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.     

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.     

Promoter polymorphism of second tryptophan hydroxylase isoform (TPH2) in schizophrenia and suicidality

Allele and haplotype frequencies of a promoter polymorphism in the gene encoding tryptophan hydroxylase (TPH2) did not differ in 83 suicidal schizophrenic patients compared with 170 non-suicidal schizophrenic patients. These findings suggest that these 5' marker haplotypes in the TPH2 gene do not influence suicidal behavior in schizophrenia.     

Tyrosine hydroxylase and POMC mRNA in the arcuate region are increased by castration and hyperprolactinemia.

We have examined the changes which occur in neuronal expression of tyrosine hydroxylase (TH) and proopiomelanocortin (POMC) mRNA in response to castration and hyperprolactinemia (HP) in male rats. Steady-state mRNA levels were determined by quantitative in situ hybridization histochemistry (ISHH) using 35S-labeled synthetic 48-base oligodeoxynucleotide probes. Castration produced a 27% increase in TH mRNA in the periventricular and arcuate nuclei. PRL-exposed rats exhibited a further 27% increase in the level of TH mRNA and a striking 48% increase in POMC mRNA in periarcuate region cell bodies. These results indicate that gonadal steroids and PRL are involved, either directly or indirectly, in regulating the biosynthesis of TH and POMC in the hypothalamus.     

Increased tryptophan hydroxylase immunoreactivity in the dorsal raphe nucleus of alcohol-dependent, depressed suicide subjects is restricted to the dorsal subnucleus

Considerable evidence suggests that alcoholics with co-occurring depressive disorder are at greater risk for developing psychosocial problems particularly suicidal behavior. Moreover, dysfunction in serotonin (5-HT) neurotransmission has been implicated in depression, suicide and alcoholism. In the present study, we measured the levels of tryptophan hydroxylase (TPH), the main synthetic enzyme of 5-HT synthesis, in specific nuclei of the dorsal raphe (DR) in depressed suicide victims with alcohol dependence and matched psychiatrically normal controls. TPH immunoreactivity (IR) was quantified in frozen tissue sections containing the DR from 8 suicide victims with a diagnosis of major depression and alcohol dependence, and 8 psychiatrically normal control subjects by using immunoautoradiographic methods. We found that the levels of TPH-IR were significantly increased by 46% in the dorsal subnucleus of the DR in depressed suicide victims with alcohol dependence when compared with controls. In contrast, TPH-IR levels did not significantly differ in the other DR subnuclei between depressed, alcoholic suicide subjects, and controls. Our results indicate that abnormalities in 5-HT biosynthesis in the brain of depressed alcoholic suicide subjects are restricted within distinct regions of the DR.     

Impaired executive control is associated with a variation in the promoter region of the tryptophan hydroxylase 2 gene

Current models of attention describe attention not as a homogenous entity but as a set of neural networks whose measurement yields a set of three endophenotypes-alerting, orienting, and executive control. Previous findings revealed different neuroanatomical regions for these subsystems, and data from twin studies indicate differences in their heritability. The present study investigated the molecular genetic basis of attention in a sample of 100 healthy subjects. Attention performance was assessed with the attention network test that distinguishes alerting, orienting, and executive control (conflict) using a simple reaction time paradigm with different cues and congruent and incongruent flankers. Two gene loci on candidate genes for cognitive functioning, the functional catechol-O-methyltransferase (COMT) VAL158MET and the tryptophan hydroxylase 2 (TPH2) -703 G/T promoter polymorphism, were tested for possible associations with attention. COMT is involved in the catabolism of dopamine, and TPH is the rate-limiting enzyme for serotonin synthesis. Results showed no effect of the COMT polymorphism on attention performance. However, the TT genotype of TPH2 -03 G/T was significantly associated with more errors (a possible indicator of impaired impulse control; p = .001) and with decreased performance in executive control (p = .001). This single-nucleotide polymorphism on the TPH2 gene explained more than 10% of the variance in both indicators of attention stressing the role of the serotonergic system for cognitive functions.     

Tyrosine hydroxylase mRNA is increased in old age and norepinephrine uptake transporter mRNA is decreased in middle age in locus coeruleus of Brown-Norway rats

In normal aging, cell loss occurs in the locus coeruleus (LC), the major noradrenergic nucleus in the brain. This study examined changes in the LC of aged rats by measuring mRNA expression for tyrosine hydroxylase (TH) and the norepinephrine uptake transporter (NET). TH and NET mRNA expression were measured by in situ hybridization in young, middle-aged and aged rats. It appears that in middle age, the transporter system responds initially to LC cell loss by decreasing NET mRNA expression. Then, with further aging and cell loss, TH mRNA expression increases which may potentially increase NE synthesis in the remaining neurons. These findings suggest that multiple regulatory components are used to maintain stable noradrenergic synaptic levels despite neuronal loss. Published by Elsevier Science B.V.     

