Anterograde or Retrograde Transsynaptic Circuit Tracing in Vertebrates with Vesicular Stomatitis Virus Vectors

Kevin T. Beier1, Nathan A. Mundell1, Y. Albert Pan1, Constance L. Cepko2

1 These authors contributed equally to this unit, 2 Department of Genetics, Department of Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.26
DOI:  10.1002/0471142301.ns0126s74
Online Posting Date:  January, 2016
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Abstract

Viruses have been used as transsynaptic tracers, allowing one to map the inputs and outputs of neuronal populations, due to their ability to replicate in neurons and transmit in vivo only across synaptically connected cells. To date, their use has been largely restricted to mammals. In order to explore the use of such viruses in an expanded host range, we tested the transsynaptic tracing ability of recombinant vesicular stomatitis virus (rVSV) vectors in a variety of organisms. Successful infection and gene expression were achieved in a wide range of organisms, including vertebrate and invertebrate model organisms. Moreover, rVSV enabled transsynaptic tracing of neural circuitry in predictable directions dictated by the viral envelope glycoprotein (G), derived from either VSV or rabies virus (RABV). Anterograde and retrograde labeling, from initial infection and/or viral replication and transmission, was observed in Old and New World monkeys, seahorses, jellyfish, zebrafish, chickens, and mice. These vectors are widely applicable for gene delivery, afferent tract tracing, and/or directional connectivity mapping. Here, we detail the use of these vectors and provide protocols for propagating virus, changing the surface glycoprotein, and infecting multiple organisms using several injection strategies. © 2016 by John Wiley & Sons, Inc.

Keywords: VSV; transsynaptic tracing; neural circuitry; axon tracing; gene delivery

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Passage and Concentration of Replication‐Competent rVSV
  • Alternate Protocol 1: Passage and Concentration of rVSVΔG
  • Basic Protocol 2: Generation of rVSVΔG Pseudotyped with Minimal Contamination of Previous Envelope G Protein
  • Basic Protocol 3: Stereotaxic Injection of rVSV into Mice
  • Basic Protocol 4: Injection of rVSV into Embryonic Chicken Visual System
  • Basic Protocol 5: Viral Tracing of Visual Circuitry in Zebrafish
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Passage and Concentration of Replication‐Competent rVSV

  Materials
  • 10% (w/v) poly‐D‐lysine hydrobromide (Sigma‐Aldrich, cat. no. P7405)
  • Tissue culture–grade H 2O
  • Cells: 293 (ATCC #CRL‐1573; Graham et al., ), 293T cells (ATCC #CRL‐3216; DuBridge et al., ), or BHK‐21 cells (ATCC #CCL‐10; Whitt, ); all can be purchased from http://www.atcc.org/
  • Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, cat. no. 12491‐015)
  • Fetal bovine serum (FBS; Thermo Fisher Scientific, cat. no. 10082139)
  • Penicillin/streptomycin, 5000 U/ml (Thermo Fisher Scientific, cat. no. 15070‐063)
  • Recombinant vesicular stomatitis virus (rVSV; see Strategic Planning)
  • 70% ethanol (optional)
  • 10% (v/v) bleach
  • Mineral oil (Sigma‐Aldrich, cat. no. M5904‐500 ml)
  • Cell culture plates: 10‐cm tissue culture–treated dishes (Corning, cat. no. 430167)
  • Fluorescence inverted microscope
  • Cell scrapers (Fisher Scientific, cat. no. 08‐100‐242)
  • Beckman ultracentrifuge tubes, Thinwall, Ultra‐Clear, 38.5 ml, 25 × 89 mm (Beckman‐Coulter, cat. no. 344058)
  • Corning bottle‐top vacuum filter system, 0.45 μm (Sigma‐Aldrich, cat. no. CLS430768)
  • Ultracentrifuge with Beckman SW28 or SW32 rotor, or equivalent
  • Shaker
  • Conical‐bottomed cryogenic vials, 0.5 ml (Sigma‐Aldrich, cat. no. Z353361)
  • 12‐ or 24‐well culture plates
NOTE: All steps need to be conducted in a BSL‐2 approved space, with proper use of personal protective equipment (PPE).

