Wickersham and Callaway reply:

Dr. Kasparov draws attention to the idiosyncrasies of the widely used human cytomegalovirus promoter, and we agree that viral vectors using this promoter cannot reliably drive strong transgene expression in neurons of particular types. His observations provide additional examples of the drawbacks of previously available viruses proposed for use as retrograde tracers. We are happy to say that we have detected no such expression difficulties using the deletion-mutant rabies virus described in our paper, which infects a wide variety of cell types in every brain region in which we have tested it. In contrast to commonly used viral vectors for which, as Dr. Kasparov points out, the choice of promoter is crucial, rabies virus transcribes its genes with its own polymerase, conferring complete independence from the host-cell transcriptional environment and allowing high-level transgene expression regardless of cell type. Also unlike typical vectors, the virus described in our paper retains the ability to replicate its core to high copy number within infected cells, leading to EGFP expression levels that are impressive in the extreme.

Finally, we would like to take this opportunity to point out another advantage, described in detail in our recent paper in Neuron1, of this glycoprotein-deleted rabies virus. When complemented by expression of its glycoprotein in trans within infected cells, this virus spreads to cells directly presynaptic to them. This should allow tracing not just of cells that project to an injection site but also of the cells presynaptic to the ones that are initially infected. This initial infection furthermore can be directed to a genetically specified population of cells, allowing tracing of direct inputs to particular neuron types or even to single neurons.

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