Brain Net Unidirectional Uptake of α-[14C]Methyl-L-Tryptophan (α-MTrp) and Its Correlation with Regional Serotonin Synthesis,Tryptophan Incorporation into Proteins,and Permeability Surface Area Products of Tryptophan and α-MTrp

The uptake and trapping constants for labeled tryptophan (Trp) via the serotonin (5-hydroxytryptamine; 5-HT) metabolic pathway and for the incorporation of Trp into proteins, and -[¹⁴C]methyl-L-tryptophan (-MTrp) were measured. Measurements were done in rats treated with either saline or probenecid (200 mg/kg). In addition, the blood-brain barrier (BBB) permeability surface area products for Trp (PS_T) and -MTrp (PS) were measured in normal rats. The results suggest that, in both groups of rats, there is a highly significant correlation (p < 0.05; Pearson Product Moment Correlation (PPMC) between the brain uptake and trapping constants for -MTrp and those of Trp via the 5-HT metabolic pathway, but there is no significant correlation (p > 0.05; PPMC) between either of these constants and the PS products of either compound. There is also no significant correlation (p > 0.05; PPMC) between the constant for the Trp incorporation into proteins with any of the other parameters. For all parameters, except Trp incorporation into proteins (-MTrp is not incorporated into proteins), there was a highly significant correlation (p < 0.001) between the quantities measured for Trp and -MTrp. The data presented here strongly suggests that the brain uptake and trapping of -MTrp relates to brain 5-HT synthesis, and does not relate to the BBB transport or protein incorporation of Trp. On the basis of these results, as well as those previously reported, we concluded that trapping (unidirectional uptake) of -MTrp can be converted to the 5-HT synthesis rates in the brain. From this also follows that labeled -MTrp is a good tracer for in vivo evaluation of the brain 5-HT synthesis.     

Serotonin Synthesis Increased in Terminals Four Days after Reserpine Treatment: An Autoradiographic Study in Rat Brain

The rate of 5-HT synthesis was determined in discrete rat brain regions 4 days after a single dose of reserpine (10 mg/kg) or reserpine carrier (controls), using an autoradiographic method with labelled -methyl-L-tryptophan as a tracer. The results show that the rate of 5-HT synthesis was unchanged in the dorsal and median raphe, significantly decreased in the raphe magnus, and significantly increased in areas rich in serotonergic nerve terminals (i.e., hypothalamus, hippocampus, median geniculate body, parietal and visual cortices). An increase in tryptophan hydroxylase activity could account for the increase in the rate of serotonin synthesis seen in some regions. Since the 5-HT synthesis rate showed regional variability there seems to be a need for regional studies of the effect of drugs on the 5-HT synthesis. In addition, the 5-HT synthesis rate was not significantly different from that in controls in many of the brain regions.     

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

Abstract— The effect of l -tryptophan loading upon the amount of 5-HT accumulating in the brains of rats pretreated with a monoamine oxidase inhibitor was studied. The amount of brain 5-HT accumulated increased with increasing tryptophan dosages and brain tryptophan concentrations up to a tryptophan dose of 120 mg/kg body wt. and a brain tryptophan of about 70 μg/g brain. Above this dose and concentration no further increase in brain 5-HT accumulation occurred. After monoamine oxidase inhibition and tryptophan loading gross hyperactivity and hyperpyrexia occurred. Monoamine oxidase inhibition, tryptophan administration and intact aromatic amino acid decarboxylase activity were all collectively essential for the production of hyperactivity and hyperpyrexia. DL-Parachlorophenyl-alanine prevented both the occurrence of hyperactivity and the increased accumulation of, brain 5-HT. Indices of hyperactivity correlated with the amount of brain 5-HT accumulating in 1 h after tryptophan loading but not with the overall concentration of brain 5-HT, suggesting that hyperactivity was dependent upon the rate of 5-HT synthesis. Reserpine and tetra-benazine pretreatment speeded the onset and rate of development of the hyperactive state without altering the synthesis of brain 5-HT. It is suggested that when monoamine oxidase is inhibited and the rate of 5-HT synthesis is increased, granular uptake and storage of 5-HT and other rate-limiting mechanisms for 5-HT inactivation are unable to prevent 5-HT 'spilling over’to produce hyperactivity. The crucial dependence of 5-HT synthesis upon brain tryptophan concentration and the ability of intraneuronal metabolism, when monoamine oxidase activity is intact, to cope with increased 5-HT synthesis and prevent ‘spillover’, raise the possibility that brain 5-HT synthesis is normally in excess of functional needs, and suggest that intraneuronal metabolism and the intraneuronal organization of 5-HT pools are of more importance than synthesis in regulating the amount of 5-HT available for functional activity.     

