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Microdissection of Neural Networks by Conditional Reporter Expression from a Brainbow Herpesvirus

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Microdissection of Neural Networks by Conditional Reporter Expression from a Brainbow Herpesvirus Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus J. Patrick Carda,1,2, Oren Kobilerb,1, Joshua McCambridgea, Sommer Ebdlahada, Zhiying Shanc, Mohan K. Raizadac, Alan F. Sveda, and Lynn W. Enquistb aDepartment of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260; bDepartment of Molecular Biology and the Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544; and cPhysiology and Functional Genomics, University of Florida, Gainesville FL 32610 Edited* by Larry W. Swanson, University of Southern California, Los Angeles, CA, and approved January 12, 2011 (received for review October 8, 2010) Transneuronal transport of neurotropic viruses is widely used works. For example, definition of the brain’s neural network, to define the organization of neural circuitry in the mature and which regulates homeostasis through autonomic outflow, has developing nervous system. However, interconnectivity within benefited enormously from temporal analysis of the replication complex circuits limits the ability of viral tracing to define con- and transneuronal passage of pseudorabies virus (PRV; a DNA α – nections specifically linked to a subpopulation of neurons within swine -herpesvirus) from peripheral tissues (e.g., refs. 10 12). fl a network. Here we demonstrate a unique viral tracing technology Functionally distinct neurons in uential in the regulation of that highlights connections to defined populations of neurons cardiovascular function (e.g., blood pressure versus heart rate) within a larger labeled network. This technology was accom- are distributed throughout this network and their activity is plished by constructing a replication-competent strain of pseu- coordinated through local circuits connecting network nodes (13–15). The reciprocity of connections that ensures integrated dorabies virus (PRV-263) that changes the profile of fluorescent activity among such functionally related components of preau- reporter expression in the presence of Cre recombinase (Cre). tonomic circuitry also promotes efficient viral spread throughout The viral genome carries a Brainbow cassette that expresses a de- the network. Thus, although viral transneuronal tracing has fault red reporter in infected cells. However, in the presence of proven to be an effective means of defining the full extent of Cre, the red reporter gene is excised from the genome and expres- preautonomic circuitry, the efficiency of viral transport through sion of yellow or cyan reporters is enabled. We used PRV-263 in the entire network undermines the fine-scale resolution of syn- combination with a unique lentivirus vector that produces Cre aptic inputs to defined populations of neurons. NEUROSCIENCE expression in catecholamine neurons. Projection-specific infection Creative approaches for limiting replication of virus to tar- of central circuits containing these Cre-expressing catecholamine geted populations of neurons have substantially improved the neurons with PRV-263 resulted in Cre-mediated recombination of ability to define the synaptology of neural circuitry. Wickersham the PRV-263 genome and conditional expression of cyan/yellow et al. developed a novel tracing approach in which replication of reporters. Replication and transneuronal transport of recombined rabies virus (an RNA virus) is restricted to targeted populations virus produced conditional reporter expression in neurons synap- of neurons and their first-order synaptic partners (16). This ap- tically linked to the Cre-expressing catecholamine neurons. This proach was achieved by deleting the rabies glycoprotein gene unique technology highlights connections specific to phenotypi- from the viral genome and pseudotyping the virus so that it cally defined neurons within larger networks infected by retro- differentially infects neurons engineered to express the deleted grade transneuronal transport of virus from a defined projection glycoprotein. Because the rabies glycoprotein is necessary for retrograde transneuronal passage of progeny virus, infection is target. The availability of other technologies that restrict Cre ex- fi pression to defined populations of neurons indicates that this ap- limited to the targeted neurons and their rst-order synaptic proach can be widely applied across functionally defined systems. inputs. DeFalco et al. constructed a PRV recombinant (PRV- 2001), whose replication is dependent upon the presence of the bacterial enzyme Cre recombinase (Cre), and used it to define autonomic | preautonomic network | sympathetic | transneuronal tracing polysynaptic circuits in transgenic mice that express Cre in neu- ropeptide