Tryptophan Hydroxylase mRNA Research Articles

Key Enzymes of the Serotonergic System - Tryptophan Hydroxylase2 and Monoamine OxidaseA - In the Brain of Rats Selectively Bred for a Reaction toward Humans: Effects of Benzopentathiepin TC-2153.

At the Institute of Cytology and Genetics (Novosibirsk, Russia) for over 85 generations, gray rats have been selected for high aggression toward humans (aggressive rats) or its complete absence (tame rats). Aggressive rats are an interesting model for studying fear-induced aggression. Benzopentathiepin TC-2153 exerts an antiaggressive effect on aggressive rats and affects the serotonergic system: an important regulator of aggression. Theaim of this study was to investigate effects of TC-2153 on key serotonergic-system enzymes- tryptophan hydroxylase2 (TPH2) and monoamine oxidaseA (MAOA)- in the brain of aggressive and tame rats. Either TC-2153 (10 or 20mg/kg) or vehicle was administered once intraperitoneally to aggressive and tame male rats. TPH2 and MAOA enzymatic activities and mRNA and protein levels were assessed. The selection for high aggression resulted in upregulation of Tph2 mRNA in the midbrain, of the TPH2 protein in the hippocampus, and of proteins TPH2 and MAOA in the hypothalamus, as compared to tame rats. MAO enzymatic activity was higher in the midbrain and hippocampus of aggressive rats while TPH2 activity did not differ between the strains. The single TC-2153 administration decreased TPH2 and MAO activity in the hypothalamus and midbrain, respectively. The drug affected MAOA protein levels in the hypothalamus: upregulated them in aggressive rats and downregulated them intame ones. Thus, this study shows profound differences in the expression and activity of key serotonergic system enzymes in the brain of rats selectively bred for either highly aggressive behavior toward humans or its absence, and the effects of benzopentathiepin TC-2153 on these enzymes may point to mechanisms of its antiaggressive action.

Glutamine increases stability of TPH1 mRNA via p38 mitogen-activated kinase in mouse mastocytoma cells

Expression changes for tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin synthesis, by environmental glutamine (GLN) were examined in mouse mastocytoma-derived P815-HTR cells. GLN-treated cells exhibited a robust increase in TPH1 mRNA after a 6 h exposure to GLN. 6-Diazo-5-oxo-L-norleucine (DON), a glutamine-utilizing glutaminase inhibitor, significantly inhibited the GLN-induction of TPH1 mRNA. Nuclear run-on assays and mRNA decay experiments demonstrated that the primary mechanism leading to increased TPH1 mRNA levels was not due to transcriptional changes, but rather due to increased TPH1 RNA stability induced by GLN. Treatment with GLN also led to activation of p38 MAP kinase, but not p42/44 MAPK. In addition, SB203580, a p38 MAP kinase specific inhibitor, completely abolished the GLN-mediated increase of TPH1 mRNA levels, suggesting the pathway stabilizing TPH1 mRNA might be mediated by the activated p38 MAP kinase pathway. Additionally, SB203580 significantly reduced the stability of TPH1 mRNA, and this reduction of the stability was not affected by GLN in the culture medium, implying a sequential signaling from GLN being mediated by p38 MAP kinase, resulting in alteration of TPH1 mRNA stability. TPH1 mRNA stability loss was also dependent on de novo protein synthesis as shown by treatment of cells with a transcriptional/translational blocker. We provide evidence that TPH1 mRNA levels are increased in response to increased exogenous GLN in mouse mastocytoma cells via a stabilization of TPH1 mRNA due to the activity of the p38 MAP kinase.

Epididymal Fat-Derived Sympathoexcitatory Signals Exacerbate Neurogenic Hypertension in Obese Male Mice Exposed to Early Life Stress.

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Serotonin in the Pathogenesis of Lymphocytic Colitis.

