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 viruses, which produce infectious progeny that cross synapses to infect other neurons within a functional circuit^3,4,13. PRV152, which is isogenic with PRV-Bartha, only crosses synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site^3,5. By precisely controlling the peripheral site of infection it is possible to limit the entry of the virus into the brain through a specific subset of motor neurons. As the virus sequentially infects chains of connected neurons, the resulting pattern of eGFP signal throughout the brain will then resolve the network of neurons that are connected to the initially infected cells.

An additional advantage of using virus for neural tracing is the amplification of the reporter protein (eGFP in this case) within infected cells. This signal amplification provides a level of sensitivity that allows detection of even sparse projections. For example, a sparse projection from vibrissa motor cortex to the facial motor neurons controlling the whiskers was found in rats using virally expressed green fluorescent protein¹⁴; previous studies failed to find this projection using classical tracers without reporter gene amplification^11,15. Unfortunately, many viral tracing vectors, like the one used in the cited study, do not cross synapses, thereby limiting their use for tracing multi-synaptic circuits.

While presenting distinct advantages for identifying the network of cells participating in a motor circuit, the distributed nature of transsynaptic tracing with PRV-152 makes interpreting specific connections within the circuit difficult. Therefore, we present a simple method for validating specific connections within circuits identified using PRV-152 by double-labeling using biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb). The combined use of BDA and CTb is a well-established approach for tracing connections between specific sets of neurons^6-8,11. When used together, these two tracers can be visualized in the same section using a two-color DAB (3, 3'-diaminobenzidine) procedure¹⁶. High molecular weight BDA (BDA10kDa) was selected for this protocol because it yields detailed labeling of neuronal processes^6,7,9. Additional advantages of BDA10kDa include the following: it is preferentially transported in the anterograde direction^6-8; it can be delivered by iontophoretic or pressure injection^6-8; it can be visualized by a simple avidin-biotinylated HRP (ABC) procedure¹⁷; and it can be imaged by light or electron microscopy^6,7,18. Immunochemical detection of CTb with peroxidase and DAB was chosen for retrograde labeling of motoneurons because it is effective at revealing cellular processes including distal dendrites^10,19. We recently used this approach to identify the vocal motor pathway in mice and to reveal a sparse connection from primary motor cortex to the laryngeal motor neurons, which was previously assumed to be absent²⁰.

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 Injections into Muscle

1. Induce general anesthesia by intramuscular injection of ketamine-xylazine (100 mg/kg ketamine; 10 mg/kg xylazine) and maintain an appropriate anesthetic plane using isofluorane.
2. Protect the corneas with an opthalmic ointment.
3. Prepare the surgical site according to aseptic technique by trimming hair and disinfecting the surgical site with alternating scrubs of Betadine and 70% alcohol (minimum of 3 cycles).  Be sure to use sterile drapes to cover surgical areas. Make sure to adhere to sterile techniques throughout the procedures.  
4. Make a skin incision and reveal the muscle of interest. For example, to access the cricothyroid laryngeal muscle it is necessary to first remove the overlying portion of the sternohyoid muscle.
5. Seal any transected muscles with VetBond tissue adhesive.

3. Injection of PRV into Muscle

1. Load the 10 µl NanoFil microsyringe system with freshly thawed PRV solution, attach a 34 G stainless steel needle, and carefully mount it on the stereotaxic device.
2. Slowly clear the dead space and verify that solution exits the microsyringe tip. Discard fluid as bioharzard waste.
3. Using a stereotaxic micropositioning device carefully place the microsyringe tip into the muscle of interest and slowly fill the muscle until slight swelling is visible. The injection rate will vary depending on the size of the muscle and volume to be injected. For example, five injections of 200 nl (1 µl total) at a rate of 4 nl/sec should be made 1 min apart at the same site to fill the cricothyroid muscle. Move the syringe to the next muscle of interest (the lateral cricoarytenoid in this case) and repeat the injection procedure. Only puncture each muscle once.
4. Retract the microsyringe after five minutes.
5. Seal the break in the fascia using VetBond tissue adhesive.
6. After all injections have been completed, close the wound using VetBond tissue adhesive. Depending on your local institional animal use guidelines, sutures or wound clips may be used. 
7. Monitor the animal until sternal recumbency and provide analgesia, food, water, and care as required by your institutional animal use guidelines.
8. After the experimentally determined survival time (in this case, 90 hr to label 2^nd order cortical neurons), sacrifice the animal by pentobarbital overdose and perfuse transcardially with 0.9% saline followed by 4% formaldehyde in 0.1 M PBS.
9. Remove and post-fix the brain in 4% formaldehyde for 24 hr.
10. Cryoprotect the brain in phosphate buffer containing 30% sucrose for at least 48 hr.

4. Immunochemical Detection of eGFP

1. Cut sections at 40 µm on a sliding microtome and save floating sections into 0.1M PBS. Thinner sections, for example 30 µm, may be used when staining mounted sections.
2. Quench sections for 30 min in 0.3% H₂O₂ in PBS protected from light.
3. Block non-specific antigens in the sections for 30 min in PBS containing 0.3% Tween 20 with normal goat serum from the VECTASTAIN Elite Kit (VE kit).
4. Incubate blocked sections for 3.5 hr in rabbit anti-eGFP (1:1,000) at RT.
5. Wash sections three times in PBS for 5 min, then incubate them for 1 hr in goat anti-rabbit secondary antibody from the VE kit at RT.
6. Prepare the ABC solution from the VE kit according to the manufacturer’s instructions.
7. Wash sections three times in PBS for 5 min, then react them for 1 hr in ABC solution from the VE kit at RT.
8. Wash sections three times in phosphate buffer for 10 min, and develop for 8 min in phosphate buffer, pH 7.4, containing 0.05% DAB (3, 3'-diaminobenzidine) and 0.015% H₂O₂.
9. Mount sections on SuperFrost Plus slides and dehydrate them through a graded alcohol series (70%, 95%, 100% and 100% for 5 min each).
10. Clear the sections through two xylene washes (5 min each) and coverslip the slides with Permount mounting medium.
11. Image the mounted sections on a microscope. The DAB reaction product inside the cells should appear brown. Digitized images can be color inverted to highlight fine processes.

