Acyclovir inhibits rat liver tryptophan-2,3-dioxygenase and induces a concomitant rise in brain serotonin and 5-hydroxyindole acetic acid levels.

Viral diseases of the brain may induce changes in neurotransmitter synthesis and metabolism. In experimental herpes simplex encephalitis, brain serotonin is reduced, whilst it's major metabolite, 5-hydroxyindole acetic acid and turnover is increased. It is well established that reduced levels of brain monoamines, serotonin and norepinephrine may contribute to the symptoms of clinical depression, which raises the possibility that this condition is prevalent in herpes simplex encephalitis. An inverse relationship exists between liver tryptophan-2,3-dioxygenase activity and brain serotonin levels and there is an interdependency between serotonin and norepinephrine levels. The aim of this study is to determine the effect of acyclovir, an antiviral used in the treatment of herpes simplex encephalitis, on rat liver tryptophan-2,3-dioxygenase activity in vitro and in vivo as well as on rat forebrain serotonin, 5-hydroxyindole acetic acid and norepinephrine levels. The results show that acyclovir inhibits tryptophan-2,3-dioxygenase activity in vitro and in vivo, with a concomitant rise in serotonin and 5-hydroxyindole acetic acid levels. However, acyclovir reduces the turnover of serotonin to 5-hydroxyindole acetic acid, without any effect on norepinephrine levels. It appears that acyclovir may have the potential to reduce the clinical symptoms of depression in herpes simplex encephalitis. However, a greater turnover of serotonin to 5-hydroxyindole acetic acid could possibly be masked by conversion of serotonin to 5-hydroxytryptophol, which needs to be investigated further.

Objective post-mortem diagnosis of chronic alcohol abuse--a review of studies on new markers.

Identification of chronic excessive alcohol consumption in living and deceased individuals is a fundamental task in forensic pathology. Reliable methods for post-mortem diagnosis of chronic alcohol abuse are required because morphological findings are unspecific and ante-mortem data are often unreliable. In clinical practice, several biochemical markers indirectly demonstrating chronic alcohol abuse are employed, but thus far these methods have not been used in routine post-mortem investigations. We reviewed publications in which these markers have been applied to autopsy material. Based on this review, some of these biochemical parameters are useful in post-mortem diagnostics, although further systematic research is required.

HPLC determination of serotonin and its metabolites from human platelet-rich plasma; shift to 5-hydroxytryptophol formation following alcohol consumption.

A sensitive, simple, and reliable high-performance liquid chromatographic method with electrochemical detection is developed for the measurement of four natural products, the serotonin-related indols from human platelet-rich plasma (PRP) using N-methylserotonin as internal standard. Separation of serotonin (5HT), 5-hydroxytryptophan (5HTP), 5-hydroxytryptophol (5HTOL), and 5-hydroxyindole-acetic acid (5HIAA) is carried out on Supelcosil LC-18DB stationary phase. A mixture of 48 mM citric acid, 28 mM sodium phosphate dibasic, 0.027 mM Na2EDTA, and 3% methanol (pH 3.18) serves as the mobile phase. Measurements are carried out at 25 degrees C at Eox=0.65 V. The calibration curves are linear through the range of 10-200 pg/mL. Method validation is performed according to internationally accepted criteria. Blood is collected from healthy controls and schizophrenic subjects. Significantly higher PRP serotonin is measured in schizophrenics; patients with recent alcohol consumption could be characterized with significantly elevated 5HTOL/5HIAA ratio.

Evaluation of 5-hydroxytryptophol and other endogenous serotonin (5-hydroxytryptamine) analogs as substrates for UDP-glucuronosyltransferase 1A6.

