Tryptophan Hydroxylase Protein 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.

Contribution of the mutation T865G in TPH1 gene to the genetic potentiality of housed Mongolian sheep to year-round breeding.

Seasonal breeding is widespread in sheep and significantly affects the development of the housed sheep industry. To improve and balance the reproduction performance of sheep, year-round breeding has the goal of modern sheep farming. The tryptophan hydroxylase (TPH), which initiates and regulates biosynthesis of melatonin, is an important player in the formation of mammalian year-round breeding. However, little is known about its role in regulation of sheep seasonal breeding. In this study, a missense mutation, T865G in TPH1 gene was detected in 328 individuals of six Mongolian sheep groups. It was positively selected among Mongolian sheep. This mutation may appear between 13,683 and 350,973years ago and only exist in Hu sheep now. In Hu sheep, the frequency of allele T was 89.66%, and that of allele G was 10.34%. The TPH1 protein structure and property analysis suggested that this mutation from T to G affect the three-dimensional structure and reduce the hydropathicity of catalytic core. When the allele is T, the protein activity is twice that of the allele G, and their difference was significant (p<.05). In conclusion, T865G is an ancient mutation of TPH1 gene and affects the function of TPH protein, which may contribute to the genetic potentiality of Mongolian sheep to year-round breeding.

Endogenous Deficiency of Brain-Derived Neurotrophic Factor Induces the Downregulation of Tryptophan Hydroxylase-2 Expression in Raphe Nuclei of Rapid Ejaculator Rats

BackgroundPremature ejaculation (PE) is one of the most common ejaculatory disorders. Recent studies have suggested a close relationship between the serotonin (5-hydroxytryptamine [5-HT]) system and brain-derived neurotrophic factor (BDNF), raising the question of whether BDNF plays a role in ejaculation regulation. To our knowledge, no previous studies have explored BDNF level of the central nervous system in ejaculatory disorders. At the same time, the interaction of central BDNF and 5-HT systems has not been undertaken in ejaculation regulation field. AimThe aim of this study was to investigate the interaction between BDNF and 5-HT levels in raphe nuclei which contains the serotonergic neurons in a rat animal model with different ejaculatory behavior. MethodsEighteen male rats were selected and classified as “sluggish,” “normal,” and “rapid” ejaculators on the basis of ejaculation frequency during copulatory behavioral testing. BDNF and 5-HT levels were determined by enzyme-linked immunosorbent assay (ELISA). Real-Time Quantitative PCR and Western blot analyses were used to measure the mRNA level of Tryptophan Hydroxylase-2 (TPH2) gene and the expression of TPH2 protein (the rate-limiting enzyme in central 5-HT synthesis) in raphe nuclei, respectively. OutcomesMale rat sexual behavior, the levels of BDNF and 5-HT in raphe nuclei of rats with different ejaculatory behavior, the mRNA level of gene encoding TPH2 and the expression of TPH2 protein in raphe nuclei. ResultsThe primary finding of our study was that BDNF concentration was significantly decreased in raphe nuclei of rapid ejaculators. There was a strong positive correlation between the levels of BDNF and 5-HT (r = 0.944, P < .001). Further results showed that decreased TPH2 gene expression accompanied by TPH2 protein was shown in rapid ejaculators with lower BDNF level. Clinical ImplicationsWith refinement of current knowledge, BDNF may eventually serve as a promising biomarker in patients with PE. Strengths & LimitationsThere are no previous studies examining the interaction of the brain BDNF and 5-HT in ejaculation regulation field. The main limitation is the limited sample size. ConclusionBDNF may act via increasing the synthesis of central 5-HT in the process of ejaculation regulation. Our results suggest lack of endogenous BDNF induces the downregulation of TPH2 gene expression and the decrease of 5-HT synthesis in raphe nuclei of rapid ejaculator rats. Huang Y, Peng D, Geng H, et al. Endogenous Deficiency of Brain-Derived Neurotrophic Factor Induces the Downregulation of Tryptophan Hydroxylase-2 Expression in Raphe Nuclei of Rapid Ejaculator Rats. J Sex Med 2021;18:1491–1499.

Genetic Background Underlying 5-HT1A Receptor Functioning Affects the Response to Fluoxetine.

