Wild-type
mice injected with H129ΔTK-TT showed no tdT expression in virally infected cells (identified by HSV-1 antigen expression; Figure 2B) as assessed both by native fluorescence and by anti-dsRED antibody staining (data not shown), indicating minimal leakage from the loxP-STOP-loxP cassette (Zinyk et al., 1998). We next examined labeling of cerebellar circuitry downstream of Purkinje cells in these mice. Purkinje cells axons, which are the main efferents from the cerebellar cortex, target the deep cerebellar nuclei (DCN). In H129ΔTK-TT infected Veliparib order mice, tdT could be detected in most of the three major subdivisions of the DCN—the fastigial nuclei (FN), the interposed nuclei (IP), and the dentate nucleus (DN) (Figures 2D–2F). We also detected labeling in known DCN targets (Ito, 1984), including the vestibular nuclei (VE; Figures 2G–2I), the inferior olive (IO; Figures 2J–2L), ventral lateral thalamus (VL; Figures 2M–2O), and the red nucleus (RN; Figures S1M–S1O).
Labeling was also observed in the interpeduncular nuclei, a target of fastigial axons (Snider et al., 1976) (Figures S1G–S1I), and in the hippocampus and cortical amygdala (Figures S1J–S1L). tdT labeling in all these structures was present in cell somata, as determined by counterstaining Galunisertib price with fluorescent Nissl (Figures 1F, 1I, 1L, and 1O). We determined the neuronal versus glial identity of these cells by costaining with antibody markers. In the inferior olive (IO), the majority of tdT expressing cells coexpressed the panneuronal marker NeuN (333/395; 85%), while only a very small percentage (1/45; 2%) coexpressed GFAP (Figures 2P–2R and Figures S2A–S2H). Qualitatively similar results were observed in the area postrema (AP) and nucleus of the solitary tract (NTS), two additional sites where anterograde labeling from infected Purkinje cells was
detected (Ross et al., 1981), and in the VPL (Figures S2I–S2X and Table S1). These data indicate oxyclozanide that the majority of tdT expressing cells labeled in the cerebellar pathway by H129ΔTK-TT virus are neurons. Unexpectedly, we observed tdT labeling in DBH+ neurons within the locus coeruleus (LC) (Figures S1A–S1C), which are known to project to Purkinje cells (Hoffer et al., 1973). Such LC labeling was not reported in previous transsynaptic labeling studies using wheat germ agglutinin (WGA) expressed from the same PCP/L7 promoter element, either in AAV (Braz et al., 2002) or in transgenic mice (Yoshihara, 2002 and Yoshihara et al., 1999). This labeling could therefore reflect retrograde transport of recombined virus released from infected Purkinje cells. However, we found that tdT-positive LC neurons ectopically expressed the cointegrated L7/PCP2-GFP/Cre transgene (Figures S1D–S1F and data not shown).