Neonatal RU-486 (mifepristone) exposure increases androgen receptor immunoreactivity and sexual behavior in male rats.

Progesterone and progestin receptors (PRs) are known to play a role in the development of brain physiology and behavior in many different species. The distribution and regulation of PRs within the developing brain suggest that they likely contribute to the organization of the brain and behavior in a sex-specific manner. We examined the role of PR signaling during development on the organization of adult sexual behavior and androgen receptor (AR) expression in the brain. We administered the PR antagonist, RU-486, subcutaneously to male and female rats on postnatal days 1-7 (0=day of birth) and examined adult sexual behavior and AR-immunoreactivity (AR-ir) in the adult brain. A typical sex difference in lordosis quotient (LQ) was observed and neonatal RU-486 treatment did not alter this behavior. In contrast, neonatal RU-486 treatment increased adult male sexual behavior and AR-ir in several brain areas in males. These data indicate that a transient disruption in PR signaling during development can have lasting consequences on the male brain and may increase male sexual behavior in part by increasing AR expression, and therefore androgen sensitivity, in adulthood.

Permanent and plastic epigenesis in neuroendocrine systems.

The emerging area of neuroepigenetics has been linked to numerous mental health illnesses. Importantly, a large portion of what we know about early gene×environment interactions comes from examining epigenetic modifications of neuroendocrine systems. This review will highlight how neuroepigenetic mechanisms during brain development program lasting differences in neuroendocrine systems and how other neuroepigenetic processes remain plastic, even within the adult brain. As epigenetic mechanisms can either be stable or plastic, elucidating the mechanisms involved in reversing these processes could aid in understanding how to reverse pathological epigenetic programming.

Neonatal MeCP2 is important for the organization of sex differences in vasopressin expression.

Several neurodevelopmental disorders are marked by atypical Methyl-CpG-binding protein 2 (MeCP2) expression or function; however, the role of MeCP2 is complex and not entirely clear. Interestingly, there are sex differences in some of these disorders, and it appears that MeCP2 has sex-specific roles during development. Specifically, recent data indicate that a transient reduction in MeCP2 within developing amygdala reduces juvenile social play behavior in males to female-typical levels. These data suggest that MeCP2 within the amygdala is involved in programming lasting sex differences in social behavior. In the present study, we infused MeCP2 or control siRNA into the amygdala of male and female rats during the first three days of postnatal life in order to assess the impact of a transient reduction in MeCP2 on arginine vasopressin (AVP), a neural marker that is expressed differentially between males and females and is linked to a number of social behaviors. The expression of AVP, as well as several other genes, was measured in two-week old and adult animals. Two-week old males expressed more AVP and galanin mRNA in the amygdala than females, and a transient reduction in MeCP2 eliminated this sex difference by reducing the expression of both gene products in males. A transient reduction in MeCP2 also decreased androgen receptor (AR) mRNA in two-week old males. In adulthood, control males had more AVP-immunoreactive (AVP-ir) cells than females in the centromedial amygdala (CMA), bed nucleus of the stria terminalis (BST) and in the fibers that project from these cells to the lateral septum (LS). A transient reduction in MeCP2 eliminated this sex difference. Interestingly, there were no lasting differences in galanin or AR levels in adulthood. Reducing MeCP2 levels during development did not alter estrogen receptorα, neurofilament or Foxg1. We conclude that a transient reduction in MeCP2 expression in the developing male amygdala has a transient impact on galanin and AR expression but a lasting impact on AVP expression, highlighting the importance of MeCP2 in organizing sex differences in the amygdala.

Progesterone impairs social recognition in male rats.

The influence of progesterone in the brain and on the behavior of females is fairly well understood. However, less is known about the effect of progesterone in the male system. In male rats, receptors for progesterone are present in virtually all vasopressin (AVP) immunoreactive cells in the bed nucleus of the stria terminalis (BST) and the medial amygdala (MeA). This colocalization functions to regulate AVP expression, as progesterone and/or progestin receptors (PR)s suppress AVP expression in these same extrahypothalamic regions in the brain. These data suggest that progesterone may influence AVP-dependent behavior. While AVP is implicated in numerous behavioral and physiological functions in rodents, AVP appears essential for social recognition of conspecifics. Therefore, we examined the effects of progesterone on social recognition. We report that progesterone plays an important role in modulating social recognition in the male brain, as progesterone treatment leads to a significant impairment of social recognition in male rats. Moreover, progesterone appears to act on PRs to impair social recognition, as progesterone impairment of social recognition is blocked by a PR antagonist, RU-486. Social recognition is also impaired by a specific progestin agonist, R5020. Interestingly, we show that progesterone does not interfere with either general memory or olfactory processes, suggesting that progesterone seems critically important to social recognition memory. These data provide strong evidence that physiological levels of progesterone can have an important impact on social behavior in male rats.

