The PLC/IP 3 R/PKC pathway is required for ethanol-enhanced GABA release

Abstract

Research on the actions of ethanol at the GABAergic synapse has traditionally focused on postsynaptic mechanisms, but recent data demonstrate that ethanol also increases both evoked and spontaneous GABA release in many brain regions. Using whole-cell voltage-clamp recordings, we previously showed that ethanol increases spontaneous GABA release at the rat interneuron-Purkinje cell synapse. This presynaptic ethanol effect is dependent on calcium release from internal stores, possibly through activation of inositol 1,4,5-trisphosphate receptors (IP(3)Rs). After confirming that ethanol targets vesicular GABA release, in the present study we used electron microscopic immunohistochemistry to demonstrate that IP(3)Rs are located in presynaptic terminals of cerebellar interneurons. Activation of IP(3)Rs requires binding of IP(3), generated through activation of phospholipase C (PLC). We find that the PLC antagonist edelfosine prevents ethanol from increasing spontaneous GABA release. Diacylglycerol generated by PLC and calcium released by activation of the IP(3)R activate protein kinase C (PKC). Ethanol-enhanced GABA release was blocked by two PKC antagonists, chelerythrine and calphostin C. When a membrane impermeable PKC antagonist, PKC (19-36), was delivered intracellularly to the postsynaptic neuron, ethanol continued to increase spontaneous GABA release. Overall, these results suggest that activation of the PLC/IP(3)R/PKC pathway is necessary for ethanol to increase spontaneous GABA release from presynaptic terminals onto Purkinje cells.

Figure 1

Ethanol increases vesicular release of GABA. A: a trace from a representative neuron illustrating the effect of 100 mM ethanol on mIPSC frequency. B: ethanol significantly increased mIPSC frequency compared to control and washout [*, p<.05; repeated measures ANOVA; F(2,42) = 18.3]. The mIPSC frequency values of the control and washout groups were not significantly different (p = 0.6). C: incubating the slice with bafilomycin A₁ (baf A₁) greatly reduced mIPSC frequency. In the presence of bafilomycin A₁, 100 mM ethanol did not significantly increase mIPSC frequency compared to control (p = 2.6). D: the data shown in B, represented as a % change (see methods for explanation). 100 mM ethanol significantly increased mIPSC frequency (see B) in contrast to weak and non-significant effects on mIPSC fast decay time (τ-fast), slow decay time (τ-slow) and amplitude.

Figure 2

GABAergic presynaptic terminals are immunopositive for IP₃R. A: micrograph shows a GABAergic presynaptic terminal immunopositive for IP₃R making a symmetric synaptic contact with the dendritic shaft of a Purkinje cell. Immediately above the terminal is a bundle of parallel fibers. B: micrograph shows two glutamatergic presynaptic terminals making asymmetric synaptic contacts with Purkinje cell spines (arrows). Note IP₃R labeling associated with endomembranes within the spines. The top terminal is immunopositive for the IP₃R. A bundle of parallel fibers is visible in the top right corner. C: presynaptic terminals likely, for their morphology, to be GABAergic, probable glutamatergic presynaptic terminals, and Purkinje cell spines had more IP₃R staining than parallel fibers (*, p<.05, t test).

Figure 3

Inhibition of PLC blocks ethanol from increasing mIPSC frequency. A: a trace from a representative neuron demonstrating the effect of 100 mM ethanol on mIPSC frequency in the presence of edelfosine (10 μM). B: Edelfosine (10 μM) and BAPTA-AM (100 μM) prevented 100 mM ethanol from increasing mIPSC frequency compared to control (*, p<.05, one-way ANOVA, Dunnett's post hoc test).

Figure 4

Inhibition of PKC prevents ethanol from increasing mIPSC frequency. A: a trace from a representative neuron showing the effect of 100 mM ethanol on mIPSC frequency when 20 μM PKC (19-36) was included in the pipette internal solution (PKC_int). B: chelerythrine (1 μM), calphostin C (1 μM), and SC9 (25 μM) blocked 100 mM ethanol from increasing mIPSC frequency compared to control (*, p<.05, one-way ANOVA, Dunnett's post hoc test). C: ethanol (50 and 100 mM) increased mIPSC frequency in the presence of 20 μM PKC_int, (*, p<.05, one-way ANOVA, Dunnett's post hoc test).