Age-related changes in sexual function and steroid-hormone receptors in the medial preoptic area of male rats.

Testosterone is the main circulating steroid hormone in males, and acts to facilitate sexual behavior via both reduction to dihydrotestosterone (DHT) and aromatization to estradiol. The mPOA is a key site involved in mediating actions of androgens and estrogens in the control of masculine sexual behavior, but the respective roles of these hormones is not fully understood. As males age they show impairments in sexual function, and a decreased facilitation of behavior by steroid hormones compared to younger animals. We hypothesized that an anatomical substrate for these behavioral changes is a decline in expression and/or activation of hormone receptor-sensitive cells in the mPOA. We tested this by quantifying and comparing numbers of AR- and ERα-containing cells, and Fos as a marker of activated neurons, in the mPOA of mature (4-5months) and aged (12-13months) male rats, assessed one hour after copulation to one ejaculation. Numbers of AR- and ERα cells did not change with age or after sex, but the percentage of AR- and ERα-cells that co-expressed Fos were significantly up-regulated by sex, independent of age. Age effects were found for the percentage of Fos cells that co-expressed ERα (up-regulated in the central mPOA) and the percentage of Fos cells co-expressing AR in the posterior mPOA. Interestingly, serum estradiol concentrations positively correlated with intromission latency in aged but not mature animals. These data show that the aging male brain continues to have high expression and activation of both AR and ERα in the mPOA with copulation, raising the possibility that differences in relationships between hormones, behavior, and neural activation may underlie some age-related impairments.

Copulation induces expression of the immediate early gene Arc in mating-relevant brain regions of the male rat.

The medial amygdala (MeA), bed nucleus of the stria terminalis (BNST), and medial preoptic area (mPOA) are important for the regulation of male sexual behavior. Sexual experience facilitates sexual behaviors and influences activity in these regions. The goal of this study was to determine whether sexual experience or copulation induces plasticity in the MeA, BNST, or mPOA of male rats, as indicated by changes in levels of Arc, which is indicative of activity-dependent synaptic plasticity in the brain. To this end, sexually naïve or experienced males were placed in mating arenas either alone, with an inaccessible estrus female, or with an accessible estrus female. Arc protein levels were then quantified in these three regions using immunohistochemistry. As expected, sexual experience facilitated copulation, as evidenced by a reduction in latencies to mount, intromit, and ejaculate. Copulation also increased the number of Arc-positive cells in the MeA, anterior BNST, posterior BNST, and the posterior mPOA, but not in the central-rostral region of the mPOA. Surprisingly, prior sexual experience did not impact levels of Arc, suggesting that copulation-induced Arc occurs in both sexually naïve and experienced males.

Aging and estradiol effects on gene expression in the medial preoptic area, bed nucleus of the stria terminalis, and posterodorsal medial amygdala of male rats.

Studies on the role of hormones in male reproductive aging have traditionally focused on testosterone, but estradiol (E2) also plays important roles in the control of masculine physiology and behavior. Our goal was to examine the effects of E2 on the expression of genes selected for E2-sensitivity, involvement in behavioral neuroendocrine functions, and impairments with aging. Mature adult (MAT, 5 mo) and aged (AG, 18 mo) Sprague-Dawley male rats were castrated, implanted with either vehicle or E2 subcutaneous capsules, and euthanized one month later. Bilateral punches were taken from the bed nucleus of the stria terminalis (BnST), posterodorsal medial amygdala (MePD) and the preoptic area (POA). RNA was extracted, and expression of 48 genes analyzed by qPCR using Taqman low-density arrays. Results showed that effects of age and E2 were age- and region-specific. In the POA, 5 genes were increased with E2 compared to vehicle, and there were no age effects. By contrast the BnST showed primarily age-related changes, with 6 genes decreasing with age. The MePD had 5 genes that were higher in aged than mature males, and 17 genes with significant interactions between age and E2. Gene families identified in the MePD included nuclear hormone receptors, neurotransmitters and neuropeptides and their receptors. Ten serum hormones were assayed in these same males, with results revealing both age- and E2-effects, in several cases quite profound. These results support the idea that the male brain continues to be highly sensitive to estradiol even with aging, but the nature of the response can be substantially different in mature and aging animals.

