Synaptic puncta quantification

Synapse quantification was calculated using custom-written plugin, Puncta Analyzer as described before [59]. Briefly, cells in primary and co-cultured neurons were fixed in 4% PFA and co-immunostained for synaptophysin (SYP), PSD-95 and MAP2. Confocal images were taken using a Leica confocal microscope SP8 (Leica Microsystems Inc.), 63X oil-immersion objective lens at 1024 × 1024 pixel resolution 2X zoom and zoom factor 1.5 corresponding to a voxel dimension 0.13 µm × 0.13 µm × 0.32 µm in X, Y, and Z plans. Cell bodies were centred in the field of view and Z-stack dimensions were set manually by tracking MAP2-positive neurons including dendrites and soma. Synapses were quantified by analysing co-localization of SYP and PSD-95 puncta using a custom based plug-in Puncta Analyzer in ImageJ. Background was removed separately from the red and green channel using the rolling ball background subtraction algorithm, then thresholding was performed for detecting discrete puncta, and puncta were identified in red and green channels using the Puncta Analyzer plugin. Co-localized puncta were counted as a synapse. Three independent experiments were performed. For each independent experiment, 15 to 20 neurons from at least two coverslips (or one 4-well chamber slide) were analysed per experimental conditions. For each independent culture, similar regions of interest (all dendrites of neurons including soma) were assessed for co-localization and multiple dendritic fragments were sampled within a particular neuron.