Effects of a D1 antagonist and of sexual experience on copulation-induced Fos-like immunoreactivity in the medial preoptic nucleus.

The medial preoptic nucleus (MPN) of the medial preoptic area (MPOA) and the medial amygdala are two brain regions in which male rat sexual behavior increased Fos-like immunoreactivity (Fos-Li). Dopamine is released in the MPOA during male rat sexual behavior and facilitates copulation. Psychostimulants, which increase dopamine levels, induce Fos-Li in the striatum through D1 receptors. We examined whether copulation-induced Fos-Li in the MPN was also mediated through D1 receptors. In Experiment 1, sexually inexperienced male rats that received the D1 antagonist Schering 39166 prior to their first sexual experience had fewer Fos-Li cells in the MPN than did those that received vehicle. In Experiment 2, no significant effect of the D1 antagonist was observed on copulation-induced Fos-Li in male rats that had received repeated sexual experiences prior to the drug test day. Sexual experience increases copulatory efficiency; the mechanisms by which this improvement occurs are unclear. In Experiment 3, copulation by highly experienced male rats led to greater Fos-Li in the MPN than did copulation by sexually naive males. Although there were no differences between groups in amygdala Fos-Li in these studies, in several groups Fos-Li in the medial amygdala was positively correlated with the post-ejaculatory interval. These experiments indicate that (1) stimulation of D1 receptors may contribute to the transient copulation-induced increase in Fos-Li in the MPN, and (2) repeated sexual experiences enhanced copulation-induced Fos-Li in the MPN, which may represent a marker of altered responsiveness of neurons in the MPN to sexual or conditioned stimuli.     

Testosterone or Oestradiol Implants in the Medial Preoptic Area Induce Mating in Noncopulating Male Rats

Noncopulating (NC) male rats are those males that do not mount, intromit or ejaculate when repeatedly tested with receptive females. The lack of sexual behaviour in these males is not associated with alterations in testosterone or oestradiol (E₂) plasma concentrations. Instead, it has been shown that androgen receptors are higher and oestrogen receptors are lower in the medial preoptic area (MPOA) of NC male rats than those observed in copulating (C) male rats. We have also observed reduced aromatase activity in the MPOA (but not in other brain regions) of NC male rats. The aim of the present study was to determine whether testosterone or E₂ implants in the MPOA of NC male rats could induce sexual behaviour. Accordingly, in Experiment 1, we evaluated the long‐term effects of testosterone or E₂ implants in the MPOA, the ventromedial nucleus of the hypothalamus or the medial amygdala with respect to inducing sexual behaviour in castrated C male rats. Male rats were bilaterally implanted with a guide cannula, either empty or containing testosterone or E₂. Starting 1 week later, all male rats were mated once weekly for 5 months. As described previously, the site where hormone implants most consistently induced sexual behaviour in castrated C male rats was the MPOA. Experiment 1 extended these findings showing that the males continued mating even 5 months after the implant. In the second experiment, NC males were implanted in the MPOA with a guide cannula empty or filled with testosterone or E_2. One week after the testosterone or E₂ implant, the percentage of males that mounted and intromitted started to increase and, 5 weeks after the implant, 50% of the subjects displayed mounts and intromissions. All NC males implanted with testosterone ejaculated consistently from week 11 after the implant until the end of testing (5 months), whereas all subjects implanted with E₂ ejaculated from week 16 after the implant until the end of testing. These results support the hypothesis that, in the MPOA of NC male rats, there is a hormonal alteration associated with the lack of sexual behaviour.     

Anatomical interrelationships of the medial preoptic area and other brain regions activated following male sexual behavior: A combined Fos and tract-tracing study

