Removal of Adrenal Steroids from the Medium Reverses the Stimulating Effect of Catecholamines on Corticotropin Releasing Hormone Neurons in Organotypic Cultures

Abstract

An organotypic culture system of anterior hypothalamic slices was developed for studying the secretory responses of corticotropin-releasing hormone (CRH) neurons to corticosteroid-catecholamine interactions. The standard culture medium included 5% horse serum containing 50 µg/l cortisol. In 1- to 3-day cultures, the tissue viability was demonstrated by the presence of arginine vasopressin immunolabeled perikarya and axons in the paraventricular nucleus and by sustained tissue concentrations of CRH (around 50 pg/mg protein). However, immunoreactive CRH neurons were not detectable in cultures in the standard medium. Exposure of cultures to high K⁺ (56 mM) in the medium induced a ten-fold increase in basal CRH release which was completely abolished in a Ca²⁺-free medium containing 2 mMEGTA. Noradrenaline (NA) triggered CRH release in a dose-dependent (1-20 µM) 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 and 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 µg/l). Under steroid-free conditions, NA (1–10 µM) not only failed to induce CRH release, but strongly inhibited the consistent baseline in CRH release. This was reminiscent of a similar corticosteroid-dependent inversion of the NA effect on the hypothalamic-pituitary-adrenal axis described in vivo. Overall, these results are direct evidence of complex corticosteroid-catecholamine interrelationships as major regulatory factors of the hypothalamic-pituitary-adrenal axis.

A Centrally Acting, Anxiolytic Angiotensin II AT1 Receptor Antagonist Prevents the Isolation Stress-Induced Decrease in Cortical CRF1 Receptor and Benzodiazepine Binding

“…administration of a CRF antagonist blunts the stress-induced increase in extracellular norepinephrine levels in the prefrontal cortex (Shimizu et al, 1994). This in turn could decrease CRF release from cortical neurons, as it is known that at least in the hypothalamus CRF release is under noradrenergic control (Szafarczyk et al, 1995).…”

Glucocorticoids Provoke a Shift from α2‐ to α1‐Adrenoreceptor Activities in Cultured Hypothalamic Slices Leading to Opposite Noradrenaline Effect on Corticotropin‐Releasing Hormone Release

“…This technique preserves the morphofunctional structure of cultured brain areas and high tissue CRH levels, as previously described (Szafarczyk et al, 1995). In brief, 9-or 10-day-old Sprague-Dawley rat pups were used.…”

“…This amplifying effect of adrenalectomy is reversed completely by a mild oral CORT supplementation, yielding a fair basal CURT plasma level [-~50ng/ml (Gaillet et al, 1993)1. From in vitro studies used to investigate further the mechanism involved in the steroid dependence of catecholamine effects on CRH release, we also demonstrated on organotypic cultures of hypothalamic slices that NA acts in a dose-dependent manner as a potent secretagogue for CRH release in the presence of low levels of glucocorticoids and that, in the absence of glucocorticoids, the basal CRH release is high and NA strongly inhibits CRH secretion (Szafarczyk et al, 1995). The mechanisms underlying this functional and steroid-dependent plasticity of CRH neurons with respect to the catecholaminergic modulation are not yet known.…”

“…Addition of dexamethasone causes a reduction in basal-, K~-,or noradrenaline (NA)-stimulated CRH release (Hu et al, 1992a,c;Bertini et al, 1993) from cultured hypothalamic cells, through type II and type I corticosteroid receptors, respectively (Hu et al, 1992a,c). The removal of steroids leads to a fivefold increase in basal CRH release from cultured hypothalamic slices (Bertini et al, 1993;Szafarczyk et al, 1995). Concerning the steroid modulation of NA-CRH interaction, we have demonstrated recently in vivo that VNAB lesions inhibit the ether stress-induced ACTH and corticosterone (CURT) surges in rats with intact adrenals and that, on the contrary, adrenalectomized and VNAB-lesioned rats show a greatly amplified ACTH stress response over the controls.…”

“…In fact, the CRH-synthesizing neurons in the parvocellular portion of the paraventricular nucleus (PVN) of the hypothalamus receive direct catecholaminergic innervation originating from the lower brainstem and conveyed by the ventral noradrenergic ascending bundle (VNAB) Lipositz et al, 1986). As demonstrated in various in vivo and in vitro approaches, a stimulatory effect predominates in the central catecholaminergic control of the CRH-ACTH axis, particularly on basal and stressinduced CRH secretion at the hypothalamic level (Axelrod and Reisine, 1984;Ganong, 1984;Guillaume et al, 1987;Plotsky, 1987;Szafarczyk et al, 1987Szafarczyk et al, , 1995Tsagarakis et al, 1988;Widmaier et al, 1989).…”