ABSTRACT
Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1 (HSV-1) strain H129 (H129), and have been successfully applied to map diverse neural circuits. Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.