Embryonic Development of Avian Pineal Secretory Activity-A Lesson from the Goose Pineal Organs in Superfusion Culture.

The embryonic ontogeny of pineal secretory activity in birds has been investigated almost exclusively in chickens. This study aimed to characterize this process in domestic geese. The pineal organs of embryos aged 18-28 days were incubated in superfusion culture under different light conditions for 4-5 days and treated with norepinephrine (NE). Melatonin (MLT) was measured by radioimmunoassay and other indoles by HPLC with fluorescence detection. Additionally, pineal organs were collected from embryos at 14-28 days of age and used to measure catecholamines by HPLC with electrochemical detection. MLT secretion increased with embryo age, most intensively between the 22nd and 24th days of life. The daily changes in MLT secretion under the 12 L:12D cycle occurred on the first day of culture, starting from an embryonic age of 24 days. MLT secretion was controlled by the light-dark cycle in all age groups studied. However, exposure to light during the scotophase did not alter the secretion of MLT. The endogenous oscillator expressed its activity in regulating MLT secretion in the pineal organs of embryos aged 24 days and older but could not generate a rhythm after one cycle. The rhythm of 5-hydroxytryptophan release during the first day of culture was found in the pineal organs of all embryos, while the rhythmic release of N-acetylserotonin and 5-methoxyindole acetic acid started at the age of 24 days. The proportion of released indoles changed with embryo age. NE caused a decrease in MLT secretion and provoked an increase in serotonin release. Incubation of the pineal organs induced the development of MLT secretory machinery and its diurnal rhythmicity. The pineal content of catecholamines increased prominently at the end of embryonic development.

Enhanced production of 5-hydroxytryptophan through the regulation of L-tryptophan biosynthetic pathway.

5-Hydroxytryptophan (5-HTP) is the precursor of the neurotransmitter serotonin and has been used for the treatment of various diseases such as depression, insomnia, chronic headaches, and binge eating associated obesity. The production of 5-HTP had been achieved in our previous report, by the development of a recombinant strain containing two plasmids for biosynthesis of L-tryptophan (L-trp) and subsequent hydroxylation. In this study, the L-trp biosynthetic pathway was further integrated into the E. coli genome, and the promoter strength of 3-deoxy-7-phosphoheptulonate synthase, which catalyzes the first step of L-trp biosynthesis, was engineered to increase the production of L-trp. Hence, the 5-HTP production could be manipulated by the regulation of copy number of L-trp hydroxylation plasmid. Finally, the 5-HTP production was increased to 1.61 g/L in the shaking flasks, which was 24% improvement comparing to the original producing strain, while the content of residual L-trp was successfully reduced from 1.66 to 0.2 g/L, which is beneficial for the downstream separation and purification. Our work shall promote feasible progresses for the industrial production of 5-HTP.

Bifidobacterium with the role of 5-hydroxytryptophan synthesis regulation alleviates the symptom of depression and related microbiota dysbiosis.

Depression disorder is rapidly advancing worldwide, and therapeutic strategy through gut-brain axis has been proven to be effective in the treatment. Here we studied the effect of lactic acid bacteria (LAB) treatment on depression. C57BL/6J mice were administered with LAB during a 5-week chronic unpredictable mild stress. Bifidobacterium longum subsp. infantis E41 and Bifidobacterium breve M2CF22M7, which improved the expression of Tph1 and secretion of 5-hydroxytryptophan (5-HTP) in RIN14B cells, significantly reduced depressive behaviors of mice in the forced swim test, sucrose preference test and step-down test, as well as increased the level of 5-hydroxytryptamine and brain-derived neurotrophic factor concentration in brain. Besides, M2CF22M7 reduced the serum corticosterone level. E41 increased cecal butyrate level, which significantly and positively correlated with some depression-related indexes. Using 16S rRNA-amplicon sequencing of faces, E41 and M2CF22M7 were found to improve the chronic-stress-induced microbial dysbiosis. They also normalized the host's pathways involving metabolism and gene information processing. These results indicate that Bifidobacterium E41 and M2CF22M7 have an antidepressant effect in mice partly in a 5-HTP dependent and microbiota-regulating manner. Nurturing the gut microbiota with these strains may become an emerging therapeutic way for mood disorder.

