Steroids, stress and the gut microbiome-brain axis.

It is becoming well established that the gut microbiome has a profound impact on human health and disease. In this review, we explore how steroids can influence the gut microbiota and, in turn, how the gut microbiota can influence hormone levels. Within the context of the gut microbiome-brain axis, we discuss how perturbations in the gut microbiota can alter the stress axis and behaviour. In addition, human studies on the possible role of gut microbiota in depression and anxiety are examined. Finally, we present some of the challenges and important questions that need to be addressed by future research in this exciting new area at the intersection of steroids, stress, gut-brain axis and human health.

Oestradiol and diet modulate energy homeostasis and hypothalamic neurogenesis in the adult female mouse.

Leptin and oestradiol have overlapping functions in energy homeostasis and fertility, and receptors for these hormones are localised in the same hypothalamic regions. Although, historically, it was assumed that mammalian adult neurogenesis was confined to the olfactory bulbs and the hippocampus, recent research has found new neurones in the male rodent hypothalamus. Furthermore, some of these new neurones are leptin-sensitive and affected by diet. In the present study, we tested the hypothesis that diet and hormonal status modulate hypothalamic neurogenesis in the adult female mouse. Adult mice were ovariectomised and implanted with capsules containing oestradiol (E2 ) or oil. Within each group, mice were fed a high-fat diet (HFD) or maintained on standard chow (STND). All animals were administered i.c.v. 5-bromo-2'-deoxyuridine (BrdU) for 9 days and sacrificed 34 days later after an injection of leptin to induce phosphorylation of signal transducer of activation and transcription 3 (pSTAT3). Brain tissue was immunohistochemically labelled for BrdU (newly born cells), Hu (neuronal marker) and pSTAT3 (leptin sensitive). Although mice on a HFD became obese, oestradiol protected against obesity. There was a strong interaction between diet and hormone on new cells (BrdU+) in the arcuate, ventromedial hypothalamus and dorsomedial hypothalamus. HFD increased the number of new cells, whereas E2 inhibited this effect. Conversely, E2 increased the number of new cells in mice on a STND diet in all hypothalamic regions studied. Although the total number of new leptin-sensitive neurones (BrdU-Hu-pSTAT3) found in the hypothalamus was low, HFD increased these new cells in the arcuate, whereas E2 attenuated this induction. These results suggest that adult neurogenesis in the hypothalamic neurogenic niche is modulated by diet and hormonal status and is related to energy homeostasis in female mice.

Nuclear receptor coactivators: regulators of steroid action in brain and behaviour.

Steroid hormones act in specific regions of the brain to alter behaviour and physiology. Although it has been well established that the bioavailability of the steroid and the expression of its receptor is critical for understanding steroid action in the brain, the importance of nuclear receptor coactivators in the brain is becoming more apparent. The present review focuses on the function of the p160 family of coactivators, which includes steroid receptor coactivator-1 (SRC-1), SRC-2 and SRC-3, in steroid receptor action in the brain. The expression, regulation and function of these coactivators in steroid-dependent gene expression in both brain and behaviour are discussed.

Convergence of multiple mechanisms of steroid hormone action.

Steroid hormones modulate a wide array of physiological processes including development, metabolism, and reproduction in various species. It is generally believed that these biological effects are predominantly mediated by their binding to specific intracellular receptors resulting in conformational change, dimerization, and recruitment of coregulators for transcription-dependent genomic actions (classical mechanism). In addition, to their cognate ligands, intracellular steroid receptors can also be activated in a "ligand-independent" manner by other factors including neurotransmitters. Recent studies indicate that rapid, nonclassical steroid effects involve extranuclear steroid receptors located at the membrane, which interact with cytoplasmic kinase signaling molecules and G-proteins. The current review deals with various mechanisms that function together in an integrated manner to promote hormone-dependent actions on the central and sympathetic nervous systems.

Anatomically-specific actions of oestrogen receptor in the developing female rat brain: effects of oestradiol and selective oestrogen receptor modulators on progestin receptor expression.

