This could be accomplished by altering the curvature of synaptic vesicles, altering the timing of membrane scission, or altering the internalization of endocytic cargo (Bai et al., 2010, Jao et al., 2010 and Suresh and Edwardson, 2010). Microisland cultures of E17.5 striatal, hippocampal, and thalamic neurons Screening Library order were prepared according to published procedures (Pyott and Rosenmund, 2002). VGLUT1 knockout mice were described previously ( Wojcik et al., 2004). VGLUT2 knockout mice were also described previously

( Moechars et al., 2006). All procedures to maintain and use these mice were approved by the Institutional Animal Care and Use Committee for Baylor College of Medicine and Affiliates. Cultures were plated on astrocytes derived from DAPT P1 cortex tissue at a density of 2000–3000 neurons per 35 mm dish. Extracellular solution contained 140 mM NaCl, 2.4 mM KCl, 10 mM HEPES, 10 mM glucose, 4 mM MgCl2, and 2 mM CaCl2, pH 7.3 (305 mOsm). Internal

solution contained 136 mM KCl, 17.8 mM HEPES, 1 mM EGTA, 0.6 mM MgCl2, 4 mM ATP, 0.3 mM GTP, 12 mM creatine phosphate, and 50 U/ml phosphocreatine kinase. All experiments were performed at room temperature (23°C–24°C). Whole-cell voltage-clamp recordings were performed on neurons from control and experimental groups in parallel on the same day (9–14 in vitro). Action potential-evoked EPSCs or IPSCs were triggered by a 2 ms somatic depolarization to 0 mV. RRP size was determined by integrating

the transient synaptic current induced by a 4 s application of hypertonic sucrose solution directly onto the neuron. To obtain Pvr, we recorded the basal evoked synaptic responses and the response to the hypertonic sucrose solution successively from the same neuron. The evoked response was integrated for 1 s to calculate the charge transfer. Pvr was calculated as the ratio of evoked response charge to RRP charge. Short-term plasticity was examined either by evoking 50 synaptic responses at 10 Hz or 2 responses with a 20 ms interval in standard external solution. Data were analyzed offline by using AxoGraph X 1.0 (AxoGraph Scientific, Sydney, Australia) and KaleidaGraph Dipeptidyl peptidase (Synergy Software, Reading, PA). Values for analysis were pooled from at least two independent cultures. Statistical significances were tested by using Student’s t test for two groups with normal distribution, the nonparametric Kolmogorov-Smirnov test for two groups that were not normally distributed, or one-way ANOVA with a Student-Newman-Keuls post hoc test for three or more groups. For values reported as normalized, the average value of the control group (either wild-type neurons or neurons rescued with the wild-type isoform) was calculated for each day and then used to normalize individual neuron values from each group for that day.