Fig. 1

Problems that can occur in monosynaptic retrograde tracing and a flowchart to control for them. (A) The aim of monosynaptic retrograde viral tracing experiments is to identify presynaptic connections (purple) to a pre-determined population of neurons (green). If there is no leakage of the Cre-dependent transgenes TVA (green receptor) and G-protein (yellow dot), the RVdG-EnvA will exclusively enter starter cells expressing Cre (Cre) and subsequently spread retrogradely one synapse. If there is TVA-leakage, the RVdG-EnvA can also infect cells lacking Cre. In cases of both TVA and G-protein leakage, there will be an increase in both starter cells (S) and traced cells (T). If there is only G-protein leakage, the possibility of additional traced cells (T2) arises if the G-protein is expressed in presynaptic cells (T1) that target the starter cells (Cre). (B) Injecting the RVdG-EnvA into wild-type mice serves as a control for the specificity of the RVdG-EnvA, while injecting it into responder mice carrying the G-protein and TVA under Cre-dependent expression serves as a control for evaluating potential leakage of TVA and G-protein. If there are labelled starter cells (S) after injection of the RVdG-EnvA into wild type mice, the RVdG-EnvA can enter cells independent of EnvA-TVA binding. If there are labelled starter cells (S) after injection of the RVdG-EnvA into responder mice, there might be TVA-leakage. The presence of traced cells (T) suggests G-protein leakage. (C) Illustration of tracing experiments using helper virus (HV). The HV enters most cells, but TVA and G-protein are expressed in a Cre-dependent manner. One-week post injection of HV the RVdG-EnvA is injected infecting cells expressing TVA and spreading from cells expressing G-protein. (D) Flowchart with proposed control experiments for monosynaptic retrograde tracing. Red colour in title boxes indicates a problem, whereas green colour indicates desired result