Development of central and peripheral serotonin-producing systems in rats in ontogenesis

The work deals with study of development of central and peripheral serotonin-producing systems in rat ontogenesis before and after formation of the blood-brain barrier. By the method of highly efficient liquid chromatography it has been shown that the serotonin level in peripheral blood before formation of the blood-brain barrier (in fetuses and neonatal rats) is sufficiently high for realization of physiological effect on target cells and organs. At the period of formation of the blood-brain barrier the serotonin level in brain sharply rises, whereas the serotonin concentration and amount in blood plasma and duodenum increase insignificantly. Completion of formation of the blood-brain barrier is accompanied by a significant increase of the serotonin content in duodenum, probably for maintenance of the high serotonin level in blood. To evaluate secretory activity, the mean rate of daily serotonin increment in the studied tissues was calculated. In brain, this parameter was maximal at the period of formation of the blood-brain barrier-from the 4th to the 16th postnatal days. This allows thinking hat brain before formation of the blood-brain barrier is the most important source of serotonin in peripheral blood.     

Determination of 5-hydroxytryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid in 20 rat brain nuclei using liquid chromatography with electrochemical detection

High-performance liquid chromatography with electrochemical detection is utilized for the simultaneous determination of serotonin, its precursor 5-hydroxytryptophan, and its major metabolite 5-hydroxyindoleacetic acid in nervous tissue samples. Tissue preparation required only homogenization in acidic solution and centrifugation prior to application to the chromatograph. Detection limits in the low picogram range were obtained for those indoles separated. This assay was used in combination with a micropunch dissection technique of 20 discrete rat brain nuclei to measure serotonin, its precursor, and major metabolite. The specificity of the assay was checked with pharmacological experiments aimed to increase or decrease serotonin levels. Pargyline, a monoamine oxidase inhibitor, led to a marked increase in serotonin and a decrease of 5-hydroxyindoleacetic acid while p-chlorophenylalanine, by blocking the conversion of tryptophan to 5-hydroxytryptophan, selectively depleted 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid.     

Brain serotonin in response to the hypothalamo-hypophyseal-adrenal system to insulin hypoglycemia

The response of hypothalamus-pituitary-adrenal system to insulin administration was studied in the male Wistar rats submitted to a strong and prolonged blockade of serotonin brain synthesis by repetitive injections of p-chlorophenylalanine (PCPA) a 5-hydroxytryptophan inhibitor. After 24, 48, 72 or 96 hours of either one or two doses of PCPA (250 mg/kg, i.p.) were administered with insulin (0.25 UI/kg s.c.), the plasmatic glucose and corticosterone levels being estimated at 0, 30 and 60 minutes. When PCPA was injected twice, the lapse between them was 48 hours. Insulin produced decrease of plasmatic corticosterone values and inhibition of the response to insulin, specially between 48 and 72 hours, for a single management of PCPA, and stronger and more prolonged for the double dose. The fall of serotonin content in brain maintained great correlation with effects referred above. The results support that stimulatory action of insulin on the pituitary-adrenal system is mediated by central serotoninergic neurons and reaffirms the hypothesis that serotonin (5HT) positively modulates the activity of hypothalamus-pituitary-adrenal system.     

Effects of pyridoxine deficiency and dl-p-chlorophenylalanine administration to rats on 5-hydroxytryptamine and noradrenaline concentrations in brain and 5-hydroxytryptamine concentration in blood

Pyridoxine deficiency in post-weanling rats caused a marked decrease in body weight and a small but significant decrease in brain weight. Although the concentration of circulating 5-hydroxytryptamine was markedly decreased, the concentrations of 5-hydroxytryptamine and noradrenaline in the brain were not affected. p-Chlorophenylalanine, an inhibitor of 5-hydroxytryptamine synthesis, decreased the 5-hydroxytryptamine content of brain to very low values in both the deficient and control animals, whereas the noradrenaline contents were not appreciably affected. The concentration of 5-hydroxytryptamine in blood, the origin of which is primarily gastrointestinal, was decreased only in the controls but not in the deficient animals after p-chlorophenylalanine treatment. These results suggest that whereas l-tryptophan hydroxylase (EC 1.14.3.2) is rate-limiting in the brain as has been reported by others, the pyridoxal 5'-phosphate-dependent enzyme 5-hydroxytryptophan decarboxylase (EC 4.1.1.28) may be more important in the gastrointestinal tract in the regulation of 5-hydroxytryptamine synthesis.     