The influence of genetic background on sensitivity to drugs represents a topical problem of personalized medicine. Here, we investigated the effect of chronic (20 mg/kg, 14 days, i.p.) antidepressant fluoxetine treatment on recombinant B6-M76C mice, differed from control B6-M76B mice by CBA-derived 102.73–110.56 Mbp fragment of chromosome 13 and characterized by altered sensitivity of 5-HT1A receptors to chronic 8-OH-DPAT administration and higher 5-HT1A receptor mRNA levels in the frontal cortex and hippocampus. Significant changes in the effects of fluoxetine treatment on behavior and brain 5-HT system in recombinant B6-M76C mice were revealed. In contrast to B6-M76B mice, in B6-M76C mice, fluoxetine produced pro-depressive effects, assessed in a forced swim test. Fluoxetine decreased 5-HT1A receptor mRNA levels in the cortex and hippocampus, reduced 5-HT1A receptor protein levels and increased receptor silencer Freud-1 protein levels in the hippocampus of B6-M76C mice. Fluoxetine increased mRNA levels of the gene encoding key enzyme for 5-HT synthesis in the brain, tryptophan hydroxylase-2, but decreased tryptophan hydroxylase-2 protein levels in the midbrain of B6-M76B mice. These changes were accompanied by increased expression of the 5-HT transporter gene. Fluoxetine reduced 5-HT and 5-HIAA levels in cortex, hippocampus and midbrain of B6-M76B and in cortex and midbrain of B6-M76C; mice. These data demonstrate that changes in genetic background may have a dramatic effect on sensitivity to classic antidepressants from the Selective Serotonin Reuptake Inhibitors family. Additionally, the results provide new evidence confirming our idea on the disrupted functioning of 5-HT1A autoreceptors in the brains of B6-M76C mice, suggesting these mice as a model of antidepressant resistance.

Maternal high-fat diet stimulates proinflammatory pathway and increases the expression of Tryptophan Hydroxylase 2 (TPH2) and brain-derived neurotrophic factor (BDNF) in adolescent mice hippocampus

Maternal high-fat diet (HFD) consumption can promote a systemic inflammatory condition that may impair the offspring brain development, damaging memory and learning, when it reaches the hippocampus. This study aims to evaluate maternal HFD effects, during pregnancy and lactation, upon dams/mice offspring nutritional status, protein and gene expression of inflammatory pathway (JNK, pJNK and TNF-α), serotonin system molecules (Tryptophan Hydroxylase 2 (TPH2), key-enzyme of serotonin synthesis, serotonin transporter (SERT); 5-HT1A serotonergic receptor (5-HT1A)) and brain derived neurotrophic factor (BDNF) on recently weaned mice offspring hippocampus. Female Swiss mice were fed a control diet (CD, 11,5% fat) or a HFD (45.0% fat) from pre-mating to lactation. After weaning, the offspring received CD up to 28 post-natal days (PND28). Body weight and visceral adiposity (retroperitoneal and gonadal adipose tissue) of dams and offspring were measured. After euthanasia, the offspring hippocampus was dissected for evaluations of BDNF, inflammatory pathway and serotonergic system molecules protein and gene expression, through the techniques of Western Blotting, RTqPCR and ELISA. Our findings show that, during pregnancy, HFD-dams and HFD-offspring exhibited an increase in body weight gain and visceral adipose tissue compared to control animals. The hippocampus of HFD-offspring showed increased protein expression of TPH2, BDNF, pJNK and increased mRNA levels of TNF-α. However, the TPH2 increase in HFD-offspring did not alter hippocampal serotonin levels quantified through ELISA. Maternal HFD promoted an obesity phenotype in its offspring with increased body weight and visceral adiposity, increased protein and gene expression of the pro-inflammatory proteins pJNK and TNF-α. These changes were accompanied by increased TPH2 and BDNF protein expression. Thus, our findings show that maternal HFD during gestation and lactation increased pJNK and TNF-α expression in their offspring hippocampus indicating a pro-inflammatory state, with increased BDNF expression and alterations in its serotonergic system reflected by increased TPH2 expression.

In silicoanalysis of the tryptophan hydroxylase 2 (TPH2) protein variants related to psychiatric disorders.

