Imunohistochemistry and in situ hybridization

For immunohistochemistry, mice were overdosed with 750–1000 mg kg⁻¹ mixture of Ketamine, Xylazine and perfused transcardially with PBS, followed by 4% cold paraformaldehyde (PFA) in PB. Extracted brains were kept in 30% sucrose in PB at 4°C overnight, then transferred to PBS. A sliding microtome (Leica SM2000R) was used to cut 40-μm coronal slices in PBS. Slices were washed with PBS-T (PBS + 0.2% Triton X-100), then incubated with PBS-T + 20% normal donkey serum, at room temperature for 1 h for blocking. Then, slices were incubated with one or more primary antibodies at 4°C for 24 h (rabbit anti-calretinin AB5054, Millipore, dilution 1:1000; goat anti c-Fos SC-52, Santa Cruz Biotechnology, dilution 1:500; rabbit anti-PV, Abcam, dilution 1:1000; or for electrophysiology slices, Streptavidin Alexa Fluor 488 conjugated (1:1000); rabbit anti HA-tag AB3724, Cell Signaling Technology, dilution 1:1000). Three washes of PBS-T for 10 min each were performed on the slices before 1 h incubation with secondary antibody at 1:1000 dilution (A-21206; A-21447, ThermoFisher Scientific). Slices were washed three more times in PBS-T for 10 min each, stained with 4′,6-diamidino-2-phenylindole (DAPI) to label cell nuclei and mounted with Prolong antifade reagent (Thermofisher) onto microscope slides. For in situ hybridization, Calb2-cre animals were injected hilus with pAAV₅-hSynDIO-mCherry in the ventral hilus. Three weeks later, they were anesthetized with isofluorane, decapitated and their brains removed immediately. The ventral hippocampus was dissected, placed in a Cryomold (Sakura, Japan) filled with O.C.T. Tissue-Tek compound (Sakura, Japan), and stored at −80 °C until 14 μm sections were cut with a cryostat (Microm HM 560, Thermo) and mounted in Superfrost plus slides (Thermo scientific). The whole in situ hybridization procedure was performed with and according to the protocol of the RNAscope Fluorescent Multiplex Assay (ACD) using probes for vGluT1 and mCherry. Slides were imaged in an upright Zeiss LSM 700 confocal microscope. Cytoarchitecture borders for cell counting were defined in accordance with the Allen Brain Institute Atlas, http://atlas.brain-map.org.