The current study demonstrates that altering cage environment via limitation of bedding material leads to significant chronic stress in the immature rat, associated with changes in gene expression at multiple levels of the HPA axis. CRH mRNA expression in PVN was reduced, yet decreased pituitary CRH binding capacity was consistent with enhanced hypothalamic CRH release during this chronic stress. The mechanisms accounting for the reduction of hypothalamic CRH mRNA expression may involve altered glucocorticoid negative feedback, as suggested by the reduced GR mRNA expression in PVN and frontal cortex of chronically stressed pups. Alternatively, or in concert, increased CRH release and a failure of acute stress-induced facilitation of CRH production may contribute to reduced hypothalamic CRH mRNA levels. Taken together, these findings indicate that chronic stress early in life leads to a profound and distinctive alterations of the molecular underpinnings of the neuroendocrine stress response.
This study demonstrated that experimental manipulation of the rearing environment led to changes in the pups that are typical for chronic stress: increased basal CORT secretion (30
), increased adrenal weight and decreased body weight (30
). This chronic stress was provoked by placing pups and dams in cages with limited bedding material. Bedding type and volume are important components of the dams’ nesting environment, and limiting the amount of available bedding constituted a continuous stressor for the dam and pups, as well as altered dam–pup interaction (9
). Intriguingly, the effects of this significant chronic stress on the molecules governing the central pathways of the stress response: hypothalamic CRH expression, expression and binding of CRH receptors, and glucocorticoid receptor expression in hypothalamus, hippocampus and frontal cortex, were distinguished from those observed in chronic stress in the adult, indicating that the stage of development of the limbic-hypothalamic-pituitary-adrenal axis played a critical role in the processes triggered by this chronic stress.
The neuroendocrine stress response of the immature rat, specifically on days 4–14 of life, is characterized by attenuated hormonal responses and altered gene regulation in response to stress as compared to the adult situation (33
). This stress hypo-responsiveness during development appears to be stressor-specific, since the HPA axis is fully capable of responding to stimuli that may be considered stressful to a neonatal rat (e.g. cold or saline injection) (4
). In addition to stress-selectivity, the neuroendocrine stress response during this period is highly regulated by maternal input, involving both feeding and sensory signals (5
The contribution of developmental stage and maternal regulation to the molecular and hormonal responses to acute stress in the 4–14-day-old rat are currently being elucidated. Thus, maternal deprivation for 24 h enables a higher adrenocorticotropic hormone (ACTH) (38
) and CORT (7
) response to acute stress. Importantly, the hypothalamic mechanisms effecting stress-induced ACTH release appear to be under maternal regulation. While basal CRH mRNA levels were found to be either unchanged (7
) or decreased (8
), we (40
) and others (5
) have shown rapid stress-induced transcription of the CRH gene throughout the ‘stress-hyporesponsive’ period in naive rats. Interestingly, at early ages (e.g. P6), no changes in CRH mRNA were found at 4 h after stress (4
), although an increase was reported at the 15-min time-point (5
). In contrast to the response to acute stress under ‘normal’ maternal–pup interaction, maternal deprivation led to augmented stress-induced synthesis of vasopressin in the 12-day-old pup, which may have contributed to the larger ACTH secretion (38
The molecular basis of the maternally dependent stress-induced transcription of hypothalamic CRH gene may involve glucocorticoid receptor activation in hypothalamus, frontal cortex and hippocampus. Indeed, reduced GR mRNA in PVN, hippocampal CA1 and frontal cortex have been reported after maternal deprivation, with likely consequences on the magnitude of negative-feedback modulation of CRH expression (20
). Thus, elements of maternal input appear to be critical to both the molecular and hormonal neuroendocrine responses of the immature rat to acute stress.
Whereas the unique, age-dependent molecular mechanisms of the response of the immature rat to acute stress have been emerging, relatively little insight has been gained into mechanisms of the molecular and hormonal response to chronic stress. It is logical to consider that these mechanisms would include both processes that govern the mature HPA axis, as well as age-specific (and potentially maternal dependent) molecular cascades. In the adult rat, chronic stress has been shown to affect CRH mRNA expression in a complex manner, and increased (41
), decreased (31
) or unchanged (45
) levels have been found, depending on the stress paradigm. In the context of the current study, chronic or intermittent stress in mature rats was found to result in relative desensitization of the CRH-neuroendocrine system, and increase of the role of vasopressin, synthesized in the parvocellular PVN, as an activator of the HPA axis (46
). Furthermore, vasopressin-induced CORT secretion has been shown to elicit downregulation of CRH mRNA expression in PVN (as found in the current study in the immature rat). In the adult, however, this enhanced role of vasopressin required at least 2 weeks of chronic or intermittent stress (31
). In this respect, the recently described rapid enhancement of vasopressin expression in immature rat PVN upon acute stress in the setting of maternal separation is intriguing (38
). These data indicate that the onset of modulation of vasopressin expression (and consequent contribution of this hormone to the stress response) may be significantly faster during the second week of postnatal life (49
Indeed, the remarkably rapid reduction of CRH mRNA levels in PVN of the chronically stressed pups in the current study, evident already by P9 (i.e. after 1 week of the modified rearing conditions), is consistent with a more rapid time-course of the adaption to chronic stress, which may be specific to this age (49
). Several processes may be involved in this suppression of CRH gene expression. First, the reduced CRH mRNA levels occurred in the face of chronically elevated glucocorticoid levels, as has been found also by area-under-the curve analysis in our previous study (9
). These findings are consistent with a negative feedback evoked by the stress-associated chronically elevated plasma glucocorticoid concentrations (39
). Glucocorticoids have been shown to depress hypothalamic CRH mRNA expression in adult PVN (26
), but earlier studies suggested a relative lack of glucocorticoid negative feedback on PVN-CRH expression in rats younger than 9 days (14
). However, more recent studies support the presence of a robust glucocorticoid-induced negative feedback in the 9-day-old rat, particularly when glucocorticoid levels are elevated persistently (6
). Thus, a reasonable explanation for the reduced hypothalamic CRH mRNA levels may derive from a glucocorticoid negative feedback.