The tryptophan hydroxylase 2 (TPH2) enzyme catalyzes the first step of serotonin biosynthesis. Serotonin is known for its role in several homeostatic systems related to sleep, mood, and food intake. As the reaction catalyzed by TPH2 is the rate-limiting step of serotonin biosynthesis, mutations in TPH2 have been associated with several psychiatric disorders (PD). This work undertakes an in silico analysis of the effects of genetic mutations in the human TPH2 protein. Ten algorithms were used to predict the functional and stability effects of the TPH2 mutations. ConSurf was used to estimate the evolutionary conservation of TPH2 amino acids. GROMACS was used to perform molecular dynamics (MD) simulations of TPH2 WT and P260S, R303W, and R441H, which had already been associated with the development of PD. Forty-six TPH2 variants were compiled from the literature. Among the analyzed variants, those occurring at the catalytic domain were shown to be more damaging to protein structure and function. The ConSurf analysis indicated that the mutations affecting the catalytic domain were also more conserved throughout evolution. The variants S364K and S383F were predicted to be deleterious by all the functional algorithms used and occurred at conserved positions, suggesting that they might be deleterious. The MD analyses indicate that the mutations P206S, R303W, and R441H affect TPH2 flexibility and essential mobility at the catalytic and oligomerization domains. The variants P206S, R303W, and R441H also exhibited alterations in dimer binding affinity and stability throughout the simulations. Thus, these mutations may impair TPH2 functional interactions and, consequently, its function, leading to the development of PD. Furthermore, we developed a database, SNPMOL (http://www.snpmol.org/), containing the results presented in this paper. Understanding the effects of TPH2 mutations on protein structure and function may lead to improvements in existing treatments for PD and facilitate the design of further experiments.

Creatine transporter knockout mice (Slc6a8) show increases in serotonin-related proteins and are resilient to learned helplessness

Approximately 20% of adults in the U.S. will experience an affective disorder during their life. While it is well established that serotonin (5-HT) is a crucial factor in mood, impaired cellular bioenergetics are also implicated. Creatine (Cr), through the Cr/Phospho-Cr (PCr) shuttle, maintains high ATP concentrations in the neuron. This system may be implicated in the etiology of affective disorders, as reduced Cr, PCr, and ATP are often seen in the brains of affected patients. To address this issue, Cr transporter (Crt) deficient male mice (Slc6a8−/y) and female mice heterozygous for Crt expression (Slc6a8+/-) were used to evaluate how a Cr deficient system would alter affective-like behaviors. Slc6a8−/y and Slc6a8+/- mice had faster escape latencies in learned helplessness, indicating a potential resilience to behavioral despair. Slc6a8−/y had decrease latency to immobility in the tail-suspension test and Slc6a8+/- had increased open entries in elevated zero maze, but all other variables matched those of wildtype mice, however. Slc6a8−/y mice have increased 5-hydroxyindoleacetic acid content in the hippocampus and striatum and increased monoamine oxidase protein and tryptophan hydroxylase-2 protein content in the hippocampus, while 5-HT levels are unchanged. This indicates an alteration to the 5-HTergic system in Cr deficient mice. Our results indicate that Cr plays a complex role in affective disorders and 5-HT, warranting further investigation.

Decreased serotonin synthesis is involved in seizure-induced respiratory arrest in DBA/1 mice.

A known cause of seizure-induced respiratory arrest is the deficiency in serotonergic neurotransmission. Tryptophan hydroxylase-2 (TPH2) is the rate-limiting enzyme of central serotonin (5-hydroxytryptamine) synthesis which converts l-tryptophan to 5-hydroxytryptophan. A recent study revealed a reduction in TPH2 protein expression in the brainstems of DBA/1 mice that developed recurrent seizure-induced respiratory arrest, whereas the activity of this protein was unexplored. Thus this study aims to investigate the association between intrinsic 5-hydroxytryptamine synthesis in the brainstem and the susceptibility for sudden unexpected death in epilepsy in DBA/1 mice. The effect of LY393558, a potent 5-hydroxytryptamine reuptake inhibitor with 5-HT1B/1D receptor antagonist properties, on seizure-induced respiratory arrest evoked by acoustic stimulation was also examined in DBA/1 mice. ELISA results showed significantly decreased TPH2 activity in the brainstems of untreated DBA/1 mice than that of C57BL/6J mice. Moreover, the concentrations of 5-hydroxytryptamine, 5-hydroxytryptophan and 5-HIAA in the brainstems of DBA/1 mice with or without acoustic stimulation were significantly lower than that of C57BL/6J mice. Acute administration of LY393558 also significantly reduced seizure-induced respiratory arrest in DBA/1 mice. These observations provide novel evidences for the hypothesis that 5-hydroxytryptamine deficiency might be a potential cause of seizure-induced respiratory arrest.

