Neural circuits look forward.

A fundamental step in linking the activity of individual neurons to circuits and animal behavior is to identify their patterns of connectivity. Traditional methods in electrophysiology and microscopy, although powerful, are limited to addressing only a neuron’s immediate neighbors (1). A further limitation of these methods is that this can be addressed only in a piecemeal manner, whereby just a few of a neuron’s many synaptic connections can be probed in a given experiment. Determining the larger network connectivity of individual neurons within the intact brain requires transsynaptic tracers. Advances in virology and molecular genetics have improved our ability to target a single neuron and to trace its inputs (2), but what is missing from this toolbox is a reliable method to trace a neuron’s output. In a remarkable study in PNAS, Beier et al. present a virusbased anterograde tracer, which targets genetically selected neurons and unambiguously maps the monoand polysynaptic outputs with relatively low cytotoxicity to the circuit it is tracing (3). At the beginning of the 20th century, the pioneers in neuroanatomy begun to lay the groundwork for mapping the connections in the central and peripheral nervous system, primarily through the use of fiber degeneration studies carried out in combination with silver impregnation methods. Improvements on this approach were made through the discovery of tracer materials that were directionally transported within the axons (4). Beginning with the finding that the plant enzyme HRP is taken up by the axons and actively transported back to the soma, neuroanatomists have used a variety of tracers ranging from plant lectins and radioactively labeled amino acids to bacterial toxins and fluorescent dyes. These chemical tracers can reliably reveal the locations of neurons projecting to or from particular brain regions, perhaps most memorably through the demonstration of ocular dominance columns in the visual cortex. However, with the exception of wheat germ agglutinin–leptin and tetanus toxin, most chemical tracers do not cross synapses and hence are limited to examining neuronal connections in a point-topoint manner. Furthermore, because of the tracer material’s dependence on cellular machinery for uptake and transport, neuroanatomical tracers produce an incomplete diagram of a labeled neuron’s afferent and efferent connectivity (5). The recognition that several viruses entered the central nervous system from the periphery by “hitchhiking” along synaptically connected neurons provided the first clue that neurotrophic viruses would be a valuable alternative to chemical tracers (6). The neurotrophic viruses function as self-amplifying tracers that can produce intense labeling in the infected recipient neurons, distinguishing them from the majority of chemical tracers that are quickly diluted. Several strains of α-herpesviruses, for which the directionality of the viral transport has been shown to depend on the specific strain, are commonly used viruses for tracing neuronal connections (7). In addition, the recognition that the attenuated strains of rabies virus (RV) have a strong affinity for neurons and travels exclusively in the retrograde direction through chemical synapses established them as an attractive retrograde transsynaptic tracer (8). Four years ago, Callaway and coworkers presented a dramatic improvement in our ability to direct the synaptic specificity of the RV-based transsynaptic tracers (9). They demonstrated a clever approach for genetically targeting neuronal subsets for RV infection while also restricting viral propagation to monosynaptic targets through RV glycoprotein (G) complementation. From a conceptual standpoint, this study also supported previous observations that the G coat particle alone is necessary and sufficient to alter the transport properties and tropism of neurotropic viruses. This finding suggested to Cepko and coworkers, who have a long history of using viruses in a creative manner in the nervous system (10), that the virus G primes the virus for retrograde or Fig. 1. Schematic description of the transneuronal tracers generated by Beier et al. (3) in PNAS. (A) The VSV genome was modified such that a fluorescent reporter (YFP) was placed upstream of viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), G, and polymerase (L). VSV with endogenous G did not display transsynaptic spread. (B) Directionally selective polysynaptic tracers were generated by replacing the VSV-G with LCMV-G (purple) for anterograde spread, or with RV-G (orange) for retrograde spread of the viral vector. (C) G protein-deficient VSV (VSVΔG) pseudotyped with LCMV-G exhibited monosynaptic anterograde spread. (D) ASLV-A/RV-G pseudotyped VSVΔG can only infect the neurons expressing the TVA receptor from biolistic transfection (blue). By cotransfecting the gene for the LCMV-G in the same neurons, the VSVΔG can produce viruses that were able to transsynaptically infect postsynaptic neurons (Upper). VSVΔG repackaged with the G coat protein expressed from RV-G plasmid (orange) displayed transsynaptic spread to the presynaptic population of neurons (Lower). Because only the initially infected neuron contains virus G protein (LCMV-G or RV-G), VSV cannot spread any further, limiting VSV infection to neurons monosynaptically connected to initially infected population. A plasmid encoding mCherry was cotransfected for the cell originally targeted for infection to be identified within the monosynaptic network of GFP-labeled cells.