L. Sl~(.atat, Table 2. Effects of short- and long-term DZP treatment on 5-HT synthesis m midbrain and P2 pellet Tissue examined Mid-brain
Parameter
Acute Control Treated
TP (,ug gl
10.29 4-0.73
7.82 _+0.52*
T P I I Inmol g hr)
8.21 _-0.N)
6.01 F_t).40*
5-HT lt~g.gl TP I/~g."gl
5-HT 0~g,gl
7.86
7.56 _+0.34* 10.71
2.16
_+0.74* 2.80
+_0.12"
± 0.(?9
_+0.19"
16.49 -:- 1.03 0.92
3.42 _+o.25 15.26 _+1.31 1.12
3.62 z0.12 1791 ..+ 1.12 t).7g
7.~6 _+0.69* 22.92 _+ 1.46" I. 17
r0.06
+0.07*
_+().(X~
4-0.1 I*
1.65 3.98 _+0.28
5-HT Synthesis "
5.73 4-0.28 _+0.45 1.61
~(1.08
P2 pellet
('hronic ('ontro] Treated
Values represent the means -4- S.E.M. of 6 rats in the group. DZP (10 mg.kg) was administered s.c. either acutel,, or chronically for 22 days. In acute study, animals were sacrificed 2 hr after the single injection whereas in chronic experiments, rats were killed 6 hr after the 22nd injection of DZP. Corresponding controls received an equal volume of the vehiclc. "pmoles ~'~CO2..mg protein25 min. * Statistically significant difference when compared with thc values of control rats (P < 0.051. phine has also been observed (Knapp & Mandell. 1972b). Whereas single injection of morphine decreased the activity of T P H in 5-HT nerve terminals, it failed to change T P H in mid-brain area. In contrast, chronic exposure to morphine by pellet implantation for 5 days resulted in a significant increase in the activity of nerve ending enzyme but not in cell body preparation. Morphine treatment at no time produced any significant change in the activity of 5-hydroxytryptophan decarboxylase, an intermediate enzyme involved in 5-HT synthesis. Cocaine
Much of the work on the effects of cocaine on the central nervous system has been concerned with central catecholaminergic systems. However, recent studies have demonstrated that cocaine manifests its behavioural effects partly by stimulating 5-HT receptors, which by a negative feedback mechanism affects the synthesis of this neurotransmitter. Taylor and Ho (1976) reported that short-term (5 days) treatment with cocaine preferentially inhibited the soluble form of TPH, presumed to be localized in the cytoplasm of cell bodies. When the treatment was extended to 45 days, the activity of soluble TPH returned to normal, and that of the particulate enzyme (localized in the nerve terminals) was significantly increased. It would appear that following short-term administration of cocaine, there is a marked stimulation of 5-HT receptors which by a negative feedback mechanism reduces the activity of T P H in cell bodies. The increase in particulate fraction of TPH after chronic cocaine treatment might represent a compensatory mechanism to control the stimulation of rcceptor.
demonstrated that over-functioning of serotoncrgic neurons is associated with anxiety and that 1.4-benzodiazepines elicit their anxiolytic effect by reducing the turnover (release) of 5-HT in brain. The view gains support from our findings in which a single injection of D Z P (10 mg/kgl increased the endogenous levels of 5-HT in several brain areas (Rastogi et al., 19771 as well as in synaptosomal preparation (P_, pellet) without producing any significant effect on the rate of 5-HT synthesis and TP level in the P2 pellet (Tablc 2). Acute D Z P treatment produced no effect on M A O activity suggesting that the elevated level of the amine was not associated with its reduced deamination. Furthermore, short-term treatment with D Z P was found to produce a significant rise in the absolute concentrations of labelled 5-HT in brains of rats pretreated with [t4C]5-HT by thc intraventricular route (Chase et al.. 1970). Since brain 5-HT also has been implicated to play a role in ,~izures (Kilian & Frey, 1973). it is postulated that D Z P produces its anticonvulsant effect by diminishing 5-HT turnover. When D Z P or a relatively new benzodiazepine, bromazepam, was injectcd repeatedly over a period of 22 days. TPH activity in mid-brain was significantly enhanced (Agarwal et al., 1977). We believe that diminished outward flow of 5-HT, after short-term treatment. results in lowering of thc amine level at the vicinity of reccptor sites. This by a feedback mechanism, results in increased T P H activity in mid-brain of rats (Agarwal et al., 1977). The compensatory rise in TPH formation in serotonergic cell bodies culminates in an increased rate of 5-HT synthesis in synaptosomal fraction of brain of rats exposed to DZP over a long period (22 days) of time (Singhal & Rastogi. 1977).
Diazepam
More recently, we have found differential effects on brain T P H activity following single and repeated injections of diazepam (DZP) in rats (Singhal & Rastogi, 1977). Using the "conflict test", Wise et al. (1972)
c O n c l . I 'SIONS The investigations briefly s u m m a r i z e d herein can
be viewed as emerging research trend in identifying
Psychotropic drugs and brain 5-hydroxytryptamine changes the role o f 5 - h y d r o x y t r y p t a m i n e r g i c n e u r o n s in the regulation o f various b e h a v i o u r a l processes as well as the m e c h a n i s m s of p s y c h o t r o p i c drug effects. O u r data d e m o n s t r a t e that thyroid a n d a d r e n o c o r t i c a l h o r m o n e s affect the m e t a b o l i s m of this i m p o r t a n t n e u r o t r a n s m i t t e r in the brain. Also, it seems i m p o r tant to investigate the influence of b o t h short- a n d long-term t r e a t m e n t of p s y c h o t r o p i c drugs on the 5 - h y d r o x y t r y p t a m i n e r g i c system. Indeed, it would be quite deceptive to p r e s u m e the c h r o n i c effects of a drug on a given n e u r o t r a n s m i t t e r system, on the basis of acute findings.
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