REMOVAL OF ADRENAL-STEROIDS FROM THE MEDIUM REVERSES THE STIMULATING EFFECT OF CATECHOLAMINES ON CORTICOTROPIN-RELEASING HORMONE NEURONS INORGANOTYPIC CULTURES

An organotypic culture system of anterior hypothalamic slices was deve loped for studying the secretory responses of corticotropin-releasing hormone (CRH) neurons to corticosteroid-catecholamine interactions. Th e standard culture medium included 5% horse serum containing 50 mu g/l cortisol. In 1- to 3-day cultures, the tissue viability was demonstra ted by the presence of arginine vasopressin immunolabeled perikarya an d axons in the paraventricular nucleus and by sustained tissue concent rations of CRH (around 50 pg/mg protein). However, immunoreactive CRH neurons were not detectable in cultures in the standard medium. Exposu re of cultures to high K+ (56 mM) in the medium induced a ten-fold inc rease in basal CRH release which was completely abolished in a Ca2+-fr ee medium containing 2 mM EGTA. Noradrenaline (NA) triggered CRH relea se in a dose-dependent (1-20 mu M) and time-dependent (0.5-6 h) manner . Removal of corticosteroids from the media by charcoal treatment led to (1) the visualization of immunolabelled CRH perikarya and fibers an d a 55% rise in CRH content of the paraventricular nucleus tissue and (2) to a five-fold increase in CRH release. Both effects were reversed by supplementation of the culture medium with corticosterone (50 mu g /l). Under steroid-free conditions, NA (1-10 mu M) not only failed to induce CRH release, but strongly inhibited the consistent baseline in CRH release. This was reminiscent of a similar corticosteroid-dependen t inversion of the NA effect on the hypothalamic-pituitary-adrenal axi s described in vivo. Overall, these results are direct evidence of com plex corticosteroid-catecholamine interrelationships as major regulato ry factors of the hypothalamic-pituitary-adrenal axis.