Maternal ethanol administration inhibits 5-hydroxytryptamine synthesis and tryptophan hydroxylase expression in the dorsal raphe of rat offspring

Maternal ethanol consumption during pregnancy has a detrimental effect on the central nervous system (CNS) development of fetus. 5-Hydroxytryptamine (5-HT) is an important neurotransmitter and/or neuromodulator in the mammalian CNS. Tryptophan hydroxylase (TPH) is the rate limiting enzyme of 5-HT synthesis. Ethanol is known to induce neuropsychiatric disorders by alteration of the central serotonergic system. In the present study, the effects of maternal ethanol intake on the 5-HT synthesis and the TPH expression in the dorsal raphe of rat offspring were investigated. The present results show that the synthesis of 5-HT and the expression of TPH in the dorsal raphe of rat offspring were suppressed by maternal ethanol intake and that the suppressive effect of alcohol was more potent in the 5 weeks old rat pups compared to the 3 weeks old rat pups. Based on the present study, it can be suggested that the pathogenesis of ethanol-induced neuropsychological disorders involves ethanol-induced suppression on the 5-HT synthesis and the TPH expression in the dorsal raphe of offspring.     

Ovarian steroids increase PSD‐95 expression and dendritic spines in the dorsal raphe of ovariectomized macaques

Estradiol (E) and progesterone (P) promote spinogenesis in several brain areas. Intracellular signaling cascades that promote spinogenesis involve RhoGTPases, glutamate signaling and synapse assembly. We found that in serotonin neurons, E ± P administration increases (a) gene and protein expression of RhoGTPases, (b) gene expression of glutamate receptors, and (c) gene expression of pivotal synapse assembly proteins. Therefore, in this study we determined whether structural changes in dendritic spines in the dorsal raphe follow the observed changes in gene and protein expression. Dendritic spines were examined with immunogold silver staining of a spine marker protein, postsynaptic density‐95 (PSD‐95) and with Golgi staining. In the PSD‐95 study, adult Ovx monkeys received placebo, E, P, or E + P for 1 month (n = 3/group). Sections were immunostained for PSD‐95 and the number of PSD‐95‐positive puncta was determined with stereology. E, P, and E + P treatment significantly increased the total number of PSD‐95‐positive puncta (ANOVA, P = 0.04). In the golgi study, adult Ovx monkeys received placebo, E or E + P for 1 month (n = 3–4) and the midbrain was golgi‐stained. A total of 80 neurons were analyzed with Neurolucida software. There was a significant difference in spine density that depended on branch order (two‐way ANOVA). E + P treatment significantly increased spine density in higher‐order (3°–5°) dendritic branches relative to Ovx group (Bonferroni, P < 0.05). In summary, E + P leads to the elaboration of dendritic spines on dorsal raphe neurons. The ability of E to induce PSD‐95, but not actual spines, suggests either a sampling or time lag issue. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and, in turn, increase serotonin neurotransmission throughout the brain. Synapse 67:897–908, 2013 . © 2013 Wiley Periodicals, Inc.     

Elevated expression of tryptophan hydroxylase-2 mRNA at the neuronal level in the dorsal and median raphe nuclei of depressed suicides

Deficient levels of serotonin are associated with suicide and depression. Paradoxically, in the dorsal raphe nucleus (DRN) there are more serotonin neurons and more neuronal tryptophan hydroxylase-2 (TPH2) expression postmortem in depressed suicides. In this study, we sought to determine whether greater TPH2 expression in depressed suicides was the result of more TPH2 expression per neuron. In situ hybridization and computer-assisted image analysis were performed on tissue sections throughout the extent of the raphe nuclei at the level of silver grains per neuron to systematically quantify TPH2 neuronal expression. Depressed suicides have 26.5% more TPH2 grain density per neuron in the DRN compared with matched controls (P=0.04). The difference in grain density is greater at mid- and caudal anatomical levels across the rostrocaudal axis of the DRN. Densitometric analysis of TPH2 expression in the DRN subnuclei showed that higher expression levels were observed at posterior anatomical levels of depressed suicides (121% of control in the caudal subnucleus). Higher TPH2 expression in depressed suicides may explain more TPH2 protein and reflect a homeostatic response to deficient serotonin levels in the brains of depressed suicides. Localized changes in TPH2 expression in specific subnuclei of the DRN suggest that the serotonergic compensatory mechanism in depression and suicide is specifically regulated within the DRN and has implications for regions innervated by this subnucleus.     

Tyrosine hydroxylase,tryptophan hydroxylase,biopterin, and neopterin in the brain of anorexia nervosa

Summary The activities of tyrosine hydroxylase and tryptophan hydroxylase and contents of biopterin and neopterin were measured for the first time in various regions of human brain from a patient with anorexia nervosa (AN). In AN as compared with controls, tyrosine hydroxylase activity was markedly reduced in all brain regions analyzed, while tryptophan hydroxylase activity and biopterin content had a tendency to increase. Neopterin content did not change dramatically. The opposite changes of tyrosine hydroxylase and tryptophan hydroxylase suggest an imbalance between the activity of catecholaminergic neurons and that of serotonergic neurons, and may be related to pathogenesis of AN.