(A) Viral constructs. Proper expression of genes carried by AAV helper viruses (TVA-mCherry, oG) depends on the correct orientation of the constructs’ exons enabled by the recombination by both Cre and Flp and the subsequent splicing of artificial introns (black boxes). Modified RV has its G protein replaced by EGFP and is pseudotyped with EnvA.
(B) Experimental timeline of virus injections.
(C) After the expression of TVA-mCh and oG in Cre- and Flp-positive, GABAergic PV cells by AAV helper viruses, RV selectively transduces these cells and spreads to first-order presynaptic neurons. PV starter cells appear yellow because of the mixture of AAV-mCh and RV-EGFP, while presynaptic partners exclusively express RV-EGFP.
(D) For injection of AAVs, the C2 whisker-related column in barrel cortex was mapped performing intrinsic signal optical imaging and targeted by the injection pipette.