PLASMA TRYPTOPHAN AND 5-HT METABOLISM IN THE CNS OF THE NEWBORN RAT

—The relationships between plasma tryptophan and 5-HT metabolism in the CNS were studied in newborn rats and compared with adults. Both the concentration of free tryptophan in plasma and that of the amino-acid in brain were much higher immediately after birth than later on. Drugs such as salicylate and chlordiazepoxide, which increased brain tryptophan concentrations in adults by displacing the plasma amino acid bound to serum albumin, were ineffective in newborn rats: most of the amino acid being already free in their plasma. The study of 5-HT metabolism in brain stem slices revealed that the affinity of the uptake process for tryptophan was higher in newborn than in adult animals, whereas the reverse situation was observed for the enzyme complex involved in 5-HT synthesis (lower apparent K_m in adults). In addition, the catabolism of newly synthesized 5-HT was more rapid in newborn than in adult tissues. Finally, the free state of tryptophan in plasma of newborn animals induced in brain both a high amino acid concentration and, in contrast to the situation observed in adults, a synthesis rate of 5-HT very near its maximal value.     

Acute flesinoxan treatment produces a different effect on rat brain serotonin synthesis than chronic treatment: An α-methyl-l-tryptophan autoradiographic study

5-HT_1A receptor agonists display anxiolytic and anti-depressant properties in clinical studies. In this study, we used the α-[¹⁴C]methyl-l-tryptophan (α-MTrp) autoradiographic method to evaluate the effects of the 5-HT_1A agonist, flesinoxan, on regional 5-HT synthesis in the rat brain, following acute or a 14-day continuous treatment. In the first series of experiments, flesinoxan (5 mg/kg; i.p.) was administered 40 min before the α-MTrp. It resulted in a significant increase of the arterial blood oxygen partial pressure (pO₂) and a reduction of the regional rate of 5-HT synthesis throughout the brain, with the exception of a few regions (medial geniculate body and thalamus). In the second series of experiments, flesinoxan (5 mg/kg day) was administered for 14 days, using an osmotic minipump implanted subcutaneously. When compared to rats treated with saline, there was an overall significant (p < 0.05) reduction in the synthesis (one-sample two-tailed t-test). However, there was no significant influence on the 5-HT synthesis rate in the dorsal and median raphe nuclei and the majority of their projection areas. A significant (p < 0.05) reduction was observed in the nucleus raphe magnus, medial caudate, ventral thalamus, amygdala, ventral tegmental area, medial forebrain bundle, nucleus accumbens, medial anterior olfactory nucleus and superior olive. The unaltered 5-HT synthesis rates in a large majority of regions following the 14-day treatment of flesinoxan may reflect the normalization (implies to not be different from salne treated control) of synthesis due to a desensitization of 5-HT_1A autoreceptors on the cell body of 5-HT neurons as well as at postsynaptic sites, which is known to occur following long-term treatment with 5-HT_1A agonists. It is of some importance to note that the normalization of the synthesis occurred in the majority of the brain limbic structures, the brain areas implicated in affective disorders and the corresponding successful treatments, as well as in the cortical regions, which are implicated in mood. However, there were some terminal regions (e.g., accumbens, anterior olfactory, lateral thalamus, raphe magnus and obscurus) in which the chronic flesinoxan treatment resulted in a significant reduction of synthesis, suggesting that there was not a full desensitization across the brain of the receptors controlling 5-HT synthesis.     

