Prenatal alcohol exposure alters adult expression of sexually dimorphic behavior in the rat.

Saccharin preference and performance in a Lashley III maze were found to be altered in adult male and female rats that had been exposed to alcohol during gestation. Specifically, the sexual dimorphism normally observed in both behaviors was absent in fetal alcohol-exposed animals. The lack of sexual dimorphism appeared to result from a masculinization of the exposed females and a feminization of the exposed males.

Colocalization of androgen receptors and mating-induced FOS immunoreactivity in neurons that project to the central tegmental field in male rats.

Bilateral lesions of the central tegmental field (CTF) in male rats virtually eliminate mating behavior. This study examined if mating-induced Fos expression (a measure of neuronal activation) and androgen receptors (AR) are colocalized in brain and spinal cord neurons which project to the CTF. Animals received unilateral injections of the retrograde tracer Fluorogold (FG) in the lateral part of the CTF (CTFl), and 10 days later were killed after ejaculating with females. Brains and spinal cords were examined for FG transport, AR-immunoreactivity (AR-ir), and Fos-immunoreactivity (Fos-ir). AR-ir and Fos-ir were visualized with fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. The CTFl received projections from AR-containing neurons in forebrain structures (bed nucleus of stria terminalis, medial preoptic area, lateral and ventromedial hypothalamus), in the central amygdala and various mid- and hindbrain structures (dorsolateral tegmentum, superior and inferior colliculi, pedunculopontine nucleus), and in the lumbosacral spinal cord (lamina X). Some of the AR-containing neurons in bed nucleus of stria terminalis and in the dorsal part of the medial preoptic area with projections to the CTFl were activated by mating. Most AR-containing neurons in spinal lamina X with projections to the CTFl were also activated by mating. Information from spinal cord and pontine nuclei and from outputs descending from the forebrain may be relayed in the CTFl. Thus, as part of a network of hormone-sensitive neurons linking brain and spinal cord mechanisms for mating, the CTFl could participate in the integration of visceral and somatic information relevant for sexual behavior.

Androgen receptor immunoreactivity and mating-induced Fos expression in forebrain and midbrain structures in the male rat.

The distribution of androgen receptor immunoreactive-neurons, mapped with the PG21 anti-androgen receptor antibody, was compared in male rat brains with the distribution of Fos-immunoreactive neurons induced by mating. In gonadally intact, but not in castrated male rats, substantial numbers of androgen receptor-containing neurons were present in a variety of forebrain and midbrain regions. The PG21 antibody apparently had a higher affinity for occupied than for non-occupied androgen receptors. Androgen receptor-immunoreactive regions included the medial preoptic area and other forebrain areas previously identified as containing androgen receptors, the dorsal and ventral periaqueductal gray, and a midbrain region that included the lateral part of the central tegmental field, part of the caudal zona incerta, the subparafascicular nucleus of the thalamus and the peripeduncular nucleus. Fos-expressive neurons were essentially absent in non-mated males but were present in the brains of rats which mated to ejaculation. All brain regions in which androgen receptor-immunoreactive neurons were counted also expressed Fos immunoreactivity after mating, and there was considerable overlap between the distributions of androgen receptor- and Fos-immunoreactive neurons. In a second experiment, we used immunofluorescent techniques to document the intraneuronal co-localization of Fos with androgen receptor immunoreactivity in the medial preoptic area, medial amygdala, and central tegmental field. In these regions mating-induced Fos immunofluorescence was exclusively localized in androgen receptor-immunofluorescent neurons. However, not all androgen receptor neurons were Fos expressive, suggesting that only some androgen-sensitive neurons were activated during mating. These results are consonant with the view that hormone actions on forebrain and midbrain structures influence the neuronal activity correlated with mating.

Distribution of androgen receptor-like immunoreactivity in the brains of cynomolgus monkeys.

