Cholesterol as a key player in the balance of evoked and spontaneous glutamate release in rat brain cortical synaptosomes

Membrane rafts are domains enriched in sphingolipids, glycolipids and cholesterol that are able to compartmentalize cellular processes. Noteworthy, many proteins have been assigned to membrane rafts including those related to the control of the synaptic vesicle release machinery, which is a important step for neurotransmission between synapses. In this work, we have investigated the role of cholesterol in key steps of glutamate release in isolated nerve terminals (synaptosomes) from rat brain cortices. Incubation of synaptosomes with methyl-beta-cyclodextrin (M beta CD) induced glutamate release in a dose-dependent fashion. H gamma CD, a cyclodextrin with low affinity for cholesterol, had no significant effect on spontaneous glutamate release. When we evaluated the effects of M beta CD on glutamate release induced by depolarizing stimuli, we observed that M beta CD treatment inhibited the KCl-evoked glutamate release. The glutamate release induced by M beta CD was not altered by treatment with EGTA nor with EGTA-AM. The KCl-evoked glutamate release was no further inhibited when synaptosomes were incubated with M beta CD in the absence of calcium. We therefore investigated whether the cholesterol removal by M beta CD changes intra-synaptosomal sodium and calcium levels. Our results suggested that the cholesterol removal effect on spontaneous and evoked glutamate release might be upstream to sodium and calcium entry through voltage-activated channels. We therefore tested if M beta CD would have a direct effect on synaptic vesicle exocytosis and we showed that cholesterol removal by M beta CD induced spontaneous exocytosis and inhibited synaptic vesicle exocytosis evoked by depolarizing stimuli. Lastly, we investigated the effect of protein kinase inhibitors on the spontaneous exocytosis evoked by M beta CD and we observed a statistically significant reduction of synaptic vesicles exocytosis. In conclusion, our work shows that cholesterol removal facilitates protein kinase activation that favors spontaneous synaptic vesicles and consequently glutamate release in isolated nerve terminals. (C) 2012 Elsevier Ltd. All rights reserved.