Transneuronal Circuit Analysis with Pseudorabies Viruses

J. Patrick Card1, Lynn W. Enquist2

1 Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, 2 Department of Molecular Biology, Princeton University, Princeton, New Jersey
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.5
DOI:  10.1002/0471142301.ns0105s68
Online Posting Date:  July, 2014
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Our ability to understand the function of the nervous system is dependent upon defining the connections of its constituent neurons. Development of methods to define connections within neural networks has always been a growth industry in the neurosciences. Transneuronal spread of neurotropic viruses currently represents the best means of defining synaptic connections within neural networks. The method exploits the ability of viruses to invade neurons, replicate, and spread through the intimate synaptic connections that enable communication among neurons. Since the method was first introduced in the 1970s, it has benefited from an increased understanding of the virus life cycle, the function of viral genome, and the ability to manipulate the viral genome in support of directional spread of virus and the expression of transgenes. In this unit, we review these advances in viral tracing technology and the way in which they may be applied for functional dissection of neural networks. Curr. Protoc. Neurosci. 68:1.5.1‐1.5.39. © 2014 by John Wiley & Sons, Inc.

Keywords: herpesvirus; rabies; transneuronal; transgene expression

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Table of Contents

  • Introduction
  • Basic Protocol 1: Retrograde Infection of CNS Circuits by Peripheral Injection of Virus
  • Basic Protocol 2: Transneuronal Analysis by Intracerebral Injection
  • Alternate Protocols
  • Alternate Protocol 1: Transneuronal Analysis with Multiple Recombinant Strains
  • Alternate Protocol 2: Conditional Replication and Spread of PRV
  • Alternate Protocol 3: Conditional Reporters of PRV Infection and Spread
  • Alternate Protocol 4: Reporters of Neural Activity in Polysynaptic Circuits
  • Support Protocol 1: Growing and Titering a Prv Viral Stock
  • Support Protocol 2: Immunohistochemical Processing and Detection
  • Support Protocol 3: Dual‐Immunofluorescence Localization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Retrograde Infection of CNS Circuits by Peripheral Injection of Virus

  • Experimental animals, e.g., rodents
  • Betadine
  • Sterile physiological saline (0.9% w/v NaCl)
  • High‐titer PRV (see protocol 7; Table 1.5.1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • PLP fixative for perfusion (see recipe)
  • Animal clippers
  • Surgical instruments (will vary depending on required surgery)
  • 10‐μl Hamilton syringe equipped with a 26‐G needle that has a sharpened beveled tip (sterilize by autoclaving; do not use cold sterilization as the solution will compromise the titer of the inoculum)
  • Sutures and/or wound clips
  • Appropriate syringes and needles for injection of analgesics into experimental animal
  • Heating pad or heat lamp
  • Additional reagents and equipment for animal anesthesia ( appendix 4B), injection of animals ( appendix 4F), and perfusion fixation (unit 1.1)

Basic Protocol 2: Transneuronal Analysis by Intracerebral Injection

  • Experimental animals, e.g., rodents
  • High‐titer PRV (see protocol 7; Table 1.5.1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • PLP fixative (see recipe)
  • Stereotaxic apparatus (David Kopf Instruments)
  • Stereotaxic atlas (e.g., Paxinos and Watson, ; Swanson, )
  • Surgical instruments
  • Syringe with needle (see note below)
  • Sterile bone wax and gel foam
  • Wound clips
  • Heating pad or heat lamp
  • Additional materials for animal anesthesia ( appendix 4B), injection of animals ( appendix 4F), monitoring of post‐surgical condition of animals ( protocol 1), perfusion fixation (unit 1.1), and immunohistochemical localization of neurochemicals (see protocol 8 and units 1.1 & 1.2)

