Principle of monosynaptic tracing with pseudotyped RV. Neurons are engineered by standard techniques (plasmid transfection, electroporation, gene gun, or transgenics) to express the EnvA receptor TVA, the RV glycoprotein G, and a fluorescent marker (dsRed), thereby allowing identification of potential target cells and infected cells. (1) Successful infection of target cells by EnvA-pseudotyped RV expressing eGFP is observed by yellow (merge) fluorescence; uninfected cells remain red. (2) The G-deficient RV is complemented in situ by the expressed G protein, allowing transsynaptic spread to presynaptic neurons exclusively. (3) The lack of G in infected (green) presynaptic cells prevents spread to secondary order neurons.