Glucocorticoid stimulation of pro-ANF mRNA expression of adult rat hypothalamic neurons in culture: a cAMP-dependent effect.

We describe here long term primary cultures of adult rat hypothalamic cells, a novel system which we have exploited to examine the effect of glucocorticoids on adult ANF neurons in vitro. ANF neurons with and without neurite outgrowths were identified by immunocytochemistry and by in situ hybridization studies, in which pro-ANF mRNA signals were localised in cultured cells. Dexamethasone (DM) augmented the abundance of pro-ANF mRNA signals in a dose-related manner with ED50 of 5 x 10(-12)M and Emax of 10(-10)M. This stimulation effect was mimicked by corticosterone but not by deoxycorticosterone. Similarly, activation of the adenylyl cyclase-cAMP system with forskolin, 8-brom-cAMP or 3-isobutyl-1-methylxanthine (IBMX), also markedly elevated the level of pro-ANF mRNA abundance. Interestingly, whereas forskolin-induced stimulation was reversed by Rp-cAMP, a cAMP antagonist, the augmentation induced by DM alone or by DM and forskolin together, was similarly suppressed by the chemical. We therefore conclude that (i) adult ANF neurons are capable of regenerating neurite outgrowths in vitro, (ii) cAMP dependent pathways play a fundamental role in regulating the expression of pro-ANF mRNA, and (iii) the stimulatory effect of glucocorticoids is indirect and mediated, at least in part, through modulating the activity of cAMP dependent pathways.