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

Abstract

1. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded in mouse Purkinje cells in the presence of 1 micro M tetrodotoxin (TTX). Under these conditions, which eliminated Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs), the contribution of Ca(2+) stores to spontaneous GABA release was examined. 2. The plant alkaloid ryanodine acts as an inhibitor of endoplasmic reticulum ryanodine-sensitive Ca(2+) release channels (ryanodine receptors) at low micromolar concentrations. Ryanodine effects were confined to a subpopulation of cells tested. At 10 micro M ryanodine, 4/12 cells showed a significant increase in mean mIPSC frequency of +19.6+/-4.0% (n=4). 3. The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump inhibitor cyclopiazonic acid (CPA) produced a more robust effect. In 8/10 cells, 25 micro M 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 micro M), another SERCA pump inhibitor. 4. Ruthenium red (RuR) has been proposed to either act directly on the release machinery or block Ca(2+) pumps on internal stores. At 10 micro M 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 Ca(2+) 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 Ca(2+) stores may contribute to spontaneous GABA release onto mouse Purkinje cells.

Figure 1

Effects of ryanodine on Purkinje cell mIPSCs. (A) Example of 10 μM ryanodine-induced increase in mIPSC frequency. Raw data are three consecutive 2 s sweeps superimposed in either TTX control or steady-state ryanodine conditions; V_H=−70 mV. (B) Time course of action of ryanodine (black bar) on mean mIPSC frequency for example cell shown in A. Ryanodine caused a slow onset increase in frequency. (C) Cumulative frequency plot for the effects of ryanodine on control mIPSC inter-event interval of example cell shown in A; bin width 5 ms. Ryanodine caused a significant shortening of the mIPSC inter-event interval (P<0.05, KS test) in this cell. (D) Effects of ryanodine on mean mIPSC amplitude. Ryanodine (10 μM, n=12) had no significant effect on control mean mIPSC amplitude (n=12, white bar).

Figure 2

Effects of cyclopiazonic acid (CPA) on Purkinje cell mIPSCs. (A) Example of 25 μM CPA-induced increase in mIPSC frequency. Raw data are three consecutive 2 s sweeps superimposed in either TTX control or steady-state CPA conditions; V_H=−70 mV. (B) Time course of action of CPA (black bar) on mean mIPSC frequency for example cell shown in A. CPA caused a slow onset increase in frequency. (C) Pooled cumulative frequency plot (n=8) for CPA effects on control mIPSC inter-event interval; bin width 5 ms. CPA caused a significant shortening of the mIPSC inter-event interval (P<0.05, KS test). (D) Pooled cumulative frequency plot (n=8) for CPA effects on control mIPSC amplitude; bin width 2 pA. CPA had no significant effect on mIPSC amplitude.

Figure 3

Effects of ruthenium red (RuR) on Purkinje cell mIPSCs. (A) Example of 10 μM RuR-induced increase in mIPSC frequency. Raw data are three consecutive 2 s sweeps superimposed in either TTX control or steady-state RuR conditions; V_H=−70 mV. (B) Time course of action of RuR (black bar) on mean mIPSC frequency for example cell shown in A. RuR caused a relatively rapid onset increase in frequency. (C) Pooled cumulative frequency plot (n=9) for RuR effects on control mIPSC inter-event interval; bin width 5 ms. RuR caused a significant shortening of the mIPSC inter-event interval (P<0.05, KS test). (D) Pooled cumulative frequency plot (n=9) for RuR effects on control mIPSC amplitude; bin width 2 pA. RuR had no significant effect on mIPSC amplitude.

Figure 4

Effects of reagents on the frequency of small, intermediate and large amplitude mIPSCs. (A) Effects of different treatments on percentage increase in control mIPSC frequency. RuR (10 μM, n=9) caused a significantly greater increase in mean mIPSC frequency than 10 μM ryanodine (n=4, ^**=P<0.01), 25 μM CPA (n=8, ^***=P<0.001) and 1–2 μM thapsigargin (n=6, ^*=P<0.05). (Bi) Effect of 10 μM RuR (n=9) on the frequency of small- (0–25 pA), intermediate- (26–50 pA) and large- (>50 pA) amplitude mIPSCs. No clear differences in RuR-induced increases were seen between each group. (Bii) Effect of modulation of Ca²⁺ stores (n=18, group data from 10 μM ryanodine, n=4; 25 μM CPA, n=8; 1–2 μM thapsigargin, n=6) on the frequency of small- (0–25 pA), intermediate- (26–50 pA) and large- (>50 pA) amplitude mIPSCs. No clear differences in Ca²⁺ store modulator-induced increases were seen between each group.

Figure 5

Effects of CPA on Purkinje cell mIPSCs in the presence of RuR. (A) Example of occlusion of effects of 25 μM CPA by 10 μM RuR. Time course of action of RuR (white bar) and subsequent application of CPA (black bar) on mean mIPSC frequency. RuR caused a relatively rapid onset increase in frequency, CPA had no further effects. (B) Pooled cumulative frequency plot (n=5) for RuR effects and RuR/CPA on control mIPSC inter-event interval; bin width 5 ms. RuR caused a significant shortening of the control mIPSc inter-event interval (P<0.05, KS test), but CPA had no further effects. (D) Pooled cumulative frequency plot (n=9) for RuR effects and RuR/CPA on control mIPSC amplitude; bin width 2 pA. Neither RuR nor RuR/CPA had any significant effect on mIPSC amplitude.