Region-specific onset of handling-induced changes in corticotropin-releasing factor and glucocorticoid receptor expression

Abstract

Early-life experience including maternal care profoundly influences hormonal stress responses during adulthood. Daily handling on postnatal day (P) 2-9, eliciting augmented maternal care upon returning pups to their cage, permanently modifies the expression of the stress neuromodulators corticotropin-releasing factor (CRF) and glucocorticoid receptor (GR). We have previously demonstrated reduced hypothalamic CRF expression already at the end of the handling period, followed by enhanced hippocampal GR mRNA levels (by P45). However, the initial site(s) and time of onset of these enduring changes have remained unclear. Therefore, we used semiquantitative in situ hybridization to delineate the spatiotemporal evolution of CRF and GR expression throughout stress-regulatory brain regions in handled (compared with undisturbed) pups. Enhanced CRF mRNA expression was apparent in the amygdaloid central nucleus (ACe) of handled pups already by P6. By P9, the augmented CRF mRNA levels persisted in ACe, accompanied by increased peptide expression in the bed nucleus of the stria terminalis and reduced expression in the paraventricular nucleus. The earliest change in GR consisted of reduced expression in the ACe of handled pups on P9, a time point when hippocampal GR expression was not yet affected. Thus, altered gene expression in ACe, bed nucleus of the stria terminalis as well as paraventricular nucleus may contribute to the molecular cascade by which handling (and increased maternal care) influences the stress response long term.

FIG. 1

CRF mRNA expression is altered already in rats handled from P2 to P5 and killed on P6, whereas GR expression is not affected. Semiquantitative analyses of CRF (A and B) and GR (C and D) in situ hybridization signals in the ACe (A and C) and the PVN (B and D) were conducted. At this age, CRF mRNA expression was increased in ACe of handled rats (A); CRF mRNA levels in PVN and GR mRNA levels in both regions did not differ between handled and undisturbed rats. Values are expressed as percent of undisturbed values (means ± sem); *, P < 0.05. n = 8–12 animals per group.

FIG. 2

CRF mRNA is differentially regulated in stress-responsive brain regions in 9-d-old (P9) rats that have experienced handling from P2 to P8. Photomicrographs of coronal brain sections—from undisturbed (Undist.) and handled rats—after in situ hybridization show typical CRF mRNA signals. Quantification of each region is located on the right. A, The ACe; B, anteriodorsal BnST (AD-BnST), at the level shown in the diagram. In both of these regions, handling led to increased CRF mRNA expression. C, The PVN, where handling led to reduced CRF expression. BLA, Basolateral nucleus of the amygdala; Piri, piriform cortex. ODs were averaged from four sections per brain-region per animal (n = 8–12 animals per group) and are expressed as percent of undisturbed values (means ± sem); *, P < 0.05.

FIG. 3

GR mRNA expression is modified in rats that have been handled from P2 to P8 and killed on P9 in a region-specific manner. Comparative bright-field photomicrographs from undisturbed and handled rats indicate unaltered GR expression levels in hippocampal CA1 region (A) and PVN (C) and significant down-regulation in ACe (B) of handled rats. BLA, Basolateral nucleus of the amygdala; DG, dentate gyrus. Quantitative analysis is shown on the right, and values are expressed as percent of undisturbed values (means ± sem); *, P < 0.05. n = 8–12 animals per group.

FIG. 4

Plasma CORT levels in 9-d-old handled rats are elevated after pups are returned to their cage. Baseline plasma CORT levels in the handled and undisturbed groups were not significantly different on P6 (A) or on P9 (C). B and D, Plasma CORT levels were measured at the end of the handling paradigm (0 min) and in rats killed 15 min or 30 min after the pups’ return to the dam. B, Transient elevation of plasma CORT was not found in rats handled from P2 to P6 and evaluated on P6. D, Plasma CORT levels in P9 pups killed 15 min after their return to the dam were increased and this elevation was transient. Values are means ± sem; *, P < 0.05. n = 6 animals per group.