A polyclonal antibody, PA1, raised in a rabbit against fusion proteins containing fragments of the human prostatic androgen receptor (AR) was used to map the distribution of AR-like immunoreactivity in the brains of adult male and female cynomolgus monkeys. PA1 AR-immunoreactive (ARir) labeling occurred in the cell nuclei and, more weakly, in the cytoplasm of brain cells. The PA1 ARir labeling occurred primarily in brain regions previously shown on the basis of gonadal steroid autoradiography to contain androgen receptors. However, the distribution of PA1 ARir staining was substantially more restricted than that of autoradiographic labeling using 3H-androgens. The pattern of PA1 ARir labeling was closely similar between animals and occurred in the lateral septum, medial preoptic area, bed nucleus of stria terminalis, anterior, cortical, accessory basal and medial amygdala, several hypothalamic nuclei including the supraoptic, anterior, paraventricular, ventromedial and arcuate nuclei, and the premammillary nucleus. No significant sex differences were observed. With the exception of the supraoptic nucleus, reported not to be labeled by autoradiography, earlier autoradiographic findings and the current immunocytochemical results, although not congruent, have noteworthy similarities.

Androgen receptor and mating-induced fos immunoreactivity are co-localized in limbic and midbrain neurons that project to the male rat medial preoptic area.

Two studies were designed to document neuronal colocalization of androgen receptor immunoreactivity and mating-induced Fos immunoreactivity (AR-ir, Fos-ir) in brain of male rats and to examine the extent to which limbic and midbrain neurons that project to the preoptic area are androgen sensitive and activated by mating. Brains from male rats, killed 1 h after ejaculating with receptive females, were examined for Fos-ir and AR-ir and compared with those from control rats not given access to females. PG21 anti-AR and anti-c-fos primary antibodies were visualized by fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. In mated males (Expt. 1), Fos-ir and AR-ir were colocalized in neurons of the medial preoptic nucleus (MPN), the dorsal medial amygdala (dMEA), the central tegmental field (CTF), the bed nucleus of the stria terminalis, the anterior hypothalamus, the lateral hypothalamus, and the ventral premamillary nucleus. In Expt. 2, male rats received a unilateral injection of the retrograde tracer FluoroGold (FG) in the preoptic area and four days later were killed after ejaculating with receptive females. Brains were subsequently examined for FG transport, Fos-ir and AR-ir. Fluorogold-containing neurons were present in dMEA and CTF as well as in other hypothalamic and limbic regions known to project to the MPN. In dMEA and CTF, nuclear colocalization of AR-ir and mating-induced Fos-ir was present in a proportion of FG-containing neurons. Sexually relevant information may be carried through the brain by an interconnected network of hormone-sensitive neurons.

Enhanced responses of nucleus accumbens neurons in male rats to novel odors associated with sexually receptive females.

One group of male rats was trained to associate novel odors with three different environmental conditions: the presence of (i) a sexually receptive female (RF), (ii) an unreceptive female (UF) and (iii) no other rat (NO). A second group of males received no training. Single units in nucleus accumbens (NAC) were then recorded in anesthetized animals and their responsiveness to various odors was tested. Odors that had been associated with receptive females during training evoked significantly more unit responses in NAC than did the same odors in untrained males. There were no differences between trained and untrained males in the numbers of units responsive to odors associated with unreceptive females and with the empty training chamber. In trained animals, both the percentage of responding units and the magnitude of olfactory-evoked responses were significantly larger with RF-associated odors than with either UF or NO odors. Both of these effects were more pronounced in rats that had ejaculated with females during training than in rats that had not. Findings demonstrated that pairing odors with the presentation of sexually receptive females enhanced the responsiveness of NAC neurons to those odors and indicated a role for NAC in associating environmental stimuli with natural reward processes.

Distribution of androgen receptor-like immunoreactivity in the brains of intact and castrated male hamsters.

The distribution of androgen receptor-like (AR) immunoreactivity was mapped in brains of (a) intact, sham-castrated and (b) castrated male hamsters. The pattern of AR-immunoreactive (AR-ir) staining was, in general, similar to that reported for gonadal steroid autoradiography of the male hamster brain. Moreover, with one exception, AR-like staining was similar in intact and castrated males, and occurred in the medial preoptic area, bed nucleus of stria terminalis, amygdala, hippocampus, thalamus, and several hypothalamic nuclei including the periventricular, supraoptic, and ventromedial nuclei, and median eminence. However, while AR-ir labeling was virtually absent in the lateral septum of intact males, it was clearly present in the lateral septum of castrated males. The view that androgen receptors in brain generally decline after castration received no support from this study.

