Mating-induced expression of c-fos in the male Syrian hamster brain: Role of experience,pheromones, and ejaculations

This study was designed to investigate the effects of pheromonal cues and specific behaviors within the male copulatory sequence on c-fos expression in the medial nucleus of the amygdala (Me), the bed nucleus of the stria terminalis (BNST), and the medial preoptic area (MPOA) of the Syrian hamster brain. Sexually experienced male hamsters were placed into clean testing arenas and were either: 1) left alone as handled controls; 2) exposed to female hamster vaginal secretion (FHVS) on cotton swabs; or mated to various end points of copulation with a sexually receptive female: 3) five intromissions, 4) one ejaculation, 5) five ejaculations, or 6) long intromissions. A seventh group of sexually naive control males 7) was left alone in the arena. The brains of these males were compared to those of the sexually experienced controls to determine whether exposure to cues associated with prior sexual experience could alter c-fos expression. In males exposed only to FHVS, Fos immunoreactivity (Fos-ir) increased within the posterodorsal Me, the anterodorsal part of the posteromedial BNST, and the magnocellular medial preoptic nucleus (MPNmag). Following one ejaculation, Fos-ir increased within the caudal posterodorsal Me, the dorsolateral MPOA, and the paraventricular nucleus of the hypothalamus. After multiple ejaculations, additional labeling was observed within the posteroventral part of the posteromedial BNST, the medial preoptic nucleus (MPN), the central tegmental field, and in cell clusters of the caudal posterodorsal Me and rostral posteromedial BNST. Fos-ir also increased within the posterodorsal Me, MPN, and MPNmag in sexually experienced control males exposed to the empty test chamber compared to sexually naive males exposed to an identical chamber. These results demonstrate that the mating-induced pattern of neuronal activation in sexually experienced males is dependent upon multiple factors, including prior sexual experience in the testing environment, investigation of FHVS, and the number of ejaculations achieved. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 481–501, 1997     

Expression of FOS, EGR-1, and ARC in the amygdala and hippocampus of female rats during formation of the intromission mnemonic of pseudopregnancy

Pseudopregnancy (PSP) in the female rat is a neuroendocrine condition that is induced by repeated and intermittent vaginocervical stimulation received during mating and involves the expression of bicircadian prolactin surges and cessation of normal estrous cyclicity for 10-12 days postmating. The temporal patterning and number of intromissions received by the female are critical for PSP initiation, and thus, short-term encoding of VCS occurs during transduction of intromissions into PSP. In this experiment, we characterized and compared the mating-induced neural activation patterns within amygdalar and hippocampal regions using expression of the immediate early genes FOS, EGR-1, and ARC. Cycling female rats mated on proestrus received 15 or 5 intromissions under paced or nonpaced mating conditions. High numbers of intromissions during nonpaced mating or low numbers received during paced mating are sufficient to induce PSP, while five nonpaced intromissions and mounts without intromission are insufficient. Here we demonstrate that the CA1 region of the hippocampus was selectively sensitive to PSP-sufficient but not PSP-insufficient mating stimulation by showing significant effects of paced mating for all three IEGs. Paced mating also stimulated the expression of ARC within the basolateral, cortical, and central nuclei of the amygdala. The posterodorsal medial amygdala also showed selective EGR-1 responses to PSP-sufficient mating stimulation. There was no effect of hemisphere on IEG expression. The postmating expression profiles of these IEGs provide evidence that limbic areas involved in encoding and consolidation of memory are involved in initiating the neuroendocrine memory of PSP.     

Increased estrogen receptor alpha immunoreactivity in the forebrain of sexually satiated rats

Estrogen receptor alpha (ERalpha) participates in the neuroendocrine regulation of male sexual behavior, primarily in brain areas located in the limbic system. Males of many species present a long-term inhibition of sexual behavior after several ejaculations, known as sexual satiety. It has been shown that androgen receptor density is reduced 24 h after a single ejaculation or mating to satiety, in the medial preoptic area, nucleus accumbens and ventromedial hypothalamus. The aim of this study was to analyze if the density of ERalpha was also modified 24 h after a single ejaculation or mating to satiety. Sexual satiety was associated with an increased ERalpha density in the anteromedial bed nucleus of the stria terminalis (BSTMA), ventrolateral septum (LSV), posterodorsal medial amygdala (MePD), medial preoptic area (MPA) and nucleus accumbens core (NAc). A single ejaculation was related to an increase in ERalpha density in the BSTMA and MePD. ERalpha density in the arcuate (Arc) and ventromedial hypothalamic nuclei (VMN), and serum estradiol levels remained unchanged 24 h after one ejaculation or mating to satiety. These data suggest a relationship between sexual activity and an increase in the expression of ERalpha in specific brain areas, independently of estradiol levels in systemic circulation.     

