Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats.

The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by intracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17ß-estradiol (free E2). In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior induced by G1 in ovariectomized (OVX), E2-benzoate (EB)-primed rats. In Experiment 1, icv injection of G1 induced lordosis behavior at 120 and 240min. In Experiment 2, icv injection of the GPER-1 antagonist G15 significantly reduced lordosis behavior induced by either G1 or free E2. In addition, Antide, a GnRH-1 receptor antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv injection of Antide blocked the stimulatory effect of E2 on lordosis behavior. In Experiment 3, systemic injection of either tamoxifen or RU486 significantly reduced lordosis behavior induced by icv administration of G1 in OVX, EB-primed rats. The results suggest that GnRH release activates both estrogen and progestin receptors and that this activation is important in the chain of events leading to the display of lordosis behavior in response to activation of GPER-1 in estrogen-primed rats.

Estrogen receptors regulate the estrous behavior induced by progestins, peptides, and prostaglandin E2.

The role of classical estrogen receptors (ERs) in priming female reproductive behavior has been studied previously; however, the participation of this receptor during activation of estrous behavior has not been extensively studied. The purpose of this work was to test the possibility that the facilitation of lordosis behavior in estrogen-primed rats by progesterone (P) and its 5α- and 5ß-reduced metabolites, gonadotropin-releasing hormone (GnRH), leptin, prostaglandin E2 (PGE2) and vagino-cervical stimulation (VCS) involves interactions with classical ERs by using the selective ER modulator, tamoxifen. To further assess the role of ERs, we also explored the effects of the pure ER antagonist, ICI182780 (ICI), on estrous behavior induced by P and GnRH. Ovariectomized, estrogen-primed rats (5µg estradiol benzoate 40h earlier) were injected intraventricularly with the above-mentioned compounds, or they received VCS. All compounds and VCS effectively facilitated estrous behavior when tested at 60, 120 or 240min after infusion or application of VCS. Intraventricular infusion of tamoxifen (5µg), 30min before, significantly attenuated estrous behaviors induced in estradiol-primed rats by P, most of its 5α- and 5ß-reduced metabolites, GnRH, and PGE2, but not by VCS. Although there was a trend for reduction, tamoxifen did not significantly decrease lordosis in females treated with 5ß-pregnan-3,20-dione. ICI also inhibited lordosis behavior induced by P and GnRH at some testing intervals. These results suggest that activation of classical ERs participates in the triggering effects on estrous behavior induced by agents with different chemical structures that do not bind directly to ERs.

A pubertal immune challenge alters the antidepressant-like effects of chronic estradiol treatment in inbred and outbred adult female mice.

Puberty is a period characterized by brain reorganization that contributes to the development of neural and behavioral responses to gonadal steroids. A single injection of the bacterial endotoxin, lipopolysaccharide (LPS), during the pubertal period decreases sexual receptivity in response to ovarian hormones in adulthood. Because chronic estradiol treatment alleviates depression-like symptoms in ovariectomized adult mice, we investigated the effect of pubertal LPS treatment on estradiol's antidepressant effects. We hypothesized that pubertal LPS treatment would decrease the antidepressant-like effect of estradiol in adult ovariectomized female mice, as it decreases other behavioral responses to ovarian hormones. As expected, chronic estradiol treatment decreased depression-like behavior, as measured by the duration of immobility, in saline-treated mice from two different strains, as well as in mice treated with LPS in adulthood. In contrast, in mice treated pubertally with LPS, estradiol strikingly increased the duration of immobility. No difference in body weight and in locomotion was found among the groups, suggesting that the differences in depression-like behavior were not due to differences in body weight or locomotor activity between LPS-treated and control mice. These results suggest that exposure to an immune challenge during the pubertal period alters the responsiveness of depression-like behavior to estradiol.

Steroid hormone receptors: long- and short-term integrators of the internal milieu and the external environment.