Metabolic pathway engineering for high-level production of 5-hydroxytryptophan in Escherichia coli.

Cellular metabolic networks should be carefully balanced using metabolic engineering to produce the desired products at the industrial scale. As the precursor for the biosynthesis of the neurotransmitter serotonin, 5-hydroxytryptophan (5-HTP) is effective in treating a variety of diseases, such as depression, fibromyalgia, obesity, and cerebellar ataxia. Due to the lack of an efficient synthetic method, commercial production of 5-HTP is only achieved by extracting from the seeds of Griffonia Smplicifolia. This study reports efficient microbial production of 5-HTP via metabolically engineered Escherichia coli. Firstly, human tryptophan hydroxylase I (TPH1) gene was functionally expressed. For endogenous supply of the cofactor tetrahydrobiopterin (BH4), human BH4 biosynthesis and regeneration pathway was reconstituted. Whole-cell bioconversion resulted in high-level production of 5-HTP (~1.2 g/L) from 2 g/L L-tryptophan in shake flasks. Further metabolic engineering efforts were employed to achieve 5-HTP biosynthesis from simple carbon sources. The whole biosynthetic pathway was divided into three functional modules, L-tryptophan module, the hydroxylation module, and the BH4 module. By reducing the copy number of L-tryptophan module, replacing TPH1 with a more stable mutant form, and promoter regulation of the BH4 module, 5-HTP was produced at a final titer of 1.3 g/L in the shake flask and 5.1 g/L in a fed-batch fermenter with glycerol as the carbon source, both of which were the highest ever reported for microbial production of 5-HTP.

Maternal Inflammation Disrupts Fetal Neurodevelopment via Increased Placental Output of Serotonin to the Fetal Brain.

UNLABELLED: Maternal inflammation during pregnancy affects placental function and is associated with increased risk of neurodevelopmental disorders in the offspring. The molecular mechanisms linking placental dysfunction to abnormal fetal neurodevelopment remain unclear. During typical development, serotonin (5-HT) synthesized in the placenta from maternal l-tryptophan (TRP) reaches the fetal brain. There, 5-HT modulates critical neurodevelopmental processes. We investigated the effects of maternal inflammation triggered in midpregnancy in mice by the immunostimulant polyriboinosinic-polyribocytidylic acid [poly(I:C)] on TRP metabolism in the placenta and its impact on fetal neurodevelopment. We show that a moderate maternal immune challenge upregulates placental TRP conversion rapidly to 5-HT through successively transient increases in substrate availability and TRP hydroxylase (TPH) enzymatic activity, leading to accumulation of exogenous 5-HT and blunting of endogenous 5-HT axonal outgrowth specifically within the fetal forebrain. The pharmacological inhibition of TPH activity blocked these effects. These results establish altered placental TRP conversion to 5-HT as a new mechanism by which maternal inflammation disrupts 5-HT-dependent neurogenic processes during fetal neurodevelopment. SIGNIFICANCE STATEMENT: The mechanisms linking maternal inflammation during pregnancy with increased risk of neurodevelopmental disorders in the offspring are poorly understood. In this study, we show that maternal inflammation in midpregnancy results in an upregulation of tryptophan conversion to serotonin (5-HT) within the placenta. Remarkably, this leads to exposure of the fetal forebrain to increased concentrations of this biogenic amine and to specific alterations of crucially important 5-HT-dependent neurogenic processes. More specifically, we found altered serotonergic axon growth resulting from increased 5-HT in the fetal forebrain. The data provide a new understanding of placental function playing a key role in fetal brain development and how this process is altered by adverse prenatal events such as maternal inflammation. The results uncover important future directions for understanding the early developmental origins of mental disorders.

Molecular basis of 5-hydroxytryptophan synthesis in Saccharomyces cerevisiae.

We report for the first time that 5-hydroxytryptophan can be synthesized in Saccharomyces cerevisiae by heterologously expressing prokaryotic phenylalanine 4-hydroxylase or eukaryotic tryptophan 3/5-hydroxylase, together with enhanced synthesis of MH4 or BH4 cofactors. The innate DFR1 gene in the folate synthesis pathway was found to play pivotal roles in 5-hydroxytryptophan synthesis.

