We thank Dr. Toshio Terashima of Kobe University, Japan, for teaching the laryngeal surgery technique, and Dr. Lynn Enquist of Princeton University for supplying PRV-Bartha. Research was supported by NIH pioneer award DP1 OD000448 to Erich D. Jarvis and an NSF Graduate Research Fellowship award to Gustavo Arriaga. Figures from appropriately credited previous work are used under the PLoS ONE open access Creative Commons license (CC-BY) in accordance with the journal&#8217;s editorial policies.

NOTE: All animal procedures have been reviewed and approved by the Duke University Institutional Animal Care &#38; Use Committee.
1. Storing Pseudorabies Virus
<ol>
	<li>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.</li>
	<li>Aliquot the virus at 20 &#181;l per tube inside a BSL-2 biosafety cabinet and store at -80 &#176;C under appropriate biosafety conditions.</li>
	<li>Thaw an aliquot of PRV immediately before injecting.</li>
</ol>
2. Surgical Preparation for Injections into Muscle
<ol>
	<li>Induce general anesthesia by intramuscular injection of ketamine-xylazine (100&#160;mg/kg ketamine; 10&#160;mg/kg xylazine) and maintain an appropriate anesthetic plane using isofluorane.</li>
	<li>Protect the corneas with an opthalmic ointment.</li>
	<li>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). &#160;Be sure to use sterile drapes to cover surgical areas. Make sure to adhere to sterile techniques throughout the procedures.&#160;&#160;</li>
	<li>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.</li>
	<li>Seal any transected muscles with VetBond tissue adhesive.</li>
</ol>
3. Injection of PRV into Muscle
<ol>
	<li>Load the 10 &#181;l NanoFil microsyringe system with freshly thawed PRV solution, attach a 34 G stainless steel needle, and carefully mount it on the stereotaxic device.</li>
	<li>Slowly clear the dead space and verify that solution exits the microsyringe tip. Discard fluid as bioharzard waste.</li>
	<li>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&#160;(1 &#181;l&#160;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.</li>
	<li>Retract the microsyringe after five minutes.</li>
	<li>Seal the break in the fascia using VetBond tissue adhesive.</li>
	<li>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.&#160;</li>
	<li>Monitor the animal until sternal recumbency and provide analgesia, food, water, and care as required by your institutional animal use guidelines.</li>
	<li>After the experimentally determined survival time (in this case, 90 hr to label 2nd 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.</li>
	<li>Remove and post-fix the brain in 4% formaldehyde for 24 hr.</li>
	<li>Cryoprotect the brain in phosphate buffer containing 30% sucrose for at least 48 hr.</li>
</ol>
4. Immunochemical Detection of eGFP
<ol>
	<li>Cut sections at 40 &#181;m on a sliding microtome and save floating sections into 0.1M PBS. Thinner sections, for example 30 &#181;m, may be used when staining mounted sections.</li>
	<li>Quench sections for 30 min in 0.3% H2O2 in PBS protected from light.</li>
	<li>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).</li>
	<li>Incubate blocked sections for 3.5 hr in rabbit anti-eGFP (1:1,000) at RT.</li>
	<li>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.</li>
	<li>Prepare the ABC solution from the VE kit according to the manufacturer&#8217;s instructions.</li>
	<li>Wash sections three times in PBS for 5 min, then react them for 1 hr in ABC solution from the VE kit at RT.</li>
	<li>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&#39;-diaminobenzidine) and 0.015% H2O2.</li>
	<li>Mount sections on SuperFrost Plus slides and dehydrate them through a graded alcohol series (70%, 95%, 100% and 100% for 5 min each).</li>
	<li>Clear the sections through two xylene washes (5 min each) and coverslip the slides with Permount mounting medium.</li>
	<li>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.</li>
</ol>