Steroid hormones largely exert their actions by activating nuclear receptors, which, as transcription factors, powerfully influence fundamental processes of neural development. Often, steroid receptor action demonstrates remarkable specificity under different developmental, anatomical or hormonal conditions. Yet, the mechanisms underlying such specificity are poorly understood. The present study examined the anatomically-specific regulation of progestin receptor (PR) expression by oestrogen receptor (ER) activation in the ventromedial nucleus (VMN) of the hypothalamus and the medial preoptic nucleus (MPN) of the neonatal female rat brain, using the selective oestrogen receptor modulators (SERMs), tamoxifen and ICI 182780 (ICI), in the presence or absence of oestradiol benzoate (EB) treatment. The results demonstrate that PR immunoreactivity (PR-ir) in the neonatal female MPN was significantly increased by EB and this increase was abolished by either tamoxifen or ICI treatment. In contrast, within the VMN of the same animals, EB had no effect on PR-ir and the SERMs only modestly decreased PR-ir. Interestingly, ICI acted as a true antagonist regardless of EB treatment, whereas tamoxifen acted as an ER agonist in the absence of EB in the MPN, but not the VMN, representing one of the first in vivo demonstrations of tissue-specific and oestradiol-independent effects of tamoxifen on ER activation. The present results indicate that PR expression is highly dependent on oestradiol and its receptor in the MPN, although it is independent of both oestradiol and ER activation within the neonatal VMN. These findings demonstrate the anatomically-specific actions of oestradiol and its receptor to induce PR in two brain regions controlling different aspects of female reproductive behaviours in adulthood.

Steroid receptor coactivator-2 expression in brain and physical associations with steroid receptors.

Estradiol and progesterone bind to their respective receptors in the hypothalamus and hippocampus to influence a variety of behavioral and physiological functions, including reproduction and cognition. Work from our lab and others has shown that the nuclear receptor coactivators, steroid receptor coactivator-1 (SRC-1) and SRC-2, are essential for efficient estrogen receptor (ER) and progestin receptor (PR) transcriptional activity in brain and for hormone-dependent behaviors. While the expression of SRC-1 in brain has been studied extensively, little is known about the expression of SRC-2 in brain. In the present studies, we found that SRC-2 was highly expressed throughout the hippocampus, amygdala and hypothalamus, including the medial preoptic area (MPOA), ventral medial nucleus (VMN), arcuate nucleus (ARC), bed nucleus of the stria terminalis, supraoptic nucleus and suprachiasmatic nucleus. In order for coactivators to function with steroid receptors, they must be expressed in the same cells. Indeed, SRC-2 and ER(alpha) were coexpressed in many cells in the MPOA, VMN and ARC, all brain regions known to be involved in female reproductive behavior and physiology. While in vitro studies indicate that SRC-2 physically associates with ER and PR, very little is known about receptor-coactivator interactions in brain. Therefore, we used pull-down assays to test the hypotheses that SRC-2 from hypothalamic and hippocampal tissue physically associate with ER and PR subtypes in a ligand-dependent manner. SRC-2 from both brain regions interacted with ER(alpha) bound to agonist, but not in the absence of ligand or in the presence of the selective ER modulator, tamoxifen. Analysis by mass spectrometry confirmed these ligand-dependent interactions between ER(alpha) and SRC-2 from brain. In dramatic contrast, SRC-2 from brain showed little to no interaction with ERbeta. Interestingly, SRC-2 from both brain regions interacted with PR-B, but not PR-A, in a ligand-dependent manner. Taken together, these findings reveal that SRC-2 is expressed in brain regions known to mediate a variety of steroid-dependent functions. Furthermore, SRC-2 is expressed in many ER(alpha) containing cells in the hypothalamus. Finally, SRC-2 from brain interacts with ER and PR in a subtype-specific manner, which may contribute to the functional differences of these steroid receptor subtypes in brain.

Nuclear Thimet oligopeptidase is coexpressed with oestrogen receptor alpha in hypothalamic cells and regulated by oestradiol in female mice.

