To measure paired pulse ratios (PPR), pairs of 20 Hz and 40 Hz were delivered and averages of at least 6 sweeps were analyzed. PPR was calculated as a ratio of EPSC2/EPSC1. Animals (postnatal day 21, mSYD1AKO and wild-type littermates) were transcardially perfused with fixative (2% paraformaldehyde,

2% glutaraldehyde Selleck Paclitaxel in 100 mM phosphate buffer [pH 7.4]) and brains were postfixed for 1 hr. Tissues were sectioned coronally at 60 μm thickness in PBS on a vibratome. Sections from the same front-caudal brain region (Bregma −1.9) were analyzed for each genotype. Sections were washed in 0.1 M cacodylate buffer [pH 7.4], postfixed in 0.1 M reduced osmium (1.5% K4Fe(CN)6, 1% OsO4 in water) and embedded in Epon resin. The stratum radiatum of

area CA1 was identified using the pyramidal cell layer and the alveus as landmarks. Images were acquired on a Transmission Electron Microscope (Fei Morgagni, 268D). Quantification of the number and distribution of vesicles was performed using XtraCount software (developed by C. Olendrowitz, Göttingen, Germany). All image acquisition and analysis was done blinded with respect to the genotype of the animals. Independent data sets were collected from 4 KO and 4 wild-type animals. For each animal, at least 35 9.3 μm2 fields were acquired and at least 83 synapses analyzed. The total number of synapses quantitatively analyzed was 404 for wild-type and 366 for KO material. Images were acquired on a LSM5 confocal Galunisertib research buy microscope (Zeiss) and assembled using Adobe Photoshop and Illustrator software. For the analysis

of dendritic arborizations, neurons were traced and analyzed with Neurolucida (MBF Bioscience). The identification of axons versus dendrites is based on the unique characteristics of cerebellar granule cells, which exhibit 3–5 short dendritic processes these and a thinner, much more elongated axon. Colocalization analysis of proteins in COS cells was performed by the Pearson’s coefficient method computed on fluorograms, using the JaCOP plugin in ImageJ (Bolte and Cordelières, 2006). Quantification of pre- and postsynaptic proteins in granule cells was performed by a wavelet-based segmentation method, using the Multidimensional Image Analysis module (Racine et al., 2007 and Izeddin et al., 2012), run in Metamorph software (Molecular Devices). Puncta on different channels were segmented and counted by thresholding the third wavelet map with a value ranging from 15 to 35 times the noise standard deviation. Some images for figures were processed by deconvolution using a theoretical PSF, a signal/noise ratio of 10 for each channel and 30 iterations of the deconvolution algorithm (Huygens remote manager v2.1.2).