Tryptophan Availability Modulates Serotonin Release from Rat Hypothalamic Slices

Application of a novel in vitro experimental system has allowed us to describe the relationship between tryptophan availability and serotonin release from rat hypothalamic slices. Superfusing hypothalamic slices with a physiologic medium containing l-tryptophan (1, 2, 5, or 10 microM) caused dose-dependent elevations in tissue tryptophan levels; the magnitude of the elevations produced by supplementing the medium with less than 5 microM tryptophan was within the physiologic range for rat brain tryptophan levels. Slice serotonin levels rose biphasically as the tryptophan concentration in the medium was increased. Superfusing the slices with medium supplemented with a low tryptophan concentration (1 or 2 microM) caused proportionally greater incremental changes in serotonin levels than the increases caused by further elevating the tryptophan concentration (5 or 10 microM). The spontaneous release of serotonin from the slices exhibited a dose-dependent relationship with the tryptophan concentration of the superfusion medium. Electrically evoked serotonin release, which was calcium-dependent and tetrodotoxin-sensitive, also increased in proportion to the medium tryptophan concentration. These data suggest that the rate at which serotonin is released from hypothalamic nerve terminals is coupled to brain tryptophan levels. Accelerations in hypothalamic serotonin synthesis, caused by elevating brain tryptophan levels, result in proportionate increases in the rates of serotonin release during rest and with membrane depolarization.     

Comparative Ontogenesis of Brain Tryptamine, Serotonin, and Tryptophan

The pattern of ontogenetic development of tryptophan (TP), tryptamine (T), indole-3-acetic acid (IAA), 5-hydroxytryptamine (5-HT; serotonin), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the brains of rats aged 1-45 days is presented. Analysis of the five components in each brain allows the calculation of the acid/amine and amine/amino acid ratios. These metabolic indexes are a useful tool to study and compare the metabolic origins and fates of both amines. The ontogenetic patterns of TP, T, and IAA are very similar, especially during the first week postpartum. The highest and lowest levels found for T were 2.2 ng/g and 0.1 ng/g at the 1st and 5th day, respectively. The temporal relationship between the T/TP and IAA/T ratios suggests the existence of mechanisms protecting T against monoamine oxidase (MAO) which develop in parallel to synaptogenesis. Significant correlations were found between TP and IAA during the whole period studied and between TP and T during the first week after birth. The 5-HT peak found during the first postpartum week could be due to a non-neuronal pool of 5-HT protected against MAO and possibly contained in mast cells. Preliminary determinations on leptomeningeal membranes suggest the existence of such a pool.     

Effect of Tetrahydrobiopterin on Serotonin Synthesis, Release, and Metabolism in Superfused Hippocampal Slices

The effects of 6R-5,6,7,8-tetrahydro-L-biopterin (6R-BH4), the in vivo cofactor for tryptophan hydroxylase, on the synthesis, release, and metabolism of serotonin were studied in superfused slices from rat hippocampus. 6R-BH4 did not alter the spontaneous release of [3H]serotonin but it did significantly increase release when slices were depolarized with 30 mM KCl. Under the same incubation conditions, 6R-BH4 altered neither the synthesis (basal or tryptophan-stimulated) nor the metabolism of serotonin in hippocampal slices. The synthetic pteridine 6-methyl-5,6,7,8-tetrahydropterin also augmented release under depolarizing conditions whereas biopterin, the oxidized form of 6R-BH4, did not. The 6S isomer of BH4, which is relatively inactive as a cofactor for tryptophan hydroxylase, was equipotent with 6R-BH4 in stimulating serotonin release. 6R-BH4 did not inhibit serotonin uptake nor did it function as a serotonin autoreceptor antagonist to increase release. A direct serotonin releasing effect of 6R-BH4, like that produced by p-chloroamphetamine, could also be ruled out. At suboptimal concentrations of extracellular calcium, the KCl-induced release of 3H was significantly reduced, yet the increase in release caused by BH4 remained the same in magnitude. It is concluded that 6R-BH4 increases the depolarization-induced release of serotonin through an interaction with the release mechanism itself, possibly by enhancing calcium influx or by increasing the sensitivity of the release mechanism to calcium. The effects of 6R-BH4 on serotonin release are independent from its function as the cofactor for tryptophan hydroxylase.     