Alternate Protocol 1: Passage and Concentration of rVSVΔG

  Materials
  • 10% (w/v) poly‐D‐lysine hydrobromide (Sigma‐Aldrich, cat. no. P7405)
  • Tissue culture–grade H 2O
  • Cells: 293 (ATCC #CRL‐1573); Graham et al., ), 293T cells (ATCC #CRL‐3216; DuBridge et al., ), TVA800 (obtained from the John Young lab or the Ed Callaway lab at the Salk Institute)
  • Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, cat. no. 12491‐015)
  • Fetal bovine serum (FBS)
  • Plasmid encoding the G protein (pCMV‐VSV‐G; Stewart et al., ; Addgene, Plasmid #845)
  • rVSVΔG (see Strategic Planning)
  • 70% ethanol (optional)
  • 10% (v/v) bleach
  • Mineral oil (Sigma‐Aldrich, cat. no. M5904‐500 ml)
  • Cell culture plates: 10‐cm tissue culture–treated dishes (Corning, cat. no. 430167)
  • Fluorescence inverted microscope
  • Beckman ultracentrifuge tubes, Thinwall, Ultra‐Clear, 38.5 ml, 25 × 89 mm (Beckman‐Coulter, cat. no. 344058)
  • Corning bottle‐top vacuum filter system, 0.45 μm (Sigma‐Aldrich, cat. no. CLS430768)
  • Ultracentrifuge with Beckman SW28 or SW32 rotor, or equivalent
  • Shaker
  • Conical‐bottomed cryogenic vials, 0.5 ml (Sigma‐Aldrich, cat. no. Z353361)
  • 12‐ or 24‐well culture plates
  • Additional reagents and equipment for transfection (Ausubel et al., , Chapter 9)
NOTE: All steps need to be conducted in a BSL‐2 approved space, with proper use of personal protective equipment (PPE).

Basic Protocol 2: Generation of rVSVΔG Pseudotyped with Minimal Contamination of Previous Envelope G Protein

  Materials
  • 10% (w/v) poly‐D‐lysine hydrobromide (Sigma‐Aldrich, cat. no. P7405)
  • Tissue culture–grade H 2O
  • Cells: 293 (ATCC #CRL‐1573); Graham et al., ), 293T cells (ATCC #CRL‐3216; DuBridge et al., ), or TVA800 (obtained from the John Young lab or the Ed Callaway lab at the Salk Institute)
  • Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, cat. no. 12491‐015)
  • Fetal bovine serum (FBS; Thermo Fisher Scientific, cat. no. 10082139)
  • Penicillin/streptomycin, 5000 U/ml (Thermo Fisher Scientific, cat. no. 15070‐063)
  • Plasmid encoding the EnvA protein
  • rVSVΔG (see Strategic Planning)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 70% ethanol (optional)
  • 10% (v/v) bleach
  • Mineral oil (Sigma‐Aldrich, cat. no. M5904‐500 ml)
  • Cell culture plates: 10‐cm tissue culture–treated dishes (Corning, cat. no. 430167)
  • Fluorescence inverted microscope
  • Rocking platform
  • Tabletop centrifuge
  • Cell scrapers (Fisher Scientific, cat. no. 08‐100‐242)
  • Beckman ultracentrifuge tubes, Thinwall, Ultra‐Clear, 38.5 ml, 25 × 89 mm (Beckman‐Coulter, cat. no. 344058)
  • Corning bottle‐top vacuum filter system, 0.45 μm (Sigma‐Aldrich, cat. no. CLS430768)
  • Ultracentrifuge with Beckman SW28 or SW32 rotor, or equivalent
  • Shaker
  • Conical‐bottomed cryogenic vials, 0.5 ml (Sigma‐Aldrich, cat. no. Z353361)
  • 24‐well plates
  • Additional reagents and equipment for transfection (Ausubel et al., , Chapter 9), basic cell culture techniques including trypsinization ( appendix 3B; Phelan, ), and titration of virus ( protocol 1, step 12)
NOTE: All steps need to be conducted in a BSL‐2 approved space, with proper use of personal protective equipment (PPE).