Precursor-directed biosynthesis of 5-hydroxytryptophan using metabolically engineered E. coli.

A novel biosynthetic pathway was designed and verified reversely leading to the production of 5-hydroxytryptophan (5-HTP) from glucose. This pathway takes advantage of the relaxed substrate selectivities of relevant enzymes without employing the unstable tryptophan 5-hydroxylase. First, high-titer of 5-HTP was produced from 5-hydroxyanthranilate (5-HI) by the catalysis of E. coli TrpDCBA. Then, a novel salicylate 5-hydroxylase was used to convert the non-natural substrate anthranilate to 5-HI. After that, the production of 5-HI from glucose was achieved and optimized with modular optimization. In the end, we combined the full pathway and adopted a two-stage strategy to realize the de novo production of 5-HTP. This work demonstrated the application of enzyme promiscuity in non-natural pathway design.

Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan.

5-Hydroxytryptophan (5-HTP) is a drug that is clinically effective against depression, insomnia, obesity, chronic headaches, etc. It is only commercially produced by the extraction from the seeds of Griffonia simplicifolia because of a lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase activity in Escherichia coli. Then, sequence- and structure-based protein engineering dramatically shifted its substrate preference, allowing for efficient conversion of tryptophan to 5-HTP. Importantly, E. coli endogenous tetrahydromonapterin (MH4) could be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion allowed the high-level production of 5-HTP (1.1-1.2 g/L) from tryptophan in shake flasks. On this basis, metabolic engineering efforts were further made to achieve the de novo 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy for expanding the native metabolism of microorganisms.

Tetrahydrobiopterin biosynthesis as an off-target of sulfa drugs.

The introduction of sulfa drugs for the chemotherapy of bacterial infections in 1935 revolutionized medicine. Although their mechanism of action is understood, the molecular bases for most of their side effects remain obscure. Here, we report that sulfamethoxazole and other sulfa drugs interfere with tetrahydrobiopterin biosynthesis through inhibition of sepiapterin reductase. Crystal structures of sepiapterin reductase with bound sulfa drugs reveal how structurally diverse sulfa drugs achieve specific inhibition of the enzyme. The effect of sulfa drugs on tetrahydrobiopterin-dependent neurotransmitter biosynthesis in cell-based assays provides a rationale for some of their central nervous system-related side effects, particularly in high-dose sulfamethoxazole therapy of Pneumocystis pneumonia. Our findings reveal an unexpected aspect of the pharmacology of sulfa drugs and might translate into their improved medical use.

Tryptamine 5-hydroxylase-deficient Sekiguchi rice induces synthesis of 5-hydroxytryptophan and N-acetyltryptamine but decreases melatonin biosynthesis during senescence process of detached leaves.

Melatonin biosynthesis was examined in Sekiguchi mutant rice lacking functional tryptamine 5-hydroxylase (T5H) activity, which is the terminal enzyme for serotonin biosynthesis in rice. During senescence process, the leaves of Sekiguchi mutant rice produced more tryptamine and N-acetyltryptamine compared with the wild-type Asahi leaves. Even though T5H activity is absent, Sekiguchi leaves produce low levels of serotonin derived from 5-hydroxytryptophan, which was found to be synthesized during senescence process. Accordingly, both rice cultivars exhibited similar levels of N-acetylserotonin until 6 days of senescence induction; however, only Asahi leaves continued to accumulate N-acetylserotonin after 6 days. In contrast, a large amount of N-acetyltryptamine was accumulated in Sekiguchi leaves, indicating that tryptamine was efficiently utilized as substrate by the rice arylalkylamine N-acetyltransferase enzyme. An increase in N-acetyltryptamine in Sekiguchi had an inhibitory effect on synthesis of melatonin because little melatonin was produced in Sekiguchi leaves at 6 days of senescence induction, even in the presence of equivalent levels of N-acetylserotonin in both cultivars. The exogenous treatment of 0.1 mmN-acetyltryptamine during senescence process completely blocked melatonin synthesis.