Pargyline and р- Chlorophenylalanine Decrease Expression of Ptpn5 Encoding Striatal-Enriched Protein Tyrosine Phosphatase (STEP) in the Mouse Striatum

Striatal-enriched protein tyrosine phosphatase (STEP), which was initially identified in the striatum, is encoded by the Ptpn5 gene and is expressed in neurons of various structures of the brain. STEP is involved in regulating neuroplasticity, and its expression abnormalities are associated with human neurodegenerative disorders. The STEP inhibitor 8-trifluoromethyl-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (TC-2153) has been shown to affect the serotoninergic system of the brain. However, the influence of the serotoninergic system on the STEP regulation has not been studied yet. The aim of the study was to investigate how pharmacologically induced changes in the brain serotonin (5-HT) level affect Ptpn5 expression and STEP activity in adult male C57BL/6J mice. To modulate the 5-HT level in the brain, the 5-HT synthesis inhibitor p-chlorophenylalanine or 5-HT degradation inhibitor pargyline was administered intraperitoneally for three successive days. Changes in 5-HT concentration in the brain were assayed using high-performance liquid chromatography. The STEP activity was determined spectrophotometrically in the supernatant by the rate of p-nitrophenyl phosphate dephosphorylation in the absence and presence of the selective STEP inhibitor TC-2153. The Ptpn5 mRNA level was determined using quantitative RT-PCR. The Ptpn5 expression level in the striatum was three times higher than in the cortex and hippocampus. Both increases and decreases in brain 5-HT were for the first time associated with a decrease in Ptpn5 mRNA in the striatum. STEP activity in the striatum and cortex was significantly higher than in the hippocampus. However, p-chlorophenylalanine and pargyline did not affect the STEP activity in the brain structures tested. Thus, a new method was proposed to study the STEP activity in the brain and p-chlorophenylalanine and pargyline were shown to decrease Ptpn5 expression in the striatum in mice.     

5-Hydroxytryptophyl peptides: potent inhibitors of a storage compartment of serotonin.

Several analogues of 5-hydroxytryptophan were tested for their ability to inhibit the binding of serotonin to serotonin-binding protein (SBP), a protein found within serotonergic neurons which has a high affinity for serotonin. An N-substituted dipeptide, N-acetyl-5-hydroxytryptophan-5-hydroxytryptophan amide, was found to be an inhibitor of this binding. The inhibition (50% at 1.0 μM) was specific, since it did not affect other known sites of serotonin binding. The binding of serotonin to its membrane receptor was not affected by the dipeptide (up to 10 μM). Uptake of serotonin by synaptosomes was only slightly affected (9% at 10 μM), and aromatic-L-amino-acid carboxy-lyase(EC 4.1.1.28) and amine: oxygen oxidoreductase (deaminating) (flavin-containing) (EC 1.4.3.4) were not inhibited (10 μM and 5 mM respectively), The peptide was not hydrolyzed by honiogenates of brain or myenteric plexus. The ¹⁴C-labelled dipeptide was shown to be taken up by synaptosomes. However, the uptake of the peptide was not affected either by drugs that inhibit serotonin uptake or by serotonin itself although the uptake was abolished by excess 5-hydroxytryptophan. Intraventricular injection of N-acetyl dipeptide caused a biphasic effect depending on dose. Lower doses (10nmol) induced a decrease in serotonin brain levels (40%). Higher doses (300 nmol) caused a 95% increase in serotonin levels. It is suggested that 5-hydroxytryptophyl peptides may be used as potent specific inhibitors of SBP, a storage compartment of serotonin.     

Analgesic effects of N-acetyl-5HTP-5HTP amide are not directly related to brain serotonin levels.

A synthetic dipeptide, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, was shown previously to inhibit the binding of serotonin to a soluble specific serotonin binding protein as well as to alter brain serotonin levels. When injected into rats intraventricularly, the dipeptide caused an increase in pain threshold, lasting for several hours, as determined by either a flinch-jump test or a tail-flick test. This effect was reversed by naloxone. The dipeptide is a very weak inhibitor of the binding of labelled naloxone or dihydromorphine to a membranous opiate receptor preparation. The analgesic activity of the dipeptide was not diminished by p-chlorophenylalanine or the setonergic neurotoxin 5,7-dihydroxytryptamine, which depleted brain serotonin levels. This implies that the analgesic action of the dipeptide is not mediated directly by its effect on serotonin concentration.     

