3 mm round glass coverslip

Mice were implanted with cranial windows at P10 using a modification of established adult cranial window protocols^{58 ,59} . Briefly, mice were anesthetized with nebulized isoflurane (2% induction, 1.5% maintenance) in 30% O₂ / 70% air. Body temperature was monitored and maintained between 36.5–38 C, and carprofen and dexamethasone were administered at 5 mg/kg and 0.1 mg/kg, respectively. Hair, skin and periosteum were removed from the skull, and a 2 mm round craniotomy was performed with a #0 dental burr. Craniotomies were positioned to allow imaging of primary somatosensory cortex⁶⁰ . A 3 mm round glass coverslip (Warner Instruments) was placed over the craniotomy and attached with cyanoacrylate glue (Vetbond, 3 M). Dental acrylic (Lang Dental) sealed the edges of the coverslip and skin. A small metal stabilization bar was implanted to later secure the mouse to the microscope stage for imaging. After recovery from anesthesia, pups were returned to their mothers or to foster mothers. Use of experienced outbred foster mothers reduced cannibalism. Cranial window implants were well-tolerated, did not appear to interfere with activity or development of the mice, and did not induce inflammation (Supplementary Fig. 3). If bleeding was observed or windows were unclear, animals were not used for imaging.

Mice were anesthetized with a ketamine/xylazine cocktail (8.0 mg ketamine + 0.6 mg xylazine/ml, 10 ml/kg, i.p.). Standard aseptic sterile surgery techniques were applied for survival surgeries. The animal was kept on a thermal pad to maintain a body temperature of 37°C. The animal was head-fixed in a stereotaxic frame with non-penetrating ear bars. Before the initial incision, the hair was removed, and the scalp was cleaned with 70% ethanol and 10% povidone-iodine solution. The scalp was removed. The injection coordinates over vS1 (1.6 mm posterior, 3.3 mm lateral) and/or vM2 (1.5 mm anterior, 0.5 mm lateral) relative to bregma were measured. A burr hole was drilled at the injection sites. An injection pipette (inner diameter ∼260 μm, tip diameter ∼15 μm, BRAND) was then filled with 400 nl of AAV1-Syn-GCamp6s-WPRE-SV40 solution (Addgene). The pipette was slowly inserted into the burr hole ∼300 μm below pia. The virus solution was infused by a micropump at a flow rate of 0.9 μl/min. A craniotomy 3 mm in diameter was drilled over the virus injection site. The cortex was cleaned and covered with a 3 mm round glass coverslip (Warner Instruments). Dental cement (C&B Metabond, Parkell) was then used to seal the coverslip to the skull and to cement a head plate onto the skull.