63× magnification (n= 4–10 Z planes, 1 μm steps). In situ expression was quantified by smFISH puncta counting in Pvalb+ cells using maximum projections through confocal stacks acquired in Frontal Cortex. Soma were manually segmented based after Gaussian-filtering Pvalb puncta. The resulting regions of interest (ROIs) were used as masks for analysis of other fluorescent channels. ROIs contained DAPI nuclei and exhibited little overlap. Manual segmentation and automated puncta detection/surface area measurements were implemented in CellProfiler 2.1.1 (Carpenter et al., 2006).
