Tonic, but not phasic corticosterone, constrains stress activatedextracellular-regulated-kinase 1/ 2 immunoreactivity within the hypothalamic paraventricular nucleus

Abstract

The negative-feedback actions of corticosterone (CORT) depend on both phasic and tonic CORT secretion patterns to regulate hypothalamic-pituitary-adrenal (HPA) axis activity. How these two different CORT secretion pattens influence specific intracellular signal transduction pathway activity within the cellular elements of the HPA axis has not been determined. For example, it is unknown whether CORT has suppressive actions over signal transduction events within medial parvocellular paraventricular nucleus (PVN) corticotrophin-releasing hormone (CRH) neurones, nor whether these suppressive actions are responsible for alterations in PVN transcriptional processes and neurohormone secretion associated with stress. The extracellular-regulated kinase (ERK) is a stress activated intracellular signalling molecule that is potentially subject to glucocorticoid negative-feedback regulation. We tested the ability of CORT to modulate levels of the active (phosphorylated) form of ERK (pERK1/2) in the PVN of rats. Acute psychological stress (restraint) produced a rapid increase in the number of PVN pERK1/2 immunopositive cells within CRH neurones. Absence of tonic CORT via adrenalectomy (ADX) produced no change in basal pERK1/2 cell counts but augmented the increased pERK1/2 cell counts elicited by acute restraint. Treatment of ADX rats with CORT in the drinking water normalised this enhanced pERK1/2 response to stress. By contrast, treatment of ADX rats with a phasic increase in CORT 1 h before restraint had no effect on pERK1/2 cell counts, despite substantially suppressing stress-induced PVN crh gene expression and adrenonocorticotrophic hormone secretion. This tonic CORT inhibition of stress-induced activation of ERK1/2 may involve both alteration of the activity of stress-dependent neural inputs to PVN CRH neurones and alteration within those neurones of stress-dependent intracellular signalling mechanisms associated with ERK activation.

Figure 1

(A–C) Restraint significantly increased ACTH and CORT secretion, and increased PVN pERK1/2 cell counts in adrenal intact rats. Rats not challenged with restraint displayed low basal ACTH, CORT and PVN pERK1/2 cell counts. Five minutes of restraint rapidly increased ACTH, CORT and pERK1/2 cell counts. The increased ACTH and CORT hormone levels and pERK1/2 cell counts remained elevated over the course of a 30 minute restraint challenge. *, represents a difference compared to unstressed rats (time 0); p < 0.05, FLSD.

Figure 2

Colocalization of basal and stress-stimulated pERK1/2 cell counts in PVN CRH neurons. (A) Both the number of single labeled (pERK1/2 alone) and double labeled (pERK1/2 and CRH) PVN pERK1/2 immunopositive cells were increased in response to 15 minute of restraint. Approximately 50% of stress-stimulated immunofluorescence pERK1/2 was colocalized within PVN CRH neurons. *, represents a significant stress effect compared to non-stressed group (p < 0.05, FLSD). (B) Displays a representative merged immunofluorescence image of a single PVN hemisphere section from either a non-stressed (left) rat or stressed (right) rat. Single labeled pERK1/2 immunoreactive cells are labeled with red immunofluorescence (gray arrows), single labeled CRH immunoreactive cells are labeled with green immunofluorescence (blue arrow heads), and pERK1/2+CRH doubled immunoreactive cells are evident by yellow/orange immunoflourescence (pink arrow heads). Scale bar = 100 µm.

Figure 3

ADX resulted in a marked increase in both basal and stress-stimulated ACTH secretion, as well as stress-stimulated PVN pERK1/2 cell counts. (A) ACTH secretion was increased by 15 minutes restraint. Both basal and stress-stimulated ACTH secretion were substantially increased in ADX rats. (B) PVN pERK1/2 cell counts were also increased by restraint. ADX rats displayed a greater increase in PVN pERK1/2 cell counts compared to sham-ADX rats. *, represents a significant stress effect compared to the no stress group within the same surgical condition (p < 0.05, FLSD); #, represents a significant tonic CORT condition (ADX) effect compared to the sham group within same stress condition (p < 0.05, FLSD).

Figure 4

Tonic CORT replacement in drinking water of ADX rats normalized basal and stress-induced ACTH secretion, and normalized stress-induced PVN pERK1/2 cell counts. (A–B) Restraint (15 minutes) increased ACTH secretion and PVN pERK1/2 cell counts, and ADX augmented these stress-stimulated effects. Tonic CORT water replacement of ADX rats normalized basal and stress-stimulated ACTH secretion to levels comparable to those displayed by sham-ADX rats. Tonic CORT water replacement also normalized the increase in stress-stimulated PVN pERK1/2 cell counts. *, represents a significant stress effect compared to no stress group within the same tonic CORT condition (p < 0.05, FLSD); #, represents a significant tonic CORT condition effect compared to sham rats within the same stress condition (p < 0.05, FLSD); +, represents a significant CORT replacement (ADX+CORT water) effect compared to non-replaced ADX rats within the same stress condition (p < 0.05, FLSD). (C) Shows representative photomicrographs from each treatment group for pERK1/2 immunoperoxidase histochemistry in one hemisphere of the PVN (3rd ventricle on the right side of no stress condition images and on the left side of stress condition images). Scale bar = 100 µm.

Figure 5

Phasic increase in CORT 1 hour prior to restraint challenge suppressed stress-stimulated ACTH secretion and PVN crh gene expression, but not stress-stimulated PVN pERK1/2 cell counts in ADX rats. (A) Basal and restraint-stimulated ACTH secretion was suppressed 1 hour after phasic CORT treatment. (B–C) Stress-induced PVN crh gene expression was suppressed by this CORT pretreatment. (C) Shows representative autoradiograms for in situ hybridization of PVN crh hnRNA expression. Images are centered over the PVN containing ventral-medial portion of coronal brain sections. (D) Phasic CORT pretreatment had no effect on stress-stimulated PVN pERK1/2 cell counts. *, represents a significant stress effect compared to non-stressed rats within the same phasic CORT condition (p < 0.05, FLSD); +, represents a significant phasic CORT effect compared to vehicle treated rats within the same stress condition (p < 0.05, FLSD).