Oregon Health & Science University
Synaptic vesicles fuse with nerve terminal plasma membrane not only in response to an action potential, but also spontaneously. Such spontaneous events contribute to neuronal baseline noise, maintain synaptic strength, and can induce and modulate action potential firing in inhibitory neurons. The mechanisms controlling this mode of synaptic transmission remain largely unknown. The overall objective of these studies was to determine mechanisms controlling spontaneous vesicle fusion. The rate of spontaneous exocytosis is enhanced by extracellular calcium, however the mechanisms are not understood. Our data indicate that extracellular calcium does not catalyze spontaneous vesicle fusion by influx through voltage-gated calcium channels and increases in the concentration of intracellular calcium ([Ca[superscript 2+][subscript i]). These data suggest that extracellular calcium catalyzes spontaneous neurotransmitter release by a distinct mechanism from that of action potential-evoked release. We hypothesized that a G-protein coupled receptor (GPCR) that is activated by extracellular divalent cations, the calcium-sensing receptor (CaSR), could transduce changes in synaptic cleft [Ca[superscript2+]] to changes in spontaneous exocytosis. Using pharmacological and genetic approaches, we find that CaSR plays a role in controlling spontaneous glutamate release from neocortical nerve terminals. We also examined how intracellular signaling controls synaptic vesicle fusion. CaSR couples to the Gq family of GPCRs in both expression systems and native tissue. Gq activates phospholipase C (PLC) and the metabolism of phosphatidylinositol-(4,5)-bisphosphate (PIP2), resulting in the production of diacylglycerol (DAG) and inositol triphosphate. DAG analogues are potent modulators of both spontaneous and action potential-evoked glutamate release. We hypothesized that PLC activation increases neurotransmitter release. In our experiments, PLC inhibition decreased spontaneous glutamate release and the size of the readily releasable pool of synaptic vesicles, suggesting that PLC activity may maintain synaptic vesicle exocytosis. We also hypothesized that extracellular calcium could stimulate spontaneous glutamate release by inducing calcium-induced calcium release (CICR) from ryanodine receptors (RyRs) on intracellular calcium stores. CICR has been reported to produce presynaptic calcium transients and spontaneous release in hippocampal boutons, and large-amplitude miniature inhibitory postsynaptic currents in cerebellar neurons. To determine the role of RyR-mediated Ca[superscript 2+] release in spontaneous glutamate release in neocortical neurons, we measured miniature excitatory postsynaptic currents (mEPSCs) upon application of caffeine. We confirmed that caffeine increases spontaneous release in neocortical neurons, but observed that caffeine also produced a robust decrease in mEPSC amplitude. Caffeine inhibited postsynaptic non-NMDA type glutamate receptors, thus explaining the decrease in mEPSC amplitude. However, CICR did not appear to account for the dependence of spontaneous release rate on [Ca[superscript 2+][subscript o], because Ca[superscript 2+] enhancement of spontaneous release was insensitive to buffering of intracellular Ca[superscript 2+] by BAPTA.
Neuroscience Graduate Program
School of Medicine
Vyleta, Nicholas Patrick, "Regulation of spontaneous glutamate release by extracellular calcium" (2010). Scholar Archive. 544.