5. Surgical Preparation for Injection of Tracers into Brain Regions Discovered by PRV Tracing
<ol>
	<li>Induce general anesthesia by intramuscular injection of ketamine-xylazine (100&#160;mg/kg ketamine; 10&#160;mg/kg xylazine) and maintain an appropriate anesthetic plane using isofluorane.</li>
	<li>Protect the corneas with an opthalmic ointment.</li>
	<li>Fix the head in an appropriate stereotaxic frame.</li>
	<li>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).</li>
	<li>Make a scalp incision and retract the skin over the brain region of interest.</li>
	<li>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.</li>
</ol>
6. Injection of Biotinylated Dextran Amines into Brain
<ol>
	<li>Prepare 7.5% biotinylated dextran amines (BDA) by dissolving 25 mg of BDA (10,000 MW) in 333 &#181;l of sterile saline.</li>
	<li>Load the Nanoject II micropipette system with sufficient BDA solution for the planned injections.</li>
	<li>Slowly clear the dead space and verify that solution is exiting the micropipette tip.</li>
	<li>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&#160;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.</li>
	<li>Seal the craniotomy using dental cement, and close the scalp wound using VetBond tissue adhesive.</li>
	<li>Monitor the animal until sternal recumbency and provide analgesia, food, water, and care as required by your institutional animal use guidelines.</li>
</ol>
7. Injection of Cholera Toxin Subunit b into Muscle
<ol>
	<li>Prepare 1% Cholera Toxin subunit b (CTb) by dissolving 1 mg of CTb in 100 &#181;l of sterile saline.</li>
	<li>Six days after BDA injection into the brain, perform the surgical preparation as described above for PRV injections into muscle.</li>
	<li>Load the 10 &#181;l&#160;NanoFil microsyringe system with CTb solution, attach a 34 G stainless steel needle, and carefully mount it on the stereotaxic device.</li>
	<li>Perform the microinjections into the muscle(s) of interest as previously described for PRV.</li>
	<li>Monitor the animal until sternal recumbency and provide analgesia, food, water, and care as required by your institutional animal use guidelines.</li>
	<li>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.</li>
	<li>Remove and post-fix the brain in 4% formaldehyde for 24 hr.</li>
	<li>Cryoprotect the brain in phosphate buffer containing 30% sucrose for at least 48 hr.</li>
</ol>
8. Detection of BDA and CTb in the Same Sections
<ol>
	<li>Cut sections at 40 &#181;m on a microtome and save floating sections into 0.1M PBS.</li>
	<li>Quench sections for 30 min in 0.3% H2O2 in PBS protected from light.</li>
	<li>Prepare the ABC solution from the VE kit according to the manufacturer&#8217;s instructions.</li>
	<li>Wash sections three times in PBS for 5 min, then react them for 1 hr in ABC solution at RT.</li>
	<li>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&#39;-diaminobenzidine), 0.015% H2O2, and 0.05% nickel chloride. The DAB reaction product inside the cells should appear black.</li>
	<li>Block sections for 30 min in PBS containing 0.3% Tween 20 with normal rabbit serum from the VECTASTAIN Elite Kit.</li>
	<li>Incubate blocked sections for 2 hr in goat anti-CTb (1:10,000) at RT.</li>
	<li>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.</li>
	<li>Prepare the ABC solution from the VE kit according to the manufacturer&#8217;s instructions.</li>
	<li>Wash sections three times in PBS for 5 min, then react them for 30 min in ABC solution at RT.</li>
	<li>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&#39;-diaminobenzidine) and 0.015% H2O2. The DAB reaction product inside the cells should appear brown.</li>
	<li>Mount sections on SuperFrost Plus slides and dehydrate them through a graded alcohol series (70%, 95%, 100% and 100% for 5 min each).</li>
	<li>Clear the sections through two xylene washes (5 min each) and coverslip the slides with Permount mounting medium.</li>
	<li>Image the mounted sections on a microscope.</li>
</ol>