Serotonin Metabolism and Release in Frontal Cortex of Rats on a Vitamin E-Deficient Diet

Abstract: Rats were fed a control or vitamin E (all- rac -α-tocopheryl acetate)-deficient diet for 3 or 12 weeks. Serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), tryptophan, and α-tocopherol concentrations were determined in the frontal cortex using HPLC. α-Tocopherol concentrations fell significantly to 27% of control values at 12 weeks. Tissue 5-HT, 5-HIAA, and tryptophan concentrations were not significantly altered by the vitamin E-deficient diet at either time point. In vivo microdialysis revealed normal basal and K⁺-stimulated concentrations of 5-HT and 5-HIAA, but extracellular concentrations of tryptophan were significantly decreased after 3 weeks on the vitamin E-deficient diet, which resulted in an increase in the tissue/extracellular ratio and suggested a change in compartmentation. However, after 12 weeks on the deficient diet these values had returned to normal. Results in general indicate that a prolonged and substantial depletion of brain vitamin E can occur without major disturbance of serotonergic function.     

Feedback control of rat brain 5-hydroxytryptamine synthesis

Abstract— The effect of increased levels of 5-hydroxytryptamine (5-HT) on the synthesis of [³H]5-HT from intracisternally injected tracer doses of [³H]tryptophan was studied in the rat brain stem. The [³H]5-HT which accumulated in the first 15 min after [³H]tryptophan injection was measured at various times after the acute intraperitoneal administration of the monoamine oxidase inhibitors Catron or Pargyline. The 5-HT levels reached two and three times control values respectively at 20 min and 180 min after monoamine oxidase inhibitor administration but [³H]5-HT accumulation was decreased (40 per cent) at 180 min when compared with 20 min. These data as well as those obtained after chronic treatment with monoamine oxidase inhibitors revealed that there is an inverse relationship between [³H]5-HT accumulation and the endogenous 5-HT level. Monoamine oxidase activity was undetectable during all the intervals in which [³H]5-HT accumulation was measured. No inhibition of [³H]5-HT accumulation was detected when [³H]5-hydroxytryptophan was injected instead of [³H]tryptophan. The results are consistent with a negative feedback of 5-HT synthesis at the rate-limiting tryptophan hydroxylation step.     

Amino acids and central fatigue

Summary. There is an increasing interest in the mechanisms behind central fatigue, particularly in relation to changes in brain monoamine metabolism and the influence of specific amino acids on fatigue. Several studies in experimental animals have shown that physical exercise increases the synthesis and metabolism of brain 5-hydroxytryptamine (5-HT). Support for the involvement of 5-HT in fatigue can be found in studies where the brain concentration of 5-HT has been altered by means of pharmacological agents. When the 5-HT level was elevated in this way the performance was impaired in both rats and human subjects, and in accordance with this a decrease in the 5-HT level caused an improvement in running performance in rats. The precursor of 5-HT is the amino acid tryptophan and the synthesis of 5-HT in the brain is thought to be regulated by the blood supply of free tryptophan in relation to other large neutral amino acids (including the branched-chain amino acids, BCAA) since these compete with tryptophan for transport into the brain. Studies in human subjects have shown that the plasma ratio of free tryptophan/BCAA increases during and, particularly, after sustained exercise. This would favour the transport of tryptophan into the brain and also the synthesis and release of 5-HT which may lead to central fatigue. Attempts have been made to influence the 5-HT level by giving BCAA to human subjects during different types of sustained heavy exercise. The results indicate that ingestion of BCAA reduces the perceived exertion and mental fatigue during exercise and improves cognitive performance after the exercise. In addition, in some situations ingestion of BCAA might also improve physical performance; during exercise in the heat or in a competitive race when the central component of fatigue is assumed to be more pronounced than in a laboratory experiment. However, more experiments are needed to further clarify the effect of BCAA and also of tryptophan ingestion on physical performance and mental fatigue. Received January 3, 2000 / Accepted February 1, 2000     

Densities of Serotonergic Projections as Revealed by In Situ Synthesised Labelled α-Methyl-Serotonin: an Autoradiographic Evaluation

