Presynaptic internal Ca2+ stores contribute to inhibitory neurotransmitter release onto mouse cerebellar Purkinje cells

1 Miniature inhibitory postsynaptic currents (mIPSCs) were recorded in mouse Purkinje cells in the presence of 1 muM tetrodotoxin (TTX). Under these conditions, which eliminated Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs), the contribution of Ca2+ stores to spontaneous GABA release was examined. 2 The plant alkaloid ryanodine acts as an inhibitor of endoplasmic reticulum ryanodine-sensitive Ca2+ release channels (ryanodine receptors) at low micromolar concentrations. Ryanodine effects were confined to a subpopulation of cells tested. At 10 muM ryanodine, 4/12 cells showed a significant increase in mean mIPSC frequency of + 19.6 +/- 4.0% (n = 4). 3 The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor cyclopiazonic acid (CPA) produced a more robust effect. In 8/10 cells, 25 muM CPA caused a significant increase in mean mIPSC frequency; the mean increase being +26.0 +/- 3.0% (n = 8). Similar results were seen with thapsigargin (1-2 muM), another SERCA pump inhibitor. 4 Ruthenium red (RuR) has been proposed to either act directly on the release machinery or block Ca2+ pumps on internal stores. At 10 muM RuR, all cells showed a rapid, large increase in mean mIPSC frequency of + 90.4 +/- 16.4% (n = 9). This increase was greater than that seen by agents known to modulate Ca2+-Stores and was more consistent with a direct action. At this concentration, RuR also occluded the effects of CPA. 5 For all reagents, there were no obvious effects on mean mIPSC amplitude. However, the effects on mIPSC frequency were consistent with a presynaptic action and indicate that Ca2+ stores may contribute to spontaneous GABA release onto mouse Purkinje cells.