Colocalization of Mating-Induced Fos and D2-Like Dopamine Receptors in the Medial Preoptic Area: Influence of Sexual Experience.

Dopamine in the medial preoptic area (mPOA) stimulates sexual activity in males. This is evidenced by microdialysis and microinjection experiments revealing that dopamine receptor antagonists in the mPOA inhibit sexual activity, whereas agonists facilitate behavior. Microdialysis experiments similarly show a facilitative role for dopamine, as levels of dopamine in the mPOA increase with mating. While the majority of evidence suggests an important role for dopamine receptors in the mPOA in the regulation of male sexual behaviors, whether sexual activity or sexual experience influence dopamine receptor function in the mPOA has not been previously shown. Here we used immunohistochemical assays to determine whether varying levels of sexual activity or experience influence the number of cells containing Fos or D2 receptor immunoreactivity. Results show that sexual experience facilitated subsequent behavior, namely experience decreased latencies. Moreover, the number of cells with immunoreactivity for Fos or D2 correlated with levels of sexual experience and sexual activity. Sexual activity increased Fos immunoreactivity. Sexually experienced animals also had significantly more D2-positive cells. Sexually inexperienced animals copulating for the first time had a larger percentage of D2-positive cells containing Fos, when compared to sexually experienced animals. Finally, regardless of experience, animals that had sex prior to sacrifice had significantly more D2-positive cells that contained Fos, vs. animals that did not copulate. These findings are noteworthy because sexually experienced animals display increased sexual efficiency. The differences in activation of D2 and changes in receptor density may play a role in this efficiency and other behavioral changes across sexual experience.

Estradiol in the Preoptic Area Regulates the Dopaminergic Response to Cocaine in the Nucleus Accumbens.

The sex-steroid hormone estradiol (E2) enhances the psychoactive effects of cocaine, as evidenced by clinical and preclinical studies. The medial preoptic area (mPOA), a region in the hypothalamus, is a primary neural locus for neuroendocrine integration, containing one of the richest concentrations of estrogen receptors in the CNS and also has a key role in the regulation of naturally rewarding behaviors. However, whether estradiol enhances the neurochemical response to cocaine by acting in the mPOA is still unclear. Using neurotoxic lesions and microdialysis, we examined whether the mPOA modulates cocaine-induced neurochemical activity in the nucleus accumbens. Tract tracing and immunohistochemical staining were used to determine whether projections from the mPOA to the ventral tegmental area (VTA) are sensitive to estrogen signaling. Finally, estradiol microinjections followed by microdialysis were used to determine whether estrogenic signaling in the mPOA modulates cocaine-induced changes of dopamine in the nucleus accumbens. Results showed that lesions of the mPOA or microinjections of estradiol directly into the mPOA increased cocaine-induced release of dopamine in the nucleus accumbens. Immunohistochemical analyses revealed that the mPOA modulates cocaine responsiveness via projections to both dopaminergic and GABAergic neurons in the VTA, and that these projections are sensitive to estrogenic stimulation. Taken together, these findings point to a novel estradiol-dependent pathway that modulates cocaine-induced neurochemical activity in the mesolimbic system.

Astrocytes in the medial preoptic area modulate ejaculation latency in an experience-dependent fashion.

While sexually experienced males copulate at a higher frequency than sexually inexperienced males, there is still a great deal of variability in their behavior. Within the medial preoptic area (mPOA) of the hypothalamus, glutamate modulates some of this variability. Glutamate levels, for example, increase during sexual activity, peaking with ejaculation and falling precipitously during the post-ejaculation interval. Whereas lower glutamate levels after ejaculation translates to longer post-ejaculatory intervals, administration of glutamate uptake inhibitors into the mPOA increases the number of ejaculations a male rat achieves over a mating bout, and reduces the latency to ejaculate once mating begins. Because astrocytes modulate the availability of neuronal glutamate, we hypothesized that differences in the number of GFAP-positive astrocytes in the mPOA may account for variability in sexual behavior. To this end, we examined whether the number of astrocytes in the mPOA related to ejaculation latency as well as to the duration of the post-ejaculatory interval (PEI) in sexually experienced and sexually inexperienced males. Results indicate that the number of astrocytes negatively correlated with latency to reach ejaculations in sexually inexperienced but not sexually experienced rats while the number of astrocytes and PEI were not related. Astrocyte numbers did not vary between inexperienced and experienced subjects indicating that astrocyte processes may differentially project to sex-relevant glutamatergic synapses or that glutamatergic innervation of the mPOA changes as a function of sexual experience.