Lymphocytic colitis (LC) is a chronic inflammatory disease associated with watery diarrhea, abdominal pain, and colonic intraepithelial lymphocytosis. Serotonin (5-hydroxytryptamine, 5-HT) is reported to increase in certain colon diseases; however, little is known regarding its metabolism in LC. In the present work, the level of 5-HT in serum and the number of enteroendocrine cells (EECs) as well as the expression of the 5-HT rate-limiting enzyme tryptophan hydroxylase 1 (TPH1) in colonic biopsies and urine 5-hydroxyindoeoacetic acid (5-HIAA) were determined in 36 LC patients that were treated with budesonide and 32 healthy controls. The 5-HT serum and 5-HIAA urine levels were measured using ELISA, the EEC number was determined immunohistochemically, and the colonic TPH1 mRNA expression was determined using RT-PCR. The levels of 5-HT and 5-HIAA and the number of EECs were higher in LC patients than in the controls, and positive correlations were observed between the 5-HT and 5-HIAA levels, 5-HT and EEC number, TPH1 mRNA and EEC number, as well as the severity of disease symptoms and 5-HIAA. Budesonide decreased the levels of 5-HT, 5-HIAA, and TPH1 expression and the number of EECs to values that did not differ from those for controls. In conclusion, the serotonin metabolism may be important for LC pathogenesis, and the urinary level of 5-HIAA may be considered as a non-invasive marker of this disease activity.

Pomegranate derivative urolithin A enhances vitamin D receptor signaling to amplify serotonin-related gene induction by 1,25-dihydroxyvitamin D

Mediated by the nuclear vitamin D receptor (VDR), the hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25D), is known to regulate expression of genes impacting calcium and phosphorus metabolism, the immune system, and behavior. Urolithin A, a nutrient metabolite derived from pomegranate, possibly acting through AMP kinase (AMPK) signaling, supports respiratory muscle health in rodents and longevity in C. elegans by inducing oxidative damage-reversing genes and mitophagy. We show herein that urolithin A enhances transcriptional actions of 1,25D driven by co-transfected vitamin D responsive elements (VDREs), and dissection of this genomic effect in cell culture reveals: 1) urolithin A concentration-dependency, 2) occurrence with isolated natural VDREs, 3) nuclear receptor selectivity for VDR over ER, LXR and RXR, and 4) significant 3- to 13-fold urolithin A-augmentation of 1,25D-dependent mRNA encoding the widely expressed 1,25D-detoxification enzyme, CYP24A1, a benchmark vitamin D target gene. Relevant to potential behavioral effects of vitamin D, urolithin A elicits enhancement of 1,25D-dependent mRNA encoding tryptophan hydroxylase-2 (TPH2), the serotonergic neuron-expressed initial enzyme in tryptophan metabolism to serotonin. Employing quantitative real time-PCR, we demonstrate that TPH2 mRNA is induced 1.9-fold by 10 nM 1,25D treatment in culture of differentiated rat serotonergic raphe (RN46A-B14) cells, an effect magnified 2.5-fold via supplementation with 10 μM urolithin A. This potentiation of 1,25D-induced TPH2 mRNA by urolithin A is followed by a 3.1- to 3.7-fold increase in serotonin concentration in culture medium from the pertinent neuronal cell line, RN46A-B14. These results are consistent with the concept that two natural nutrient metabolites, urolithin A from pomegranate and 1,25D from sunlight/vitamin D, likely acting via AMPK and VDR, respectively, cooperate mechanistically to effect VDRE-mediated regulation of gene expression in neuroendocrine cells. Finally, gedunin, a neuroprotective natural product from Indian neem tree that impacts the brain derived neurotropic factor pathway, similarly potentiates 1,25D/VDR-action.

Photoperiod regulates the daily profiles of tryptophan hydroxylase-2 gene expression the raphe nuclei of rats

ِAim Tryptophan hydroxylase-2 mRNA (TPH, the rate limiting enzyme in 5-HT synthesis) expression levels display circadian variations in the median and dorsal raphe nuclei. This circadian pattern is under the control of the suprachiasmatic nuclei (SCN), the master clock. Photoperiodic cue is encoded by the SCN which convey the seasonal message to target sites. In the present study, we have investigated the effect of photoperiodic changes on the serotonergic neurons of the raphe nuclei. We have assessed the daily expression of TPH2 mRNA in both median and dorsal raphe nuclei of rats housed either under long photoperiod (18 h light/6 h dark cycle, LP18:6) or short photoperiod (SP6:18). ٌ Results Our results demonstrate that under LP18:6, TPH2 mRNA levels display a progressive decrease during the dark period and a maximal expression is reported at the beginning of the light period. The expression pattern of TPH2 mRNA under SP6:18 remains unchanged during the dark period and increases significantly before the light/dark transition. This latter expression pattern is in line with the daily profiles of TPH2 mRNA reported previously under standard lighting regimen (12 h light/12 h dark cycle). Conclusion The present results suggest that TPH2 mRNA expression pattern within DR and MR is affected by photoperiod which might in turn affect TPH content and 5‐HT release within the circadian structures, but also in all the serotonergic projection areas of the brain.

