Small animal stereotactic frame

Female severe combined immunodeficient hairless outbred (SHO) mice (weight 20 to 27 g) were obtained from Charles River (Sulzfeld, Germany) and were 7 to 9 weeks of age at initiation of experiments. Mice were anesthetized with i.p. injection of 100 mg/kg ketamine (WDT, Garbsen, Germany) and 10 mg/kg xylazine (Rompun®, Bayer Vital GmbH, Leverkusen, Germany). Scalp was disinfected and removed over the whole right hemisphere of the brain to expose the cranium. The periosteum was removed by a bone scraper. Next, a 0.7 mm burr hole in diameter was drilled 2 mm right of midline and 0.5 mm posterior to bregma with a dental drill. Mice were then placed in a small-animal stereotactic frame (David Kopf Instruments, Tujunga, CA) to allow proper cell inoculation. A 25 μl 22-gauge needle (Hamilton, Höchst, Germany) filled with D54-Caspase-3/7 GloSenor cell suspension (8×10⁷ cells per ml) was adjusted in a 60 degree ankle over the burr hole and penetrated 3 mm into the brain tissue. Three μl of the cell suspension (240,000 cells) was slowly injected into the brain. After injection, needle was slowly withdrawn and hole was sealed with Cyano veneer tissue glue (Hager Werken, Duisburg, Germany). All animal studies were approved by the local government (file number 55.2-1-54-2532.2-42-11, Government of Upper Bavaria).

Mice were anesthetized with a mixture of ketamine (80mg/kg) and xylazine (10 mg/kg) injected intraperitoneally (i.p.), and placed on a small animal stereotactic frame (David Kopf Instruments). Recombinant Hcrt::ChR2-mCherry or control Hcrt::mCherry lentivirus (1 μl), or DIO-ChR2-eYFP or control DIO-eYFP AAV virus (0.2 μl; Fig. 5a) were bilaterally injected within the LHA (anteroposterior (AP), 1.5 mm; mediolateral (ML), ± 1.13 mm; dorsoventral (DV) 5.20 mm) through a 33 gauge injector cannula (Plastics One) using a syringe pump (World Precision Instruments) at a rate of 0.1 μl min⁻¹. The injection cannula was left in place for 15 min following the injection and then slowly removed. After the viral injection, a 26 gauge bilateral cannula (Plastics One) with 2 mm between individual cannulae and 5 mm shaft length was slowly lowered into the brain, placed above the LHA (AP, 1.5 mm; ML, ± 1.0 mm; DV, 4.7 mm), and affixed to the skull with C&B Metabond (Parkell) and dental acrylic. To allow time for recovery after surgery and viral expression, we housed animals for 2 to 3 weeks following injection in individual cages before any experiments were initiated. Optical fibers (200 μm, Thorlabs) were inserted into the implanted cannula guide, extending 0.3 mm below the guide, at least 3 days prior to the experimental procedures.