Meal ingestion, amino acids and brain neurotransmitters: effects of dietary protein source on serotonin and catecholamine synthesis rates.

Carbohydrate ingestion raises tryptophan uptake and serotonin synthesis in rat brain. The addition of protein is generally believed only to block such increases. However, some recent evidence suggests dietary protein may not be limited to this action. In the present studies, we fed rats single meals containing one of 5 proteins (zein, wheat gluten, soy protein isolate, casein, lactalbumin, 17% by weight) or no protein, and killed them 2.5 h later, 30 min after the injection of m-hydroxybenzylhydrazine, to allow serotonin and catecholamine synthesis rates to be measured in brain. Blood and cerebral cortex samples were analyzed for tryptophan and other large, neutral amino acids; 5-hydroxytryptophan and dihydroxyphenylalanine were measured in hypothalamus, hippocampus and cerebral cortex as indices of serotonin and catecholamine synthesis, respectively. An 8-fold variation occurred in cortex tryptophan: a marked decline followed zein ingestion, and modest reductions after casein or gluten. A large rise in cortex tryptophan occurred after lactalbumin consumption, and smaller increases after soy protein or carbohydrate (no protein). In the brain regions examined, a 4-8-fold range in serotonin synthesis occurred which closely followed the tryptophan alterations. No effects were observed in regional catecholamine synthesis rates. Cortical concentrations of leucine showed small changes; leucine has been linked to mTOR (mammalian target of rapamycin) signaling in brain circuits regulating food intake. The data suggest that tryptophan concentrations and serotonin synthesis in brain neurons are remarkably sensitive to which protein is present in a meal. Conceivably, this relationship might inform the brain about the nutritional quality of the protein ingested.

Chronic treatment and withdrawal of the cannabinoid agonist WIN 55,212-2 modulate the sensitivity of presynaptic receptors involved in the regulation of monoamine syntheses in rat brain.

Brain monoamines are involved in many neurochemical and behavioral effects of cannabinoids, but little is known on the regulation of noradrenaline, dopamine, and serotonin (5-HT) synthesis in cannabinoid addiction. This study investigated in rat brain the chronic effects of the potent cannabinoid agonist WIN 55,212-2 and of rimonabant-precipitated withdrawal, as well as the sensitivity of synthesis-modulating inhibitory receptors, on the accumulation of L-3,4-dihydroxyphenylalanine (DOPA) and 5-HTP after decarboxylase inhibition. Acute WIN (8 mg/kg; 1 h) increased DOPA synthesis in cortex (52%), hippocampus (51%), and cerebellum (56%) and decreased DOPA accumulation in striatum (31%). Acute WIN also decreased the synthesis of 5-HTP in all brain regions (40-53%). Chronic WIN (2-8 mg/kg; 5 days) and/or antagonist-precipitated withdrawal induced tolerance to the acute effects of WIN on the accumulation of DOPA (cortex and striatum) and 5-HTP (all brain regions). The inhibitory effect of clonidine (alpha2-agonist; 1 mg/kg) on the accumulation of DOPA (15-41%) and 5-HTP (22-41%) was markedly decreased or abolished after chronic WIN and precipitated withdrawal, mainly in noradrenergic and serotonergic brain regions, which indicated desensitization of alpha2-autoreceptors and alpha2-heteroreceptors regulating the synthesis of noradrenaline and 5-HT. In WIN-dependent rats (chronic and withdrawal states), the effect of a low dose of (+/-)-8-hydroxy-2-(di-n-propylamino)-tetralin (5-HT1A agonist; 0.1 mg/kg) on the accumulation of precursor amino acids was markedly potentiated in cerebellum and striatum, indicating the induction of supersensitivity of 5-HT1A-autoreceptors and 5-HT1A-heteroreceptors that regulate the synthesis of 5-HT, noradrenaline, and dopamine in these brain regions. These chronic adaptations in presynaptic receptor function could play a relevant role in cannabinoid addiction.

Glucocorticoid modulation of tryptophan hydroxylase-2 protein in raphe nuclei and 5-hydroxytryptophan concentrations in frontal cortex of C57/Bl6 mice.

