In general, infected structures, such as the ventromedial hypothalamic nucleus (VMH), which are more synapses removed from the MOE, exhibited a smaller percentage of tdT-positive cells than those separated by fewer synapses, such as the AON (Figures S5C and S5D), consistent with the idea that spread is predominantly synaptic. Several viral systems for conditional retrograde transsynaptic tracing have been developed (reviewed in Callaway, 2008 and Ekstrand et al., Screening Library price 2008), but an analogous system for conditional anterograde transsynaptic

viral tracing in vivo has not been implemented. Here we have developed such a method by using homologous recombination (Weir and Dacquel, 1995) to manipulate the genome of the H129 strain of HSV (Dix et al., 1983), a well-characterized anterograde transsynaptic tracer virus (Zemanick et al., 1991). Using lines of transgenic mice specifically expressing Cre recombinase, we tested this recombinant virus in the visual, cerebellar, and olfactory systems, respectively. In each case, the pattern of labeling obtained was Cre-dependent, concordant with previously described patterns of connectivity, and consistent with an anterograde mode of transneuronal transfer. The use of alpha herpesvirus-based

transneuronal tracers, such as pseudorabies virus (Ekstrand et al., 2008), has been criticized based not only on their toxicity, but also on the contention that the virus can spread

in a nonsynaptic manner to fibers-of-passage or even PF-01367338 nmr to glial cells (Ugolini, 2008 and Ugolini, 2010). In our studies, the overall pattern of labeling observed in the three systems examined was remarkably specific and consistent with patterns of connectivity revealed by classical methods. We found little or no evidence of spread to glia (Figure 2R and Figure S2), even in regions where glia were closely juxtaposed with tdT-labeled neurons (e.g., sustentacular cells in the MOE and Muller Ergoloid glia in the retina). While it is difficult to completely exclude nonsynaptic spread, little or no labeling of photoreceptors, or of oculomotor neurons in the Edinger-Westphal nuclei, was obtained in our retinal injections. We also failed to detect labeling of neuromodulatory afferents to the olfactory bulb at early time points. All of these data are consistent with the reported anterograde-specific pattern of labeling by the H129 strain (Rinaman and Schwartz, 2004, Sun et al., 1996 and Zemanick et al., 1991). The lack of specificity reported by others for HSV (Ugolini, 2008 and Ugolini, 2010) probably reflects the use of different strains of these Herpes viruses. The pattern of labeling obtained in each of the three test systems employed here was complex, as would be expected given the polysynaptic nature of the labeling method.