Uptake and Release of Tryptophan and Serotonin: An HPLC Method to Study the Flux of Endogenous 5-Hydroxyindoles Through Synaptosomes

An HPLC assay with fluorometric detection has been developed that is sensitive enough to measure simultaneously endogenous levels of tryptophan, serotonin (5-hydroxytryptamine, or 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) inside synaptosomes as well as that released into the incubation medium. Using this assay, we have observed that tryptophan is rapidly taken up by synaptosomes and turned over to 5-HIAA without a concurrent release of 5-HT. Exogenous 5-HT is also rapidly taken up, and, within 20-30 min, 80% of the 5-HT is deaminated. Veratridine induces release of both tryptophan and 5-HT from synaptosomes. Changes in the disposition of exogenous tryptophan or 5-HT can be completely accounted for by uptake or by stoichiometric changes in metabolites. This assay method should be valuable in the study of 5-HT pools and in the determination of from which pool 5-HT release occurs.     

Stimulation of the Serotonin Autoreceptor Prevents the Calcium-Calmodulin-Dependent Increase of Serotonin Biosynthesis in Rat Raphe Slices

The role of the serotonin (5-hydroxytryptamine) autoreceptor in the regulation of the activity of tryptophan hydroxylase was investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measuring the accumulation of 5-hydroxytryptophan in the presence of inhibition of aromatic L-amino acid decarboxylase using 3-hydroxy-4-bromobenzyloxy-amine by HPLC with fluorescence detection. Serotonin and its agonists N,N-dimethyl-5-methoxytryptamine and 1-(m-chlorophenyl)-piperazine reduced the formation of 5-hydroxytryptophan to 50-60% at 10(-5) M. The effect of serotonin was reversed by 10(-5) M methiothepin, an antagonist of the serotonin autoreceptor. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), dose-dependently reduced the basal formation of 5-hydroxytryptophan to 40-50% at 10(-6) and 10(-4) M, respectively. W-7 also reduced the activated formation by A-23187 or dibutyryl cyclic AMP in a dose-dependent manner. W-7 had no effect on 5-hydroxytryptophan formation reduced by serotonin at 10(-5) M. These results suggest that the role of the serotonin autoreceptor was related to the prevention of the calcium-calmodulin-dependent activation of tryptophan hydroxylase.     

Modulation of Tryptophan Hydroxylase Activity by Phospholipids: Stimulation Followed by Inactivation

The activity of tryptophan hydroxylase from the rat brainstem was stimulated rapidly three- to fourfold by the addition of phosphatidylinositol or phosphatidylserine. However, the activity of the enzyme once stimulated was decreased gradually by subsequent incubation with the phospholipid at 37 degrees C, reaching a level below the original activity after 1 h of incubation. The presence of ferrous ion almost perfectly protected the enzyme against this phospholipid inactivation. The activity of the enzyme inactivated by incubation with the phospholipid was not only restored, but also increased further by incubation at 37 degrees C with ferrous ion and dithiothreitol. Gel filtration analysis revealed that the enzyme stimulated by phosphatidylinositol was eluted in a void volume together with the phospholipid vesicles, but the enzyme inactivated by incubation with phosphatidylinositol was eluted at a later region apart from the vesicles. These results, taken together, suggest the possible involvement of cellular membranes in the regulation of tryptophan hydroxylase in the central nervous system.     

