Polymorphism screening and haplotype analysis of the tryptophan hydroxylase gene (TPH1) and association with bipolar affective disorder in Taiwan.
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BACKGROUND: Disturbances in serotonin neurotransmission are implicated in the etiology of many psychiatric disorders, including bipolar affective disorder (BPD). The tryptophan hydroxylase gene (TPH), which codes for the enzyme catalyzing the rate-limiting step in serotonin biosynthetic pathway, is one of the leading candidate genes for psychiatric and behavioral disorders. In a preliminary study, we found that TPH1 intron7 A218C polymorphism was associated with BPD. This study was designed to investigate sequence variants of the TPH1 gene in Taiwanese and to test whether the TPH1 gene is a susceptibility factor for the BPD. METHODS: Using a systematic approach, we have searched the exons and promoter region of the TPH1 gene for sequence variants in Taiwanese Han and have identified five variants, A-1067G, G-347T, T3804A, C27224T, and A27237G. These five variants plus another five taken from the literature and a public database were examined for an association in 108 BPD patients and 103 controls; no association was detected for any of the 10 variants. RESULTS: Haplotype constructions using these 10 SNPs showed that the 3 most common haplotypes in both patients and controls were identical. One of the fourth common haplotype in the patient group (i.e. GGGAGACCCA) was unique and showed a trend of significance with the disease (P = 0.028). However, the significance was abolished after Bonferroni correction thus suggesting the association is weak. In addition, three haplotype-tagged SNPs (htSNPs) were selected to represent all haplotypes with frequencies larger than 2% in the Taiwanese Han population. The defined TPH1 htSNPs significantly reduce the marker number for haplotype analysis thus provides useful information for future association studies in our population. CONCLUSION: Results of this study did not support the role of TPH1 gene in BPD etiology. As the current studies found the TPH1 gene under investigation belongs to the peripheral serotonin system and may link to a cardiac dysfunction phenotype, a second TPH gene that functions predominantly in the brain (i.e., nTPH or TPH2) should be the target for the future association study. (+info)
Two populations of glutamatergic axons in the rat dorsal raphe nucleus defined by the vesicular glutamate transporters 1 and 2.
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Most glutamatergic neurons in the brain express one of two vesicular glutamate transporters, vGlut1 or vGlut2. Cortical glutamatergic neurons highly express vGlut1, whereas vGlut2 predominates in subcortical areas. In this study immunohistochemical detection of vGlut1 or vGlut2 was used in combination with tryptophan hydroxylase (TPH) to characterize glutamatergic innervation of the dorsal raphe nucleus (DRN) of the rat. Immunofluorescence labeling of both vGlut1 and vGlut2 was punctate and homogenously distributed throughout the DRN. Puncta labeled for vGlut2 appeared more numerous then those labeled for vGlut1. Ultrastructural analysis revealed axon terminals containing vGlut1 and vGlut2 formed asymmetric-type synapses 80% and 95% of the time, respectively. Postsynaptic targets of vGlut1- and vGlut2-containing axons differed in morphology. vGlut1-labeled axon terminals synapsed predominantly on small-caliber (distal) dendrites (42%, 46/110) or dendritic spines (46%, 50/110). In contrast, vGlut2-containing axons synapsed on larger caliber (proximal) dendritic shafts (> 0.5 microm diameter; 48%, 78/161). A fraction of both vGlut1- or vGlut2-labeled axons synapsed onto TPH-containing dendrites (14% and 34%, respectively). These observations reveal that different populations of glutamate-containing axons innervate selective dendritic domains of serotonergic and non-serotonergic neurons, suggesting they play different functional roles in modulating excitation within the DRN. (+info)
Serotonin and its antidepressant-sensitive transport in mouse cumulus-oocyte complexes and early embryos.
