Pulled glass pipette

Intracranial injection was performed as described previously [[28] , [29] (link), [30] ]. Anterograde labeling using AAV injections was performed using a stereotaxic device (David Kopf, USA) under isoflurane gas anesthesia (1.5–2%). Following a scalp incision, the skull was exposed and drilled to create a tiny injection hole. Utilizing a pulled glass pipette (Drummond Scientific, Wiretrol, Broomall, PA, USA) with a 50 μm tip diameter, 300 nL pAAV-FLEX-GFP/pAAV-hSyn-DIO-hM3D(Gq)-mCherry viruses were intracranially injected into each side according to groups. Virus injections were performed on the ARC bilaterally (AP: 1.35 mm; ML: 0.35 mm; DV: 5.75 mm) [31 (link)] for 40 nL/min by a micromanipulator allowing 15 min for each injection. After taking out the pipette, the scalp was sutured. Mice were allowed to recover for at least two weeks before performing any procedure.

Stereotaxic surgeries were performed as described previously.^{64 (link)} Briefly, under anesthesia with 1.5% isoflurane in the stereotaxic instrument (David Kopf instruments, Tujunga-CA), scalp was incised to expose skull, a small hole was opened with a drill and 150 to 600 nL virus (pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE (AAV1, Addgene 104495), AAV9-syn-deltaLight3.0¹⁵ and AAV9-syn-delta-Light0¹⁵ (Canadian Neurophotonics, sensor: 300nL of 3.3 × 10¹² vg/mL and mutant sensor: 300nL of 9.5 × 10¹² vg/mL respectively), AAV-CAG-GFP (AAV5, Addgene 37825), AAV-ChR2) was injected bilaterally and intracranially using a pulled glass pipette (Drummond Scientific, Wiretrol, Broomall-PA). Viral injections were performed in the ARC (bregma: −1.25 mm, midline: ±0.25 mm, dorsal surface: −5.6 mm) by a micromanipulator (Narishige, East Meadow, NY). Scalp was stitched, or ferrule placement was performed after viral injections. For in vivo fiber photometry recording, ferrule capped metal optical fiber (200 μm core diameter, NA = 0.48, Thorlabs) was implanted above the ARC using the same coordinates, except for the dorsal surface, which was ~100–200 mm above the viral injection. Ferrules were fixed with dental cement. At least 2 weeks were given for animal recovery and transgene expression before further experiments.

All surgeries were performed under aseptic conditions. Mice (> 8 weeks old) were anesthetized with isoflurane (3.5% induction, followed by 1–2% maintenance at 1 L/min), and local anesthetic (lidocaine, 2%) was administered subcutaneously at the incision site. Analgesia (buprenorphine for pre-operative treatment, 0.1 mg/kg, intraperitoneal (i.p.); ketoprofen for post-operative treatment, 5 mg/kg i.p.) was administered for two days after surgery. After leveling, cleaning, and drying the skull, we affixed a custom-made titanium head plate to the skull with adhesive cement^{20 (link)} (C&B Metabond, Parkell).
For all injections, the solution (6-OHDA or virus) was backfilled into a pulled glass pipette (Drummond, 5–000-1001-X), followed by mineral oil and a plunger. A small craniotomy (< 1 mm diameter) was made using a dental drill, and then the pipette assembly was mounted on the stereotaxic holder, lowered to the desired coordinate, and injected slowly (~100 nL/min) to minimize damage to the surrounding tissue (Narishige, MO-10). After each injection, we waited at least 10 minutes to allow the solution to diffuse away from the pipette tip before slowly going up to the next coordinate or retracting the pipette from the brain. Target coordinates (in mm) for the lAcbSh were the same across experiments: AP 1.1 from bregma, ML 1.7, and DV 4.2 from the pial surface.