Epigenetic control of vasopressin expression is maintained by steroid hormones in the adult male rat brain.

Although some DNA methylation patterns are altered by steroid hormone exposure in the developing brain, less is known about how changes in steroid hormone levels influence DNA methylation patterns in the adult brain. Steroid hormones act in the adult brain to regulate gene expression. Specifically, the expression of the socially relevant peptide vasopressin (AVP) within the bed nucleus of the stria terminalis (BST) of adult brain is dependent upon testosterone exposure. Castration dramatically reduces and testosterone replacement restores AVP expression within the BST. As decreases in mRNA expression are associated with increases in DNA promoter methylation, we explored the hypothesis that AVP expression in the adult brain is maintained through sustained epigenetic modifications of the AVP gene promoter. We find that castration of adult male rats resulted in decreased AVP mRNA expression and increased methylation of specific CpG sites within the AVP promoter in the BST. Similarly, castration significantly increased estrogen receptor α (ERα) mRNA expression and decreased ERα promoter methylation within the BST. These changes were prevented by testosterone replacement. This suggests that the DNA promoter methylation status of some steroid responsive genes in the adult brain is actively maintained by the presence of circulating steroid hormones. The maintenance of methylated or demethylated states of some genes in the adult brain by the presence of steroid hormones may play a role in the homeostatic regulation of behaviorally relevant systems.

Mu-opioid receptor densities are depleted in regions implicated in agonistic and sexual behavior in male European starlings (Sturnus vulgaris) defending nest sites and courting females.

Social status and resource availability can strongly influence individual behavioral responses to conspecifics. In European starlings, males that acquire nest sites sing in response to females and dominate other males. Males without nest sites sing, but not to females, and they do not interact agonistically with other males. Little is known about the neural regulation of status- or resource-appropriate behavioral responses to conspecifics. Opioid neuropeptides are implicated in birdsong and agonistic behavior, suggesting that opioids may underlie differences in the production of these behaviors in males with and without nest sites. Here, we examined densities of immunolabeled mu-opioid receptors in groups of male starlings. Males that defended nest boxes dominated other males and sang at higher rates when presented with a female than males without nest boxes, independent of testosterone concentrations. Multiple regression analyses showed nest box ownership (not agonistic behavior or singing) predicted the optical density of receptor labeling in the medial bed nucleus of stria terminalis, paraventricular nucleus, ventral tegmental area and the medial preoptic nucleus. Compared to males without nest boxes, males with nest boxes had lower densities of immunolabeled mu-opioid receptors in these regions. Singing additionally predicted the area covered by labeling in the ventral tegmental area. The results suggest that elevated opioid activity in these regions suppresses courtship and agonistic behavioral responses to conspecifics in males without nest boxes. The findings are consistent with a dynamic role for opioid receptors in adjusting social behavior so that it is appropriate given the resources available to an individual.

Epigenetic turn ons and turn offs: chromatin reorganization and brain differentiation.

The study of epigenetics allows for the understanding of gene × environmental interactions and provides a mechanism by which brief internal or external environmental changes can shape lasting differences in gene function and behavior. Epigenetic processes appear to impact a wide variety of physiological processes within the developing brain, including neuroendocrine function. An epigenetic model is proposed by which steroid hormones and the social environment induces appropriate masculinization of the brain by turning on and off gene transcriptional events. This minireview will discuss how epigenetic events influence sexual differentiation of the brain and point at examples suggesting that some epigenetic events can be quite dynamic.

Winning territorial disputes selectively enhances androgen sensitivity in neural pathways related to motivation and social aggression.

Winning aggressive disputes can enhance future fighting ability and the desire to seek out additional contests. In some instances, these effects are long lasting and vary in response to the physical location of a fight. Thus, in principle, winning aggressive encounters may cause long-term and context-dependent changes to brain areas that control the output of antagonistic behavior or the motivation to fight (or both). We examined this issue in the territorial California mouse (Peromyscus californicus) because males of this species are more likely to win fights after accruing victories in their home territory but not after accruing victories in unfamiliar locations. Using immunocytochemistry and real-time quantitative PCR, we found that winning fights either at home or away increases the expression of androgen receptors (AR) in the medial anterior bed nucleus of the stria terminalis, a key brain area that controls social aggression. We also found that AR expression in brain regions that mediate motivation and reward, nucleus accumbens (NAcc) and ventral tegmental area (VTA), increases only in response to fights in the home territory. These effects of winning were likely exclusive to the neural androgenic system because they have no detectible impact on the expression of progestin receptors. Finally, we demonstrated that the observed changes in androgen sensitivity in the NAcc and VTA are positively associated with the ability to win aggressive contests. Thus, winning fights can change brain phenotype in a manner that likely promotes future victory and possibly primes neural circuits that motivate individuals to fight.

