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Keywords:

  • rat;
  • calcium channel;
  • presynaptic;
  • exocytosis;
  • cerebellum;
  • FM1-43;
  • imaging

Abstract

The free calcium concentration, [Ca2+]c, in fura-2-loaded rat cerebellar granule cells was investigated by digital imaging during trains of uniform field stimuli in order to compare the ability of calcium channels in somata and neurites to respond to brief, physiologically relevant depolarizations. Very few somata responded to 20 Hz trains of 1 ms pulses, while virtually all neurites showed an extensive increase which was rapidly reversed when stimulation was terminated. In contrast, both somata and neurites responded when cells were depolarized with 50 mM KCl. The field stimuli evoked a tetrodotoxin-sensitive increase in Na+ concentration in both somata and neurites. When 4-aminopyridine, which inhibits delayed K+ currents in these cells, was present during the field stimulus both somata and neurites increased their [Ca2+]c, suggesting that prolongation of the duration of depolarization is required for somatic Ca2+ channel activation. The neurite response did not depend on the orientation of the neurite relative to the applied field. The neurite response was insensitive to nifedipine (1 μM) and ω-agatoxin-IVA (30 nM) but was uniformly inhibited by ω-conotoxin-GVIA (30% inhibition at 1 μM) and ω-conotoxin-MVIIC (44% inhibition at 5 μM). The two inhibitors were not additive. The neurite [Ca2+]c response was insensitive to the combination of ionotropic glutamate receptor antagonists. Field stimulation caused the exocytosis of the fluorescent probe FM1-43 previously loaded during KCl depolarization, suggesting that presynaptic Ca2+ channels contribute to the field-evoked neurite response.