Basic Protocol 3: Stereotaxic Injection of rVSV into Mice

  Materials
  • Mineral oil (Sigma‐Aldrich, cat. no. M5904‐500 ml)
  • Purified rVSV (titer should be >109 pfu/ml; stored in aliquots at −80°C; see protocol 1, Alternate Protocol, or protocol 3)
  • Mice of any strain (optimal age should be determined empirically; stereotactic coordinates change over time, with 6 weeks of age sufficient for adult coordinates)
  • 0.01 mg/ml buprenorphine in 0.9% (w/v) NaCl
  • Anesthetic: inhalable isoflurane, or 10 mg/ml ketamine and 1 mg/ml xylazine in 0.9% (w/v) NaCl ( appendix 4B; Davis, )
  • 70% (v/v) ethanol in spray bottle
  • Refresh Lacri‐Lube Lubricant Eye Ointment
  • Betadine surgical scrubs, 7.5% povidone‐iodine
  • Mouse brain atlas (e.g., Franklin and Paxinos, ; or Allen Institute Brain Atlas; http://www.brain‐map.org/)
  • Stereotaxic apparatus (e.g., David Kopf Instruments)
  • Injection pump (UMP‐3, World Precision Instruments)
  • Dual small hub RN coupler (Hamilton, cat. no. 55752‐01) to hold glass capillary in place during injection
  • Glass capillaries (Drummond Scientific, cat. no. 5‐000‐2005) pulled into fine‐tipped injection needles; both the plunger and glass capillaries made to fit the plunger are provided
  • Dumont #5 forceps (Fine Science Tools, cat. no. 11251‐20)
  • Dumont #55 forceps (Fine Science Tools, cat. no. 11255‐20)
  • Fine scissors (Fine Science Tools, cat. no. 14060‐09)
  • Heating pad
  • Animal clippers
  • Scalpel handle (Fine Science Tools, cat. no. 10003‐12)
  • Scalpel blades (Fine Science Tools, cat. no. 10010‐00)
  • Dental drill (Stoelting, cat. no. 51449)
  • Drill bits (Stoelting, cat. no. 514551)
  • Coated Vicryl (polyglactin 910) sutures, size 4–0 (Ethicon, product code J214H)
  • Additional reagents and equipment for injection ( appendix 4F; Donovan and Brown, ) and anesthesia ( appendix 4B; Davis, 2007) of rodents
NOTE: Personal protective equipment (PPE) should be used for this protocol

Basic Protocol 4: Injection of rVSV into Embryonic Chicken Visual System

  Materials
  • Fertilized White Leghorn chicken eggs (Charles River, specific antigen free); store at 16°C (range = 13° to 21°C) at 70% to 80% relative humidity for up to 1 week to prevent development
  • 70% (v/v) ethanol in spray bottle
  • Purified rVSV (titer should be >109 pfu/ml, stored in aliquots at −80°C; see protocol 1, Alternate Protocol, and protocol 3)
  • DMEM (see protocol 1)
  • PBS ( appendix 2A)
  • 10× (1% w/v) Fast Green (Fisher Scientific, cat. no. BP123; optional)
  • 4% (v/v) formaldehyde (w/v) in PBS
  • Dispase I (Sigma‐Aldrich, cat. no. D4818)
  • Egg rack (we use egg packaging from Charles River)
  • Egg incubator
  • 18‐G needles
  • 10‐ml syringes
  • 1.9 mil (thickness = 0.0019 in.) clear acrylic packaging tape (Duck Brand, cat. no. DUC0007567)
  • Curved scissors (Fine Science Tools, cat. no. 14091‐09)
  • 5‐μl Hamilton syringe (Hamilton, cat. no. 87931)
  • 30‐G, 15 mm beveled needle tip for Hamilton syringe (Hamilton, cat. no. 7803‐07)
  • Stereomicroscope with zoom optics and illuminated stage
  • Dumont #5 forceps (Fine Science Tools, cat. no. 11251‐20)
  • Dumont #55 forceps (Fine Science Tools, cat. no. 11255‐20)
  • Spring scissors (Roboz, cat. no. RS‐5606)
NOTE: Personal protective equipment (PPE) should be used for this protocol.NOTE: Aseptic technique should be used throughout. All surgical tools should be sterilized by an autoclave or bead sterilizer.