Molecular Evolution of Tryptophan Hydroxylases in Vertebrates: A Comparative Genomic Survey.

Serotonin is a neurotransmitter involved in various physiological processes in the central and peripheral nervous systems. Serotonin is also a precursor for melatonin biosynthesis, which mainly occurs in the pineal gland of vertebrates. Tryptophan hydroxylase (TPH) acts as the rate-limiting enzyme in serotonin biosynthesis and is the initial enzyme involved in the synthesis of melatonin. Recently, two enzymes—TPH1 and TPH2—were reported to form the TPH family in vertebrates and to play divergent roles in serotonergic systems. Here, we examined the evolution of the TPH family from 70 vertebrate genomes. Based on the sequence similarity, we extracted 184 predicted tph homologs in the examined vertebrates. A phylogenetic tree, constructed on the basis of these protein sequences, indicated that tph genes could be divided into two main clades (tph1 and tph2), and that the two clades were further split into two subgroups of tetrapods and Actinopterygii. In tetrapods, and some basal non-teleost ray-finned fishes, only two tph isotypes exist. Notably, tph1 in most teleosts that had undergone the teleost-specific genome duplication could be further divided into tph1a and tph1b. Moreover, protein sequence comparisons indicated that TPH protein changes among vertebrates were concentrated at the NH2-terminal. The tertiary structures of TPH1 and TPH2 revealed obvious differences in the structural elements. Five positively selected sites were characterized in TPH2 compared with TPH1; these sites may reflect the functional divergence in enzyme activity and substrate specificity. In summary, our current work provides novel insights into the evolution of tph genes in vertebrates from a comprehensive genomic perspective.

Antidepressant mechanisms of venlafaxine involving increasing histone acetylation and modulating tyrosine hydroxylase and tryptophan hydroxylase expression in hippocampus of depressive rats.

Venlafaxine (VEN) is a widely used antidepressant as a serotonin-reuptake and norepinephrine-reuptake inhibitor. It is used primarily in depression, especially with generalized anxiety disorder or chronic pain. This medicine is of interest because its mechanisms involved multiple aspects. In the current study, the antidepressant action of VEN was investigated by studying the histone acetylation and expression of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) in rats exposed to chronic unpredicted stress (CUS) for 28 days. Male Sprague-Dawley rats were divided into a control group, VEN-treated control group, CUS group, and VEN-treated CUS group. VEN (23.4 mg/kg once daily) was administered to rats by intragastric gavage, whereas the same volume of vehicle was given to rats in the control and model groups. Rat behaviors, acetylated H3 at lysine 9 (acH3K9), acetylated H3 at lysine 14 (acH3K14), acetylated H4 at lysine 12 (acH4K12), histone deacetylase 5, and TH and TPH expression in the hippocampus were determined. Chronic VEN treatment significantly relieved the anxiety- and depression-like behaviors, prevented the increase of histone deacetylase 5 expression and decrease of acH3K9 level, and promoted TH and TPH protein expression in the hippocampus of CUS rats. The results suggest that the preventive antidepressant mechanism of VEN is partly involved in the blocking effects on histone de-acetylated modification and then increasing TH, TPH expression.

Physiological dosages of estradiol and diarylpropionitrile decrease depressive behavior and increase tryptophan hydroxylase expression in the dorsal raphe nucleus of rats subjected to the forced swim test.

The dorsal raphe nucleus (DR) is a crucial source of serotonin (5-HT) neurons involved in the regulation of stress-induced depression. Estrogen receptors have been identified in the DR, yet the role of estrogen in modulating this adaptive response is incompletely understood. The current study investigated the effects of different dosages of estradiol (E2, 10 and 50 μg/rat/day for 11 consecutive days) and selective estrogen receptor modulators: Diarylpropionitrile (DPN, 10 μg/rat/day for 11 consecutive days) and propyl pyrazole triol (PPT, 10 μg/rat/day for 11 consecutive days) on behavior and the expression of tryptophan hydroxylase (TPH) and glucocorticoid receptor in the DR of ovariectomized rats subjected to the forced swim test (FST). 10 μg E2 and DPN, an estrogen receptor β agonist, increased swimming and decreased immobility in the FST, while 50 μg E2 and PPT, an estrogen receptor α agonist, failed to influence the behavior of the rats in the FST. Similarly, 10 μg E2 and DPN increased TPH protein expression in the DR, while 50 μg E2 and PPT did not. Both 10 μg E2 and 50 μg E2 increased glucocorticoid receptor protein expression in the DR. Interestingly, 50 μg E2 led to a greater increase in plasma corticosterone levels compared with 10 μg E2. These observations suggest that a physiological dosage of E2 reduces depressive behavior and enhances TPH expression. High dosage of E2 lacks antidepressant activity in part due to heightened effects on corticosterone levels, which may conversely decrease TPH expression in the DR.