Serotonin is a specific in vitro substrate for human UDP-glucuronosyltransferase (UGT) 1A6. In this study, the contribution of UGT1A6 to the glucuronidation of endogenous structural analogs of serotonin, including 5-hydroxytryptophol, N-acetylserotonin, and 6-hydroxymelatonin, was evaluated using available recombinant human UGT isoforms, human liver microsomes, and liver microsomes from animals that do not express functional UGT1A6 (Gunn rats and cats). Only UGT1A6 and UGT1A9 were found to glucuronidate 5-hydroxytryptophol at a concentration of 2 mM, although the glucuronidation rate with UGT1A6 was over 10 times that of UGT1A9. K(m) values for human liver microsomes (156, 141, and 134 microM) were most similar to that of expressed UGT1A6 (135 microM) but vastly different from that of UGT1A9 (3674 microM). 5-Hydroxytryptophol glucuronidation by human liver microsomes (n = 54) correlated well with serotonin glucuronidation (R(s) = 0.83) and UGT1A6 protein content (R(s) = 0.85). 5-Hydroxytryptophol also competitively inhibited serotonin glucuronidation by human liver microsomes (K(i) = 291 microM) and UGT1A6 (K(i) = 200 microM). N-acetylserotonin was glucuronidated most extensively by UGT1A6, although UGT1A9 and UGT1A10 showed moderate catalysis. 6-Hydroxymelatonin was glucuronidated largely by UGT1A9 and UGT1A10 but not at all by UGT1A6. Gunn rat liver glucuronidation rates for serotonin, 5-hydroxytryptophol, N-acetylserotonin, and 6-hydroxymelatonin were 11, 5, 32, and 3%, respectively, of that of normal rat liver. Cat liver microsomes did not glucuronidate serotonin, whereas relatively low activities were observed for the other indole substrates. In conclusion, these results indicate that human UGT1A6 plays a predominant role in the glucuronidation of 5-hydroxytryptophol and N-acetylserotonin, whereas 6-hydroxymelatonin is not a substrate for this enzyme.

Acute effect of simultaneous administration of tryptophan and ethanol on serotonin metabolites in the locus coeruleus in rats.

Using the microdialysis method, we investigated whether the levels of serotonin (5-hydroxytryptamine, 5-HT) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTPL), in the locus coeruleus are influenced by tryptophan alone or simultaneous administration of tryptophan and ethanol. Tryptophan (50 mg/kg, i.p.) led to a significant increase in the levels of 5-HIAA, but not 5-HT in the locus coeruleus. However, ethanol (1.25 g/kg) had no effect on the levels of 5-HT and its metabolites. Combined administration of tryptophan and ethanol caused very marked increases in 5-HIAA and 5-HTPL levels in the locus coeruleus. A time lag in the increased 5-HIAA levels between tryptophan alone and tryptophan plus ethanol was observed. Moreover, 5-HIAA levels in the locus coeruleus induced by tryptophan were abolished by microinjection of 5,7-dihydroxytryptamine (150 microg/4 microl) into the dorsal raphe nucleus. Judging from the present results, the serotonergic afferents to the locus coeruleus may originate for about 20-30% from cell bodies located in the dorsal raphe nucleus. Teeth-chattering was significantly detected in the tryptophan plus ethanol-treated rats when compared with the tryptophan-treated rats, but not in the saline-treated controls. These results may suggest that the increased levels of 5-HIAA and 5-HTPL in the locus coeruleus induced by tryptophan are potentiated by ethanol, and that these levels are partly responsible for behavioral activation.

The ethanol conjugate ethyl glucuronide is a useful marker of recent alcohol consumption.

