Viral and compound 21 (C21) injections

At PN10, wildtype and LE-Tg (Pvalb-iCre)20ttc male and female pups were anesthetized with isoflurane mixed with oxygen. The skull was exposed and holes were drilled in the skull bilaterally above the dHPC. A Hamilton syringe with a 33-gauge needle mounted onto a nanopump (KD Scientific, Holliston, MA) was stereotactically inserted into the dHPC (2.3 mm posterior to bregma, 2.0 mm lateral from midline and 2.8 mm ventral). AAV9/hSyn-DIO-hM4Di-mCherry, AAV9/hSyn-DIO-hM3Dq-mCherry, or AAV9/hsyn-DIO-mCherry (2.3 × 10¹³ genomic copies/mL, 1 μL per side; Addgene) was microinjected at a rate of 0.2 μL/min. The needle was left in place an additional 6 min following microinjection to ensure complete diffusion of the AAV and then slowly retracted. The scalp was sutured. After recovery from the surgery, pups were returned to the dam and littermates for a 7-day recovery period prior to experimental manipulations. C21 (HelloBio, cat# HB6124) was dissolved in PBS pH 7.4 and 0.5mg/kg injected intraperitoneally, 6 h prior to, 30 min prior to, or immediately after experimental manipulations. This dosage of C21 is known to activate both DREADD (hM4di/hM3dq) receptors and does not cause any off-target effects (Thompson et al., 2018; Jendryka et al., 2019; Goutaudier et al., 2020). After behavioral experiments, the rats were anesthetized with an i.p. injection of 750 mg/kg chloral hydrate and transcardially perfused with 4% paraformaldehyde in PBS pH 7.4, and their brains were postfixed in this solution overnight at 4°C, followed by PBS pH 7.4 with 30% sucrose for at least 48 h. We collected 35-μm brain sections by cryosection. The sections were then incubated with the blocking solution (PBS pH 7.4 with 0.25% Triton X-100, 4% normal goat serum, 1% BSA) for 2 h at room temperature. Then, stainings were performed diluted in the blocking solution for 48 h at 4°C: rabbit anti-parvalbumin (1:10,000, Abcam, cat#ab11427) or rabbit anti-cFos antibodies (1:500, Cell Signaling Technology; mab#2250). Subsequently, the brain sections were stained with goat anti-rabbit Alexa Fluor 488 (1:800, Invitrogen, cat# A11029) for 2 h at room temperature and mounted with Prolong Diamond antifade mountant with DAPI (Invitrogen, cat# {"type":"entrez-protein","attrs":{"text":"P36962","term_id":"547832","term_text":"P36962"}}P36962). Images were collected by an Olympus VS120 virtual slide microscope (Olympus, Tokyo, Japan) and Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) under nonsaturating conditions. Quantification was performed using ImageJ software (US National Institutes of Health) by experimenters blind to the experimental conditions. All images of an experiment were processed using the same parameters to remove background and outlier noise. Regions of interest (ROIs) were manually drawn around mCherry, parvalbumin, or cFos expressing cells by an experimenter blind to treatment conditions, and ROIs were then used as masks to overlay and quantify parvalbumin/mcherry or mCherry/cFos colocalization in the exact same location.