The medial preoptic nucleus (MPN) is an essential site for the regulation of male sexual behavior. Previous studies using c-fos as a marker for neural activation have shown that copulation increased c-fos expression in the MPN. Neural activation was also present in brain regions that are connected with the MPN and are involved in male sexual behavior, including the posteromedial bed nucleus of the stria terminalis (BNSTpm), posterodorsal preoptic nucleus (PD), posterodorsal medial amygdala (MEApd), and parvocellular subparafascicular thalamic nucleus (SPFp). The present study investigated whether the copulation-induced, activated neurons in these brain regions are involved in the bidirectional connections with the MPN. Therefore, mating-induced Fos expression was combined with application of anterograde (biotinylated dextran amine) or retrograde (cholera toxin B subunit) tracers in the MPN. The results demonstrated that neurons in the BNSTpm, PD, MEApd, and SPFp that project to the MPN were activated following copulation. However, in males that displayed sexual behavior but did not achieve ejaculation, few double-labeled neurons were evident, although both retrogradely labeled neurons and Fos-immunoreactive cells were present. In addition, retrograde neurons that expressed Fos were located in discrete subdivisions within the brain regions studied, where Fos is induced after ejaculation. Likewise, anterogradely labeled fibers originating from the MPN were not distributed homogeneously but were particularly dense in these discrete subdivisions. These results demonstrate that copulation-induced Fos-positive neurons in specific subdivisions of the BNSTpm, PD, MEApd, and SPFp have bidirectional connections with the MPN. Taken together with previous findings, this supports the existence of a discrete subcircuit within a larger neural network underlying male sexual behavior. J. Comp. Neurol. 397:421–435, 1998. © 1998 Wiley-Liss, Inc.     

The medial and lateral cell groups of the sexually dimorphic area of the gerbil hypothalamus are essential for male sex behavior and act via separate pathways

Male reptiles, birds and mammals do not copulate if the medial preoptic area (MPOA) is destroyed but the MPOA cell groups necessary for male sexual behavior were not known. Here, two cell groups essential for copulation are identified in the sexually dimorphic area (SDA) of the gerbil (Meriones unguiculatus) MPOA. Bilateral cell-body lesions of either the medial or lateral SDA eliminated mating in sexually experienced male gerbils given testosterone. Nearby MPOA lesions did not. The medial and lateral SDA affect sex behavior via separate pathways since lesioning the medial SDA on one side of the brain and the lateral SDA on the other did not stop sexual behavior.     

Distribution of galanin-immunoreactive cells within sexually dimorphic components of the medial preoptic area of the male and female rat

A high percentage of galanin-immunoreactive (GAL-I) cells within sexually dimorphic components of the medial preoptic area (MPOA) of the rat also concentrate estrogen and GAL microinjected within the medial preoptic nucleus (MPN) facilitates masculine sexual behavior after testosterone priming. Thus, we determined the distribution of GAL-I cells within the MPOA and their response to gonadal steroids. We report significantly greater numbers of GAL-I cells within the central division of the medial preoptic nucleus (MPNc) and fewer within the anteroventral periventricular nucleus (AVPv), of the gonadectomized male than the gonadectomized female; that GAL-I cell numbers and densities within the AVPv are increased significantly in the intact, testosterone- or estrogen-treated male compared to the gonadectomized male and that GAL-I cell numbers and densities within the MPNc and GAL-I cell densities within the medial division of the MPN (MPNm), are increased significantly by gonadal steroids in rats of both sexez. The results suggest an involvement of galaninergic cells within the MPOA in the regulation of sexually dimorphic, gonadal steroid-sensitive functions.     

Partial antagonism of 8-OH-DPAT'S effects on male rat sexual behavior with a D2, but not a 5-HT1A, antagonist

The serotonin agonist 8-hydroxy-di-propylaminotetralin (8-OH-DPAT), injected systemically or directly into the medial preoptic area (MPOA), reduces the ejaculatory threshold in male rats. While 8-OH-DPAT has been characterized as an agonist at the 5-HT_1A receptor, it also acts at other receptor sites including the dopamine D₂ receptor. The current experiments investigated whether 8-OH-DPAT injected into the MPOA facilitates male sexual behavior through stimulation of the 5-HT_1A receptor or the dopamine D₂ receptor. Experiment 1 co-administered 8-OH-DPAT (6 μg) with either the 5-HT_1A antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride (MPPI) (10 μg) or the D₂ antagonist raclopride (10 μg). Raclopride blocked 8-OH-DPAT's facilitative effects on ejaculation frequency and latency, while the 5-HT_1A antagonist was ineffective. In Experiment 2, 8-OH-DPAT (500 μM), retrodialyzed into the MPOA through a microdialysis probe, enhanced male copulatory behavior similarly to the microinjection, increasing ejaculation frequency and decreasing ejaculation latency, postejaculatory interval and mount frequency. Retrodialyzing 8-OH-DPAT through a microdialysis probe in the MPOA had been previously shown to increase extracellular levels of dopamine and serotonin. The data from the present studies suggest that the effects of 8-OH-DPAT in the MPOA on male rat copulatory behavior may be mediated, at least in part, either directly through 8-OH-DPAT's activity at D₂ receptors or indirectly through 8-OH-DPAT's ability to increase extracellular dopamine.     

