We sparsely seeded potential starter cells in each culture well by using the Syn1-EBFP-Cre rAAV at low titer to recombine rAAVs encoding TVA (CAG-Flex-TVA-mCherry) and the rabies glycoprotein (G; CAG-Flex-B19G) at high-titer (Methods), thus enabling efficient EnvA-mediated rabies founder infections and G-dependent presynaptic spread. Here, SYN1 is linked to infection.