Fluoromount

Selected sections at the level of the SuM were first treated with 1% H₂O₂ rinsed in PB mounted on SuperFrost Plus slides (Fisher Scientific) and air dried at RT. They were then processed for fluorescent RNAscope in situ hybridization according to the manufacter’s protocol (Advanced Cell Diagnostics). Briefly, sections were treated with 100% ethanol and protease III for 30 min at 40 °C. They were incubated in a solution containing both RNAscope^® Probe-Mm-S1c17a6 for detection of VGLUT2 mRNA and Mm-S1c32a1-C3 for detection of VGAT mRNA. After hybridization, sections were processed for visualization using the RNA-scope Multiplex Fluorescent reagent Kit v2 (Advanced Cell Diagnostics) and the Tyramide Signal Amplification (TSA™) Plus Cyanine 3 and TSA Plus Cyanine 5 systems (Perkin Elmer).
After the RNAscope assay, sections were rinsed in KPBS and processed for immnohistofluorescent detection of the RV using the MOM kit as described above. After the overnight incubation in the mouse monoclonal antibody directed against RV (1:3000), sections were rinsed in KPBS and incubated for 2 h in Alexa488-conjugated donkey anti-mouse (1:200; Invitrogen) diluted in MOM diluent. After several rinses in KPBS, all sections were coverslipped with Fluoromount (Electron Microscopy Sciences). The specimens were analyzed with a Zeiss laser-scanning confocal microscope.

For labeling of PV- and CB1R-positive inhibitory synapses, P14 mice were stereotaxically injected with AAV-Cre-GFP into CA1 of the right hemisphere and AAV-RFP into CA1 of the left hemisphere. After 4 days of recovery from surgery and 4–7 days in an enriched environment or standard housing, mice were anesthetized briefly with isoflurane and decapitated. Hippocampi were rapidly dissected in ice-cold dissection media consisting of (in mM): 1 CaCl₂, 5 MgCl₂, 10 glucose, 4 KCl, 26 NaHCO₃, 218 sucrose, 1.3 NaH₂PO₄∙H₂O, 30 HEPES. Hippocampi were immediately drop fixed in 4% paraformaldehyde in PBS at 4°C for 2 hr followed by overnight incubation in 30% sucrose in PBS. Cryoprotected tissue was stored in Tissue-Tec O.C.T. at −20°C, sectioned at 20 μM (Leica CM1950 cryostat) and mounted on slides (Superfrost/Plus, Fisher Scientific).
For NPAS4 and inhibitory synapse immunostaining, hippocampal sections were blocked in 5% goat serum and 0.2% Triton X-100 in PBS overnight at 4°C. Sections were incubated in primary antibody overnight at 4°C in blocking solution, washed three times in PBS, and incubated overnight in a species-matched secondary at 4°C, and washed again three times in PBS. Slices were briefly dipped in ddH₂O and cover slipped with Fluoromount (Electron Microscopy Sciences). See 'Key Resources Table' for antibodies and concentrations used for all IHC experiments.

Brains were incubated in 4% paraformaldehyde with 0.1 M phosphate-buffered saline (PBS; pH 7.4) for 24 h, 30% sucrose in PBS for 24 h, and then held in PBS (all at 4 °C). Next, 50 µm coronal sections were prepared with a vibratome and washed × 3 in PBS for 5 min. Sections were incubated in a blocking solution containing PBS with 2% bovine serum albumen and 0.2% Triton X-100 for 1 h before incubation with anti-choline acetyltransferase antibody (1:800 in blocking solution; AB144P, Millipore) overnight at 4 °C. Sections were washed × 4 in PBS for 5 min and incubated in Alexa Flour 488 antibody (1:200; A-11001, Invitrogen) for 2 h at room temperature. Following another round of washes in PBS (5 min × 4), sections were placed on a glass slide and cover-slipped with Fluoromount (Electron Microscopy Sciences) before imaging on a fluorescent microscope (Zeiss).