Considerable attention has focused on regulation of central tryptophan hydroxylase (TPH) activity and protein expression. At the time of these earlier studies, it was thought that there was a single central TPH isoform. However, with the recent identification of TPH2, it becomes important to distinguish between regulatory effects on the protein expression and activity of the two isoforms. We have generated a TPH2-specific polyclonal antiserum (TPH2-6361) to study regulation of TPH2 at the protein level and to examine the distribution of TPH2 expression in rodent and human brain. TPH2 immunoreactivity (IR) was detected throughout the raphe nuclei, in lateral hypothalamic nuclei and in the pineal body of rodent and human brain. In addition, a prominent TPH2-IR fiber network was found in the human median eminence. We recently reported that glucocorticoid treatment of C57/Bl6 mice for 4 days markedly decreased TPH2 messenger RNA levels in the raphe nuclei, whereas TPH1 mRNA was unaffected. The glucocorticoid-elicited inhibition of TPH2 gene expression was blocked by co-administration of the glucocorticoid receptor antagonist mifepristone (RU-486). Using TPH2-6361, we have extended these findings to show a dose-dependent decrease in raphe TPH2 protein levels in response to 4 days of treatment with dexamethasone; this effect was blocked by co-administration of mifepristone. Moreover, the glucocorticoid-elicited inhibition of TPH2 was functionally significant: serotonin synthesis was significantly reduced in the frontal cortex of glucocorticoid-treated mice, an effect that was blocked by mifepristone co-administration. This study provides further evidence for the glucocorticoid regulation of serotonin biosynthesis via inhibition of TPH2 expression, and suggest that elevated glucocorticoid levels may be relevant to the etiology of psychiatric diseases, such as depression, where hypothalamic-pituitary-adrenal axis dysregulation has been documented.

Effect of the beta3 adrenoceptor agonist CL 316243 on hypothalamic 5-HT synthesis and suppression of REM sleep in the rat.

Beta3 adrenoceptor agonists show an antidepressant-like profile in preclinical rodent assays and improve mood in clinically-obese patients. These observations suggest a possible antidepressant utility for beta3 adrenoceptor agonists. The present study examined the effects of acute and chronic administration of the beta3 adrenoceptor agonist CL 316243 on two physiological indicators of antidepressant activity in the rat: hypothalamic 5-HT synthesis and suppression of REM sleep. 5-HT synthesis was estimated by the accumulation of 5-hydroxytryptophan (5-HTP) after treatment with the L-aromatic acid decarboxylase inhibitor NSD 1015. Sleep-wake patterns were monitored using electroencephalogram and electromyogram signals collected by radiotelemetry. Rats were administered CL 316243 acutely or once daily for 11 days. Acute administration of CL 316243 significantly increased hypothalamic 5-HT synthesis, as indicated by increased levels of 5-HTP, and reduced the amount of REM sleep. However, chronic administration produced no changes in 5-HTP or REM compared with vehicle treatment. The present observations suggest that acute administration of CL 316243 causes antidepressant-like effects on REM sleep, possibly mediated by increased central 5-HT synthesis. However, these effects are not maintained with repeated dosing.

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.

Withdrawal from chronic ethanol increases the sensitivity of presynaptic 5-HT(1A) receptors modulating serotonin and dopamine synthesis in rat brain in vivo.

The in vivo sensitivity of presynaptic 5-HT(1A) receptors (autoreceptors and heteroreceptors) modulating the synthesis of 5-hydroxytryptophan/serotonin (5-HTP/5-HT) and 3,4-dihydroxyphenylalanine/dopamine (DOPA/DA) in rat brain was investigated after ethanol treatment and withdrawal. In saline-treated rats as well as in acute ethanol (2 g/kg, intraperitoneally (i.p.), 2 h)- and chronic ethanol (2 g/kg for 7 days)-treated rats, a low dose of the 5-HT(1A) receptor agonist 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT; 0.1 mg/kg, i.p., 1 h) did not decrease the synthesis of 5-HTP in brain (except modestly in striatum; 20% after the chronic treatment) or that of DOPA in striatum. In contrast, in chronic ethanol-withdrawn rats (24 h), 8-OH-DPAT significantly decreased the synthesis of 5-HTP in the hippocampus (29%), cerebral cortex (41%) and striatum (33%) and that of DOPA in the striatum (28%). Similar effects were induced by the mixed 5-HT(1A) agonist/D(2) antagonist buspirone (1 mg/kg, i.p., 1 h) which also decreased 5-HTP synthesis in the hippocampus (24%), cerebral cortex (36%) and striatum (35%) of chronic ethanol-withdrawn rats. These results indicate that chronic ethanol and more clearly the spontaneous withdrawal from chronic ethanol induce supersensitivity of 5-HT(1A)-auto/heteroreceptors modulating the synthesis of 5-HT and DA in rat brain.