Increased Extracellular Serotonin in Rat Brain After Systemic or Intraraphe Administration of Morphine

Abstract: The effect of morphine on serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the CNS of unanesthetized rats was investigated by microdialysis. Morphine was administered either subcutaneously, by local perfusion into the diencephalon, or by intraraphe microinjection. Systemic administration of morphine resulted in a significant increase in both extracellular 5-HT and 5-HIAA in the diencephalon. The effect of morphine on 5-HT was dose dependent during local perfusion of the diencephalon with inhibitors of uptake or monoamine oxidase. Systemic morphine also produced significant increases in extracellular 5-HT in the striatum and hippocampus during uptake inhibition. The site of opioid effects on 5-HT was tested by locally perfusing morphine into the diencephalon. This had no effect on 5-HT or 5-HIAA. In contrast, intraraphe injection of morphine caused a dose-dependent increase in extracellular 5-HT and 5-HIAA in the diencephalon. These results suggest that systemic morphine induces an increase in 5-HT release in widespread areas of the forebrain. This appears to be due to an effect on 5-HT cell bodies and not on 5-HT nerve endings in projection sites.     

Characteristics of Tryptophan Accumulation by Isolated Rat Forebrain Synaptosomes

Uptake of 10 microM L-tryptophan into isolated rat brain synaptosomes was studied to assess its effect on the rate of serotonin synthesis from tryptophan. The initial rate of uptake was rapid, being two orders of magnitude above the rate of tryptophan hydroxylation. Uptake was highly concentrative, the concentration ratio across the plasma membrane at equilibrium being approximately 9. This concentration ratio was decreased to about 1 in the presence of high concentrations of amino acids transported by the L-type neutral amino acid uptake system. A mixture of the large neutral amino acids at physiological concentrations decreased the internal tryptophan concentration to 58% of that in their absence. Large tryptophan concentration ratios were observed in experiments in which Na+ in the medium was replaced with choline+. The concentrative uptake of tryptophan was energy-dependent, being decreased by inclusion of cyanide and omission of glucose. The concentration gradient was abolished by veratridine or rotenone. Time courses of the changes in ATP content and tryptophan concentration ratio on addition of these and other agents established that tryptophan uptake is probably not driven by ATP hydrolysis or efflux of other amino acids, but by the plasma membrane potential.     

Stress-and Endotoxin-Induced Increases in Brain Tryptophan and Serotonin Metabolism Depend on Sympathetic Nervous System Activity

Stressful treatments and immune challenges have been shown previously to elevate brain concentrations of tryptophan. The role of the autonomic nervous system in this neurochemical change was investigated using pharmacological treatments that inhibit autonomic effects. Pretreatment with the ganglionic blocker chlorisondamine did not alter the normal increases in catecholamine metabolites, but prevented the increase in brain tryptophan normally observed after footshock or restraint, except when the duration of the footshock period was extended to 60 min. The footshock- and restraint-related increases in 5-hydroxyindoleacetic acid (5-HIAA) were also prevented by chlorisondamine. The increases in brain tryptophan caused by intraperitoneal injection of endotoxin or interleukin-1 (IL-1) were also prevented by chlorisondamine pretreatment. The footshock-induced increases in brain tryptophan and 5-HIAA were attenuated by the beta-adrenergic antagonist propranolol but not by the alpha-adrenergic antagonist phenoxybenzamine or the muscarinic cholinergic antagonist atropine. Thus the autonomic nervous system appears to be involved in the stress-related changes in brain tryptophan, and this effect is due to the sympathetic rather than the parasympathetic limb of the system. Moreover, the main effect of the sympathetic nervous system is exerted on beta- as opposed to alpha-adrenergic receptors. We conclude that activation of the sympathetic nervous system is responsible for the stress-related increases in brain tryptophan, probably by enabling increased brain tryptophan uptake. Endotoxin and IL-1 also elevate brain tryptophan, presumably by a similar mechanism. The increase in brain tryptophan appears to be necessary to sustain the increased serotonin catabolism to 5-HIAA that occurs in stressed animals, and which may reflect increased serotonin release.     