Basic Protocol 5: Viral Tracing of Visual Circuitry in Zebrafish

  Materials
  • Larval zebrafish, 2 to 5 days‐post fertilization
  • Embryo water: mix 20 ml methylene blue (1 g/liter stock; Fisher Scientific, cat. no. BP117), 6 g Instant Ocean Aquarium Sea Salt Mixture, and 20 liters H 2O
  • 100× (20 mM) 1‐phenyl‐2‐thiourea (PTU) stock (Fisher Scientific, cat. no. AC207250250; dilute in water, heat to dissolve)
  • 70% (v/v) ethanol in spray bottle
  • 30× (0.4% w/v) Tricaine (Acros Organics, cat. no. A00004; adjusted to pH 7 to 7.5 with 1 M Tris base, pH 9)
  • 1.5% low‐melting point agarose (Fisher Scientific, cat. no. BP1360; dilute in water and keep at 42°C)
  • Purified recombinant VSV (rVSV; titer should be >109 pfu/ml, stored in 10‐μl aliquots at −80°C; see protocol 1, Alternate Protocol, and protocol 3)
  • 10× (1% w/v) Fast Green (Fisher Scientific, cat. no. BP123)
  • Halocarbon Oil Series 27 (Sigma‐Aldrich, cat. no. H8773)
  • Bleach
  • 4% formaldehyde in PBS ( appendix 2A) with 0.25% (v/v) Triton X‐100
  • PBT: 0.25% (v/v) Triton X‐100 in phosphate‐buffered saline (PBS; appendix 2A)
  • Glass pipets (100 mm length, 1 mm outer diameter, 0.75 mm inner diameter, with filament; e.g., World Precision Instruments, cat. no. TW100F‐4)
  • Micropipet puller (Sutter Instruments, cat. no. P‐97)
  • Fine forceps, no. 5 (Fine Science Tools, cat. no. 11251‐20)
  • Stereomicroscope with zoom optics and illuminated stage
  • 100‐mm petri dishes
  • Glass Pasteur pipets (Fisher Scientific, cat. no. 13‐678‐30)
  • Pipet pump (Bel‐Art Products, cat. no. F37898‐0000)
  • Pneumatic pump with foot switch (World Precision Instruments, cat. no. PV820 and 3260)
  • Source of pressurized gas, e.g., nitrogen tank
  • Glass‐bottom dishes (MatTek Corporation, cat. no. P50G‐1.5‐14‐F)
  • Microloader tips (Eppendorf, cat. no. 5242956003)
  • Microelectrode holder (World Precision Instruments, cat. no. 5430-ALL)
  • Micromanipulator (Narishige, cat. no. MN‐151)
  • Stage micrometer (Fisher Scientific, cat. no. 50‐753‐2911)
  • Incubator with lighting timer
  • Fluorescent stereomicroscope
  • Rocking platform
NOTE: PPE should be used for this protocol
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Figures