Thimet oligopeptidase (EC 3.4.24.15; also called EP24.15 and TOP; referred to here as TOP) is a neuropeptidase involved in the regulation of several physiological functions including reproduction. Among its substrates is gonadotrophin-releasing hormone (GnRH), an important hypothalamic hormone that regulates the synthesis and release of oestradiol and facilitates female sexual behaviour. Using immunohistochemistry, we found that TOP is expressed in the nucleus of cells throughout the female mouse brain, and in high levels in steroid-sensitive regions of the hypothalamus, which is consistent with previous findings in male rats. Furthermore, dual-label immunofluorescence revealed that TOP and oestrogen receptor alpha (ERalpha) coexpress in several reproductively-relevant brain regions, including the medial preoptic area (mPOA), arcuate nucleus (ARC), ventrolateral portion of the ventromedial hypothalamic nucleus (VMNvl) and the midbrain central grey (MCG). Previous studies in rats have shown that oestradiol decreases hypothalamic TOP levels or activity, possibly potentiating the effects of GnRH. In the present study, analysis by immunohistochemistry revealed that oestradiol decreased TOP immunoreactivity in the VMNvl, whereas no differences were detected in the mPOA, ARC or median eminence. Overall, the present findings indicate that TOP is coexpressed with ERalpha, and oestradiol regulates TOP expression in a brain region-specific manner in female mice, providing neuroanatomical evidence that TOP may function in reproductive physiology and/or behaviour.

Nuclear receptor coactivators: essential players for steroid hormone action in the brain and in behaviour.

Steroid hormones act both in the brain and throughout the body to influence behaviour and physiology. Many of these effects of steroid hormones are elicited by transcriptional events mediated by their respective receptors. A variety of cell culture studies reveal that nuclear receptor coactivators are critical for modulating steroid receptor-dependent transcription. Thus, in addition to the availability of the hormone and the expression of its receptor, nuclear receptor coactivators are essential for steroid-dependent transactivation of genes. This review discusses the mounting evidence indicating that nuclear receptor coactivators are critical for modulating steroid hormone action in the brain and in the regulation of behaviour.

Cells in behaviourally relevant brain regions coexpress nuclear receptor coactivators and ovarian steroid receptors.

Oestradiol and progesterone act in the brain to elicit profound effects on behaviour and physiology. One physiological function of oestradiol is the induction of progesterone receptor (PR) expression in a variety of behaviourally relevant brain regions, including the ventromedial nucleus of the hypothalamus (VMN), the medial preoptic nucleus of the preoptic area (MPOA), the arcuate nucleus (ARC) and the medial central grey (MCG). Ligand-dependent transcriptional activity of steroid receptors, including oestrogen receptors (ER) and Pr, is dramatically influenced by nuclear receptor coactivators. In previous studies, we have found that two of these nuclear receptor coactivators, steroid receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP), are important in ER-mediated induction of PR in the VMN and in steroid-dependent behaviours. For nuclear receptor coactivators to function in hormone-dependent transcription in the brain and regulate behaviour, both receptor and coactivator must be expressed in the same cell. In the present study, we used a dual-label immunohistochemical technique to investigate if individual cells in behaviourally relevant brain regions coexpress nuclear receptor coactivators and steroid receptors. Confocal analysis revealed that in oestrogen-primed rats, most of the E-induced PR cells in the VMN (89.6%), MPOA (63%), ARC (82.6%), and many in the MCG (39%), also express SRC-1. In addition, the majority of the cells containing E-induced PR in the VMN (78.3%), MPOA (83.1%), ARC (83.6%), and MCG (60%) also express CBP. These results, taken together with the findings that virtually all oestradiol-induced PR containing cells in the brain express ER, suggest that these neurones represent sites of functional interaction of nuclear receptor coactivators with ovarian steroid receptors in the brain. The present findings provide neuroanatomical evidence that nuclear receptor coactivators are integral in mediating steroid hormone action in behaviourally relevant brain regions.

Expression of the nuclear receptor coactivator, cAMP response element-binding protein, is sexually dimorphic and modulates sexual differentiation of neonatal rat brain.

Recent studies indicate that the transcriptional activity of steroid receptors is governed by proteins called nuclear receptor coactivators. Using immunocytochemistry, we found that on the day of birth (postnatal d 0) males express higher levels of the nuclear receptor coactivator, cAMP response element binding protein-binding protein (CBP), within the ventromedial hypothalamus, medial preoptic area, and arcuate nucleus. Using Western immunoblots, we confirmed that males have higher levels of CBP on postnatal d 0, 1, and 5; however, there was no sex difference on postnatal d 11. To examine the functional role of CBP, we infused oligodeoxynucleotides that were antisense to CBP mRNA or a scrambled sequence as a control into the hypothalamus of female rats on postnatal d 0, 1, and 2. On postnatal d 1, all rats were injected with 100 microg testosterone propionate to both masculinize (increase male) and defeminize (decrease female) sexual behavior. Rats were ovariectomized in adulthood and tested for adult sexual behavior. Neonatal CBP antisense oligodeoxynucleotides treatment interfered with the defeminizing, but not the masculinizing, actions of testosterone. These results indicate that CBP expression in developing rat brain is sexually dimorphic and an important modulator for steroid hormone action.

