A second tryptophan hydroxylase isoform, TPH-2 mRNA, is increased by ovarian steroids in the raphe region of macaques

Recently, a second gene that codes for the rate-limiting enzyme in serotonin synthesis was found in brain, named tryptophan hydroxylase-2 (TPH-2). We sequenced overlapping segments (251 and 510 bp) of 5' monkey TPH-2 and questioned whether TPH-2 is regulated by estrogen (E) and progesterone (P) in serotonin neurons of macaques. Monkey TPH-2 was 97% homologous to human TPH-2 and 65% homologous to monkey TPH-1 in the coding region. Spayed monkeys were administered placebo, E-only, P-only, or E + P for 1 month via Silastic implants (n = 4/treatment) and the midbrain was utilized for TPH-2 in situ hybridization (ISH). Additional monkeys (n = 3/treatment) were used to determine the relative abundance of TPH-2 mRNA with quantitative (q) RT-PCR. In the ISH assay, all of the hormone treatments caused a significant and similar increase in TPH-2 mRNA optical density (fourfold; P < 0.004) and positive pixel area (twofold; P < 0.002) over spayed controls. Treatment with E or E + P for 1 month increased the relative abundance of TPH-2 mRNA over spayed controls in the qRT-PCR assay (ANOVA P < 0.05 and P < 0.007, respectively). In conclusion, ovarian steroids stimulate TPH-2 mRNA expression, which could in turn cause an increase in serotonin synthesis. This would impact many of the neural functions that are governed by serotonin.     

Effects of oral estrogen, raloxifene and arzoxifene on gene expression in serotonin neurons of macaques

The serotonin neural system contributes to cognition and affect, both of which exhibit pathologies with gender bias. We previously showed that estrogen (E) treatment of female macaques via Silastic implant alters gene expression for tryptophan hydroxylase (TPH), the serotonin reuptake transporter (SERT) and the 5HT1A autoreceptor. In addition, we have found that serotonin neurons of macaques express ER beta (ER beta). Together these studies suggest that the serotonin neural system could transduce the action of estrogen via ER beta on aspects of mood and cognition. However, estrogen replacement therapy can increase the risk for breast and uterine cancer. Therefore, we questioned whether the selective estrogen receptor modulators, raloxifene and arzoxifene, act in a manner similar to E on gene expression in serotonin neurons of a nonhuman primate model. Female rhesus macaques were ovariectomized and orally dosed with vehicle, estradiol 17beta, raloxifene or arzoxifene once per day by sipper bottles for 30 days. The animals were then euthanized and the midbrains were prepared for in situ hybridization for TPH, SERT and 5HT1A receptor mRNAs followed by densitometric analysis. There was a significant increase in TPH total signal (positive pixelsxOD) with E, raloxifene and arzoxifene, respectively. There was a significant decrease in SERT mRNA optical density with all treatments. 5HT1A autoreceptor mRNA did not change with any treatment. If these changes in gene expression are reflected by similar changes in the functional proteins, then raloxifene or arzoxifene could increase serotonin neurotransmission with little or no negative action in peripheral tissues. In conclusion, the selective estrogen receptor modulators, raloxifene and arzoxifene, act in a manner similar to natural E on TPH and SERT mRNA expression in serotonin neurons. This suggests that raloxifene and arzoxifene are agonists at ER beta in the context of the serotonin neuron. However, the responses to E were more variable and less robust with the oral dosing paradigm compared to a chronic implant paradigm.     

Steroid regulation of tryptophan hydroxylase protein in the dorsal raphe of macaques.