Alternate Protocol 1: Transneuronal Analysis with Multiple Recombinant Strains

Alternate Protocol 2: Conditional Replication and Spread of PRV

Alternate Protocol 3: Conditional Reporters of PRV Infection and Spread

Alternate Protocol 4: Reporters of Neural Activity in Polysynaptic Circuits

Support Protocol 1: Growing and Titering a Prv Viral Stock

  • PK15 cells grown in 100‐mm dishes containing DMEM/10% FBS/pen‐strep (see recipe). PK15 cells can be obtained from investigators who routinely work with PRV (e.g., Lynn Enquist) or from the American Type Tissue Culture Collection (ATCC).
  • PRV‐Bartha—and other strains of PRV—are not commercially available but, like PK15 cells, can be readily obtained from investigators who study PRV or through the CNNV (
  • Trypsin‐EDTA (see recipe)
  • Unsupplemented DMEM containing 2% FBS
  • DMEM/2% FBS/pen‐strep (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe), 37°C
  • DMEM/1% methocel/sodium bicarbonate/2% FBS/pen‐strep (see recipe)
  • 0.5% methylene blue in 70% methanol
  • 100‐mm sterile plastic dishes
  • Plastic cell scraper
  • Sterile 50‐ml screw‐cap plastic tubes
  • Sterile 1.7‐ml microcentrifuge tubes, snap cap
  • Inverted microscope
  • Cup sonicator
  • Screw‐cap cryovials
  • Centrifuge with Fotodyne 24‐place rotor
  • Cryoguard M‐40 thermal exposure indicators (Controlled Chemicals)
  • 6‐well tissue culture plates
  • Rocking platform
  • Additional reagents and equipment for tissue culture ( appendix 3B)

Support Protocol 2: Immunohistochemical Processing and Detection

  • Perfused tissue from experimental animal (see Basic Protocol protocol 11 or protocol 22)
  • PLP fixative (see recipe)
  • 0.1 M and 10 mM sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • 20% to 30% (w/v) sucrose in 0.1 M sodium phosphate buffer, pH 7.4
  • Glycol‐based cryoprotectant (see recipe)
  • 0.5% (w/v) sodium borohydride in PBS (prepare fresh; optional)
  • 0.5% (v/v) H 2O 2/30% (v/v) methanol in PBS (prepare fresh; optional)
  • Primary antibody solution (see recipe and Table 1.5.1)
  • Biotinylated, affinity‐purified secondary antibody against IgG of species used to raise primary antibody
  • Normal serum generated in same species as secondary antibody
  • 10% (v/v) Triton X‐100
  • Vectastain Elite kit (Vector Laboratories)
  • Diaminobenzidine (DAB)
  • Tris‐buffered saline (TBS), pH 7.6 ( appendix 2A)
  • 30% H 2O 2
  • Bleach
  • 50%, 70%, 95%, and 100% ethanol
  • Xylene
  • Resin for mounting coverslips (Cytoseal 60; Stephens Scientific)
  • Rocking platform
  • Freezing microtome, chucks, and freezing medium (Shandon M‐1 Embedding Matrix; Thermo Scientific)
  • Plexiglas compartments with porous nets on the bottom (Brain Research Laboratories)
  • Paintbrush with fine bristles
  • Gelatin‐coated (subbed) microscope slides (unit 1.1)
  • Coverslips

Support Protocol 3: Dual‐Immunofluorescence Localization

  • Sectioned tissue in cryoprotectant ( protocol 8)
  • Primary antibodies generated against PRV or reporter proteins (see note below)
  • Primary antibodies generated against phenotypic markers of neurons that contribute to the circuit of interest. These antibodies should be generated in a species different from those raised against PRV (see recipe).
  • Secondary antibodies generated against the IgG of the two species used for the primary antibodies, conjugated, respectively to Cy2 and Cy3 (Jackson ImmunoResearch Laboratories)
  • Light‐proof vials
  • Fluorescence microscope (unit 2.1)
  • Additional reagents and equipment for immunohistochemical processing and detection of tissues (see protocol 8) and fluorescence microscopy (unit 2.1)
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