Altered 3D Structure of Human Tryptophan Hydroxylase-2 Caused by Change in the amino acid 341: In Silico Analysis

Introduction Neuropsychiatric syndromes have an important connection with disorders in the regulation of serotonin, with human tryptophan hydroxylase-2 being one of the related biosynthetic enzymes of this neurotransmitter. Evidence-based genetic studies suggest a possible involvement of this enzyme in neuropsychiatric disorders caused by abnormalities in the synthesis and regulation of serotonin. Objective To analyze the structural effects of single nucleotide polymorphism (SNP) in the enzyme tryptophan hydroxylase-2 and the changes that lead to functional alterations. Materials and Methods In this study, we performed an in silico analysis of SNPs associated with abnormal folding of the tryptophan hydroxylase-2 protein. Different programs were used to identify amino acid changes evidencing pathogenic effects and possible functional impairments. Results A change in the amino acid 341 (lysine [L]for phenylalanine [F]) (L341F) of the protein chain affects the total enthalpy of the protein. The enthalpy turned positive due to the energy required for the amino acid to return to its original condition. The protein function is also affected negatively because of the altered structured. Conclusion The change in the L341F leads to serious structural defects in the tryptophan hydroxylase-2. Those defects can be further related with functional instability and associated to the etiology of neuropsychiatric diseases.

Vaccinium bracteatum Leaf Extract Reverses Chronic Restraint Stress-Induced Depression-Like Behavior in Mice: Regulation of Hypothalamic-Pituitary-Adrenal Axis, Serotonin Turnover Systems, and ERK/Akt Phosphorylation.

The leaves of Vaccinium bracteatum Thunb. are a source of traditional herbal medicines found in East Asia. The present study aimed to evaluate the mechanisms underlying the antidepressant-like effects of water extract of V. bracteatum Thunb. leaves (VBLW) in a mouse model of chronic restraint stress (CRS) and to identify the possible molecular in vitro mechanisms of the neuroprotective effects. The CRS-exposed mice were orally administered VBLW (100 and 200 mg/kg) daily for 21 days consecutively. The behavioral effects of VBLW were assessed through the forced swim test (FST) and the open field test (OFT). The levels of serum corticosterone (CORT), corticotropin releasing hormone (CRH), and adrenocorticotropin hormone (ACTH), brain monoamines, such as serotonin, dopamine, and norepinephrine, and serotonin turnover by tryptophan hydroxylase 2 (TPH2), serotonin reuptake (SERT), and monoamine oxidase A (MAO-A) were evaluated, in addition to the extracellular signal-regulated kinases (ERKs)/protein kinase B (Akt) signaling pathway. CRS-exposed mice treated with VBLW (100 and 200 mg/kg) showed significantly reduced immobility time and increased swimming and climbing times in the FST, and increased locomotor activity in the OFT. Moreover, CRS mice treated with VBLW exhibited significantly decreased CORT and ACTH, but enhanced brain monoamine neurotransmitters. In addition, CRS mice treated with VBLW had dramatically decreased protein levels of MAO-A and SERT, but increased TPH2 protein levels in the hippocampus and the PFC. Similarly, VBLW significantly upregulated the ERKs/Akt signaling pathway in the hippocampus and the PFC. Furthermore, VBLW showed neuroprotective effects via increased CREB phosphorylation in CORT-induced cell injury that were mediated through the ERK/Akt/mTOR signaling pathways. These results suggested that the antidepressant-like effects of VBLW might be mediated by the regulation of the HPA axis, glucocorticoids, and serotonin turnover, such as TPH2, SERT, and MAO-A, as well as the concentration of monoamine neurotransmitters, and the activities of ERK and Akt phosphorylation, which were possibly associated with neuroprotective effects.