BACKGROUND: Current biological state markers remain suboptimal with regard to sensitivity and specificity for monitoring alcohol consumption. The currently used state markers can be influenced by age, sex, and a variety of substances and non-alcohol-associated diseases and do not fully cover the time axis for alcohol intake. Ethyl glucuronide (EtG) is a promising, nonvolatile, water-soluble marker of recent alcohol consumption that is stable during storage and can be detected for an extended time period (up to 80 hr) after alcohol is completely eliminated from the body. PATIENTS AND METHODS: In the WHO/ISBRA Study of State and Trait Markers of Alcohol Use and Dependence, EtG was determined in urine samples from 304 patients with an liquid chromotography, electrospray ionization double mass spectrometry (ESI-LC/MS-MS) method. Deuterium labeled EtG was used as internal standard. Determination limit was 0.1 mg/liter. All measurements were performed in duplicate. A calibration solution was measured after each 10 samples. RESULTS: The following significant correlations were found for the Spearman rank correlation for the total sample between EtG and other variables: sobriety in days (r = -0.6), 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid (HTOL/HIAA) ratio (r = 0.58), ethanol level (r = 0.433), methanol level (r = 0.198), carbohydrate-deficient transferrin (r = 0.458), gamma-glutamyltransferase (r = 0.428), aspartate aminotransferase (r = 0.260), age (r = 0.264), and total grams of ethanol consumed in the previous month (r = 0.467). In a subsample of 277 subjects in whom no ethanol was detectable in urine, the following correlations with EtG levels were found: sobriety (days; r = -0.597), HTOL/HIAA ratio (r = 0.478), gamma-glutamyltransferase (r = 0.422), total grams of ethanol consumed last month (r = 0.395), and carbohydrate-deficient transferrin (r = 0.366; all significant at p < 0.05). When we compared results between EtG levels and the HTOL/HIAA ratio, 68.8% (n = 119) of those positive for EtG did not have elevated values for the HTOL/HIAA ratio. Thirty-one percent (31.2%) of these 119 subjects were positive for both parameters, but of those negative for EtG, only 4.4% had an elevated HTOL/HIAA ratio. CONCLUSIONS: EtG is a good candidate for a sensitive, specific, and reliable marker of recent alcohol intake. The complementary use of this marker with other biological state markers should significantly improve treatment outcome and therapy effectiveness and reduce costs.

Distribution of serotonin, its metabolites and 5-HT transporters in the neostriatum of Lurcher and weaver mutant mice.

Serotonin (5-HT) uptake sites, or transporters, were measured in the neostriatum (caudate putamen) of wild type (+/+) mice and heterozygous (wv/+) and homozygous (wv/wv) weaver, as well as in heterozygous Lurcher (Lc/+) mutants. These topological surveys were carried out by quantitative ligand binding autoradiography using the uptake site antagonist [3H]-citalopram as a probe of innervation densities in four quadrants of the rostral neostriatum and in two halves of the caudal neostriatum. In addition, tissue concentrations of 5-HT, 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophol were measured by high-performance liquid chromatography with electrochemical detection in these neostriatal divisions. In +/+ mice and in Lc/+ mutants there was a dorso-ventral gradient of increasing 5-HT levels, and they exhibited a similar heterogeneity of [3H]-citalopram labeling. In contrast, the gradients of 5-HT concentrations and [3H]-citalopram binding disappeared in the weaver mutants, suggesting a rearrangement of the 5-HT innervation. This reorganization of the 5-HT system in the neostriatum was more obvious in the wv/wv and is compatible with the hypothesis that the postnatal dopaminergic deficiencies that characterize weaver mutants lead to a sprouting of fibers and thus constitute a genetic model of dopaminergic denervation that leads to a 5-HT hyperinnervation.

Central serotonin system in Dystonia musculorum mutant mice: biochemical, autoradiographic and immunocytochemical data.

The autosomal recessive mutation dystonia musculorum (dt(J)/dt(J)) causes degenerative alterations of peripheral and central sensory pathways that lead to ataxia. To investigate possible changes in the central serotonin system of these mice, HPLC measurements of 5-hydroxytryptophan, 5-hydroxy-tryptamine (serotonin; 5-HT), and 5-HT metabolites were obtained from 22 brain regions and the spinal cord of wild type and dt(J)/dt(J) mutant mice. Also, 5-HT transporters were quantified by [(3)H]citalopram autoradiography in 72 brain regions, subregions, and nuclei, and the 5-HT innervation visualized by immunocytochemistry throughout the brain and spinal cord. In all brain regions measured for indoleamine content, there were no significant differences between the two genotypes. In the spinal cord, an increased tissue concentration of 5-HT (+34%), 5-hydroxyindole-3-acetic acid (+33%), 5-hydroxytryptophol (+21%), and 5-hydroxytryptophan (+45%) in dt(J)/dt(J) actually corresponded to the same total amount of each of these indoleamines in the entire spinal cord, when taking into account its reduced size in the mutants. Quantification of the binding to 5-HT transporters showed increases in the medial geniculate nucleus (+14%), medial (+24%) and lateral (+18%) hypothalamus, interpeduncular (+13%), vestibular (+22%), and deep cerebellar nuclei (+37%) of dt(J)/dt mice, and decreases in the ventral tegmental area (-13%), median and linear raphe nuclei (-20%), as well as in the solitary complex (-35%). There were no apparent differences in the distribution of 5-HT-immunostained fibers in these and other regions of brain and in the spinal cord of dt(J)/dt(J) compared to wild type mice. The bulk of these results indicates a relative sparing of the central 5-HT system in the dt(J)/dt(J) mice, even though alterations in 5-HT transporters could justify attempts at improving the sensorimotor dysfunction by administration of serotoninergic agents in these mice.

