Altered regulation of gene and protein expression of hypothalamic-pituitary-adrenal axis components in an immature rat model of chronic stress

Abstract

Chronic stress early in postnatal life influences hormonal and behavioural responses to stress persistently, but the mechanisms and molecular cascades that are involved in this process have not been clarified. To approach these issues, a chronic stress paradigm for the neonatal rat, using limited bedding material to alter the cage environment, was devised. In 9-day-old rats subjected to this chronic stress for 1 week, significant and striking changes in the expression and release patterns of key molecules that govern the neuroendocrine stress responses were observed. The presence of sustained stress was evident from enhanced activation of peripheral elements of the neuroendocrine stress response, i.e. increased basal plasma corticosterone concentrations, high adrenal weight and decreased body weight. Central regulatory elements of the neuroendocrine stress response were perturbed, including reduced expression of hypothalamic corticotropin-releasing hormone that, surprisingly, was accompanied by reduced glucocorticoid receptor expression. Thus, the effects of chronic sustained stress in the neonatal rat on the hypothalamic-pituitary-adrenal axis included substantial changes in the expression and activity of major regulators of this axis. Importantly, the changes induced by this chronic stress differed substantially from those related to acute or recurrent stress, providing a novel model for studying the long-term effects of chronic, early life stress on neuroendocrine functions throughout life.

FIG. 1

Indicators of the induction of chronic stress by experimental manipulation of the rearing environment of neonatal rats. (A) Basal plasma corticosterone (CORT) concentrations were significantly increased in the group reared with limited cage-bedding (U-NB, n = 11 animals per group) compared to the ‘normally’ reared undisturbed controls (U; n = 12). (B) A more specific marker of chronic stress, increased adrenal weight, was found in the experimental group (n = 12 per group). (C) Chronic stress was also evident from modest, but significant, reduction in body weight in the experimental group (n = 48 per group). (D) Adrenal weight expressed per 100 g body weight better reflects the significant hyperplasia of adrenal tissue associated with the chronic-stress in the U-NB group (P < 0.01). All parameters were measured on postnatal day 9, at the termination of the experiment. Values are expressed as means ± SEM. *P < 0.05.

FIG. 2

(A) Pituitary corticotropin releasing hormone (CRH) receptor binding capacity was decreased by 75.63% in chronically stressed (U-NB) immature rats. Binding capacity was determined in pituitary homogenates as described in the Methods section, using pooled (4–8 per tube) pituitaries from 32 rats per group, and includes both receptor types. (B) mRNA concentration of the CRF₂ receptor in the hypothalamic ventromedial nucleus (VMH), shown to be influenced by some subacute stressors in the immature rat (10, 16), did not differ between chronically stressed (U-NB) and undisturbed rats (U; n = 6 per group). (C) mRNA expression of the other member of the CRH receptor family, CRF₁, was reduced in discrete hippocampal fields [CA1 and dentate gyrus (DG)] of the experimental (U-NB) pups, compared to the controls (n = 10 per group). No change was evident in CRF₁ mRNA expression in the CA3. Values are expressed as means ± SEM. *P < 0.05.

FIG. 3

Corticotropin releasing hormone (CRH) mRNA expression in paraventricular nucleus (PVN) of 9-day-old rats that had experienced chronic stress during postnatal days 2–9 (U-NB) was reduced compared to that of the undisturbed (U) group (n = 12 animals per group). (A) Semi-quantitative analysis of optical density signal over PVN was performed after in situ hybridization of matched coronal sections using unbiased techniques, as described in the Methods section. Values are expressed as means ± SEM. *P < 0.05. (B) Decreased CRH mRNA signal over PVN is evident in a photomicrograph derived from a chronically stressed rat, compared with a matched section from a control.

FIG. 4

Glucocorticoid receptor (GR) gene expression in paraventricular nucleus (PVN) and frontal cortex (FC), but not in hippocampal CA1, were reduced by early life chronic stress (n = 12 per group). (A) Semi-quantitative analysis was performed on sections subjected to in situ hybridization for GR mRNA. Significantly (*) lower GR mRNA signal is evident over PVN and FC of the experimental group (U-NB) compared with the undisturbed controls (U). Values are expressed as means ± SEM. (B) Representative sections of the PVN illustrate the quantitative data shown in (A).

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 acutely by combined physical/psychological stress (4, 6, 7, 10, 11, 40). Increased corticotropin releasing hormone (CRH) release and compensatory enhancement of CRH expression result in increased CRH-induced adrenocorticotropic hormone (ACTH) and corticosterone (CORT) production. The elevated plasma CORT levels result in downregulation of glucocorticoid receptor (GR) mRNA in hippocampus and paraventricular nucleus (PVN). The panel on the right demonstrates the achievement of a new chronic stress ‘steady-state’. In this state, ACTH and CORT production and release is likely driven not only by the depleted hypothalamic CRH (the functions of which may be further minimized by reduced pituitary CRH receptors), but potentially also by vasopressin (AVP) or other secretagogues. The augmented drive of the adrenals leads to their hypertrophy, and to chronically high plasma glucocorticoid levels. These downregulate GR mRNA expression in frontal cortex (not shown) and PVN, and contribute to the depression of hypothalamic CRH expression. Filled arrows denote a positive, facilitatory action whereas open arrows denote negative influences.