An anterogradely transported, cell-targetable variant of VSV has shown promise in hippocampal slice cultures (Beier et al., 2011), but this conditional variant has not yet been tested and validated in vivo. The HSV-1 strain H129 (Dix et al., 1983) is an attractive candidate for developing a conditional anterograde transneuronal tracer virus (Zemanick et al., 1991). In its native form, H129 has been utilized to trace circuitry in the rodent visual (Archin et al., 2003 and Sun et al., 1996), viscerosensory (Rinaman and Schwartz, 2004), trigeminal

(Barnett et al., 1995), and white adipose sensory pathways (Song et al., 2009), as well as primary motor cortex (Kelly and Strick, 2003 and Zemanick et al., 1991), and spinothalamic (Dum et al., 2009) pathways in nonhuman primates. However, a conditional, selleck chemicals Cre-dependent version of H129 that can be used to trace neural circuitry in vivo has not previously been reported. Here we develop, characterize, and validate such a virus in vivo. Our results provide a method for mapping the synaptic outputs of genetically PD0325901 in vivo marked neuronal

subsets. To develop a conditional H129 strain-based tracer, we simultaneously inactivated the endogenous H129 viral HTK gene and replaced its coding sequence with a Cre-dependent loxP-STOP-loxP-tdTomato-2A-TK cassette ( Figure 1A) via homologous recombination ( Archin et al., 2003 and Weir and Dacquel, 1995), using a codon-modified form of HTK to prevent recombination within the coding sequence (cmHTK; Supplemental Experimental Procedures, available online). After cotransfection of the HTK targeting vector and native H129 genomic DNA into host cells, H129 recombinants were selected by picking acyclovir-resistant plaques ( Figure 1B; see Experimental Procedures) and validated using PCR ( Figure 1C). The resulting H129 recombinant was named H129ΔTK-TT (tdT HTK). Infection of cultured Vero cells with this virus revealed specific expression of tdT only in the presence of Cre ( Figures 1D and 1E). Recombined virus recovered from such cells and used to infect naive Vero cells rendered the latter sensitive

to only acyclovir-dependent killing, indicating that the cmHTK was enzymatically active (data not shown). As an initial test of the Cre-dependent H129ΔTK-TT system in vivo, virus was injected intracranially into the medial cerebellar vermis of PCP2/L7-Cre transgenic mice (JAX Stock #006207), which express Cre and GFP specifically in Purkinje cells (Barski et al., 2000, Oberdick et al., 1990 and Zhang et al., 2004). Four days after infection, GFP-positive Purkinje cells in PCP2/L7-Cre/GFP mice coexpressed tdT, and all tdT-positive cells were GFP positive (Figure 2C). We rarely saw tdT expression in other cell types in the cerebellar cortex, except in regions close to the site of injection exhibiting substantial tissue necrosis, where we observed some labeled granule cells (not shown).