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.     

Estrogen is more than just a "sex hormone": novel sites for estrogen action in the hippocampus and cerebral cortex

For decades estrogen was thought of only as a "sex hormone," as it plays a fundamental role in regulating behavioral and physiological events essential for successful procreation. In recent years, estrogen has been shown to exert effects on the structure and function of the hippocampus and cortex. The discovery of a new estrogen receptor (ER-beta) and localization of ER-alpha and ER-beta mRNAs in the pyramidal cells of the rat hippocampus and ER-beta mRNA in rat cortex have provided new insight into how estrogen may directly modulate the structure and function of these neurons. Moreover, recent in vivo (125)I-estrogen binding studies have shown that nuclear estrogen binding sites are widely distributed in the pyramidal cells throughout CA1-3 of the hippocampus and laminae II-VI of the isocortex, demonstrating that ER mRNAs are translated into biologically active protein. The functional impact of estrogen receptor localization in the cortex and hippocampus may prove relevant to the emerging role for estrogen as a protective factor in neurodegenerative injury. This potential role is further highlighted by the recent findings that the expression of ER-alpha and ER-beta changes following ischemic brain injury and that these changes correlate with the hormonal modulation of protective factors. These data provide the first evidence that the expression of ERs in the adult cortex is not static, but instead, responsive to neuronal injury and perhaps additional factors that influence the cortical environment and status of these neurons. Together, these data indicate that estrogen has a far greater effect on the hippocampus and isocortex than previously thought. Furthermore, these new findings challenge our current thinking about steroid hormones and their mechanism(s) of action in regions associated with learning and memory and affected by the neurodegenerative conditions of aging.     

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.     

Levels of estrogen receptors alpha and beta in frontal cortex of patients with Alzheimer's disease: relationship to Mini-Mental State Examination scores

Estrogen exerts beneficial effects on the brain throughout life. Studies demonstrate that estrogen is neuroprotective and that reduced brain estrogen activity may influence the clinical course of Alzheimer's disease (AD). Changes in levels of estrogen receptors have been detected in postmortem brain tissue of AD patients. Very little is known about the relationship between clinical stage and levels of estrogen receptors in postmortem brain. We hypothesized that estrogen receptor levels would be related to severity of cognitive impairment assessed proximate to death. Western blotting was used to quantify ER-alpha and ER-beta in nuclear, cytosolic, and crude membrane fractions of superior frontal cortex from 25 AD patients. Multiple linear regression analyses adjusted for age, sex, and education showed a significant linear relationship between Mini-Mental State Examination score (MMSE) and wild-type nuclear ER-alpha (a = 5.463, p = 0.03), but none between MMSE and wild-type nuclear ER-beta (a = 2.29, p = 0.36). We incidentally observed additional higher and lower molecular mass bands for ER-alpha in study subjects. Additional experiments performed on frontal cortex nuclear fractions prepared from subjects enrolled in a different study confirmed that these same bands are present in female and males with and without AD. Together our data show a relationship between wild-type ER-alpha and level of cognitive impairment in AD, and also suggest the possibility that variant isoforms of ER-alpha may be present in frontal cortex of patients with and without AD.     

Estrogen receptor-beta, but not estrogen receptor-alpha, is expressed in prolactin neurons of the female rat paraventricular and supraoptic nuclei: comparison with other neuropeptides

Estrogen receptor-alpha (ER-alpha) and ER-beta exhibit fine differences in their distributions in the rodent forebrain, and one such difference is observed in the paraventricular (PVN) and supraoptic (SON) nuclei. To investigate the functional significance of ER in these brain areas, we examined the neuropeptide characteristics of ER-expressing neurons in the PVN and SON of female rats by using dual-label immunocytochemistry. The distributions of ER-alpha immunoreactivity (ir) and ER-beta ir were nonoverlapping in the PVN and SON. Nuclear ER-alpha ir was found in a population of thyrotropin-releasing hormone (TRH)-expressing neurons in the PVN (5.93% +/- 1.20% SEM), but not in any other identified cell phenotype of the PVN and SON. The phenotype of neurons with the highest percentage expressing ER-beta was found to be prolactin (PRL) immunoreactive in both the parvocellular (84.95% +/- 4.11%) and the magnocellular (84.76% +/- 3.40%) parts of the PVN as well as the SON (87.57% +/- 4.64%). Similarly, most vasopressin-immunoreactive neurons were also ER-beta positive in the PVN (66.14% +/- 2.47%) and SON (72.42% +/- 4.51%). In contrast, although a high percentage of oxytocin (OXY) neurons coexpressed ER-beta in the PVN (84.39% +/- 2.99%), there was very little ER-beta/OXY colocalization in the SON. Low levels of corticotropin-releasing hormone neurons also expressed ER-beta ir in the PVN (12.57% +/- 1.99%), but there was no ER-beta colocalization with TRH. In summary, these findings further support the possibility of direct effects of estrogen on neuropeptide expression and implicate estrogen involvement in the regulation of various aspects of neuroendocrine function.     