The effects of mating stimulation on c-Fos immunoreactivity in the female hamster medial amygdala are region and context dependent

During mating in hamsters, both tactile and nontactile sensory stimulation experienced by the female affect sexual behavior and progestational neuroendocrine reflexes. To test the interactions of these types of mating stimulation, c-Fos immunohistochemistry measured brain cellular activity during sexual behavior under conditions that included combinations of tactile and nontactile mating stimulation. Test groups received: (1) mating stimulation from a male, females being either fully mated or mated while wearing a vaginal mask, or (2) experimenter applied manual vaginocervical stimulation (VCS)-with or without males present, or (3) handling similar to VCS but without insertions-with or without males present. Numbers of c-Fos immunoreactive cells were counted in specific subdivisions of the posterior medial amygdala (MeP) and ventromedial hypothalamus (VMH). The medial amygdala dorsal and ventral subdivisions responded differentially to components of mating stimulation. The posterodorsal Me (MePD) cellular activation was greatest during mating conditions that included VCS and/or males present. However, the posteroventral Me (MePV) was sensitive to male exposure and not to VCS. Also, MePV and VMH shell responses mirrored each other, both being primarily sensitive to male exposure. In separate tests, manual VCS induced pseudopregnancy, though the procedure was most effective with additional nontactile stimulation from males present. In summary, contextual cues provided by nontactile male stimulation enhance the effect of vaginocervical and other tactile stimulation on reproductive processes. Furthermore, c-Fos expression in the female hamster medial amygdala is region and context dependent.     

Expression of estrogen receptor-alpha and -beta mRNA in the brain of Japanese quail embryos

The present study was conducted to investigate the mRNA expression of the two estrogen receptor (ER) subtypes ERalpha and ERbeta in the brain of Japanese quail embryos. We found expression of both ERalpha and ERbeta mRNA in homogenate of whole head from 6-day-old embryos, and in brain homogenate from 9- and 12-day-old embryos using real-time PCR. In 9- and 12-day-old embryos the ERalpha expression was higher in females than in males. We used in situ hybridization to examine the localization of the ERs in sections from male and female brains on day 9 and day 17 of incubation. On day 9, ERbeta mRNA was detected in the developing medial preoptic nucleus (POM), in the medial part of the bed nucleus of the striae terminalis (BSTm), and in the tuberal region of the hypothalamus. ERalpha signal could not be detected in the POM, the BSTm or the tuberal region in 9-day-old embryos. In 17-day-old embryos, ERbeta was highly expressed in the preoptic area, the nucleus Taeniae of the Amygdala (TnA) and the BSTm. Expression of ERalpha mRNA was detected in parts of the preoptic area and in the telencephalic TnA. No ERalpha expression was found in the BSTm, an area known to be sexually dimorphic in adults. The high embryonic expression of ERbeta in brain areas linked to sexual behavior indicates that ERbeta plays a role in sexual differentiation of the Japanese quail brain.     

Elevated Fos-like immunoreactivity in the brains of postpartum female prairie voles, Microtus ochrogaster

This study investigated the expression of the protein product of the immediate early gene c-fos in the brains of female prairie voles (Microtus ochrogaster) in association with pregnancy and postparturient activities including maternal behavior, lactation and postpartum estrus. Fos expression was assessed in female voles that were late in pregnancy, nonpregnant or at one of three different times postpartum (0-8, 12-24, and 24-48 h, respectively). A significant increase in the number of cells displaying Fos immunoreactivity (Fos-ir) was observed during the 0-8 h and 12-24 h postpartum time periods in the accessory olfactory bulbs, medial preoptic area, hypothalamus (specifically, the supraoptic nucleus, ventro-medial hypothalamus, and paraventricular nucleus), lateral septum, bed nucleus of the stria terminalis, and primary somatosensory area of the brain. The number of Fos-ir cells decreased after 24 h postpartum. There were no significant changes in Fos-ir cell numbers in the primary olfactory bulbs, hippocampus, or caudate putamen. The neural activation of the medial preoptic area, accessory olfactory bulbs, hypothalamus, and bed nucleus is consistent with reports in rats of Fos induction associated with the onset of maternal behavior. In voles postpartum estrous behavior begins and ends 0-12 h after parturition. Maternal behavior, including lactation, is initiated at the same time but persists for several weeks. The highest Fos-ir cell numbers reported here coincide with the timing of postpartum estrous behavior in this species.     