5. Surgical Preparation for Injection of Tracers into Brain Regions Discovered by PRV Tracing

1. Induce general anesthesia by intramuscular injection of ketamine-xylazine (100 mg/kg ketamine; 10 mg/kg xylazine) and maintain an appropriate anesthetic plane using isofluorane.
2. Protect the corneas with an opthalmic ointment.
3. Fix the head in an appropriate stereotaxic frame.
4. Prepare the surgical site according to aseptic technique by trimming hair and disinfecting the site with alternating scrubs of Betadine and 70% alcohol (minimum of 3 cycles).
5. Make a scalp incision and retract the skin over the brain region of interest.
6. Perform a small craniotomy at the appropriate stereotaxic coordinates. For example, the coordinates for laryngeally connected motor cortex identified by PRV tracing in an adult mouse are 1.2 mm lateral and 0.2 mm rostral to Bregma.

6. Injection of Biotinylated Dextran Amines into Brain

1. Prepare 7.5% biotinylated dextran amines (BDA) by dissolving 25 mg of BDA (10,000 MW) in 333 µl of sterile saline.
2. Load the Nanoject II micropipette system with sufficient BDA solution for the planned injections.
3. Slowly clear the dead space and verify that solution is exiting the micropipette tip.
4. Using a stereotaxic micropositioning device carefully lower the micropipette tip into the brain region of interest and slowly inject BDA. Adjust the injection rate depending on the size of the region to be labeled. For example, 12 injections of 4.6 nl should made be at four different locations 0.2 mm apart (rostro-caudal direction) to cover the laryngeally connected motor cortex in adult mice. The final injection volume should be adjusted according to the size of the brain region of interest, keeping in mind that label may spread from the injection core by diffusion through brain tissue and along the processes of labeled neurons.
5. Seal the craniotomy using dental cement, and close the scalp wound using VetBond tissue adhesive.
6. Monitor the animal until sternal recumbency and provide analgesia, food, water, and care as required by your institutional animal use guidelines.

7. Injection of Cholera Toxin Subunit b into Muscle

1. Prepare 1% Cholera Toxin subunit b (CTb) by dissolving 1 mg of CTb in 100 µl of sterile saline.
2. Six days after BDA injection into the brain, perform the surgical preparation as described above for PRV injections into muscle.
3. Load the 10 µl NanoFil microsyringe system with CTb solution, attach a 34 G stainless steel needle, and carefully mount it on the stereotaxic device.
4. Perform the microinjections into the muscle(s) of interest as previously described for PRV.
5. Monitor the animal until sternal recumbency and provide analgesia, food, water, and care as required by your institutional animal use guidelines.
6. Three days after CTb injection, sacrifice the animal by pentobarbital overdose and perfuse transcardially with 0.9% saline followed by 4% formaldehyde in 0.1 M PBS.
7. Remove and post-fix the brain in 4% formaldehyde for 24 hr.
8. Cryoprotect the brain in phosphate buffer containing 30% sucrose for at least 48 hr.

8. Detection of BDA and CTb in the Same Sections

1. Cut sections at 40 µm on a microtome and save floating sections into 0.1M PBS.
2. Quench sections for 30 min in 0.3% H₂O₂ in PBS protected from light.
3. Prepare the ABC solution from the VE kit according to the manufacturer’s instructions.
4. Wash sections three times in PBS for 5 min, then react them for 1 hr in ABC solution at RT.
5. Wash sections three times in phosphate buffer for 10 min, and develop for 8 min in phosphate buffer, pH 7.4, containing 0.05% DAB (3, 3'-diaminobenzidine), 0.015% H₂O₂, and 0.05% nickel chloride. The DAB reaction product inside the cells should appear black.
6. Block sections for 30 min in PBS containing 0.3% Tween 20 with normal rabbit serum from the VECTASTAIN Elite Kit.
7. Incubate blocked sections for 2 hr in goat anti-CTb (1:10,000) at RT.
8. Wash sections three times in PBS for 5 min, then incubate them for 1 hr in rabbit anti-goat secondary antibody from the VE kit at RT.
9. Prepare the ABC solution from the VE kit according to the manufacturer’s instructions.
10. Wash sections three times in PBS for 5 min, then react them for 30 min in ABC solution at RT.
11. Wash sections three times in phosphate buffer for 10 min, and develop for up to 8 min in phosphate buffer, pH 7.4, containing 0.05% DAB (3, 3'-diaminobenzidine) and 0.015% H₂O₂. The DAB reaction product inside the cells should appear brown.
12. Mount sections on SuperFrost Plus slides and dehydrate them through a graded alcohol series (70%, 95%, 100% and 100% for 5 min each).
13. Clear the sections through two xylene washes (5 min each) and coverslip the slides with Permount mounting medium.
14. Image the mounted sections on a microscope.

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
[header]
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