Hamilton syringe

Mice were anesthetized with isoflurane and placed on a stereotaxic frame (Neurostar, Tübingen, Germany) for viral injection. Two AAV mixtures were injected into the mPFC for gene overexpression; AAV1.CamKII0.4.eGFP.WPRE.rBG (serotype 2.1, ddTiter: 6.028e13 GC/ml) and AAV-EFα1-loxP-OGA-HA-loxP (serotype 2, ddTiter: 8.59e12 GC/ml) mixture was used for the control. 0.2 μL of the mixture was injected to a targeted site for control condition. CamKII.HI.eGFP-Cre.WPRE.SV40 (serotype 2, ddTiter: 1.76e13 GC/ml) and AAV-EFα1-loxP-OGA-HA-loxP mixture was used for the experimental group. 0.45 μL of the mixture was injected to a targeted site for the overexpression condition. Virus was delivered into the prelimbic mPFC (AP: + 2.0 mm ML: ± 0.3 mm DV: −2.3 mm) using a 10-mL Hamilton syringe fitted with a 33-gauge needle (World Precision Instruments, Sarasota, FL, USA). Mice were subsequently returned to a home cage to recover, and used for electrophysiology, immunohistochemistry or behavioral experiments 2–3 weeks after the surgery.

Mice were anesthetized by intraperitoneal (i.p.) injection of 100 mg/kg ketamine and 10 mg/kg xylazine. A small burr hole was drilled in the skull and mice were stereotactically injected with a mixture of AAV1-Mecp2-Cas9 (5 × 10⁹ vg) and AAV1-U6-gRNA^Mecp2-hSyn-GFP or AAV1-U6-gRNA^Dnmt3b-hSyn-GFP (3 × 10⁹ vg into the left ventral dentate gyrus (coordinates: anterior/posterior (y): −3.5, mediolateral (x): + 2.7, dorsal/ventral (z): −3). We injected using a Hamilton syringe fitted with a 30 gauge needle at a rate of 0.2 µl/min using a pump and controller (World Precision Instruments, Sarasota, FL). Six weeks after injection, genomic DNA was isolated directly from hippocampus using the Qiagen Blood and Tissue Kit (Qiagen). Briefly, we added 360 μL of ATL buffer and 40 μL of proteinase K to dissected hippocampus tissue. Samples were then digested for 5 min at 56 °C and were homogenized using disposable pestles. Next, samples were digested for another 10 min at 56 °C. After vortexing, we added 400 μL of the AL buffer from the kit and incubated the samples for another 5 min at 56 °C. After the addition of 400 μL of 100% ethanol, we purified the genomic DNA according to the manufacturers’ instructions.