Bruce S. Cushing

Mechanisms underlying epigenetic effects of early social experience: the role of neuropeptides and steroids.

In mammals the neonatal period is a time of significant social interaction. This is true even in solitary species as females spend a significant amount of time nursing and caring for their offspring. In social species interactions may also include the father, older siblings and extended family members. This period is a time of significant development, including organization of the central nervous system, and therefore a time when the degree and type of social interaction influences the development and expression of social behavior in adulthood. The purpose of this review is to examine the possible mechanisms for the epigenetic effects of early social experience on the subsequent expression of social behavior. We propose that social interactions during the neonatal period organize the subsequent expression of behavior by altering sensitivity to neuropeptides and steroids. Both neuropeptides (e.g. oxytocin and arginine vasopressin) and steroids (e.g. estrogen) regulate or influence the expression of behaviors such as affiliation, aggression, sociosexual behavior, parental behavior, and responses to stress. Therefore, changes in sensitivity to these hormones via reorganization of receptors or changes in hormone production and secretion are potentially powerful mechanisms through which early social experience can mold subsequent social behaviors.

NEONATAL MANIPULATIONS OF OXYTOCIN ALTER EXPRESSION OF OXYTOCIN AND VASOPRESSIN IMMUNOREACTIVE CELLS IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS IN A GENDER-SPECIFIC MANNER

Early postnatal manipulations of oxytocin have long-term behavioral and physiological consequences; the present study examined the hypothesis that oxytocin or its absence influences the subsequent expression of either oxytocin or arginine vasopressin in the CNS. On postnatal day 1 female and male prairie voles (Microtus ochrogaster) received a single i.p. injection of oxytocin (3 microg), oxytocin antagonist (0.3 microg), or 50 microl of isotonic saline or were only handled. On postnatal days 1, 8 and 21, brains were fixed, sectioned and stained for oxytocin or vasopressin immunoreactivity and analyzed as a function of age, treatment and sex. Both oxytocin and vasopressin immunoreactivity were observed on day 1 in the supraoptic and paraventricular nuclei (PVN) of the hypothalamus. Numbers of oxytocin and vasopressin neurons increased with age in both nuclei. Females treated on postnatal day 1 with oxytocin or oxytocin antagonist displayed a significant increase in oxytocin immunoreactivity on day 21 in the PVN. In contrast, males treated with antagonist tended to have decreased vasopressin immunoreactivity in the same region. These results revealed that the effects of neonatal manipulation of oxytocin are age-dependent, site-specific and sexually dimorphic. The long-lasting effects of neonatal exposure to exogenous oxytocin and oxytocin antagonist indicate a role for oxytocin in the development of the CNS during the neonatal period, affecting the development of the oxytocinergic system in females and the vasopressinergic system in males. The developmental effects observed suggest one possible mechanism by which neonatal exposure to oxytocin or neonatal inhibition of endogenous oxytocin produces long-lasting behavioral and physiological alterations and could play a role in the development of male- and female-typical behavior.

Developmental effects of oxytocin on stress response: single versus repeated exposure.

Both exogenous and endogenous oxytocin (OT) are associated with an attenuated stress response. Increased levels of OT in the early postnatal period have been shown to affect behavior and physiology in rodents, and these effects last into adulthood, suggesting an organizational role for OT during development. We investigated the effects of neonatal exposure to OT on the development of the stress response in male and female prairie voles (Microtus ochrogaster). OT or an OT antagonist (OTA) was administered either on postnatal day 1 (single, D1) or days 1-7 (repeated, D1-7) and then on day 8 the response to social isolation was assessed by quantifying ultrasonic vocalizations (USVs) and measuring plasma corticosterone (CORT). Treatment effects were observed only in females. A single treatment with OTA was associated with a decrease in vocalizations, while repeated treatment produced an increase in vocalizations. A single treatment with either saline or OTA increased basal CORT. The results suggest that endogenous OT may be involved in the development of the stress response in females.

Estrogen receptor‐α distribution in male rodents is associated with social organization

