Ab11427

Frontal cortex, anterior pituitary, and posterior pituitary from 3-mo-old Sprague Dawley rats were homogenized separately with a Dounce homogenizer in 300 µl of lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% IGEPAL CA-630; I8896; Sigma-Aldrich), with protease inhibitor cocktail (P8340; Sigma-Aldrich) and 1 mM PMSF. The homogenates were sonicated for 5 min on ice and centrifuged at 16,000 g for 20 min in 4°C to collect the supernatant. Protein concentrations were determined by BCA assay and adjusted to 1 µg/µl for all samples. A 15-µl aliquot of each sample was resolved on 15% SDS-PAGE gel, transferred to nitrocellulose membranes, and probed with rabbit anti-calbindin D28K (1:1,000, ab108404; Abcam), rabbit anti-calretinin (1:500, ab92341; Abcam), and rabbit anti-parvalbumin (1:1,000, ab11427; Abcam) antibodies, followed by secondary horse radish peroxidase conjugated antibody, and developed with enhanced chemiluminescence (Pierce Chemicals). The same membrane was then stripped with stripping buffer (1.5% glycine, 0.1% SDS, and 1% Tween 20, pH 2.2) for 15 min at room temperature, probed with mouse anti-actin (1:2,000; Abcam), goat anti-oxytocin (1:1,000, EB09854; Everest), or guinea pig anti–growth hormone antibodies (1:2,000). The experiments were repeated twice.

Immunohistochemistry was performed to validate the expression of AAV1-hSyn-GCaMP6f, AAV8-hSyn-hM3D(Gq)-mCherry, and the proportion of PV neurons in the population of GCaMP6f-positive neurons driven by synapsin promotor. Animals were deeply anesthetized with ketamine and xylazine and transcardially perfused with 4% paraformaldehyde solution in PBS. Fixed brains were extracted from the skull and equilibrated in 30% sucrose solution in PBS. The brains were cut into 30-μm-thick sections using a microtome (Leica Microsystems, Wetzlar, Germany). After blocking and permeabilization for 1 hour in 5% bovine serum albumin and 0.5% Triton X-100 in PBS, the slices were incubated at 4°C overnight with primary antibody diluted in PBS. After PBS wash, slices were subsequently incubated with secondary antibody in PBS at room temperature for 3 hours. Slices were mounted on glass slides in Fluoromount-G (Southern Biotech, Birmingham, AL). Fixed tissue was imaged using a Zeiss LSM510 Meta confocal microscope (Carl Zeiss) with a 20× objective (N.A. 1.0; Carl Zeiss). The following antibodies were used for staining: anti-PV antibody (1:500; ab11427, Abcam) and goat anti-rabbit Alexa Fluor 647 (1:500; A-21245, Thermo Fisher Scientific).

To confirm the loss of PNNs, rats were transcardially perfused with saline, followed by formaldehyde, and brains were postfixed and cryopreserved for at least 24 hours. Coronal sections (50 μm) through the vHipp were blocked (2% normal goat serum), incubated with lectin from Wisteria floribunda (4 μg mL^-1), then AlexaFluor594 conjugated to streptavidin. A subset of sections was also incubated with a rabbit anti-PV antibody (Abcam:AB11427), then anti-rabbit AlexaFluor488. Sections were mounted and coverslipped with Prolong Gold Antifade reagent (Molecular Probes). The representative images were acquired using an Olympus IX81 Motorized inverted confocal microscope and FV10-ASW software and enhanced using ImageJ.

Mice were perfused with 4% PFA, sliced on a cryostat at 40 μm, and immunolabeled with rabbit anti‐NeuN (1:500, #24307; Cell Signaling), rabbit anti‐PV (1:500, #ab11427; Abcam), rat anti‐CTIP2 (1:500, #ab18465; Abcam), mouse anti‐MBP (1:500, #ab62631; Abcam), and secondary antibodies were Invitrogen, 1:500, as previously described.²¹ Confocal images were taken on Nikon Eclipse Ti‐U at 10x/0.45 NA or 20x/0.95 NA, 1024 × 1024 or 512 × 512 frame size, 5 μm z‐interval. Myelinated axons were imaged on Leica SP8 STED at 93x/1.3 NA, glycerol. GFP FLEX callosum fibers were imaged on CSU‐W1 SoRa Yokogawa Spinning Disk Confocal, Nikon at 100x/1.35 NA, silicon, or 60x/1.49 NA, oil, 2304 × 2304, 0.5 μm step size. Excitation lasers were 405, 488, and 561. Imaris version 9.8 (Bitplane) was used for visualization and Adobe Photoshop CC for figure display.
We injected AAV9 CamKII0.4.eGFP.WPRE.rBG (#105541‐AAV9; Addgene), Cre‐driven AAV9 pCAG‐FLEX‐EGFP‐WPRE (#51502‐AAV9; Addgene), or green retrograde Lumafluor RetroBeads IX in the right premotor cortex of TRAP2 or C57Bl/6 mice (AP +2.6 mm, ML −1.8 mm, DV −0.5 mm; 100 nl) or in the right VL (anterior VL: AP −1.05 mm, posterior VL: AP: −1.40 mm, ML −1.00 mm, DV −3.70 mm) with Hamilton syringe (#7634‐01) and allowed 2 weeks for the viral expression.