Many of the influences of estrogens and progestins on the brain and behavior are mediated by estrogen receptors and progestin receptors, acting as transcriptional regulators. The homologous and heterologous regulation of the concentrations of these receptors by cognate hormones is well established. However, although they were discovered and characterized based on their binding to cognate hormone and their role in transcriptional regulation, steroid hormone receptors have a more complex role and serve many more functions than originally suspected. First, besides being regulated by steroid hormones, the intracellular concentrations of brain steroid hormone receptors are regulated by neurotransmitters, a pathway by which stimuli from the environment, including from conspecific animals, can modulate the concentration of particular steroid hormone receptors in subsets of cells. Further, besides being activated by cognate steroid hormones, the receptors can be activated by a variety of neurotransmitters and phosphorylation pathways, providing a route through which environmental stimulation can activate steroid-receptor-dependent functions in specific cells. In addition, the transcription factor, estrogen receptor-α, produced from the estrogen receptor-α gene, can be modified to be targeted to membranes, where it can signal via kinase pathways. Finally, developmental experiences, such as particular stressors during the pubertal period, can permanently remodel the brain's response to ovarian hormones, most likely by long-term changes in regulation of the receptors mediating those responses. In addition to their function in responding to cognate ligand, it is now more appropriate to think of steroid hormone receptors as integrators of a wide variety of signaling pathways.

Absence of progestin receptors alters distribution of vasopressin fibers but not sexual differentiation of vasopressin system in mice.

Perinatal estrogens increase the number of vasopressin-expressing cells and the density of vasopressin-immunoreactive fibers observed in adult male rodents. The mechanism of action of estrogens on sexual differentiation of the extra-hypothalamic vasopressin system is unknown. We hypothesized that the sexually dimorphic expression of progestin receptors (PRs) during development would masculinize vasopressin expression in mice. We compared the number of vasopressin-expressing cells in the bed nucleus of the stria terminalis (BNST) and medial amygdala and the density of vasopressin-immunoreactive fibers in several brain regions of male and female wild type and PRKO mice using in situ hybridization and immunohistochemistry. As expected, sex differences in vasopressin cell number were observed in the BNST and medial amygdaloid nucleus. Vasopressin-immunoreactive fiber density was sexually dimorphic in the lateral septum, lateral habenular nucleus, medial amygdaloid nucleus, and mediodorsal thalamus. Sex differences were also observed in the principal nucleus of the BNST and medial preoptic area but not in the dorsomedial hypothalamus, which are thought to receive vasopressin innervation from the suprachiasmatic nucleus. Deletion of PRs did not alter the sex difference in vasopressin mRNA expression and vasopressin fiber immunoreactivity in any area examined. However, deletion of PRs increased the density of vasopressin fiber immunoreactivity in the lateral habenular nucleus. Our data suggest that PRs modulate vasopressin levels, but not sexual differentiation of vasopressin innervation in mice.

Immunocytochemical investigation of nuclear progestin receptor expression within dopaminergic neurones of the female rat brain.

Progesterone influences most processes involved in female reproduction, including ovulation, sexual behaviour, pregnancy, parturition, lactation and maternal behaviour. One neurotransmitter through which progesterone might regulate many of these functions is dopamine. To determine where in the brain progesterone might alter dopaminergic activity necessary for these and other processes in rats via cell nuclear progestin receptors, ovariectomized rats were injected subcutaneously with either 4 micro g oestradiol benzoate to induce high levels of hypothalamic progestin receptor expression, or oil, and perfused 48 h later. Dual-label immunocytochemistry was used to visualize cells having immunoreactivity (ir) for progestin receptors and tyrosine hydroxylase, a rate-limiting enzyme for dopamine synthesis. Many cells containing both progestin receptor-ir and tyrosine hydroxylase-ir were found throughout the periventricular hypothalamus of oestradiol-treated females. Conversely, very few cells in the hypothalamus of oil-treated controls contained progestin receptor-ir and, consequently, few dual-labelled cells were found in this group. The greatest percentage of tyrosine hydroxylase immunoreactive cells expressing progestin receptors in oestradiol-treated females was in, or near, the arcuate nucleus (A12 group), where up to 55% of tyrosine hydroxylase-expressing cells coexpressed progestin receptors. Notably, dual-labelled cells in oestradiol-treated females were also found more rostrally than previously reported, with approximately 15-20% of tyrosine hydroxylase-ir cells in the preoptic area/anterior hypothalamus (A14 group) also containing progestin receptor-ir. No dual-labelled cells were found for either group in the posterodorsal hypothalamus (A11 group), zona incerta (A13 group), retrorubral field (A8 group), ventral tegmental area (A10 group) or substantia nigra (A9 group) because little or no progestin receptor-ir was found in these sites. These data provide new information about the neural substrate where progesterone might regulate dopamine release in the preoptic area/anterior hypothalamus. Using more sensitive techniques than those used previously, they also confirm the relationship between progestin receptor and tyrosine hydroxylase in the arcuate nucleus, which could be important for the regulation of prolactin release throughout the female reproductive cycle. Additionally, although progesterone alters mesolimbic and nigrostriatal dopamine release, and the numerous behaviours that these pathways influence, these data again suggest that it does not do so via nuclear progestin receptor in dopaminergic cells of the ventral tegmental area and substantia nigra.