The spatial organization of the peripheral olfactory system of the hamster. Part I: Receptor neuron projections to the main olfactory bulb.

The spatial organization of projections from olfactory receptor neurons to the main olfactory bulb (MOB) was studied in hamsters by using fluorescent stilbene isothiocyanates as retrograde tracers. Injections confined to small sectors of the MOB produce labeling of receptor neurons that is more restricted circumferentially (i.e., with respect to the medial-lateral and dorsal-ventral axes) than longitudinally (i.e., with respect to the rostral-caudal axis) along the mucosal sheet. This restricted labeling is also discontinuous, giving an initial impression that the peripheral input is only crudely organized with respect to the medial-lateral and dorsal-ventral axes of the nasal cavity. However, from analyses of serial sections, it is apparent that each set of mucosal segments shares convergent projections to a circumferential quadrant of the MOB with other segments that are positioned around a common domain of the nasal cavity airspace. The primary afferent projections to the MOB, thus, are organized rhinotopically (i.e., with respect to the three-dimensional position of receptor neurons in olfactory space) rather than mucosotopically.

The spatial organization of the peripheral olfactory system of the hamster. Part II: Receptor surfaces and odorant passageways within the nasal cavity.

The spatial organization of olfactory receptor surfaces and odorant passageways within the nasal cavity was studied in hamsters through descriptive and morphometric analyses of a complete stereotaxically defined series of coronal, sagittal, and horizontal sections through the snout. These analyses reveal that the caudal two-thirds of each cavity is divided into two longitudinally oriented medial and lateral channels. The olfactory mucosa that lines these two channels projects selectively onto the medial and lateral halves of the main olfactory bulb (MOB), respectively. Moreover, the ethmoturbinates of the caudal recesses create highly convoluted channels, lined by ventrally projecting mucosa, that lie ventral, lateral, and dorsal to a relatively smooth central channel lined by dorsally projecting mucosa. The rhinotopic map makes equivalent representations of medial and lateral olfactory space to the MOB but gives the smooth space lined by dorsally projecting mucosa a disproportionately larger representation on the MOB than the convoluted space lined by the more expansive ventrally projecting mucosa. Recent descriptions of the spatial distribution of probes for odorant receptor proteins conform closely to this organization, giving credence to the idea that rhinotopy is a basis for representing to the MOB the specific molecular features of odorant molecules.

Immunohistochemical labeling of androgen receptors in the brain of rat and monkey.

Androgen receptor antibodies have recently been developed using fusion proteins containing fragments of human prostatic androgen receptor. We have used a polyclonal antibody raised in rabbits to label androgen receptors in brain sections from male and female rats and monkeys. Free-floating frozen sections were incubated in primary antibody, and processed by the peroxidase-avidin-biotin complex method using biotinylated anti-rabbit IgG. Nickel intensified diaminobenzidine was used as the chromagen, and neurons were labeled in the amygdala, hippocampus, bed nucleus of stria terminalis, septum, preoptic area, in several hypothalamic nuclei including the supraoptic and paraventricular nuclei, in several brain stem motor nuclei and in cerebral cortex. Staining was most intense in cell nuclei but also occurred in cytoplasm and in some neuronal processes. Labeling was more restricted in monkey than in rat brain. Omitting the primary antibody or pre-incubating the primary antibody with rat prostatic cytosol for control purposes demonstrated the specificity of staining.

Prenatal alcohol exposure alters enkephalin levels, without affecting ethanol preference.

Pregnant dams were pair-fed a liquid diet containing 35% ethanol derived calories or isocaloric sucrose during the last two trimesters of pregnancy. No differences were observed in adult ethanol preference between fetal alcohol exposed (FAE) animals and pair-fed controls. However, Met- and Leu-enkephalin levels were significantly elevated in globus pallidus of adult FAE animals. Pituitary levels were unaffected.

Effects of progesterone on the sexual behavior of castrated, testosterone-treated male cynomolgus monkeys (Macaca fascicularis).