Effects of histamine and cimetidine on the levels of serotonin and 5-hydroxyindoleacetic acid in various parts of the digestive tract and in the blood and brain of rats

In the investigations on male Wistar rats it was demonstrated that histamine (0.05 and 0.5 mg/kg) decreased the serotonin level, without affecting the level of 5-HIAA in the stomach and duodenum. Contrary to this, cimetidine (15, 75 and 150 mg/kg) raised slightly the level of serotonin and decreased the 5-HIAA level in the stomach and duodenum. In the jejunum histamine in the lower dose raised the levels of serotonin and 5-HIAA, and in the higher dose it decreased only the concentration of serotonin. Cimetidine, on the other hand, only in the highest dose increased the serotonin level and decreased significantly the level of 5-HIAA. In the brain a rise of the serotonin level was observed only after histamine. No effects were observed of histamine and cimetidine on the blood serotonin level. Histamine reduced the number of enterochromaffinocytes in the duodenum. These results point to an evident interaction between the histaminergic and the serotoninergic structures in the digestive tract of rats.     

On the presence of serotonin in mammalian cardiomyocytes

Pleiotropic effects of serotonin (5-HT) in the cardiovascular system are well documented. However, it remains to be elucidated, whether 5-HT is present in adult mammalian cardiomyocytes. To address this issue, we investigated the levels of 5-HT in blood, plasma, platelets, cardiac tissue, and cardiomyocytes from adult mice and for comparison in human right atrial tissue. Immunohistochemically, 5-HT was hardly found in mouse cardiac tissue, but small amounts could be detected in renal preparations, whereas adrenal preparations revealed a strong positive immunoreaction for 5-HT. Using a sensitive HPLC detection system, 5-HT was also detectable in the mouse heart and human atrium. Furthermore, we could identify 5-HT in isolated cardiomyocytes from adult mice. These findings were supported by detection of the activity of 5-HT-forming enzymes-tryptophan hydroxylase and aromatic L-amino acid decarboxylase-in isolated cardiomyocytes from adult mice and by inhibition of these enzymes with p-chlorophenylalanine and 3-hydroxybenzyl hydrazine. Addition of the first intermediate of 5-HT generation, that is 5-hydroxytryptophan, enhanced the 5-HT level and inhibition of monoamine oxidase by tranylcypromine further increased the level of 5-HT. Our findings reveal the presence and synthesis of 5-HT in cardiomyocytes of the mammalian heart implying that 5-HT may play an autocrine and/or paracrine role in the heart.     

Brain and blood indole metabolites after peripheral administration of14C-5-HT in rat

Radioactive techniques were used to reexamine the reports that pharmacological quantities of peripheral serotonin (5-hydroxytryptamine or 5-HT) gain access to brain parenchyma. Intravenous injection of 0.108–4.19 mg/kg of¹⁴C-5-HT (3.55 Ci/100 g weight) produced significant metabolic differences in brain but not blood as a function of dose at up to 10 min after injection. Neither of the metabolites, 5-hydroxyindoleacetic acid nor 5-hydroxytryptophol, were detectable in brain following their intravenous injection, suggesting that when such metabolites are found in brain they represent central metabolism. It has also been shown that peripheral compartments in general, and specifically blood in the cerebral vasculature and the adrenergic nerve endings in the cerebral blood vessels, contribute to the uptake and metabolism of 5-HT. We conclude that doses up to 0.435 mg/kg 5-HT do not cross the blood-brain barrier in the rat but are being totally metabolized in nonneuronal tissues that are invariably removed and assayed along with brain parenchyma. The level at which 5-HT actually passes the blood-brain barrier was found to be at least 0.863 mg/kg. This value is one-third lower than that previously reported.     

