Long-term Cre-mediated retrograde tagging of neurons using a novel recombinant pseudorabies virus.

Hassana K Oyibo,Lynn W Enquist,Hysell V Oviedo,Petr Znamenskiy,Anthony M Zador

doi:10.3389/fnana.2014.00086

Abstract

Brain regions contain diverse populations of neurons that project to different long-range targets. The study of these subpopulations in circuit function and behavior requires a toolkit to characterize and manipulate their activity in vivo. We have developed a novel set of reagents based on Pseudorabies Virus (PRV) for efficient and long-term genetic tagging of neurons based on their projection targets. By deleting IE180, the master transcriptional regulator in the PRV genome, we have produced a mutant virus capable of infection and transgene expression in neurons but unable to replicate in or spread from those neurons. IE180-null mutants showed no cytotoxicity, and infected neurons exhibited normal physiological function more than 45 days after infection, indicating the utility of these engineered viruses for chronic experiments. To enable rapid and convenient construction of novel IE180-null recombinants, we engineered a bacterial artificial chromosome (BAC) shuttle-vector system for moving new constructs into the PRV IE180-null genome. Using this system we generated an IE180-null recombinant virus expressing the site-specific recombinase Cre. This Cre-expressing virus (PRV-hSyn-Cre) efficiently and robustly infects neurons in vivo and activates transgene expression from Cre-dependent vectors in local and retrograde projecting populations of neurons in the mouse. We also generated an assortment of recombinant viruses expressing fluorescent proteins (mCherry, EGFP, ECFP). These viruses exhibit long-term labeling of neurons in vitro but transient labeling in vivo. Together these novel IE180-null PRV reagents expand the toolkit for targeted gene expression in the brain, facilitating functional dissection of neuronal circuits in vivo.

Highlights

Neuroinvasive viruses have become an essential part of the systems neuroscience toolkit as a vehicle for delivery of genetic material into neurons
We have developed a novel set of reagents based on Pseudorabies Virus (PRV) for efficient and long-term genetic tagging of neurons based on their projection targets
Herpes Simplex Virus-1 (HSV-1) encodes five immediate early genes, and the recombinant herpes simplex virus 1 (HSV-1) used for retrograde tagging has deletions in two of the essential IE genes; ICP4 and ICP27, and carries a mutation in the tegument protein VP16 that blocks transactivation of the other 3 IE genes (Lilley et al, 2001)

Summary

Introduction

Neuroinvasive viruses have become an essential part of the systems neuroscience toolkit as a vehicle for delivery of genetic material into neurons Their applications range from anatomical tracing with fluorescent proteins to monitoring and manipulating the activity of defined populations of cells (Boyden et al, 2005; Deisseroth, 2011; Sasaki et al, 2011; Akerboom et al, 2012). One powerful approach to labeling neuronal subpopulations exploits the ability of certain viruses to infect axon terminals and undergo retrograde transport to the soma, thereby labeling neurons that project to the injection site. This approach can target populations of projection neurons without the need for a cell-type specific promoter to direct gene expression. Our goal was to produce a set of retrograde viral tools that would be suitable for the mapping of connectivity, but for the long-term manipulation of specific anatomically and genetically defined neuronal populations

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