GABAERGIC CELLS AND SIGNALS APPEAR TOGETHER IN THE EARLY POSTMITOTIC PERIOD OF TELENCEPHALIC AND STRIATAL DEVELOPMENT

Single cell suspensions derived from embryonic telencephala taken from embryos of gestational day 13 (E13) as well as rat striatal tissue from E14, 15 and 17 were prepared by tissue digestion with papain. Cell suspensions were analyzed by flow cytometry or plated onto poly-D-lysine-coated culture dishes for either nuclear staining or immunocytochemistry. Experiments on functional Na+ channels and GABA(A) receptor expression were carried out using a fluorescence-activated cell sorter (FACS) and a negatively charged fluorescent indicator dye (oxonol). FACS analysis of embryonic cell suspensions at E13-17 consistently revealed one major subpopulation accounting for 85-90% of the events and one minor subpopulation (10-15% of the total). When sorted, the major subpopulation consisted of phase-bright cells of 5-7 mum diameter some of which had neurites. The minor population consisted of phase-dark cells and resealed membranes of 0.5-4 mum diameter as well as debris. Almost all the cells obtained in the high FALS (forward-angle light scatter) subpopulation at E17 expressed 200-kDa neurofilament and tetanus toxin antigens while the small diameter cells seldom expressed tetanus toxin and particles never did. A small number of GABA-containing neurons were detected in the telencephalon at E13 (3%) and in the developing striatum at E14 (6%). All of the GABA-containing neurons expressed neurofilament. In the embryonic rat striatum, nanomolar concentrations of muscimol (GABA(A) agonist) induced depolarizing responses. A small number of cells in the high FALS subpopulation were responsive to muscimol starting at embryonic day 14, and the number of responsive cells increased at E15. At E17, both FALS subpopulations (high and low) were responsive to muscimol. A modest response to muscimol was observed in E13 telencephalic cells. A small percentage of GABA-containing neurons was present in the cell suspension at this time. Fully developed GABA(A)-mediated responses at E15 coincided with an approximate 5-fold increase in the percentage of GABA-containing neurons. Veratridine responses were already detectable in the high FALS subpopulation of E13 telencephalic cells.