The inhibition of tryptophan hydroxylase, not protein synthesis, reduces the brain trapping of α-methyl-l-tryptophan: an autoradiographic study

The effects of the tryptophan hydroxylase (TPH) inhibitor p-chlorophenylalanine (PCPA; 200mg/kg; 3 days), and of the protein synthesis inhibitor cycloheximide (CXM, 2mg/kg), on regional serotonin (5-HT) synthesis were studied using the alpha-[14C]methyl-L-tryptophan (alpha-[14C]MTrp) autoradiographic method. The objectives of these investigations were to evaluate the changes, if any, on 5-HT synthesis, as measured with alpha-MTrp method, following the inhibition of TPH by PCPA, or the inhibition of proteins synthesis by CXM. The rats were used in the tracer experiment approximately 24h after the last dose of PCPA was administered, and in the CXM experiments, they were used 30 min following a single injection of CXM. In both experiments, the control rats were injected with the same volume of saline (0.5 ml/kg; s.c.) and at the same times as the drug injections. The results demonstrate that trapping of alpha-MTrp, which is taken to be related to brain 5-HT synthesis, is drastically reduced (40-80%) following PCPA treatment. The inhibition of protein synthesis with CXM did not have a significant effect on the global brain trapping of alpha-MTrp and 5-HT synthesis. These findings suggest that the brain trapping of alpha-[14C]MTrp relates to brain 5-HT synthesis, but not to brain protein synthesis.     

Production and effects of platelet-activating factor in the rat brain

The synthesis of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in rat brain was evaluated. Extracted PAF was characterized using standard HPLC and TLC techniques, and by correlation of its bioactivity with the acetylation state of the 2-position of the molecule. PAF was quantified by bioassay, its ability to cause [3H]serotonin release from washed rabbit platelets. The low basal level of PAF (0.25 +/- 0.15 pmol/g wet wt., mean +/- S.E.) in the brain of the intact rat was greatly increased by intraperitoneal injection of the chemoconvulsant drugs picrotoxin or bicuculline, to levels of 10.68 +/- 2.18 and 4.97 +/- 0.75 pmol/g wet wt., respectively. Electroconvulsion also increased brain PAF, to 1.76 +/- 0.30 pmol/g wet wt. Equivalent experiments using bicuculline in the isolated perfused rat brain yielded qualitatively similar results, indicating that the production of PAF in the brain is independent of systemic metabolism. When a 32P-labeled nerve-ending (synaptosome) preparation from rat brain was challenged with synthetic PAF (denoted AGEPC) at 0.1 nM concentration, responses were observed consistent with accelerated turnover of polyphosphoinositides. AGEPC also caused an increase in the Na+-Ca2+ exchange of synaptic membrane vesicles. Furthermore, AGEPC infused into the vasculature of the isolated perfused rat brain caused changes consistent with an increase in blood-brain barrier permeability, although AGEPC did not itself significantly penetrate the blood-brain barrier. It is concluded from these studies that PAF is synthesized within the rat brain in response to convulsant stimuli and that one of its effects is to accelerate synaptic polyphosphoinositide turnover. In addition, circulating PAF can influence blood-brain barrier permeability without itself penetrating the blood-brain barrier.     

Peripheral 5-carboxamidotryptamine induces hindlimb scratching by stimulating 5-HT1A receptors in rats.

Treatment of rats with 5-carboxamidotryptamine (5-CT) or 5-methoxy-tryptamine (5-MeOT) induces a hindlimb scratch response. These compounds have high affinity for 5-HT1A and 5-HT1D receptors. The selective 5-HT1A receptor agonist N,N-dipropyl-5-CT (DP-5-CT) also induced hindlimb scratching while the selective 5-HT1D receptor agonist, sumatriptan, did not. 5-CT-induced hindlimb scratching was inhibited dose-dependently by several 5-HT1A antagonists (BMY 7378, NAN-190, MDL 73005EF and pindobind-5-HT1A) as well as the non-selective 5-HT antagonist, methiothepin. Pretreatment of rats with the serotonin (5-HT) synthesis inhibitor, p-chlorophenylalanine (PCPA) or the 5-HT depleting agent, reserpine, markedly attenuated 5-CT-induced hindlimb scratching. These data suggest that hindlimb scratching induced by 5-HT agonists may not be centrally mediated but rather may be mediated by a neuronal 5-HT1A receptor localized outside the blood-brain barrier.     