In male cynomolgus monkeys the synthetic progestin, medroxyprogesterone acetate (MPA), decreases testosterone (T) levels and sexual behavior, binds to progestin receptors in brain, and reduces by about 70% the uptake of [3H]androgens by both brain and genital tract tissues. To examine the behavioral effects of progesterone (P) itself, eight castrated, T-treated males were each tested twice weekly with an estrogenized female before, during, and after they were treated with two SC Silastic P implants. Data from six 4-week treatment periods were analyzed to facilitate comparisons with our previous data using MPA: i) baseline, ii) weeks 4-7 of P treatment, iii) weeks 8-11 of P treatment, iv) weeks 1-4 after P implants were removed, v) weeks 5-8 after P withdrawal, and finally vi) weeks 9-12 after P withdrawal (384 1 h behavior tests). Weekly blood samples (N = 192) were analyzed by radioimmunoassay to determine plasma levels of both T and P. P treatment, which resulted in high plasma P levels (about 44 ng/ml), produced decrements in measures of male sexual behavior and motivation that were both qualitatively and quantitatively similar to those produced by MPA treatment but, unlike MPA, P did not decrease plasma T levels or change them in any way (about 850 ng/100 ml throughout). The findings suggest that P implants may be preferable to weekly MPA injections in the treatment of male sex offenders because they require less patient compliance and may not have MPA's troubling side effects.

Progesterone decreases mating and estradiol uptake in preoptic areas of male monkeys.

Synthetic progestins such as medroxyprogesterone acetate (MPA) are used widely in the treatment of male sex offenders. In male cynomolgus monkeys (Macaca fascicularis) treated with testosterone (T), both MPA and progesterone (P) had comparable inhibitory effects on male sexual motivation and behavior. To determine if P, like MPA, decreases endogenous T levels, plasma T and P levels were analyzed in weekly blood samples (N=186) from eight intact males, each paired with a sexually receptive female before, during, and after treatment with subcutaneous Silastic P implants (336 behavior tests). P treatment decreased sexual activity but not plasma T levels. To ascertain if P, like MPA, acts by decreasing the nuclear uptake of T by brain, four P-treated and four control males were euthanized 60 min after intravenous injection of 3 mCi of [3H]T. The nuclear uptake of unchanged [3H]T and its metabolites [3H]E(2) and [3H]DHT was measured in samples of brain, pituitary gland, genital tract, and liver. P, unlike MPA, did not affect the nuclear uptake of [3H]androgens by brain, but reduced by 80% the nuclear accumulation of [3H]E(2) in tissue samples containing preoptic area and the anterior part of the bed nucleus of stria terminalis, although not in samples from hypothalamus or amygdala.

Effects of mating on c-fos expression in the brains of male macaques.

The c-fos polyclonal anti-c-fos antibody was used to examine the effects of mating on Fos expression in brain neurons of 11 male macaques. Behavior tests were for 30 min, five males were unmated, four were mated, and two were social controls. Mated males were killed 60 min after ejaculation. Social controls were paired with females, but mating did not occur. Fos immunoreactive (Fos-ir) neuronal nuclei were counted in nine brain regions extending from the medial preoptic to the mammillary body area of all males. In contrast to previous reports on nonprimate laboratory species, overall there was as much Fos-ir in unmated as in mated males. Moreover, there was significantly less Fos expression in four brain regions (known to contain steroid receptors), namely, ventromedial hypothalamus, arcuate nucleus, lateral mammillary area, and bed nucleus of stria terminalis, of mated than of unmated males. There were no significant differences between mated and unmated males in the 5 other brain regions studied. These findings may reflect taxonomic differences between primates and nonprimates, or result from greater neural activation in feral animals maintained in a laboratory than in domesticated, inbred laboratory species. The simplest interpretation would be that neural activity in the male primate is turned off by mating in some brain sites but not in others.

Behavioral responses to Depo-Provera, Fadrozole, and estradiol in castrated, testosterone-treated cynomolgus monkeys (Macaca fascicularis): the involvement of progestin receptors.