An estimate of serotonergic innervation density and regional serotonin (5-HT) concentration was performed from the distribution of in situ produced labelled α-methyl-serotonin. Rats were injected with (³H) labelled α-methyl-l-tryptophan and the tracer distribution was measured using the autoradiographic method 14 days following the injection. In a separate experiment, the total brain concentration of 5-HT in the rat brain was found to be 2.4 ± 0.2 nmol/g. Based on this, and the assumption that the specific activity of in situ produced α-methyl-serotonin is the same as that of the injected tracer, it was possible to estimate the regional concentrations of 5-HT and the relative concentration of regional serotonergic innervations. It was found, and reported for the first time here, that the highest concentration of serotonergic innervation is present in the solitary nucleus. Regionally measured 5-HT concentrations accord well with previously reported concentrations of 5-HT.     

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.     

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.     

TRYPTOPHAN LOADING: CONSEQUENT EFFECTS ON THE SYNTHESIS OF KYNURENINE AND 5-HYDROXYINDOLES IN RAT BRAIN

Abstract— Tryptophan loading of rats resulted in a continuous non-linear uptake of l -tryptophan from plasma into the brain. The optimum tryptophan load for increasing cerebral 5-hydroxytryptamine (5-HT) level was 25 mg/kg. Above this, there was a gradual decrease both in the levels and synthesis of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) as assessed from simultaneous intraperitoneal or intraventricular injections of l [¹⁴C]tryptophan. A 5–10 fold increase in cerebral tryptophan produced a limited stimulation of 5-HT synthesis. When the cerebral tryptophan level reached 1 ± 10 -4 , substrate inhibition in vivo of the tryptophan monooxygenase (tryptophan-5-hydroxylase) but not of the indoleamine-2,3-dioxygenase occurred. Cerebral synthesis of kynurenine increased linearly with increasing tryptophan load. At a plasma ratio of 50:1 tryptophan to kynurenine, tryptophan loading interfered with the entry of peripheral kynurenine. Tryptophan loading also increased the efflux of 5-hydroxyindoles from the brain. One hour after intraperitoneal injection of l -kynurenine sulfate (5 mg/kg) into rats, there was a shift in the plasma ratio of l -tryptophan to l -kynurenine to 4:1. In these rats, a 20% reduction of cerebral tryptophan was noted.     

Rapid repletion of brain serotonin in malnourished corn-fed rats following L-tryptophan injection.

The concentration of tryptophan in serum, and the levels of tryptophan, serotonin (5-HT), and 5-hydroxyindole-acetic acid (5-HIAA) in brain are substantially reduced in rats that consume for 6 weeks a diet in which corn is the only source of protein. Single injections of L-tryptophan (25, 50, or 100 mg/kg) cause dose-related increases in brain tryptophan, 5-HT, and 5-HIAA in corn-fed animals. At each dose, brain tryptophan content rises to a proportionately greater extent in corn-fed rats than in well-nourished controls, even though serum tryptophan concentrations attain higher levels in controls. This difference may reflect the greatly reduced serum concentrations in corn-fed rats of other large neutral amino acids that compete with tryptophan for uptake into the brain (tyrosine, phenylalanine, leucine, isoleucine, and valine). However, the substantial decrease in serum albumin levels also diminishes the binding of tryptophan to serum albumin; thus it is not yet possible to state which of these changes is responsible for the much greater increments in brain tryptophan observed in corn-fed rats after tryptophan injection. The fact that tryptophan administration rapidly restores brain 5-hydroxyindole levels in corn-fed animals suggests that the reductions in 5-HT and 5-HIAA levels associated with this type of malnutrition may be largely caused by inadequate availability of substrate.     

Regulation of serotonin synthesis

The regulation of serotonin synthesis in the central nervous system seems to involve mechanisms which control the intracellular concentration of tryptophan and the hydroxylation step. Numerous situations are reviewed which demonstrate that changes in the concentration of free tryptophan in plasma and/or in the uptake of the amino acid in brain may alter tryptophan concentration and 5-HT synthesis. In other cases, modifications in 5-HT synthesis were still detected after tryptophan loading, clearly indicating that the tryptophan concentration is not the only critical parameter. In particular, changes in 5-HT synthesis dependent on nerve impulse flow seem to be regulated at the hydroxylation step. In addition, long term regulations of 5-HT synthesis involving changes in tryptophan hydroxylase concentration have been recently demonstrated. How serotoninergic neurons integrate all these regulating factors to modulate 5-HT synthesis is still an open question.     