Testosterone, Preoptic Dopamine, and Copulation in Male Rats

Steroid hormones prime neural circuits for sexual behavior, in part by regulating enzymes, receptors, or other proteins affecting neurotransmitter function. Dopamine facilitates male sexual behavior in numerous species and is released before and/or during copulation in three integrative neural systems. The nigrostriatal system enhances readiness to respond; the mesolimbic system promotes many appetitive behaviors; the medial preoptic area (MPOA) contributes to sexual motivation, genital reflexes, and copulation. We have reported a consistent relationship between precopulatory dopamine release in the MPOA, when an estrous female was behind a perforated barrier, and the ability to copulate after the barrier was removed. Recent, but not concurrent, testosterone was necessary for the precopulatory dopamine response and copulation. The deficit in MPOA dopamine release in castrates was observed in basal conditions as well as the sexual context. However, dopamine in tissue punches from castrates was higher than in intact males. Because tissue levels represent primarily stored neurotransmitter, dopamine appeared to have been synthesized normally, but was not being released. Amphetamine induced greater dopamine release in castrates, again suggesting excessive dopamine storage. The decreased release may result from decreased activity of nitric oxide synthase in the MPOA of castrates. A marker for this enzyme showed lower activity in castrates than in intact males. Finally, blocking nitric oxide synthase in intact males blocked the copulation-induced release of dopamine in the MPOA. Therefore, one means by which testosterone may promote copulation is by upregulating nitric oxide synthesis in the MPOA, which in turn enhances dopamine release.     

Effects of discrete lesions of the sexually dimorphic nucleus of the preoptic area or other medial preoptic regions on the sexual behavior of male rats

The sexually dimorphic nucleus of the rat medial preoptic area (SDN-POA) has a volume five times larger in the adult male compared with that of the adult female. In the present study, the effects of discrete electrolytic destruction of the SDN-POA or other specific medial preoptic (MPOA) regions on masculine sexual behavior were determined in adult, sexually experienced male rats. Small lesions encompassing the SDN-POA had no effect on the maintenance of copulatory behavior. Lesions of similar size placed within the ventral or anterio-dorsal MPOA also did not consistently affect the display of masculine sexual behavior. However, animals that received small lesions within their dorsal MPOA showed a substantial, long-term decrease in number of mounts, intromissions, and ejaculations compared to these parameters in sham-lesioned control rats, thus indicating a lesion-induced disruption of those neural mechanisms mediating these behaviors. Collectively these data suggest that the SDN-POA is not critical for a full expression of male sexual behavior and that the dorsal MPOA may be more important than other MPOA regions for copulatory behavior.     

Stimulation of the medial amygdala enhances medial preoptic dopamine release: implications for male rat sexual behavior

Increased dopamine (DA) in the medial preoptic area (MPOA) facilitates male sexual behavior. A major source of innervation to the MPOA is the medial amygdala (MeA). We now report that chemical stimulation of the MeA enhanced levels of extracellular MPOA DA in anesthetized male rats. These results suggest that DA activity in the MPOA can be regulated by input from the MeA to the MPOA.     

Identification of a sexually dimorphic neural population immunoreactive for calcitonin gene-related peptide (CGRP) in the rat medial preoptic area.

