Chemosignals related to reproductive and social status (pheromones) carry messages between opposite-sex and same-sex individuals in many species. Each individual must distinguish signals relevant to its own social behavior with conspecifics from signals used by other (heterospecific) species relevant to their social behavior. In male hamsters, the medial amygdala responded in a categorically different way to conspecific stimuli (socially relevant) and heterospecific stimuli (not socially relevant but serving similar purposes for other species), and may play an important role in this decision. Immediate-early gene responses to conspecific chemosignals and heterospecific chemosignals were characteristically different. The categorical responses, generated by chemosensory input from the vomeronasal organ and (probably) GABA inhibition within the amygdala, were not apparent at more peripheral sensory levels. This is the first evidence for an important role of the amygdala, a limbic structure known to be involved in social and emotional behavior, in discrimination of species specificity in chemosignals.

Estrogens play a critical role in brain development by acting on areas that express estrogen receptors. In the rodent cortex, estrogen receptor alpha (ER alpha) mRNA expression is high early in postnatal development but declines starting at postnatal day (PND) 10 and is virtually absent in the adult cortex. The mechanisms controlling this regulation are largely unknown. Methylation is important for gene silencing during development in many tissues, including the brain. In the present study, we examined the methylation status of ER alpha 5' untranslated exons during early postnatal development in male and female mice using methylation-specific PCR and pyrosequencing. Several regions of ER alpha promoter displayed a significant increase in methylation at PND 18 and 25 compared with PND 4. DNA methyltransferases (DNMT) are important for the initiation and maintenance of methylation. Real-time PCR showed that DNMT3A, the de novo DNMT peaked at PND 10 and was decreased by PND 25. DNMT1, which is important for maintenance of methylation, increased across development and stayed high in adult cortex. The methyl-CpG-binding protein 2 (MeCP2) is also important for stabilization of methylation. A chromatin immunoprecipitation assay showed a correlation between association of MeCP2 with ER alpha promoter and the increase in methylation and decrease in ER alpha expression after PND 10. In mice containing a mutant MeCP2 protein, ER alpha mRNA expression and promoter methylation patterns across development were different compared with wild-type mice. These data suggest that methylation of ER alpha promoters regulates ER alpha mRNA expression in the cortex during postnatal development in a MeCP2-dependent fashion.

Permanent middle cerebral artery occlusion (MCAO) causes neuronal cell death in the striatum and cortex. In rodents, estradiol treatment protects the cortex from cell death in an estrogen receptor alpha (ERα) dependent manner. ERα is only transiently expressed in the cortex during neonatal development and is very low in uninjured adult cortex. Following MCAO, ERα mRNA expression is upregulated in the cortex of female rats, but the mechanism of this increase is still unknown. It is also unknown whether a similar increase in ERα expression in seen in males. In the following studies, male and vehicle or estradiol-treated ovariectomized (OVX) female rats underwent MCAO to investigate the regulation of ERα expression after ischemia. 24 hours after surgery, mRNA or genomic DNA was collected from 1mm micropunches taken from 300µm brain sections for quantitative RT-PCR or methylation-specific (MSP) PCR, respectively. Additionally, adjacent 20µm sections were processed for ERα immunohistochemistry. In OVX females, ERα mRNA and protein were increased in the ischemic cortex, but unchanged in males. We hypothesized that this increase in ERα in females is due to a reversal of gene silencing by DNA methylation. Using MSP targeting of CpG islands within the 5' UTR of the rat ERα gene, we found that ischemia decreased methylation in the ischemic cortex of both groups of females, but there was no change in methylation in males. Using chromatin immunoprecipitation, we found that MeCP2 associates with ERα 5'UTR corresponding with the methylation status of the promoter. These data are the first to demonstrate a difference in the regulation of ERα expression in response to MCAO between males and females and that methylation of the ERα gene corresponds with mRNA levels in the brain.

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Abstract17β-estradiol is a hormone with far-reaching organizational, activational and protective actions in both male and female brains. The organizational effects of early estrogen exposure are essential for long-lasting behavioral and cognitive functions. Estradiol mediates many of its effects through the intracellular receptors, estrogen receptor-alpha (ERα) and estrogen receptor-beta (ERβ). In the rodent cerebral cortex, estrogen receptor expression is high early in postnatal life and declines dramatically as the animal approaches puberty. This decline is accompanied by decreased expression of ERα mRNA. This change in expression is the same in both males and females in the developing isocortex and hippocampus. An understanding of the molecular mechanisms involved in the regulation of estrogen receptor alpha (ERα) gene expression is critical for understanding the developmental, as well as changes in postpubertal expression of the estrogen receptor. One mechanism of suppressing gene expression is by the epigenetic modification of the promoter regions by DNA methylation that results in gene silencing. The decrease in ERα mRNA expression during development is accompanied by an increase in promoter methylation. Another example of regulation of ERα gene expression in the adult cortex is the changes that occur following neuronal injury. Many animal studies have demonstrated that the endogenous estrogen, 17β-estradiol, is neuroprotective. Specifically, low levels of estradiol protect the cortex from neuronal death following middle cerebral artery occlusion (MCAO). In females, this protection is mediated through an ERα-dependent mechanism. ERα expression is rapidly increased following MCAO in females, but not in males. This increase is accompanied by a decrease in methylation of the promoter suggesting a return to the developmental program of gene expression within neurons. Taken together, during development and in adulthood, regulation of ERα gene expression in the cortex can occur by DNA methylation and in a sex-dependent fashion in the adult brain.