Sexual experience influences mating-induced activity in nitric oxide synthase-containing neurons in the medial preoptic area.

Nitric oxide (NO) acts in the medial preoptic area (mPOA) of the hypothalamus to facilitate the expression of male sexual behavior and has also been widely implicated in mechanisms of experience, learning, and memory. Using immunohistochemistry for Fos, as a marker for neural activity, and nitric oxide synthase (NOS), the enzyme that catalyzes the production of nitric oxide (NO), we examined whether sexual activity and sexual experience influence Fos co-expression in NOS-containing neurons in the mPOA of male rats. Consistent with previous findings, results indicate that mating increased activity in the mPOA, and that sexual experience facilitated the expression of sexual behaviors, together with increased mating-induced Fos and NOS in the mPOA. Results also indicate that mating increased co-expression of Fos in NOS-containing neurons, and that this increase was highest in animals undergoing their first sexual encounter, indicating that initial sexual experience increases NO production in the mPOA of male rats.

The medial preoptic area modulates cocaine-induced activity in female rats.

Drugs of abuse exert their effects by exploiting natural neurobiological reward mechanisms, especially the mesolimbic dopamine (DA) system. However, the mesolimbic system does not operate in isolation, and input from other reward-relevant structures may play a role in cocaine's rewarding effects. The medial preoptic area (mPOA) of the hypothalamus is involved in the regulation of two essential and naturally rewarding behaviors: sexual and maternal behaviors. It also makes strong neuroanatomical connections with areas of the mesolimbic system, particularly the ventral tegmental area (VTA). As such, the mPOA is a logical candidate for a neuroanatomical locus modulating activity in the mesolimbic system and emergent behavioral expressions of drug reward, yet the role of this structure is largely unexplored. Here, using a female rat model, we show that the mPOA innervates the VTA in a region-specific manner, that lesions of the mPOA augment cocaine-induced Fos expression in the nucleus accumbens (NAc) and cocaine-induced conditioned place preference. We also show that approximately 68% of mPOA-VTA efferents release γ-aminobutyric acid (GABA), over 75% are sensitive to DA as evidenced by colocalization with DA receptors, and nearly 60% of these contain both DA receptors and GABA, which suggests a novel key role for the mPOA in the inhibition of the mesolimbic DA circuit. Combined, these results reveal the mPOA as a critical modulating structure in cocaine-induced mesolimbic activity and behavioral manifestation of reward, at least in part, via GABAergic output that is sensitive to DA input.

Mating-relevant olfactory stimuli activate the rat brain in an age-dependent manner.

Chemosensory stimulation is vital for the expression of rodent sexual behavior. As sexual activity decreases with aging, this study investigated whether aging also impacts the integration of sex-relevant chemosensory cues. To this end, several measures were obtained from adult (10-12 months) and aged (30-36 months) male rats after exposure to a conspecific estrous female. These included rates of investigatory behaviors, levels of stimulus-induced Fos immunoreactivity, activation of gonadotropin-releasing hormone-containing cells, and levels of circulating testosterone and corticosterone. The results indicated no significant differences in investigatory behaviors, levels of corticosterone, or activation of gonadotropin-releasing hormone-containing cells between the two groups. As has been reported previously, the levels of testosterone were lower in the aged rats. However, stimulus-induced neural activity was higher in the bed nucleus of the stria terminalis and the medial preoptic area of aged rats, whereas no differences were found in the main olfactory bulb, accessory olfactory bulb, medial amygdala, ventral tegmental area, or nucleus accumbens. These findings suggest the presence of a compensatory mechanism in the hypothalamus of aged animals versus adults, whereby more cells are recruited to elicit a sexual response in the presence of a sexually exciting stimulus.