Photoperiod regulates the daily profiles of Tryptophan Hydroxylase-2 gene expression the raphe nuclei of rats

Tryptophan hydroxylase-2 mRNA (TPH, the rate limiting enzyme in 5-HT synthesis) expression levels display circadian variations in the median and dorsal raphe nuclei. This circadian pattern is under the control of the suprachiasmatic nuclei (SCN), the master clock. Photoperiodic cue is encoded by the SCN which convey the seasonal message to target sites. In the present study, we have investigated the effect of photoperiodic changes on the serotonergic neurones of the raphe nuclei. We have assessed the daily expression of TPH2 mRNA in both median and dorsal raphe nuclei of rats housed either under long photoperiod (18 h light/6 h dark cycle, LP18:6) or short photoperiod (SP6:18). Our results demonstrate that under LP18:6, TPH2 mRNA levels display a progressive decrease during the dark period and a maximal expression is reported at the beginning of the light period. The expression pattern of TPH2 mRNA under SP6:18 remains unchanged during the dark period and increases significantly before the day/night transition. This latter expression pattern is in line with the daily profiles of TPH2 mRNA reported previously under standard lighting regimen (12 h light/12 h dark cycle). The present results suggest that TPH2 mRNA expression pattern within DR and MR is affected by photoperiod which might in turn affect TPH content and 5­HT release within the circadian structures, but also in all the serotonergic projection areas of the brain.

Effect of the Tryptophan Hydroxylase Inhibitor Telotristat on Growth and Serotonin Secretion in 2D and 3D Cultured Pancreatic Neuroendocrine Tumor Cells

Serotonin, a biologically active amine, is related to carcinoid syndrome in functioning neuroendocrine tumors (NETs). Telotristat ethyl is a novel inhibitor of the tryptophan hydroxylase (TPH), a key enzyme in the production of serotonin. While its use in patients with carcinoid syndrome and uncontrolled diarrhea under somatostatin analogs (SSAs) has been recently approved, in vitro data evaluating its effectiveness are lacking. For this reason, we aimed to evaluate the effect of telotristat as monotherapy, and in combination with SSAs, on proliferation and secretion in a NET cell line model. The human pancreatic NET cell lines BON-1/QGP-1 were used as 2D and 3D cultured models; somatostatin receptor and TPH mRNA expression, as well as the potential autocrine effect of serotonin on tumor cell proliferation using a 3D culture system were evaluated. Telotristat decreased serotonin production in a dose-dependent manner at a clinically feasible concentration, without affecting cell proliferation. Its combination with pasireotide, but not with octreotide, had an additive inhibitory effect on serotonin secretion. The effect of telotristat was slightly less potent, when BON-1 cells were co-treated with octreotide. Octreotide and pasireotide had no effect on the expression of TPH. Telotristat did not have an effect on mRNA expression of somatostatin receptor subtypes. Finally, we showed that serotonin did not have an autocrine effect on NET cell proliferation on the 3D cell model. These results suggest that telotristat is an effective drug for serotonin inhibition, but the effectiveness of its combination with SST₂ (somatostatin receptor subtype 2)-preferring SSA should be evaluated in more detail.

Effect of Nyoshinsan on decreased voluntary activity of OVX mice

Nyoshinsan is one of the kampo medicine used in the treatment for menopausal disorder. We have already reported that ovariectomized (OVX) rats were seen in the decreases of voluntary activity at the dark time and serotonin level in amygdala (Behav. Brain. Res. 227(1)1-6(2012)). In this study, we examined effects of Nyosihnsan on a voluntary activity in OVX mice. The female ICR mice of 9-week old were OVX or sham operation. Either Nyoshinsan (750 mg/kg/day, p.o.) or water were administered to each group for 8 weeks (6 times/week) starting from 8 weeks after OVX. The voluntary activity of mice was evaluated by using an activity sensor (model NS-AS01 neuroscience, Inc.) at dark time (19:00-7:00). Moreover, RNA expression level of tryptophan hydroxylase (TPH) was measured in hippocampus and prefrontal cortex by Real-time PCR. In the result, Nyoshinsan significantly prevented decreased voluntary activity at dark periods induced by OVX. In addition Nyoshinsan suppressed down-regulation of TPH mRNA expression level induced by OVX. These results revealed that these OVX induced despair-like behaviors were improved by administration of Nyoshinsan. Moreover, it was also thought that the voluntary activity mediated by serotonin level in hippocampus.

