The method of rabies-based retrograde tracing.

<p><b>(A)</b> AAV virus and rabies virus for transsynaptic retrograde tracing. Double-floxed EGFP-TVA and rabies glycoprotein (RG) under control of CAG were respectively packaged into AAV viral vectors. The rabies virus without glycoprotein (SADΔG) was equipped with the mCherry fluorescent protein. <b>(B)</b> Experimental design. On Day 1, AAV-DIO-EGFP-TVA and AAV-DIO-RG viral vectors were injected into the dorsal striatum of ChAT-Cre, D1-Cre, or D2-Cre mice. Two weeks later, SADΔG-mCherry was injected into the same site of the dorsal striatum. Histology was performed after one more week. The starter cells are labeled both in green and red, whereas the input neurons are labeled only in red. <b>(C)</b> Confirmation of effective transsynaptic labeling using rabies virus. Sequential injections of AAV-DIO-EGFP-TVA, AAV-DIO-RG and SADΔG-mCherry viruses into the dorsal striatum of a D1-Cre mouse labeled starter cells (green) and input neurons (red; left panel). Same virus injections did not label any neurons in a wild-type C57BL/6J mouse (right panel). <b>(D)</b> Zoom-in view of starter cells and transsynaptically labeled cells in the dorsal striatum of the D1-Cre mouse shown in (C). <b>(E)</b> Transsynaptic retrograde labeling neurons in the cingulate cortex (cg).