Sexual motivation and behavior decreased in male cynomolgus monkeys given either Depo-Provera (medroxyprogesterone acetate, MPA), which reduces androgen uptake by brain, or the nonsteroidal aromatase inhibitor, Fadrozole, which virtually eliminates the conversion of testosterone (T) to estradiol (E2) in brain. This suggested that both unchanged T and E2 are important for the control of male primate sexual behavior, but combined treatment with MPA and Fadrozole did not have the anticipated summatory effects in intact males: the behavioral decrements when MPA-treated males were given Fadrozole were about half those observed when Fadrozole was given alone. The present study tested the hypothesis that Fadrozole suppressed the behavioral effects of MPA by preventing the induction by E2 of progestin receptors in the brain to which MPA binds. Eight castrated, T-treated males were each tested with an estrogenized female i) during baseline, ii) during MPA treatment, iii) during treatment with MPA and Fadrozole together, and iv) with E2 treatment added to condition (iii) (256 1-h behavior tests). All dosages were those used in previous studies. Sexual motivation, as reflected in mounting attempts and mounting attempt latencies, was further diminished by E2 treatment in males receiving both MPA and Fadrozole, but ejaculatory activity was not changed. Immunocytochemistry demonstrated that the distributions of progestin and androgen receptors were little affected by MPA treatment, and that progestin receptor immunoreactivity was almost completely abolished in the brains of males receiving both MPA and Fadrozole but present in those receiving additional E2 treatment, findings that supported the hypothesis.

Male hamster investigatory and copulatory responses to vaginal discharge: an attempt to impart sexual significance to an arbitrary chemosensory stimulus.

Hamster vaginal discharge elicits intense genital investigation and facilitates overt copulatory behavior toward anesthetized males (female surrogates) whose hindquarters have been scented with this material. The ability of an arbitrary chemosensory stimulus to acquire behavioral activity like that of vaginal discharge through association with maternal stimuli and/or adult sexual experience was examined in male hamsters. Vanillin was used as the arbitrary stimulus because it is attractive to hamsters, is not likely to be a natural constituent of hamster scents, is not known to exert any adverse physiological effects, and is a subliming solid with an extremely long persistence when used as an artificial scent. The males were reared by vanillin-scented or control solvent (water)-scented foster mothers, and in adulthood were paired repeatedly with vanillin- or solvent-scented receptive females. Behavioral testing with scented surrogates was performed one week preceding, and again following, the sexual pairings. Rearing by vanillin-scented mothers modestly but significantly increased the amount of time sexually naive males spent investigating the hindquarters as compared to other body regions of vanillin-scented surrogates. However, neither neonatal nor adult interactions with vanillin-scented females imparted to this stimulus the capacity to facilitate overt copulatory behavior. Also, regardless of the males' exposure history, only vaginal discharge caused the males to direct their investigatory behavior predominantly toward the hindquarters. The characteristic investigatory and copulatory responses exhibited by male hamsters toward vaginal discharge thus do not appear to be readily developed toward arbitrary chemosensory stimuli associated with particular females to which the males have been exposed.

Chemical studies of hamster vaginal discharge: male behavioral responses to a high molecular weight fraction require physical contact.

This report describes the isolation and behavioral testing in normal male hamsters of a high molecular weight fraction (HMF) of vaginal discharge which accounts for much of the aphrodisiac activity in the discharge. The HMF encompasses a group of proteins which elute as a relatively narrow, major peak upon agarose gel filtration of estrous vaginal discharge. The crude fraction from gel filtration retains a variety of volatiles including sulfur-containing compounds which we have previously found to account for much of the initial attraction of males to the female but which do not, themselves, facilitate overt copulatory behavior. Procedures for markedly reducing the presence of such volatiles to yield the HMF are described. In behavioral assays using anesthetized males as surrogate females, scenting the hindquarters of the surrogates with the HMF elicits intense genital investigation by experimental males, although this effect on investigatory behavior is not as dramatic as that of the unfractionated vaginal discharge. Like the unfractionated discharge, the HMF significantly increases the incidence of intromission attempts toward scented surrogates. To assess whether physical contact with the HMF is required for behavioral activity, as would be expected if the active material is proteinaceous, series of preference tests were performed using vanillin as a competing stimulus under conditions in which physical contact with the stimuli either was possible or was prevented. The unfractionated discharge was preferred in both conditions, whereas significant preferences for the HMF were exhibited only if it could be contacted by the snout of experimental males. The active material in the HMF thus appears to be of extremely low volatility, at least prior to physical contact with it by the male.

Male hamster copulatory responses to a high molecular weight fraction of vaginal discharge: effects of vomeronasal organ removal.