The medial preoptic area (MPOA) of the rat exhibits morphological sex differences and is implicated in sex-specific functioning and behaviour. Using immunocytochemistry, the distribution and numbers of cells containing calcitonin gene-related peptide (CGRP) were examined in the MPOA of adult male and female rats. In the intact female rat, CGRP-immunoreactive (-IR) cells were found in a continuum within the MPOA extending from the caudal aspects of the organum vasculosum of the lamina terminalis through the anteroventral periventricular nucleus (AVPv) to the region of the medial preoptic nucleus (MPN). An additional small group of CGRP-IR cells was noted at the level of the caudal MPNin the ventrolateral (VL) region. Compared with males, the AVPv and MPN regions of the female contained over 25-fold more CGRP-IR cells (P < 0.01). The VL region contained similar numbers of CGRP-IR cells in both sexes. Ovariectomy 1 month earlier, with or without subsequent 17-beta estradiol treatment, had no effect on the numbers or distribution of CGRP-IR cells in the MPOA. Gonadectomy of male rats resulted in a significant increase (P < 0.01) in the numbers of CGRP-IR cells in the AVPv and MPN regions. Subsequent administration of testosterone propionate for 1 week reduced (P < 0.05) numbers of CGRP-IR cells to levels observed in the intact male. Neurones containing CGRP in the VL group were not altered by gonadal steroid manipulation. This study shows that CGRP neurones in the AVPv/MPN region are sexually dimorphic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microinjection of cis-flupenthixol, a dopamine antagonist, into the medial preoptic area impairs sexual behavior of male rats

Systemically administered dopamine agonists have been shown to facilitate copulation in male rats. Microinjection of the dopamine agonist apomorphine into the medial preoptic area has also been reported to facilitate sexual behavior. The present experiments investigated the effects of medial preoptic microinjections of the dopamine antagonist cis-flupenthixol on male rat copulatory behavior. Fewer males initiated copulation and fewer ejaculated following flupenthixol administration. Those males that did ejaculate following flupenthixol injections had fewer ejaculations and longer interintromission intervals. Flupenthixol also antagonized the facilitative effects of apomorphine injections into the medial preoptic area. Flupenthixol and apomorphine produced only minor alterations in noncopulatory behaviors. The results suggest that dopamine receptors within the medial preoptic area are important in the regulation of masculine sexual behavior in the rat.     

Neuronal nitric oxide synthase and gonadal steroid interaction in the MPOA of male rats: co-localization and testosterone-induced restoration of copulation and nNOS-immunoreactivity

Neuronal nitric oxide synthase (nNOS) in the medial preoptic area (MPOA) has been implicated in various physiological functions, including male rat copulation. Based on their apparent sensitivity to gonadal steroid manipulation, we hypothesized that nNOS cells contain steroid receptors, and the testosterone-induced restoration of nNOS-immunoreactivity in castrates should accompany the restoration of copulation. In Experiment 1, we investigated co-localization of nNOS with the androgen receptor (AR) and the estrogen receptor alpha (ERalpha) using immunocytochemistry. We found regionally specific co-localizations of nNOS-AR and nNOS-ERalpha. In Experiment 2, we investigated the relationship between MPOA nNOS-immunoreactivity (ir) and copulatory measures in the testosterone-induced restoration paradigm in castrates. The restoration of various copulatory measures was accompanied by an increase in optical density of nNOS-ir, but not in the number of nNOS-ir cells. These data provide additional evidence supporting the role of MPOA nitric oxide in male rat copulation.     

Homogeneity of intracellular electrophysiological properties in different neuronal subtypes in medial preoptic slices containing the sexually dimorphic nucleus of the rat

The sexually dimorphic nucleus of the preoptic area (SDN-POA) is larger in male than in female rats, the male phenotype requiring the presence of circulating androgens perinatally. These experiments investigated the intracellular electrophysiology and morphology of SDN-POA neurons and compared these properties with those of other medial preoptic area (MPOA) neurons. Biocytin-injected cells in the SDN-POA either had one or two primary dendrites, or they had multipolar dendritic arrays; dendrites were aspiny or sparsely spiny and displayed limited branching. Neurons in other parts of the MPOA were similar morphologically. Regardless of morphology, neurons situated in either the SDN-POA or surrounding MPOA had low-threshold potentials and linear or nearly linear current-voltage relations. In most (73%) cells, stimulation of the dorsal preoptic region evoked a fast excitatory postsynaptic potential followed by a fast inhibitory postsynaptic potential (IPSP). Bicuculline blocked the fast IPSPs, which reversed near the Cl² equilibrium potential (-71 ± 5mV), indicating their mediation by gamma-aminobutyric acid (GABA)_A receptors. Neurons in the SDN-POA have electrophysiological properties similar to those of other medial preoptic cells. When compared with the hypothalamic paraventricular nucleus, the MPOA appears relatively homogeneous electrophysiologically. This is despite the morphological variability within this population of neurons and heterogeneities that are also apparent at other levels of analysis. Finally, GABA-mediated, inhibitory synaptic contacts are widespread among medial preoptic neurons, consistent with indications from earlier reports that GABA provides a link in the feedback actions of gonadal steroids on the release of gonadotropic hormones. © 1994 Wiley-Liss, Inc.     

