Quantitative immunocytochemistry of corticotropin-releasing factor (CRF). Studies on nonbiological models and on hypothalamic tissues of rats after hypophysectomy, adrenalectomy and dexamethasone treatment

Abstract

Indirect immunofluorescence cytochemistry of ovine corticotropin releasing factor (oCRF) was performed by use of an antiserum raised against a conjugate of oCRF and bovine thyroglobulin. The staining intensity was quantitated by use of an automated microfluorimeter. In cryostat sections of formaldehyde fixed oCRF containing gelatine models, the staining intensity was dependent on the concentration of oCRF (1–100 μM) added to the gel. Immunoinhibition experiments showed that oCRF induced identical concentration-dependent (0.001–1 μM) quenching of the immunostaining of oCRF containing models and rat median eminence (ME) preparations. Comparison of immunoinhibition of oCRF and ME extracts indicates that approximately 1.5 ng of CRF immunoreactivity (CRF i) is present in the ME of intact adult male Wistar rats. In the hypothalamus of rats, the majority of CRF i nerve fibers are localized in the external zone of the median eminence, whereas a large population of CRF i cell bodies is present in the paraventricular nucleus. Manipulations of the pituitary-adrenal system result in changes in the distribution of CRF i in these neurons. One week after extirpation of the adrenals or of the pituitary gland, the CRF i in the ME was reduced to 32 ± 3%and48 ± 6% respectively. This decrease in CRF i in the median eminence can be largely prevented by treatment of rats with dexamethasone in doses that effectively reduce plasma ACTH and corticosterone levels. In contrast to intact rats, CRF i cell bodies can be visualized in the paraventricular nucleus of non-colchicine-treated rats after adrenalectomy or hypophysectomy. These data support the view that the hypothalamic CRF i neurons play a central role in the control of pituitary-adrenal activity. We postulate that quantitative evaluation of the immunocytochemical staining represents a tool to study changes in activity of CRF i neurons.