Supersensitivity of 5-HT1A autoreceptors and alpha2-adrenoceptors regulating monoamine synthesis in the brain of morphine-dependent rats.

The sensitivity of 5-HT1A serotonin receptors and alpha2-adrenoceptors (autoreceptors and heteroreceptors) modulating brain monoamine synthesis was investigated in rats during morphine treatment and after naloxone-precipitated withdrawal. The accumulation of 5-hydroxytryptophan (5-HTP) and 3,4-dihydroxyphenylalanine (DOPA) after decarboxylase inhibition was used as a measure of the rate of tryptophan and tyrosine hydroxylation in vivo. Acute morphine (3-100 mg/kg, 1 h) increased the synthesis of 5-HTP/5-HT in various brain regions (15%-35%) and that of DOPA/dopamine (DA) in striatum (28%-63%), but decreased the synthesis of DOPA/noradrenaline (NA) in hippocampus and cortex (20%-33%). Naloxone (2-60 mg/kg, 1 h) did not alter the synthesis of 5-HTP or DOPA in brain. Tolerance to the inhibitory effect of morphine on DOPA/NA synthesis and a sensitization to its stimulatory effects on DOPA/DA and 5-HTP/5-HT synthesis were observed after chronic morphine and/or in morphine-withdrawn rats. In morphine-dependent rats (tolerant and withdrawn states) the inhibitory effects of the 5-HT1A agonists 8-OH-DPAT and buspirone (0.1 mg/kg, 1 h), and that of the alpha2-adrenoceptor agonist clonidine (0.1 mg/kg, 1 h), on the synthesis of 5-HTP/5-HT were potentiated (25%-50%). Moreover, the effect of 8-OH-DPAT was antagonized by WAY 100135, a selective 5-HT1A antagonist. In morphine-dependent rats (tolerant state), the inhibitory effects of clonidine on the synthesis of DOPA/NA (hippocampus, hypothalamus) and DOPA/DA (striatum) also were potentiated (35%-55%). In summary, we conclude that morphine addiction is associated with supersensitivity of 5-HT1A serotonin receptors and alpha2-adrenoceptors (autoreceptors and heteroreceptors) that modulate the synthesis of monoamines in brain.

Effects of imidazoline receptor ligands on monoamine synthesis in the rat brain in vivo.

