Quantifying Co-localization of Neuronal Markers

For cFos, GFP, and Npas4 co-localization analyses, flat images were obtained using a 10x objective on a Leica microscope with a Leica DFC450 C digital camera (28 (link)). All images were converted to binary format, and watershed segmentation was used to separate overlapping cells. Cell counts from hippocampal subregions were performed using two sections per mouse (1.8 mm posterior to bregma and 2.2 mm posterior to bregma) and averaged between two sections. Within hippocampal sections, a 200 μm² area of DG, a 200 μm² area of CA3, and a 100 μm² area of CA1 were defined. Only cells in a specific size range were counted, and a constant threshold was used to distinguish cells based on maximal signal to noise ratio. JACoP (Image J Plugin) object-based co-localization was used to determine co-localization by comparing the position of the centroids of the nuclei of the color channels. Their respective coordinates were then used to define structures separated by distances equal to or below the optical resolution. All quantifications were performed by an experimenter blind to treatment condition.