Response to male odours in progestin receptor- and oestrogen receptor-containing cells in female rat brain.

Sensory cues from male rats, such as odours and vaginal-cervical stimulation (VCS), play a modulatory role in female rat sexual behaviour. For example, exposure to male odours and VCS appears to be at least partially responsible for increases in sexual behaviour following repeated mating of oestradiol-primed female rats. Although there is evidence that VCS influences sexual behaviour via a ligand-independent progestin receptor (PR)-dependent mechanism, the mechanism by which odours influence sexual behaviour is not known. We tested the hypothesis that, similar to VCS, the effects of male odours on sexual behaviour are mediated by progestin receptors. Female rats were injected with the progestin antagonist, RU486, or oil vehicle and were then exposed to male-soiled bedding or clean bedding. Although exposure to male-soiled bedding resulted in higher levels of Fos immunoreactivity in brain areas associated with female sexual behaviour, the progestin antagonist did not reduce this effect. Furthermore, there was minimal coexpression of odour-induced Fos and progestin receptors in brain areas associated with female sexual behaviour. Together, these results suggest that the effects of male odours are not mediated by a PR-dependent mechanism. Therefore, we tested the hypothesis that oestrogen receptor (ER)-containing cells are involved in the effects of olfactory cues. Although there was virtually no coexpression of ERbeta and odour-induced Fos in brain areas associated with female sexual behaviour, exposure to male odours slightly increased the number of cells coexpressing ER(alpha) and odour-induced Fos in the posterodorsal medial amygdala. Although, these results do not support the hypothesis that the effects of odours are mediated by a PR-dependent mechanism, they suggest that integration of male odours and hormonal cues may occur in ER(alpha)-containing cells in the posterodorsal medial amygdala.

A progestin antagonist blocks vaginocervical stimulation-induced fos expression in neurones containing progestin receptors in the rostral medial preoptic area.

Vaginocervical stimulation (VCS) has a variety of effects on the brain, physiology and behaviour. Previous work demonstrated that a progestin antagonist blocked neuronal response to VCS (i.e. Fos expression) in the absence of progesterone in some neurones, and suggested that some of the effects of VCS on the brain are mediated by ligand-independent activation of progestin receptors (PRs). Although it had been reported previously that some of the cells in which VCS induces Fos expression also contain PRs, it had not been determined if a progestin antagonist blocked Fos expression in these particular neurones. The purpose of this experiment was to determine if a progestin antagonist decreases Fos expression specifically in cells that also express PRs in the preoptic area and ventromedial hypothalamus. As has been shown previously, VCS increased Fos-immunoreactive (ir) expression in the particular areas studied. In the rostral medial preoptic area, VCS increased Fos expression in cells that coexpressed PRs, as well as in cells that do not. However, in the caudal medial preoptic area, VCS only increased Fos expression in cells that did not coexpress PRs. Injection of the progestin antagonist, RU 486, decreased Fos expression in the rostral, but not caudal medial preoptic area, and it decreased Fos expression only in cells that coexpressed PR-ir. In contrast to a previous report, in the present study, the progestin antagonist did not inhibit VCS-induced Fos expression in the ventromedial hypothalamic area. The results of this experiment suggest that the progestin antagonist inhibits VCS-induced Fos expression in some neurones by blocking PRs, and they provide further support for the idea that VCS influences neuronal response in some cells by ligand-independent activation of PRs in those cells.