Olfactory contribution to Fos expression during mating in inexperienced male hamsters

Male hamsters are very dependent on chemosensory cues for normal mating behavior. We have previously reported that central, vomeronasal pathways are intensely and selectively activated during mating or pheromonal stimulation. The contribution of main olfactory sensory input to the patterns of c-fos activation was investigated in this study. Sexually inexperienced male hamsters were either made anosmic by intranasal infusion of zinc sulfate or remained intact. Fos protein immunoreactivity was analyzed in main olfactory and vomeronasal pathways of the zinc sulfate-treated, anosmic animals after mating with receptive females for 45 min, and compared with Fos patterns seen in intact mating animals, some of which have been described in a previous publication. The zinc sulfate-treated anosmic males described here all mated when given access to receptive females. Whether mated or unstimulated, anosmic males had little or no Fos expression in main olfactory pathways; significantly less even than in unstimulated intact animals. Mating did not increase Fos expression in main olfactory pathways of intact animals over that of unstimulated intact controls. However, Fos expression in central vomeronasal pathways was significantly higher in mating anosmic males, as in intact males, compared with appropriate non-mating controls. Fos expression was significantly different between intact and zinc sulfate-treated anosmic mating males in only one area studied. The rostral anterior medial amygdala, known to receive a small olfactory terminal field, had significantly lower Fos expression in zinc sulfate-treated anosmic males that mated when compared with intact-mating animals. Thus, functional main olfactory input to the rostral vomeronasal amygdala can be demonstrated but does not appear to be critical for mating behavior in previously inexperienced male hamsters with intact vomeronasal organs. Other main olfactory input appears to have a negligible contribution to Fos-patterns in such animals.      

Clitoral stimulation induces conditioned place preference and Fos activation in the rat

The present study examined the ability of clitoral stimulation (CLS) to induce conditioned place preference (CPP) and Fos protein in the brain. Ovariectomized, hormone-primed Long-Evans rats were randomly assigned to receive either distributed CLS (1 stimulation every 5 s for 1 min prior to being placed in one distinctive side of a nonbiased CPP box for 2 min, after which the cycle of stimulation and CPP exposure were repeated for 4 more cycles, totaling 60 stimulations) or continuous CLS (1 stimulation per second for 1 min with 2 min in one side of the CPP box, repeated for 4 more cycles, totaling 300 stimulations). Two days later, females were placed into the other side of the CPP box without prior stimulation. CPP was tested after 5 sequential exposures each of CLS and no stimulation. Females given distributed stimulation developed a significant CPP whereas females given continuous stimulation did not. CLS induced Fos in hypothalamic and limbic structures, including the nucleus accumbens, piriform cortex, arcuate nucleus, and dorsomedial portion of the ventromedial hypothalamus, compared to no stimulation. However, distributed CLS induced more Fos in the medial preoptic area than continuous CLS or no stimulation. In contrast, continuous CLS induced more Fos in the posteroventral medial amygdala compared to no stimulation. These data indicate that CLS induces a reward state in the rat and a pattern of Fos activation in regions of the brain that process genitosensory input, incentive salience, and reward.     

FOS expression in grafted gonadotropin-releasing hormone neurons in hypogonadal mouse: Mating and steroid induction

We used FOS expression, widely accepted as a marker for neuronal activation, to evaluate physiologically induced activation of gonadotropin-releasing hormone (GnRH) neurons within intraventricular preoptic area grafts in hypogonadal (hpg) female mice. Hpg mice lack endogenous GnRH due to a mutated gene, but can respond to grafted GnRH neurons with reproductive development. The purpose of this study was to determine the degree to which the host brain regulates grafted GnRH neurons. FOS expression in grafted GnRH neurons was induced in progesterone-primed female mice paired with sexually active males. The degree of sexual activity did not affect the outcome, with 40.9 ± 12.2% of the grafted GnRH cells expressing FOS when male partners performed intromissions, and 47.5 ± 10.2% when they also ejaculated. There was little or no FOS expression in the grafts of unprimed hpg mice paired with sexually active males, in unpaired mice primed with progesterone or sequential estradiol benzoate and progesterone, or in controls. The pattern of FOS expression in the brains of the female hpg mice engaged in mating behavior was similar to that reported in other species, with moderate to high expression in the medial preoptic area, ventromedial nucleus, and medial amygdala in females paired with males that ejaculated. The present results support the hypothesis that host-derived activation of grafted GnRH neurons underlies aspects of reproductive responses seen in hpg mice with grafts, and further, that at least a portion of the host-graft connectivity is steroid sensitive. © 1996 John Wiley & Sons, Inc.     

