Jenne M. Westberry

Distinctive Responses in the Medial Amygdala to Same-Species and Different-Species Pheromones

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.

Estrogen receptor-alpha gene expression in the cortex: Sex differences during development and in adulthood

17β-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.

The influence of chemosensory input and gonadotropin releasing hormone on mating behavior circuits in male hamsters.

Chemosensory input is important for mating behavior in male hamsters. Chemosignals found in female hamster vaginal fluid activate regions of the brain that receive input from the vomeronasal/accessory olfactory system and are important for mating behavior. Mating or exposure to these chemosignals produces increased Fos protein expression in the amygdala, bed nucleus of the stria terminalis, and medial preoptic area (MPOA). These brain regions contain cell bodies and/or fibers of gonadotropin releasing hormone (GnRH) neurons, suggesting potential relationships between chemosensory systems and GnRH. GnRH is released naturally when male rodents (mice and hamsters) encounter female chemosignals, and intracerebrally injected GnRH restores mating behavior in sexually naive male hamsters after removal of the vomeronasal organs. We report here that the combination of pheromone exposure and intracerebrally-injected GnRH increases Fos expression in the MPOA above the increase seen in pheromone-exposed males, or in males given only the exogenous GnRH. In males with vomeronasal organs removed (VNX), there was an also an increment in Fos expression in the MPOA when these pheromone exposed males were injected with GnRH, provided they had previous sexual experience. Males with vomeronasal organs removed and without sexual experience showed increased Fos expression in the medial amygdala when pheromone exposure and GnRH injection were combined, but not in the medial preoptic area.

Dynamic Regulation of Estrogen Receptor-Alpha Gene Expression in the Brain: A Role for Promoter Methylation?

Estrogen has long been known to play an important role in coordinating the neuroendocrine events that control sexual development, sexual behavior and reproduction. Estrogen actions in other, non-reproductive areas of the brain have also been described. It is now known that estrogen can also influence learning, memory, and emotion and has neurotrophic and neuroprotective properties. The actions of estrogen are largely mediated through at least two intracellular estrogen receptors. Both estrogen receptor-alpha and estrogen receptor-beta are expressed in a wide variety of brain regions. Estrogen receptor-alpha (ERalpha), however, undergoes developmental and brain region-specific changes in expression. The precise molecular mechanisms that regulate its expression at the level of gene transcription are not well understood. Adding to the complexity of its regulation, the estrogen receptor gene contains multiple promoters that drive its expression. In the cortex in particular, the ERalpha mRNA expression is dynamically regulated during postnatal development and again following neuronal injury. Epigenetic modification of chromatin is increasingly being understood as a mechanism of neuronal gene regulation. This review examines the potential regulation of the ERalpha gene by such epigenetic mechanisms.

Regulation of estrogen receptor alpha gene expression in the mouse prefrontal cortex during early postnatal development

Estrogens have many functions in the developing rodent brain, and most of these depend on the presence of estrogen receptors. Understanding how expression of these receptors are regulated is crucial for understanding the roles of estradiol in the male and female brain during development In rodents, the prefrontal cortex (PFC) has been shown to be involved in working memory, attention, and behavioral inhibition. Many studies have demonstrated an effect of estradiol on sex difference in these functions attributed to differences in the PFC. We have previously demonstrated that estrogen receptor alpha (ERα) expression decreases in the isocortex across early postnatal development. This decrease corresponds with an increase in methylation of many sites along the ERα promoter. Here we have examined both ERα and ERβ mRNA expression in the PFC to determine if methylation also plays a role in this important brain region. We investigated expression of alternate promoters for ERα and methylation of CpG sites along two of these promoters. We found that the pattern of ERα mRNA expression in PFC was similar to the pattern of ERα expression in the isocortex and that there were no sex differences in the level of expression across development. We did, however, find subtle differences in promoter expression and methylation that may indicate a sex-specific difference in PFC during development resulting in a difference in adult response.

Regulation of neuropeptide Y in the rat amygdala following unilateral olfactory bulbectomy

While the mechanisms are not fully understood, olfactory bulbectomy (OBX) is a well-known rat model of depression and depression-related disorders such as anxiety and aggression. Alterations in neuropeptide Y (NPY) levels in the brain have been linked to depression and have been shown to be involved in the response to stress. This study explored the possible regulation of NPY immunoreactivity in specific regions of the amygdala 14 days after OBX in adult male Sprague-Dawley rats (n=6). Unilateral OBX and immunohistochemistry permitted comparisons of NPY in the ipsilateral amygdala with NPY in the contralateral (sham) amygdala. OBX resulted in significant increases (P<0.05) in NPY immunoreactivity in the anterior medial amygdala (threefold) and the posterior medial amygdala (2.5-fold). These regions receive projections from the accessory olfactory bulb (AOB). In contrast, the anterior and posterolateral cortical nuclei of the amygdala receive projections from the main olfactory bulb (MOB). NPY was not increased in these nuclei. These data show that not only does OBX increase NPY immunoreactivity in the amygdala, but also suggest that the AOB plays a prominent role in this regulation.