Cigarette Smoking and Tryptophan Hydroxylase 2 mRNA in the Dorsal Raphe Nucleus in Suicides

Cigarette smoking is associated with suicide and mood disorders and stimulates serotonin release. Tryptophan hydroxylase (TPH2) synthesizes serotonin and is over-expressed in suicides. We determined whether smoking is associated with TPH2 mRNA in suicides and controls. TPH2 mRNA was measured postmortem in the dorsal raphe nucleus (DRN) of controls (N = 26, 17 nonsmokers and nine smokers) and suicides (N = 23, 5 nonsmokers and 18 smokers). Psychiatric history was obtained by psychological autopsy. TPH2 mRNA was greater in suicide nonsmokers than suicide smokers, control smokers and control nonsmokers (p = 0.006). There was more TPH2 mRNA throughout the DRN. Smoking interferes with the TPH2 mRNA increase observed in suicide nonsmokers. The absence of altered TPH2 expression in non-suicide smokers suggests no pharmacological effect of smoking.     

Effects of perinatal exposure to low-dose cadmium on thyroid hormone-related and sex hormone receptor gene expressions in brain of offspring

Perinatal cadmium (Cd) exposure has been shown to alter behaviors and reduce learning ability of offspring. A few studies have shown that Cd reduced serum thyroid hormones (THs), which are important for brain development during the perinatal period. Brain specific genes, neurogranin (RC3) and myelin basic protein (BMP), are known to be regulated by TH through TH receptors (TR). It has been suggested that RC3 may play roles in memory and learning. In addition, Cd has been suggested to have estrogen-like activity. To evaluate the effects of perinatal low-dose exposure to Cd on thyroid hormone-related gene (RC3, TR-beta1, MBP, RAR-beta) and sex hormone receptor gene (ER-alpha, ER-beta and PgR) expressions in the brain and on behaviors of offspring, mice were administered with 10ppm Cd (from gestational day 1 to postnatal day 10) and/or 0.025% methimazole (MMI; anti-thyroid drug) (from gestational day 12 to postnatal day 10) in drinking water. Also, 0.1% MMI was administered as a positive control (high MMI group). RC3 mRNA expression was reduced in the female brain of combined exposure and high MMI groups and was negatively correlated with the activity in the open-field. ER-alpha, ER-beta and PgR mRNA expressions were decreased in male and female Cd, and female Cd+MMI groups, respectively; among these changes the reduced expression of PgR was opposite to estrogenic action. These results suggested that perinatal exposure to Cd disrupted the gene expressions of sex hormone receptors, which could not be considered to be a result of estrogenic action. Our study indicates that alteration in the gene expressions of RC3 and sex hormone receptors in the brain induced by perinatal Cd and MMI exposure might be one mechanism of developmental toxicity of Cd.     

Regulation of the content of progesterone and estrogen receptors, and their cofactors SRC-1 and SMRT by the 26S proteasome in the rat brain during the estrous cycle

In this work we have determined the role of the 26S proteasome in the regulation of the content of progesterone receptors (PR-A and PR-B), estrogen receptors (ER-alpha and ER-beta), the coactivator SRC-1 and the corepressor SMRT in the rat brain during the estrous cycle. The 26S proteasome inhibitor MG132 was injected once into the lateral ventricle on proestrous day; and 24h later, on estrous day we evaluated the content of PR and ER isoforms, SRC-1 and SMRT in the hypothalamus, the preoptic area and the hippocampus by Western blot. A significant increase in the content of both PR isoforms, ER-beta and SRC-1 was observed after the administration of MG132 in the three studied cerebral regions. SMRT content was increased in the hypothalamus and the preoptic area and a significant increase in ER-alpha content was only observed in the preoptic area. These results suggest that essential proteins that participate in progesterone and estrogen actions in the brain should be regulated by the 26S proteasome in a tissue-specific manner in physiological conditions.     