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Literature Cited

Literature Cited
  Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). 2015. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
  Baer, A. and Kehn‐Hall, K. 2014. Viral concentration determination through plaque assays: Using traditional and novel overlay systems. J. Vis. Exp. 93:1‐10. doi: 10.3791/52065.
  Beier, K.T., Saunders, A.B., Oldenburg, I.A., Sabatini, B.L., and Cepko, C.L. 2013b. Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo. Front. Neural. Circuits 7:11. doi: 10.3389/fncir.2013.00011.
  Beier, K.T., Borghuis, B.G., El‐Danaf, R.N., Huberman, A.D., Demb, J.B., and Cepko, C.L. 2013a. Transsynaptic tracing with vesicular stomatitis virus reveals novel retinal circuitry. J. Neurosci. 33:35‐51. doi: 10.1523/JNEUROSCI.0245-12.2013.
  Beier, K.T., Saunders, A., Oldenburg, I. A, Miyamichi, K., Akhtar, N., Luo, L., and Cepko, C.L. 2011. Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors. PNAS 108:15414‐15419. doi: 10.1073/pnas.1110854108.
  Boussif, O., Lezoualc'h, F., Zanta, M. A, Mergny, M.D., Scherman, D., Demeneix, B., and Behr, J.P. 1995. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. PNAS 92:7297‐7301. doi: 10.1073/pnas.92.16.7297.
  Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G., and Deisseroth, K. 2005. Millisecond‐timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8:1263‐1268. doi: 10.1038/nn1525.
  Davis, J. A. 2008. Mouse and rat anesthesia and analgesia. Curr. Protoc. Neurosci. 42:A.4B.1‐A.4B.21.
  Donovan, J. and Brown, P. 2005. Parenteral injections. Curr. Protoc. Neurosci. 33:A.4F.1‐A.4F.9.
  DuBridge, R.B., Tang, P., Hsia, H.C., Leong, P.M., Miller, J.H., and Calos, M.P. 1987. Analysis of mutation in human cells by using an Epstein‐Barr virus shuttle system. Mol. Cell. Biol. 7:379‐387. doi: 10.1128/MCB.7.1.379.
  Dulbecco, R. and Vogt, M. 1953 Some problems of animal virology as studied by the plaque technique. Cold Spring Harb. Symp. Quant. Biol. 18:273‐279. doi: 10.1101/SQB.1953.018.01.039.
  Franklin, K. and Paxinos, G. 2007. The Mouse Brain in Stereotaxic Coordinates, 3rd Ed. Academic Press, San Diego.
  Goodpasture, E.W. and Teague, O. 1923. Transmission of the virus of herpes febrilis along nerves in experimentally infected rabbits. J. Med. Res. 44:139‐184.7.
  Graham, F.L., Smiley, J., Russell, W.C., and Nairn, R. 1977. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36:59‐74. doi: 10.1099/0022-1317-36-1-59.
  Huang, A. and Baltimore, D. 1970. Defective viral particles and viral disease processes. Nature 226:325‐327. doi: 10.1038/226325a0.
  Iannacone, M., Moseman, E.A., Tonti, E., Bosurgi, L., Junt, T., Henrickson, S.E., Whelan, S.P., Guidotti, L.G., and von Andrian, U.H. 2010. Subcapsular sinus macrophages prevent CNS invasion on peripheral infection with a neurotropic virus. Nature 465:1079‐1083. doi: 10.1038/nature09118.
  Inoue, D. and Wittbrodt, J. 2011. One for all‐a highly efficient and versatile method for fluorescent immunostaining in fish embryos. PLoS ONE 6:1‐7. doi: 10.1371/journal.pone.0019713.