Epigenetic regulation of estrogen receptor beta expression in the rat cortex during aging

During aging, there is an increase in neurodegenerative diseases and a decrease in cognitive performance. Postmenopausal women are more vulnerable as their estrogen levels decline, but most hormone replacement therapies do not prevent cognitive decline. One potential reason is that the timing of hormone replacement is critical and changes in the estrogen receptor expression may over-ride hormonal intervention. In rodents, estrogen receptor &bgr; (ER&bgr;) mRNA decreases in the cortex with age. One mechanism by which ER&bgr; mRNA could be regulated is by epigenetic modification of ER&bgr; promoter. Here, we show an increase in methylation of ER&bgr; promoter corresponding to decrease in ER&bgr; mRNA in the cortex of an aging female.

Selective Response of Medial Amygdala Subregions to Reproductive and Defensive Chemosignals from Conspecific and Heterospecific Species

In hamsters and inbred mice, pheromone-containing chemosensory signals originating from the animal’s own species (conspecific) and other species (heterospecific) produce differential patterns of immediate early gene (IEG = Fos/FRAs) expression in the medial amygdala. In males of both species, conspecific stimuli, regardless of gender or putative function, activated neurons in both anterior and posterior medial amygdala (MeA, MeP). With heterospecific stimuli, MeA was activated but MeP appeared to be suppressed. MeP neurons expressing GABA-receptor were selectively suppressed by heterospecific stimuli at the same time as the GABAergic caudal intercalated nucleus (ICNc) of the amygdala was activated, suggesting suppression of MeP by ICN. We propose that information on conspecific chemosignals with preprogrammed meaning (pheromones) is analyzed by MeP neurons, probably influenced by gonadal steroid status. Information about heterospecific stimuli that activate anterior medial amygdala via the vomeronasal organ appears to have restricted access to MeP. Signals from conspecific males that are potentially threatening elicit different patterns of activation in MeP than other conspecific signals. In hamsters, male flank gland secretion activates predominantly GABA-immunoreactive neurons and mainly in ventral MeP (MePv). Male mouse urine also activates predominantly MePv in mice. This region responds to predator odors in rats and is reported to do so in mice. These findings, with other data, support a division of labor in medial amygdala according to the reproductive or defense-related potential of the stimuli. There is some evidence for a convergence of information on conspecific and heterospecific threatening stimuli but, so far, the details are not entirely consistent. In our experiments with hamsters and mice, stimuli from potential predators (cat urine, cat collar) like other heterospecific stimuli, activated MeA and not MeP. Others studying mice found activation in ventral MeA (MeAv) during male-male interactions and in MePv by cat collar stimuli. Since the submission of this paper we have also found activation in mouse MePv by stronger cat collar stimuli (see note at end of text).

GABAergic mechanisms contributing to categorical amygdala responses to chemosensory signals

Chemosensory stimuli from conspecific and heterospecific animals, elicit categorically different immediate-early gene response-patterns in medial amygdala in male hamsters and mice. We previously showed that conspecific signals activate posterior (MeP) as well as anterior medial amygdala (MeA), and especially relevant heterospecific signals such as chemosensory stimuli from potential predators also activate MeP in mice. Other heterospecific chemosignals activate MeA, but not MeP. Here we show that male hamster amygdala responds significantly differentially to different conspecific signals, by activating different proportions of cells of different phenotype, possibly leading to differential activation of downstream circuits. Heterospecific signals that fail to activate MeP do activate GABA-immunoreactive cells in the adjacent caudal main intercalated nucleus (mICNc) and elicit selective suppression of MeP cells bearing GABA-Receptors, suggesting GABA inhibition in MeP by GABAergic cells in mICNc. Overall, work presented here suggests that medial amygdala may discriminate between important conspecific social signals, distinguish them from the social signals of other species and convey that information to brain circuits eliciting appropriate social behavior.

Characteristic Response to Chemosensory Signals in GABAergic Cells of Medial Amygdala Is Not Driven by Main Olfactory Input

Chemosensory stimuli from same species (conspecific) and different species (heterospecific) elicit categorically different immediate-early gene (IEG) response patterns in medial amygdala in male hamsters and mice. All heterospecific stimuli activate anterior medial amygdala (MeA) but only especially salient heterospecific stimuli, such as those from predators activate posterior medial amygdala (MeP). We previously reported that characteristic patterns of response in separate populations of cells in MeA and MeP distinguish between different conspecific stimuli. Both gamma aminobutyric acid (GABA)-immunoreactive (ir) cells and GABA-receptor-ir cells make this distinction. Here, using zinc sulfate lesions of the main olfactory epithelium, we show evidence that main olfactory input does not contribute to the characteristic patterns of response in GABA-ir cells of male hamster amygdala, either for conspecific or heterospecific stimuli. Some GABAergic cells are output neurons carrying information from medial amygdala to behavioral executive regions of basal forebrain. Thus, the differential response to different conspecific signals can lead to differential activation of downstream circuits based on nonolfactory input. Finally, we show that an intact vomeronasal organ is necessary and sufficient to produce the characteristic patterns of response to conspecific and heterospecific chemosensory stimuli in hamster medial amygdala. Although main olfactory input may be critical in species with less prominent vomeronasal input for equivalent medial amygdala responses, work presented here suggests that hamster medial amygdala uses primarily vomeronasal input to discriminate between important unlearned conspecific social signals, to distinguish them from the social signals of other species, and may convey that information to brain circuits eliciting appropriate social behavior.