Prenatal flutamide treatment eliminates the adult male rat's dependency upon vasopressin when forming social-olfactory memories.

The sexually dimorphic number of cells expressing arginine vasopressin (AVP) in the bed nucleus of the stria terminalis and the density of AVP fibers within the lateral septum appear to be organized by pre- and postnatal androgens. Social recognition behaviors are also sexually dimorphic and AVP-dependent. Whereas AVP antagonists prevent males from recognizing familiar intruders by olfactory investigation of the anal-genital area, they have no effect in females. To test the hypothesis that the male's dependency upon AVP to form social recognition memories begins prior to birth, we compared the effectiveness of an AVP antagonist to block social recognition in control males and females with that seen in male offspring whose mothers were treated prenatally with an androgen antagonist (flutamide). In an initial study we showed that while sexual experience may enhance social recognition in males, virgin males exhibit the ability to recognize conspecifics and are sensitive to the memory blocking actions of AVP antagonists. In a second experiment, pregnant rats were treated daily for the last 10 days of gestation with either flutamide (10 mg) or control vehicle. Within 12 h of birth, male offspring from flutamide litters were injected with either testosterone proprionate (50 microg TP) or vehicle control. AVP-antagonist treatment in adults eliminated the ability of control males to recognize familiar juvenile intruders, but had no effect on males exposed prenatally to flutamide, regardless of whether these males were treated with TP or vehicle on day 1 of life. These data support the hypothesis that the development of the male's dependency upon AVP to express social recognition memories begins with the organizational actions of prenatal androgens.

Sex differences in the distribution of estrogen receptors in the septal area and hypothalamus of the domestic pig (Sus scrofa).

Recently in the pig hypothalamus a vasopressin- and oxytocin-containing nucleus was identified which, like the supraoptic nucleus, becomes sexually dimorphic after puberty. Following the increase in circulating steroids at puberty, the vasopressin- and oxytocin-containing nucleus becomes twice as large in both males and females. In adulthood, the vasopressin- and oxytocin-containing nucleus of females is approximately twice as large as that in males. Because these alterations are possibly due to an influence of gonadal steroids, i.e. estrogens, the vasopressin- and oxytocin-containing nucleus cells were tested for the presence of estrogen receptors. In addition to the area of the vasopressin- and oxytocin-containing nucleus, the present study documented the distribution of estrogen receptors in the septal area and other parts of the hypothalamus of intact post-pubertal male and female pigs, by utilizing immunocytochemical methodology. Intense nuclear estrogen receptor staining was found in a number of areas, i.e. the medial preoptic area, the oxytocin-containing dorsomedial extension of the supraoptic nucleus, a possible homologue of the sexually dimorphic nucleus of the preoptic area, the median preoptic nucleus, the medial and lateral part of the bed nucleus of the stria terminalis, the ventromedial hypothalamus and the arcuate nucleus. In the ventral part of the lateral septum, the septohypothalamic nucleus, the nucleus subfornicalis and the stigmoid nucleus estrogen receptor immunoreactivity was less intense. Dorsolaterally of the vasopressin- and oxytocin-containing nucleus, estrogen receptor positive cells were observed, but the vasopressin- and oxytocin-containing nucleus itself lacked such receptors. In the magnocellular supraoptic nucleus and paraventricular nucleus no nuclear estrogen receptor staining was found. However, a weak cytoplasmic staining was present in all cells. There was a clear sex difference in the estrogen receptor-immunoreactive cell number in a possible homologue of the sexually dimorphic nucleus of the preoptic area. Compared to male pigs, in female pigs the number of cells showing estrogen receptor immunoreactivity in this area, which is known to be sexually dimorphic in various species, was twice as high. In other areas, such as the medial part of the bed nucleus of the stria terminalis, the medial preoptic area, the arcuate and ventromedial hypothalamic nucleus, a similar sex difference was found. In addition estrogen receptor immunoreactivity was generally more intense in females. No sex differences were noted in the overall distribution of estrogen receptor cells in the areas studied.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical localization of estrogen receptors within neurotensin cells in the rostral preoptic area of the rat hypothalamus.