Modulation of midazolam 1-hydroxylation activity in vitro by neurotransmitters and precursors.

OBJECTIVE: The aim of this study was to find whether endogenous substances could modulate CYP3A activity. There is evidence that CYP3A, a major phase-I xenobiotic metabolizing enzyme, is present in human brain but, at the present time, endogenous substrates for such an enzyme remain to be identified. A possible linkage between the CYP2D6 enzyme and serotonergic transmission has been recently reported by our group. In the same manner, structurally related enzymes such as CYP3A could also be related to endogenous compounds. METHODS: CYP3A activity was measured using the enzyme-specific substrate midazolam in human liver microsomes. Several neurotransmitters, precursors, and their metabolites, corresponding to three different metabolic routes, were assayed as putative modulators of CYP3A enzyme activity. These comprised serotonergic, catecolaminergic, and GABAergic transmitters and precursors. The inhibitory capacity of ketoconazole, a competitive inhibitor of CYP3A, was also analyzed for comparison. RESULTS: The kinetic analysis of the midazolam 1-hydroxylase activity measured in microsomes from five human liver samples indicated Km values (mean +/- SD) of 5.8 +/- 4.9 microM, and Vmax values of 1.7 +/- 1.4 nmol min(-1) per mg microsomal protein in all the samples used in the study. Of the 14 substances analyzed, adrenaline, serotonin, and 5-hydroxytriptofol were full inhibitors of CYP3A enzyme activity (Ki values of 42.3, 26.4, and 43 microM, respectively). The remaining substances were weak inhibitors or had no inhibitory effect. CONCLUSION: Brain CYP3A activity could be modulated by some neurotransmitters and precursors.

Studies on the interaction between ethanol and serotonin metabolism in rat, using deuterated ethanol and 4-methylpyrazole.

The metabolic interaction between ethanol and serotonin (5-hydroxytryptamine) via alcohol dehydrogenase (ADH; EC 1.1.1.1) was studied in tissue homogenates of Sprague-Dawley rats by following the transfer of deuterium from deuterated ethanol over endogenous NADH to 5-hydroxytryptophol (5HTOL). Homogenates of whole brain, lung, spleen, kidney, liver, stomach, jejunum, ileum, colon, and caecum were incubated in the presence of [2H2]ethanol and 5-hydroxyindole-3-acetaldehyde (5HIAL), and the [2H]5HTOL formed was identified and quantified using gas chromatography-mass spectrometry. ADH activity was most abundant in liver, kidney, and within the gastrointestinal tract. The highest incorporation of deuterium was obtained in homogenates of kidney, lung, and colon, whereas in brain, which contains very low ADH activity, no incorporation could be demonstrated. Addition of extra NAD+ (2.4 mM) increased the formation of [2H]5HTOL 2.6-fold in liver homogenates, but only 1.2-fold in kidney homogenates. 4-Methylpyrazole, a potent inhibitor of class I ADH, inhibited the 5HIAL reduction in homogenates of lung, kidney, jejunum, ileum, and colon, and caused a marked drop in 5HTOL oxidation in all tissues except stomach and spleen. These results demonstrate that in the rat a metabolic interaction between ethanol and serotonin via the ADH pathway may take place in several tissues besides the liver, which is the main tissue for ethanol detoxification.

Activities of human alcohol dehydrogenases in the metabolic pathways of ethanol and serotonin.

