COCULTURE OF HYPOTHALAMIC NEURONS AND MELANOTROPE CELLS - A MODEL TO STUDY SYNAPTOGENESIS BETWEEN CENTRAL NEURONS AND ENDOCRINE-CELLS

As a first step towards elucidating mechanisms involved in neuroendocr ine synaptogenesis, we developed a model of co-culture based on hypoth alamic-intermediate pituitary interactions. Dissociated hypothalamic n eurons from fetal rats at embryonic day lj were cultured in a defined medium together with melanotrope cells of the pituitary intermediate l obe from neonatal rats. In these co-cultures, establishment of synapti c contacts between GABAergic or dopaminergic neurons and an endocrine target cell, the melanotrope cell, was studied by morphofunctional app roaches. Using double immunostaining with antibodies directed against glutamate decarboxylase or tyrosine hydroxylase and alpha-melanocyte-s timulating hormone, we demonstrated morphological contacts between GAB Aergic or dopaminergic neurons and melanotrope cells as early as three days in vitro. Furthermore, using an antibody directed against synaps in I, we showed a modification of synapsin I immunoreactivity from dif fuse to punctate distribution correlated with the establishment of con tacts and the observation of characteristic neuroendocrine synapses by electron microscopy. These results were further confirmed by electrop hysiological studies. Patch-clamp recordings demonstrated that, at six days in vitro, some melanotrope cells displayed GABAergic synaptic cu rrents, which occurred either spontaneously and/or could be evoked che mically by 50 mM KCI or 100 mu M kainate. The proportion of the melano trope cells receiving functional synaptic inputs increased until 10 da ys in culture, a stage al which virtually all melanotrope cells in con tact with neurons possessed functional synapses. The results presented here describe the establishment of neuroendocrine synapses in vitro, studied by combining morphofunctional and electrophysiological approac hes. Copyright (C) 1996 IBRO.