Agonistic encounters and brain activation in dominant and subordinate male greater long-tailed hamsters

During an agonistic encounter test, dominant male greater long-tailed hamsters (Tscheskia triton) initiated attacks sooner and displayed higher levels of aggression and flank marking behavior than their subordinate counterparts. Accordingly, subordinate males exhibited more defensive behavior than dominant ones. Specific patterns of neuronal activation, measured by Fos-immunoreactive staining (Fos-ir), were found in the hamster brain following agonistic interactions. Increased Fos-ir was observed in the bed nucleus of the stria terminalis (BST), ventromedial hypothalamus (VMH), and medial (MeA) and anterior cortical (ACo) nuclei of the amygdala (AMYG) in both dominant and subordinate males. In contrast, dominant males had significantly higher Fos-ir densities in the medial preoptic area (MPOA) than subordinate males, whereas subordinate males expressed higher densities of Fos-ir in the anterior hypothalamus (AH) and central nucleus of the amygdala (CeA). Additionally, Fos-ir levels in the MPOA were significantly correlated with aggression and Fos-ir levels in the AH and CeA were correlated with defensive behavior. Together, our data indicate distinct patterns of neuronal activation associated with agonistic encounters in a behavior-specific manner in male greater long-tailed hamsters.     

Role of septum and preoptic area in regulating masculine and feminine sexual behavior in male rats

The effects of septal or preoptic lesions on both masculine and feminine sexual behaviors were examined in castrated adult male rats. Three weeks after brain surgery, animals were implanted with Silastic tubes containing testosterone (T) and observations of masculine sexual behavior were carried out four times every 5 days. T tubes were removed immediately after the end of the masculine behavioral tests. Two weeks later, animals implanted with Silastic tubes containing estradiol-17 beta(E2) were subjected to three feminine sexual behavioral tests at 5-day intervals. The bilateral lateral septal lesion (LSL) and the medial preoptic lesion (MPOL) effectively suppressed the performance of mounts, intromissions, and ejaculations, whereas the medial septal lesion (MSL), the dorsolateral preoptic lesion (DPOL), and the sham operation did not show any significant suppression of these behaviors. In the feminine sexual behavioral tests, intact and sham-operated control males showed only a low lordotic activity. However, the performance of the lordosis reflex was markedly facilitated by LSL or DPOL, while the lordotic activity of MSL and MPOL males was not significantly different from that of control males. These results suggest that the lateral septum exerts not only a facilitatory influence on masculine sexual behavior but also an inhibitory influence on feminine sexual behavior in male rats. On the other hand, the medial preoptic area may play a critical role in regulating masculine sexual behavior in male rats.     

Comparison of in vivo and in vitro subcellular localization of estrogen receptors alpha and beta in oligodendrocytes

The existence of estrogen receptors (ERs) in oligodendrocytes (OLGs) in vivo and in vitro is unresolved, as their presence has been reported in some studies and their absence in others. Using molecular and immunocytochemical techniques, we describe the subcellular localization of ERalpha and ERbeta in OLGs in vivo and in vitro. Both ERalpha and ERbeta are detected in an immortalized OLG cell line and in enriched OLG cultures by RT-PCR and western blot. Immunocytochemistry of OLGs from enriched cultures shows ERalpha receptors are nuclear, whereas ERbeta receptors are cytoplasmic. Confocal and deconvolution microscopy of enriched OLG cultures reveals ERbeta immunoreactivity is concentrated in perikarya and veins of OLG membrane sheets; lesser reactivity is present in their plasma membranes and nuclei. In vivo, we readily detect ERalpha in neurons but not in OLGs, even though we used different fixation procedures and different ERalpha antibodies. The presence of ERalpha in cultured OLGs may be due to culture media that contains factors stimulating ERalpha expression but are reduced in normal brain. In vivo, ERbeta immunoreactivity is readily detectable in OLG cytoplasm and in myelin sheaths. Incubation of glial cultures without or with increasing concentrations of 17beta-estradiol (E2) shows that E2 significantly accelerates OLG process formation.     

Localization of estrogen receptor-alpha and -betamRNA in brain areas controlling sexual behavior in Japanese quail

Two estrogen receptors (ERs), denoted ERalpha and ERbeta, have been identified in humans and various animal species, including the Japanese quail. Estrogens play a key role in sexual differentiation and in activation of sexual behavior in Japanese quail. The distribution of ERalpha in the brain of male and female adult quail has previously been studied using immunohistochemistry, whereas in situ hybridization has been employed to study the distribution of ERbeta mRNA in males only. In this article, we used in situ hybridization to study the distribution of mRNAs for both ERalpha and ERbeta in brain areas controlling sexual behavior of Japanese quail. Our results show that both ERalpha mRNA and ERbeta mRNA are localized in areas important for sexual behavior, such as the preoptic area and associated limbic areas, in both males and females. Moreover, we found differences in distribution of mRNA for the two receptors in these areas. The results of this article support previously reported data and provide novel data on localization of ER mRNAs in adult quail brain of both sexes.     