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.

Nuclear receptor coactivators in neuroendocrine function.

Steroid hormones influence a variety of neuroendocrine events, including brain development, sexual differentiation and reproduction. Hormones elicit many of these effects by binding to neuronal steroid receptors, which are members of a nuclear receptor superfamily of transcriptional activators. However, the mechanisms by which activated steroid receptors regulate gene expression in brain are not well understood. Recently, a new class of proteins, known as nuclear receptor coactivators, have been found to dramatically enhance steroid receptor mediated transactivation of genes in vitro. Here, the proposed molecular mechanisms of how these coactivators enhance the transcriptional activity of steroid receptors are summarized. While much is known about the mechanisms of these coactivators in vitro, it is unclear how these cofactors function in hormone action in vivo or in the brain. This paper discusses some of the initial and enticing investigations into the role of these important coregulatory proteins in neuroendocrine events. Finally, some of the critical issues and future directions in nuclear receptor coactivator function in neuroendocrinology are highlighted.

Steroid receptor coactivator-1 (SRC-1) mediates the development of sex-specific brain morphology and behavior.

Steroid hormone action during brain development exerts profound effects on reproductive physiology and behavior that last into adulthood. A variety of in vitro studies indicate that steroid receptors require nuclear receptor coactivators for efficient transcriptional activity. To determine the functional significance of the nuclear receptor coactivator SRC-1 in developing brain, we investigated the consequence of reducing SRC-1 protein during sexual differentiation of the brain. We report that reducing SRC-1 protein interferes with the defeminizing actions of estrogen in neonatal rat brain. Our data indicate that SRC-1 protein expression is critically involved in the hormone-dependent development of normal male reproductive behavior and brain morphology.

Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo.

Full transcriptional activation by steroid hormone receptors requires functional synergy between two transcriptional activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2) terminal regions. One possible mechanism for achieving this functional synergy is a physical intramolecular association between amino (N-) and carboxyl (C-) domains of the receptor. Human progesterone receptor (PR) is expressed in two forms that have distinct functional activities: full-length PR-B and the amino-terminally truncated PR-A. PR-B is generally a stronger activator than PR-A, whereas under certain conditions PR-A can act as a repressor in trans of other steroid receptors. We have analyzed whether separately expressed N- (PR-A and PR-B) and C-domains [hinge plus ligand-binding domain (hLBD)] of PR can functionally interact within cells by mammalian two-hybrid assay and whether this involves direct protein contact as determined in vitro with purified expressed domains of PR. A hormone agonist-dependent interaction between N-domains and the hLBD was observed functionally by mammalian two-hybrid assay and by direct protein-protein interaction assay in vitro. With both experimental approaches, N-C domain interactions were not induced by the progestin antagonist RU486. However, in the presence of the progestin agonist R5020, the N-domain of PR-B interacted more efficiently with the hLBD than the N-domain of PR-A. Coexpression of steroid receptor coactivator-1 (SRC-1) and the CREB binding protein (CBP), enhanced functional interaction between N- and C-domains by mammalian two-hybrid assay. However, addition of SRC-1 and CBP in vitro had no influence on direct interaction between purified N- and C-domains. These results suggest that the interaction between N- and C-domains of PR is direct and requires a hormone agonist-induced conformational change in the LBD that is not allowed by antagonists. Additionally, coactivators are not required for physical association between the N- and C-domains but are capable of enhancing a functionally productive interaction. In addition, the more efficient interaction of the hLBD with the N-domain of PR-B, compared with that of PR-A, suggests that distinct interactions between N- and C-terminal regions contribute to functional differences between PR-A and PR-B.

Hinge and amino-terminal sequences contribute to solution dimerization of human progesterone receptor.

