The development of Ca 2+ channel responses and their coupling to exocytosis in cultured cerebellar granule cells

Abstract

Using single-cell imaging, we investigate developmental changes in the modulation of KCl-evoked Ca 2+ entry by various voltage-dependent Ca 2+ channels and the coupling of these channels to exocytosis in cultured cerebellar granule neurons. A component of the KCl-evoked Ca 2+ elevation sensitive to nifedipine and localized at cell somata, decreases with culture age. A component blocked by 200 nM ω-Agatoxin-IVA increases with age and whilst localized primarily at the cell somata, also becomes evident at the neurites. The change in activity between nifedipine-sensitive Ca 2+ channels and ω-Agatoxin-IVA-sensitive Ca 2+ channels occurs at 13 days in vitro at cell somata. A component of Ca 2+ entry insensitive to nifedipine and 200 nM ω-Agatoxin-IVA is localized primarily at the neurites and is apparent at all ages. Single-cell imaging of exocytosis using FM1–43 destaining indicates that the residual, but not the nifedipine- or ω-Agatoxin-IVA-sensitive components of Ca 2+ entry, modulates exocytosis. However cells cultured for 20–26 days develop a component of Ca 2+ entry at the neurites which is sensitive to 200 nM ω-Agatoxin-IVA and ω-Conotoxin-MVIIC and which partially controls release. Immunolocalization studies reveal that binding sites for ω-Conotoxin-GVIA are present throughout development, even though this toxin does not inhibit KCl-evoked [Ca 2+] c elevations or exocytosis. 300 nM ω-Agatoxin-IVA labels both somata and, at later developmental stages, neurites, consistent with the functional studies.