STIMULATION OF BRAIN SEROTONIN SYNTHESIS BY DIBUTYRYL-CYCLIC AMP IN RATS

Cyclic AMP and dibutyryl-cyclic AMP, a derivative of cyclic AMP resistant to phosphodiesterase inactivation, were injected into the lateral ventricles of rats. These nucleotides did not change the level of brain 5-HT but increased the brain level of its principal metabolite, 5-hydroxyindoleacetic acid. Cyclic AMP was less potent than dibutyryl-cyclic AMP. Butyrate and 5′-AMP were inactive. The effect of dibutyryl cyclic AMP on 5-HT metabolism was studied both in vivo and in vitro. The rate of synthesis of 5-HT was measured by the rate of accumulation of 5-hydroxyindoleacetic acid after the transport of this acid out of the brain was blocked with probenecid. The rate of synthesis of brain 5-HT increased from 0-38 μg/g/h in control rats to 0-65 μg/g/h after dibutyryl-cyclic AMP. In addition cyclic AMP and dibutyryl-cyclic AMP markedly increased brain tryptophan, while AMP was inactive. Since brain tryptophan hydroxylase has a K_m for its substrate that is much higher than the concentrations of tryptophan normally present in the brain, it is likely that the increase in the rate of synthesis of brain 5-HT is secondary to the cyclic AMP induced increase in the levels of brain tryptophan. In vitro studies revealed that dibutyryl-cyclic AMP increased the uptake of radioactive labelled tryptophan into slices of rat brain stem and the formation of 5-HT and 5-hydroxyindoleacetic acid.     

Effect of orally administered l-tryptophan on serotonin,melatonin, and the innate immune response in the rat

To assess the effects of external administration of L-tryptophan on the synthesis of serotonin and melatonin as well as on the immune function of Wistar rats, 300 mg of the amino acid were administered through an oral cannula either during daylight (08:00) or at night (20:00) for 5 days. Brain, plasma, and peritoneal macrophage samples were collected 4 h after the administration. The accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used to measure the rate of tryptophan hydroxylation in vivo. Circulating melatonin levels were determined by radioimmunoassay, and the phagocytic activity of macrophages was measured by counting, under oil-immersion phase-contrast microscopy, the number of particles ingested. The results showed a diurnal increase (p < 0.05) in the brain 5-HTP, serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindolacetic acid (5-HIAA) of the animals which had received tryptophan at 08:00 and were killed 4 h later. In the animals which received tryptophan during the dark period, the 5-HT declined but the 5-HT/5-HIAA ratio remained unchanged. There was also a significant increase (p < 0.05) in nocturnal circulating melatonin levels and in the innate immune response of the peritoneal macrophages in the animals which had received tryptophan at 20:00. The results indicated that the synthesis of serotonin and melatonin, as well as the innate immune response, can be modulated by oral ingestion of tryptophan.     

Effects of desipramine on regional serotonin synthesis in the rat brain: acute and chronic autoradiographic studies

Various studies have implicated the involvement of noradrenaline (NA) and/or serotonin (5-hydroxytryptamine (5-HT)) in the pathogenesis and treatment of depression. The aim of the present study was to investigate the effects of acute and 7 days of administration of desipramine, a NA re-uptake inhibitor, on the rate of 5-HT synthesis in the rat brain. The study was done by an autoradiographic method using alpha-[14C]-methyl-L-tryptophan as a tracer. The acute (10mg/kg, i.p., 2h before i.v. infusion of the tracer) or 7 days of desipramine (10mg/kg per day, i.p.) did not affect plasma tryptophan (Trp) concentrations, as compared to control (saline treated) rats. Acute treatment with desipramine decreased the rate of 5-HT synthesis in the brain regions that contain 5-HT cell bodies between 19 and 28%, and increased the rate of 5-HT synthesis in the majority of areas containing 5-HT terminals between 21 and 65%. In contrast to the acute treatment, a 7-day administration increased 5-HT synthesis rates in the dorsal raphe (24%), but decreased it in raphe magnus (35%), superior olive (45%), caudate (31%), superior (38%) and inferior (53%) colliculus, and in the auditory cortex (35%). This suggests that the effect of desipramine on 5-HT synthesis rate is time-dependent and differs in the cell bodies and structures containing 5-HT nerve terminals.     