Optimal vitamin D spurs serotonin: 1,25-dihydroxyvitamin D represses serotonin reuptake transport (SERT) and degradation (MAO-A) gene expression in cultured rat serotonergic neuronal cell lines

BackgroundDiminished brain levels of two neurohormones, 5-hydroxytryptamine (5-HT; serotonin) and 1,25-dihydroxyvitamin D3 (1,25D; active vitamin D metabolite), are proposed to play a role in the atypical social behaviors associated with psychological conditions including autism spectrum disorders and depression. We reported previously that 1,25D induces expression of tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway to 5-HT, in cultured rat serotonergic neuronal cells. However, other enzymes and transporters in the pathway of tryptophan metabolism had yet to be examined with respect to the actions of vitamin D. Herein, we probed the response of neuronal cells to 1,25D by quantifying mRNA expression of serotonin synthesis isozymes, TPH1 and TPH2, as well as expression of the serotonin reuptake transporter (SERT), and the enzyme responsible for serotonin catabolism, monoamine oxidase-A (MAO-A). We also assessed the direct production of serotonin in cell culture in response to 1,25D.ResultsEmploying quantitative real-time PCR, we demonstrate that TPH-1/-2 mRNAs are 28- to 33-fold induced by 10 nM 1,25D treatment of cultured rat serotonergic neuronal cells (RN46A-B14), and the enhancement of TPH2 mRNA by 1,25D is dependent on the degree of neuron-like character of the cells. In contrast, examination of SERT, the gene product of which is a target for the SSRI-class of antidepressants, and MAO-A, which encodes the predominant catabolic enzyme in the serotonin pathway, reveals that their mRNAs are 51–59% repressed by 10 nM 1,25D treatment of RN46A-B14 cells. Finally, serotonin concentrations are significantly enhanced (2.9-fold) by 10 nM 1,25D in this system.ConclusionsThese results are consistent with the concept that vitamin D maintains extracellular fluid serotonin concentrations in the brain, thereby offering an explanation for how vitamin D could influence the trajectory and development of neuropsychiatric disorders. Given the profile of gene regulation in cultured RN46A-B14 serotonergic neurons, we conclude that 1,25D acts not only to induce serotonin synthesis, but also functions at an indirect, molecular-genomic stage to mimic SSRIs and MAO inhibitors, likely elevating serotonin in the CNS. These data suggest that optimal vitamin D status may contribute to improving behavioral pathophysiologies resulting from dysregulation of serotonergic neurotransmission.

Improvement effects of a mixed extract of flowers of Pueraria thomsonii Benth. and peels of Citrus unshiu Markovich on postmenopausal symptoms of ovariectomized mice

BackgroundMenopausal hot flushes occur frequently in postmenopausal women. In the present study, we investigated a regulatory effect of a mixed extract of flowers of Pueraria thomsonii Benth. and peels of Citrus unshiu Markovich (PCE17), an extract of flowers of Pueraria thomsonii Benth. (PE), an extract of peels of Citrus unshiu Markovich (CE), a mixture of tectorigenin 7-O-xylosylglucoside, tectoridin, and tectorigenin (Tec, the active compounds of PE), and hesperidin (Hes, an active compound of CE) on menopausal hot flushes. MethodsWe examined the anti-hot flushes properties of PCE17, PE, CE, Tec, or Hes using a mouse model of ovariectomy-induced hot flushes. ResultsThe ovariectomy-induced rise in the tail skin temperature was significantly prevented by PCE17, PE, CE, Tec, or Hes. PCE17, PE, CE, Tec, or Hes significantly enhanced 5-HT levels and attenuated RANKL levels in the hypothalamus of ovariectomized (OVX) mice. Treatment with PCE17, PE, CE, Tec, or Hes significantly enhanced the levels of estrogen receptor (ER)-β, 5-HT1A, 5-HT2A, and tryptophan hydroxylase mRNA expression in the hypothalamus of OVX mice. PCE17, PE, or CE decreased follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, but did not increase estrogen levels in the serum of OVX mice. Tec or Hes decreased FSH or LH levels and increased estrogen levels. Treatment with PCE17, PE, CE, or Tec ameliorated vaginal atrophy in OVX mice. Finally, PCE17, PE, CE, Tec, or Hes significantly increased norepinephrine and dopamine levels in the hypothalamus of OVX mice. ConclusionThus, these results imply that PCE17 has protective effects against hot flushes.