It should be noted that the decreased GR mRNA expression in PVN of chronically stressed rats in the current study (), might predict reduced transduction of the glucocorticoid signal onto CRH-producing PVN neurones. However, GR mRNA levels were analysed at the termination of the chronic stress, and their reduction may therefore be a consequence of the chronically elevated glucocorticoid plasma levels, rather than a reflection of the acute, early events induced by the application of the chronic stress paradigm in the immature pup brain. In addition, the net effects of these region-specific changes in GR expression would further influence the magnitude of the neuroendocrine response to future stresses. The reduced cortical GR mRNA expression [consistent with the implication of cortical-GR inhibitory signalling onto the HPA axis response to stress (29
)], would be expected to lead to enhanced HPA reactivity to subsequent stressors (23
A second possible mechanism for the reduced CRH mRNA in PVN may result from persistent increased CRH release, without compensatory enhanced synthesis, leading to depleted mRNA stores (6
). Increased CRH release is suggested from the large (76%) reduction in pituitary CRH receptor binding, consistent with receptor desensitization by enhanced secretion of hypothalamic CRH, although an alternative possibility, that absence of the ‘trophic’ effect of CRH may reduce corticotroph receptor synthesis, cannot be excluded. Chronic stressors have been found to downregulate pituitary CRH receptors in adult rat (32
), and potential mechanisms may include internalization and thus reduction of receptor binding capacity resulting directly from CRH binding, as shown after infusion of CRH intermittently for 3 days (50
) or continuously for 48–50 h (51
). Alternatively, chronic stress-related increase of plasma glucocorticoids may affect pituitary CRH receptors: chronic CORT decreased the relative density of CRH receptors in corticotrophs in vitro
, by increasing receptor internalization rate (52
). In the immature rat, pituitary CRH receptors may be even more sensitive to circulating glucocorticoids (53
). Thus, the reduced CRH receptor binding found here may derive from both increased CRH secretion and high glucocorticoid levels, both of which may also contribute to the observed reduction of hypothalamic CRH mRNA levels.
Thus, a dynamic chain of events may be provoked by the persistent stress imposed on 2-day-old rats, resulting in the spectrum of molecular changes of the HPA axis noted above. A schematic presentation of this putative sequence of events is depicted in . In this scenario, bedding limitation initially acts as an acute stress, activating the immature rat’s HPA axis in the context of abnormal maternal interaction. Acutely, CRH release (4
) and enhanced ‘compensatory’ transcription occur, leading to elevated plasma stress hormones. However, during the first few postnatal days, CRH transcription does not result in persistent elevation of CRH mRNA expression (4
). Indeed, CRH transcription and steady-state mRNA levels may be further downregulated by the chronic, GR mediated negative feedback resulting from elevated plasma steroids. By day 9, CRH mRNA levels are reduced, and the persistently elevated plasma CORT levels are driven also by other ACTH secretagogues, most likely vasopressin (38
). These chronically high CORT levels eventually downregulate GR mRNA in regions involved in HPA and hypothalamic CRH mRNA regulation (frontal cortex and the PVN itself) but, by that time, a new steady-state has been reached (vide infra).
FIG. 5 Schematic presentation of the putative changes in gene expression and hormonal release patterns during the evolution of the chronic stress state in the neonatal rat. The normal steady-state is shown on the left. The middle panel depicts changes induced (more ...)
In this new steady state, hypothalamic neuroendocrine functions are governed by altered ‘higher’ limbic structures and pathways. This is evident not only from the selective reduction of GR mRNA, noted above, but also from the remarkable reduction of the expression of the stress-mediating CRH receptor, CRF1
, in hippocampal CA1 and dentate gyrus. It might be noted that in adult studies of chronic stress, hippocampal CRF1
mRNA expression was found to increase (54
), decrease (55
) or remain unchanged (50
) depending on the stress paradigm. In the context of the current study, CRF1
binding capacity and CRF1
mRNA expression in the immature rat have been shown to be regulated by CRH itself (24
), so that reduced CRF1
mRNA expression in hippocampus here is consistent with reduced local hippocampal CRH. While the current study did not examine CRH levels in this region, other work suggests that, during the developmental age discussed in this study, hippocampal CRH expression is at least twice as high as in mature hippocampus (56
), and may be regulated by selective acute stressors (58
). Whether hippocampal CRH expression is regulated by chronic stress, such as the current paradigm, and influences CRF1
mRNA levels, requires further study.
In summary, chronic stress resulting from alteration of cage environment leads to profound alterations of the molecular effectors of the neuroendocrine stress response at multiple hierarchial levels. These include CRH mRNA expression in PVN, binding capacity and mRNA concentrations of CRH receptors, and glucocorticoid receptor (GR) mRNA expression in central nervous system regions involved in the regulation of the neuroendocrine stress response. These findings may pertain to the human situation, where early life stress has been shown to lead to persistent alterations of HPA axis function including elevated basal glucocorticoid concentrations, impaired glucocorticoid feedback, and alterations in CRH-receptor regulation, not unlike those found here. Therefore, this model may provide a useful tool for understanding the molecular mechanisms involved in the reciprocal interactions of early life chronic stress and the HPA axis, and of the long-term alterations of the neuroendocrine stress response induced by early life stress.