BACKGROUND: Tryptophan hydroxylase (TPH) is the rate-limiting enzyme for the synthesis of serotonin, and serotonin is a pivotal neurotransmitter in the regulation of mood, affective behavior, pituitary hormone secretion, and numerous autonomic functions. We previously demonstrated that estradiol (E) and progesterone (P) increase TPH mRNA levels in the dorsal raphe of macaques. METHODS: This study employed western blotting and densitometric quantitation to determine whether the changes observed at the level of gene expression were manifested by changes in TPH protein expression and whether modified estrogens or progestins had actions similar to the native ligands. In addition, the effect of the antiestrogen tamoxifen was examined. Ovariectomized (ovx) rhesus and cynomolgus macaques were untreated or treated with E, P, E+P, equine estrogens (EE), medroxyprogesterone (MPA), EE+MPA, or tamoxifen. The dorsal raphe region was subjected to Western analysis. RESULTS: E treatment for 28 days increased TPH protein mass four to six fold over ovariectomized controls. Addition of P to the E regimen or treatment with P for 28 days after E priming did not alter TPH from E treatment alone. Treatment of ovx macaques with a low dose of P caused a two-fold increase in TPH protein. Treatment of ovariectomized macaques for 30 months with EE alone or MPA alone significantly increased TPH protein; however, unlike P, the addition of MPA to the EE regimen blocked the stimulatory effect of EE. Tamoxifen treatment significantly reduced TPH protein compared to EE and ovariectomized control animals. CONCLUSION: The stimulatory effect of E and P on TPH protein in the dorsal raphe of macaques correlates with the previously observed effect at the level of mRNA expression. P had no effect on the stimulatory action of E, whereas MPA blocked the stimulatory effect of EE. Tamoxifen acted as a potent antiestrogen on TPH protein expression. If TPH protein mass influences serotonin synthesis, then these steroids will impact many autonomic systems that are regulated by serotonin.     

Alteration of serotonin transporter messenger RNA level in the peripheral blood mononuclear cells from simian/human immunodeficiency virus infected Chinese rhesus macaques (Macaca mulatta)

Serotonin transporter (SERT, 5-HTT) is a key element in the serotonergic system which is probably involved in the psychiatric disorders commonly observed in people living with HIV/AIDS. However, no information is available about the effects of HIV infection on SERT expression. In this study, a TaqMan® real-time RT-PCR method was established, levels of SERT mRNA in the peripheral blood mononuclear cells (PBMCs) and various tissues from normal Chinese rhesus macaques, in PBMCs from 32 SHIV-sf162p4 infected rhesus macaques and from 8 rhesus macaques before and 7, 14, 21, 28 and 196 days after SHIV-sf162p4 infection, and in PBMCs before and after in vitro phytohemagglutinin (PHA) stimulation were examined. It was found that SERT mRNA was widely distributed in lymphoid tissues; the level of SERT mRNA was significantly reduced in PBMCs from SHIV infected rhesus macaques and in PBMCs stimulated with PHA. The most evident decrease (to about one-tenth) in SERT mRNA level was observed at day 7 after SHIV infection. Difference in PBMC SERT mRNA level between 5-HTTLPR genotypes was not statistically significant. These data indicated that, in addition to previously observed abnormality in serotonin metabolism, SERT expression might be affected in HIV/AIDS, which might be associated with depression and other psychiatric disorders in HIV/AIDS. Besides, this study provided a basis for quantitative analysis of SERT gene expression under effects of host and environmental factors, such as 5-HTTLPR genotypes, SERT targeting drugs or other infectious agents.     

Epsilon-sarcoglycan immunoreactivity and mRNA expression in mouse brain

Myoclonus dystonia (M-D) is a hereditary movement disorder caused by a maternally imprinted gene that is often associated with psychiatric symptoms. Most cases of M-D are believed to result from mutations of the epsilon-sarcoglycan protein. The neuroanatomical distribution of epsilon-sarcoglycan-like immunoreactivity in mouse was investigated by using an antiserum against the epsilon-sarcoglycan protein. The expression of epsilon-sarcoglycan mRNA was studied by a sensitive fluorescence in situ hybridization (FISH) method. Immunohistochemistry and FISH revealed a wide distribution of epsilon-sarcoglycan protein and mRNA throughout the mouse brain. High expression levels of epsilon-sarcoglycan mRNA and immunoreactivity were found in the mitral cell layer of the olfactory bulb, the Purkinje cell layer in cerebellum, and the monoaminergic neurons in the mouse midbrain. Immunohistochemistry revealed a similar distribution of epsilon-sarcoglycan protein. Double-labeling FISH showed colocalization of tyrosine hydroxylase and epsilon-sarcoglycan mRNAs within all the midbrain dopaminergic (DAergic) cell groups. By combining FISH with fluorescence immunohistochemistry, coexpression of epsilon-sarcoglycan mRNA and tryptophan hydroxylase immunoreactivity was found in the serotonergic (5-HTergic) neurons within the dorsal raphe nucleus. The distribution of epsilon-sarcoglycan in the mouse brain suggests that the symptom complex of M-D may be related to the effects of decreased epsilon-sarcoglycan activity on the development or function of monoaminergic neurons.     

