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Social isolation in male rats at weaning results in reduced basal levels of the neuroactive steroid 3α,5α-tetrahydroprogesterone (3α,5α-TH PROG) in the brain and plasma as well as increased anxiety-like behavior. We now show that socially isolated female rats also manifest a reduced basal cerebrocortical concentration of 3α,5α-TH PROG as well as an anxiety-like profile in the elevated plus-maze and Vogel conflict tests compared with group-housed controls. In contrast, despite the fact that they were raised under normal conditions, adult male offspring of male and female rats subjected to social isolation before mating exhibited an increased basal cerebrocortical level of 3α,5α-TH PROG but no difference in emotional reactivity compared with the offspring of group-housed parents. These animals also showed an increased basal activity of the hypothalamic-pituitary-adrenal axis as well as reduced abundance of corticotropin-releasing factor in the hypothalamus and of corticotropin-releasing factor receptor type 1 in the pituitary. Moreover, negative feedback regulation of hypothalamic-pituitary-adrenal axis activity by glucocorticoid was enhanced in association with up-regulation of glucocorticoid receptor expression in the hippocampus. There was also attenuation of corticosterone release induced by foot-shock stress in the offspring of socially isolated parents. The increase in the brain concentration of 3α,5α-TH PROG induced by acute stress was also blunted in these animals. Our results thus show that a stressful experience before mating can influence neuroendocrine signaling in the next generation.
Early life experiences are thought to have a profound impact on development and maturation of the CNS, including the induction of persistent plasticity of the neuroendocrine stress system (Levine 1967; Heim and Nemeroff 2002). Indeed, exposure to stress hormones during the perinatal period is thought to be a determinant of interindividual differences in emotionality, cognitive function, and stress responsiveness in adult animals and humans (Heim and Nemeroff 2001; Lupien et al. 2009; Veenema 2009). The developmental timing and context of stressful stimuli are important, however, in determining the adaptive or maladaptive consequences. Exposure to pre-natal stress has programming effects on the hypothalamic-pituitary-adrenal (HPA) axis and the brain as a result of the increase in maternal glucocorticoid secretion (Cottrell and Seckl 2009). Given the key role of glucocorticoids in brain maturation, elevated levels impair brain development and function and can render individuals more vulnerable to stress (Seckl 2008).
In spite of the many studies that have examined the effects of perinatal stress on the adult brain and emotionality, few have addressed the effects of stress experienced by parents in early life on the emotionality and stress responsiveness of offspring. Human studies have described intergeneration transmission of behavior (Harper 2005; Kim et al. 2009), and animal studies have shown transgenerational effects of environmental experiences (Anway et al. 2005; Champagne 2008; Curley et al. 2008). This type of inheritance is thought to represent a means through which adaptive changes in one generation can be transmitted across generations as an essential and dynamic component of evolution (Badyaev and Uller 2009). Recent studies have shown that stress vulnerability can influence behavior, social abilities, and serotonergic function in offspring across two generations, suggesting the possibility that exposure to environmental stress may produce epigenetic alterations (via DNA methylation) in germ cells that might then affect the phenotype of offspring (Franklin et al. 2010, 2011).
Social isolation is a model of prolonged mild stress and is associated with marked behavioral alterations—such as increased locomotor activity, anxiety, depression, and aggression in laboratory animals (Fone and Porkess 2008). In male rats, social isolation results in a decrease in the brain and plasma concentrations of neuroactive steroids that act as positive modulators at GABAA receptors (Majewska et al. 1986) and is accompanied by an enhanced response to the acute administration of ethanol as well as by enhanced neurosteroidogenesis in response to acute stressful stimuli (Serra et al. 2000, 2003). It also increases the sensitivity of the pituitary to corticotropin-releasing factor (CRF) and impairs negative feedback regulation of the HPA axis (Serra et al. 2005).
We have now investigated whether environmental influences in early life affect the stress phenotype of the next generation, in particular whether there is a transgenerational transmission of functional changes induced by chronic stress that alters the basal activity and reactivity of the HPA axis. Our previous studies on the effects of social isolation on emotional and neuroendocrine states were performed with male rats. We therefore first examined the effects of social isolation on these parameters in females. In the offspring of socially isolated parents, we then evaluated basal emotional state and measured the brain concentration of the neuroactive steroid 3α,5α-tetrahydroprogesterone (3α,5α-TH PROG). Basal activity of the HPA axis was evaluated by measurement of the plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone as well as the expression of CRF in the hypothalamus, the type 1 receptor for CRF (CRFR1) in the pituitary, and the glucocorticoid receptor (GR) in the hippocampus. To evaluate stress reactivity, we determined the effect of acute foot-shock stress on the plasma corticosterone level as well as the efficiency of negative feedback regulation of the HPA axis with the dexamethasone suppression test. All the experiments were performed in male offspring to compare the results with our previous studies in socially isolated males.
