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Transneuronal Circuit Analysis With Pseudorabies Viruses

J. Patrick Card¹, Lynn W. Enquist²

¹University of Pittsburgh, Pittsburgh, Pennsylvania
²Princeton University, Princeton, New Jersey

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 1.5

DOI:

10.1002/0471142301.ns0105s09

Online Posting Date:

May, 2001

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Abstract

Over the past decade there has been a dramatic increase in the use of viruses as transneuronal tracers of neuronal circuitry. The method exploits the propensity of neurotropic viruses to invade neurons and then produce infectious progeny that cross synapses to infect other neurons within a circuit. The protocols and commentaries included in this unit focus upon the use of the swine alpha herpesvirus known as pseudorabies virus (PRV) for polysynaptic analysis. Here, the aspects of experimental design that have the greatest import for successful use of viruses in circuit definition are presented. Accordingly, the protocols included in this unit can be applied in concert with methods in which the use of classical tract tracers has been detailed. A procedure for retrograde infection of CNS circuits in the rat CNS by peripheral injection of virus is detailed, while transneuronal analysis by intracerebral injection is also described. A variant of these procedures, transneuronal analysis with multiple recombinant strains, is also described along with methods for growing and titering viral stocks, and procedures for single and dual immunohistochemical localization of viral antigens in fixed brain tissue.

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Unit Introduction
Basic Protocol 1: Retrograde Infection of CNS Circuits by Peripheral Injection of Virus
Basic Protocol 2: Transneuronal Analysis by Intracerebral Injection
Alternate Protocol: Transneuronal Analysis with Multiple Recombinant Strains
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
Bibliography
Figures
Tables

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Materials

Basic Protocol 1: Retrograde Infection of CNS Circuits by Peripheral Injection of Virus

Materials

Experimental animal
Ketamine
Xylazine
Sterile physiological saline (0.9% w/v NaCl)
Titered PRV-Bartha virus (see Support Protocol 1)
PLP fixative (see recipe)

Appropriate syringes and needles for injection of anesthetic into experimental animal
Surgical instruments (will vary depending on required surgery)
Sutures and/or wound clips
Heating pad or heat lamp
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)

Additional reagents and equipment for perfusion fixation (unit 1.1)

NOTE: PRV and other neurotropic viruses are class 2 infectious agents that require a laboratory which meets Biosafety Level 2 (BSL-2) regulations as defined in Health and Human Services Publication 88-8395 (see Critical Parameters). Among these regulations is the requirement that infected animals be confined to the BSL-2 laboratory throughout the experiment and that the laboratory be dedicated to the viral studies. Thus, one must ensure that the laboratory contains all of the reagents and equipment necessary for the experiment. Aseptic surgical procedures should be used, but it is important to include precautions that protect against compromising the concentration of the inoculum (see Critical Parameters).

Basic Protocol 2: Transneuronal Analysis by Intracerebral Injection

Materials

Experimental animal
Ketamine
Xylazine
Sterile physiological saline (0.9% w/v NaCl)
Titered PRV-Bartha virus (see Support Protocol 1)
PLP fixative (see recipe)

Stereotaxic apparatus
Surgical instruments
Syringe with needle (see note below)
Bone wax or gel foam
Wound clips
Heating pad or heat lamp

Additional materials for perfusion fixation (unit 1.1) and immunohistochemical localization of neurochemicals (see Support Protocol 2 and units 1.1 & 1.2)

NOTE: The needle or cannula used for injection will influence the zone of viral diffusion. Hamilton microliter syringes equipped with fixed needles of 26- to 32-G are generally adequate for injection of large cell groups. The needles affixed to these syringes are either blunt or have a sharpened tip with the opening on the beveled surface. If a beveled needle is used, the opening should be directed towards the cell group of interest. This is quite important, since the affinities of virions for extracellular matrix molecules restrict virus diffusion to the immediate vicinity of the injection. More restricted injections can be made with glass pipets. The pipets are pulled using standard procedures (see Chapter 6) and the tip broken back so that the internal diameter is 15 to 20 µm. The shaft of the pipet is placed over the needle of a Hamilton syringe preloaded with virus, and the interface is sealed with beeswax. It is important to keep the length of the glass sleeve as short as possible, since the space between the glass and the needle becomes a reservoir for virus that must be filled before virus can be ejected from the pipet tip.