Vesicular glutamate transporter 3‐expressing nonserotonergic projection neurons constitute a subregion in the rat midbrain raphe nuclei

We previously reported that about 80% of vesicular glutamate transporter 3 (VGLUT3)‐positive cells displayed immunoreactivity for serotonin, but the others were negative in the rat midbrain raphe nuclei, such as the dorsal (DR) and median raphe nuclei (MnR). In the present study, to investigate the precise distribution of VGLUT3‐expressing nonserotonergic neurons in the DR and MnR, we performed double fluorescence in situ hybridization for VGLUT3 and tryptophan hydroxylase 2 (TPH2). According to the distribution of VGLUT3 and TPH2 mRNA signals, we divided the DR into six subregions. In the MnR and the rostral (DRr), ventral (DRV), and caudal (DRc) parts of the DR, VGLUT3 and TPH2 mRNA signals were frequently colocalized (about 80%). In the lateral wings (DRL) and core region of the dorsal part of the DR (DRDC), TPH2‐producing neurons were predominantly distributed, and about 94% of TPH2‐producing neurons were negative for VGLUT3 mRNA. Notably, in the shell region of the dorsal part of the DR (DRDSh), VGLUT3 mRNA signals were abundantly detected, and about 75% of VGLUT3‐expressing neurons were negative for TPH2 mRNA. We then examined the projection of VGLUT3‐expressing nonserotonergic neurons in the DRDSh by anterograde and retrograde labeling after chemical depletion of serotonergic neurons. The projection was observed in various brain regions such as the ventral tegmental area, substantia nigra pars compacta, hypothalamic nuclei, and preoptic area. These results suggest that VGLUT3‐expressing nonserotonergic neurons in the midbrain raphe nuclei are preferentially distributed in the DRDSh and modulate many brain regions with the neurotransmitter glutamate via ascending axons. J. Comp. Neurol. 518:668–686, 2010. © 2009 Wiley‐Liss, Inc.     

5-ht5B receptor mRNA in the raphe nuclei: coexpression with serotonin transporter

We used double-label in situ hybridization to examine the cellular localization of 5-ht(5B) receptor mRNA in relation to serotonin transporter mRNA in the rat dorsal raphe (DR) and central superior nucleus (CS, median raphe nucleus). 5-ht(5B) receptor mRNA hybridization signal was often found on serotonin transporter mRNA-positive neuron profiles. The degree of cellular colocalization of these mRNAs notably varied among the different regions of the raphe nuclei. In the DR, cell bodies showing 5-ht(5B) receptor mRNA expression were abundant in the medial portions of the nucleus, all of them being also labeled for serotonin transporter mRNA. In contrast, in the ventrolateral regions (lateral wings) of the DR, we observed serotonin transporter mRNA-positive cells, but they were devoid of 5-ht(5B) receptor mRNA signal. In the CS, the level of coexpression of 5-ht(5B) receptor mRNA with serotonin transporter mRNA was high in the intermediate portions of the nucleus; however, we were unable to detect specific 5-ht(5B) receptor mRNA hybridization signal in its caudal extent. Our results support the presence of 5-ht(5B) receptor in serotonergic neurons in the DR and CS, suggesting an autoreceptor role for this receptor subtype.     

Decreased 5-HT transporter mRNA in neurons of the dorsal raphe nucleus and behavioral depression in the obese leptin-deficient ob/ob mouse

The neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) is an important regulator of feeding behavior. A hypothalamic site of action for 5-HT in body weight control is supported by the presence of 5-HT receptors in hypothalamic regions which are intimately associated with regulation of food intake. In the present study we have investigated whether there may be an interaction between the hormone leptin, an adipose tissue-derived cytokine signaling factor that inhibits food intake and lowers body weight, and the brain serotonergic system. Immunohistochemical analysis of colchicine-treated rats showed colocalization of 5-HT transporter- and leptin receptor-immunoreactivity in cell bodies of the dorsal raphe nucleus, suggesting that dorsal raphe neurons are targets for circulating leptin. Levels of 5-HT transporter mRNA expression were compared in neurons of the dorsal raphe nucleus of obese leptin-deficient ob/ob mice and their lean littermates using in situ hybridization. 5-HT transporter mRNA levels were significantly down-regulated in neurons of the dorsal raphe nucleus of obese ob/ob mice as compared to lean control mice. Behavioral analysis showed that obese ob/ob mice had significantly lower locomotor activity and exhibited increased immobility in Porsolt's test, a model for depression. Taken together, these results suggest that serotonergic cell bodies in the rodent dorsal raphe nucleus possess leptin receptors and that the serotonergic system, as reflected by expression levels of 5-HT transporter mRNA, is down-regulated in the obese behaviorally depressed ob/ob mouse.     

