Hypophysiotropic CRF neurons display a sustained immediate-early gene response to chronic stress but not to adrenalectomy

Abstract

Immediate-early genes (IEGs) are now widely used to identify neurons acutely activated by extracellular stimuli. Though challenge-induced IEG expression is typically transient, examples of sustained elevation have been reported, including in hypothalamic magnocellular neurosecretory neurons of osmotically challenged rats. Another chronic stimulus, adrenalectomy (ADX), targets parvocellular neurosecretory neurons in the paraventricular nucleus of the hypothalamus (PVH), resulting in a persistent elevation in the synthesis and secretion of corticotropin-releasing factor (CRF). In the present study we used hybridization histochemical methods to compare the effects of ADX on the induction over time of two independent IEG markers, c-fos and NGFI-B, in hypophysiotropic CRF neurons. The induction of both c-fos and NGFI-B mRNA was greatest 3 to 6 after ADX, diminished by 12 h, and no longer detectable at 24, 48, 72 h, or 6 days after surgery. Immunohistochemical analyses using a Fos-specific N-terminally directed antiserum also revealed Fos immunoreactivity (-ir) in the PVH at early time points (3 h), but not later than 12 h after ADX. Combined immuno- and hybridization histochemistry on tissue from 3 h ADX rats localized Fos-ir and NGFI-B mRNA to parvocellular CRF-expressing neurons, the majority of which expressed both Fos and NGFI-B. These early IEG responses, however, were not paralleled by increases in CRF mRNA, which was not significantly elevated until 48 h after ADX. In the chronically ADX rat, CRF neurons are capable of c-fos expression since animals subjected to an acute injection of hypertonic saline 5 days after ADX displayed a robust induction of Fos-ir in the parvocellular PVH. Furthermore, a chronic challenge, insulin-induced hypoglycemia, provoked comparable c-fos mRNA and protein expression in CRF neurons in the PVH of ADX and sham-operated rats, which was observed both acutely (2 h) and chronically (5 days) after the onset of hypoglycemia. The maintenance of Fos expression in parvocellular CRF neurons following chronic hypoglycemia, but not ADX, suggests an involvement of distinct signaling pathways in the maintenance of hypophysiotropic neuron responses to chronic steroid withdrawal and stress.