Fig. 9
From: Differences in neurotropism and neurotoxicity among retrograde viral tracers
Higher-order circuit tracing using a combination of different tracing viruses. (a) Strategy for mapping the higher-order input circuits of target nuclei by combining different viral tracers. (b) Diagram of higher-order circuit mapping of the LHA by combining rAAV2-retro-Cre-tagBFP, rAAV9-DIO-EGFP-TVA, rAAV9-DIO-RVG, and EnvA pseudotyped RV-∆G-DsRed. (c) Injected site for rAAV2-retro-Cre-tagBFP in LHA. (d) Direct upstream neurons from the LHA (first order) were labeled with both green and red fluorescence in the PrL and IL of the mPFC (also called starter neurons). Green, TVA-positive neuron; red, EnvA-RV-∆G-DsRed-positive neuron; yellow, starter neurons. (e-g) Nuclei upstream from the starter neurons (second order) were labeled with only red fluorescence in the AI, LO, Pir (E), AM (F), and BLA (G). (h-i) Diagram of the higher-order circuit map of the LHA obtained by combining rAAV9-DIO-RVG and RV-∆G-Cre-tagBFP labeling in the PVN. (j) Injected site for RV-∆G-Cre-tagBFP. (k) RV-∆G-Cre-tagBFP retrogradely labeled only neurons in the ipsilateral PVN. (l) Complementing RV-G by injecting AAV-DIO-RVG into the ipsilateral PVN resulted in the labeling of second-order neurons in the contralateral PVN. Scale bars = 200 μm.