Maternal high n-6 polyunsaturated fatty acid intake during pregnancy increases voluntary alcohol intake and hypothalamic estrogen receptor alpha and beta levels among female offspring

Identification of nongenetic biological factors that predispose to alcohol abuse is central to attempts to prevent alcoholism. Since an exposure to estradiol in utero increases voluntary alcohol intake in adulthood, we investigated whether an increase in pregnancy estradiol levels, caused by feeding pregnant mice a high-fat corn oil diet, also influences voluntary alcohol intake among female offspring. In addition, the effect on estrogen receptor alpha (ER-alpha) and ER-beta protein levels in the brain using Western blot assay, was determined. Pregnant CD-1 mice were kept on a high n-6 polyunsaturated fatty acid (PUFA; 43% calories from corn oil) or low n-6 PUFA diet (16% calories from corn oil) throughout gestation, and switched to a Purina laboratory chow after the pups were born. When 4 months of age, the female offspring were given a choice between 5% alcohol and tap water. The offspring of high n-6 PUFA mothers voluntarily consumed more alcohol than the offspring of low n-6 PUFA mothers. ER-alpha and ER-beta protein levels in the hypothalamus were 1.5- and 2-fold higher, respectively, in the female offspring of high n-6 PUFA mothers than in the low n-6 PUFA offspring. No significant changes in the protein levels of ER-alpha and ER-beta were seen in the frontal brain. Our findings indicate that a maternal exposure to a high n-6 PUFA diet during pregnancy increases alcohol intake among female offspring. This behavioral change, together with previously observed increase in aggressiveness and reduction in depressive-like behavior in these offspring, may be linked to an increase in the hypothalamic ER-alpha and ER-beta levels.     

鱼藤酮处理大鼠脑内VMAT2及TPH、5-HT的表达改变

目的观察鱼藤酮处理大鼠脑内单胺囊泡转运体2(VMAT2)。色氨酸羟化酶(TPH)及5—羟色胺(5—HT)的表达变化;探讨其对5—HT能神经元的影响及可能机制。方法选用健康、成年雄性Wistar大鼠,背部皮下注射鱼藤酮3d或28d制作大鼠动物模型;利用免疫细胞化学分析VMAT2在大鼠黑质、中缝背核和尾壳核以及TPH、5—HT在中缝背核的表达变化;以透射电镜观察轴突超微结构的改变,探讨鱼藤酮毒性作用的可能机制。结果鱼藤酮3d组:(1)鱼藤酮组大鼠黑质、中缝背核VMAT2的表达增加,免疫反应强度吸光度值显著高于对照组;尾壳核VMAT2的表达减弱,免疫反应强度吸光度值明显低于对照组(P0.01);(2)鱼藤酮组大鼠中缝背核TPH、5—HT的表达减少,免疫反应强度吸光度值显著低于对照组(P0.01);(3)透射电镜观察显示鱼藤酮处理大鼠中缝背核轴突变性、其内微管解聚。鱼藤酮28d组:(1)鱼藤酮组大鼠黑质、中缝背核及尾壳核VMAT2的表达均减弱,免疫反应强度吸光度值显著低于对照组(P0.01或P0.05);(2)与对照组相比,鱼藤酮组大鼠中缝背核TPH、5—HT的表达降低,免疫反应强度吸光度值显著低于对照组(P0.01)。结论鱼藤酮降低5—HT能神经元TPH、5—HT的表达,其毒性作用可能与轴突微管解聚导致突触囊泡VMAT2轴浆顺行转运障碍有关。     

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.     