Androgen actions on central serotonin neurotransmission: relevance for mood, mental state and memory.

Sex steroids exert potent effects on mood and mental state in the human. Our previous experimental findings in female rats suggest that these effects may be mediated, in part, by the action of estrogen on the 5-hydroxytryptamine2A receptor (5-HT(2A)R) and serotonin transporter (SERT) in brain. Here we review our recent findings on the effect of acute (approximately 32 h) testosterone manipulation on central 5-HT(2A)R and SERT in male rats. Castration decreased while testosterone or estrogen, but not 5alpha-dihydrotestosterone (5alpha-DHT), increased significantly the content of 5-HT(2A)R mRNA and SERT mRNA in the dorsal raphe nucleus (DR) and the density of 5-HT(2A)R and SERT binding sites in higher centers of the brain. The lack of effect of 5alpha-DHT, a potent androgen which cannot be converted to estrogen, suggests that the action of testosterone depends upon its conversion to estrogen by aromatase. This may also explain why estrogen, but not testosterone or 5alpha-DHT, increased the density of 5-HT(2A)R binding sites in the caudate-putamen, a brain region where aromatase is scarce. The estrogen induction of SERT mRNA is most prominent in the rostral DR and this together with the correlation between sensitivity of DR serotonin neurons to estrogen and neurotoxic amphetamine derivatives provides a potential topochemical handle with which to investigate testosterone/estrogen regulation of SERT gene expression. These findings are discussed in relation to the possible role of interactions between sex steroids and serotonin mechanisms in mood disorders, schizophrenia and Alzheimer's disease.     

Estrogen selectively increases tryptophan hydroxylase-2 mRNA expression in distinct subregions of rat midbrain raphe nucleus: association between gene expression and anxiety behavior in the open field.

BACKGROUND: Ovarian steroids modulate anxiety behavior, perhaps by regulating the serotonergic neurons in the midbrain raphe nucleus. The regulation of the brain-specific isoform of rat tryptophan hydroxylase (TPH2) by ovarian hormones has not yet been investigated. Therefore, we examined the effects of estrogen and progesterone on TPH2 mRNA in the rat dorsal and median raphe nuclei (DRN and MRN, respectively) and whether TPH2 mRNA levels correlated with anxiety behavior. METHODS: Ovariectomized rats were treated for two weeks with placebo, estrogen or estrogen plus progesterone, exposed to the open field test, and TPH2 mRNA was quantified by in situ hybridization histochemistry. RESULTS: Estrogen increased TPH2 mRNA in the mid-ventromedial and caudal subregions of the DRN and the caudal MRN. Combined estrogen and progesterone treatment did not change TPH2 mRNA relative to ovariectomized controls. TPH2 mRNA in caudal DRN was associated with lower anxiety-like behavior, whereas TPH2 mRNA in rostral dorsomedial DRN was associated with increased anxiety-like behavior. CONCLUSIONS: These results suggest that estrogen may increase the capacity for serotonin synthesis in discrete subgroups of raphe neurons, and reinforce previous observations that different subregions of DRN contribute to distinct components of anxiety behavior.     

Comparison of alpha2 nicotinic acetylcholine receptor subunit mRNA expression in the central nervous system of rats and mice

The nicotinic acetylcholine receptor (nAChR) alpha2 subunit was the first neuronal nAChR to be cloned. However, data for the distribution of alpha2 mRNA in the rodent exists in only a few studies. Therefore, we investigated the expression of alpha2 mRNA in the rat and mouse central nervous systems using nonradioactive in situ hybridization histochemistry. We detected strong hybridization signals in cell bodies located in the internal plexiform layer of the olfactory bulb, the interpeduncular nucleus of the midbrain, the ventral and dorsal tegmental nuclei, the median raphe nucleus of the pons, the ventral part of the medullary reticular nucleus, the ventral horn in the spinal cord of both rats and mice, and in a few Purkinje cells of rats, but not of mice. Cells that moderately express alpha2 mRNA were localized to the cerebral cortex layers V and VI, the subiculum, the oriens layer of CA1, the medial septum, the diagonal band complex, the substantia innominata, and the amygdala of both animals. They were also located in a few midbrain nuclei of rats, whereas in mice they were either few or absent in these areas. However, in the upper medulla oblongata alpha2 mRNA was expressed in several large neurons of the gigantocellular reticular nucleus and the raphe magnus nucleus of mice, but not of rats. The data obtained show that a similar pattern of alpha2 mRNA expression exists in both rats and mice, with the exception of a few regions, and provide the basis for cellular level analysis.     