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Serotonin (5-hydroxytryptamine [5-HT]), is a neurohormone found in various nonneural tissues, including the gonads of many invertebrates, in which it regulates spawning and oocyte meiotic maturation. The possibility that a local serotonergic network might also exist in the female gonads of vertebrate species, including mammals, remains poorly documented. To clarify this possibility, we investigated mouse cumulus cells, oocytes, and embryos for three key serotonergic components, namely, 5-HT itself; the rate-limiting enzyme for its production, tryptophan hydroxylase 1 (TPH1); and the 5-HT-specific transporter (SLC6A4) required for modulating its cellular effects. Using a combination of reverse transcription-polymerase chain reaction analysis and immunofluorescence confocal microscopy, we showed that mouse cumulus cells, oocytes, and embryos contain 5-HT and SLC6A4, while only cumulus cells possess the 5-HT-producing enzyme TPH1 and may thus be the local source of 5-HT observed in their neighboring cells. With a semiquantitative assay in single cells, we demonstrated that 5-HT can actively be taken up by isolated oocytes when it is supplied exogenously in vitro. This 5-HT transport in isolated oocytes is driven by a classical serotonin transporter, expressed up to the blastocyst stage, that is sensitive to the antidepressants fluoxetine and fluvoxamine, which belong to the selective serotonin reuptake inhibitor family. All together, our results show that 5-HT may be produced locally by cumulus cells and that it can be actively taken up by mammalian oocytes and embryos as part of a likely larger serotonergic network possibly regulating various developmental processes much earlier than previously thought. (+info)
(+)-Norfenfluramine-induced arterial contraction is not dependent on endogenous 5-hydroxytryptamine or 5-hydroxytryptamine transporter.
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(+)-Norfenfluramine, the major metabolite of fenfluramine, causes vasoconstriction dependence on the 5-hydroxytryptamine (5-HT)(2A) receptor in rat. (+)-Norfenfluramine was reported as a 5-hydroxytryptamine transporter (5-HTT) substrate and 5-HT releaser. Because the arterial 5-HTT exists and is functional in the rat, we hypothesized that (+)-norfenfluramine causes vasoconstriction by releasing 5-HT from vascular smooth muscle via 5-HTT. The released 5-HT, in turn, activates the 5-HT(2A) receptor. Isometric contractility experiments showed that (+)-norfenfluramine-induced mouse aortic contraction was reduced by the 5-HTT inhibitor fluoxetine (1 micriM) but not by fluvoxamine (1 microM). Tryptophan hydroxylase (TPH)-deficient (Tph1-/-) mice lack peripheral 5-HT. (+)-Norfenfluramine (10 nM-100 microM)-contracted aorta from wild-type and Tph1-/- mice with equivalent potency (-log EC(50) [M], wild type = 5.73 +/- 0.02, Tph1-/- = 5.62 +/- 0.09), and these contractions were inhibited by the 5-HT(2A) receptor antagonist ketanserin (3 nM) by a similar magnitude in aorta from wild-type and Tph1-/- mice (wild type = 19.4, Tph1-/- = 15.4-fold rightward shift versus control), as did fluoxetine (1 microM) (wild type = 22.4, Tph1-/- = 28.8-fold rightward shift versus control). To further test the role of 5-HTT in (+)-norfenfluramine-induced aortic contraction, the 5-HTT-targeted mutation mouse was used. (+)-Norfenfluramine induced similar aortic contraction in wild-type and 5-HTT-targeted mutation mice, and these contractions were inhibited by fluoxetine (1 microM). Thus, (+)-norfenfluramine vasoconstriction is not dependent on 5-HTT-mediated release of endogenous 5-HT but by activating membrane 5-HT(2A) receptors directly. Understanding of the mechanism by which (+)-norfenfluramine induces vasoconstriction is important to characterize and understand the function of the serotonergic system in peripheral arterial vasculature. (+info)
Serotonergic-like progenitor cells propagated from neural stem cells in vitro: survival with SERT protein expression following implantation into brains of mice lacking SERT.
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Neural stem cells (NSCs) obtained from the midbrain region of embryonic (E14) mice were initially cultured with basic fibroblast growth factor (bFGF), Sonic hedgehog, and FGF-8 in a serum-free N-2 culture medium to foster differentiation into a serotonergic-like phenotype. During the initial differentiating phase, these progenitor cells expressed En1, Pax3, and Pax5 mRNA. Subsequently, a single serotonin [5-hydroxytryptamine (5-HT)] and tryptophan hydroxylase-positive clone was isolated, which gave rise to cells that developed serotonergic properties. Sixty percent of these progenitor cells expressed the serotonin transporter (SERT), as indicated by specific ligand binding of [125I]-RTI-55. To further evaluate SERT functionality, we showed that these progenitor cells possessed specific [3H]-5-HT uptake activity. Implantation of the serotonergic-like progenitors into the hippocampus of adult mice genetically lacking SERT was followed by migration of these cells into adjacent brain regions, and survival of the cells at 8 weeks was accompanied by a gradual increase in density of SERT protein expression, which was not found in vehicle-injected, control mice. These findings suggest that this serotonergic-like NSC model will be a useful contribution to the development of cell biotechnology in regard to the expression of missing genes such as SERT in the adult brain. (+info)
Neonatal antidepressant exposure has lasting effects on behavior and serotonin circuitry.