Coexpression of ER beta with ER alpha and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment.

Estrogen and progestin receptors (ER, PgR) play a critical role in the regulation of neuroendocrine functions in females. The neuroanatomical distribution of the recently cloned, ER beta, overlaps with both ER alpha and PgR. To determine whether ER beta is found within ER alpha- or PgR-containing neurons in female rat, we used dual label immunocytochemistry. ER beta-immunoreactivity (ER beta-ir) was primarily detected in the nuclei of cells in the periventricular preoptic area (PvPO), the bed nucleus of the stria terminalis (BNSTpr), the paraventricular nucleus, the supraoptic nucleus, and the medial amygdala (MEApd). Coexpression of ER beta-ir with ER alpha-ir or PgR-ir was observed in the PvPO, BNSTpr, and MEApd in ovariectomized rats. E2 treatment decreased the number of ER beta-ir cells in the PvPO and BNSTpr and the number of ER alpha-ir cells in the MEApd and paraventricular nucleus, and therefore decreased the number of cells coexpressing ER beta-ir and ER alpha-ir in the PvPO, BNSTpr, and MEApd. E2 treatment increased the amount of PgR-ir in cells of the PvPO, BNSTpr, and MEApd, a portion of which also contained ER beta. These results demonstrate that ER beta is expressed in ER alpha- or PgR-containing cells, and they suggest that E can modulate the ratios of these steroid receptors in a brain region-specific manner.

A dopamine antagonist blocks vaginocervical stimulation-induced neuronal responses in the rat forebrain.

During mating in rats, the male provides vaginocervical stimulation (VCS) to the female via intromissions. VCS, provided manually, mimics many aspects of mating, including facilitation of lordosis, induction of sexual receptivity, abbreviation of the period of sexual receptivity, and induction of twice-daily prolactin surges, which result in pseudopregnancy. VCS also induces the expression of Fos, the protein product of the immediate early gene c-fos, which has been used as a marker for neurons that are responsive to mating stimuli. Because VCS induces the release of dopamine in the forebrain, as well as phosphorylation of DARPP-32, a phosphoprotein associated with activation of the D(1) subtype of dopamine receptor, we tested the hypothesis that VCS induces Fos expression by acting on the D(1) class of dopamine receptors. Injection of SCH 23390, an antagonist of the D(1) class of dopamine receptors, virtually eliminated VCS-induced Fos expression without affecting constitutive levels of Fos-Immunoreactivity (Fos-IR) in all brain areas in which VCS induced Fos expression. In a follow-up experiment, expression of a second immediate early protein, egr-1, was blocked as well, suggesting that these results are not specific to Fos. Therefore, the results are consistent with the idea that VCS induces dopamine release, causing activation of D(1) dopamine receptors, which in turn, results in neuronal response, as seen by both Fos and egr-1 expression.

Oestradiol increases phosphorylation of a dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32) in female rat brain.

Recent studies suggest that oestrogen and progestin receptors may be activated by the neurotransmitter dopamine, as well as by their respective ligands. Because intracerebroventricular infusion of D(1), but not D(2), dopaminergic receptor agonists increases oestrous behaviour in oestradiol-primed rats, we wanted to determine if treatment with oestradiol alters the activity of D(1) receptor-associated processes in steroid receptor-containing areas in female rat brain. One D(1) receptor-associated phosphoprotein that may be influenced by oestradiol is a dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000 (DARPP-32). Because DARPP-32 is phosphorylated in response to dopamine acting via a cAMP-dependent protein kinase, it provides a useful marker to examine where in the brain a particular stimulus might be altering the activity of D(1) receptor-containing neurones. To determine if oestradiol alters the phosphorylation of DARPP-32, we stained immunocytochemically brain sections of female rats treated with behaviourally relevant doses of oestradiol or oil vehicle with an antibody that detects only the threonine 34-phosphorylated form of DARPP-32. Behaviourally effective doses of oestradiol increase the phosphorylation of DARPP-32 within the medial preoptic nucleus, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus and the ventromedial nucleus of the hypothalamus, 48 h after treatment. These data suggest that oestradiol increases the activity of D(1) dopamine receptor-associated processes in oestrogen receptor-containing areas of female rat forebrain.