<ol>
	<li>Card, J. P., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transneuronal circuit analysis with pseudorabies viruses.Multiple values selected. Unit 1.5, 1.51-1.5.28 (2001).</li><li>Smith, B. N., Banfield, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudorabies+virus+expressing+enhanced+green+fluorescent+protein:+a+tool+for+in+vitro+electrophysiological+analysis+of+transsynaptically+labeled+neurons+in+identified+central+nervous+system+circuits.">Pseudorabies virus expressing enhanced green fluorescent protein: a tool for in vitro electrophysiological analysis of transsynaptically labeled neurons in identified central nervous system circuits.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (16), 9264-9269 (2000).</li><li>Aston Jones, G., Card, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+pseudorabies+virus+to+delineate+multisynaptic+circuits+in+brain+opportunities+and+limitations.">Use of pseudorabies virus to delineate multisynaptic circuits in brain opportunities and limitations.</a> Journal of Neuroscience Methods. 103 (1), 51-61 (2000).</li><li>Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pseudorabies+virus+impact+on+neurovirology+and+veterinary+medicine.">Molecular biology of pseudorabies virus impact on neurovirology and veterinary medicine.</a> Microbiology and Molecular Biology Reviews. 69 (3), 462-500 (2005).</li><li>Brittle, E. E., Reynolds, A. E., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+pseudorabies+virus+neuroinvasion+and+lethality+in+mice.">Two modes of pseudorabies virus neuroinvasion and lethality in mice.</a> Journal of Virology. 78 (23), 12951-12963 (2004).</li><li>Reiner, A., Veenman, C. L., Medina, L., Jiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway+tracing+using+biotinylated+dextran+amines.">Pathway tracing using biotinylated dextran amines.</a> Journal of neuroscience. 103, 23-37 (2000).</li><li>Reiner, A., Honig, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylated+dextran+a+versatile+anterograde+and+retrograde+neuronal+tracer.">Biotinylated dextran a versatile anterograde and retrograde neuronal tracer.</a> Brain Research. 607 (1-2), 47-53 (1993).</li><li>Dederen, P. J. W. C., Gribnau, A. A. M., Curfs, M. H. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+neuronal+tracing+with+cholera+toxin+B+subunit:+comparison+of+three+different+visualization+methods.">Retrograde neuronal tracing with cholera toxin B subunit: comparison of three different visualization methods.</a> Histochemical Journal. 26 (11), 856-862 (1994).</li><li>Hattox, A. M., Priest, C. 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M., Soban, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+modification+of+diaminobenzidine+(DAB)+precipitation+by+metallic+ions+and+its+application+for+double+immunohistochemistry.">Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry.</a> Journal of Histochemistry and Cytochemistry. 30 (10), 1079-1082 (1982).</li><li>Hsu, S. M., Raine, L., Fanger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+avidin+biotin-peroxidase+complex+(ABC)+in+immunoperoxidase+techniques+a+comparison+between+ABC+and+unlabeled+antibody+(PAP)+procedures.">Use of avidin biotin-peroxidase complex (ABC) in immunoperoxidase techniques a comparison between ABC and unlabeled antibody (PAP) procedures.</a> Journal of Histochemistry and Cytochemistry. 29 (4), 577-580 (1981).</li><li>Wouterlood, F. G., Jorritsma Byham, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anterograde+neuroanatomical+tracer+biotinylated+dextran+amine+comparison+with+the+tracer+Phaseolus+vulgaris+leucoagglutinin+in+preparations+for+electron+microscopy.">The anterograde neuroanatomical tracer biotinylated dextran amine comparison with the tracer Phaseolus vulgaris leucoagglutinin in preparations for electron microscopy.</a> Journal of Neuroscience Methods. 48 (1-2), 75-87 (1993).</li><li>Altschuler, S. M., Bao, X. M., Miselis, R. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+considerations+for+the+use+of+pseudorabies+virus+in+transneuronal+studies+of+neural+circuitry.">Practical considerations for the use of pseudorabies virus in transneuronal studies of neural circuitry.</a> Neuroscience and Biobehavioral Reviews. 22 (6), 685-694 (1998).</li><li>Zuckerman, F. A., Zsak, L., Mettenleiter, T. C., Ben Porat, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudorabies+virus+glycoprotein+gIII+is+a+major+target+antigen+for+murine+and+swine+virus-specific+cytotoxic+T+lymphocytes.">Pseudorabies virus glycoprotein gIII is a major target antigen for murine and swine virus-specific cytotoxic T lymphocytes.</a> Journal of Virology. 64 (2), 802-812 (1990).</li><li>Card, J. P., Enquist, L. W., Moore, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinvasiveness+of+pseudorabies+virus+injected+intracerebrally+is+dependent+on+viral+concentration+and+terminal+field+density.">Neuroinvasiveness of pseudorabies virus injected intracerebrally is dependent on viral concentration and terminal field density.</a> The Journal of Comparative Neurology. 407 (3), 438-452 (1999).</li><li>Pickard, G. E., Smeraski, C. 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No conflicts of interest declared.

