Stereotaxic surgery for virus injection and optical fiber implantation

Mice were anesthetized by inhalation of a 3% isoflurane mix in O₂ gas (produced by the CombiVet animal gas anesthesia system; Rothacher Medical, Switzerland) and maintained under inhalation of 1 to 1.5% isoflurane in O₂. Mice were then placed in a stereotactic apparatus (Kopf Instruments, model 940, USA) on a heating pad (Harvard Instruments, USA) and local subcutaneous analgesia using 2% lidocaine was applied. The coordinates of the craniotomy were determined by a mouse brain atlas (49). Following exposure of the skull, a hole was drilled on each side with a 0.5-mm bore (Komet Dental, Germany) using a Tech2000 drill handpiece (Ram Products Inc, USA). Viral suspension was microinjected using 10- to 20-μm tip diameter glass pipettes (PCR capillaries, Drummond Scientific, USA; pulled on a P-97 puller, Sutter Instruments, USA) and an oil hydraulic micromanipulator (MO-10, Narishige, Japan) at the following coordinates (AP, posterior from the bregma/ML, ± lateral from the midline/DV, ventral from the skull surface at bregma, in mm): for insula: −0.9/±3.9/−3.8; for LA: −1.15/±3.45/−4.45; for CeA: −1.22/±2.65/−4.70. For in vivo optogenetic manipulations, we implanted optical fibers bilaterally in the skull as described by (50) using dental UV curing cement (Tetric EvoFlow; Ivoclar Vivadent, Liechtenstein). The implants were inserted at the following stereotaxic coordinates: posterior insula (AP: −0.95 mm; ML: ±3.80 mm; DV: −3.40 mm); LA (AP: −1.15 mm; ML: ±3.48 mm; DV: −3.60 mm); CeA (AP: −1.22 mm; ML: ±2.60 mm; DV: −4.17 mm). To prevent interference with the implants by other animals, the mice were singly housed after the surgery. Behavioral experiments were performed 3 to 5 weeks later, to allow sufficient time for the injected AAV to express the opsins.