Serotonin synthesis and release in brain slices: Independence of tryptophan

doi:10.1016/0006-8993(79)90580-8

Brain Research

Volume 172, Issue 3, 31 August 1979, Pages 471-486

https://doi.org/10.1016/0006-8993(79)90580-8 Get rights and content

Abstract

The purpose of the present study was to examine the role of substrate availability in the regulation of the release and synthesis of serotonin by brain slices. Electrical field depolarization of the brain slices stimulated the synthesis and release of serotonin in the absence of changes in intracellular tryptophan concentration, in the absence of tryptophan in the incubation bath, and in the absence of changes in total tryptophan uptake. Furthermore, electrical stimulation decreased the apparent K_m for tryptophan required for synthesis of serotonin by the slices. Several conclusions were drawn.

(1) Rates of serotonin release and synthesis in brain slices may increase independently of the tissue tryptophan concentration or tryptophan uptake.
(2) There is little difference in the synthetic rate of serotonin in tissues exposed to pargyline or not at all.
(3) Blockade of monoamine oxidase by pargyline appears to give estimates of the synthetic rates of serotonin which are comparable to estimates derived from [³H]-tryptophan incorporation.
(4) Newly synthesized serotonin is preferentially released.
(5) Only in tissue pretreated with pargyline do increasing concentrations of tryptophan increase the releasable pool of 5-HT.
(6) Electrical depolarization may allosterically activate tryptophan hydroxylase; an effect which may be mimicked by homogenization of the tissue.

