Model 963

For specific infection and glycoprotein complementation of pseudotyped RV-∆G, helper viruses (120 nl, 1:1 mixture of AAV2/8-CAG-Flex-TVA-mCherry and AAV2/8-CAG-Flex-oG) were stereotaxically injected into the lateral part of the facial motor, hypoglossus, or trigeminal motor nuclei using a stereotaxic instrument (model 963, David Kopf Instruments, Tujunga, CA, USA) 3 weeks or longer after the peripheral tissue injection. The viruses were injected at the rate of 30 nl/min with the injection system described above. The stereotaxic coordinates used were for the lateral part of the FN: 5.8 mm posterior, 1.38 mm lateral to the Bregma, and 5.2 mm below the brain surface; for the hypoglossus nucleus: 5.8 mm posterior, 0.05 mm lateral to the Bregma, and 5.15 mm below the brain surface with an anteroposterior 20° angle from vertical; and for the trigeminal motor nucleus: 4.1 mm posterior, 1.27 mm lateral to the Bregma, and 4.6 mm below the brain surface with an anteroposterior 20° angle from vertical. Before suturing the skin, the craniotomy was filled with kwik-sil (World Precision Instruments, Inc, Sarasota, FL, USA) and covered with cyanoacrylate glue (Super Glue, Loctite, Westlake, OH, USA).

Mice aged 44–49 days were anaesthetized with 1%–2% isoflurane in oxygen and injected with analgesia (Carprofen, 5 mg/kg IP). Ophthalmic ointment (Alcon, UK) was applied to the eyes and sterile saline (0.1 ml) injected subcutaneously as needed to maintain hydration. A circular piece of scalp was removed and the underlying skull was cleaned and dried. A custom machined aluminum head-plate was cemented onto the skull using dental adhesive (Superbond C&B, Sun Medical, Japan). Three miniature female connectors (853-87-008-10-001101, Preci-Dip, Switzerland) were fixed to the implant with dental adhesive to enable stable connection of two cameras and an inertial measurement unit (IMU) sensor during experimental sessions. The positions and angles of the two eye tracking cameras and IR mirrors were adjusted using a stereotaxic instrument (Model 963, Kopf Instruments, USA) to align the view of the camera with the eye axis. Additionally, the pitch and roll axes of the IMU sensor were aligned to coincide with the plane spanned by the horizontal eye axes. Mice were allowed to recover from surgery for at least five days and handled before the experiments began.

Extracellular recordings were implemented using 32-channel 10-mm single shank electrodes, where electrode sites were arranged in three columns, spaced 50 microns apart from each other (A1x32-Poly3-10mm-50-177, NeuroNexus, Ann Arbor, MI, USA). Electrodes were inserted into the rodent skull using a small animal stereotaxic frame with 10-micron precision manipulators (Model 963, David Kopf Instruments, Tujunga, CA, USA). Electrodes were inserted at a 40-degree angle in the sagittal plane.
Rodents were sedated using either ketamine/xylazine cocktail for results presented in Figs. 3b–g and 4 or isoflurane for results in Figs. 3a, 5, 6, and 7. All animals were skin prepared with hair removal gel. Rodent heart rate, respiration rates and rectal temperatures were monitored throughout recordings. Cranial windows, 1–2 mm in diameter, were opened in the skull using a high-speed micro drill (Model 1474, David Kopf Instruments, Tujunga, CA, USA) under stereotaxic surgery assisted with microscope system (V-series otology microscope, JEDMED, St. Louis, MO, USA). Skull suture lines were used to identify brain structure locations. Recordings were obtained using the NeuroNexus Smartbox recording system (20 kHz sampling frequency, NeuroNexus, Ann Arbor, MI, USA) and the TDT Neurophysiology Workstation (24 kHz sampling frequency, Tucker-Davis Technologies, Alachua, FL, USA).