Sensory cues mediating mating-induced potentiation of sexual receptivity in female rats.

Repeated mating of estradiol-primed female rats increases sexual receptivity. Two studies were conducted to determine the contribution of vaginal--cervical stimulation (VCS) to this increase. In the first study, female rats were repeatedly mated for 165 min. The vaginas of half of the females were covered with tape (masked) to prevent intromissions by the males. The remaining females were unmasked. Only females receiving intromissions (unmasked) showed a significant increase in sexual receptivity during repeated mating, suggesting that VCS from intromissions is necessary for repeated mating to increase sexual receptivity. In the second experiment, female rats received either experimentally administered VCS or control scapular stimulation administered with a plastic probe 1 h prior to testing for sexual receptivity. VCS applied in this manner significantly increased sexual receptivity. Together, these findings suggest that VCS from intromissions is one of the primary factors responsible for increases in sexual receptivity following repeated mating.

Maternal behavior stimulates c-fos activity within estrogen receptor alpha-containing neurons in lactating rats.

Estradiol and other hormones are thought to be critical for the onset, but not maintenance, of maternal behavior in rats. Maternal behavior is instead maintained postpartum by tactile stimulation that dams receive during interactions with pups, and many neural sites implicated in the control of maternal behavior show elevated c-fos activity in response to this stimulation. Many of these sites also contain neurons that express the alpha subtype of the estrogen receptor (ERalpha). Because of possible interactions between tactile stimulation from pups, c-fos, and ERalpha in the lactating rat brain, we determined if populations of cells that show increased c-fos activity after maternal behavior in lactating rats also contain ERalpha. Dams were separated from their pups for 48 h beginning on day 5 postpartum. On day 7 postpartum, experimental dams were reunited with pups and mother-litter interactions were observed for 60 min. Control dams received no pup stimulation. Subjects were sacrificed 60 min later and brain sections were double immunolabeled for the Fos and ERalpha proteins. As expected, the number of ERalpha-immunoreactive (ERalpha-ir) neurons did not differ between the two groups in the eight areas analyzed (lateral region of the lateral septum, posterodorsal medial amygdala, dorsal and ventral medial preoptic area, dorsal and ventral bed nucleus of the stria terminalis, lateral habenula, and ventrolateral caudal periaqueductal gray). Consistent with previous reports, maternal dams had 2- to 7-fold more Fos-immunoreactive (Fos-ir) neurons in these sites compared with nonstimulated controls. Maternal dams had significantly more Fos-ir neurons that also contained ERalpha-ir in all sites, with the greatest increases in the ventral medial preoptic area, lateral habenula, and ventral bed nucleus of the stria terminalis. Between approximately 25 and 45% of the Fos-ir cells in the sites examined also expressed ERalpha. Thus, a substantial number of neurons that are genomically activated during maternal behavior contain ERalpha, raising the possibility that the postpartum display of maternal behavior can be influenced by ERalpha activity.

Cells containing immunoreactive estrogen receptor-alpha in the human basal forebrain.

The distribution of estrogen receptor protein-alpha (ER-alpha)-containing cells in the human hypothalamus and adjacent regions was studied using a monoclonal antibody (H222) raised against ER-alpha derived from MCF-7 human breast cancer cells. Reaction product was found in restricted populations of neurons and astrocyte-like cells. Neurons immunoreactive for ER-alpha were diffusely distributed within the basal forebrain and preoptic area, infundibular region, central hypothalamus, basal ganglia and amygdala. Immunoreactive astrocyte-like cells were noted within specific brain regions, including the lamina terminalis and subependymal peri-third-ventricular region. These data are consistent with the location of estrogen receptors in the basal forebrain of other species and the known effects of estrogens on the cellular functions of both neurons and supporting elements within the human hypothalamus and basal forebrain.