Support Protocol 1: Growing and Titering a PRV Viral Stock

Materials

PK15 cells grown in 100-mm dishes containing DMEM/10% FBS/pen-strep (see recipe). Although not commercially available, these cells can be easily obtained from investigators who routinely work with PRV (e.g., Patrick Card)
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 use it for transneuronal analysis (e.g., Patrick Card)
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

Plastic cell scraper
Sterile 1.7-ml microcentrifuge tubes, snap cap
Sterile 50-ml screw-cap plastic tubes
Cup sonicator
Screw-cap cryovials vials
6-well tissue culture plates
Rocking platform

Additional reagents and equipment for tissue culture (appendix 3B)

Support Protocol 2: Immunohistochemical Processing and Detection

Materials

Perfused tissue from experimental animal (see Basic Protocol 1 or 2)
PLP fixative (see recipe; McLean and Nakane, 1974)
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; Watson et al., 1986)
0.5% (w/v) sodium borohydride in PBS (prepare fresh; optional)
0.5% (v/v)H₂O₂/30% (v/v) methanol in PBS (prepare fresh; optional)
Primary antibody solution (see recipe)
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% H₂O₂
50%, 70%, 95%, and 100% ethanol
Xylene

Bleach
Resin for mounting coverslips (Cytoseal 60; Stephens Scientific)
Freezing microtome and chucks
Rocking platform
Plexiglas compartments with porous nets on the bottom (Brain Research Laboratories)
Gelatin-coated (subbed) microscope slides (unit 1.1)
Coverslips

Support Protocol 3: Dual Immunofluorescence Localization

Materials

Sectioned tissue in cryoprotectant (Support Protocol 2)
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
50%, 70%, 95%, 100% ethanol
Xylene
Mounting media (Cytoseal 60; Stephens Scientific)
Coverslips
Fluorescence microscope (unit 2.1)

Additional reagents and equipment fir immunohistochemical processing and detection of tissues (see Support Protocol 2) and fluorescence microscopy (unit 2.1)

NOTE: Antibodies against reporter proteins should be generated in different species. The reporters most commonly used in the construction of recombinant strains of PRV are -galactosidase and the enhanced green fluorescent protein (EGFP). Antibodies generated against both of these reporters are commercially available from a variety of vendors.

Looking for materials? Suppliers Guide

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Figures

Figure 1.5.1

(A) The structural characteristics of alpha hepesviruses. Viral DNA is sequestered within a capsid composed of virally encoded proteins. The capsid and a surrounding tegument of each virion is contained within a viral envelope acquired from the host cell. The envelope contains a second set of virally encoded proteins that are important for target cell recognition, attachment, and the receptor-mediated fusion event that leads to the release of the capsid into a permissive cell. (B) A model of virion assembly postulated for pseudorabies virus (Card and Enquist, 1995). Assembly of virions is a multistep process that leads to retrograde transynaptic passage of virions through polysynaptic circuits. Adapted with permission from Card (1998b).

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Figure 1.5.2

Morphological correlates of the events shown in Figure 1.5.1, panel B. (A) Formation and aggregation of capsids in the cell nuclei produces the classical nuclear inclusions characteristic of herpesvirus-infected neurons (arrows). (B) In electron micrographs, individual capsids appear as spherical structures containing electron-dense viral DNA. (C) In the model illustrated in Figure 1.5.1, capsids are enveloped in a two-stage process involving sequential passage of capsids through the nuclear membrane (arrow) and endoplasmic reticulum (ER). (D) Release of capsids from the ER is followed by final envelopment at the trans face of the Golgi complex. Envelopment at the trans face of the Golgi provides the two membranes that are necessary for progeny virus to leave the parent cell and gain access to the next permissive cell. Reprinted with permission from Card (1998b).

