Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling

Reviewer #1 (Recommendations for the authors):

The authors should examine the time course of health of the retrogradely infected cells and starter cells with electrophysiological measurements of membrane potential or a histological stain.

We thank the reviewer for this suggestion. We have added resting potential information of retrogradely infected cells and starter cells at ~ 7 days post injection (Figure 6D).

Regarding the time course of neuronal health beyond this point, the situation is more complex. Reardon et al., 2016 reported anecdotal evidence that channelrhodopsin stimulation was possible until 28 days after infection. However, other assays of integrity in this paper showed signs of degeneration at much earlier stages (e.g. increase of propidium iodide-positive cells 4 days after infection). Although we have obtained patch recordings at later times after injection, we had the impression that the – number – of labeled cells decreased, and that it is more difficult to get recordings. However, this is difficult to quantify, because of the high variability of labelling across animals. Furthermore, it is well known that neurons become less healthy and more difficult to record by electrophysiology in older animals, even in wild-type. Thus, after careful consideration, we arrived at the conclusion that obtaining compelling data will be difficult.

We also want to emphasize that the rabies virus-mediated expression of both ChIEF and GCaMP8 is highly powerful and has a fast onset. Therefore, we are sure that both actuator and/or indicator expression reach sufficiently high levels after ~5 days. Thus, although the health of neurons may decline at later times after infection, there is a large window in which meaningful functional connectivity measurements can be obtained. We have now mentioned this in the Results / Discussion section of the revised version (p. 11, top of the revised manuscript).

We hope that the reviewer can accept our reply, which represents an honest answer to a tricky question.

Extended Data Figure 7 seems errantly labeled in C. Number of neurons?

ED Figure 7c (now Figure 7 —figure supplement 9C) indeed shows the development of fluorescence SNR over time. To avoid confusion, the text describing the results has been revised to enable easier interpretation.

Abbreviations in figures should be defined in figure legends.

We have carefully reviewed all figure legends and made sure that all abbreviations are properly defined.