Vitamin D Stimulates Serotonin Production via Induction of the Tryptophan Hydroxylase 2 Isoform in B14 Rat Medullary Neurons

The active form of vitamin D3, 1,25‐dihydroxyvitamin D (1,25D), and serotonin (5‐HT) are postulated to play significant roles in abnormal social behavior associated with psychological conditions including autism spectrum disorders (ASDs) and depression. Moreover, we have observed that 1,25D regulates synthesis of two isoforms of tryptophan hydroxylase (TPH), the rate‐limiting enzyme in the 5‐HT biosynthetic pathway, including induction of the tryptophan hydroxylase 2 (TPH2) gene in the central nervous system (CNS) and suppression of tryptophan hydroxylase 1 (TPH1) expression in some peripheral tissues. Interestingly, major characteristics associated with autism may include low concentrations of serotonin in the CNS and elevated levels of serotonin in the periphery. Thus, in addition to assessing the effect of 1,25D on TPH gene expression, we also examined, for the first time, the ability of 1,25D to stimulate production of 5‐HT in neuronal cells. Rat serotonergic medullary cells (RN46A‐B14; B‐14) were cultured to confluency and plated in six‐well plates. After allowing cells to attach in the plates overnight, the cells were cultured in differentiation medium, followed by treatment of differentiated B‐14 cells with ethanol vehicle or 10−8 M 1,25D. At time zero, a 200 μL sample of media from each well was collected to serve as a baseline for 5‐HT concentration. The cells were then incubated over the next three days, and additional 200 μL samples were collected from each well at the 24‐, 48‐ and 72‐hour time points. A 5‐HT ELISA assay was then conducted using the collected samples. The results of the ELISA revealed a statistically significant increase (57%, p < 0.05) in 5‐HT production following treatment with 1,25D for 24 hours versus vehicle, with subsequently larger percent‐increases in 5‐HT synthesis observed at 48 and 72 hours (64% and 103%, respectively, p < 0.05). These results suggest that 1,25D stimulates the production of serotonin, likely through the induction of the neuronal TPH2 gene, and the time‐course of this effect appears to lag behind the induction of TPH mRNA which peaks at 24 hours. In summary, this study illuminates a novel coordinated biochemical and regulatory interaction between two neuromodulators, vitamin D and serotonin, thereby identifying a potential connection between serum vitamin D levels and the occurrence of psychiatric disorders commonly linked to an imbalance in CNS serotonin production, including autism and depression.Support or Funding InformationThis project was funded by the New College of Interdisciplinary Arts and Sciences at Arizona State University and the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exposure to a High-Fat Diet during Early Development Programs Behavior and Impairs the Central Serotonergic System in Juvenile Non-Human Primates.

Perinatal exposure to maternal obesity and high-fat diet (HFD) consumption not only poses metabolic risks to offspring but also impacts brain development and mental health. Using a non-human primate model, we observed a persistent increase in anxiety in juvenile offspring exposed to a maternal HFD. Postweaning HFD consumption also increased anxiety and independently increased stereotypic behaviors. These behavioral changes were associated with modified cortisol stress response and impairments in the development of the central serotonin synthesis, with altered tryptophan hydroxylase-2 mRNA expression in the dorsal and median raphe. Postweaning HFD consumption decreased serotonergic immunoreactivity in area 10 of the prefrontal cortex. These results suggest that perinatal exposure to HFD consumption programs development of the brain and endocrine system, leading to behavioral impairments associated with mental health and neurodevelopmental disorders. Also, an early nutritional intervention (consumption of the control diet at weaning) was not sufficient to ameliorate many of the behavioral changes, such as increased anxiety, that were induced by maternal HFD consumption. Given the level of dietary fat consumption and maternal obesity in developed nations these findings have important implications for the mental health of future generations.

