Extra-nuclear estrogen receptor GPR30 regulates serotonin function in rat hypothalamus.

Selective serotonin reuptake inhibitors (SSRIs), such as Prozac, are used to treat mood disorders. SSRIs attenuate (i.e. desensitize) serotonin 1A (5-HT(1A)) receptor signaling, as demonstrated in rats through decreased release of oxytocin and adrenocorticotropin hormone (ACTH) following 5-HT(1A) receptor stimulation. Maximal therapeutic effects of SSRIs for treatment of mood disorders, as well as effects on hypothalamic 5-HT(1A) receptor signaling in animals, take 1 to 2 weeks to develop. Estradiol also attenuates 5-HT(1A) receptor signaling, but, in rats, these effects occur within 2 days; thus, estrogens or selective estrogen receptor modulators may serve as useful short-term tools to accelerate desensitization of 5-HT(1A) receptors in response to SSRIs if candidate estrogen receptor targets in the hypothalamus are identified. We found high levels of GPR30, which has been identified recently as a pertussis-toxin (PTX) sensitive G-protein-coupled estrogen receptor, in the hypothalamic paraventricular nucleus (PVN) of rats. Double-label immunohistochemistry revealed that GPR30 co-localizes with 5-HT(1A) receptors, corticotrophin releasing factor (CRF) and oxytocin in neurons in the PVN. Pretreatment with PTX to the PVN before peripheral injections of 17-beta-estradiol 3-benzoate completely prevented the reduction of the oxytocin response to the 5-HT(1A) receptor agonist, (+)-8-hydroxy-2-dipropylaminotetralin (DPAT). Treatment with the selective GRP30 agonist, G-1, attenuated 5-HT(1A) receptor signaling in the PVN as measured by an attenuated oxytocin (by 29%) and ACTH (by 31%) response to DPAT. This study indicates that a putative extra-nuclear estrogen receptor, GPR30, may play a role in estradiol-mediated attenuation of 5-HT(1A) receptor signaling, and potentially in accelerating the effects of SSRIs in treatment of mood disorders.

Treatment of cycling female rats with fluoxetine induces desensitization of hypothalamic 5-HT(1A) receptors with no change in 5-HT(2A) receptors.

Although women constitute the majority of patients who receive treatment with selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, most animal studies of SSRIs are conducted on males. The present study investigated whether long-term treatment of cycling female rats with fluoxetine alters their estrous cycle and the sensitivity of hypothalamic serotonin (5-HT) 5-HT(1A) and 5-HT(2A) receptor systems. Adult female rats received daily injections of fluoxetine (10 mg/kg, i.p.) for three consecutive estrous cycles (15.2+/-0.2 days) with the first injection beginning on metestrus (when circulating estrogen levels are low and stable). Fluoxetine did not alter basal plasma estradiol levels at metestrus, nor did it alter the pattern of estrous cyclicity. Rats treated with fluoxetine showed a loss in body weight. On the morning of metestrus of the fourth cycle (18 h after the last fluoxetine injection), the rats were injected with a sub-maximal dose of the 5-HT(1A) agonist (+/-)-8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT, 50 MICRO/kg, s.c.) or a maximal dose of the 5-HT(2A) agonist [(+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl] (DOI). Plasma levels of oxytocin, ACTH and corticosterone were measured as peripheral indicators of hypothalamic 5-HT(1A) and 5-HT(2A) receptor sensitivity. Injecting 8-OH-DPAT to saline pretreated rats produced a significant increase in plasma oxytocin (299%), ACTH (1456%) and corticosterone (170%) levels but not in plasma prolactin or renin concentrations. Greater increases in plasma levels of these hormones were observed after injecting DOI. Fluoxetine treatment completely blocked the oxytocin, ACTH and corticosterone responses to 8-OH-DPAT, but did not inhibit the effect of DOI on any hormone, thus confirming that fluoxetine treatment did not produce a deficit in the functioning of corticotropin releasing hormone or oxytocin containing neurons. These results indicate that in cycling female rats, fluoxetine treatment desensitizes hypothalamic post-synaptic 5-HT(1A) receptor signaling. Understanding the pharmacological effects of fluoxetine in females may lead to more effective treatment of women with mood disorders.