There are a number of issues that must be taken into consideration when planning an experiment using PRV1524,21. 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...

Transsynaptic tracing&#160;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 circuit3,4,13. PRV152, which is isogenic with PRV-Bartha, only crosses synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site3,5. 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 amplification11,15. 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 neurons6-8,11. When used together, these two tracers can be visualized in the same section using a two-color DAB (3, 3&#39;-diaminobenzidine) procedure16. High molecular weight BDA (BDA10kDa) was selected for this protocol because it yields detailed labeling of neuronal processes6,7,9. Additional advantages of BDA10kDa include the following: it is preferentially transported in the anterograde direction6-8; it can be delivered by iontophoretic or pressure injection6-8; it can be visualized by a simple avidin-biotinylated HRP (ABC) procedure17; and it can be imaged by light or electron microscopy6,7,18. 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 dendrites10,19. 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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NanoFil Microinjection System</td>
			<td>World Precision Instruments</td>
			<td>IO-Kit</td>
			<td>34 gauge option</td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>David Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II Auto-Nanoliter Injector</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Sliding microtome</td>
			<td>Leica</td>
			<td>SM2010 R</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>VetBond</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane (Forane)</td>
			<td>Baxter&#160;</td>
			<td>1001936060</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine Swab Stick</td>
			<td>Cardinal Health</td>
			<td>2130-01</td>
			<td>200 count</td>
		</tr>
		<tr>
			<td>Permount Mounting Medium</td>
			<td>Fisher Scientific</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated dextran amines</td>
			<td>Invitrogen</td>
			<td>D-1956</td>
			<td>10,000 MW</td>
		</tr>
		<tr>
			<td>Pseudorabies virus</td>
			<td>Laboratory of Dr. Lynn Enquist (Princeton University)</td>
			<td>PRV152</td>
			<td>Titer &#62; 1 x 107</td>
		</tr>
		<tr>
			<td>Anti-Cholera Toxin B Subunit (Goat)</td>
			<td>List Biological Laboratories</td>
			<td>703</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin B Subunit</td>
			<td>List Biological Laboratories</td>
			<td>103B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-eGFP</td>
			<td>Open Biosystems</td>
			<td>ABS4528</td>
			<td></td>
		</tr>
		<tr>
			<td>3, 3&#39;-diaminobenzidine</td>
			<td>Sigma-Aldrich</td>
			<td>D5905</td>
			<td>10 mg tablets</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>200 proof</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Dilute to 4%</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma-Aldrich</td>
			<td>H3410</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Ketamine HCl &#38; Xylazine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>K4138</td>
			<td>80 mg/mL &#38; 6 mg/mL</td>
		</tr>
		<tr>
			<td>Nickel chloride</td>
			<td>Sigma-Aldrich</td>
			<td>339350</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>P3619</td>
			<td>1.0 M; pH 7.4</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>10X; pH 7.4</td>
		</tr>
		<tr>
			<td>Sodium Pentobarbital</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>50 mg/mL dose</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Sigma-Aldrich</td>
			<td>534056</td>
			<td>Histological grade</td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-6101 (rabbit); PK-6105 (goat)</td>
			<td></td>
		</tr>
		<tr>
			<td>Optixcare opthalmic ointment</td>
			<td>Vet Depot</td>
			<td>1017992</td>
			<td></td>
		</tr>
	</tbody>
</table>

transsynaptic tracing from peripheral targets with pseudorabies virus followed by cholera toxin and biotinylated dextran amines double labeling

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 site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;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&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Staining for eGFP should begin showing weak signal in primary motor neurons approximately 72 hr&#160;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 2nd order infected cells. Longer survival times will reveal 3rd and higher order cells but survival times are limited by the lethality of PRV at approximately 5 days after inoculation.
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Watch this Scientific Journal Video about Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling at JoVE.com

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

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.

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

transsynaptic-tracing-from-peripheral-targets-with-pseudorabies-virus

Research

JoVE Journal

Neuroscience

12.6K Views.  Duke University Medical Center. 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.

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

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...