Effect of ketamine on expression of tryptophan hydroxylase 2 in median raphe nuclei of mentally depressed mice

Objective To evaluate the effect of ketamine on the expression of tryptophan hydroxylase 2 (TPH2) in the median raphe nuclei of mentally depressed mice. Methods Thirty-six healthy SPF male C57BL/6J mice, aged 8-12 weeks, weighing 20-26 g, were divided into 3 groups (n=12 each) using a random number table: control group (C group), depression group (D group) and depression plus ketamine group (D+ K group). Mental depression was induced by forcing the animals to swim in a narrow cylinder from which they can not escape.Ketamine 15 mg/kg was intraperitoneally injected once a day for 7 consecutive days starting from 1 day after successful establishment of the model in group D+ K.The equal volume of normal saline was given instead of ketamine in C and D groups.Forced swimming test was performed again at 30 min after the last administration, and the immobility time was recorded.Open field test was also performed at 30 min after the last administration, and the total horizontal distance and the number of standing on the back legs were recorded.The mice were sacrificed after the end of the behavioral testing, and the hippocampi and median raphe nuclei were isolated.High-performance liquid chromatography-electrochemical detection assay was used to measure the content of 5-hydroxytryptamine (5-HT) in hippocampi.The expression of TPH2 protein and mRNA in the median raphe nuclei was detected using Western blot and real-time polymerase chain reaction, respectively. Results Compared with group C, the immobility time was significantly prolonged, the total horizontal distance was shortened, the number of standing on the back legs and content of 5-HT in hippocampi were deceased, and the expression of TPH2 protein and mRNA in the median raphe nuclei was down-regulated in group D, and the total horizontal distance was significantly shortened, the number of standing on the back legs was decreased (P 0.05). Compared with group D, the immobility time was significantly shortened, the content of 5-HT in hippocampus was increased, the expression of TPH2 protein and mRNA in the median raphe nuclei was up-regulated (P 0.05). Conclusion The mechanism by which ketamine produces anti-depressant effect may be related to up-regulation of TPH2 expression in the median raphe nuclei and increase in the synthesis of 5-HT in hippocampi of mice. Key words: Ketamine; Depression; Tryptophan hydroxylase; Median raphe nuclei

Effects of dietary L-tryptophan supplementation on intestinal response to chronic unpredictable stress in broilers.

Stress has been recognized as a critical risk factor for gastrointestinal diseases in both humans and animals. However, nutritional strategies to attenuate stress-induced intestinal barrier function and underlying mechanisms remain largely unknown. This study tested the hypothesis that L-tryptophan enhanced intestinal barrier function by regulating mucosal serotonin metabolism in chronic unpredictable stress-exposed broilers. One-day-old male broilers (Arbor Acres) were fed a basal diet supplemented with or without L-tryptophan in the absence or presence of chronic unpredictable stress. Feed intake, body weight gain, plasma corticosterone and 5-hydroxytryptamine (5-HT), intestinal permeability, mucosal secretory IgA (sIgA), and mRNA levels for tryptophan hydroxylase 1 (TPH1), IL-1β, IL-6, TNF-α, IL-10, protein abundance for claudin-1, occludin, and ZO-1 were determined. Stress exposure led to elevated plasma corticosterone (P<0.05), increased intestinal permeability (P<0.05), reduced growth performance (P<0.05), and decreased sIgA secretion compared with the controls. These effects were largely reversed (P<0.05) by L-tryptophan supplementation. Western blot analysis showed that stress exposure resulted in decreased protein abundance for occludin, claudin-1, and ZO-1, which was attenuated by L-tryptophan. mRNA levels for IL-1β, IL-6, and TNF-α were increased, but those for IL-10 were decreased, in the jejunal tissue of broilers subjected to stress. This effect of stress on cytokine expression was abolished by L-tryptophan treatment. The effects of stress were associated with decreased plasma concentration of 5-HT (P<0.05), and reduced (P<0.05) mRNA levels for TPH1. L-Tryptophan supplementation markedly attenuated stress-induced alterations in 5-HT and TPH1 mRNA level in jejunal tissues of broilers. Collectively, these results indicate that L-tryptophan supplementation alleviates chronic unpredictable stress-induced intestinal barrier dysfunction by regulating 5-HT metabolism in broilers.