Y neurons (17). In this approach, Cre mediates recom- nowledge of the synaptic organization of neural circuitry is bination of the viral genome to eliminate a floxed stop cassette Kfoundational for understanding the way in which the ner- that prevents transcription of thymidine kinase, a gene essential vous system functions, or malfunctions, in health, disease, and for viral replication in nonmitotic cells. Once the stop cassette is injury. Experimental approaches for circuit definition have long eliminated, thymidine kinase can be expressed and the resulting exploited the axonal transport capabilities of neurons (1). A vast viral genome is permanently replication competent and passes literature continues to define systems organization of brain cir- retrogradely to infect neurons synaptically linked to the Cre- cuitry through localization of “classic” tracers that are trans- expressing neurons. In addition to neuropeptide Y-containing ported through axons but do not cross synapses. Such studies neurons (17), this approach has been used to define circuits define regional associations but, in the absence of transmission synaptically linked to gonadotropin- and serotonin-containing electron microscopic analysis, do not define the synaptology of neurons (17–20). The considerable power inherent in both of the system under study. The ability of neurotropic viruses to these adaptations of the viral tracing method lies in restricting replicate and spread through neural circuits has brought a polysynaptic perspective to circuit analysis (2–6). Recent ge- netic engineering of such viruses has produced increasingly Author contributions: J.P.C., O.K., M.K.R., A.F.S., and L.W.E. designed research; J.P.C., O.K., powerful probes that have provided novel insights into the iden- J.M., S.E., Z.S., and A.F.S., performed research; O.K., Z.S., M.K.R., and L.W.E. contributed tity, organization, and activity of neural networks in a variety of new reagents/analytic tools; J.P.C., O.K., J.M., S.E., A.F.S., and L.W.E. analyzed data; and J.P.C., O.K., A.F.S., and L.W.E. wrote the paper. systems (7–9). fl The core strengths of the viral transneuronal tracing method The authors declare no con ict of interest. lie in the ability of neurotropic viruses to cross synapses and *This Direct Submission article had a prearranged editor. generate infectious progeny in each neuron of a circuit. In effect, Freely available online through the PNAS open access option. these viruses represent self-amplifying neural tracers that effi- 1J.P.C. and O.K. contributed equally to this work. ciently label synaptically linked neurons in a time-dependent 2To whom correspondence should be addressed. E-mail: [email protected]. manner. However, these desirable attributes also limit the ability This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. to define details of synaptic connectivity within complex net- 1073/pnas.1015033108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1015033108 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 the ability of the virus to replicate or move transneuronally our strategy. The first element is to maintain the ability of the virus between neurons. In addition to conferring important advan- to replicate in all permissive neurons. As a result, it will be possible tages to directed analysis of defined neural circuits, these ap- to introduce the virus to a circuit in a projection-specific fashion: proaches also place limitations upon the way in which the for example, a circuit can be infected on the basis of its target (e.g., method can be applied. Chief among these limitations is the kidney) rather than depending on the presence of a molecule es- need for targeted delivery of virus to neurons permissive to viral sential for viral invasiveness or replication. The second element is to replication (Table S1). provide a genetic means to change the profile of reporter-gene ex- In this study, we describe a viral-tracing technology based on pression from the virus in a controlled, permanent, and highly re- PRV, where the virus is replication-competent in all neurons, but producible fashion. The Brainbow technology using Cre-promoted, expresses unique reporters in response to Cre-mediated re- site-specific recombination provides a well-characterized system combination of the viral genome. This technology involves in- for achieving this goal. The third element is to provide a method sertion of the Brainbow 1.0L cassette developed by Lichtman for Cre expression in a targeted population of neurons within the and colleagues (21) into the genome of a PRV recombinant circuit. Lentivirus-mediated gene delivery is well suited for this (PRV-152) commonly used for viral transneuronal tracing (Fig. purpose in that Cre expression can be restricted to phenotypically 1A). The resulting virus (PRV-263) is replication-competent
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