Model 1900

Procedures for intracranial viral infusion targeting the VTA were described previously (DeBaker et al., 2023 (link)). In brief, mice (>50 d) were placed in a stereotaxic frame (Model 1900, David Kopf Instruments; Tujunga, CA) under isoflurane anesthesia. Bilateral microinjectors consisting of a 33-gage stainless steel tube within a shorter 26-gage stainless steel tube were attached to polyethylene-20 tubing affixed to a 10 μL Hamilton syringe. Microinjectors were lowered through bilateral burr holes in the skull to the VTA (A/P: −2.75 mm, M/L: ±0.55 mm, D/V: −5.0 mm). AAV vectors (400 nL) were infused at a rate of 100 nL/min and microinjectors were left in place for 5 min to reduce backflow along the injection track upon removal. Behavioral experiments occurred 2–3 wk. after viral infusion.

Buprenorphine (0.1 mg/kg) was subcutaneously injected ~30 min before surgery for analgesia. Then, mice were anesthetized with isoflurane (1.5−2.0 % maintenance) through the oxygen-enriched air (95 %, 1–3 l/min, Oxymat III, Weinmann). Anesthesia level was monitored via breathing rates and foot and tail reflexes before and during surgery. Mice were placed in a stereotaxic apparatus (Model 1900, Kopf Instruments), and their body temperature was maintained through a heating pad (Rodent warmer, 53800 M, Stoelting). Their eyes were covered by an eye protective cream (Bepanthen Augen und Nasensalbe, Bayer). A mixture of Lidocaine (10 mg/kg) and Ropivacaine (3 mg/kg) was injected under the skin over the skull for local anesthesia. Stereotaxic viral injections were performed as previously described^{5 (link)}. Briefly, a small craniotomy was performed above the medial geniculate body (MGB, AP: −3.28, ML: −1.9, DV: −3.0 mm) by using a stereotaxic drill (Model 1911, Kopf) with a burr drill bit (105-0135-225, Kyocera). A pulled glass pipette (2-000-001, Drummond Scientific) filled with AAV vector was slowly lowered into the brain with the help of a micropositioner (Model 2650, Kopf). AAV2/1.syn.jGCaMP7s^{56 (link)} (Addgene, 104487-AAV1, ca 500 nl, diluted by sterile PBS, 1-2x) was injected into MGB with a pressure ejection system (Picospritzer, Parker). One to two weeks after viral injection, a gradient refractive index (GRIN) lens (0.6 mm diameter, 7.3 mm length or 1.0 mm diameter, 4 mm length, Inscopix) was implanted during the second surgery (anesthesia and analgesia, see above). 0.6 mm lenses were implanted as previously described^{5 (link)}. Briefly, a 0.8 mm diameter craniotomy was performed above MGB (drill: 105-0709.400, Kyocera) and a small track was cut with a 0.7 mm sterile needle. Next, the GRIN lens was slowly advanced into the brain using the Micropositioner (Model 2650). For the implantation of 1.0 mm diameter lenses, a 1.2−1.3 mm craniotomy was performed above MGB using a hand drill (503599, World Precision Instrument) with a burr drill bit (200 µm diameter, C1.104.002, Bösch Dental). Tissue above MGB was slowly aspirated through a sterile blunt needle (27 G, Endo irrigation cannula) connected to a suction system. Sterilized phosphate buffer saline (PBS) was used to irrigate the brain until the bleeding stopped around the aspirated site. Next, the 1.0 mm lens was slowly advanced into the brain with the micropositioner. Both, 0.6 and 1.0 mm lenses were fixed to the skull with light curable glue (Loctite 4305, Henkel). A custom-made head bar was attached to the skull next to the GRIN lens, and the skull was sealed with Scotchbond (3 M), Vetbond (3 M) and dental acrylic (Paladur, Kulzer, Orth Jet, Lang Dental and/or C&B Super-Bond, Sun Medical). Meloxicam (5 mg/kg) was injected subcutaneously after the surgery for post-operative analgesia.
For optogenetic experiments, either AAV2/5-hsyn-Jaws-KGC-GFP-ER2^{57 (link)} (Addgene, 65014-AAV5, ca 500 nl, diluted by sterile saline, 2x) or AAV2/5-hSyn-EGFP (Addgene, 50465-AAV5, ca 500 nl, diluted by sterile saline, 2x) was bilaterally injected into MGB (AP: −3.2, ML: ±2, DV: −3.0/−3.3 mm) using the same methods as described above. Following the AAV injection, optical fibers were bilaterally implanted above MGB (AP: −3.2, ML: ±2, DV: −3 mm) using the Micropositioner on the same surgery. Other surgical procedures including anesthesia and local analgesia were the same as for GRIN lens implantations. Systemic analgesia was provided via carprofen in the drinking water (0.067 mg/ml) from ca. 12–24 h pre-surgery to ca. 72 h post-surgery.