Effects of baldrinal of Valeriana jatamansi on expression of CRF, TPH1 mRNA and 5-HT in rats with irritable bowel syndrome

This study aimed to investigate the effects of baldrinal of Valeriana jatamansi on the expression of corticotropin releasing factor (CRF) and tryptophan hydroxylase 1 (TPH1) mRNA and levels of 5-hydroxytryptamine (5-HT) in colon of rats with irritable bowel syndrome (IBS), and to explain its therapeutic mechanism on IBS through 5-HT pathway. Fifty-four male SD rats were randomly divided into 6 groups: blank group, model group, baldrinal high, medium and low dose groups, and pinaverium bromide group, n=9 in each group. The IBS rat models were established by using unpredictable chronic stress for 3 weeks followed by 1-hour acute restraint stress (CAS) after 7 days of rest and independent feeding. CRF expression was detected by IHC-P; TPH1 mRNA expression was detected by using RT-PCR and the 5-HT level was measured by high performance liquid chromatography(HPLC). The results indicated that the method of chronic stress with acute restrain stress method and independent feeding could lead to the increase in expressions of CRF and TPH1 mRNA and levels of 5-HT in IBS rats(P<0.05). The expressions of CRF, TPH1 mRNA and 5-HT in baldrinal groups were significantly lower than those in model group(P<0.05). The experimental results showed that IBS could result in increase in the expressions of CRF, TPH1 mRNA and levels of 5-HT, and the baldrinal of V. jatamansi could improve the symptoms of IBS by reducing the expressions of CRF, TPH1 mRNA and levels of 5-HT in colon of rats.

Benefits of Hormone Therapy Estrogens Depend on Estrogen Type: 17β-Estradiol and Conjugated Equine Estrogens Have Differential Effects on Cognitive, Anxiety-Like, and Depressive-Like Behaviors and Increase Tryptophan Hydroxylase-2 mRNA Levels in Dorsal Raphe Nucleus Subregions

Decreased serotonin (5-HT) function is associated with numerous cognitive and affective disorders. Women are more vulnerable to these disorders and have a lower rate of 5-HT synthesis than men. Serotonergic neurons in the dorsal raphe nucleus (DRN) are a major source of 5-HT in the forebrain and play a critical role in regulation of stress-related disorders. In particular, polymorphisms of tryptophan hydroxylase-2 (TpH2, the brain-specific, rate-limiting enzyme for 5-HT biosynthesis) are implicated in cognitive and affective disorders. Administration of 17β-estradiol (E2), the most potent naturally circulating estrogen in women and rats, can have beneficial effects on cognitive, anxiety-like, and depressive-like behaviors. Moreover, E2 increases TpH2 mRNA in specific subregions of the DRN. Although conjugated equine estrogens (CEE) are a commonly prescribed estrogen component of hormone therapy in menopausal women, there is a marked gap in knowledge regarding how CEE affects these behaviors and the brain 5-HT system. Therefore, we compared the effects of CEE and E2 treatments on behavior and TpH2 mRNA. Female Sprague-Dawley rats were ovariectomized, administered either vehicle, CEE, or E2 and tested on a battery of cognitive, anxiety-like, and depressive-like behaviors. The brains of these animals were subsequently analyzed for TpH2 mRNA. Both CEE and E2 exerted beneficial behavioral effects, although efficacy depended on the distinct behavior and for cognition, on the task difficulty. Compared to CEE, E2 generally had more robust anxiolytic and antidepressant effects. E2 increased TpH2 mRNA in the caudal and mid DRN, corroborating previous findings. However, CEE increased TpH2 mRNA in the caudal and rostral, but not the mid, DRN, suggesting that distinct estrogens can have subregion-specific effects on TpH2 gene expression. We also found differential correlations between the level of TpH2 mRNA in specific DRN subregions and behavior, depending on the type of behavior. These distinct associations imply that cognition, anxiety-like, and depressive-like behaviors are modulated by unique serotonergic neurocircuitry, opening the possibility of novel avenues of targeted treatment for different types of cognitive and affective disorders.