It has been hypothesized that site‐specific reduction of estrogen receptor‐α (ERα) is associated with the expression of male prosocial behaviors. Specifically, highly social males are predicted to express significantly lower levels of ERα than females and less social males in brain regions associated with prosocial behavior including the bed nucleus of the stria terminalis (BST) and the medial amygdala (MeA). This hypothesis was tested by comparing ERα immunoreactivity (IR) in three species of microtines, the polygynous montane (Microtus montanus) and meadow (M. pennsylvanicus) voles and the monogamous pine vole (M. pinetorum), and two species of cricetines that differ in the extent of social pair‐bond formation, Siberian (Phodopus sungorus) and Djungarian (P. campbelli) hamsters. As predicted, ERα‐IR was sexually dimorphic in the BST and MeA of the highly social species, with females expressing more ERα‐IR cells than males. Male and female montane voles did not differ. Male and female meadow voles differed in the ventromedial hypothalamus, with females expressing more ERα‐IR cells. Male pine voles expressed lower levels of ERα‐IR in the MeA than male montane and meadow voles and in the BST relative to montane males. Male Djungarian hamsters, which show higher levels of parental care, had fewer ERα‐IR cells in the BST than male Siberian hamsters. Results indicate that the distribution of ERα differs relative to the continuum of species‐typical affiliative behavior and supports the hypothesis that ERα has a significant role in regulating species‐specific social organization. J. Comp. Neurol. 494:595–605, 2006.

Neonatal manipulation of oxytocin affects expression of estrogen receptor alpha

In adult females many of the effects of the neuropeptide oxytocin are steroid, and especially estrogen dependent. Here we demonstrate for the first time that neonatal manipulation of oxytocin can affect the expression of estrogen receptor alpha. On the first day of postnatal life male and female prairie voles (Microtus ochrogaster) were randomly assigned to receive one of four treatments; (a) 50 microl i.p. injection of 3 microg oxytocin (approximately 1 microg/g), (b) 0.3 microg of an oxytocin antagonist (approximately 0.1 microg/g), or (c) isotonic saline. A fourth group was handled, but not injected. On postnatal day 8 or 21, brain tissue was collected, fixed and sectioned. Free-floating sections were stained for estrogen receptor alpha using immunocytochemistry, and estrogen receptor alpha immunoreactive neurons were compared by age, treatment, and sex. To compare the temporal expression of estrogen receptor alpha an additional set of brains was collected from untreated males and females on the day of birth. The effects of oxytocin manipulations were age dependent, sexually dimorphic, and site-specific. While there were no significant treatment effects on postnatal day 8, by postnatal day 21 females that received oxytocin showed a significant increase in the number of cells expressing estrogen receptor alpha-immunoreactivity in the ventromedial nucleus of the hypothalamus. Treatment with oxytocin antagonist resulted in a significant decrease in estrogen receptor alpha-immunoreactivity in the medial preoptic area in postnatal day 21 females. While there were no significant effects in males, males treated with oxytocin antagonist trended toward a reduction in estrogen receptor alpha-immunoreactivity in the medial amygdala. The results indicate that oxytocin can have organizational effects on the expression of estrogen receptor alpha, that these effects are sexually dimorphic, and finally that during the preweaning period the development of estrogen receptor alpha is sexually dimorphic.

Cohabitation induced Fos immunoreactivity in the monogamous prairie vole.

Cohabitation of sexually nai;ve male and female prairie voles (Microtus ochrogaster) triggers a cascade of physiological changes that result in the formation of stable pair bonds. In the present study we used the expression of c-Fos protein to identify CNS regions activated during initial social contact in heterosexual, male/male and female/female pairs. Sexually naive males and females were randomly assigned to one of five groups: control- no cohabitation, or cohabitation for 1 h with an unrelated opposite sex, an unrelated same sex, an unfamiliar same sex sibling, or removal for 24 h and then repaired with the familiar sibling. Heterosexual pairing resulted in significant increases in c-Fos immunoreactivity (IR) in the posterodorsal and posteroventral medial amygdala (MeA), bed nucleus of the stria terminalis, medial preoptic nucleus, ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMN-VL) in males and females, and the periventricular nucleus of the thalamus in males only. c-Fos IR during intrasexual cohabitation varied with the relationship of the experimental animal to the stimulus animal. Males cohabited with an unfamiliar unrelated male expressed significantly more c-Fos IR in the central amygdala (CeA). While females cohabited with an unfamiliar female (related or unrelated) also displayed increased c-Fos IR in the CeA, this increase was accompanied by an increase in c-Fos IR in the VMN-VL and MeA. The results from this study suggest that early neuronal activation associated with heterosexual cohabitation is similar in both sexes, while neuronal activation is sexually dimorphic in response to intrasexual cohabitation.