Expression of 5-HT1A receptor mRNA in rat dorsal raphe nucleus and ventrolateral periaqueductal gray neurons after peripheral inflammation

In the present study we observed the expression of 5-hydroxytryptamine (5-HT)1A receptor mRNA in the dorsal raphe nucleus (DRN) and ventrolateral periaqueductal gray (vlPAG) neurons, especially in 5-HT immunoreactive neurons (5-HT-IR), using in situ hybridization (ISH) and double staining with fluorescent ISH (FISH) and immunohistochemical (FIH) techniques. The findings of this study demonstrated that 5-HT1A receptor mRNA was expressed with moderate to high level in the DRN and vlPAG neurons. Following carrageenan inflammation, the expression of 5-HT1A receptor mRNA in the DRN and bilateral vlPAG neurons was significantly increased. The peak occurred at 3-8h followed by a clear decrease at 24 h, which basically corresponded to the time-course of behavioral hyperalgesia. Moderate 5-HT1A receptor mRNA and 5-HT immunoreactive (5-HT-IR) double-labeled cells were observed in the DRN and vlPAG, suggesting that some of 5-HT1A receptors in the DRN and vlPAG may be autoreceptors. Eight hours after carrageenan injection, the number of the double labeled cells was significantly increased. These results suggest that the synthesis of 5-HT1A receptors, including autoreceptors, is increased in the DRN and vlPAG during peripheral inflammation.     

Oestrogen upregulates noradrenaline release in the mediobasal hypothalamus and tyrosine hydroxylase gene expression in the brainstem of ovariectomized rhesus macaques

Noradrenaline plays a key role in the initiation of ovulation in nonprimate species. A similar noradrenaline role in the primate has not been established experimentally. We utilized the ovariectomized-oestrogen-supplemented (OVX + E) rhesus macaque to examine the effects of intravenous (i.v.) infusion of oestradiol-17beta (E2) on the activity of the brain noradrenaline system. Experiment 1 established the induction of a preovulatory surge-like release of luteinizing hormone in OVX + E monkeys by i.v. infusion of E2 (OVX + E + E2). In experiment 2, a marked increase in hypothalamic microdialysate noradrenaline concentrations occurred after identical E2 infusion into the OVX + E monkeys that were used in experiment 1. In experiment 3, tyrosine hydroxylase (TH) mRNA expression in the locus coeruleus of the brainstem increased at various times after E2 infusion as determined by semiquantitative in situ hybridization. The amount of TH mRNA in OVX + E + E2 animals was higher (P < 0.05) than that in either the OVX + E or OVX monkeys; no difference was found in the latter two groups. Moreover, selected locus coeruleus sections from E2-infused monkeys were examined for the localization of oestrogen receptors (ER) by in situ hybridization. Both ER-alpha and ER-beta mRNAs were expressed in the locus coeruleus, although the expression was greater for ER-alpha than for ER-beta. We conclude that i.v. infusion of E2, which induces a preovulatory surge-like release of LH, stimulates brain noradrenaline activity; this enhanced activity likely involves an ER-mediated process and is reflected by hypothalamic noradrenaline release and locus coeruleus TH mRNA expression. The results support the concept that noradrenaline can influence the E2-stimulated ovulation in nonhuman primates and that the brainstem is one of the components in this neuroendocrine process.     

Estrogen receptor-alpha is associated with the plasma membrane of astrocytes and coupled to the MAP/Src-kinase pathway

Estrogens influence CNS development and a broad spectrum of neural functions. Several lines of evidence also suggest a neuroprotective role for estrogen. Different modes of estrogen action have been described at the cellular level involving classical nuclear estrogen receptor (ER)-dependent and nonclassical membrane ER-mediated rapid signaling. We have previously shown that nonclassical estrogen signaling is implicated in the control of dopamine cell function and protection. Since nonclassical interactions between estrogens and glia may contribute to these effects, our aim was to demonstrate the presence of membrane-associated ERs and their putative coupling to intracellular signaling pathways in astrocytes. Confocal image analysis and fluorescence-activated cell sorting (FACS) studies indicated the attachment of ER-alpha but not ER-beta to the plasma membrane of astrocytes. ERs were located in the cell soma region and glial processes. FACS analysis revealed that only a subpopulation of midbrain astrocytes possesses membrane ER-alpha. In FACS studies on ER-alpha knockout astrocytes, only a few membrane ER-positive cells were detected. The activation of membrane ERs appears to be coupled to the MAP-kinase/Src signaling pathway as shown by Western blotting. In conclusion, our data provide good evidence that nonclassical estrogen action in astrocytes is mediated by membrane ER-alpha. The physiological consequence of this phenomenon is not yet understood, but it might have a pivotal role in estrogen-mediated protective effects on midbrain dopamine neurons.