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We have shown that female rats isolated for 1 month immediately after weaning, without any additional stressor, manifested a significant decrease in the basal cerebrocortical concentration of 3α,5α-TH PROG compared with group-housed animals. Moreover, socially isolated females exhibited an anxiety-like profile in the elevated plus-maze and Vogel tests. These characteristics are thus similar to those of male rats subjected to social isolation (Serra et al. 2000), indicating that there is no apparent sex difference in the effects of this chronic mild stress.
We also found that adult male offspring of socially isolated parents showed an increased basal level of 3α,5α-TH PROG in the cerebral cortex compared with those of group-housed parents. However, the offspring of socially isolated rats showed no difference in emotional reactivity compared to those of group-housed rats, as evidenced by their similar conditioned (Vogel's test) and non-conditioned (elevated plus-maze test) anxiety responses as well as their similar locomotor activity. 3α,5α-TH PROG is one of the most potent and efficacious positive allosteric modulators of GABAA receptor function (Majewska 1992; Lambert et al. 1995), and its administration in animals, either systemically or intracerebroventricularly, induces marked anxiolytic, sedative-hypnotic, and anti-convulsant effects (Kokate et al. 1994; Bitran et al. 1995; Concas et al. 1996). These observations suggest that the increase in the brain concentration of 3α,5α-TH PROG in the offspring of socially isolated parents may be relevant for the basal emotional state. This conclusion is consistent with the observation that acute administration of 3α,5α-TH PROG attenuated the elevation of ACTH and corticosterone levels in response to emotional stress in rats (Patchev et al. 1996). Indeed, the HPA axis appears to be subject to tonic GABA-mediated inhibition at the hypothalamic level (Cullinan et al. 2008), and peripheral injection of positive modulators of the GABAA receptor (such as benzodiazepines) reduces HPA axis activity (Imaki et al. 1995; Grottoli et al. 2002). On the other hand, neuroactive steroids, including 3α,5α-TH PROG, induced CRF or arginine vasopressin (AVP) synthesis and release at the hypothalamic level, thereby increasing plasma ACTH and corticosterone concentrations (Naert et al. 2007). Consistent with these latter observations, the offspring of socially isolated parents in this study manifested increased circulating levels of ACTH and corticosterone, indicative of an activated HPA axis, as well as a reduced abundance of CRF in the hypothalamus, possibly indicative of increased CRF release (Naert et al. 2007), under the basal condition compared with the offspring of group-housed rats. This conclusion is consistent with the evidence that social isolation of male rats, in which cerebrocortical and plasma concentration of 3α,5α-TH PROG is reduced (Serra et al. 2000), showed decrease of plasma levels of both ACTH and corticosterone, as well as increase sensitivity of the pituitary to CRF (Serra et al. 2005). CRF interacts with CRFR1 in the pituitary (De Souza and Kuhar 1986; Aguilera 1988) to regulate ACTH secretion, and both CRF and glucocorticoids down-regulate CRFR1 mRNA levels in the rat anterior pituitary (Makino et al. 1995; Pozzoli et al. 1996; Sakai et al. 1996). The significant reduction in the expression of CRFR1 apparent in the pituitary of the offspring of socially isolated rats compared with those of group-housed rats is thus consistent with the notion that the basal activity of the HPA axis is increased in these animals. It remains to be determined whether adaptation of the AVP system may contribute to this altered activity of the HPA axis. Indeed, CRF and AVP act synergistically to stimulate ACTH release (Gillies et al. 1982), and such synergism was recently attributed to heterodimerization of CRFR1 and the V1b receptor for AVP (Murat et al. 2012). A reduced level of V1b expression may be consistent with some behavioral and endocrine features of the offspring of isolated rats, given that a V1b antagonist showed anti-depressive and anxiolytic properties in rats (Serradeil-Le Gal et al. 2005) and that V1b knockout mice exhibit a reduced level of ACTH secretion in response to stress (Lolait et al. 2007).
Abnormal CRF neurotransmission and CRFR1 signal transduction has been proposed to contribute to stress pathophysiology that leads to major depression. Individuals with major depressive and anxiety disorders thus manifest hyperactivity of the HPA axis with elevated plasma ACTH and cortisol concentrations under basal conditions and a blunted ACTH response in the CRF stimulation test (Condren et al. 2002; Gillespie and Nemeroff 2005; Abelson et al. 2007), the latter of which has been attributed to reduced CRFR1 expression in the anterior pituitary secondary to chronic hypersecretion of CRF from the hypothalamus.
