Aav9 hsyn ach3

Mice were anesthetized with isoflurane (3-4%) and placed in a stereotaxic frame (Kopf instruments) on an electric heating pad (Physitemp instruments) and administered buprenorphine extended release for pre-operative analgesia (3.25 mg kg-1 subcutaneous, Ethiqa XR). Following induction, isoflurane was held at 1-2% (in 0.8-1 L min⁻¹ pure oxygen) and body temperature maintained at 37°C, throughout the surgical procedure. The scalp was shaved and cleaned with iodine solution prior to exposing the skull. For experiments to record extracellular acetylcholine release using multi-fiber arrays, a large craniotomy was performed over the right hemisphere with a surgical drill (Midwest Tradition 790044, Avtec Dental RMWT) to expose the brain surface from −2.3 to 2 mm in the anterior-posterior (AP) direction and from 0.49 to 3.4 mm in the medial-lateral (ML) direction relative to bregma. AAV9-hSyn-ACh3.0 (WZ Biosciences^{45 (link)}), 2.07x10¹³ GC ml⁻¹ diluted 1:2 in PBS was pressure-injected into the striatum of WT mice (n = 8) through a pulled glass pipette (tip diameter 30-50 μm) at 20-40 separate striatum locations chosen to maximize expression around fiber tips (200 nl at each location at a rate of 100 nl/min). For control experiments, a 1:2 mixture of AAV9-hysn-ACh3.0-mut^{45 (link)} (WZ Biosciences), 2.54x10¹³ GC ml⁻¹ was injected into the striatum of WT mice (n = 3) using the same strategy. For experiments to record extracellular DA release using multi-fiber photometry, AAV5-CAG-dlight1.3b^{67 (link)} (Addgene, # 111067), 1.7x x10¹³ GC ml⁻¹ diluted 1:3 in PBS was injected into the striatum of WT mice (n = 4) at 10-40 total locations (200-800nl at each location) using the same procedure. Following injections, the multi-fiber array was mounted onto the stereotaxic manipulator, the dura gently removed, and the array slowly lowered into position. The craniotomy was sealed with a thin layer of Kwik-Sil (WPI), and the array was secured to the skull surface using Metabond (Parkell). To allow head fixation, a metal head plate and ring (Atlas Tool and Die Works) were next secured to the skull with Metabond, and the implant surface was covered with a mixture of Metabond and carbon powder (Sigma Aldrich) to reduce optical artifacts. The fiber bundle was protected by a cylindrical plastic tube, extending ~ 1-2 mm above the fiber bundle, and secured around the bundle using a mixture of Metabond and carbon powder.
To drive the suppression of ACh release from cholinergic interneurons with tetanus toxin light chain (TelC, Fig. 6), small, circular craniotomies were drilled bilaterally above the injection sites (from bregma, in mm; AP: 1, ML: ± 1.4). Then either pAAV2/8-hSyn-FLEX-TeLC-P2A-EYFP-WPRE (Addgene, 135391,^{84 (link)}, a gift from Bernando Sabatini’s Lab, 5.14x10¹³ GC ml⁻¹) or ssAAV-5/2-hSyn1-dlox-TeTxLC_2A_NLS_dTomato(rev)-dlox-WRPE-hGHp (Viral Vector Facility University of Zurich, 4.1x10¹² VG ml⁻¹) diluted 1:1 in PBS was bilaterally injected in the anterior dorsal medial striatum of ChAT-cre mice (n = 6) at 4-12 sites per hemisphere (300nl/site at a rate of 100nl/min) at the following coordinates in mm; AP: 0.8, ML: ± 1.25, DV: −2.5 and −3; AP:1, ML: ±1.4, DV:−2.75 and −3. Control ChAT-cre mice (n = 8 Chat-cre mice) were injected with saline using the same strategy. To validate that TelC expression in the aDS leads to a reduction in ACh release, two ChAT-cre mice were bilaterally injected in the aDS using the same coordinates with ssAAV-5/2-hSyn1-dlox-TeTxLC_2A_NLS_dTomato(rev)-dlox-WRPE-hGHp diluted 1:1 in PBS. Additionally, the left hemisphere of each mouse was co-injected with AAV9-hSyn-ACh3.0 diluted 1:2 in PBS. Following the injections, the craniotomies were sealed with Kwik-Sil (WPI), and the skull was sealed with Metabond (Parkell) and a metal head plate.
To measure extracellular glutamate release into cholinergic interneurons (Fig. 8), craniotomies were drilled above the injection sites in the right hemisphere (from bregma, in mm; AP: 1, ML: 1.4). A 1:1 mixture in PBS of the genetically encoded glutamate sensor AAV9.hSyn-FLEX.8F-iGluSnFR.A184S^{68 (link)} (Adgene, #106174), 1.8x10¹³ GC ml⁻¹ was injected in aDS of ChAT cre mice (n = 2) at 6 sites (300nl/site at a rate of 100nl/min) at the following coordinates in mm: AP: 0.8, ML: 1.5, DV: −2.75, −3.25 and −3.75; AP: 1.1, ML: 1.5, DV: −2.75, −3.25 and −3.75. Then, a 100 μm core diameter optical fiber (MFC_100/125- 0.37NA) attached to a zirconia ferrule (Doric) was slowly lowered into the medial region of the aDS (AP:1, ML:1.4) to a final depth of 3 mm from bregma. The craniotomies were sealed with Kwik-Sil (WPI), the optical fiber and a head plate were secured to the skull with Metabond (Parkell). After the surgeries, mice were placed in a cage with a heating pad and received postoperative injections of meloxicam (5 mg kg⁻¹ subcutaneous, Covertus) and 1 mL of saline per day subcutaneously for 4 days after surgery. Mice were individually housed and allowed to recover in their cages for at least 2 weeks after surgery.

We injected 200 nl of AAV9-hSyn-ACh3.0 (diluted 2.5% in sterile saline from 3.6 × 10¹³ genome copies/mL, WZ Biosciences YL001003-AV9-PUB) into BLA and 300 nl of AAV9-hSyn-ACh3.0 (diluted 10% in sterile saline from 3.6 × 10¹³ genome copies/mL, WZ Biosciences YL001003-AV9-PUB) into HO-AC. An optic fiber was implanted following each injection such that the distal tip of the fiber terminated 0.15 – 0.25mm above the injection depth. We implanted a flat fiber into BLA (Doric, NA 0.37, 2mm length) and an angled fiber (Doric, NA 0.37, 5mm length, 45 deg angle) into HO-AC. Both fibers featured a 0.2mm core diameter and zirconia ferrule receptive (outer diameter 1.25mm). Fibers were fixed into place and optically sealed by applying dental cement mixed with black India Ink to the exposed skull and head plate. We allowed 3 weeks for the virus incubation before performing bulk fiber measurements.