Alcohols and aldehydes in the metabolic pathways of ethanol and serotonin are substrates for alcohol dehydrogenases (ADH) of class I and II. In addition to the reversible alcohol oxidation/aldehyde reduction, these enzymes catalyse aldehyde oxidation. Class-I gammagamma ADH catalyses the dismutation of both acetaldehyde and 5-hydroxyindole-3-acetaldehyde (5-HIAL) into their corresponding alcohols and carboxylic acids. The turnover of acetaldehyde dismutation is high (kcat = 180 min-1) but saturation is reached first at high concentrations (Km = 30 mm) while dismutation of 5-HIAL is saturated at lower concentrations and is thereby more efficient (Km = 150 microm; kcat = 40 min-1). In a system where NAD+ is regenerated, the oxidation of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid proceeds with concentration levels of the intermediary 5-HIAL expected for a two-step oxidation. Butanal and 5-HIAL oxidation is also observed for class-I ADH in the presence of NADH. The class-II enzyme is less efficient in aldehyde oxidation, and the ethanol-oxidation activity of this enzyme is competitively inhibited by acetate (Ki = 12 mm) and 5-hydroxyindole-3-acetic acid (Ki = 2 mm). Reduction of 5-HIAL is efficiently catalysed by class-I gammagamma ADH (kcat = 400 min-1; Km = 33 microm) in the presence of NADH. This indicates that the increased 5-hydroxytryptophol/5-hydroxyindole-3-acetic acid ratio observed after ethanol intake may be due to the increased NADH/NAD+ ratio on the class-I ADH.

Evidence for an endogenous clock in the retina of rainbow trout: II. Circadian rhythmicity of serotonin metabolism.

The purpose of the present study was to investigate patterns of circadian rhythmicity in the retina of a salmonid fish, the rainbow trout (Oncorhynchus mykiss). Our data present the first demonstration of intraretinal variations of serotonergic substances under light/dark-conditions (LD) and during continuous darkness (DD). All substances examined (serotonin, N-acetyl serotonin, 5-hydroxytryptophol, 5-hydroxyindole acetic acid) were rhythmic in LD. Serotonin, N-acetyl-serotonin, and 5-hydroxyindole acetic acid showed a preservation of specific features of rhythmicity in DD indicating the involvement of an endogenous pacemaker in the regulation of serotonin metabolism in the rainbow trout eye.

Elevated brain 5-hydroxytryptophol levels in experimental portal-systemic encephalopathy.

Brain tissue levels of the two serotonin metabolites 5-hydroxytryptophol and 5-hydroxyindole-3-acetic acid (5-HIAA) were measured in porta-caval shunted rats, an in vivo model of portal-systemic encephalopathy. An intraperitoneal challenge of L-tryptophan (280 mg/kg body weight) to sham-operated rats was also instituted to increase the brain serotonin metabolism in these rats. The results revealed significant increases in 5-hydroxytryptophol (by 31% and 5-HIAA (by 87%) brain levels in porta-caval shunted rats as compared to sham-operated controls. The brain 5-hydroxytryptophol-to-5-HIAA ratio was lower in the porta-caval shunted rats. The 5-hydroxytryptophol levels in sham rats after the L-tryptophan challenge were intermediate between the porta-caval shunted and sham rats but not statistically significant for either group. These results suggest that increased brain 5-hydroxytryptophol levels might be associated with the pathogenesis of portal-systemic encephalopathy. Further, the elevated brain 5-hydroxytryptophol levels in experimental portal-systemic encephalopathy are probably a result of the increased brain serotonin metabolism prevailing in this condition rather than changes in the brain redox potential.

Acute interaction between ethanol and serotonin metabolism in the rat.