In situ hybridization procedures indicate that estrogen selectively increases neurotensin and neuromedin (NT/N) mRNA levels in the rostral preoptic area of the rat hypothalamus (RPH). Using the co-localization procedures of Axelson and Van Leeuwen, J. Neuroendocrinol., 2 (1990) 209-216, the present study examined whether NT cells in the RPH contained estrogen receptors (ER). Vibratome sections of brains from adult ovariectomized, colchicine-treated rats were first incubated with estrogen receptor antibody and stained with diaminobenzidine (DAB)-Ni+ producing a blue-black nucleus. Subsequently, NT antisera were used to provide a brown reaction product with DAB as chromogen. Approximately 25% of the NT cells in the RPH contained ER. These data support the hypothesis that NT cells in the RPH that play a role in luteinizing hormone release from the pituitary are, in part, influenced directly by estrogen feedback via nuclear ER and may act as interneurons controlling luteinizing hormone releasing hormone turnover.

Kainic Acid lesion of vasopressinergic neurons in the hypothalamus disrupts flank marking behavior in golden hamsters.

Abstract It is well established that flank marking behavior in the Golden hamster is controlled by vasopressin-sensitive neurons localized to the anterior hypothalamus; however, the source(s) of vasopressinergic innervation to this area is unknown. Previous analysis by immunocytochemistry showed distinct populations of vasopressinergic magnocellular neurons localized to the supraoptic nucleus, paraventricular nucleus and the nucleus circularis that did not project to the neurohypophysis. In the present study, these same hypothalamic nuclei were lesioned by microinjection of kainic acid to determine which, if any, of these populations of vasopressin neurons are involved in the control of flank marking. Unilateral lesions in the areas of the nucleus circularis and supraoptic nucleus at the rostro-caudal plane of the anterior hypothalamus abolished odor-induced flank marking behavior. Lesions in the paraventricular nucleus at the level of the anterior hypothalamus did not consistently inhibit flank marking, while lesions of magnocellular neurons rostral or caudal to the anterior hypothalamus were ineffective. The microinjection of vasopressin into the anterior hypothalamus following lesion of the paraventricular and supraoptic nuclei stimulated flank marking, evidence that treatment with kainic acid did not damage the efferent component of this behavior. However, animals with lesions in the nucleus circularis did not respond to the microinjection of vasopressin; hence, it is uncertain whether lesions in this area disrupt vasopressinergic innervation to the anterior hypothalamus or simply destroy the motor neurons controlling flank marking. In summary, the data clearly demonstrate that vasopressin neurons localized primarily to the medial aspect of the supraoptic nucleus are necessary for sensory integration of odor-induced flank marking, and as such, may be one possible source of neurotransmitter controlling this behavior.

Differential localization of estrogen receptors in various vasopressin synthesizing nuclei of the rat brain.

Abstract Vasopressin (VP) cells in the bed nucleus of the stria terminalis, medial amygdaloid nucleus and supraoptic and paraventricular nuclei are influenced by gonadal steroids. The present paper examined whether VP cells in the bed nucleus of the stria terminalis, medial amygdaloid nucleus, and supraoptic and paraventricular nuclei contain estrogen receptors. Brains from adult short-term castrated, colchicine-treated male rats were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde. In the immunocytochemical double-staining procedure Vibratome sections were first incubated with an estrogen receptor antibody (#H222) and stained with diaminobenzidine-Ni(+). Following methanol-hydrogen peroxide washes, sections were incubated with anti-neurophysin and stained with diaminobenzidine. Parvocellular cells in the bed nucleus of the stria terminalis and medial amygdaloid nucleus were double-stained with a blue-black nucleus (indicating the estrogen receptors) surrounded by brown cytoplasm (resulting from VP-neurophysin-immunoreactivity). Our results provide the first direct anatomical evidence supporting the hypothesis that gonadal steroids' influence of parvocellular VP cells in the bed nucleus of the stria terminalis and medial amygdaloid nucleus is mediated directly via estrogen receptors localized in nuclei of VP neurons. We were unable to co-localize any estrogen receptors in VP and oxytocin cells of magnocellular size in the supraoptic, paraventricular and anterior commissural nuclei, suggesting that estrogen indirectly affects these magnocellular hypothalamic cells.

Regulation of the rat oxytocin gene by estradiol.

Abstract Oxytocin (OT) plays a role in reproduction at the level of the pituitary and mammary glands and uterus. This OT is synthesized in the hypothalamo-neurohypophyseal system (HNS). A number of observations have suggested that estrogens regulate the production of OT in the HNS. In this study the effect of 17beta-estradiol on the activity of the OT gene promoter was examined as well as the effect of 17beta-estradiol in vivo on OT messenger ribonucleic acid (mRNA) and peptide revels in the rat HNS. Vasopressin (VP) and its mRNA were also determined in the in vivo studies. The direct transcriptional stimulation of OT gene expression by 17beta-estradiol was studied in two different heterologous expression systems. When a plasmid having nucleotides -363 to +16 of the rat OT gene fused to the firefly luciferase reporter gene was co-transfected with an estrogen receptor expression vector in P19 embryonal carcinoma cells, luciferase activity was stimulated 80-fold by 17beta-estradiol. In estrogen receptor containing MCF-7 cells transfected with a plasmid having nucleotides -188 to +16 of the rat OT gene fused to the chloramphenicol acetyl transferase gene, 17beta-estradiol induced the expression of the chloramphenicol acetyl transferase gene through the cloned promoter element. After in vivo treatment of ovariectomized rats with 17beta-estradiol, levels of OT mRNA and VP mRNA were measured in microdissected supraoptic and paraventricular nuclei as well as VP and OT levels in these nuclei and the pituitary gland. As compared to non-treated ovariectomized rats there was no difference in contents of OT mRNA and VP mRNA in these hypothalamic nuclei and in levels of the peptides in paraventricular nuclei and the pituitary gland. A 30% reduction of the OT content of the supraoptic nuclei only was found, while the VP content did not change. To explain the results immunocytochemical analyses of the hypothalamus were performed, showing that the estrogen receptor was absent in the magnocellular neurons of the supraoptic and paraventricular nuclei. The results demonstrate that the 5'flanking region of the OT gene confers estrogen-sensitivity to transcription of the OT gene. This potential to respond to estrogens is not used in the OT-producing neurons of supraoptic and paraventricular nuclei probably due to the absence of the estrogen receptor.

Vasopressin immunoreactivity in the anterior hypothalamus is altered during the establishment of dominant/subordinate relationships between hamsters.

When paired for 15-min periods for 5-8 consecutive days, castrated, testosterone-treated hamsters consistently assumed the dominant status, based on a higher aggression index (18 +/- 3) and frequency of flank marking (15 +/- 3) as compared to their castrated, untreated subordinate partners (-1.3 +/- 1 and 2.4 +/- 1, respectively). In addition to these hamsters with established dominant/subordinate relationships, control hamsters with no social interactions were killed, and in all animals the vasopressin level in the anterior hypothalamus-medial preoptic area was assessed by counting vasopressin immunoreactive perikarya following immunocytochemistry, or by radioimmunoassay of vasopressin from tissue punches. In the socialized pairs the subordinate hamsters had a significantly (P less than 0.01) lower number of vasopressin staining perikarya in the anterior hypothalamus, specifically the area of the nucleus circularis, than their dominant partners (n = 6 pairs). There was also a significantly (P less than 0.001) lower level of vasopressin immunoreactivity in punches taken from the area of the nucleus circularis in subordinate hamsters as compared to their dominant partners (n = 14 pairs). However, there were no significant differences in the number of perikarya or the concentration of immunoreactive vasopressin between subordinate and dominant hamsters in the supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus or bed nucleus of the stria terminalis. The number of perikarya (n = 5 pairs) and concentration of vasopressin (n = 8 pairs) for all vasopressin immunoreactive sites, including the nucleus circularis, were similar for testosterone-treated and untreated hamsters that remained isolated and not subjected to daily aggressive encounters.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of voluntary and forced exercise on lordosis behavior in female rats.

We measured sexual behavior (lordosis) in ovariectomized, steroid-treated, exercising female rats. When estradiol-treated animals exercised voluntarily, lordosis was directly related to the amount of exercise. Forced exercise (swimming) following treatment with estradiol 17 beta and progesterone significantly increased lordosis scores but only after animals swam for 2.5 hr per day. Lordosis behavior returned to baseline levels 7 weeks after forced exercise was stopped. Circulating levels of estrogen and progesterone measured immediately after behavioral testing did not differ between animals forced to exercise and their controls. The mechanisms responsible for the increase in lordosis behavior following exercise are unclear. It is possible that a change in body composition, a change in neural sensitivity to gonadal steroids, and/or changes in pituitary or adrenal function contribute to the increased lordosis behavior in exercised animals.

Scent marking and the maintenance of dominant/subordinate status in male golden hamsters.

Since it is thought that flank marking communicates dominance status, experiments were designed to look at changes in aggression and flank marking behaviors in pairs of male hamsters with intact flank glands (Experiment One) or when one (Experiment Two) or both (Experiment Three) members of a pair had their flank glands surgically removed. In Experiment One the dominant members of twelve pairs of hamsters had a mean daily frequency of flank marks that was over two-fold greater than their subordinate partners, F(1,11) = 17.59, p less than 0.001. Over the course of five consecutive daily tests there was a significant decrease in the aggression index of both the dominant, t(44) = 4.49, p less than 0.01, and subordinate, t(44) = 3.33, p less than 0.01, hamsters. Accompanying the decrease in aggression was a significant increase in the flank marking of both dominant, t(44) = 7.8, p less than 0.01, and subordinate, t(44) = 3.59, p less than 0.01, hamsters. In Experiment Two, six out of eleven flank glandectomized hamsters were dominant over their sham operated partners while the remaining five were subordinate. Unlike Experiment One there was no significant difference in the flank marking between dominant and subordinate hamsters, in fact, in seven pairs the subordinate hamsters flank marked more than their dominant partners. In Experiment Three both hamsters had their flank glands removed, and as in Experiment Two, there was no significant difference in flank marking between dominant and subordinate hamsters, neither was there any significant change in their aggression and flank marking behaviors over the course of the five test periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Forced swimming alters vaginal estrous cycles, body composition, and steroid levels without disrupting lordosis behavior or fertility in rats.

The purpose of the present studies was to examine how exercise affects reproductive physiology and behavior. In four separate experiments, exercise regimens of forced swimming or swimming plus running, were gradually increased in duration to a maximum of 2.5 hours. Vaginal cycles were monitored daily and after eight weeks animals were tested for sexual behavior, ability to breed, changes in body composition, circulating levels of estradiol, progesterone, and corticosterone. Total body lipids were lowered in animals that both ran and swam and parametrial fat pad weight was reduced in all exercising animals. Although exercise lowered circulating levels of ovarian steroids, elevated corticosterone levels and disrupted vaginal cycles, exercised animals exhibited normal sexual behavior and bred successfully. These data indicate that intense exercise can, under certain conditions, disrupt the mechanisms controlling vaginal cycles while the neuroendocrine mechanisms controlling sexual receptivity, ovulation, and fertility remain intact.

A quantitative cytochemical analysis of large antral follicles in two types of rat polycystic ovaries.

Ovaries from normal mature rats, rats injected with testosterone propionate (TP), and from aged rats were removed, and large antral follicles examined by quantitative cytochemical techniques in order to analyze possible enzymatic defects that relate to follicular steroidogenesis. The ovaries from the TP-injected and the aged rats were polycystic. Lipid deposition was analyzed in frozen sections stained with Sudan black. A microdensitometer was used to measure delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta OHD) activity and G-6-PD type IH generation in the theca, and in peripheral region, antral region, and corona radiata of large antral follicles. 3 beta OHD is the enzyme that converts pregnenolone to progesterone. Type IH generation is related to the conversion of androstenedione to estradiol. Lipid droplet deposition was comparable in the three types of follicles. Compared to that in normal preovulatory follicles, 3 beta OHD activity was similar in identical regions of large antral follicles in TP-injected rats, but less in the theca and peripheral region of the membrana granulosa of the aged rat. G-6-PD type IH generation was less in the peripheral region of large antral follicles of both TP-injected and aged rats than in preovulatory follicles. Type IH generation was also less in the theca of TP-injected rats than in the theca of normal rats. This study provides evidence that in spite of their normal appearance, large antral follicles in polycystic ovaries are not physiologically sound. Furthermore, the enzymatic disturbance appears to be different in different types of polycystic ovaries.

A vasopressin antagonist can reverse dominant/subordinate behavior in hamsters.

Golden hamsters communicate dominance status by flank marking, a behavior that is dependent upon vasopressin-sensitive neurons in the anterior hypothalamus-medial preoptic area (AH-MPOA). The purpose of the present study was to investigate whether arginine vasopressin (AVP) and an antagonist of AVP could alter or reverse dominant/subordinate relationships in pairs of hamsters. Microinjection of AVP into the AH-MPOA of subordinate hamsters dramatically increased their flank marking despite the presence of their dominant partners. Conversely, microinjection of the AVP antagonist into the AH-MPOA of dominant hamsters blocked flank marking in the presence of their subordinate partners. Surprisingly, the untreated subordinate hamsters significantly increased their own flank marking when tested with their dominant partners treated with the AVP antagonist, thereby reversing the pattern of flank marking normally seen in dominant/subordinate relationships. However, the effect of AVP and the AVP antagonist were limited to the day of treatment. When flank marking behavior was reversed in a pair of hamsters by treatments for three consecutive days, the pair immediately displayed the original dominant/subordinate behavior when treatment was stopped.

Effects of Silastic progesterone implants on activity cycles and steroid levels in ovariectomized and intact female rats.

We have previously shown that although Silastic implants of progesterone reduce the amount of running of animals living in activity wheels, progesterone-treated animals continue to show periodic fluctuations or peaks in activity. We hypothesized that although progesterone treatment inhibited estrous cycles, ovaries of animals treated with Silastic implants of progesterone continued to secrete estradiol in amounts adequate to stimulate moderate levels of running. In the present study we tested this hypothesis by removing ovaries from progesterone-treated animals and comparing their running behavior and steroid levels to progesterone-treated animals who received sham ovariectomies. Although progesterone treatment significantly inhibited running activity, removal of ovaries in progesterone-treated animals further suppressed running activity. In addition, both estradiol and progesterone levels were significantly reduced following removal of ovaries in progesterone-treated animals. We conclude that although Silastic progesterone implants inhibit normal ovarian and estrous activity cycles, ovaries produce sufficient estradiol to stimulate running behavior.

Effects of voluntary exercise on sexual behavior in female rats.

In two experiments, ovariectomized rats given access to activity wheels showed significantly higher lordosis ratings following estradiol treatment than animals maintained in individual hanging cages. Estrogen-induced decreases in body weight also were greater in wheel-housed animals. We suggest that access to running wheels may alter responsiveness to estradiol.

Circadian rhythm dissociation in the rat: effects of long-term constant illumination.

Long-term exposure to constant dim illumination dissociated the circadian activity rhythms of female rats. Evaluation of this phenomenon by visual and spectral analysis indicated that ultradian rhythms survive the breakdown of circadian organization.