, 2010), using calcium phosphate, and cell-culture supernatants containing the viruses were collected 48 hr after transfection and directly used for infection of neurons. All steps were performed under level II biosafety conditions. Neurons were fixed and permeabilized at −20°C in 100% methanol, incubated with antisynaptobrevin-2 (mouse monoclonal; CL69.1, Synaptic Systems)

and antisynapsin (rabbit polyclonal; E028) primary antibodies in PBS with 4% BSA and 1% goat serum, washed, and stained with monoclonal antisynaptobrevin-2 and polyclonal antisynapsin and visualized using Alexa Fluor 633 goat antimouse and Alexa Fluor 546 goat antirabbit secondary antibodies (Molecular Probes). Images were acquired by using a Leica DMIRE2 confocal microscope equipped with a 63× oil-immersion objective with numerical aperture of 1.32. Identical learn more settings selleck inhibitor were applied to all samples in each experiment. Stacks of z-section images were acquired and converted to maximal projection images by using Leica Confocal Software, and analyzed blindly with ImageJ 1.44p software (NIH, Bethesda). Images were thresholded by intensity to exclude the diffuse/intracellular pool, and then puncta were quantified by counting the number of suprathreshold areas of sizes between 0.25 and 4 μm2. Pearson’s correlation coefficients

were calculated using ImageJ plugin of Mander’s coefficients. Representative images were merged using ImageJ, with presynaptic terminals (visualized via synapsin staining) presented in red and synaptobrevin-2

in green. Electrophysiological ADP ribosylation factor recordings were performed in whole-cell patch-clamp mode using concentric extracellular stimulation electrodes (Yang et al., 2010). Evoked synaptic responses were triggered by a bipolar electrode placed 100–150 μm from the soma of neurons recorded. Patch pipettes were pulled from borosilicate glass capillary tubes (Warner Instruments) using a PC-10 pipette puller (Narishige). The resistance of pipettes filled with intracellular solution varied between 3 and 5 MOhm. After formation of the whole-cell configuration and equilibration of the intracellular pipette solution, the series resistance was adjusted to 8–10 MOhm. Synaptic currents were monitored with a Multiclamp 700B amplifier (Molecular Devices). The frequency, duration, and magnitude of the extracellular stimulus were controlled with a Model 2100 Isolated Pulse Stimulator (A-M Systems) synchronized with Clampex 10 data acquisition software (Molecular Devices). The whole-cell pipette solution contained (120 mM CsCl, 5 mM NaCl, 1 mM MgCl2, 10 mM HEPES, 10 mM EGTA, 0.3 mM Na-GTP, 3 mM Mg-ATP, and 5 mM QX-314 (pH 7.2, adjusted with CsOH). The bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.