Basic Neuroscience
Anterograde neuronal circuit tracing using a genetically modified herpes simplex virus expressing EGFP

https://doi.org/10.1016/j.jneumeth.2012.05.035Get rights and content

Abstract

Insights into the anatomical organization of complex neural circuits provide important information about function, and thus tools that facilitate neuroanatomical studies have proved invaluable in neuroscience. Advances in molecular cloning have allowed the production of novel recombinant neuroinvasive viruses for use in transynaptic neural tracing studies. However, the vast majority of these viruses have motility in the retrograde direction only, therefore limiting their use to studies of synaptic input circuitry. Here we describe the construction of an EGFP reporting herpes simplex virus, strain H129, which preferentially moves along synaptically connected neurons in the anterograde direction. In vitro and in vivo characterization studies confirm that the HSV-1 H129–EGFP retains comparable replication and neuroinvasiveness as the wildtype H129 virus. As a proof of principle we confirm anterograde movement of the H129–EGFP along polysynaptic pathways by inoculating the upper airways and tracking time-dependent EGFP expression in previously described ascending sensory pathways. These data confirm a genomic locus for recombining HSV-1 H129 such that normal viral function and replication is maintained. Novel viral recombinants such as HSV-1 H129–EGFP will be useful tools for delineating the central organization of peripheral sensory pathways as well as the synaptic outputs from central neuronal populations.

Highlights

► HSV1 H129 travels anterogradely along synaptically connected neurons. ► Here we describe the construction of a novel EGFP reporting HSV1 H129 virus. ► Replication and neuroinvasiveness of H129–EGFP is comparable to wildtype H129. ► Time dependent anterograde motility is preserved in H129–EGFP. ► Synaptic organization of neural circuits can be determined with H129–EGFP.

Introduction

Neurotropic viruses, including pseudorabies virus (PRV) and herpes simplex virus type 1 (HSV-1) from the alphaherpesvirus sub-family and rabies virus from the rhabdovirus family, are widely used in neuroanatomical tracing studies for defining the organization of complex neural circuits in the central nervous system (Kuypers and Ugolini, 1990, Card and Enquist, 2001, Callaway, 2008, Ekstrand et al., 2008). Transneuronal viral tracing techniques exploit the natural properties of neurotropic viruses to be transported retrogradely and/or anterogradely along axons, travel across synaptically connected neurons and replicate in each neuron along the circuit (i.e., non-diluting and self-amplifying). PRV in particular has been used extensively as a retrograde circuit mapping tool, ideally suited to studies of the organization of neural networks innervating peripheral autonomic motor neurons or the input circuitry to CNS nuclei (Ding et al., 1993, Jansen et al., 1995, Standish et al., 1995a, Cano et al., 2001).

The utility of neurotropic viruses can be greatly increased by genetically manipulating the viral genome, for example, to produce viral recombinants that drive fluorescent reporter protein expression in infected cells (Standish et al., 1995b, Balliet et al., 2007, Wickersham et al., 2007, Granstedt et al., 2010, Osakada et al., 2011). Fluorescent reporter constructs have been described for the retrograde viral traces PRV, HSV-1 and rabies virus and these tools have greatly facilitated the identification of components of the neural circuit under study. Furthermore, simultaneous identification of multiple neuronal networks in one animal has been made possible by using isogenic viral strains expressing different reporter proteins (Billig et al., 2000, Cano et al., 2004, Tóth et al., 2008). However, most of the recombinant viral tools reported to date possess minimal or no ability to move anterogradely along neural circuitry and therefore have limited use for studying the organization of sensory circuitry or the output projections of defined CNS sites. Indeed the only neurotropic virus that has been clearly shown to move preferentially in the anterograde direction is the H129 strain of HSV-1 (Zemanick et al., 1991, Barnett et al., 1995, Sun et al., 1996, LaVail et al., 1997, Garner and LaVail, 1999, Rinaman and Schwartz, 2004, Song et al., 2009, McGovern et al., 2012), yet only two studies have genetically manipulated this virus for possible use in neuroanatomical tracing purposes (Archin et al., 2003, Lo and Anderson, 2011). Therefore the aim of the present study was to construct a HSV-1 H129–EGFP recombinant virus that drives fluorescent protein expression in infected cells and characterize its utility as a novel anterograde circuit tracing tool.

Section snippets

Cells and virus

The wildtype HSV-1 H129 strain used in this study was originally isolated from a patient with encephalitis (Dix et al., 1983) and was generously provided by Prof. Lynn Enquist (Princeton University, Princeton, NJ). Viral stocks were grown on Vero cells maintained in minimum essential media (MEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 Units/ml), streptomycin (100 μg/ml) and GlutaMAX (1 mM). Standard plaque assays were performed by serially diluting virus in MEM supplemented

Production and in vitro characterization of H129–EGFP

Co-transfection of Vero cells with pH129–EGFP and wildtype H129 viral DNA produced several candidate viral recombinants for subsequent characterization (not shown) and one of these was plaque purified six times. After each round of plaque purification the frequency of EGFP positive plaques increased and no EGFP negative plaques were visible after round 4 of plaque picking. The titer of the recombinant virus increased following each round of plaque purification to a final titer of 7.4 × 107 pfu/ml.

Discussion

A variety of recombinant viral retrograde neuronal tracers have been developed and these have proved invaluable for studying the organization of complex neural circuits. However, comparable anterograde tracing tools are largely not available. In this study we constructed a novel EGFP reporter herpes virus for use in anterograde tracing studies. Our data show that insertion of a HCMV-EGFP expression cassette into the intergenic region between genes UL26/26.5 and UL27 in the H129 strain of HSV-1

Acknowledgements

The authors would like to thank Professor Lynn Enquist and Dr. Halina Staniszewska Goraczniak (Princeton University, Princeton, NJ) for their generosity supplying the H129 strain of HSV-1.

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    These studies were funded by grants awarded to Dr. Mazzone from the National Health and Medical Research Council of Australia (566734; 454776; 1025589). The NHMRC played no role in the design, conduct or reporting of this study.

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