A1r si confocal laser

Images were obtained from one set of brain sections (1/6th of the brain) for each immunostaining. For single stainings (c-Fos or GFP), brain regions of interest were identified at various Bregma coordinates. Images were acquired bilaterally with an epifluorescence microscope (Nikon) using a 10× objective. High-resolution reconstruction was achieved by combining multiple images with overlapping fields of view (NIS Elements software). Quantifications were performed manually using an image analysis software (ImageJ 1.49v, NIH), taking into account cells with immunofluorescence above background. For dual immunostainings (PV co-labeled with tdTomato, GFP co-labeled with mCherry, c-Fos co-labeled with SST), z stacks images were acquired bilaterally with a Nikon A1R Si confocal laser, a TiE inverted research microscope using a 20× objective. Images (1024 resolution) were acquired as 14 μM z stacks with a step size of 2 μM. For c-Fos intensity in SST cells (Figure S7), we measured c-Fos immunoreactivity in SST cells and expressed the data as a percentage of background in the same field of view. All analyses were performed by an investigator blinded to treatment and/or genotype.

Images were obtained from one set of brain sections (1/6^th of the brain) for each immunostaining, except for Rabies tracing analysis which was performed on the whole brain. For single stainings (c-Fos, tdTomato, GFP, CB, SST, CR, PV, NPY, Chat,), brain regions of interest were identified at various Bregma coordinates. Images were acquired bilaterally with an epifluorescence microscope (Nikon) using a 10x objective. High-resolution reconstruction was achieved by combining multiple images with overlapping fields of view (NIS Elements software). Quantifications were performed manually using an image analysis software (ImageJ 1.49v, NIH), taking into account cells with immunofluorescence above background. For dual immunostainings, z-stacks images were acquired bilaterally with a Nikon A1R Si confocal laser, a TiE inverted research microscope using a 20x objective. Images (1024 resolution) were acquired as 14 μM z-stacks with a step size of 2 μM. For c-Fos intensity in SST cells (Supplementary Fig. 9l-n), we measured c-Fos immunoreactivity in SST cells and expressed the data as a percentage of background in the same field of view. All analyses were performed by an investigator blinded to treatment and/or genotype.