Progesterone, but not progesterone-independent activation of progestin receptors by a mating stimulus, rapidly decreases progestin receptor immunoreactivity in female rat brain.

Recent studies suggest that progestin receptors may be activated in vivo by neurotransmitters in the absence of ligand. More specifically, vaginal-cervical stimulation (VCS) can influence sexual behavior by activating progestin receptors in the absence of progesterone. Another way to test if progestin receptors are influenced by particular stimuli is to examine progestin receptor immunostaining. We report that progestin receptor immunoreactivity is decreased in the forebrain of estradiol-primed ovariectomized (OVX) rats within 1 h after a subcutaneous injection of progesterone, a time by which rapid down-regulation of progestin receptors does not seem to have occurred. In estradiol-primed OVX rats, VCS also decreased progestin receptor immunoreactivity within 1 h in the medial preoptic area, but not in any other area examined. To determine if the decrease in immunoreactivity by VCS was due to adrenal secretions or by ligand-independent activation of progestin receptors, we repeated the experiment in estradiol-primed OVX/adrenalectomized rats. Prior removal of the adrenal glands blocked the rapid decrease in progestin receptor immunoreactivity, even though data from other experiments suggest that progestin receptors are activated by VCS at this time. These studies suggest the possibility that progestin receptors may be affected differentially by progesterone-dependent or by progesterone-independent pathways. This raises the possibility that activation of progestin receptors by these two distinct pathways may lead to different neuronal consequences.

Oestrogen receptor-alpha-immunoreactive neurones project to the suprachiasmatic nucleus of the female Syrian hamster.

Ovarian steroid hormones regulate circadian period and phase, but classical receptors for these hormones are absent in the circadian pacemaker localized in the suprachiasmatic nucleus of the hypothalamus (SCN). In order to determine whether effects of oestrogen may be exerted through steroid-binding systems afferent to the SCN we have performed double label immunocytochemistry for oestrogen receptor-alpha(ER-alpha) and the retrograde tracer cholera toxin B subunit (CtB) after its application to the SCN. Most of the areas that contain ER-alpha-immunoreactive (ERalpha-ir) cells also contained cells afferent to the SCN. The percentage of neurones afferent to the SCN which show ERalpha-immunoreactivity varies between areas. As many as one-third of the neurones afferent to the SCN in some parts of the preoptic area and the corticomedial amygdala are ERalpha-ir. Very few of the afferent neurones from the septum and the central grey are ERalpha-ir, whereas an intermediate proportion of afferents from the bed nucleus of the stria terminalis and the arcuate nucleus are ERalpha-ir. Our retrograde tracing results were compared with results of anterograde tracing from some of the sites containing SCN afferents. Using a combined retrograde and anterograde tracing technique we tested the possibility that single ERalpha-ir neurones afferent to the SCN could receive reciprocal innervation by SCN efferents. Although we found SCN input to some SCN afferent neurones, we found no evidence of reciprocity between single ERalpha-ir cells and the SCN. Our results indicate the existence of oestrogen binding systems afferent to the SCN. These neuroanatomical pathways may mediate effects of gonadal steroid hormones on circadian rhythms.

Projections of the estrogen receptor-immunoreactive ventrolateral hypothalamus to other estrogen receptor-immunoreactive sites in female guinea pig brain.

The ventrolateral hypothalamus in female guinea pigs includes an estrogen receptor dense region adjacent to the ventromedial hypothalamus. This region is reciprocally connected with other estrogen receptor-containing areas suggesting that steroid hormone receptor-containing cells may be directly linked. Phaseolus vulgaris leucoagglutinin, an anterograde tract tracer, was specifically placed in this region with the aim of labeling some projections from estrogen receptor-containing neurons. These projections were colocalized immunocytochemically with the distribution of estrogen receptor-containing cells. Dense ventrolateral hypothalamic innervation was observed in some regions also containing a high concentration of estrogen receptor-containing cells. These regions included the medial preoptic area, the bed nucleus of the stria terminalis, the ventrolateral hypothalamus anterior and posterior to the injection site, and the midbrain central gray. A low density of ventrolateral hypothalamic fibers and terminals was observed in two regions rich in estrogen receptors, the amygdala and the arcuate nucleus. In general, ventrolateral hypothalamic fibers and terminals were present in all regions where estrogen receptors were found except the medial thalamus and habenular region. Labeled terminal boutons and perineuronal baskets were found around estrogen receptor-containing cells in most regions which contained estrogen receptor-containing cells. These close appositions were suggestive of synaptic contacts, suggesting that the ventrolateral hypothalamus may influence steroid-dependent behaviors via the modulation of estrogen receptor-containing cells. Furthermore, ventrolateral hypothalamic projections may include direct connections with estrogen receptor-containing cells, suggesting the presence of a network of interconnected estradiol-sensitive neurons involved in the regulation of estradiol-dependent functions.

