ROLE FOR GLIAL-CELLS IN REGULATING THE FUNCTIONAL EXPRESSION OF NEUROPEPTIDE-Y (NPY) NEURONS IN AGGREGATE CULTURES DERIVED FROM DISSOCIATED FETAL BRAIN-CELLS

A series of studies from our laboratory have established an aggregate culture system of fetal rat brain cells expressing neuropeptide Y (NPY) which can serve as a model to study the role of glia-neuron paracrine interactions in the developmental expression of NPY neurons. In this system, NPY production increases progressively with culture-age and it is induced by forskolin (FOR) and phorbol 12-myristate 13-acetate (PMA). We addressed the following question: Is the functional expression of the NPY neurons impaired in the absence of glial cells (particularly astrocytes) and if so, can secretory products of aggregates composed of the full complement of brain cells (intact aggregates) restore the function of the impaired NPY neurons? Aggregates were generated from 17-day-old fetal rat cortex and maintained in serum-free medium for 13-15 days. Cytosine arabinoside (CA; doses of 0.5-8 mu M) was added to the cultures on day 1 and the effectiveness in elimination of glial cells was verified on day 15 by measuring the incorporation of H-3 thymidine into DNA and by immunostaining for the astrocyte marker glial fibrillary acidic protein (GFAP). Basal NPY production and FOR (10 mu M) + PMA (20 nM) stimulated production of NPY on days 13-15 were taken as functional criteria. FOR + PMA induced approximate to 2-fold increase in NPY production in control cultures (no CA). CA inhibited both basal and FOR + PMA induced production of NPY and DNA synthesis in a dose-dependent manner: at 6 mu M CA, basal NPY production was reduced by about 50%, FOR + PMA stimulated production of NPY and DNA synthesis were completely inhibited, and astrocytes were essentially eliminated. A competing dose of 100 mu M deoxycytidine completely reversed the effects of 6 mu M CA on DNA synthesis and basal and FOR + PMA stimulated production of NPY. Conditioned media derived from intact aggregates completely restored basal and partially (approximate to 50%) restored FOR + PMA stimulated production of NPY in CA-treated aggregates. These results implicate secretory products of glial cells (astrocytes?) and/or glial-neuron paracrine interactions in regulating the developmental expression of NPY neurons in culture such that activation of the cAMP and protein kinase C pathways leads to increased production of NPY. (C) 1994 Wiley-Liss, Inc.