Serotonergic Neurones in the Dorsal Raphe Nucleus That Project into the Medial Preoptic Area Contain Oestrogen Receptor β

Serotonin is involved in female sexual behaviour in which the medial preoptic area (MPA) has a pivotal role. The present study used immunohistochemistry, in situ hybridization and retrograde transport analysis to investigate whether serotonin neurones in the dorsal raphe nucleus (DRN) of females projecting into the MPA contained oestrogen receptor alpha or beta. The projection of serotonin neurones from the DRN to the MPA was confirmed using the microinjection of Fluoro-Gold (FG), a fluorescent retrograde tracer, into the MPA of ovariectomized (OVX-group) and OVX-rats treated with oestradiol benzoate (E2-group). A number of serotonin neurones in the DRN were labelled with FG, indicating that these serotonin neurones in DRN project their terminals into the MPA. FG-labelled serotonin neurones expressed ERbeta mRNA in the DRN, and the number of the serotonin neurones containing ERbeta mRNA between the OVX-group and the E2-treated group was not significantly different. Serotonin neurones in the DRN did not express ERalpha-immunoreactivity. Since previous findings showed that the density of serotonin-immunoreactive fibres and the concentration of serotonin within the MPA was significantly lower in the E2-group than the OVX-group, our present observations suggested that the regulatory effects of E2 on the serotonergic neurone system in the MPA may be via ERbeta within the serotonin-containing cells in the DRN of female rats.     

Axonal transport in serotonin neurons of the midbrain raphe.

The projections of serotonin-containing neurons of the midbrain raphe nuclei (nucleus raphe dorsalis, nucleus centralis superior) are studied by analysis of axonal transport of labeled amino acids. These results are correlated with regional alterations of serotonin content following midbrain raphe lesions which produce significant serotonin depletion in nearly all regions of the central nervous system. Twenty-four hours following injection of 100 muCi [3H]proline, raphe neurons have taken up labeled material and transported it, presumably as protein, to telencephalon, diencephalon, brain stem, the cerebellum and the spinal cord. This transport appears to take place predominantly in serotonin neurons. After injection of 100 muCi [3H]5-HTP into nucleus raphe dorsalis or nucleus centralis superior, the pattern of regional distribution of transported material is very similar to that obtained with tritiated proline. Selective lesions of serotonin terminals with 5.6-DHT result in greatly diminished axonal transport of proteins to all telencephalic, diencephalic and mesencephalic areas as well as to cerebellum, pons-medulla and spinal cord. Unilateral destruction of the medial forebrain bundle results in significant reduction in axonal transport of labeled material to ipsilateral telencehalon and thalamus. These results provide further support for the view that serotonin neurons of the midbrain raphe nuclei project widely throughout the neuraxis to telencephalon, diencephalon, brain stem, cerebellum and spinal cord.     

Increased tryptophan hydroxylase mRNA in raphe serotonergic neurons spared by 5,7-dihydroxytryptamine

Neurons expressing the tryptophan hydroxylase (TPH) mRNA within the raphe nuclei of control rats showed a distribution similar to that observed using an antibody for TPH. Numerous packed cells expressing the TPH mRNA were observed in the ventral and dorsal zone of the nucleus raphe dorsalis (NDR) and in the pars dorsalis of the nucleus centralis superior (NCS) whereas fewer and more scattered neurons were found in the pars medialis of NCS. Five days after the intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), which markedly reduced the serotonin (5-HT) content in the hippocampus, caudate putamen and cortex, the hybridization signal had completely disappeared in the dorsal region of the NDR. In the ventromedial region, above and between the medial longitudinal fasciculus (MLF), which includes the pars dorsalis of NCS, there was a partial decrease of cell number and a marked increase of the grain density over spared neurons. No significant change was noted in the number of TPH-positive cells and hybridization signal in individual neurons of the pars medialis of NCS. Consistent with previous evidence of increased TPH activity in the residual 5-HT terminals, the present study shows that synthesis of the TPH mRNA may be augmented in some neurons surviving the lesion.