  Junt, T., Moseman, E.A., Iannacone, M., Massberg, S., Lang, P. A, Boes, M., Fink, K., Henrickson, S.E., Shayakhmetov, D.M., Di Paolo, N.C., van Rooijen, N., Mempel, T.R., Whelan, S.P., and von Andrian, U.H. 2007. Subcapsular sinus macrophages in lymph nodes clear lymph‐borne viruses and present them to antiviral B cells. Nature 450:110‐114. doi: 10.1038/nature06287.
  Kelly, R.M. and Strick, P.L. 2000. Rabies as a transneuronal tracer of circuits in the central nervous system. J. Neurosci. Methods 103:63‐71. doi: 10.1016/S0165-0270(00)00296-X.
  Lawson, N.D., Stillman, E. A, Whitt, M. A, and Rose, J.K. 1995. Recombinant vesicular stomatitis viruses from DNA. Proc. Natl. Acad. Sci. U.S.A. 92:4477‐4481. doi: 10.1073/pnas.92.19.9009c.
  Lein, E.S., Hawrylycz, M.J., Ao, N., Ayres, M., Bensinger, A., Bernard, A., Boe, A.F., Boguski, M.S., Brockway, K.S., Byrnes, E.J., Chen, L., Chen, L., Chen, T.M., Chin, M.C., Chong, J., Crook, B.E., Czaplinska, A., Dang, C.N., Datta, S., Dee, N.R., Desaki, A.L., Desta, T., Diep, E., Dolbeare, T.A., Donelan, M.J., Dong, H.W., Dougherty, J.G., Duncan, B.J., Ebbert, A.J., Eichele, G., Estin, L.K., Faber, C., Facer, B.A., Fields, R., Fischer, S.R., Fliss, T.P., Frensley, C., Gates, S.N., Glattfelder, K.J., Halverson, K.R., Hart, M.R., Hohmann, J.G., Howell, M.P., Jeung, D.P., Johnson, R.A., Karr, P.T., Kawal, R., Kidney, J.M., Knapik, R.H., Kuan, C.L., Lake, J.H., Laramee, A.R., Larsen, K.D., Lau, C., Lemon, T.A., Liang, A.J., Liu, Y., Luong, L.T., Michaels, J., Morgan, J.J., Morgan, R.J., Mortrud, M.T., Mosqueda, N.F., Ng, L.L., Ng, R., Orta, G.J., Overly, C.C., Pak, T.H., Parry, S.E., Pathak, S.D., Pearson, O.C., Puchalski, R.B., Riley, Z.L., Rockett, H.R., Rowland, S.A., Royall, J.J., Ruiz, M.J., Sarno, N.R., Schaffnit, K., Shapovalova, N.V., Sivisay, T., Slaughterbeck, C.R., Smith, S.C., Smith, K.A., Smith, B.I., Sodt, A.J., Stewart, N.N., Stumpf, K.R., Sunkin, S.M., Sutram, M., Tam, A., Teemer, C.D., Thaller, C., Thompson, C.L., Varnam, L.R., Visel, A., Whitlock, R.M., Wohnoutka, P.E., Wolkey, C.K., Wong, V.Y., Wood, M., Yaylaoglu, M.B., Young, R.C., Youngstrom, B.L., Yuan, X.F., Zhang, B., Zwingman, T.A., and Jones AR. 2007. Genome‐wide atlas of gene expression in the adult mouse brain. Nature 445:168‐176. doi: 10.1038/nature05453.
  Lundh, B. 1990. Spread of vesicular stomatitis virus along the visual pathways after retinal infection in the mouse. Acta Neuropathol. 79:395‐401. doi: 10.1007/BF00308715.
  Matsuda, T. and Cepko, C.L. 2004. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. U.S.A. 101:16‐22. doi: 10.1073/pnas.2235688100.
  Mundell, N., Beier, K.T., Pan, Y., Lapan, S., Göz‐Aytürk, D., Berezovskii, V.K., Wark, A., Drokhlyansky, E., Bielecki, J., Born, R.T., Schier, A., and Cepko, C.L. 2015. Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms. J. Comp. Neurol. 523:1639‐1663. doi: 10.1002/cne.23761.
  Narayan, S., Barnard, R.J. O., and Young, J.A. T. 2003. Two retroviral entry pathways distinguished by lipid raft association of the viral receptor and differences in viral infectivity. J. Virol. 77:1977‐1983. doi: 10.1128/JVI.77.3.1977-1983.2003.
  Nassi, J., Cepko, C.L., Born, R.T., and Beier, K.T. 2015. Neuroanatomy goes viral! Front. Neuroanat. 9:80. doi: 10.3389/fnana.2015.00080.