The effect of acute ethanol on peripheral serotonin (5HT) metabolism was studied in Sprague-Dawley rats. Four hours after a single dose of ethanol (1.0 g/kg) administered into the stomach, a significant increase in the 5HT level in stomach tissue and a decrease in ileum was observed. The level of 5-hydroxyindole-3-acetic acid (5HIAA) was increased in urine, while increased concentrations of 5-hydroxytryptophol (5HTOL) occurred in jejunum, ileum, spleen and urine. After 7-9 h when the blood ethanol concentration had returned to zero, 5HTOL levels were still higher than control values in jejunum, ileum and urine. At 4 h, an elevated ratio of 5HTOL to 5HIAA was observed in urine and ileum (by approximately 2-fold), liver (approximately 3-fold), and spleen (approximately 5-fold), whereas the ratio was reduced in stomach. In urine and spleen, this metabolic shift persisted after 7-9 h. The 5HTOL level in bile was increased by approximately 3.5-fold after 8 h. 5HIAA was not detectable in bile. The present results indicate that the rat has a much higher proportion of 5HTOL formation than man under normal conditions. The rat does not appear to be an ideal model for studying the interaction between ethanol and 5HT metabolism in man.

Fusaric acid in Fusarium moniliforme cultures, corn, and feeds toxic to livestock and the neurochemical effects in the brain and pineal gland of rats.

Fusaric acid is produced by several species of Fusarium, which commonly infect corn and other agricultural commodities. Since this mycotoxin may augment the effects of other Fusarium toxins, a gas chromatography/mass spectrometry method of analysis in feeds was developed. Fusaric acid was analyzed as the trimethylsilyl-ester from F. moniliforme-cultures, -contaminated corn screenings, and feeds toxic to livestock. The mycotoxin was found in all samples and ranged from 0.43 to 12.39 micrograms/g sample. Also, fusaric acid was tested for its neurochemical effects in the brain and pineal gland of rats. Animals were dosed intraperitoneally (100 mg/kg body weight) 30 min prior to the onset of the dark phase (lights out) and the effects were studied at 1.5, 3.5, and 5.5 h after treatment. Brain serotonin (5HT), 5-hydroxyindoleacetic acid (5HIAA), tyrosine (TYRO), and dopamine (DA) were increased (P < 0.05) by fusaric acid, and norepinephrine (NEpi) was decreased (P < 0.05). Analogously, DA in the pineal gland increased and NEpi decreased (P < 0.05). Pineal N-acetylserotonin (NAc5HT) was increased (P < 0.05), whereas pineal 5HT and its two major metabolites 5HIAA and 5-hydroxytryptophol (5HTOL) decreased (P < 0.05). Elevated brain TYRO and brain and pineal DA, with decreased NEpi, may be consistent with fusaric acid's partial inhibitory effect on tyrosine-hydroxylase and its inhibitory effect on dopamine-beta-hydroxylase, respectively. Elevated pineal Nac5HT is consistent with decreased pineal 5HT and the increased pineal DA, and support the dopaminergic stimulatory activity of the enzyme responsible for the conversion of 5HT to NAc5HT. This is the first report of fusaric acid's in vivo effect on pineal DA, NEpi, 5HT, and NAc5HT in rats, and a relation for the effects on TYRO, 5HT, and 5HIAA in brain tissue. The results indicate fusaric acid alters brain and pineal neurotransmitters and may contribute to the toxic effects of Fusarium-contaminated feeds.

Occurrence in vivo of 5-hydroxytryptophol in the brain of rats treated with ethanol.

The effect of ethanol (EtOH) on the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) and the efflux of their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindol-3-ylacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOL) from the striatum of the freely moving rat were studied in vivo using brain microdialysis. Striatal DA and 5-HT release was maximally enhanced at first fraction after the administration of EtOH (2 g/kg, i.p.). The level of the DA-oxidized metabolite, DOPAC, decreased significantly. In the 5-HT metabolic pathway, the oxidized metabolite, 5-HIAA, did not show significant changes, whereas levels of the biogenic alcohol 5-HTOL were increased to 180% at 90 min following EtOH administration. It is suggested that EtOH, most probably via acetaldehyde, could shift 5-HT metabolism from the oxidative to the reductive pathway in the rat brain.

Changes in biogenic amines in rat hippocampus during development and aging.