We and others have shown previously that progesterone receptors (PR) form homodimers in solution in the absence of DNA and that dimers are the preferential form of receptor that binds with high affinity to target DNA. To determine the sequence regions involved in solution homodimerization, wild type PR and truncated PR proteins were expressed in an insect baculovirus system. The expression constructs included the ligand-binding domain [LBD, amino acids (aa) 688-933], the LBD plus hinge (hLBD, aa 634-933), the hLBD plus the DNA-binding domain (DhLBD, aa 538-933), and the full- length A and B isoforms of PR. PR-PR interactions were detected by three methods, coimmunoprecipitation of the PR fragments with full-length PR-A, pull-down of PR-polypeptides with polyhistidine-tagged versions of the same polypeptides immobilized to metal affinity columns and cooperative ligand-binding assays (Hill coefficient, n(H) > 1 indicating PR-PR interaction). By all three assays, the LBD alone was not sufficient to mediate protein-protein interaction. However, the LBD did exhibit other properties ascribed to this domain, including binding to steroids with a relatively good affinity and specificity, ligand-induced conformational changes at the carboxyl terminus tail and binding of heat shock protein 90 and its dissociation in response to hormone. Thus, failure of the expressed LBD to mediate dimerization does not appear to be due to an extensively misfolded or unstable polypeptide. The minimal carboxyl-terminal fragment capable of mediating PR-PR interaction was the hLBD construct. However, by immobilized metal affinity chromatography assay, self-association of PR-A was 3.5-fold more efficient than that of either the DhLBD or hLBD constructs. An expressed amino-terminal domain (aa 165-535) lacking the DNA-binding domain, hinge, and LBD was found to physically associate with PR-A or with another amino-terminal fragment lacking the LBD, but retaining the DNA-binding domain. These results provide evidence for direct amino-terminal interactions in the more efficient PR-PR interaction exhibited by wild-type PR-A, as compared with DhLBD and hLBD constructs. The overall results of this paper are consistent with the conclusion that the carboxyl-terminal LBD is not sufficient for mediating PR dimerization and that multiple regions, including the hinge and amino-terminal sequences, contribute either directly or indirectly to homodimerization of PR.

Estradiol and progesterone influence the response of ventromedial hypothalamic neurons to tactile stimuli associated with female reproduction.

Stimulation of the vagina and cervix, provided by the male during copulation or manually with a probe, causes many behavioral and endocrine changes associated with female reproduction in rats. Previously, we found that vaginal-cervical stimulation (VCS), by mating or manual probing, increases the expression of Fos-immunoreactivity (Fos-IR) in discrete populations of neurons in the preoptic area, mediobasal hypothalamus and midbrain, suggesting that these neurons respond to VCS. The purpose of the present study was to determine if hormonal priming would increase the number of Fos-IR cells following VCS. Contrary to our hypothesis, in Experiment 1 priming animals with a behaviorally effective dose of 17 beta-estradiol benzoate followed 48 h later by progesterone caused a trend towards a decrease in the number of VCS-induced Fos-IR cells in the ventromedial hypothalamus. In Experiment 2, which was done to confirm this decrease in VCS-induced Fos-IR neurons by hormones, this effect was found to be statistically significant. Furthermore, this hormone-induced decrease in VCS-responsive cells was localized to the ventromedial nucleus of the hypothalamus, an area rich in estrogen and progestin receptors. No effects of hormone treatment on VCS-induced Fos-IR were observed in any other brain regions analyzed. These findings suggest that steroid hormones may elicit some of their effects on female reproductive behavior and physiology by altering the responsiveness of ventromedial nucleus neurons to vaginal-cervical stimulation.

Intraneuronal convergence of tactile and hormonal stimuli associated with female reproduction in rats.

Stimulation of the vagina and cervix, by mating or manual probing, elicits many behavioral and endocrine changes associated with female reproduction in rats. We and others have identified neurons in the medial preoptic area, medial division of the bed nucleus of the stria terminalis, posterodorsal portion of the medial amygdala, ventromedial hypothalamus, dorsomedial hypothalamus and midbrain central gray that increase Fos expression in response to vaginal-cervical stimulation (VCS). In the present study, we used a double-label immunofluorescent technique to determine if any of these VCS-responsive neurons also contained estrogen receptor-immunoreactivity. We found that over 80% of the VCS-induced Fos-IR neurons in the medial division of the bed nucleus of the stria terminalis also contained estrogen receptor-immunoreactivity. Furthermore, high percentages of VCS-responsive neurons in the medial preoptic area, posterodorsal medial amygdala, ventromedial hypothalamus and midbrain central gray contained estrogen receptor-immunoreactivity as well. These results suggest that sensory and hormonal information associated with female reproduction converge on specific populations of neurons and may be integrated at the molecular level within these neurons.