The inhibition of tryptophan hydroxylase, not protein synthesis, reduces the brain trapping of alpha-methyl-L-tryptophan: an autoradiographic study

The effects of the tryptophan hydroxylase (TPH) inhibitor p-chlorophenylalanine (PCPA; 200mg/kg; 3 days), and of the protein synthesis inhibitor cycloheximide (CXM, 2mg/kg), on regional serotonin (5-HT) synthesis were studied using the alpha-[14C]methyl-L-tryptophan (alpha-[14C]MTrp) autoradiographic method. The objectives of these investigations were to evaluate the changes, if any, on 5-HT synthesis, as measured with alpha-MTrp method, following the inhibition of TPH by PCPA, or the inhibition of proteins synthesis by CXM. The rats were used in the tracer experiment approximately 24h after the last dose of PCPA was administered, and in the CXM experiments, they were used 30 min following a single injection of CXM. In both experiments, the control rats were injected with the same volume of saline (0.5 ml/kg; s.c.) and at the same times as the drug injections. The results demonstrate that trapping of alpha-MTrp, which is taken to be related to brain 5-HT synthesis, is drastically reduced (40-80%) following PCPA treatment. The inhibition of protein synthesis with CXM did not have a significant effect on the global brain trapping of alpha-MTrp and 5-HT synthesis. These findings suggest that the brain trapping of alpha-[14C]MTrp relates to brain 5-HT synthesis, but not to brain protein synthesis.     

Brain serotonin and blood pressure regulation: studies using in vivo electrochemistry and direct tissue assay

Hypotensive responses to tryptophan and 5-hydroxytryptophan infusions were studied in normotensive male Sprague-Dawley rats. Results showed that 5-hydroxytryptophan but not tryptophan lowered pressure in a dose dependent way in direct relation to the production of brain serotonin and 5-HIAA. Intrinsic release of serotonin from brain was also studied during periods of induced hypertension and hypotension. Brain monoamine responses to blood pressure changes induced by intravenous phenylephrine and nitroprusside were measured in dorsal raphe nucleus and nucleus tractus solitarius by in vivo electrochemistry. Results showed that 5-HIAA was increased during drug induced hypertension and during reflex hypertension which followed a period of hypotension. These changes were blocked by sinoaortic denervation indicating that these central serotonergic neurons are responding to increased pressure sensed by baroreceptors. Therefore, serotonin has a role in blood pressure regulation as a pharmacologic agent and as a neurotransmitter in homeostatic control of pressure.     

Streptozotocin-Induced Diabetes Reduces Brain Serotonin Synthesis in Rats

The rate of brain 5-hydroxytryptamine (serotonin) synthesis and turnover in streptozotocin-diabetic rats was assessed using three separate methods: the rate of 5-hydroxytryptophan accumulation following decarboxylase inhibition with Ro 4-4602; the decline in 5-hydroxyindoleacetic acid levels following monoamine oxidase inhibition with pargyline; and the rate of 5-hydroxyindoleacetic acid accumulation following blockade of acid transport with probenecid. Each of the three methods revealed that 5-hydroxytryptamine synthesis and turnover is decreased by 44-71% in diabetic rats with plasma glucose levels of between 500 and 600 mg%. In addition, the levels of free and bound plasma tryptophan were measured and the levels of the free amino acid were found to be the same in control and diabetic rats. Since diabetic rats exhibit a 40% decrease in brain tryptophan, the free tryptophan level in plasma does not predict brain tryptophan levels in diabetic rats. These data are discussed within the context of psychiatric disturbances experienced by diabetic patients.     