The influence of chemosensory input and gonadotropin releasing hormone on mating behavior circuits in male hamsters

Chemosensory input is important for mating behavior in male hamsters. Chemosignals found in female hamster vaginal fluid activate regions of the brain that receive input from the vomeronasal/accessory olfactory system and are important for mating behavior. Mating or exposure to these chemosignals produces increased Fos protein expression in the amygdala, bed nucleus of the stria terminalis, and medial preoptic area (MPOA). These brain regions contain cell bodies and/or fibers of gonadotropin releasing hormone (GnRH) neurons, suggesting potential relationships between chemosensory systems and GnRH. GnRH is released naturally when male rodents (mice and hamsters) encounter female chemosignals, and intracerebrally injected GnRH restores mating behavior in sexually naive male hamsters after removal of the vomeronasal organs. We report here that the combination of pheromone exposure and intracerebrally-injected GnRH increases Fos expression in the MPOA above the increase seen in pheromone-exposed males, or in males given only the exogenous GnRH. In males with vomeronasal organs removed (VNX), there was an also an increment in Fos expression in the MPOA when these pheromone exposed males were injected with GnRH, provided they had previous sexual experience. Males with vomeronasal organs removed and without sexual experience showed increased Fos expression in the medial amygdala when pheromone exposure and GnRH injection were combined, but not in the medial preoptic area.     

Activation of noradrenergic neurons projecting to the diencephalon following central administration of histamine is mediated by H1 receptors

The effect of histamine on the activity of noradrenergic neurons terminating in discrete regions of the diencephalon was examined in male rats. Noradrenergic neuronal activity was estimated by measuring the concentration of norepinephrine and its metabolite 3-methoxy-4-hydroxyphenylethyleneglycol [MHPG] in the medial zona incerta [MZI] and in the dorsomedial [DMN], periventricular [PeVN] and medial preoptic hypothalamic nuclei [MPN]. The intracerebroventricular administration of histamine effected a time-related increase in MHPG concentrations in the MZI, DMN, PeVN and MPN; these effects were blocked by the H₁ antagonist mepyramine but not the H₂ antagonist zolantidine. Neither mepyramine nor zolantidine affected basal MHPG concentrations in any of the brain regions examined. These results indicate that central administration of histamine increases the activity of noradrenergic neurons projecting to the diencephalon via an action at H₁ but not H₂ receptors.     

Partial antagonism of 8-OH-DPAT'S effects on male rat sexual behavior with a D2, but not a 5-HT1A, antagonist

The serotonin agonist 8-hydroxy-di-propylaminotetralin (8-OH-DPAT), injected systemically or directly into the medial preoptic area (MPOA), reduces the ejaculatory threshold in male rats. While 8-OH-DPAT has been characterized as an agonist at the 5-HT_1A receptor, it also acts at other receptor sites including the dopamine D₂ receptor. The current experiments investigated whether 8-OH-DPAT injected into the MPOA facilitates male sexual behavior through stimulation of the 5-HT_1A receptor or the dopamine D₂ receptor. Experiment 1 co-administered 8-OH-DPAT (6 μg) with either the 5-HT_1A antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride (MPPI) (10 μg) or the D₂ antagonist raclopride (10 μg). Raclopride blocked 8-OH-DPAT's facilitative effects on ejaculation frequency and latency, while the 5-HT_1A antagonist was ineffective. In Experiment 2, 8-OH-DPAT (500 μM), retrodialyzed into the MPOA through a microdialysis probe, enhanced male copulatory behavior similarly to the microinjection, increasing ejaculation frequency and decreasing ejaculation latency, postejaculatory interval and mount frequency. Retrodialyzing 8-OH-DPAT through a microdialysis probe in the MPOA had been previously shown to increase extracellular levels of dopamine and serotonin. The data from the present studies suggest that the effects of 8-OH-DPAT in the MPOA on male rat copulatory behavior may be mediated, at least in part, either directly through 8-OH-DPAT's activity at D₂ receptors or indirectly through 8-OH-DPAT's ability to increase extracellular dopamine.     