The nuclear receptor corepressor has organizational effects within the developing amygdala on juvenile social play and anxiety-like behavior.

Nuclear receptor function on DNA is regulated by the balanced recruitment of coregulatory complexes. Recruited proteins that increase gene expression are called coactivators, and those that decrease gene expression are called corepressors. Little is known about the role of corepressors, such as nuclear receptor corepressor (NCoR), on the organization of behavior. We used real-time PCR to show that NCoR mRNA levels are sexually dimorphic, that females express higher levels of NCoR mRNA within the developing amygdala and hypothalamus, and that NCoR mRNA levels are reduced by estradiol treatment. To investigate the functional role of NCoR on juvenile social behavior, we infused small interfering RNA targeted against NCoR within the developing rat amygdala and assessed the enduring impact on juvenile social play behavior, sociability, and anxiety-like behavior. As expected, control males exhibited higher levels of juvenile social play than control females. Reducing NCoR expression during development further increased juvenile play in males only. Interestingly, decreased NCoR expression within the developing amygdala had lasting effects on increasing juvenile anxiety-like behavior in males and females. These data suggest that the corepressor NCoR functions to blunt sex differences in juvenile play behavior, a sexually dimorphic and hormone-dependent behavior, and appears critical for appropriate anxiety-like behavior in juvenile males and females.

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.

Mecp2 organizes juvenile social behavior in a sex-specific manner.

Methyl-CpG-binding protein 2 (MeCP2) binds methylated DNA and recruits corepressor proteins to modify chromatin and alter gene transcription. Mutations of the MECP2 gene can cause Rett syndrome, whereas subtle reductions of MeCP2 expression may be associated with male-dominated social and neurodevelopmental disorders. We report that transiently decreased amygdala Mecp2 expression during a sensitive period of brain sexual differentiation disrupts the organization of sex differences in juvenile social play behavior. Interestingly, neonatal treatment with Mecp2 small interfering RNA within the developing amygdala reduced juvenile social play behavior in males but not females. Reduced Mecp2 expression did not change juvenile sociability or anxiety-like behavior, suggesting that this disruption is associated with subtle behavioral modification. This suggests that Mecp2 may have an overlooked role in the organization of sexually dimorphic behaviors and that male juvenile behavior is particularly sensitive to Mecp2 disruption during this period of development.

Evidence that female endocrine state influences catecholamine responses to male courtship song in European starlings.

Little is known about the neural control of female responses to male courtship. Female European starlings in breeding condition with high concentrations of estrogen select mates based on variation in song and approach nest boxes broadcasting male song. In contrast, outside of the breeding season (when estrogen is low) females do not display the same response to male song. The catecholamines dopamine and norepinephrine regulate behaviors important for mate choice such as arousal, attention, sexual motivation, and goal-directed approach responses, suggesting a role for catecholamines in female responses to male song. In the present study, treating females with a dopamine agonist inhibited, whereas an antagonist stimulated female interest in nest boxes broadcasting male song. In a second study immunocytochemistry was used to examine the distribution of the phosphorylated (i.e., active) form of tyrosine hydroxylase (pTH), the rate-limiting enzyme for catecholamine synthesis. Exposure to male song in breeding condition females reduced pTH density in brain regions involved in social behavior (lateral septum, ventromedial nucleus of the hypothalamus) and a region involved in visual processing (nucleus of Edinger-Westphal) but not song control regions. Opposite patterns of pTH labeling densities were observed in the same regions in response to song in non-breeding condition females. pTH in the ventral tegmental area was also affected by song and female endocrine condition. Overall, the present data support an inhibitory role for dopamine in female responses to courtship and suggest that endocrine state and catecholamines interact to regulate this behavior.

Microarray profiling of gene expression patterns in adult male rat brain following acute progesterone treatment.

