Aav9 cag flex gfp

All viral vectors used in these studies have been extensively used in neuroscience. For anterograde tracing, AAV9-CAG-FLEX-GFP (Addgene, 51502) and AAV5-DIO-Synaptophysin-venus-GFP (Addgene, generated from plasmid 137188) were used. For monosynaptic rabies input circuit tracing, AAV5-Ef1A-DIO-GTB (Addgene, 27056), AAV5-Ef1A-DIO-TVA-mCherry (Addgene, generated from plasmid 37084) and EnvA-SAD-Rb-ΔG-GFP (32635, Salk Institute) were used. For chemogenetic studies, AAV5-EF1a-DIO-hM3Dq-mCherry (Addgene, 44631), AAV5-EF1a-DIO-hM4Di-mCherry (Addgene, 44632) or AAV5-EF1a-DIO-mCherry (Addgene, generated from plasmid 50462) were used. For optogenetic activation studies, AAV5-EF1a-DIO-hChR2(H134R)-EYFP (Addgene, 20298) or AAV5-EF1a-DIO-EYFP (Addgene, 27056) were used.

For the retrograde and anterograde light microscopic experiments in the GAD2-Cre mice, we used AAVrg-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (titer >1 × 10¹² vg/ml, 84445; Addgene) or AAV9-CAG-FLEX-GFP (titer >1 × 10¹² vg/ml, AV5220C; UNC vector core), respectively. To optimally visualize the injection site of the retrograde virus, we co-injected cholera toxin subunit B (CTB; mix 1:1, final concentration 0.5%). For the transsynaptic experiments in wild types, we used adeno-associated virus AAV1-CMV-HI-eGFP-Cre-WPRESV40 (titer 8 × 10¹² vg/ml, 105545-AAV1; Addgene) that expresses Cre and GFP in the nucleus, while applying AAV9-hsyn-DIO-mCherry (>1 × 10¹³ vg/ml, No 50459-AAV9; Addgene) in the SC as a Cre-dependent tracer. For the mesodiencephalic junction (MDJ) tracing experiment, we injected a retrograde Cre virus AAVrg-pAAV-EF1a-Cre (titer >1 × 10¹², No 55636-AAVrg; Addgene) in IO and a Cre-dependent virus pAAV9-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA (titer >1 × 10¹³, No 20298-AAV9; Addgene) in MDJ, labeling MDJ fibers in IO. For the anterograde tracing at the electron microscopic level, we used WGA-HRP (L3892; Sigma-Aldrich), which was diluted to 7% in saline, or pAAV9-hSyn-eGFP (titer >1 × 10¹², 50465-AAV9; Addgene).