Aav5 hsyn dio hm3d gq mcherry

Stereotaxic injections were performed on adult mice (8–12 weeks; 22 ± 3 g) under anesthesia (1.5–3% isoflurane and 1 l/min O₂) using a stereotactic apparatus (Kopf Instruments). Cre dependent expression of control or chemogenetic products was achieved using AAV5-hSyn-DIO-mCherry (8.4e12 vg/ml, Addgene #50459), AAV5-hSyn-DIO-hM3D(Gq)-mCherry (2.3e13 vg/ml, Addgene #44361) and AAV5-hSyn-DIO-hM4D(Gi)-mCherry (2.5e13 vg/ml, Addgene #44362). Vectors were a kind gift from Brian Roth (Addgene catalog #44362, #44361, #50459).
The following coordinates were used to target the vlPAG: bregma Anterior-Posterior (AP), −4.7 to −4.9 mm; Medial-Lateral (ML), ±0.3–0.4 mm; Dorsal-Ventral (DV), 2.7–2.9 mm (Samineni et al., 2017 (link)). Injections were made with a motorized microinjector (UMP3T-2 with SMARTouch, WPI) and 200 nl of the vector was infused at the speed of 100 nl/min. Then, the nanovolum needle (SEG Syringe, volume 1 nl, 0.63 mm OD, part #000500) was kept in place for 5 min, retracted 0.1 mm and left for a further 3 min before complete withdrawal. 6–0 black braided silk sutures and iodine sterilization were used to aid in wound healing, and pain relief was administered (buprenorphine 0.05–0.1 μg/g mice, s.c). Following surgery, the mice were individually housed until recovery from the procedure (max 2 d) before being returned to their home cage.

The AAV2/9-CaMKIIa-YFP-difopein (titer at 7.82 × 10¹² v.g./ml), AAV2-CaMKIIa-tdTomato (titer at 4.42 × 10¹² v.g./ml), and AAV2/9-CMV-DIO-EGFP (titer at 9.05 × 10¹² v.g./ml) were constructed and produced by OBiO Technology (Shanghai) Corp., Ltd. Briefly, cDNA encoding YFP-difopein, tdTomato, or DIO-EGFP was subcloned into a rAAV vector. The viral vectors were then produced using the triple transfection method in HEK 293 cells and AAV titers were determined by real-time PCR. Control vector (AAV2-CaMKIIa-YFP, titer at 5.1 × 10¹² v.g./ml) was purchased from the UNC viral core facility. For chemogenetic manipulations, AAV5-hSyn-hM4D(Gi)-mCherry (Addgene #50475, titer at 1.2 × 10¹³ v.g./ml), AAV5-hSyn-DIO-hM4D(Gi)-mCherry (Addgene #44362, titer at 8 × 10¹² v.g./ml), AAV5-hSyn-mCherry (Addgene #114472, titer at 2.8 × 10¹³ v.g./ml), AAV5-hSyn-hM3D(Gq)-mCherry (Addgene #50474, titer at 1 × 10¹³ v.g./ml), AAV5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene #44361, titer at 1 × 10¹³ v.g./ml), and AAVretro-hSyn-Cre-WPRE-hGH (Addgene #105553, titer at 2.1 × 10¹³ v.g./ml) were purchased from Addgene. Upon arrival, all viral vectors were aliquoted and stored at −80°C prior to stereotaxic injections.

For the chemogenetic manipulations of KOR neurons in the CeA, a Cre-inducible viral vector encoding Gi-DREADD (AAV5-hSyn-DIO-hM4D(Gi)-mCherry, 0.3–0.5 μL, titer ≥ 7 × 10¹² vg/mL, Addgene (Watertown, MA, USA, Catalog #: 44362-AAV5) or Gq-DREADD (AAV5-hSyn-DIO-hM3D(Gq)-mCherry, 0.3–0.5 μL, titer ≥ 7 × 10¹² vg/mL, Addgene, Catalog #: 44361-AAV5) was injected stereotaxically into the right CeA of heterozygous KOR-Cre mice for specific chemogenetic inhibition or activation of KOR neurons in the CeA. A selective DREADD actuator (deschloroclozapine, DCZ) was used for behavioral (100 μg/kg DCZ in saline, i.p., administered for 7 days) or electrophysiological brain slice (0.5 μM DCZ in artificial cerebrospinal fluid, ACSF) experiments. Appropriate controls were used, such as Cre-dependent mCherry-expression (Dio-mCherry), vehicle (saline, i.p.), or ACSF (brain slice).