Extended Data Fig. 1: Comparison of different AAV helper viruses and rabies viruses for monosynaptic retrograde tracing.

(a–c) Comparison of spurious rabies infection from AAV helper viruses expressing wild-type TVA and mutant TVA^66T. Tricistronic AAV helper viruses were constructed to conditionally express either the wild-type TVA or TVA^66T, together with dTomato and RG (a). Cre-negative wild-type mice were sequentially injected with AAV helper viruses and EnvA-pseudotyped recombinant rabies viruses expressing EGFP. Each AAV helper virus/rabies virus pair was tested in two wild-type mice. Top-down view of whole brains (b) and observation of the injection sites under the confocal microscope (c) revealed fewer spurious rabies infection from AAV helper virus expressing TVA^66T. (d–f) Comparison of monosynaptic retrograde tracing in VISp using SAD B19 strain of recombinant RV expressing EGFP (d, similar results observed in 6 independent experiments) or H2B-EGFP (e, similar results observed in 7 independent experiments) or CVS N2c strain of recombinant RV expressing H2B-EGFP (f, similar results observed in 303 experiments). Note that in H2B-EGFP expressing SAD B19 experiment there is still green fluorescence in the processes of infected and transmitted neurons (e), due to the very high-level transgene expression in this rabies strain, whereas in CVS N2c experiment H2B-EGFP is strictly contained within nuclei of infected and transmitted neurons (f). Scale bars, 100 µm in b, 1 mm in panels showing full brain sections in d–f, and 500 µm in panels showing selected brain areas in d–f.