ERbeta protein expression in female cynomolgus monkey and CF-1 mouse brain: Western analysis

In humans and rodents, multiple ERbeta variants with sizes ranging from 477-549 amino acids (aa) have been described. The identification of these variants in target tissues has important implications for estrogen signaling and cellular responsiveness. Western blot analysis using two anti-ERbeta antibodies specific for mammalian ERbeta sequences (PA1-310B and PA1-311) was employed to examine ERbeta protein expression in neural tissues from ovariectomized (OVX) cynomolgus macaques and CF-1 mice as well as to assess potential regulatory effects of acute and extended estradiol (E(2)) treatment. In hypothalamic extracts from both species, a single ERbeta immunoreactive (ERbeta-ir) band was detected at approximately 54 kDa, corresponding to the expected molecular weight for ERbeta477 and/or 485. In cynomolgus females, oral E(2) administration for 16 weeks had no apparent effect on hypothalamic ERbeta protein expression. In mouse, a single injection of E(2) did not change hypothalamic ERbeta protein levels 1.5, 4, 8, 16, or 24 h after injection. Extending the hormonal treatment to 4 or 21 days in OVX female mice also had no effect on the level of hypothalamic ERbeta protein. Additional regional analyses in female mouse brain with PA1-310B antibody showed that a second, 59 kDa ERbeta-ir band was present in cortex, striatum, hippocampus, and amygdala that could represent one or both of the larger ERbeta variants (530 and 549aa). The expression level of the second ERbeta isoform exhibited regional variation, with the strongest immunoreactivity detected in cortex and amygdala. Elucidating the functions of these ERbeta isoforms in the CNS will facilitate our understanding of the tissue- and promoter-specific actions of estrogen.     

Effects of intracranial implants of dihydrotestosterone on the reproductive physiology and behavior of male guinea pigs

An experiment was conducted to determine whether the intracranial application of dihydrotestosterone (DHT), a nonaromatizable androgen, would stimulate male guinea pig mating. Of three castrate groups studied, one was a control group in which subjects were implanted both in the medial preoptic area (MPOA) and under the skin with cannulae containing cholesterol (NoDHT). Males in one of the experimental groups received implants of cholesterol in the MPOA plus subcutaneous implants containing DHT (ScDHT). Members of the other experimental group were subcutaneously implanted with cholesterol and simultaneously given intracranial implants of DHT (IcDHT). Compared to either the NoDHT control group of the ScDHT experimental group, greater numbers of males in the IcDHT group displayed mounts (P less than 0.01), intromissions (P less than 0.01), and ejaculations (P less than 0.001). Additionally, the hypothalamic implants of DHT had no significant effects on peripheral target tissues. These data indicate that androgenic stimulation of the guinea pig brain is sufficient to activate masculine sexual behavior in this species.     

Obesity-inducing amygdala lesions: examination of anterograde degeneration and retrograde transport

Small lesions centered in the posterodorsal region of the medial amygdala resulted in excessive weight gains in female rats. Unilateral lesions were nearly as effective as bilateral lesions in the first 48 h after surgery (+21 to +32 g). Assessment of lesion damage was done by both qualitative evaluation and by a quantitative grid-point counting method. The critical sites for weight gain were the intra-amygdaloid bed nucleus of the stria terminalis and the posterodorsal medial amygdaloid nucleus. Incidental damage to the overlying globus pallidus was negatively related to weight gain. The cupric silver method for demonstrating axonal degeneration was applied to brains with obesity-inducing lesions. A dense pattern of degenerating terminals was found in the lateral septum, amygdala, ventral striatum, and ventromedial hypothalamus. Degeneration in the paraventricular nucleus of the hypothalamus was scarce or absent. Small retrograde tracer injections made in either the intra-amygdaloid bed nucleus of the stria terminalis or in the posterodorsal medial amygdaloid nucleus labeled cells in the amygdala, lateral septum, and hypothalamus, reciprocating the anterograde projections from the amygdala to these areas. The data suggest that subdivisions of the posterodorsal amygdala participate in the regulation of feeding in a manner that is similar to the better-known role of this part of the brain in mediating reproductive behavior. Although topographical differences may exist within the amygdaloid and hypothalamic subdivisions regulating these two sexually dimorphic behaviors, the relays engaged by feeding-related connections and those related to reproduction are remarkably parallel.