Induction of progestin receptors by estradiol in the forebrain of estrogen receptor-alpha gene-disrupted mice.

Mice, rats, and humans have two types of estrogen receptors, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). Estrogen receptor-alpha gene-disrupted (ERalpha-disrupted) mice bear two nonfunctional copies of the ERalpha gene. This mutation blocks the synthesis of full-length ERalpha, renders the animals infertile, and inhibits the induction of female sexual behaviors by estradiol and progesterone. It is likely that many of the processes contributing to the regulation of sexual receptivity by estradiol and progesterone are compromised in ERalpha-disrupted mice. However, given the importance of progesterone in the regulation of sexual receptivity and given the importance of progestin receptors (PRs) in mediating the responses of females to progesterone, we investigated the effects of ERalpha disruption on the induction of PRs by estradiol in the forebrain. We hypothesized that estradiol would induce PRs in wild-type mice but not in ERalpha-disrupted mice. Ovariectomized wild-type and ERalpha-disrupted mice were implanted with either estradiol-filled capsules or empty capsules for 5 d, after which their brains were processed for the immunocytochemical detection of PR. Estradiol increased the number of PR-immunoreactive cells in both wild-type and ERalpha-disrupted mice. The residual responsiveness of ERalpha-disrupted mice to estradiol could be accounted for by an ERbeta-dependent mechanism or another as yet unidentified estrogen receptor; however, because ERalpha-immunoreactivity and PCR product representing the 3' end of ERalpha mRNA were found in at least one PR-containing region of the ERalpha-disrupted mice, an ERalpha splice variant may also mediate the induction of PR-immunoreactivity in ERalpha-disrupted mice.

Mating-related stimulation induces phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein-32 in progestin receptor-containing areas in the female rat brain.

Vaginal-cervical stimulation induces a number of physiological and behavioral events, including the facilitation of mating behavior. Although the facilitation of one component of mating behavior, lordosis, by vaginal-cervical stimulation does not require the presence of progesterone, it appears to be mediated by neural progestin receptors. Abundant evidence suggests that dopamine may play a role in the neural circuitry activated by vaginal-cervical stimulation, including the mating-induced release of dopamine in progestin receptor-containing areas of the brain, changes in the activational state of progestin receptors because of dopamine D1 receptor stimulation, facilitation of lordosis by D1 receptor stimulation in estradiol-primed rats via progesterone-independent events, and D1 agonist-induced neuronal responses in progestin receptor-containing areas and cells. We tested the hypothesis that vaginal-cervical stimulation induces phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32; Mr = 32,000), a protein phosphorylated predominantly in response to the stimulation of D1 receptors. At 9 d after ovariectomy, female rats were injected subcutaneously with a behaviorally effective dose of estradiol benzoate. At 48 hr later they received vaginal-cervical or control (perineal) stimulation, and they were perfused 1 hr later. Vaginal-cervical stimulation increased the number of cells expressing pDARPP-32 immunoreactivity by 92% in the medial preoptic nucleus, 134% in the caudal ventromedial hypothalamic nucleus, 123% in the posterodorsal medial amygdala, and 103% in the bed nucleus of the stria terminalis. These results suggest that some of the neuronal effects of vaginal-cervical stimulation, and perhaps other social or environmental stimuli, are mediated by phosphorylation of DARPP-32, perhaps via stimulation of D1 receptors, within progestin receptor-containing areas.