Endocrine functions of brain in adult and developing mammals

The main prerequisite for organism’s viability is the maintenance of the internal environment despite changes in the external environment, which is provided by the neuroendocrine control system. The key unit in this system is hypothalamus exerting endocrine effects on certain peripheral organs and anterior pituitary. Physiologically active substances of neuronal origin enter blood vessels in the neurohemal parts of hypothalamus where no blood-brain barrier exists. In other parts of the adult brain, the arrival of physiologically active substances is blocked by the blood-brain barrier. According to the generally accepted concept, the neuroendocrine system formation in ontogeny starts with the maturation of peripheral endocrine glands, which initially function autonomously and then are controlled by the anterior pituitary. The brain is engaged in neuroendocrine control after its maturation completes, which results in a closed control system typical of adult mammals. Since neurons start to secrete physiologically active substances soon after their formation and long before interneuronal connections are formed, these cells are thought to have an effect on brain development as inducers. Considering that there is no blood-brain barrier during this period, we proposed the hypothesis that the developing brain functions as a multipotent endocrine organ. This means that tens of physiologically active substances arrive from the brain to the systemic circulation and have an endocrine effect on the whole body development. Dopamine, serotonin, and gonadotropin-releasing hormone were selected as marker physiologically active substances of cerebral origin to test this hypothesis. In adult animals, they act as neurotransmitters or neuromodulators transmitting information from neuron to neuron as well as neurohormones arriving from the hypothalamus with portal blood to the anterior pituitary. Perinatal rats—before the blood-brain barrier is formed—proved to have equally high concentration of dopamine, serotonin, and gonadotropin-releasing hormone in the systemic circulation as in the adult portal system. After the brain-blood barrier is formed, the blood concentration of dopamine and gonadotropin-releasing hormone drops to zero, which indirectly confirms their cerebral origin. Moreover, the decrease in the blood concentration of dopamine, serotonin, and gonadotropin-releasing hormone before the brain-blood barrier formation after the microsurgical disruption of neurons that synthesize them or inhibition of dopamine and serotonin synthesis in the brain directly confirm their cerebral origin. Before the blood-brain barrier formation, dopamine, serotonin, gonadotropin-releasing hormone, and likely many other physiologically active substances of cerebral origin can have endocrine effects on peripheral target organs—anterior pituitary, gonads, kidney, heart, blood vessels, and the proper brain. Although the period of brain functioning as an endocrine organ is not long, it is crucial for the body development since physiologically active substances exert irreversible effects on the targets as morphogenetic factors during this period. Thus, the developing brain from the neuron formation to the establishment of the blood-brain barrier functions as a multipotent endocrine organ participating in endocrine control of the whole body development.     

Improved Production of Human Immunoglobulin γ1 Chain Fc Polypeptides in Escherichia coli and Their Properties

Exposure to some xenobiotics (pentobarbital, 3-terf-butyl-4-methoxyphenol (BHA), chloretone (acetone chloroform), 1, l-bis-(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and polychlorinated biphenyls (PCB)) for a 5 hr period increased the concentrations of brain serotonin and 5-hydroxyindole acetic acid (5HIAA). The decrease in the brain serotonin level elicited by /7-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, was prevented by the concomitant administration of chloretone. The administration of both chloretone and pargyline (an inhibitor of monoamine oxidase) caused significant elevation of the brain 5HIAA level as compared with that in a pargyline control, however, the concentration of brain serotonin was not different between pargyline alone and chloretone plus pargyline. These results show that the increase in the brain serotonin level caused by chloretone is not due to acceleration of brain serotonin synthesis, but to retardation of the degradation of brain serotonin, and the increase in brain 5HIAA caused by chloretone may be due to the reduced removal of 5HIAA from the brain. Chloretone plus pargyline caused significant elevation of hypothalamus catecholamines, as compared to in the pargyline control, so the catecholamine turnover rates may be accelerated by the administration of chloretone.     

Effect of 8-hydroxy-2-(di-n-propylamino)-tetralin on the tryptophan-induced increase in 5-hydroxytryptophan accumulation in rat brain

The injection of 8-hydroxy-2-(di-n-propylamino)-tetralin [8-OH-DPAT]reduced 5-hydroxytryptophan accumulation in vivo in rat cerebral cortex, hypothalamus and brainstem. Brain tryptophan levels were unaffected. Dose-related increases in 5-hydroxytryptophan accumulation produced by single injections of L-tryptophan (0, 25, 75 mg/kg ip) were substantially diminished by pretreatment with 8-OH-DPAT. The drug did not affect the tryptophan-induced increments in brain tryptophan level. Since 8-OH-DPAT is known to reduce the activity of serotonin neurons in vivo, these results suggest that when serotonin neurons are relatively inactive, the ability of an injection of tryptophan to stimulate serotonin synthesis is greatly attenuated.     

Serotonin synthesis and its light-dark variation in the rat retina

Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas alpha-methyltryptophan, a competitive substrate inhibitor, was hydroxylated into alpha-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT-PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis.