Progestin receptor-mediated reduction of anxiety-like behavior in male rats

Background

It is well known progesterone can have anxiolytic-like effects in animals in a number of different behavioral testing paradigms. Although progesterone is known to influence physiology and behavior by binding to classical intracellular progestin receptors, progesterone''s anxiety reducing effects have solely been attributed to its rapid non-genomic effects at the GABA_A receptor. This modulation occurs following the bioconversion of progesterone to allopregnanolone. Seemingly paradoxical results from some studies suggested that the function of progesterone to reduce anxiety-like behavior may not be entirely clear; therefore, we hypothesized that progesterone might also act upon progestin receptors to regulate anxiety.

Methodology/Principal Findings

To test this, we examined the anxiolytic-like effects of progesterone in male rats using the elevated plus maze, a validated test of anxiety, and the light/dark chamber in the presence or absence of a progestin receptor antagonist, RU 486. Here we present evidence suggesting that the anxiolytic-like effects of progesterone in male rats can be mediated, in part, by progestin receptors, as these effects are blocked by prior treatment with a progestin receptor antagonist.

Conclusion/Significance

This indicates that progesterone can act upon progestin receptors to regulate anxiety-like behavior in the male rat brain.     

Estrogenic effects of zearalenone on the expression of progestin receptors and sexual behavior in female rats

Zearalenone is a resorcylic acid lactone compound that is produced by fungal infection of edible grains and is believed to influence reproduction by binding to estrogen receptors. In order to study the potential estrogenic effects of this compound in the brain, we examined the effects of zearalenone on the expression of neuronal progestin receptors and feminine sexual behavior in female rats. Ovariectomized rats were treated with zearalenone (0.2, 1.0, or 2.0 mg), estradiol benzoate, or vehicle daily for 3 days. They were then either perfused, and progestin receptors visualized by immunocytochemistry, or injected with progesterone and tested for sexual receptivity with male rats. Progestin receptor-containing cells were counted in the medial preoptic area and ventromedial hypothalamus. The two highest doses of zearalenone increased the concentration of neuronal progestin receptors, as did 10 microg of estradiol. The highest dose of zearalenone (2 mg) also induced progestin receptor staining density comparable to that of 10 microg of estradiol benzoate. In behavioral tests, ovariectomized animals treated with 2 mg of zearalenone followed by progesterone showed levels of sexual receptivity comparable to females treated daily with estradiol benzoate (2 microg) followed by progesterone. These studies suggest that, although structurally distinct and less potent than estradiol, zearalenone can act as an estrogen agonist in the rat brain.     

An assessment of agonist/antagonist effects of tamoxifen in the female mouse brain.

Ovariectomized CFW mice were treated with tamoxifen (TAM) alone or in combination with estradiol benzoate (EB) to determine its ability to promote/block lordotic behavior and the induction of hypothalamic progestin receptors (PR). Across a range of doses, TAM plus progesterone treatment did not activate female sexual behavior. When given with EB, TAM suppressed lordotic behavior in a dose-dependent fashion. TAM did not induce PR when given alone and it completely blocked the ability of EB to induce PR. It therefore appears that for these responses TAM functioned as a pure antagonist in the female mouse brain, although the degree of its antiestrogenicity varied with the response under consideration. A potential mechanism mediating this differential effectiveness is discussed.     

Vasopressin regulates social recognition in juvenile and adult rats of both sexes, but in sex- and age-specific ways

In adult male rats, vasopressin (AVP) facilitates social recognition via activation of V1a receptors within the lateral septum. Much less is known about how AVP affects social recognition in adult females or in juvenile animals of either sex. We found that administration of the specific V1a receptor antagonist d(CH(2))(5)[Tyr(Me)(2)]AVP into the lateral septum of adult rats impaired, whereas AVP extended, social discrimination in both sexes. In juveniles, however, we detected a sex difference, such that males but not females showed social discrimination. Interestingly, administration of the V1a receptor antagonist to juveniles (either intracerebroventricularly or locally in the lateral septum) did not prevent social discrimination, but instead significantly decreased the investigation of a novel as opposed to a familiar animal in both sexes, with stronger effects in males. V1a receptors were found to be abundantly expressed in the lateral septum with higher binding density in females than in males. These findings demonstrate that activation of V1a receptors in the lateral septum is important for social recognition in both sexes, and that the roles of septal V1a receptors in social recognition change during development.     

Co-localization of midbrain projections, progestin receptors, and mating-induced fos in the hypothalamic ventromedial nucleus of the female rat

In female rats, sexual behavior requires the convergence of ovarian hormone signals, namely estradiol and progesterone, and sensory cues from the male on a motor output pathway. Estrogen and progestin receptors (ER and PR) are found in neurons in the hypothalamic ventromedial nucleus (VMH), a brain region necessary for lordosis, the stereotypic female copulatory posture. A subset of VMH neurons sends axonal projections to the periaqueductal gray (PAG) to initiate a motor output relay, and some of these projection neurons express PR. Previous studies showed that VMH neurons are activated during mating, based on the expression of the immediate early gene Fos. Many of the activated neurons expressed ER; however, it is not known if such activated neurons co-express PR. Fluorogold, a retrograde tracer, was injected into the PAG of ovariectomized rats to label neurons projecting from the VMH. Hormone-treated animals then were mated, and their brains were immunohistochemically stained for PR and Fos. Of the Fos-positive neurons, 33% were double-labeled for PR, 19% were double-labeled with Fluorogold, and 5% were triple-labeled for Fos, PR, and the retrograde tracer. The majority of triple-labeled neurons were found in the rostral, rather than caudal, portion of the VMH. These results show that PR-containing neurons are engaged during sexual behavior, which suggests that these neurons are the loci of hormonal-sensory convergence and hormonal-motor integration.     

Sexual differentiation of mammalian frontal cortex

The pattern of distribution of the progesterone binding sites was examined in selected nuclei of the brain of male and female rat. In female rats the frontal cortex resulted to be the region with the highest concentration of 3H R5020 binding sites. However, in male rats the same region showed very little progestin binding activity. When female rats were androgenized via neonatal exposure to testosterone, the progestin binding activity of the frontal cortex became similar to that we observed in male rats. The present investigation indicates that sexual differentiation of the rat brain may include also brain regions not clearly involved in sex related functions like the frontal cortex.     

The regulation of social recognition, social communication and aggression: vasopressin in the social behavior neural network

Neuropeptides in the arginine vasotocin/arginine vasopressin (AVT/AVP) family play a major role in the regulation of social behavior by their actions in the brain. In mammals, AVP is found within a circuit of recriprocally connected limbic structures that form the social behavior neural network. This review examines the role played by AVP within this network in controlling social processes that are critical for the formation and maintenance of social relationships: social recognition, social communication and aggression. Studies in a number of mammalian species indicate that AVP and AVP V1a receptors are ideally suited to regulate the expression of social processes because of their plasticity in response to factors that influence social behavior. The pattern of AVP innervation and V1a receptors across the social behavior neural network may determine the potential range and intensity of social responses that individuals display in different social situations. Although fundamental information on how social behavior is wired in the brain is still lacking, it is clear that different social behaviors can be influenced by the actions of AVP in the same region of the network and that AVP can act within multiple regions of this network to regulate the expression of individual social behaviors. The existing data suggest that AVP can influence social behavior by modulating the interpretation of sensory information, by influencing decision making and by triggering complex motor outputs. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.     

Challenges to defining a role for progesterone in breast cancer

Progesterone is an ovarian steroid hormone that is essential for normal breast development during puberty and in preparation for lactation. The actions of progesterone are primarily mediated by its high affinity receptors, including the classical progesterone receptor (PR) -A and -B isoforms, located in diverse tissues such as the brain where progesterone controls reproductive behavior, and the breast and reproductive organs. Progestins are frequently prescribed as contraceptives or to alleviate menopausal symptoms, wherein progestin is combined with estrogen as a means to block estrogen-induced endometrial growth. Estrogen is undisputed as a potent breast mitogen, and inhibitors of the estrogen receptor (ER) and estrogen producing enzymes (aromatases) are effective first-line cancer therapies. However, PR action in breast cancer remains controversial. Herein, we review existing evidence from in vitro and in vivo models, and discuss the challenges to defining a role for progesterone in breast cancer.     

Progesterone, but not progesterone-independent activation of progestin receptors by a mating stimulus, rapidly decreases progestin receptor immunoreactivity in female rat brain

Recent studies suggest that progestin receptors may be activated in vivo by neurotransmitters in the absence of ligand. More specifically, vaginal-cervical stimulation (VCS) can influence sexual behavior by activating progestin receptors in the absence of progesterone. Another way to test if progestin receptors are influenced by particular stimuli is to examine progestin receptor immunostaining. We report that progestin receptor immunoreactivity is decreased in the forebrain of estradiol-primed ovariectomized (OVX) rats within 1 h after a subcutaneous injection of progesterone, a time by which rapid down-regulation of progestin receptors does not seem to have occurred. In estradiol-primed OVX rats, VCS also decreased progestin receptor immunoreactivity within 1 h in the medial preoptic area, but not in any other area examined. To determine if the decrease in immunoreactivity by VCS was due to adrenal secretions or by ligand-independent activation of progestin receptors, we repeated the experiment in estradiol-primed OVX/adrenalectomized rats. Prior removal of the adrenal glands blocked the rapid decrease in progestin receptor immunoreactivity, even though data from other experiments suggest that progestin receptors are activated by VCS at this time. These studies suggest the possibility that progestin receptors may be affected differentially by progesterone-dependent or by progesterone-independent pathways. This raises the possibility that activation of progestin receptors by these two distinct pathways may lead to different neuronal consequences.     

Sex differences and similarities in the neuroendocrine regulation of social behavior in an African cichlid fish

An individual's position in a social hierarchy profoundly affects behavior and physiology through interactions with community members, yet little is known about how the brain contributes to status differences between and within the social states or sexes. We aimed to determine sex-specific attributes of social status by comparing circulating sex steroid hormones and neural gene expression of sex steroid receptors in dominant and subordinate male and female Astatotilapia burtoni, a highly social African cichlid fish. We found that testosterone and 17β-estradiol levels are higher in males regardless of status and dominant individuals regardless of sex. Progesterone was found to be higher in dominant individuals regardless of sex. Based on pharmacological manipulations in males and females, progesterone appears to be a common mechanism for promoting courtship in dominant individuals. We also examined expression of androgen receptors, estrogen receptor α, and the progesterone receptor in five brain regions that are important for social behavior. Most of the differences in brain sex steroid receptor expression were due to sex rather than status. Our results suggest that the parvocellular preoptic area is a core region for mediating sex differences through androgen and estrogen receptor expression, whereas the progesterone receptor may mediate sex and status behaviors in the putative homologs of the nucleus accumbens and ventromedial hypothalamus. Overall our results suggest sex differences and similarities in the regulation of social dominance by gonadal hormones and their receptors in the brain.     

Regulation of estrogen-stimulated lordosis behavior and hypothalamic progestin receptor induction by antiestrogens in female rats

Two estrogen antagonists, CI-628 (CI) and tamoxifen (TX), were used to examine the relationship between estrogen priming of lordosis behavior and progestin receptor induction in the hypothalamus-preoptic area (HPOA) of ovariectomized female rats. Lordosis behavior was assessed by measuring lordosis quotients (LQ) in response to injection of 2 micrograms of estradiol benzoate (EB) followed 48 hr later by 500 micrograms of progesterone (P). Behavior testing began 4 hr after P injection. The effects of antiestrogens were assessed by injecting CI and TX (1-2 mg) from 0 to 48 hr prior to EB. Levels of cytosol progestin receptor in the HPOA were determined by quantifying the specific binding of 0.5 nM [3H]R5020 to cytosols from animals receiving the same EB and antiestrogen treatments used in behavioral testing. TX given concurrently with or CI given 2 hr before EB abolished both lordosis behavior and induction of HPOA progestin receptors. In contrast, CI given 12 hr prior to EB abolished lordosis but permitted a 95% elevation in the concentration of progestin binding sites in the HPOA. TX or CI given 48 hr before EB resulted in moderate levels of lordosis (mean LQs from 56 to 69) and induction of HPOA progestin receptors from 85 to 130% above noninjected controls. However, CI given 24 hr prior to EB produced less than a 40% increase in brain R5020 binding even though lordosis behavior was equivalent to that seen in the 48-hr animals (mean LQ = 53). These data indicate that the effects of antiestrogens on female sexual behavior and on the synthesis of brain progestin receptors depend on which antiestrogen is used and the time interval between administration of estrogen and antiestrogen. They also demonstrate that under some conditions estrogen induction of cytosol progestin receptors in the HPOA can be dissociated from estrogen priming of lordosis behavior in rats.     

Vasopressin Inhibits LTP in the CA2 Mouse Hippocampal Area

Growing evidence points to vasopressin (AVP) as a social behavior regulator modulating various memory processes and involved in pathologies such as mood disorders, anxiety and depression. Accordingly, AVP antagonists are actually envisaged as putative treatments. However, the underlying mechanisms are poorly characterized, in particular the influence of AVP on cellular or synaptic activities in limbic brain areas involved in social behavior. In the present study, we investigated AVP action on the synapse between the entorhinal cortex and CA2 hippocampal pyramidal neurons, by using both field potential and whole-cell recordings in mice brain acute slices. Short application (1 min) of AVP transiently reduced the synaptic response, only following induction of long-term potentiation (LTP) by high frequency stimulation (HFS) of afferent fibers. The basal synaptic response, measured in the absence of HFS, was not affected. The Schaffer collateral-CA1 synapse was not affected by AVP, even after LTP, while the Schaffer collateral-CA2 synapse was inhibited. Although investigated only recently, this CA2 hippocampal area appears to have a distinctive circuitry and a peculiar role in controlling episodic memory. Accordingly, AVP action on LTP-increased synaptic responses in this limbic structure may contribute to the role of this neuropeptide in controlling memory and social behavior.     

Activational effects of estradiol and dihydrotestosterone on social recognition and the arginine-vasopressin immunoreactive system in male mice lacking a functional aromatase gene

In rodents, parts of the arginine-vasopressin (AVP) neuronal system are sexually dimorphic with males having more AVP-immunoreactive cells/fibers than females. This neuropeptide neuronal system is highly sensitive to steroids and has been proposed to play an important role in the processing of olfactory cues critical to the establishment of a social memory. We demonstrate here that gonadally intact male aromatase knockout (ArKO) mice, which cannot aromatize androgens into estrogens due to a targeted mutation in the aromatase gene, showed severe deficits in social recognition as well as a reduced AVP-immunoreactivity in several brain regions. To determine whether this reduction is due to a lack of organizational or activational effects of estrogens, we assessed social recognition abilities and AVP-immunoreactivity in male ArKO and wild-type (WT) mice when treated with estradiol benzoate (EB) in association with dihydrotestosterone propionate (DHTP) in adulthood. Adult treatment with EB and DHTP restored social recognition abilities in castrated ArKO males since they showed normal female-oriented ultrasonic vocalizations and were able to recognize an unfamiliar female using a habituation-dishabituation paradigm. Furthermore, adult treatment also restored AVP-immunoreactivity in the lateral septum of ArKO males to levels observed in intact WT males. These results suggest that social recognition in adulthood and stimulation of AVP expression in the adult mouse forebrain depend predominantly on the estrogenic metabolite of testosterone. Furthermore, our results are in line with the idea that the organization of the AVP system may depend on androgen or sex chromosomes rather than estrogens.     

Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor alpha expression

Around the time of birth, male rats express higher levels of progesterone receptors in the medial preoptic nucleus (MPN) than female rats, suggesting that the MPN may be differentially sensitive to maternal hormones in developing males and females. Preliminary evidence suggests that this sex difference depends on the activation of estrogen receptors around birth. To test whether estrogen receptor alpha (ER alpha) is involved, we compared progesterone receptor immunoreactivity (PRir) in the brains of male and female neonatal mice that lacked a functional ER alpha gene or were wild type for the disrupted gene. We demonstrate that males express much higher levels of PRir in the MPN and the ventromedial nucleus of the neonatal mouse brain than females, and that PRir expression is dependent on the expression of ER alpha in these regions. In contrast, PRir levels in neocortex are not altered by ER alpha gene disruption. The results of this study suggest that the induction of PR via ER alpha may render specific regions of the developing male brain more sensitive to progesterone than the developing female brain, and may thereby underlie sexual differentiation of these regions.     

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.     

Genomic regulation of sexual behavior

Estrogen receptors are distributed in discrete areas of the hypothalamus, preoptic area and amygdala of the rat brain, and in some of these areas estrogens induce progestin receptor sites. Estradiol (E), followed by progesterone (P), induce feminine sexual behavior in female, but not in male, rats. This induction takes time (on the order of hours, not minutes, so that the hormone may be cleared from the body) and is dependent on RNA and protein synthesis. Within the hypothalamic ventromedial nuclei (VMN), E and P induce changes in RNA and protein synthesis and also induce morphological changes indicative of cellular growth, genomic activation, and either new synapse formation or morphological rearrangement of existing synapses. Neurochemically, a number of neurotransmitter systems are implicated in the control of feminine sexual behavior, including acetylcholine, serotonin, GABA, and the neuropeptides, oxytocin and CCK. One of the means by which E and P may exert their influence on sexual behavior, aside from the morphological alterations, is by regulating levels of receptors for certain of these neurotransmitters. The critical differences which underlie the inability of male rats to display high levels of feminine sexual behavior after E plus P priming may depend on sex differences in the ability of E to induce particular neurochemical products as well as P receptors and upon differences in neural circuitry in the VMN.