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Another interesting aspect of the study by Elks and colleagues was the observation that increases in tryptophan increased both basal and stimulation-induced release of 5-HT only when the MAO inhibitor pargyline was present in the incubation medium. Elks et al. (1979) concluded that 5-HT synthesis and release is independent of tryptophan and their results point out the importance of MAO in determining the release of 5-HT into the synapse (i.e., into functional activity). Third, we developed a method for measuring the flux of tryptophan and 5-HT into and out of superfused synaptosomes and observed that the addition of tryptophan to the medium resulted in large increases in intra-synaptosomal tryptophan and 5-HT but did not result in the release of 5-HT from synaptosomes (Wolf & Kuhn, 1986).
Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT). Increases or decreases in TPH catalytic activity can therefore lead to corresponding changes in the neuronal content of 5-HT. TPH requires a reduced pteridine cofactor, molecular oxygen, and nonheme iron to hydroxylate its substrate l-tryptophan. Alterations in the tissue content of any of these four factors could change TPH activity. Posttranslational modifications of TPH, including phosphorylation and cysteine oxidation, cause significant changes in its catalytic activity. The discovery of a brain-specific isoform of TPH, termed TPH2 to distinguish this enzyme from the peripheral form TPH1, opened new avenues of research that allowed in-depth studies of TPH molecular function, solutions of 3D crystal structures, creation of TPH1 and TPH2 knockout mouse lines, and the successful search for specific inhibitors of TPH isoforms some of which are entering clinical trials for the treatment of disorders linked to excess peripheral 5-HT.
Evidence for serotonin synthesis-dependent regulation of in vitro neuronal firing rates in the midbrain raphe complex
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Evidence suggests that 5-hydroxytryptamine 1A (5-HT_1A) receptor-mediated autoregulation of serotonergic neuronal firing rates is impaired in stress-related neuropsychiatric disorders. In vitro models may provide insight into neural mechanisms underlying regulation of serotonergic systems. However, serotonin synthesis and tonic autoregulation of serotonergic neuronal firing rates are impaired in in vitro preparations lacking tryptophan. We describe the effects of perfusion of living rat brain slices with tryptophan on both 1) tissue concentrations of serotonin metabolites and 2) neuronal firing rates within the dorsal raphe nucleus. Brain slices were perfused with artificial cerebrospinal fluid lacking tryptophan for 4 h, followed by exposure to 1) 40 μM tryptophan (0–60 min) or 2) 0–400 μM tryptophan (23 min) and microdissected for analysis of indole concentrations. Parallel studies examined effects of tryptophan on neuronal firing rates and interactions with drugs expected to alter synaptic concentrations of serotonin. Tryptophan resulted in time-dependent and concentration-dependent increases in serotonin and serotonin metabolites, effects that were correlated with restoration of tonic autoinhibition of dorsal raphe nucleus neuronal firing rates. Inhibition of serotonin synthesis resulted in time-dependent and concentration-dependent increases in 5-hydroxtryptophan that correlated with reversal of the tryptophan-mediated autoinhibition of neuronal firing rates. Tryptophan modulated effects of several drugs on neuronal firing rates, including a selective 5-HT_1A receptor antagonist (WAY-100635), a monoamine oxidase inhibitor (pargyline), a selective serotonin reuptake inhibitor (fluoxetine), and a serotonin-releasing agent (methylenedioxymethamphetamine). These studies support the hypothesis that tonic autoregulation of serotonergic neuronal firing rates is dependent on tryptophan availability and characterise conditions necessary to study this process in vitro.
Correlation between serotonin synthesis and 5-HT<inf>1A</inf> receptor binding in the living human brain: A combined α-[<sup>11</sup>C]MT and [<sup>18</sup>F]MPPF positron emission tomography study
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Serotonin (5-HT) is one of the major neurotransmitters and has been implicated in a wide variety of cerebral functions. Several lines of evidence indicate that 5HT_1A receptors exert a negative feedback in the synthesis and release of serotonin. While most of what is known about serotonin comes from studies in animals, much less empirical evidence exists about the serotonergic system in the living human brain. This study aims to assess the correlation between serotonin synthesis and 5-HT_1A receptor binding using positron emission tomography (PET) in humans. Six healthy male volunteers underwent 2 PET scans in the same day: one measuring α-[¹¹C]MT K⁎ [ml/g/min] trapping constant (a measure of serotonin synthesis) and one measuring 5-HT_1A receptor binding potential BP_ND with [¹⁸F]MPPF. Volumes of interest (VOIs) selected a priori included: anterior cingulate cortex (ACC), anterior insula, hippocampus, amygdala, thalamus, hypothalamus and midbrain raphe nuclei. Correlation analyses were conducted voxel-by-voxel and with manually traced VOIs. A significant negative correlation between serotonin synthesis and 5-HT_1A binding potential was found bilaterally in hippocampus and anterior insula and in the left ACC. The combination of [¹⁸F]MPPF and α-[¹¹C]MT PET offers a means to investigate key determinants of 5-HT neurotransmission under physiological and psychopathological conditions in the human brain in vivo.
Altered serotonin synthesis, turnover and dynamic regulation in multiple brain regions of mice lacking the serotonin transporter
2005, Neuropharmacology
Citation Excerpt :
Hints of these findings in SERT −/− mice came from similar studies showing a significant decrease in 5-HT synthesis rates in mice and rats following chronic administration of the selective serotonin reuptake inhibiting antidepressant, citalopram, which was similarly considered a likely result of changes in autoreceptor sensitivity, although direct studies of these receptors were not available (Moret and Briley, 1992). While experiments using monoamine oxidase inhibition in rat brain have suggested that 5-HT synthesis may be regulated by end-product inhibition in vivo (Tappaz and Pujol, 1980; Moret and Briley, 1992), other studies using brain slices exposed to a MAO inhibitor were in disagreement (Elks et al., 1979). End-product inhibition has been well known for TH (Spector et al., 1967; Haavik et al., 1991; Wu et al., 1992) but may not apply to TPH as TPH was inhibited in vitro by only a high, non-physiological concentration (>1 mM) of 5-HT (Park et al., 1994), suggesting that 5-HT does not inhibit TPH by a direct interaction at the active site of the enzyme (Martinez et al., 2001; Wang et al., 2002).
