Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Summary

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Abstract

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)¹. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site^2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)^4,5 . This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3'-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites^6-11.

Introduction

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the rodent brain by utilizing the swine pathogen pseudorabies virus (PRV), especially the attenuated strain PRV-Bartha first described in 1961¹². Here, we present a protocol for identifying the motor cortical representation of specific muscles or muscle groups using a recombinant pseudorabies virus strain (PRV152) expressing the enhanced green fluorescent protein (eGFP) reporter gene². The described method exploits the behavior of neurotropic virus....

Protocol

NOTE: All animal procedures have been reviewed and approved by the Duke University Institutional Animal Care & Use Committee.

1. Storing Pseudorabies Virus

1. We obtain live virus (PRV152) from the laboratory of Dr. Lynn Enquist at Princeton University at a titer of 1 x 10⁹ pfu/m. The protocol to generate the virus has been published².
2. Aliquot the virus at 20 µl per tube inside a BSL-2 biosafety cabinet and store at -80 °C under appropriate biosafety conditions.
3. Thaw an aliquot of PRV immediately before injecting.

2. Surgical Preparation for In....

Results

Staining for eGFP should begin showing weak signal in primary motor neurons approximately 72 hr after injecting PRV152 into muscle. The replication and transsynaptic transport of virus are titer- and time-dependent⁴. Approximately 90 hr after injection, eGFP staining will reveal robust signal in 2^nd order infected cells. Longer survival times will reveal 3^rd and higher order cells but survival times are limited by the lethality of PRV at approximately 5 days after inoculation.

Discussion

There are a number of issues that must be taken into consideration when planning an experiment using PRV152^4,21. Most importantly, pseudorabies virus is lethal. As mentioned previously, great apes, including humans are not susceptible to infection, but appropriate care must be exercised to protect other animals. Adult mice typically survive five to seven days after inoculation with the attenuated PRV152 strain. Therefore, PRV152 is not appropriate for experiments that require survival times longer than one wee.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
NanoFil Microinjection System	World Precision Instruments	IO-Kit	34 G option
Stereotaxic frame	David Kopf Instruments	Model 900
Nanoject II Auto-Nanoliter Injector	Drummond Scientific Company	3-000-204
Sliding microtome	Leica	SM2010 R
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VetBond	3M	1469SB
Isofluorane (Forane)	Baxter	1001936060
Betadine Swab Stick	Cardinal Health	2130-01	200 count
Permount Mounting Medium	Fisher Scientific	SP15-500
SuperFrost Plus slides	Fisher Scientific	12-550-15
Biotinylated dextran amines	Invitrogen	D-1956	10,000 MW
Pseudorabies virus	Laboratory of Dr. Lynn Enquist (Princeton University)	PRV152	Titer >1 x 107
Anti-Cholera Toxin B Subunit (Goat)	List Biological Laboratories	703
Cholera Toxin B Subunit	List Biological Laboratories	103B
Anti-eGFP	Open Biosystems	ABS4528
3, 3'-diaminobenzidine	Sigma-Aldrich	D5905	10 mg tablets
Ethanol	Sigma-Aldrich	E7023	200 proof
Formaldehyde	Sigma-Aldrich	F8775	Dilute to 4%
Hydrogen peroxide	Sigma-Aldrich	H3410	30%
Ketamine HCl & Xylazine HCl	Sigma-Aldrich	K4138	80 mg/ml & 6 mg/ml
Nickel chloride	Sigma-Aldrich	339350
Phosphate buffer	Sigma-Aldrich	P3619	1.0 M; pH 7.4
Phosphate buffered saline	Sigma-Aldrich	P5493	10x; pH 7.4
Sodium Pentobarbital	Sigma-Aldrich	P3761	50 mg/ml dose
Sucrose	Sigma-Aldrich	S9378
Tween 20	Sigma-Aldrich	P1379
Xylenes	Sigma-Aldrich	534056	Histological grade
VECTASTAIN Elite ABC Kit	Vector Laboratories	PK-6101 (rabbit); PK-6105 (goat)
Optixcare opthalmic ointment	Vet Depot	1017992