Aav5 hsyn hm3d gq mcherry

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.

The commercial AAV5-hSynhM3D(Gq)-mCherry or the control virus AAV5-hSyn-mCherry (Addgene) were injected into the sciatic nerve of C57BL/6J mice (1 µl/sciatic nerve) 4–5 weeks before the experiment. For chemogenetic stimulation, the animals received two daily intraperitoneal (i.p.) injections of 5 mg/kg CNO (Tocris). After DCA, injections were delivered starting from 3DPI and lasted 7 consecutive days. In the case of SNC, the injections were given prior to injury: from 3 days pre-injury to the same day of the injury (in total 4 days of chemogenetic stimulation).