The effects of postnatal development and aging on the concentration of dopamine, noradrenaline, serotonin and their principle metabolites have been studied in the hippocampus of the rat. During development the concentration of dopamine increases 1.5 fold during the first 90 days. 3-methoxytyramine was found in low concentrations. The homovanillic acid and DOPAC concentrations showed no changes apart from a decrease at day 15 and an increase at day 8, respectively. From birth up to 30 months, the noradrenaline concentration increased by a factor of about 10. Their metabolites each showed a different profile. The concentration of tryptophan was always the highest among the compounds studied. It decreased from birth to day 15, while the concentration of serotonin and 5-hydroxyindolacetic acid increased 3 and 5 fold respectively during this time. However, 5-hydroxytryptophan and 5-hydroxytryptophol concentrations were very low and unchanged at all stages. These findings led to the conclusion that the neurotransmitters: noradrenaline and serotonin, are developed in the hippocampus during the first three months. During aging, the serotonin concentration is increased without significant change in the other compounds studied.

Effect of selective monoamine oxidase inhibitors on rat pineal melatonin synthesis in vitro.

Freshly cultured pineal glands respond to the monoamine oxidase A (MAO A) inhibitor clorgyline and to high concentrations of the MAO B inhibitor, deprenyl, with an increase in serotonin N-acetyltransferase activity and N-acetylated indoles and a fall in 5-hydroxylated serotonin degradation products. Glands cultured for 48 hours before challenge respond less. Response is absent in glands cultured for 72 hours and in glands from ganglionectomized animals cultured for 48 hours before challenge. These data are consistent with the hypothesis that these MAO inhibitors stimulate melatonin synthesis by protecting norepinephrine from degradation.

Hydroxyindole-O-methyltransferase catalyses production of methoxyindoles in rat pineal gland dependent on the concentration of hydroxy precursors and their affinity for the enzyme.

Indole metabolites were separated by thin-layer chromatography following organ culture of rat pineal glands with tritiated tryptophan. Methoxyindole production was shown to differ substantially from results obtained when pineal glands were incubated with radiolabelled serotonin. The correlation between hydroxy- and corresponding methoxyindoles was, however, remarkably similar to previous results, and indicated that in the pineal gland, it is probable that production of methoxyindoles is dependent on the concentration of the various hydroxyindoles and their relative affinities for hydroxyindole-O-methyltransferase. It is also probable that although several forms of the enzyme may exist in the pineal gland, the catalytic sites are homogeneous in their activity.

5-Hydroxytryptophol and 5-hydroxyindoleacetic acid levels in rat brain: effects of various drugs affecting serotonergic transmitter mechanisms.

The effect of various pharmacological treatments on the levels of free and total 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) in rat brain were investigated using a gas chromatographic-mass spectrometric technique. The disappearance of 5-HTOL following monoamine oxidase (MAO) inhibition induced by pargyline was more rapid (t1/2 10-15 min) than that of 5-HIAA (t1/2 30-40 min) in all regions investigated, indicating a rapid turnover of 5-HTOL. The selective MAO-A inhibitor chlorgyline produced a more pronounced reduction of 5-HTOL than of 5-HIAA, while the MAO-B inhibitor deprenyl was without effect on both serotonin metabolites. The MAO-A inhibitor amiflamine which is selective for serotonin neurons, was also more effective in reducing free 5-HTOL levels than of 5-HIAA levels, suggesting that the formation of 5-HTOL is closely associated with serotonin neurons. Neonatal treatment with the serotonin neurotoxin 5, 7-dihydroxytryptamine (5,7-HT) led to a more pronounced reduction of 5-HTOL levels in cerebral cortex than that of 5-HIAA levels, while the increase of 5-HTOL levels in pons-medulla was more marked than of 5-HIAA levels. Probenecid treatment increased several fold both conjugated 5-HTOL and 5-HIAA levels in brain tissue. An increase was also noted for free 5-HTOL, although of less magnitude. Treatment with the serotonin receptor active agents methiothepin and d-lysergic acid diethylamide produced similar alterations of free 5-HTOL and 5-HIAA. The present results have demonstrated that free 5-HTOL has a rapid turnover in rat brain and that free 5-HTOL levels may serve as a useful index for serotonin turnover.