Ims 20

AAV carrying the tdTomato and SypEGFP genes [27 (link)] was a generous gift from Dr. Hongkui Zeng (Addgene, #51509-AAV1; Watertown, MA, USA). Brn3a^Cre/+ mice (2–3 months of age) were deeply anesthetized with a mixture of medetomidine (37.5 μg/kg, Nippon Zenyaku Kogyo), midazolam (2 mg/kg, Sandoz), and butorphanol (0.25 mg/kg, Meiji Seika Pharma), and their backs were shaved using an electric shaver. The intervertebral spaces rostral to the L1 vertebra, which correspond to the L5 spinal segment [28 (link)], were exposed by carefully removing the connective tissues. The AAV injection tool included a fine glass capillary (G-1; Narishige) and a Hamilton syringe (80330-701RN; Hamilton Co. Reno, NV, USA) connected by an RN compression fitting (55750–01; Hamilton Co.). The AAV solution was loaded into the glass capillary and injected into three different regions of the L5 spinal dorsal horn on the right side using a microinjector (IMS-20, Narishige). For each injection, 300 μL of AAV solution was used. Following injection, the glass capillary was maintained at the injection site for 3 min to minimize leakage. The wounds were sutured immediately after the operation, and the mice were placed on a heating pad until recovery. Three weeks post operation, the mice were transcardially perfused with 4% paraformaldehyde solution to obtain brain and spinal cord samples.

Subject male rats were anesthetized with an intraperitoneal injection of a mixture of medetomidine (0.375 mg/kg), midazolam (2.0 mg/kg), and butorphanol (2.5 mg/kg) or using a combination of intraperitoneal sodium pentobarbital (15 mg/kg) and isoflurane gas (2%). To record neural signals, a custom microdrive array (Kloosterman et al., 2009 (link)) including 16 independently adjustable tetrodes (twisted bundles of four 12.7 μm polyimide-coated nichrome wires, gold plated to an impedance of ∼300 kΩ; Sandvik) was implanted in the left cortical areas including the primary and secondary motor cortex (M1 and M2; AP, −2.0 to 4.5 mm; ML, 0.5 to 4.5 mm). For optogenetic tagging of recorded neurons, in some rats, five optic fibers were implanted above the tetrodes (0.2 mm from cortical surface), and AAV-retro-hSyn-hChR2-EYFP (0.1 μl; 26973-AAVrg, Addgene) was injected into the left PAG (AP, −7.8 mm; ML, 0.7 mm; DV, 5.6 mm; Paxinos and Watson, 2007 ) using a motorized stereotaxic microinjector (IMS-20; Narishige) through a pipette angled 15° to the lateral. In a separate group of male rats, to label neurons projecting to the PAG, AAV-retro-hSyn-EGFP (0.1 μl; 50465-AAVrg, Addgene) was similarly injected to the left PAG. Stimulus adult female rats were ovariectomized under anesthesia with an intraperitoneal injection of the mixture described above.

AAV injections were performed when Cre⁻ and Cre⁺ mice were 12–14 weeks old. Animals were anesthetized with isoflurane (3.0% for induction and 2.0–2.5% for maintenance, wt/vol) and placed in a stereotaxic apparatus (Narishige Scientific Instrument) with bregma and lambda skull landmark levels. According to the experimental protocols of Sherman and colleagues^{22 (link)}, two holes were drilled in the skull at predefined coordinates relative to bregma to allow the insertion of glass pipettes containing a mixture of two virus solutions (ChR2: 0.9–1.7 × 1013 genome copies per ml; eNpHR: 1.4 × 1013 genome copies per ml), which were held by a stereotaxic micromanipulator (SMM-100, Narishige) and connected to a Hamilton syringe with a pressure injection system (Motorized Stereotaxic Microinjector, IMS-20, Narishige). Each animal had bilateral injections targeted precisely to a location between the PreBötC and BötC (6.70 mm caudal to bregma, 1.25 mm lateral to the midline, and 4.65 mm ventral from the dorsal surface of the brain) according to the mouse brain atlas⁶⁰ . Once situated, 500 nl per side was slowly injected (100 nl/min) and the pipette was left in place for at least 5 min after injection to minimize backflow. The wound was closed with instant adhesives. AAV injections were performed by an investigator blinded to the animals.