This study was designed to assess the effects of imidazoline drugs on putative presynaptic imidazoline receptors modulating brain monoamine synthesis in vivo. The accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used as a measure of the rate of tyrosine and tryptophan hydroxylation in various brain regions of naive rats and after irreversible alpha2-adrenoceptor inactivation with EEDQ (1.6 mg/kg, i.p., 6 h). Clonidine (1-3 mg/kg), moxonidine (1-10 mg/kg) and rilmenidine (10 mg/kg) (mixed I1/alpha2 agonists) decreased dopa and 5-HTP synthesis in the cerebral cortex (14%-81%), hippocampus (27%-84%) and/or striatum (29%-56%), but these inhibitory effects were abolished in N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)-treated rats. Similarly, the stimulatory effect of efaroxan (mixed I1/alpha2 antagonist; 10 mg/kg) on dopa synthesis in the cortex (77%) and hippocampus (57%) was abolished by EEDQ. The selective I1-ligand 2-endo-amino-3-exoisopropylbicyclo-heptane (AGN-192403; 5-10 mg/kg) did not modify dopa or 5-HTP synthesis in any brain region in naive or EEDQ-treated rats. Idazoxan (mixed I2/alpha2 antagonist; 20 mg/kg) increased dopa synthesis in the cortex (111%) and hippocampus (87%), but the stimulatory effects were abolished by EEDQ. Moreover, idazoxan and efaroxan decreased 5-HTP synthesis in the cortex (12%-34%) and hippocampus (30%-34%) in a manner sensitive to blockade by the 5-HT1A receptor antagonist WAY 100135. The selective I2-igands 2-(2-benzofuranyl)-2-imidazoline (2-BFI; 20 mg/kg) and 2-styryl-2-imidazoline (LSL 61122; 10 mg/kg) did not alter the synthesis of dopa or 5-HTP in the cortex or hippocampus. In striatum, 2-BFI (1-20 mg/kg) dose-dependently decreased dopa synthesis (ED50: 5.9 mg/kg), reduced dopamine levels (6%-36%) and increased those of its metabolites DOPAC (15%-95%) and HVA (24%-74%). The inhibitory effect of 2-BFI on dopa/dopamine synthesis in striatum remained unchanged after alkylation of imidazoline receptors with isothiocyanatobenzyl imidazoline (IBI; 60 mg/kg, 6 h) or blockade of these receptors with 2-(2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole (KU-14R; 7-20 mg/kg). Therefore, most imidazoline drugs modulated the synthesis of brain monoamines through interaction with alpha2-adrenoceptors or 5-HT1A receptors. The results do not provide functional evidence for the existence of presynaptic imidazoline receptors regulating the synthesis of monoamines in the rat brain.

Brain tryptophan concentrations and serotonin synthesis remain responsive to food consumption after the ingestion of sequential meals.

The response of brain tryptophan concentration and serotonin synthesis to the ingestion of two sequential meals was examined in rats. Fasted rats ingested a carbohydrate meal followed 2 h later by a protein-containing meal and were examined 2 or 4 h after the first meal. Other rats ingested a protein meal first, followed by a carbohydrate meal. When the carbohydrate meal was fed first, brain tryptophan concentrations and serotonin synthesis increased at 2 h; these changes were reversed at 4 h if the second meal contained protein. When the protein meal was fed first, there were no changes in brain tryptophan or serotonin at 2 h, and a second carbohydrate meal at 2 h did not raise brain tryptophan or serotonin 2 h later. Carbohydrate ingestion 3 h after a protein meal, however, did raise brain tryptophan and serotonin 2 h later. Brain tryptophan concentrations and serotonin synthesis are thus responsive to the sequential ingestion of protein and carbohydrate meals if there is a sufficient interval between meals.

Promotion of sleep by prostaglandin D2 in rats made insomniac by pretreatment with para-chlorophenylalanine.

The correlation between the somnogenic effect of prostaglandin (PG) D2 and the serotoninergic system was examined in freely-moving rats (n = 64) by use of a continuous infusion method. Rats pretreated with para-chlorophenylalanine (PCPA: 450 mg/kg body weight, i.p.) or non-PCPA-pretreated rats received infusion of PGD2, serotonin, or its direct precursor, 5-hydroxytryptophan (5HTP), into their third cerebral ventricle at a rate of 100 pmol/0.2 microliter/min between 11:00 and 17:00 h. In the PCPA-pretreated insomniac rats, PGD2 infusion resulted in an immediate increase in slow-wave sleep (SWS) and an increase with a 2-h latency in paradoxical sleep (PS). The total amounts of SWS and PS during the PGD2-infusion period were 151% and 154% of the respective control values. These results indicate that inhibition of the biosynthesis of serotonin and 5HTP by PCPA marginally affects the sleep-promoting effect of PGD2. The transient sleep restoration produced by 5HTP infusion into PCPA-pretreated rats was hardly affected by the simultaneous infusion (200 pmol/0.2 microliter/min; 07:00-17:00 h) of diclofenac sodium, an inhibitor of cyclo-oxygenase, suggesting that PGD2 production is not critically involved in the sleep restoration by 5HTP. The sleep-promoting property of PGD2 is thus probably independent of the serotoninergic modulation of sleep-wake activity.