THE DEVELOPMENT OF CA2+ CHANNEL RESPONSES AND THEIR COUPLING TO EXOCYTOSIS IN CULTURED CEREBELLAR GRANULE CELLS

Using single-cell imaging, we investigated developmental changes in th e modulation of KCl-evoked Ca2+ entry by various voltage-dependent Ca2 + channels and the coupling of these channels to exocytosis in culture d cerebellar granule neurons. A component of the KCl-evoked Ca2+ eleva tion sensitive to nifedipine and localized at cell somata, decreases w ith culture age. A component blocked by 200 nM omega-Agatoxin-IVA incr eases with age and whilst localized primarily at the cell somata, also becomes evident at the neurites. The change in activity between nifed ipine-sensitive Ca2+ channels and omega-Agatoxin-IVA sensitive Ca2+ ch annels occurs at 13 days in vitro at cell somata. A component of Ca2entry insensitive to nifedipine and 200 nM omega-Agatoxin-IVA is local ized primarily at the neurites and is apparent at all ages. Single-cel l imaging of exocytosis using FM1-43 destaining indicates that the res idual, but not the nifedipine- or omega-Agatoxin-IVA-sensitive compone nts of Ca2+ entry, modulates exocytosis. However cells cultured for 20 -26 days develop a component of Ca2+ entry at the neurites which is se nsitive to 200 nM omega-Agatoxin-IVA and omega-Conotoxin-MVIIC and whi ch partially controls release. Immunolocalization studies reveal that binding sites for omega-Conotoxin-GVIA are present throughout developm ent, even though this toxin does not inhibit KCl-evoked [Ca2+](c) elev ations or exocytosis. 300 nM omega-Agatoxin-IVA labels both somata and , at later developmental stages, neurites, consistent with the functio nal studies. (C) 1997 IBRO. Published by Elsevier Science Ltd.