We therefore decided to test the hypothesis that the three VGLUT isoforms confer specific properties of glutamatergic neurotransmission upon the synapses at which they are present. We used whole-cell voltage clamp to record synaptic currents from primary cultured neurons expressing different endogenous or virally expressed VGLUT isoforms and measured basic parameters of synaptic function. Our results demonstrate that expression of any VGLUT, including VGLUT3, gives a neuron the ability to release glutamate and that neurons expressing VGLUT1 exhibit lower vesicular release probability (Pvr) and altered short-term plasticity compared to VGLUT2- or VGLUT3-expressing neurons. In exploring the mechanism

by which VGLUT isoforms regulate exocytosis, we identified endophilin click here A1 as a positive regulator of release efficiency and propose that VGLUT1′s effects result from binding and inhibiting endophilin A1. We wanted to directly

compare the basic functions of VGLUT1, VGLUT2 and VGLUT3 in an otherwise identical cellular environment. Previous studies demonstrated that hippocampal VGLUT1−/− neurons and thalamic VGLUT2−/− neurons have very low or undetectable levels of VGLUT protein, virtually Inhibitor Library clinical trial no evoked or spontaneous glutamate release, and a very small readily releasable pool (RRP) of filled synaptic vesicles ( Fremeau et al., 2004, Moechars et al., 2006 and Wojcik et al., 2004). We prepared primary autaptic cultures of these neurons and used lentiviruses to induce expression of each of the three VGLUT isoforms. We then performed whole-cell voltage-clamp analysis to test for rescue of the synaptic response. Evoked responses were measured in knockout neurons and neurons infected with VGLUT1, VGLUT2, and VGLUT3-expressing lentiviruses. Expression of all three isoforms rescued the deficit in EPSC peak amplitude and EPSC charge in both VGLUT1−/− hippocampal neurons ( Figures 1A and 1D) and VGLUT2−/− thalamic neurons ( Figures 1B and 1E). The EPSC amplitudes of neurons rescued with VGLUT1, VGLUT2 and VGLUT3 were not significantly different from hippocampal VGLUT+/+ neurons infected

with a lentivirus expressing only GFP, nor were they significantly no different from each other ( Figures 1D and 1E). The charge contained in the EPSC of VGLUT1, VGLUT2 and VGLUT3 expressing hippocampal neurons were slightly larger, but not significantly different from, control neurons, and were not significantly different from each other ( Figure 1F, left panel). We also measured the size of the charge contained in the RRP by applying 500 mM sucrose ( Rosenmund and Stevens, 1996). Again all three VGLUT isoforms rescued the severe deficit seen in both VGLUT1−/− hippocampal neurons ( Figures 1C and 1F) and VGLUT2−/− thalamic neurons (data not shown) to levels not significantly different from VGLUT+/+ thalamic neurons, suggesting that the three isoforms perform the basic function of filling synaptic vesicles with glutamate in a similar manner.