Progesterone can influence various behaviors in adult male rats, however, little is known about which particular genes are regulated by progesterone in the male rat brain. Using focused microarray technology, we where able to define a subset of genes that are responsive to progesterone. Nylon membrane-based cDNA microarrays were used to profile gene expression patterns in the preoptic area/mediobasal hypothalamus (POA/MBH) of male rat brain 7 h following a single injection of progesterone. RNA was isolated from the brains of 6 male rats injected with progesterone and 6 male rats injected with sesame oil. Next, we hybridized the RNA from each animal to individual cDNA microarrays that contained more than 100 target genes, all of which are involved in cAMP and or calcium signaling pathways. Direct side-by-side comparison of all 12 arrays revealed differences in the expression patterns of 12 different genes. We confirmed the data gathered from the arrays on 4 different genes using Real-Time PCR. These data begin to outline the important role played by progesterone in mediating changes in gene expression within the male brain.

Dopaminergic activation of estrogen receptors in neonatal brain alters progestin receptor expression and juvenile social play behavior.

Steroid receptor activation in developing brain influences a variety of cellular processes that endure into adulthood, altering both behavior and physiology. We report that estrogen receptors can be activated in a ligand-independent manner within developing brain by membrane dopamine receptors. Neonatal treatment with either estradiol or a dopamine D1 receptor agonist can increase the expression of an estrogen receptor-regulated gene (i.e. progestin receptors) and later juvenile social play. More importantly, increases in social play behavior induced by neonatal treatment with estradiol or a dopamine D1 receptor agonist can be prevented by prior treatment with an estrogen receptor antagonist. This suggests that changes in dopamine transmission in developing brain can activate estrogen receptors in a ligand-independent manner to influence gene expression and have lasting consequences on social behavior.

Evidence for opioid involvement in the regulation of song production in male European starlings (Sturnus vulgaris).

Many social animals vocalize at high rates, suggesting that vocal communication is highly motivated and rewarding. In songbirds, much is known about the neural control of vocal behavior; however, little is known about neurobiological mechanisms regulating the motivation to communicate. This study examined a possible role for opioid neuropeptides in motivation and reward associated with song production in male European starlings (Sturnus vulgaris). Peripheral opioid blockade facilitated male song production. Furthermore, methionine-enkephalin immunolabeled fiber densities within brain regions in which opioids are known to regulate motivation and reward (i.e., the medial preoptic nucleus and ventral tegmental area) related positively to male song production. These data suggest that song production might be regulated by opioid activity within motivation and reward neural systems.

Seasonal plasticity in the song control system: multiple brain sites of steroid hormone action and the importance of variation in song behavior.

Birdsong, in non-tropical species, is generally more common in spring and summer when males sing to attract mates and/or defend territories. Changes in the volumes of song control nuclei, such as HVC and the robust nucleus of the arcopallium (RA), are observed seasonally. Long photoperiods in spring stimulate the recrudescence of the testes and the release of testosterone. Androgen receptors, and at times estrogen receptors, are present in HVC and RA as are co-factors that facilitate the transcriptional activity of these receptors. Thus testosterone can act directly to induce changes in nucleus volume. However, dissociations have been identified at times among long photoperiods, maximal concentrations of testosterone, large song control nuclei, and high rates of song. One explanation of these dissociations is that song behavior itself can influence neural plasticity in the song system. Testosterone can act via brain-derived neurotrophic factor (BDNF) that is also released in HVC as a result of song activity. Testosterone could enhance song nucleus volume indirectly by acting in the preoptic area, a region regulating sexual behaviors, including song, that connects to the song system through catecholaminergic cells. Seasonal neuroplasticity in the song system involves an interplay among seasonal state, testosterone action, and behavioral activity.

A model system for study of sex chromosome effects on sexually dimorphic neural and behavioral traits.

We tested the hypothesis that genes encoded on the sex chromosomes play a direct role in sexual differentiation of brain and behavior. We used mice in which the testis-determining gene (Sry) was moved from the Y chromosome to an autosome (by deletion of Sry from the Y and subsequent insertion of an Sry transgene onto an autosome), so that the determination of testis development occurred independently of the complement of X or Y chromosomes. We compared XX and XY mice with ovaries (females) and XX and XY mice with testes (males). These comparisons allowed us to assess the effect of sex chromosome complement (XX vs XY) independent of gonadal status (testes vs ovaries) on sexually dimorphic neural and behavioral phenotypes. The phenotypes included measures of male copulatory behavior, social exploration behavior, and sexually dimorphic neuroanatomical structures in the septum, hypothalamus, and lumbar spinal cord. Most of the sexually dimorphic phenotypes correlated with the presence of ovaries or testes and therefore reflect the hormonal output of the gonads. We found, however, that both male and female mice with XY sex chromosomes were more masculine than XX mice in the density of vasopressin-immunoreactive fibers in the lateral septum. Moreover, two male groups differing only in the form of their Sry gene showed differences in behavior. The results show that sex chromosome genes contribute directly to the development of a sex difference in the brain.