Organoids as an ex vivo model for studying the serotonin system in the murine small intestine and colon epithelium

Intestinal organoids were recently established as an ex vivo model of the intestinal epithelium. The present study investigated the serotonin (5-hydroxytryptamine, 5-HT) system using organoids. Organoids from murine small intestinal and colonic crypts were successfully cultured. Reverse transcription–polymerase chain reaction (RT-PCR) analysis showed that small intestinal and colonic organoids express mRNAs encoding tryptophan hydroxylase-1 (TPH1) (the rate-limiting enzyme of 5-HT synthesis), serotonin reuptake transporter (SERT), 5-HT receptor (HTR)2A, HTR2B, and HTR4. SERT mRNA levels were significantly higher in the small intestine than in the colon in both the mucosal tissues and organoids, as estimated by quantitative real-time RT-PCR. Although the 5-HT concentration and levels of chromogranin A (CgA) (an enteroendocrine cell marker), TPH1, and HTR4 mRNAs were significantly higher in the colonic mucosa than the small intestinal mucosa, they were the same in small intestinal and colonic organoids. There were no significant differences in HTR2A and HTR2B mRNA levels between the small intestine and colon in either the mucosal tissues or organoids. Immunofluorescence staining showed that the number of CgA-positive cells in the colonic organoids appeared to increase upon culturing with acetate. Acetate supplementation significantly increased CgA, TPH1, and HTR4 mRNA levels in the colonic organoids. We propose that organoids are useful for investigating the 5-HT system in the intestinal epithelium, even though colonic organoids may require gut microbiota–derived factors such as short-chain fatty acids.

Effects of moxibustion on the expression of tryptophan hydroxylase 2 mRNA and the activity of monoamine oxidase in depression model rats

Objective To investigate the effects of moxibustion on the expression of tryptophan hydroxylase 2(TPH2) mRNA in median raphe nuclei and the activity of monoamine oxidase (MAO) in serum and cortex in depression model rats and to explore its mechanism. Methods Female SD rats were randomly divided into control group(n=6), model group(n=7) and moxibustion group(n=7) .The rat depression model was induced by giving isolation-housing in combination with chronic unexpected mild stress(CUMS) for 21 days.The hemi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) was employed to detect the relative expression of TPH2 mRNA, colorimetry was used to test the activity of MAO. Results The expression of TPH2 mRNA in median raphe nuclei in rats of the moxibustion group was higher than that in the model group(relative dentisty value: (0.3759±0.0272)vs(0.2573±0.0581), P<0.05). The activity of MAO in serum and cortex was reduced in rats of the moxibustion group((1.919±1.243 ) U/ml, (1292.048±90.072)U/mg protein, respectively) compared with that in the model group((4.117±2.727)U/ml, (1441.345 ±117.050)U/mg protein, respectively). Conclusion Moxibustion has the effects of improving depression, increasing the expression of TPH2 mRNA in median raphe nuclei and reducing the activity of MAO in serum and cortex might be one of its mechanisms. Key words: Depression; Moxibustion; Tryptophan hydroxylase 2; Monoamine oxidase

Sex-dependent programming effects of prenatal glucocorticoid treatment on the developing serotonin system and stress-related behaviors in adulthood

Prenatal stress and overexposure to glucocorticoids (GC) during development may be associated with an increased susceptibility to a number of diseases in adulthood including neuropsychiatric disorders, such as depression and anxiety. In animal models, prenatal overexposure to GC results in hyper-responsiveness to stress in adulthood, and females appear to be more susceptible than males. Here, we tested the hypothesis that overexposure to GC during fetal development has sex-specific programming effects on the brain, resulting in altered behaviors in adulthood. We examined the effects of dexamethasone (DEX; a synthetic GC) during prenatal life on stress-related behaviors in adulthood and on the tryptophan hydroxylase-2 (TpH2) gene expression in the adult dorsal raphe nucleus (DRN). TpH2 is the rate-limiting enzyme for serotonin (5-HT) synthesis and has been implicated in the etiology of human affective disorders. Timed-pregnant rats were treated with DEX from gestational days 18–22. Male and female offspring were sacrificed on the day of birth (postnatal day 0; P0), P7, and in adulthood (P80–84) and brains were examined for changes in TpH2 mRNA expression. Adult animals were also tested for anxiety- and depressive- like behaviors. In adulthood, prenatal DEX increased anxiety- and depressive- like behaviors selectively in females, as measured by decreased time spent in the center of the open field and increased time spent immobile in the forced swim test, respectively. Prenatal DEX increased TpH2 mRNA selectively in the female caudal DRN at P7, whereas it decreased TpH2 mRNA selectively in the female caudal DRN in adulthood. In animals challenged with restraint stress in adulthood, TpH2 mRNA was significantly lower in rostral DRN of prenatal DEX-treated females compared to vehicle-treated females. These data demonstrated that prenatal overexposure to GC alters the development of TpH2 gene expression and these alterations correlated with lasting behavioral changes found in adult female offspring.