Image acquisition and quantification

Immunocytochemical staining for Npas4 experiments was acquired on a Nikon Spinning Disk Confocal CSU-X microscope equipped with an air 20× Plan Apo objective lens (NA = 0.8), controlled via NIS-Elements software (Nikon). Images were then processed using ImageJ (RRID: SCR_003070). ROIs were manually drawn around cell somas. After background subtraction, the mean intensity values of the ROIs were measured and normalized to the untreated control.

hNR1 labeling and staining for inhibitory presynaptic markers were analyzed using a spinning disk confocal microscope (Carl Zeiss Axio Oberserver.Z1 with Andor spinning disk and cobalt, omricron, i-beam laser; Carl Zeiss, Andor) using a 63× (1.4 NA) or 100× (1.4 NA) Plan-Apochromat oil objectives and an iXon ultra (Andor) camera controlled by iQ software (RRID: SCR_014461; Andor). Only dendrites in proximity to the soma were imaged, and inhibitory and excitatory dendrites were distinguished based on GFP signal. Images were processed using ImageJ (RRID: SCR_003070) and OpenView software (written by Prof. Dr. Noam Ziv, Technion Institute, Haifa, Israel). In brief, for synaptic protein levels at GAD65 and VGAT puncta along dendrites were detected with set parameters: 6 × 6-pixel boxes were placed over puncta and the mean fluorescence intensities were measured, followed by subtraction of background values. For hNR1 labeling within excitatory synapses, hNR1-positive puncta were detected in a similar manner and then VGLUT1 intensities within hNR1-positive puncta were measured. A value above a threshold of 5× background intensity was considered VGLUT1-positive and thus synaptic. The remaining hNR1 puncta were considered extrasynaptic. In a similar manner, boxes around detected gephyrin puncta served to measure hNR1 intensity for localization of hNR1 puncta to inhibitory synapses. Z-plane images were analyzed using Volume Viewer plugin in ImageJ. Values of all puncta per region of interest (individual dendrites) were averaged and considered one data point.