Central expression of c-Fos in neonatal male and female prairie voles in response to treatment with oxytocin

Early postnatal exposure to both exogenous and endogenous oxytocin (OT) can have long-term effects on behavior and physiology, although the mechanisms of these effects are not known. c-Fos expression was used to investigate the immediate neural effects of neonatal manipulations of OT in male and female prairie voles. On the day of birth prairie vole pups received an intraperitoneal injection of OT, a selective OT antagonist (OTA), or saline (vehicle control), while an additional group was handled but not injected. One hour after treatment brains were collected and fixed via spinning immersion and immunocytochemistry was then used to label for c-Fos immunoreactivity (IR). There were significant differences between males and females. Handled only females displayed significantly higher levels of c-Fos IR in the mediodorsal thalamic nucleus (MD) than males while handled males had higher c-Fos IR in the paraventricular nucleus of the hypothalamus than females. Exogenous OT stimulated neuronal activity in the supraoptic nucleus (SON) in males, while treatment with OTA increased Fos IR in the SON and was associated with reduced Fos IR in the MD in females. The results indicate that neuronal activity and responses to OT are sexually dimorphic in newborn prairie voles. In females changes in Fos expression were stimulated by treatment with OTA, suggesting that endogenous OT affects cellular activity while males responded to exogenous OT.

Developmental effects of oxytocin on neural activation and neuropeptide release in response to social stimuli.

Previous studies have revealed that the neuropeptide hormone oxytocin (OT) has developmental effects on subsequent social behavior and on mechanisms underlying social behavior such as OT neurons and estrogen receptor alpha. This suggests that OT might also have developmental effects on neural responses to social stimuli. This was tested in socially monogamous prairie voles (Microtus ochrogaster) by manipulating OT on the first day of life and then assessing the response to a heterosexual pairing in adulthood. The response to cohabitation was assessed by quantifying neural activation in regions of the brain associated with sociosexual behavior and anxiety using c-Fos immunoreactivity. Additionally, immunocytochemistry was used to label OT and vasopressin neurons and plasma was assayed for both neuropeptides. Treatment effects were evident in females, but not in males. Blockade of OT receptors with an OT antagonist on the first day of life resulted in neural activation of the central amygdala in response to a pairing with a novel male in adulthood. The central amygdala does not normally express c-Fos after a heterosexual pairing in reproductively naïve prairie voles. Treatment effects also were observed in vasopressin immunoreactivity in the SON with OT-treated females showing a decrease.

Intraspecific variation in estrogen receptor alpha and the expression of male sociosexual behavior in two populations of prairie voles.

Estrogen (E) regulates a variety of male sociosexual behaviors. We hypothesize that there is a relationship between the distribution of estrogen receptor alpha (ERalpha) and the degree of male social behavior. To test this hypothesis, ERalpha immunoreactivity (IR) was compared in prairie voles (Microtus ochrogaster) from Illinois (IL), which are highly social, and Kansas (KN), which are less social. The expression of androgen receptors (AR) in males also was compared between populations. The expression of ERalpha and AR were compared in brains from KN and IL males and females using immunocytochemistry (ICC). There were significant intrapopulational differences, with males expressing less ERalpha-IR than females in the medial preoptic area, ventromedial nucleus, ventrolateral portion of the hypothalamus, and bed nucleus of the stria terminalis (BST). IL males also displayed less ERalpha-IR in the medial amygdala (MeA) than IL females. While IL males expressed significantly less ERalpha-IR in the BST and MeA than KN males, there was no difference in AR-IR. Differences in the pattern of ERalpha-IR between KN and IL males were behaviorally relevant, as low levels of testosterone (T) were more effective in restoring sexual activity in castrated KN males than IL males. The lack of difference in AR combined with lower expression of ERalpha-IR in IL males suggests that behavioral differences in response to T are associated with aromatization of T to E and that reduced sensitivity to E may facilitate prosocial behavior in males.

Social factors regulate female–female aggression and affiliation in prairie voles

Although patterns of aggression and affiliation may play a major role in social organization, the mechanisms underlying these behaviors are not well understood. The purpose of this study was to examine the effects of social and hormonal experience on female-female aggression and affiliation in prairie voles, Microtus ochrogaster. Prairie voles exhibit the traits of social monogamy and tend to live in communal families structured around a male-female pair. It is rare for two unrelated females within a family to successfully reproduce. In this study, the social and/or hormonal experiences of female prairie voles were varied and female-female aggression and affiliation were measured during dyadic encounters with unfamiliar, nonaggressive females. An increase in aggression and decline in affiliative behaviors toward a stimulus female was observed during pregnancy and following male-cohabitation, with or without mating. Pairing with another female did not result in changes in either aggressive or affiliative behaviors toward the stimulus female. Female-female aggression increased and affiliative behaviors declined, with a maximal effect following approximately 8-12 days of male cohabitation. Similar patterns of change were seen in both ovariectomized and gonadally intact females, and treatment with estradiol and subsequent sexual experience did not significantly alter the tendency of females to show aggression or affiliative contact. Social experiences associated with prolonged cohabitation with a male facilitate the induction of female-female aggression; however, ovarian hormones, pregnancy or mating are not essential to induce aggression.