GoKawiilMice
All mice were group-housed and experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committees at the Massachusetts General Hospital and the Tufts University School of Medicine and NIH guidelines. All mice were housed in a 12-h (7:00 to 19:00) light–dark colony room at 22–24 °C, 30–50% humidity and with ad libitum access to food and water. Cntnap2−/−, Pvalbcre, Amigo2cre and Rpl22HA mice were obtained from the Jackson Laboratory (strain 017482, 017320, 030215 and 011029, respectively).
Viruses and virus constructs
AAV-S5E2-dTom-nlsdTom plasmid (plasmid 135630), AAV5-CamKIIa-hChR2(H134R)-eYFP (26969), AAV-S5E2-ChR2-mCherry (135634) and AAV PHP.eB virus were purchased from Addgene. AAV-S5E2-XPGs-nlsdTom viruses was generated by subcloning mouse Meis2 (NM_001346036.1), Bcl11a (NM_016707.3), Tbr1 (NM_009322.3) or dSACas9/VP64 into AAV-S5E2-dTom-nlsdTom plasmid. AAV-U6-sagRNA#1#2-Syn1-P2A-mCherry and AAV-Herc1gRNA#1-CAGGAAAAAGCCTGGTCTTCA-#2-AAAACAAATTCATGTGTATGT-Syn1-P2A-mCherry were generated by VectorBuilder. AAV-EF1a-DIO-mCherry virus was purchased from UNC. AAV-EF1a-DIO-Meis2-mCherry virus was generated by VectorBuilder. The Cal-Light plasmids pAAV-pCMV-Myc-TM-KA2-CaM-NES-TEV-N-AsLOV2-TEVseq-tTA, pAAV-hSYN-M13-TEV-C-P2A-tdTomato and pAAV-TRE-eGFP were gifts from the Hyun Laboratory. AAV.PHP.eB viruses were produced by Boston Children’s Hospital Viral Core. Note, because of differences in constructs, S5E2-dTom-P2A-nlsdTom (control) results in dTomato distributed throughout the neuron, whereas S5E2-Meis2-P2A-nlsdTom is localized to the nucleus.
Immunohistochemistry
Mice were injected with viruses 2 weeks before perfusion. Mice were anaesthetized with ketamine and xylazine (10 mg ml–1 and 1.6 mg ml–1, respectively, intraperitoneally) and transcardially perfused with 4% paraformaldehyde (PFA), and their brains were removed and incubated in 4% PFA at 4 °C overnight. Brains were placed in 30% sucrose–PBS for 2 days and then embedded in medium (OCT, Fisher HealthCare). Cryosections (35 μm) were obtained (Leica) and stored in PBS (0.01% sodium azide) at 4 °C. For immunostaining, floating sections were permeabilized, blocked in blocking solution for 2 h (PBS containing 0.3 % Triton X-100 and 10% normal donkey serum) and then incubated with primary antibodies (PBS containing 10% normal donkey serum and 0.1% Triton X-100) at 4 °C overnight. Sections were then washed with PBS 3 times, 10 min each, then incubated with secondary antibodies in PBS for 2 h at room temperature. Sections were then washed with PBS 3 times, 10 min each, mounted on glass slides and coverslipped with DAPI Fluoromount-G (SouthernBiotech).
Image analysis
For PV puncta and SYT2 puncta, images were obtained from three sections per mouse hippocampus blind to treatment and genotype. A Leica SP8 confocal laser microscope and LAS software were used to capture images in the stratum lucidum at high-resolution (2,048 × 2,048 pixels). Single confocal-plane images were captured in the CA2 and CA3ab subfields using a ×63 oil objective plus ×43 digital zoom. For quantification of sample sizes of PV+ puncta and SYT2+ puncta, densities were averaged from 18 images per mouse for CA3 and 12 images per mouse for CA2. Puncta were analysed using the StarDist 2D plugin and particle analysis tools in Fiji ImageJ. Threshold values were held constant across images. For Cal-Light, dCA3a cells were manually counted. To reduce the contribution of background fluorescence, a threshold based on maximizing Yen entropy62 was applied to GFP images during quantification.
Antibodies
The following antibodies were used in the study: PV (rabbit, Swant PV25, 1:5,000, RRID: AB_10000344; goat, Swant PVG213, RRID: AB_2721207, 1:1,000); RGS14 (mouse, NeuroMab 75-170, RRID: AB_2179931, 1:500; rabbit, Proteintech 16258-1-AP, RRID: AB_2179918, 1:500); SYT2 (mouse, Abcam AB154035-1001, RRID: AB_2916272, 1:250); RFP (rabbit, Rockland 600-401-370, RRID: AB_2209751, 1:1,000; goat, Sicgen AB1140-100, RRID: AB_2877097, 1:500); FOS (guinea pig, Synaptic Systems 226-004, RRID: AB_2619946, 1:3,000); GFP (chicken, Invitrogen A10262, RRID: AB_2534023, 1:500); gephyrin (rabbit, Synaptic Systems 147-008, RRID: AB_2619834, 1:500); MEIS2 (rabbit, Protein Tech 11550-1-AP, RRID: AB_2143028, 1:400); BCL11A (mouse, Abcam ab19487, RRID: AB_444947, 1:500); TBR1 (rabbit, Abcam ab31940, RRID: AB_2200219, 1:200); GFAP (Chicken, Millipore AB5541, RRID:AB_177521, 1:2,000); IBA1 (rabbit, FujiFilm 019-19741, RRID: AB_839504, 1:500); and SST (mouse, Santa Cruz G-10, RRID: AB_831726, 1:500). The following fluorescent-label-coupled secondary antibodies from Jackson Immuno Research were used at 1:500 dilution: Alexa-Fluor-488-conjugated donkey anti-rabbit IgG (711-545-152); Cy3-conjugated donkey anti-rabbit-IgG (711-165-152); Alexa-Fluor-488-conjugated donkey anti-mouse-IgG (715-545-151); Cy3-conjugated donkey anti-mouse-IgG (715-165-151); Alexa-Fluor-488-conjugated donkey anti-chicken-IgG (703-545-155); Cy3-conjugated donkey anti-goat-IgG (705-165-147); 647-conjugated donkey anti-guinea pig-IgG (706-605-1481); and Cy3-conjugated donkey anti-guinea-pig-IgG (706-165-148).
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