Another important finding is that VGLUT3 expression suffices for the induction of vesicular glutamate uptake and release in nonglutamatergic neurons. While Imatinib supplier recent data from VGLUT3-deficient neurons demonstrated the necessity of VGLUT3 function for glutamatergic neurotransmission in auditory hair cells and pain pathways (Obholzer et al.,

2008, Ruel et al., 2008, Seal et al., 2008 and Seal et al., 2009), our demonstration that this effect is a direct result of VGLUT3′s ability to function as a classical vesicular transporter is critical in interpreting morphological data showing that VGLUT3 localizes to terminals from serotonergic, cholinergic, and GABAergic neurons. Based on our findings, these synapses IPI 145 are very likely coreleasing glutamate along

with their classical neurotransmitters, which implies a fast excitatory signaling component at these classically modulatory synapses. Previous studies have shown that VGLUT levels are endogenously and bidirectionally regulated during development (Boulland et al., 2004 and Nakamura et al., 2005), in disease states (Eastwood and Harrison, 2005, Kashani et al., 2007 and Smith et al., 2001), with pharmacological manipulation (De Gois et al., 2005 and Wilson et al., 2005), and according to circadian rhythms (Yelamanchili et al., 2006). Our data suggest these alterations would be accompanied by changes in neuronal firing patterns and perhaps circuit behavior. For example, Ribonucleotide reductase differences between VGLUT1 and VGLUT2/3 could be important during development, where the early, transient expression of VGLUT2 and VGLUT3 in neurons that later express VGLUT1 could increase the chance of glutamate release at synapses that may contain fewer synaptic vesicles

than mature synapses. It is possible that neurons or networks of neurons actively use specific VGLUT isoform expression to regulate the efficiency of glutamate release. The mechanism by which endophilin levels regulate release efficiency is still unknown. Because endophilin is a protein known primarily for its role in endocytosis, it is possible that it acts by altering either the size of the RRP or its rate of replenishment. However, overexpression and knockdown of endophilin did not affect the RRP. Instead they increased and decreased the EPSC charge, suggesting that endophilin directly alters the fusion efficiency of synaptic vesicles. Because this effect does not require the SH3 domain, it is not likely to involve increased recruitment of dynamin or synaptojanin. The effect is, however, dependent on membrane binding and dimerization. Although it is likely that many of endophilin’s actions are dependent on interactions with synaptojanin and dynamin, recent evidence suggests endophilin’s main endocytic function requires only the BAR domain and occurs at the plasma membrane prior to vesicle scission (Bai et al., 2010).