To evaluate the consequences of inactivation of the serotonin transporter (SERT) gene on 5-HT homeostasis and function, 5-HT synthesis and turnover rates were measured using the decarboxylase inhibition method in multiple brain regions (frontal cortex, striatum, brainstem, hippocampus and hypothalamus) from mice with a genetic disruption of SERT. 5-HT synthesis rates were increased 30–60% in the different brain regions of SERT −/− mice compared to littermate +/+ control mice despite 55–70% reductions in tissue 5-HT concentrations. Brain regions that possessed a greater capacity to increase synthesis and turnover (frontal cortex, striatum) demonstrated lesser reductions in tissue 5-HT. Female SERT −/− mice had greater increases (79%) in brain 5-HT synthesis than male −/− mice did (25%), a finding associated with higher brain tryptophan concentrations in females. Despite increased 5-HT synthesis, there was no change in either TPH2 or TPH1 mRNA levels or in maximal in vitro TPH activity in the brainstem of SERT −/− mice. Catecholamine homeostasis as reflected in brain tissue concentrations and in synthesis and turnover of dopamine and norepinephrine was unchanged in SERT −/− mice. Taken together, the results demonstrate a markedly altered homeostatic situation in SERT −/− mice that lack 5-HT reuptake, resulting in markedly depleted tissue stores that are inadequately compensated for by increased 5-HT synthesis, with brain region and gender specificity observed.
Effects of tryptophan and/or acute running on extracellular 5-HT and 5-HIAA levels in the hippocampus of food-deprived rats
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The present microdialysis study has examined whether exercise-elicited increases in brain tryptophan availability (and in turn 5-HT synthesis alter 5-HT release in the hippocampus of food-deprived rats. To this end, we compared the respective effects of acute exercise, administration of tryptophan, and the combination of both treatments, upon extracellular 5-HT and 5-hydroxyindoleacetic acid (5-HLAA) levels. All rats were trained to run on a treadmill before implantation of the microdialysis probe and 24 h of food deprivation. Acute exercise (12 m/min for 1 h) increased in a time-dependent manner extracellular 5-HT levels (maximal increase: 47%). these levels returning to their baseline levels within the first hour of the recovery period. Besides, exercise-induced increases in extracellular 5-HIAA levels did not reach significance. Acute administration of a tryptophan dose (50 mg/kg i.p.) that increased extracellular 5-HIAA (but not 5-HT) levels in fed rats, increased within 60 min extracellular 5-HT levels (maximal increase: 55%) in food-deprived rats. Whereas 5-HT levels returned toward their baseline levels within the 160 min that followed tryptophan administration, extracellular 5-HIAA levels rose throughout the experiment (maximal increase: 75%). Lastly, treatment with tryptophan (60 min beforehand) before acute exercise led to marked increases in extracellular 5-HT and 5-HIAA levels (maximal increases: 100% and 83%, respectively) throughout the 240 min that followed tryptophan administration. This study indicates that exercise stimulates 5-HT release in the hippocampus of fasted rats, and that a pretreatment with tryptophan (at a dose increasing extracellular 5-HT levels) amplifies exercise-induced 5-HT release.
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The present review tries to delineate some mechanisms through which the sympathetic nervous system (SNS) and the hypothalamo-pituitary-adrenal (HPA) interact with central serotonergic systems. The recent progress in 5-hydroxytryptamine (5-HT) receptor pharmacology has helped to define the means by which central serotonergic activity may alter the respective activities of the SNS (sympathetic nerves and adrenomedulla) and of the HPA axis. These pharmacological findings have also helped to characterize the differential effects of central 5-HT upon different branches of the SNS and the numerous sites at which 5-HT exerts stimulatory influences upon the HPA axis. Although relevant to stress-related neuroendocrinology, the extent to which these interactions are involved in the antidepressant/anxiolytic properties of some serotonergic agents still remains to be clarified. Beside these findings, there is also abundant evidence for a tight control of central serotonergic systems by stress hormones. Activation of the SNS increases, by numerous means, central availability of tryptophan, whereas glucocorticoids exert differential actions upon the intra- and the extraneuronal regulation of 5-HT function. Actually, a significant number of these mechanisms is involved in the maintenance of homeostasis during stressful events, thereby confering to these mechanisms a key role in adaptation processes.

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Serotonin synthesis and release in brain slices: Independence of tryptophan

Abstract

J. biol. Chem.

Biochem. Pharmacol.

Brain Research

J. biol. Chem.

Biochem. Pharmacol.

The effects of 5-OH-tryptophan and 5-OH-tryptamine on dopamine synthesis and release in rat brain striatal synaptosomes

J. Neurochem.

In vivo conversion of 3H-tryptophan into 3H-serotonin in brain areas of adrenalectomized rats

Science

Effects of some psychotropic drugs on TH activity in different structures of the rat brain

Europ. J. Pharmacol.

Short and long term regulation of tyrosine hydroxylase

The determination of tryptophan in plasma, liver, and urine

J. Lab. clin. Med.

Brain serotonin content: physiological dependence on plasma tryptophan levels

Science

Studies in vivo on the relationship between brain tryptophan, brain 5-HT synthesis, and hyperactivity in rats treated with an MAOI and L-tryptophan

J. Neurochem.

Effect of dietary phenylalanine and tryptophan upon rat brain amine levels

J. Pharmacol. exp. Ther.

In vivo conversion of ³H-tryptophan into ³H-serotonin in brain areas of adrenalectomized rats