Effect of Precursor Loading on the Synthesis Rate and Release of Dopamine and Serotonin in the Striatum: A Microdialysis Study in Conscious Rats

The effects of systemic administration of tyrosine and phenylalanine on the extracellular levels of tyrosine and dopamine were determined by microdialysis in the striatum of awake rats. In addition, the effects of these precursors on in vivo 3,4-dihydroxyphenylalanine (DOPA) formation were determined during continuous infusion of a decarboxylase inhibitor. Both precursors increased the dialysate levels of tyrosine sixfold, but only phenylalanine administration stimulated DOPA formation. However, neither precursor affected the release of dopamine. When the precursor administration was repeated in rats in which the release of dopamine was stimulated by haloperidol pretreatment, again no effect was seen on the release of dopamine. Systemic administration of tryptophan (100 mg/kg, i.p.) during continuous infusion of a decarboxylase inhibitor induced a threefold increase in the formation of 5-hydroxytryptophan and caused an increase in the release of serotonin during infusion of an uptake inhibitor to about 150% of controls. Finally, we investigated whether dietary precursors were able to influence neurotransmitter formation and release. Rats trained to consume their daily food in a period of 2 h were implanted with microdialysis probes. Scheduled eating induced a small increase in the extracellular levels of tyrosine (135% of controls), but the release of dopamine and the formation of 5-hydroxytryptophan during continuous infusion of a decarboxylase inhibitor were not affected.     

Postmortem and Regional Changes of Serotonin, 5-Hydroxyindoleacetic Acid, and Tryptophan in Brain

Using a specific and sensitive high pressure liquid chromatographic technique for the measurement of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and tryptophan (TRP), we found that there were no changes in 5-HT or 5-HIAA in the rat cortex when left in situ for 6 h at room temperature or 24 h at 4 degrees C. Only a minimal 14% increase in 5-HT was observed after 24 h at 4 degrees C in the striatum of the same animals. Concentrations of TRP, however, were increased significantly in both brain regions by these postmortem delay procedures. A second study revealed that there were significant regional 5-HT and 5-HIAA concentration differences within the cerebral cortex. The frontal cortex was shown to have the highest concentrations of 5-HT and 5-HIAA. Further, within the frontal cortex, 5-HIAA levels varied, showing apparent progressive rostral to caudal increases. 5-HT concentrations, however, remained constant within the frontal cortex. These results are discussed in reference to the conflicting reports of the previous human suicide and postmortem studies.     

Effect of Tryptophan on Extracellular Concentrations of Tryptophan and 5-Hydroxyindoleacetic Acid in the Striatum and Cerebellum

The effects of L-tryptophan (50 mg/kg i.p.) on extracellular concentrations of tryptophan and the 5-hydroxytryptamine (5-HT) metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined in the rat striatum and cerebellum, regions with rich and poor 5-HT innervation, respectively. Determinations were on perfusates from dialysis probes in the brains of conscious, freely moving rats. The pharmacokinetic profiles of dialysate tryptophan after tryptophan load (peak concentration, time to peak concentration, area under curve, and half-life) in the two regions did not differ significantly. The dialysate 5-HIAA concentration in the striatum rose two- to threefold after the administration of tryptophan. Therefore, as 5-HIAA was undetectable in the cerebellum either before or after the administration of tryptophan, the increase of 5-HIAA in the striatum is unlikely to depend appreciably on its production within the cerebral vasculature or outside the brain or on its entering the striatum through a blood-brain barrier damaged by placement of the dialysis probe. Overall, the findings strengthen previous evidence that extracellular 5-HIAA concentrations determined by cerebral dialysis are a valid measure of the metabolism of 5-HT of brain neuronal origin.     