EVIDENCE FOR THE SYNTHESIS AND STORAGE OF 5-HYDROXYTRYPTAMINE IN TWO SEPARATE POOLS IN THE BRAIN

Abstract— Evidence is presented to support the hypothesis that 5-hydroxytryptamine (5-HT) in the rat brain is synthesized by two separate pathways and stored in two or more compartments. Lysergic acid diethylamide in doses down to 50 μg/kg was shown to reduce the formation of 5-[³H]HT from [³H]tryptophan in the presence of a monoamine oxidase inhibitor, although the total rate of accumulation of 5-HT was unchanged. Conversely, adrenalectomy was found to increase the total synthesis of 5-HT measured in the same way, although the amount of 5-[³H]HT formed suggested that there was no increase in the synthesis of the amine. In a third experiment it was found that electrical stimulation of 5-HT-containing nerves following labelling of 5-HT stores with [³H]tryptophan led to a biphasic disappearance of 5-[³H]HT. It is suggested that the method of measuring 5-HT synthesis by measuring 5-[³H]HT formed from[³H]tryptophan in the presence of a monoamine oxidase inhibitor may be a way of selectively measuring the turnover of the functional pool of 5-HT.     

EFFECTS OF ELECTRICAL STIMULATION OF RAT MIDBRAIN ON 5-HYDROXYTRYPTAMINE SYNTHESIS AS DETERMINED BY A SENSITIVE RADIOISOTOPE METHOD

Abstract— 5-Hydroxytryptamine (5-HT) synthesis has been determined in the rat brain by measuring the 5-[³H]HT formed from [³H]tryptophan in the presence of monoamine oxidase inhibitor. Electrical stimulation in the region of the midbrain raphe nucleus increased formation of 5-[³H]HT by over 100 per cent, although the level of endogenous 5-HT and the concentration and specific activity of tryptophan were unchanged; the results are interpreted in terms of a two-compartment model. The optimum stimulation parameters were determined. Three days after a single dose of the tryptophan hydroxylase inhibitor p -chlorophenylalanine, stimulation increased 5-HT synthesis by the same percentage as in untreated animals. It was also found that after the end of an hour's stimulation, synthesis returned to control values in under an hour. These results suggest that the rise in synthesis of the amine on stimulation is not due to induction of tryptophan hydroxylase, but more likely to an increase in the activity of existing enzyme.     

SEROTONIN DEFICIENCY IN EXPERIMENTAL HYPERPHENYLALANINEMIA

Abstract— The mechanism of serotonin depletion was studied in the preweanling rat in which a chemical simulation of phenylketonuria had been induced by injections of p-CPA and l -PA. Experimental conditions were selected to effectively minimize the contribution by deficient tryptophan hydroxylation and 5-HTP transport. Excessive degradation of 5-HT in the hyperphenylalaninemic brain could be eliminated as a possible mechanism. The observed levels of cerebral 5-HTP, 5-HT, 5-HIAA before and 1 h after 5-HTP loading, with and without pargyline pretreatment, clearly demonstrate greatly diminished in vivo synthesis of 5-HT in the hyperphenylalaninemic animal. This deficient synthesis could largely be accounted for by decreased activity of aromatic l -amino acid decarboxylase measured in the high speed soluble supernatant extracts of whole brain. Decreased storage of 5-HT in the particulate subcellular fraction of whole brain was also noted in the hyperphenylalaninemic animal. Significant lowering of bound serotonin levels in the brain occurred with injections of PEA into normal animals.