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A significant fraction of infants born to mothers taking selective serotonin reuptake inhibitors (SSRIs) during late pregnancy display clear signs of antidepressant withdrawal indicating that these drugs can penetrate fetal brain in utero at biologically significant levels. Previous studies in rodents have demonstrated that early exposure to some antidepressants can result in persistent abnormalities in adult behavior and indices of monoaminergic activity. Here, we show that chronic neonatal (postnatal days 8-21) exposure to citalopram (5 mg/kg, twice daily, s.c.), a potent and highly selective SSRI, results in profound reductions in both the rate-limiting serotonin synthetic enzyme (tryptophan hydroxylase) in dorsal raphe and in serotonin transporter expression in cortex that persist into adulthood. Furthermore, neonatal exposure to citalopram produces selective changes in behavior in adult rats including increased locomotor activity and decreased sexual behavior similar to that previously reported for antidepressants that are nonselective monoamine transport inhibitors. These data indicate that the previously reported neurobehavioral effects of antidepressants are a consequence of their effects on the serotonin transporter. Moreover, these data argue that exposure to SSRIs at an early age can disrupt the normal maturation of the serotonin system and alter serotonin-dependent neuronal processes. It is not known whether this effect of SSRIs is paralleled in humans; however, these data suggest that in utero, exposure to SSRIs may have unforeseen long-term neurobehavioral consequences. (+info)
Genotype-dependent activity of tryptophan hydroxylase-2 determines the response to citalopram in a mouse model of depression.
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Polymorphism of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of brain serotonin (5-HT), is associated with less synthesis of brain 5-HT in DBA/2J and BALB/c than in C57BL/6J and 129/Sv mice. We selected the forced swimming test, a mouse model used to assess the antidepressant potential of drugs, and neurochemical techniques to study strain differences in the response to citalopram, a selective 5-HT reuptake inhibitor. Citalopram reduced immobility time in C57BL/6J and 129/Sv mice but had no such effect in DBA/2J and BALB/c mice. The drug reduced accumulation of 5-hydroxytryptophan (5-HTP), an indicator of 5-HT synthesis, in C57BL/6J and 129/Sv mice but much less in DBA/2J and BALB/c mice. Pretreatment with tryptophan raised 5-HTP accumulation and reinstated the antidepressant-like effect of citalopram in DBA/2J and BALB/c mice, whereas pharmacological inhibition of 5-HT synthesis prevented the effect of citalopram in C57BL/6J and 129/Sv mice. Because there were no strain differences in catecholamine synthesis, locomotor activity, and brain levels of citalopram at the end of the behavioral test, the results suggest that the failure of citalopram to reduce immobility time in DBA/2J and BALB/c mice is attributable to genotype-dependent impairment of 5-HT synthesis. Interstrain comparisons could probably be a useful strategy for understanding the mechanisms underlying the response to selective serotonin reuptake inhibitors. (+info)
Neuronal tryptophan hydroxylase mRNA expression in the human dorsal and median raphe nuclei: major depression and suicide.
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Major depressive disorder (MDD) and suicide are associated with deficient serotonergic neurotransmission. Tryptophan hydroxylase (TPH) is the rate-limiting biosynthetic enzyme for serotonin. Previously, we reported elevated levels of TPH protein in the dorsal raphe nucleus (DRN) of depressed suicides and now examine expression of neuronal TPH2 mRNA in a cohort of matched controls and depressed suicides (n = 11 pairs). DRN TPH2 mRNA was measured by densitometric analysis of autoradiograms from in situ hybridization histochemistry experiments. TPH2 mRNA is confirmed as the raphe-specific isoform of TPH in human brain, and is expressed in neurons throughout the anteroposterior extent of the DRN and median raphe nucleus (MRN). TPH2 mRNA expression correlates with TPH protein distribution in the DRN, and has a negative correlation with age. In drug-free suicides, TPH2 expression is 33% higher in the DRN and 17% higher in the MRN as compared to matched nonpsychiatric controls. Higher levels of TPH2 mRNA were found throughout the entire extent of the rostrocaudal axis of the DRN, and were not specific to any single subnucleus. Higher TPH2 mRNA expression may explain more TPH protein observed in depressed suicides and reflect a homeostatic response to deficient brain serotonergic transmission. (+info)