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Figure 1.5.3

Use of recombinant viruses for definition of complex circuitry. The method is based upon the ability to detect unique reporter proteins produced by the two recombinant viruses. (A) In this case, either bacterial -galactosidase or jellyfish enhanced green fluorescent protein (EGFP) genes have been engineered into the same locus of the PRV-Bartha genome. Importantly, EGFP is fused to a viral gene (Us9) that is lacking in PRV-Bartha and which has been shown to differentially traffic to the Golgi complex (Brideau et al., 1999). (B) Injection of the two recombinants into different peripheral targets leads to retrograde transynaptic passage of virus through parallel circuits, and, ultimately, to coinfection to neurons that collateralize to innervate both circuits.

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Figure 1.5.4

An example of a neuron coinfected with both of the viruses described in Figure 1.5.3. Dual-labeling immunofluorescence was used to localize the two transgenes. Through the use of appropriate filters, the fluorophores can be visualized either simultaneously (A) or individually (B,C). Yellow fluorescence marks the colocalization of the red (Cy3) and green (Cy2) fluorescence. Note that -galactosidase immunoreactivity extends throughout the soma and dendrites of the neuron (panel B) whereas EGFP, which is fused to Us9, is differentially concentrated in paranuclear membranes of the cell soma (panel C).

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Figure 1.5.5

Differential distribution of viral antigens in pyramidal neurons infected with different strains of PRV. In both instances, the neurons were infected by retrograde transport of virus injected into the medial prefrontal cortex (see Card et al., 1995, for more detail). (A) Dense and extensive staining of the somatodendritic compartment of a pyramidal neuron 36 hr following injection of PRV-Bartha into the prefrontal cortex. (B) In contrast, viral immunoreactivity is sequestered within the somata and proximal dendrites of pyramidal neurons infected with a wild-type strain of PRV, even though the postinoculation interval is >10 hr longer.

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Figure 1.5.6

The sequential retrograde transynaptic passage of PRV-Bartha through hippocampal circuitry of the rat. The figures illustrate the distribution of infected neurons 45 hr (Panels A to C) and 50 hr (Panels D to F) following injection of 100 nl of virus into the junction of the CA3 and CA1 subdivisions of Ammon's Horn. In both cases, the brains were sectioned in the horizontal plane. At 45 hr, there is first-order infection of CA3 pyramidal cells (panels A and C) as well as transynaptic infection of neurons in the dentate gyrus (DG) and entorhinal cortex (ERC) as illustrated in panels A and B. The distribution of viral antigen in these neurons provides further insight into the extent of infection of individual neurons. For example, in panel B, neurons are in the early stages of infection, where viral immunoreactivity is confined to cell nuclei and perikarya and other cells, where viral antigen fills the polarized dendrites of individual granule cells. Sites at which virus leaves neurons can often be discriminated by the dense punctate staining of the somata and proximal dendrites of neurons at early stages of infection (panel C). With longer post-inoculation survival, the number of infected neurons in all three areas is substantially increased, but staining is confined to this well-established circuitry.

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Literature Cited

Literature Cited
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Key References
	Card, 1995. See above.
This reference provides a comprehensive overview of the life cycle of PRV.
	Enquist, J.P. and Enquist, L.W. 1996. Pseudorabies virus: A tool for tracing neuronal connections. In Protocols for Gene Transfer in Neuroscience: Towards Gene Therapy of Neurological Disorders (P.R. Lowenstein and L.W. Enquist, eds.) pp. 333-348. John Wiley & Sons, New York.
This chapter describes the use of PRV in a variety of systems.