Neuronal connectivity was assayed using trans-neuronal spread of rabies; in vivo, rabies transmission occurs via synaptic contacts and is strongly correlated with synaptic input strength8. Primary infection was restricted by replacing the rabies coat protein with envelope A (ENVA), which infects only via the avian tumor virus A (TVA) receptor; viral spread was limited to monosynaptically connected neurons by deleting the rabies glycoprotein gene (ΔG)9. Neurons were first transduced with a lentivirus expressing Histone 2B (H2B)-green fluorescent protein (GFP) fusion protein, TVA and G from the synapsin (SYN) promoter (LV-SYNP-HTG). One week later, neurons were transduced with modified rabies (Rabies-ENVAΔG-RFP). Primary infected cells were positive for both H2BGFP and RFP; neurons monosynaptically connected to primary cells were GFP-negative but RFP-positive (SI Fig. 4A). Transduction with Rabies-ENVAΔG-RFP alone resulted in no RFP-positive cells, whereas transduction with Rabies-ENVAΔG-RFP following lentiviral transduction without rabies glycoprotein (SYNP-HT) led to only single GFP+RFP+ cells, indicating that in vitro rabies infection and spread are dependent on TVA expression and G trans-complementation, respectively (SI Fig. 4C,D).
