Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus

Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2011; 108(8):3377-82. DOI: 10.1073/pnas.1015033108
Source: PubMed


Transneuronal transport of neurotropic viruses is widely used to define the organization of neural circuitry in the mature and developing nervous system. However, interconnectivity within complex circuits limits the ability of viral tracing to define connections specifically linked to a subpopulation of neurons within a network. Here we demonstrate a unique viral tracing technology that highlights connections to defined populations of neurons within a larger labeled network. This technology was accomplished by constructing a replication-competent strain of pseudorabies virus (PRV-263) that changes the profile of fluorescent reporter expression in the presence of Cre recombinase (Cre). The viral genome carries a Brainbow cassette that expresses a default red reporter in infected cells. However, in the presence of Cre, the red reporter gene is excised from the genome and expression of yellow or cyan reporters is enabled. We used PRV-263 in combination with a unique lentivirus vector that produces Cre expression in catecholamine neurons. Projection-specific infection of central circuits containing these Cre-expressing catecholamine neurons with PRV-263 resulted in Cre-mediated recombination of the PRV-263 genome and conditional expression of cyan/yellow reporters. Replication and transneuronal transport of recombined virus produced conditional reporter expression in neurons synaptically linked to the Cre-expressing catecholamine neurons. This unique technology highlights connections specific to phenotypically defined neurons within larger networks infected by retrograde transneuronal transport of virus from a defined projection target. The availability of other technologies that restrict Cre expression to defined populations of neurons indicates that this approach can be widely applied across functionally defined systems.

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    • "The individual neurons and their axons have thus been traced in transgenic systems such as mouse [21], Drosophila [33], [34] and zebrafish [35]. Otherwise, the mosaic expression of fluorescent proteins was attained using viral vectors encoding the Brainbow cassette [36]–[38]. "
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    • "Sections were mounted on gelatin-coated slides, air dried, and coverslipped using Vectashield Hard Set mounting medium (Vector Laboratories, Burlingame, CA). The fluorophor profile of infected neurons was determined using an Olympus BX51 epifluorescence microscope equipped with filters specific for reporter proteins encoded by the dTomato, mCerulean, and EYFP genes as described previously [18]. Digital micrographs of each region were captured with a Hamamatsu camera (Hamamatsu Photonics, Hamamatsu, Japan) and analyzed using the procedures detailed in the next section. "
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