The Expression of Brain-Derived Neurotrophic Factor and Tryptophan Hydroxylase in the Dorsal Raphe Nucleus during Repeated Stress

The mechanisms of development of stress-induced depression symptoms may include interaction between the elements of the serotonergic (5-HT) system and brain-derived neurotrophic factor (BDNF) at the level of the cell bodies of 5-HT neurons. In order to evaluate this situation, we used immunofluorescent staining of BDNF protein and the enzyme of serotonin synthesis tryptophan hydroxylase (TPH) in the dorsal raphe nucleus (DRN) of adult male rats that were subjected daily to a 15-min forced swim stress for 1, 2, 7, or 14 days. The expression of the BDNF protein was substantially decreased in the DRN after 7- or 14-day stress, whereas the expression of the TPH protein significantly increased after the 14-day stress and did not differ from the control after the shorter duration of repeated stress. The absence of correlation between some indices indicates their independence from each other at the level of the cell bodies of 5-HT neurons.

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

5-Hydroxytryptophan, a precursor for serotonin synthesis, reduces seizure-induced respiratory arrest.

The DBA/1 mouse is a relevant animal model of sudden unexpected death in epilepsy (SUDEP), as it exhibits seizure-induced respiratory arrest (S-IRA) evoked by acoustic stimulation, followed by cardiac arrhythmia and death. Defects in serotonergic neurotransmission may contribute to S-IRA. The tryptophan hydroxylase-2 (TPH2) enzyme converts L-tryptophan to 5-hydroxytryptophan (5-HTP), a precursor for central nervous system (CNS) serotonin (5-HT) synthesis; and DBA/1 mice have a polymorphism that decreases TPH2 activity. We, therefore, hypothesized that supplementation with 5-HTP may bypass TPH2 and suppress S-IRA in DBA/1 mice. TPH2 expression was examined by Western blot in the brainstem of DBA/1 and C57BL/6J mice both with and without acoustic stimulation. Changes in breathing and cardiac electrical activity in DBA/1 and C57BL/6J mice that incurred sudden death during generalized seizures evoked by pentylenetetrazole (PTZ) were studied by plethysmography and electrocardiography. The effect of 5-HTP administration on seizure-induced mortality evoked by acoustic stimulation or by PTZ was investigated in DBA/1 mice. Repetitive acoustic stimulation resulted in reduced TPH2 protein in the brainstem of DBA/1 mice as compared with C57BL/6J mice. S-IRA evoked by acoustic stimulation in DBA/1 mice was significantly reduced by 5-HTP. Following S-IRA, cardiac electrical activity could be detected for minutes before terminal asystole and death in both DBA/1 and C57BL/6J mice after PTZ treatment. The incidence of S-IRA by PTZ administration was greater in DBA/1 than in C57BL/6J mice, and administration of 5-HTP also significantly reduced S-IRA by PTZ in DBA/1 mice. Our data suggest that S-IRA is the primary event leading to death incurred in most DBA/1 and some C57BL/6J mice during PTZ-evoked seizures. Suppression of S-IRA by 5-HTP suggests that 5-HT transmission contributes to the pathophysiology of S-IRA, and that 5-HTP, an over-the-counter supplement available for human consumption, may be clinically useful in preventing SUDEP.

Melatonin Synthesis: Acetylserotonin O-Methyltransferase (ASMT) Is Strongly Expressed in a Subpopulation of Pinealocytes in the Male Rat Pineal Gland.

The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the cellular localization of melatonin synthesis in this species has not been investigated. Here we focus on the localization of melatonin synthesis using immunohistochemical methods to detect the last enzyme in melatonin synthesis, acetylserotonin O-methyltransferase (ASMT), and in situ hybridization techniques to study transcripts encoding ASMT and two other enzymes in melatonin synthesis, tryptophan hydroxylase (TPH)-1 and aralkylamine N-acetyltransferase. In sections of the rat pineal gland, marked cell-to-cell differences were found in ASMT immunostaining intensity and in the abundance of Tph1, Aanat, and Asmt transcripts. ASMT immunoreactivity was localized to the cytoplasm in pinealocytes in the parenchyma of the superficial pineal gland, and immunopositive pinealocytes were also detected in the pineal stalk and in the deep pineal gland. ASMT was found to inconsistently colocalize with S-antigen, a widely used pinealocyte marker; this colocalization was seen in cells throughout the pineal complex and also in displaced pinealocyte-like cells of the medial habenular nucleus. Inconsistent colocalization between ASMT and TPH protein was also detected in the pineal gland. ASMT protein was not detected in extraepithalamic parts of the central nervous system or in peripheral tissues. The findings in this report are of special interest because they provide reason to suspect that melatonin synthesis varies significantly among individual pinealocytes.