Ontogeny of region-specific sex differences in androgen receptor messenger ribonucleic acid expression in the rat forebrain.

Testosterone and its metabolites are the principal gonadal hormones responsible for sexual differentiation of the brain. However, the relative roles of the androgen receptor (AR) vs. the estrogen receptor in specific aspects of this process remain unclear due to the intracellular metabolism of testosterone to active androgenic and estrogenic compounds. In this study, we used an 35S-labeled riboprobe and in situ hybridization to analyze steady state, relative levels of AR messenger RNA (mRNA) expression in the developing bed nucleus of the stria terminalis, medial preoptic area, and lateral septum, as well as the ventromedial and arcuate nuclei of the hypothalamus. Each area was examined on embryonic day 20 and postnatal days 0, 4, 10, and 20 to produce a developmental profile of AR mRNA expression. AR mRNA hybridization was present on embryonic day 20 in all areas analyzed. In addition, AR mRNA expression increased throughout the perinatal period in all areas examined in both males and females. However, between postnatal days 4 and 10, sharp increases in AR mRNA expression in the principal portion of the bed nucleus of the stria terminalis and the medial preoptic area occurred in the male that were not paralleled in the female. Subsequently, males exhibited higher levels of AR mRNA than females in these areas by postnatal day 10. There was no sex difference in AR mRNA content in the lateral septum, ventromedial nucleus, or arcuate nucleus at any age. These results suggest that sex differences in AR mRNA expression during development may lead to an early sex difference in sensitivity to the potential masculinizing effects of androgen.

Region-specific effects of ovarian hormones on estrogen receptor immunoreactivity.

The cell groups in which nuclear estrogen receptor (ER) expressing neurons are found have unique, often coordinated, functions. Regulation of ER content may be one mechanism through which feedback responses can be adjusted to match the function of a specific brain region and physiological circumstance. In these immunocytochemical experiments, estrogen decreased staining intensity for ER in the ventrolateral hypothalamus and bed nucleus of the stria terminalis, but not in the periventricular preoptic area. ER staining intensity was further decreased by progesterone, following estrogen, but not in all brain regions. These results suggest that ER is regulated by estrogen in a region-specific manner. Furthermore, inhibition of responses to estrogen by progesterone may involve progesterone-induced down-regulation of ERs.

Distribution of estrogen receptor-immunoreactive cells in the forebrain of the female guinea pig.

We mapped the distribution of estrogen receptor-containing cells in the forebrain of the adult female guinea pig. Cellular estrogen receptor content was detected using monoclonal antibody H222, directed against the estrogen receptor, and the avidin-biotin method with nickel-intensified diaminobenzidine as the chromagen. A complete set of deletion, titration, and adsorption controls established the specificity of the staining. The most dense collections of estrogen receptor-immunoreactive cells were found in medial preoptic, medial hypothalamic, and limbic nuclei (amygdala, bed nucleus of the stria terminalis, lateral septum). Numerous estrogen receptor-immunoreactive cells were also found in additional, specific subregions of the remainder of the preoptic area, hypothalamus, and limbic system, and also in the midbrain (central gray). Elsewhere, estrogen receptor-immunoreactive cells were present in smaller numbers or were absent. This map confirms and extends previous maps based on estrogen binding. The majority of estrogen receptor-immunoreactive cells are found in areas known to be involved in some aspect of reproduction. In addition, many estrogen receptor-immunoreactive cells are found in areas not typically considered to have a primary role in reproductive behavior or neuroendocrine function.

A subset of progesterone target neurons have axonal projections to the midbrain.