  Phelan, M.C. 2007. Techniques for mammalian cell tissue culture. Curr. Protoc. Neurosci. 38:A.3B.1‐A.3B.19.
  Sabin, A.B. and Olitsky, P.K. 1937. Influence of host factors on neuroinvasiveness of vesicular stomatitis virus: I. effect of age on the invasion of the brain by virus instilled in the nose. J. Exp. Med. 66:15‐34. doi: 10.1084/jem.66.1.15.
  Stewart, S.A., Dykxhoorn, D.M., Palliser, D., Mizuno, H., Yu, E.Y., An, D.S., Sabatini, D.M., Chen, I.S. Y., Hahn, W.C., Sharp, P.A., Weinberg, R.A., and Novina, C.D. 2003. Lentivirus‐delivered stable gene silencing by RNAi in primary cells. RNA 9:493‐501. doi: 10.1261/rna.2192803.
  Tian, L., Hires, S.A., Mao, T., Huber, D., Chiappe, M.E., Chalasani, S.H., Petreneau, L., Akerboom, J., McKinney, S.A., Schreiter, E.R., Bargmann, C.I., Jayaraman, V., Svoboda, K., and Looger, L.L. 2009. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6:875‐881. doi: 10.1038/nmeth.1398.
  Ugolini, G., Kuypers, H.G., and Strick, P.L. 1989. Transneuronal transfer of herpes virus from peripheral nerves to cortex and brainstem. Science 243:89‐91. doi: 10.1126/science.2536188.
  van den Pol, A.N., Dalton, K.P., and Rose, J.K. 2002. Relative neurotropism of a recombinant rhabdovirus expressing a green fluorescent envelope glycoprotein. J. Virol. 76:1309‐1327. doi: 10.1128/JVI.76.3.1309-1327.2002.
  Viney, T.J., Balint, K., Hillier, D., Siegert, S., Boldogkoi, Z., Enquist, L.W., Meister, M., Cepko, C.L., and Roska, B. 2007. Local retinal circuits of melanopsin‐containing ganglion cells identified by transsynaptic viral tracing. Curr. Biol. 17:981‐988. doi: 10.1016/j.cub.2007.04.058.
  Whelan, S.P., Ball, L.A., Barr, J.N., and Wertz, G.T. 1995. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc. Natl. Acad. Sci. U.S.A. 92:8388‐8392. doi: 10.1073/pnas.92.18.8388.
  Whitt, M.A. 2010. Generation of VSV pseudotypes using recombinant dG‐VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines. J. Virol. Methods 169:365‐374. doi:10.1016/j.jviromet.2010.08.006.
  Wickersham, I.R., Lyon, D.C., Barnard, R.J. O., Mori, T., Finke, S., Conzelmann, K.‐K., Young, J.A.T., and Callaway, E.M. 2007. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53:639‐647. doi:10.1016/j.neuron.2007.01.033.
  Witko, S., Kotash, C., Nowak, R., Johnson, J., Boutilier, L., Melville, K., Heron, S.G., Clarke, D.K., Abramovitz, A.S., Hendry, R.M., Sidhu, M.S., and Parks, C. 2006. An efficient helpervirus‐free method for rescue of recombinant paramyxoviruses and rhadoviruses from a cell line suitable for vaccine development. J. Virol. Methods 1:91‐101. doi: 10.1016/j.jviromet.2006.02.006.
  Zimmer, B., Summermatter, K., and Zimmer, G. 2013. Stability and inactivation of vesicular stomatitis virus, a prototype rhabdovirus. Vet. Microbiol. 162:78‐84. doi:10.1016/j.vetmic.2012.08.023.
Internet Resources
  http://www.atcc.org/
  Many cell lines, including 293 and BHK‐21 cells, can be purchased from the ATCC Web site.
  http://vectorcore.salk.edu/
  The Salk vector core sells aliquots of multiple viral vectors, including rVSV vectors.
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