Smm 100

After inducing deep anesthesia with ketamine/xylazine, the mice were positioned in a stereotactic frame and the skin covering the frontal bone was cut. The tip of a Hamilton syringe (33G) with a stereotaxic micromanipulator (SMM-100; Narishige, Tokyo, Japan) was inserted into the primary motor cortex (coordinates from bregma: anterior and posterior, +1.0 mm; midline, ±1.0 mm; dorsal and ventral, 0.8 mm). Viral solution (1 × 10¹² vg/mL, 0.5 μL) was injected at a rate of 25 nL/min. After the injection, the needle remained in place for an additional 2 min and was withdrawn slowly.

AAV injections were performed when Cre⁻ and Cre⁺ mice were 12–14 weeks old. Animals were anesthetized with isoflurane (3.0% for induction and 2.0–2.5% for maintenance, wt/vol) and placed in a stereotaxic apparatus (Narishige Scientific Instrument) with bregma and lambda skull landmark levels. According to the experimental protocols of Sherman and colleagues^{22 (link)}, two holes were drilled in the skull at predefined coordinates relative to bregma to allow the insertion of glass pipettes containing a mixture of two virus solutions (ChR2: 0.9–1.7 × 1013 genome copies per ml; eNpHR: 1.4 × 1013 genome copies per ml), which were held by a stereotaxic micromanipulator (SMM-100, Narishige) and connected to a Hamilton syringe with a pressure injection system (Motorized Stereotaxic Microinjector, IMS-20, Narishige). Each animal had bilateral injections targeted precisely to a location between the PreBötC and BötC (6.70 mm caudal to bregma, 1.25 mm lateral to the midline, and 4.65 mm ventral from the dorsal surface of the brain) according to the mouse brain atlas⁶⁰ . Once situated, 500 nl per side was slowly injected (100 nl/min) and the pipette was left in place for at least 5 min after injection to minimize backflow. The wound was closed with instant adhesives. AAV injections were performed by an investigator blinded to the animals.

STZ was purchased from Sigma-Aldrich, St Louis, MO, USA. Under anesthesia with 4% isoflurane vapor, mice were fixed using the brain stereotaxic apparatus SR-5M-HT (Narishige, Tokyo, Japan) and administrated a single injection of STZ (6.6 mg/kg, dissolved in saline 5 µl) into the right lateral ventricle with the microinjector, IMS-20, and micromanipulator SMM-100 (Narishige). The stereotaxic coordinates were + 0.3 mm anterior, + 1.0 mm lateral (right) and + 2.5 mm ventral from bregma. After the skin suture, antibiotic ointment (20 mg/g Chloramphenicol, 5 mg/g Fradiomycin, 100,000 U/g Nystatin, Daiichi Sankyo Healthcare Co., Ltd, Tokyo, Japan) was dabbed on the wound. Control sham mice were administrated 5 µl saline into the right lateral ventricle. After the surgery, mice were monitored daily for pain/discomfort and infections in accordance with the guidelines described above. We checked for proper placement of the needle by delivering 7 μl of 5% Trypan Blue Dye (Nacalai, Kyoto, Japan) (Supplementary Figure 1).