Androgenic and Oestrogenic Influences on Tyrosine Hydroxylase‐Immunoreactive Cells of the Prairie Vole Medial Amygdala and Bed Nucleus of the Stria Terminalis

The medial amygdala (MeA) is one component of a densely interconnected, steroid hormone-sensitive network of brain sites regulating an array of sociosexual behaviours in rodents (1). Numerous subregions of the MeA receive direct projections from the main olfactory bulbs, the accessory olfactory bulbs, or both (2-6), and this chemosensory input is requisite for appropriate social interactions. In male rats, large lesions of the MeA suppress non-contact erections, alter copulation rate, or severely inhibit copulation in toto (7-10). In female rats, MeA lesions reduce their preference for odors of gonadally intact males over those of castrated males (11) and alleviate the olfactory suppression of maternal behaviours (12). In male and female hamsters, MeA lesions impair social odor recognition, social odor preferences, and/or copulatory behaviours (see 1,13). The MeA projects broadly within and outside the sociosexual behaviour network, but of particular interest are its projections to the bed nucleus of the stria terminalis (BST). The BST of laboratory rats and hamsters receives some of the densest projections emanating from the MeA (14,15), and in some species also receives limited projections directly from the accessory olfactory bulb (3,5,16). Because of their dense connections with each other, similar projections to other areas of the sociosexual network, and olfactory processing abilities, the MeA and medial BST are considered by some to be components of a continuous neural structure termed the “extended olfactory amygdala” (17). Not surprisingly, then, the BST is also implicated in the social behaviours in rodents (18-21). Prairie voles (Microtus ochrogaster) have become an important rodent model in which to study the neural networks involved in the olfactory and steroid hormone control of social behaviours because their social organization differs from most rodents, and even most mammals. In either wild or laboratory settings prairie voles are highly gregarious, socially monogamous after mating, and biparental towards their offspring (22). Neural sites mediating their unusual social behaviours include many of the sites traditionally involved in the more typical social behaviours of other rodents, including the MeA and BST. Indeed, lesions encompassing the MeA decrease general social contact and paternal behaviours in male prairie voles (23), while exposure to conspecific urinary cues, cohabitation with or without mating, or interactions with pups increase immediate-early gene (IEG) expression in both the MeA and BST (24-28). Although many of the same neural sites are involved in the social behaviour of prairie voles as in other rodents, it has often been suggested that species differences in neurochemistry underlie species differences in social structure and behaviours. In support, the distribution and density of receptors for oxytocin, vasopressin, corticotrophin-releasing hormone, and some gonadal steroids differ between monogamous voles and non-monogamous rodents, and their social behaviours are dramatically altered by manipulating these systems (29). In addition to neuropeptides and hormones, central dopamine (DA) systems differ between monogamous and non-monogamous voles. Research has focused on the mesolimbocortical system, where prairie voles have higher D2 receptor expression in the medial prefrontal cortex and lower D1 receptor expression in the medial prefrontal cortex and nucleus accumbens than do meadow voles (30,31). Furthermore, mating increases DA release in the nucleus accumbens of both male and female prairie voles, which is necessary for the establishment and maintenance of pairbonds (32), but this release does not occur in non-monogamous voles (32). The existence of species differences in dopamine systems outside the mesolimbocortical system are not often investigated, but could also be expected to contribute to species differences in social behaviour. Our laboratory has described an interesting species difference in the catecholamine innervation of the MeA and BST. We find that prairie voles have hundreds of cells and a dense plexus of fibres containing tyrosine hydroxylase (TH – the rate-limiting enzyme necessary for catecholamine release) centered in the posterodorsal MeA (MeApd) and principal nucleus of the BST (pBST), but relatively few or none of these cells exist in the MeApd and pBST of the non-monogamous species examined (laboratory rats, hamsters, and meadow voles) (33). Because these cells do not contain dopamine-beta-hydroxylase (DBH), they are probably not noradrenergic and instead may be a unique dopaminergic pathway influencing social behaviours in prairie voles. Not only is there a species difference in TH-immunoreactive (TH-ir) cells in the extended olfactory amygdala, but also a sex difference in prairie voles. Males have hundreds of these cells in both the MeApd and pBST, while females have relatively few TH-ir cells in these sites and most of these cells are weakly immunoreactive. This sex difference is almost completely generated by sex differences in circulating gonadal hormones during adulthood, because we found that castrating adult male prairie voles reduced the number of TH-ir cells in both sites to the low levels found in unmanipulated females, while giving ovariectomized female prairie voles a subcutaneous implant of testosterone raised the number of TH-ir cells in the MeApd and pBST almost to the level found in gonadally intact males (33). It is well understood that testosterone can affect neural function by acting as either an androgen or an oestrogen, depending on the route of its intracellular metabolism and a cell's sensitivity to these metabolites conferred through expression of androgen and oestrogen receptors. Similar to other rodents, the prairie vole MeA and BST express oestrogen receptors and androgen receptors (34,35). Understanding how changes in circulating gonadal hormones in adult prairie voles could affect the number and function of TH-synthesizing cells of the MeApd and pBST, and the social behaviours relying on these cells in prairie voles, requires a better understanding of the sensitivity of these cells to androgens and oestrogens. To accomplish this, we examined immunoreactivity for androgen receptors (ARs) and oestrogen receptor alpha (ERα) within the TH-ir cells of the adult male and female prairie vole extended olfactory amygdala. We then examined whether the ability of exogenous testosterone (T) to maintain the number of TH-ir cells in castrated male prairie voles, and greatly increase the number of these cells in female prairie voles, could be reproduced by either the non-aromatizable androgen, dihydrotestosterone (DHT), or by oestradiol benzoate (EB).