On the other hand, offspring of socially isolated parents may be classified as resilient. It is generally accepted concept that resilience is the ability to avoid deleterious behavioral changes in response to chronic stress that can be achieved by molecular adaptations that promote normal behavioral function (see for review Russo et al. 2012). While, offspring of socially isolated parents were not challenged by chronic stress, we found that acute stress (foot shock) induced lower percentage increases in the brain concentration of 3α,5α-TH PROG and the plasma level of corticosterone in the offspring of socially isolated parents than in those of group-housed rats. Our observations that the offspring of socially isolated rats did not show either anxiety-like behavior (Fig. 2) or depressive-like behavior in the forced swim test (data not shown) seem consistent with this scenario. Thus, the constitutively high basal cerebrocortical level of 3α,5α-TH PROG found in these animals might normalize their emotional state and represent a protective mechanism against stress; 3α,5α-TH PROG might thus regulate HPA axis activity by increasing hormone levels in the basal condition and attenuating stress-induced axis activation, as suggested by Naert et al. (2007). Accordingly, up-regulation of 3α,5α-TH PROG biosynthesis appears to be one of the mechanism for the anxiolytic and anti-aggressive effects of anti-depressant drugs (Pinna et al. 2009) and molecules that exert anxiolytic effects by increasing the brain level of 3α,5α-TH PROG has been proposed as new anxiolytic drugs (Serra et al. 1999; Schüle et al. 2001). In addition, the attenuated response of the HPA axis to acute stress apparent in the offspring of socially isolated parents seems to be well correlated with the increased negative feedback sensitivity to glucocorticoid, as revealed by the increased suppressive effect of dexamethasone on the basal and stress-induced increase in plasma corticosterone level. This increased sensitivity in turn may be related to the observed up-regulation of GR expression in the hippocampus, which mediates the inhibitory effect of glucocorticoids on HPA axis activity (Jacobson and Sapolsky 1991; Malkoski and Dorin 1999), thus reflecting an increased capability of this structure to convert a hormonal signal into a feedback action (Reul and de Kloet 1985). As proposed, the endogenous levels of 3α,5α-TH PROG play a crucial role in the HPA function, thus, reduced 3α,5α-TH PROG brain levels following social isolation leads to hyperresponsive rats to stress and impair negative feedback regulation of the HPA axis (Serra et al. 2005). In offspring of socially isolated parents negative feedback regulation of HPA axis was enhanced in association with an increased basal concentration of 3α,5α-TH PROG. This conclusion is consistent with the evidence that the impairment of the negative feedback regulation of the HPA axis apparent in isolated rats (Serra et al. 2005) was prevented or normalized by administration of exogenous 3α,5α-TH PROG either during or following social isolation (Evans et al. 2012).
In addition, the negative feedback regulation of HPA axis activity, the response to stress, and the hippocampal GR expression observed in the offspring of isolated parents resemble those in adult rats that were exposed to a brief daily period of separation from their mother during the first few weeks of life (Levine et al. 1957; Levine 1967). These animals show reduced fearfulness, attenuated ACTH, and corticosterone responses to stress, and increased GR binding capacity in the hippocampus (Levine 1967; Meaney et al. 1989, 1992). All of these changes were suggested to be because of altered dam-pup interactions. Indeed, the quality of maternal care provided to offspring early in life is a direct determinant of developmental programming and calibration of the HPA axis (Liu et al. 1997; Francis et al. 1999). Our preliminary observations indicate that socially isolated dams did not differ in the total frequency of licking-grooming and arched-back nursing from group-housed dams during the first two post-partum weeks. More detailed studies are necessary, however, to rule out the possibility that maternal behavior of socially isolated dams is a key determinant of the stress responsiveness and HPA function in their offspring.
Both corticosteroids and stress have a pronounced epigenetic impact on the brain and behavior not only within individual life spans but across generations in both humans and animals (Hunter 2012). Stress vulnerability was recently proposed to be transmitted epigenetically by DNA methylation in the germ line, thus influencing behavior of offspring in the next generation (Franklin et al. 2010). In contrast, epigenetic mechanisms were found to play a limited role in the behavioral adaptations of offspring of fathers subjected to chronic social defeat (Dietz et al. 2011). We have shown that environmental perturbation can lead not only to alterations in individuals who experience such change (Serra et al. 2000, 2003, 2005, 2006; Pisu et al. 2011) but also to altered neuroendocrine responses of future generations who were not directly exposed to the challenge (present study). Further studies should provide insight into the role of epigenetic modification of the maternal or paternal germ line in this transgenerational effect.