Effects of hydroxyflutamide in the medial preoptic area or lateral septum on reproductive behaviors in male rats

We examined whether androgen receptors in the medial preoptic area (MPOA) and lateral septum (LS) are required for the expression of copulation and sexual motivation. Castrated males received testosterone-filled silastic capsules to restore behavior, and were implanted with the antiandrogen hydroxyflutamide (OHF) or blank cannulae. One group was implanted in either the anteroventral MPOA or LS (ANT group). Another group was implanted in the posterodorsal MPOA or LS (POST group). Copulation was tested on days 2, 6, 10, and 14 of OHF exposure; partner preference, a measure of sexual motivation, was tested on day 15. The results showed that sexual behavior was significantly suppressed by OHF in the MPOA of the ANT group, but not the POST group. However, sexual motivation was significantly reduced by OHF in the MPOA of the POST group, but not the ANT group. In the LS, OHF had no effect on sexual behavior and partner preference regardless of implant site. The data suggest site specificity within the MPOA for androgen receptor activation of male reproductive behaviors.     

Sexual behavior increases c-fos expression in the forebrain of the male rat.

The ability of a wide variety of pharmacological and physiological stimuli to increase neuronal expression of Fos has led to the suggestion that it might serve as a marker of neuronal activation. Psychomotor stimulants increase the release of dopamine from the terminals of nigrostriatal and mesolimbic neurons and enhance Fos immunoreactivity in the striatum and nucleus accumbens (NAc). Because sexual behavior also increases dopamine release in these and other forebrain regions, the present study examined the effect of copulation on Fos immunoreactivity in the forebrain of intact, sexually active male rats. Sexual behavior produced a striking increase in Fos immunoreactivity in the medial preoptic area (MPOA), NAc, bed nucleus of the stria terminalis and piriform cortex. However, no increase in Fos immunoreactivity was observed in the striatum. These results are consistent with neurochemical, physiological, and behavioral data suggesting that the MPOA and NAc are important substrates of sexual behavior.     

Expression of mu-opioid receptor mRNA in the medial preoptic area of juvenile rats

Previous studies indicate that the latency to initiate parental behavior in both male and female rats increases with age; weanling (21 days old) rats display parental behavior 0-2 days after exposure to newborn pups, while older juveniles (30 days old) require 5-6 days of pup exposure before they express the behavior. Furthermore, activation of mu-opioid receptors inhibits parental behavior in juvenile and adult rats. We hypothesized that the age-related increase in behavioral latency could be modulated by the induction of mu-receptor expression in the medial preoptic area (MPOA), a region in which mu-receptors regulate parental behavior. In situ hybridization histochemistry was used to measure mu-receptor mRNA expression in the MPOA of male and female Sprague-Dawley rats that were 21, 24, 27, 30, or 33 days old. Using autoradiographic film analysis, we observed that neurons within part of the MPOA expressed very dense mu-receptor mRNA. Comparison of mRNA distribution with histological boundaries indicated that neurons within the medial preoptic nucleus (MPN), excluding the central part, exhibited the highest density of mu-receptor mRNA within the MPOA. High densities of mu-receptor mRNA extended dorsolaterally and caudally from the MPN toward the bed nucleus of the stria terminalis. MPN mu-receptor mRNA expression was not altered with age and no sex difference was observed. The dense presence of mu-receptor mRNA in the MPN suggests that ample substrate exists on which mu-receptor ligands could modulate the latency to begin parental behavior in juvenile rats, but such behavioral expression apparently is not mediated by a change in mu-receptor mRNA production.