Anti-Apoptotic Protein Bcl-xL Expression in the Midbrain Raphe Region Is Sensitive to Stress and Glucocorticoids.

Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with Bcl-xL, caused a significant increase in tph2 mRNA levels and slightly enhanced tryptophan hydroxylase immunoreactivity in the midbrain. The increasing effect on the bcl-xl expression was specific to the short-term stress. Forced swim repeated daily for 2 weeks led to a decrease in bcl-xl mRNA in the midbrain without any effects on the Bcl-xL protein expression in the 5-HT neurons. In chronically stressed animals, an increase in tph2 gene expression was not associated with any changes in tryptophan hydroxylase protein levels. Our findings are the first to demonstrate that both short-term stress and acute glucocorticoid exposures induce Bcl-xL protein expression in the midbrain 5-HT neurons concomitantly with the activation of the 5-HT synthesis pathway in these neurons.

Tryptophan hydroxylase 2 and Bcl-xL in the rat raphe nucleus after acute and chronic forced swim stress

Stressful events induce changes in the expression of numerous genes and their protein products in the brain. However, the mechanisms mediating these changes, as well as their significance for the development of stress-induced depression or for coping with stress, remain obscure. Evidence for the sensitivity of tryptophan hydroxylase-2 (TPH2), the rate-limiting enzyme of the serotonin (5-HT) pathway, to stress is concisely reviewed, as well as the neuroprotective function of the antiapoptotic protein Bcl-xL in the brain. The goal of our experiments was to investigate the gene and protein expression of TPH2 and Bcl-xL in the dorsal (DRN) and median (MRN) raphe nuclei during repeated stress events. Gene (RT PCR) and protein (immunohistochemistry) expression was assessed 24 hours after the second and fourteenth forced swim sessions. The increase in TPH2 protein expression observed after the second swim stress exposure might reflect the protective action of Bcl-xL. During the subsequent stressful events, the stress-induced increase in Bcl-xL expression decreased. This effect was associated with the weakening of serotonergic neuron function evidenced by the compensatory activation of the TPH2 gene expression without TPH2 protein increase. Thus, shortand long-term forced swimming resulted in qualitatively different alterations in brain expression of TPH2 and Bcl-xL, suggesting their specific roles during acute and chronic stages in the development of stress-induced psychopathology. These changes may constitute a component of the mechanisms underlying elevated tph2 gene expression in depressed patients.

Differences in Anxiety-Like Behavior within a Batch of Wistar Rats Are Associated with Differences in Serotonergic Transmission, Enhanced by Acute SRI Administration, and Abolished By Serotonin Depletion.

Background:The anxiety-reducing effect of long-term administration of serotonin reuptake inhibitors is usually seen only in subjects with anxiety disorders, and such patients are also abnormally inclined to experience a paradoxical anxiety-enhancing effect of acute serotonin reuptake inhibition. These unique responses to serotonin reuptake inhibitors in anxiety-prone subjects suggest, as do genetic association studies, that inter-individual differences in anxiety may be associated with differences in serotonergic transmission.Methods:The one-third of the animals within a batch of Wistar rats most inclined to spend time on open arms in the elevated plus maze were compared with the one-third most inclined to avoid them with respect to indices of brain serotonergic transmission and how their behavior was influenced by serotonin-modulating drugs.Results:“Anxious” rats displayed higher expression of the tryptophan hydroxylase-2 gene and higher levels of the tryptophan hydroxylase-2 protein in raphe and also higher levels of serotonin in amygdala. Supporting these differences to be important for the behavioral differences, serotonin depletion obtained by the tryptophan hydroxylase-2 inhibitor p-chlorophenylalanine eliminated them by reducing anxiety in “anxious” but not “non-anxious” rats. Acute administration of a serotonin reuptake inhibitor, paroxetine, exerted an anxiety-enhancing effect in “anxious” but not “non-anxious” rats, which was eliminated by long-term pretreatment with another serotonin reuptake inhibitor, escitalopram.Conclusions:Differences in an anxiogenic impact of serotonin, which is enhanced by acute serotonin reuptake inhibitor administration, may contribute to differences in anxiety-like behavior amongst Wistar rats.