Isoflurane

Surgical and experimental procedures were similar to those described previously.^{13 (link),15 (link),21 (link),30 (link),38 (link)} Anesthesia was induced by inhalation of 2.5–3.0% isoflurane (Zoetis Inc., Kalamazoo, MI, USA) in oxygen until the rat was unresponsive to toe pinch, and this level of anesthesia was maintained throughout surgery. A stainless steel, bipolar electrode (Plastics One, Roanoke, VA, USA) was implanted via stereotaxic surgery by inserting the cathode into the left medial forebrain bundle at the lateral hypothalamus (2.8 mm posterior to bregma, 1.7 mm lateral to the midsagittal suture, and 8.8 mm ventral to the exterior surface of the skull). The anode was grounded by coiling around one of three screws anchored into the dorsal surface of the skull. The electrode, screws, and grounding wire were permanently affixed to the skull using dental acrylic resin. Ketoprofen (5 mg/kg) was administered for post-operative analgesia immediately and 24 h after surgery, and rats were allowed to recover for at least 7 days prior to commencing ICSS training.

All tissues were dissected 4h after the last post-drug treatment, passing the KXA half-life of 2–3 h (Zhang et al., 2018 ). For histological analysis, animals were quickly anesthetized with isoflurane (Zoetis, Cat#6089373) and secured to the perfusion plate. The chest was open to expose the heart. The left ventricle was cannulated and the inferior vena cava cut. The animals were initially perfused with 20 ml of phosphate-buffered saline (PBS) with heparin (100 mg/l, Sigma, Cat#H0878), followed by 20 ml of 4% (w/v) paraformaldehyde (PFA, Sigma, Cat#P6148) in PBS using a peristaltic pump (Behr, Cat#PLP 380, speed: 25 rpm). The animals were decapitated, the retina and brain explanted and post-fixed in 4% (w/v) PFA/PBS for 30 minutes and overnight (16h), respectively. Then the tissues were washed in PBS and stored at 4°C with 0.025% (w/v) sodium azide (VWR, Cat#786–299). For cryoprotection, the tissue was transferred to 30% (w/v) sucrose (Sigma, Cat#84097) in PBS and incubated overnight at 4°C. To increase antibody permeability, the brain slices were frozen over dry-ice and thawed at room temperature for three cycles. Then, the brain was sliced in 100 μm coronal slices on a vibratome (Leica VT 1200S), if not otherwise indicated. For parasagittal sections, the brain was divided along the midline and the same vibratome settings were used.