Progestin-concentrating neurons in the preoptic area and hypothalamus that project to the midbrain in the female rat were identified using the combined steroid hormone autoradiography-retrograde axonal tracing technique. Progesterone target neurons were most abundant in the periventricular preoptic area and the medial preoptic nucleus, and in the ventromedial and arcuate nuclei of the hypothalamus. In the medial preoptic area as a whole, about 14% of the progestin-concentrating cells were afferent to the midbrain. More specifically, 23% of medial preoptic nucleus progesterone target neurons communicated directly with midbrain cell groups, whereas a much smaller percentage (2%) of periventricular preoptic target neurons projected to the midbrain. In the medial basal hypothalamus as a whole, 11% of the progestin-concentrating cells detected sent axons to the midbrain. This proportion was slightly higher in the ventromedial nucleus (15%), and much lower in the arcuate nucleus (3%). In the dorsal and lateral hypothalamic areas, close to 30% of the progesterone target neurons sent axons to the midbrain, although the total number and density of target cells in the two latter areas was low. These data support the idea that transduction by forebrain target neurons of the progesterone signal into altered synaptic transmission in the midbrain is one route through which progesterone can influence a variety of behaviors.

Estrogen plus progesterone increases progestin receptor immunoreactivity in the brain of ovariectomized guinea pigs.

The goal of these experiments was to determine the number and distribution of brain cells that contain progestin receptors (PR) and to determine the effect of estrogen and estrogen plus progesterone on PR content of those cells. Ovariectomized adult female guinea pigs were treated with oil (control), or estrogen followed by oil, or estrogen followed by progesterone. As expected, only those animals treated with estrogen plus progesterone became sexually receptive. The cellular content of PR was determined using a monoclonal antibody to the receptor, and standard immunocytochemical techniques. Analysis of the PR-immunoreactive (PR-IR) cells consisted of: (1) mapping the anatomical distribution of PR-IR cells; (2) analyzing the effect of steroid hormones on PR-IR cell number, and (3) determining the effect of steroid hormones on PR immunoreactivity per cell. PR immunoreactivity was located exclusively in the nuclei of cells in the preoptic area and hypothalamus. The most dense collections of PR-IR cells were found in the preoptic area, ventrolateral nucleus of the hypothalamus, and infundibular nucleus. Estrogen caused a dramatic increase in the number of PR-IR cells in these cell groups. Sequential treatment with estrogen plus progesterone further increased PR-IR cell number, in the preoptic area by 65%, in the ventrolateral nucleus by 38%, and in the infundibular nucleus by 49%. A cell-by-cell rating of the PR immunoreactivity was carried out in these three cell groups. We found that the staining intensity across the populations of PR-IR cells was increased by estrogen and further increased by sequential estrogen plus progesterone. Alterations in cellular PR content may contribute importantly to the ability of progesterone target cell groups to perform their specialized roles in steroid-regulated activity.

Immunocytochemical analysis of somatostatin in the hypothalamus of obese and non-obese Zucker rats.

Levels of growth hormone (GH) are reduced in the genetically obese Zucker rat, fa/fa, in comparison to lean littermates. In normal rats, GH release is regulated by stimulatory and inhibitory factors of hypothalamic origin. The present experiment focuses on hypothalamic somatostatin (SOM; growth hormone release inhibiting factor) in order to determine if abnormal hypothalamic SOM may be a correlate of depressed GH secretion in fa/fa rats. We compared immunocytochemical localization of hypothalamic SOM between 5 obese (fa/fa) Zucker rats and 5 non-obese littermates. Brain sections from pairs of animals were processed simultaneously. The distribution of SOM immunoreactive cell bodies in the hypothalamus agreed with previous reports. SOM-containing neurons in the periventricular area were counted and analyzed at 4 hypothalamic levels: (1) anterior to the suprachiasmatic nucleus (SCN); (2) through SCN; (3) between SCN and the ventromedial hypothalamic nucleus (VMH); and (4) through VMH. The greatest number of SOM-immunoreactive cell bodies was observed at levels (2) and (3). The numbers of SOM-containing cells did not differ significantly between obese and lean animals. No apparent difference in density of fiber staining was observed in the median eminence.

Hypothalamo-spinal projections in the golden hamster.

The distribution of hypothalamic projections to the spinal cord in hamsters was determined using the retrograde tracers horseradish peroxidase (HRP) and wheat germ agglutinin-HRP (WGA-HRP). Large injections of HRP or WGA-HRP were made into the thoracic spinal cord of adult male golden hamsters. HRP-labeled neurons were observed primarily in the parvocellular division of the paraventricular nucleus and in the lateral hypothalamus. The organization of hypothalamo-spinal connections appears to be highly conserved in mammalian species.