The importance of the vomeronasal (accessory olfactory) system for the copulatory responses of male hamsters to a high molecular weight fraction (HMF) of vaginal discharge was assessed in animals that had their vomeronasal organs (VNO) removed. These organs were extirpated bilaterally using an oral approach through the palate so as to eliminate the peripheral afferents to the accessory olfactory bulb (AOB) with minimal or no damage to the main olfactory system. The selective peripheral deafferentation procedure was verified by applying horseradish peroxidase intranasally following intraperitoneal injections of epinephrine to facilitate the vomeronasal pumping mechanism that draws fluids into the VNO. Heavy, bilateral anterograde labeling was evident in the olfactory nerve afferents within the main olfactory bulb of males that had their VNO removed and of animals that received sham surgery. Sham-operated males also had heavy, bilateral labeling in the vomeronasal nerve afferents within the AOB, whereas no such labeling occurred among animals with bilateral removal of the VNO. In sham-operated animals, both the HMF and the unfractionated discharge significantly increased the incidence of intromission attempts toward anesthetized males (surrogate females) whose hindquarters were scented with these stimuli. The unfractionated discharge also produced a significant elevation of overt copulatory behavior in males with selective peripheral deafferentation of the vomeronasal system, whereas the HMF did not facilitate copulatory behavior in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical studies of hamster vaginal discharge: effects of endocrine ablation and protein digestion on behaviorally active macromolecular fractions.

Our previous studies have demonstrated that macromolecular fractions of hamster vaginal discharge elicit intense genital investigation and facilitate copulatory behavior toward anesthetized males (female surrogates) whose hindquarters have been scented with these fractions, and that the aphrodisiac activity is significantly reduced in the vaginal discharge of ovariectomized (OVEX) or hypophysectomized (HYPOX) females. The present series of studies compared the protein compositions of estrous, diestrous, OVEX, and HYPOX vaginal discharge, and assessed whether protein digestion of estrous vaginal discharge affects its aphrodisiac activity. Polyacrylamide gel electrophoresis of vaginal lavages showed that the endocrine status of females has a profound effect on the protein composition of the vaginal discharge. The concentration of the major proteins dropped by about ten-fold in going from the estrous to the diestrous condition. The concentration of these same proteins appear to be at least another order of magnitude lower in the vaginal discharges of OVEX and HYPOX females. These major proteins had molecular weights greater than 10,000 Daltons. A macromolecular fraction of estrous vaginal discharge isolated by gel filtration at elevated temperature (conditions known to separate some protein-bound steroids) and containing proteins of molecular weight greater than 10,000 Daltons was found to be modestly but significantly less active than estrous discharge in its ability to elicit intense genital investigation, and not significantly different from estrous discharge in its ability to facilitate overt copulatory behavior toward scented surrogates. Pronase digestion of the proteins in this fraction did not alter its effects on investigatory behavior toward scented surrogates but significantly reduced its ability to facilitate copulatory behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

Male hamster investigatory and copulatory responses to vaginal discharge: relationship to the endocrine status of females.

The ability of vaginal discharge from ovariectomized (OVEX) or hypophysectomized (HYPOX) female hamsters to elicit intense genital investigation and to facilitate overt copulatory behavior in males was compared with that of estrous vaginal discharge. The discharges were collected by vaginal lavage with water. In order to avoid exposure of experimental males to female stimuli other than vaginal discharge, the behavioral tests employed anesthetized males (female surrogates) whose hindquarters were scented with the collected vaginal material or with control solvent (water). Both the OVEX and HYPOX discharges elicited intense genital investigation and significantly increased the incidence of intromission attempts toward the scented surrogates. However, both types of discharge had significantly less behavioral activity than estrous vaginal discharge. In a subsequent experiment, a water dilution series of estrous vaginal discharge was tested for the ability to promote genital investigation and copulatory behavior toward scented surrogates. The amount of discharge typically extruded by one estrous female in response to tactile genital stimulation can be diluted one hundred-fold without appreciable reductions in its behavioral activity. These results demonstrate that the behavioral activity of hamster vaginal discharge is related to the endocrine status of females, and suggest that previous failures to demonstrate clear dependence on ovarian function might have been due to ceiling effects in laboratory tests.