Oxindole, a Sedative Tryptophan Metabolite, Accumulates in Blood and Brain of Rats with Acute Hepatic Failure

Abstract: Rats treated with oxindole (10–100 mg/kg i.p.), a putative tryptophan metabolite, showed decreased spontaneous locomotor activity, loss of the righting reflex, hypotension, and reversible coma. Brain oxindole levels were 0.05 ± 0.01 nmol/g in controls and increased to 8.1 ± 1.7 or 103 ± 15 nmol/g after its administration at doses of 10 or 100 mg/kg i.p., respectively. To study the role that oxindole plays in the neurological symptoms associated with acute liver failure, we measured the changes of its concentration in the brain after massive liver damage, and we investigated the possible metabolic pathways leading to its synthesis. Rats treated with either thioacetamide (0.2 and 0.4 g/kg i.p., twice) or galactosamine (1 and 2 g/kg i.p.) showed acute liver failure and a large increase in blood or brain oxindole concentrations (from 0.05 ± 0.01 nmol/g in brains of controls to 1.8 ± 0.3 nmol/g in brains of thioacetamide-treated animals). Administration of tryptophan (300–1,000 mg/kg p.o.) caused a twofold increase, whereas administration of indole (10–100 mg/kg p.o.) caused a 200-fold increase, of oxindole content in liver, blood, and brain, thus suggesting that indole formation from tryptophan is a limiting step in oxindole synthesis. Oral administration of neomycin, a broad-spectrum, locally acting antibiotic agent able to reduce intestinal flora, significantly decreased brain oxindole content. Taken together, our data show that oxindole is a neurodepressant tryptophan metabolite and suggest that it may play a significant role in the neurological symptoms associated with acute liver impairment.     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

Serotonergic Regulation of Acetylcholine Release in Rat Frontal Cortex

Abstract: The extent to which serotonin regulates the activity of cortically projecting cholinergic neurons was studied using in vivo microdialysis to monitor interstitial concentrations of acetylcholine in the frontal cortex of freely moving rats. Systemic administration of the serotonin release-inducing agent fenfluramine (3 or 10 mg/kg, i.p.) increased acetylcholine release by 110–130%. The fenfluramine-induced increase in acetylcholine release was significantly attenuated by pretreatment with the selective serotonin uptake inhibitor fluoxetine (10 mg/kg, i.p.). Pretreatment with the selective dopamine D₁ receptor antagonist SCH-23390 (0.3 mg/kg, s.c.) failed to prevent the fenfluramine-induced increase in acetylcholine release. In contrast, the serotonin 5-HT_2A receptor antagonist ketanserin (5 mg/kg, i.p.) blocked fenfluramine-induced increases in acetylcholine release. In contrast to previous studies that have concluded that serotonin has inhibitory actions on cortical acetylcholine release, the present results indicate that fenfluramine increases cortical acetylcholine release in vivo by its ability to enhance serotonin transmission and that serotonin produces these effects at least in part via actions at serotonin 5-HT_2A receptors.     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

Influence of Fluoxetine on Regional Serotonin Synthesis in the Rat Brain

Abstract: The aim of the present study was to test the hypothesis that there should be a difference between the effects of an acute and an 8-day (chronic) administration of fluoxetine (10 mg/kg) on the rate of serotonin [5-hydroxytryptamine (5-HT)] synthesis. The 5-HT synthesis rate was measured in discrete regions of the rat brain using the α-[¹⁴C]methyl- l -tryptophan autoradiographic method. The results show that the acute and chronic fluoxetine treatments influence the 5-HT synthesis rate in different ways. A single dose of fluoxetine induced a significant increase in 5-HT synthesis in the visual, auditory, and parietal cortices, substantia nigra, hypothalamus, ventral thalamus, and dorsal hippocampus. In contrast, after a chronic treatment a decrease was observed in the substantia nigra, caudate, and nucleus accumbens, the auditory, parietal, sensorimotor, and frontal cortices, and ventral tegmental area. A significant decrease in the rate of 5-HT synthesis was observed in the dorsal raphe after both the single and chronic treatments. The results suggest that extracellular 5-HT has a delayed influence on the brain 5-HT synthesis rate in structures with serotonergic terminals. The findings from the acute study could be important for patients who have just started receiving fluoxetine treatment, as an increase in the 5-HT synthesis rate might occur in the acute phase of their treatment. In addition, the findings from the chronic treatment study might give us a better understanding of how the brain serotonergic system adapts during a prolonged exposure to extracellular 5-HT.