Nevertheless, the engagement of a large fraction of the brain by olfactory afferents is consistent with the widespread ramification of fibers revealed by labeling of individual OB glomeruli with nontransneuronal anterograde tracers (Sosulski et al., 2011) and probably reflects ERK inhibitor solubility dmso the importance of olfaction for both learned and innate behaviors in mice. One obvious application of

this technology is the transneuronal labeling of pathways engaged by neurons expressing a specific, behaviorally relevant olfactory receptor, e.g., those involved in pheromone detection (Mombaerts et al., 1996). However, the efficiency of labeling in the MOE and VNO was low, presumably due to the inhibition of viral spread by mucus, and in pilot experiments we were unable to detect labeling in mice expressing Cre recombinase under the control of a specific olfactory receptor, MOR28, that is expressed in ∼1% of ORNs (Mombaerts, 2006). Nevertheless, our preliminary data suggest that this problem may be overcome by injection of the virus into the olfactory bulb, where infection of ORN axons can occur followed by retrograde transport to the cell body and recombination in ORNs (Figures S1R and S1S). The methodology described here, while powerful, has certain limitations.

First, like other replicating transneuronal tracers (Callaway, 2008 and Ekstrand selleck chemicals llc et al., 2008), HSV-based tracers are toxic and kill infected neurons, as well as eventually the whole animal (see Table S2). This limits the number of days that an injected animal can be maintained before analysis. Furthermore, there is unpredictable variability in survival times, reflecting variability in the initial level of infection. This makes it currently difficult to perform prospective time course studies of the progression of labeling in a given pathway, except in a retrospective manner (Figures S5C and S5D). In addition, due to viral cytotoxicity, in animals sacrificed after longer incubation times the initial sites of infection have often been cleared

from the brain by macrophages, obscuring until the identification of initial relays in a pathway. This limitation may be overcome by analyzing animals exhibiting mild symptoms and/or after shorter survival times, in order to identify the pattern of labeling in early structures. Second, although virus released endogenously from infected neurons appears to be taken up exclusively by dendrites and transported in the anterograde direction (Zemanick et al., 1991), exogenously injected virus can clearly infect nerve terminals and undergo retrograde transport to the cell body, as reported previously (Barnett et al., 1995, Rinaman and Schwartz, 2004 and Song et al., 2009) and confirmed here in the olfactory and cerebellar systems (Figures S1P–S1S).