Hypothalamic ovarian steroid hormone-sensitive neurons involved in female sexual behavior.

Estradiol and progesterone regulate sexual behaviors in guinea pigs and rats, at least in part, through interaction with intracellular steroid hormone receptors. In the present review of work from the laboratory of the authors, we summarize recent work which has focused on one of the sites of hormone action in female guinea pigs--the ventrolateral hypothalamus. We summarize results of earlier experiments in which the regulation of steroid hormone receptors in this area was assessed after hormonal treatments with predictable effects on female sexual behavior. We then review the results of recent tract-tracing experiments in which we have examined the projections from the steroid receptor-immunoreactive neurons in this region, as well as the afferent projections from other neuroanatomical areas, including neurons which themselves contain estrogen receptors. We also present studies on the afferent input into these neurons by noradrenergic neurons and discuss the possibility that noradrenergic input influences steroid hormone sensitivity in these neurons. Finally, we discuss the results of experiments in which Fos-immunocytochemistry was used in rats to identify the neurons responding to a particular tactile stimulus associated with female reproduction, i.e., vaginal-cervical stimulation. These experiments further define a complex neuroanatomical system, in which many of the elements are estradiol or progesterone-responsive, that is involved in the hormonal regulation of sexual behavior in guinea pigs and rats.

Fos expression in the rat brain following vaginal-cervical stimulation by mating and manual probing.

Vaginal-cervical stimulation (VCS), provided by mating or manual probing, induces many reproductive behavioral and endocrine changes in female rats. These changes include an increase in lordosis duration, heat termination and pseudopregnancy. Electrophysiological and [14C]2-deoxy-D-glucose studies collectively show that neurons in the medial preoptic area, ventromedial hypothalamus and midbrain central gray respond to manual VCS. In the present study we immunocytochemically labeled brain sections for Fos, the protein product of the immediate early gene c-fos, to detect VCS-responsive neurons in hormone-primed animals receiving VCS by mating or manual probing. In Experiment 1, females receiving mounts and intromissions were compared to: 1) vaginally-masked females receiving mounts but no VCS, 2) females exposed to an intact anesthetized male or 3) females not exposed to males or the testing arena. Those animals receiving VCS showed a dramatic increase in the number of Fos-immunoreactive cells in the medial preoptic area, posterodorsal portion of the medial amygdala and bed nucleus of the stria terminalis, as well as the dorsomedial hypothalamus, ventromedial hypothalamus and midbrain central gray. These effects of VCS were confirmed in Experiment 2 in animals receiving manual vaginal-cervical probing. These findings extend previous electrophysiological and [14C]2-deoxy-D-glucose studies by providing evidence that additional brain areas respond to VCS by mating, as well as manual probing.

Immunocytochemical evidence for noradrenergic regulation of estrogen receptor concentrations in the guinea pig hypothalamus.

The noradrenergic system interacts with steroid hormones to influence female sexual behavior and gonadotropin release in rodents. Using a double label immunocytochemical procedure for estrogen receptors and dopamine-beta-hydroxylase, we investigated the anatomical relationships between noradrenergic neurons and estrogen receptor-immunoreactive (ER-IR) cells in the brain of ovariectomized female guinea pigs. Dopamine-beta-hydroxylase-immunoreactive (DBH-IR) varicosities were closely associated with ER-IR cells throughout the hypothalamus and preoptic area. This anatomical relationship was observed with almost 80% of the ER-IR cells in the ventrolateral hypothalamus (VLH), an area involved in the regulation of female sexual behavior in guinea pigs. Furthermore, the presence of closely associated DBH-IR varicosities was related to staining intensity of ER-IR neurons. In the rostral VLH, ER-IR neurons with closely associated DBH-IR varicosities stained more darkly than ER-IR neurons lacking this association, suggesting noradrenergic regulation of basal levels of cellular estrogen receptors. These findings provide neuroanatomical evidence suggestive of noradrenergic regulation of estrogen receptor levels in the hypothalamus of female guinea pigs.