Cloning and Packaging of Recombinant AAV Constructs

The hM4D(Gi) plasmid was purchased from Addgene (#45548). Standard molecular biology techniques were used to clone GFP-T2A into an AAV2 transfer plasmid (GeneOptimizer, GeneArt; Thermo Fisher Scientific). The codon-optimized version of HA-hM4D(Gi) full sequence is available upon request) was linked to GFP via a viral 2A peptide, and contained a woodchuck hepatitis posttranscriptional regulatory element (WPRE). For all in vivo experiments, the CMV promoter was replaced with a 1.3 kb CamKIIα promoter to allow expression in excitatory neurons (27 (link)), the antibiotic resistance was changed from ampicillin to kanamycin, and a restriction site after the 2A peptide was removed. A stop codon and restriction site after the hM4D(Gi) reading frame was inserted using polymerase chain reaction methods to facilitate excision of a nontagged hM4D(Gi) from an hM4D(Gi) mCherry plasmid (Addgene; #50477). The untagged hM4D(Gi) fragment was cloned into an AAV expression plasmid under the control of the human CamKIIα promoter and containing a WPRE and bovine growth hormone polyA and flanked by AAV2-inverted terminal repeats. AAV8 or AAV5 serotype vectors were packaged using methods described previously (36 (link)).