Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses

Dušica Curanović1, Toh Hean Ch'ng2, Moriah Szpara1, Lynn Enquist1

1 Department of Molecular Biology, Princeton University, Princeton, New Jersey, 2 University of California at Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.4
DOI:  10.1002/0471143030.cb2604s43
Online Posting Date:  June, 2009
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Abstract

Compartmented neuronal cultures allow experimenters to establish separate fluid environments for neuronal axons and the soma from which they emanate. Physical isolation of cell bodies and axons is achieved by culturing neurons in tri‐chambered Teflon rings. Dissociated ganglia are plated in one end compartment of the trichamber, and axonal growth is guided underneath watertight silicone grease barriers into a separate compartment. Since the axons and cell bodies are located in different compartments, they can be infected and assayed separately. We describe the assembly and use of compartmented neuronal cultures for in vitro study of directional infection of neurons by alpha herpesviruses. Selective application of viral inoculum to only one compartment ensures that the remainder of the neuron is not contaminated by input inoculum. This allows for quantification of viral spread, and unambiguous interpretation of immunofluorescence and electron microscopy images. Curr. Protoc. Cell Biol. 43:26.4.1‐26.4.23. © 2009 by John Wiley & Sons, Inc.

Keywords: compartmented neuron culture; Campenot chambers; tri‐chamber; anterograde; retrograde; alpha herpesvirus; virus

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assembling the Trichamber System
  • Basic Protocol 2: Plating and Maintaining Dissociated Neurons in the Trichamber System
  • Support Protocol 1: Disassembling and Cleaning the Trichambers: Routine Cleaning
  • Support Protocol 2: Disassembling and Cleaning the Trichambers: Occasional Cleaning
  • Basic Protocol 3: Studying the Spread of Viral Infection: Anterograde Transport and Neuron‐To‐Cell Transmission of Infection
  • Alternate Protocol 1: Studying the Spread of Viral Infection: Retrograde Transport and Axon‐Mediated Infection of Neurons
  • Basic Protocol 4: Visualizing Infection in the Trichamber System: Immunofluorescence
  • Alternate Protocol 2: Visualizing Infection in the Trichamber System: Electron Microscopy of Trichamber System Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Assembling the Trichamber System

  Materials
  • Poly‐DL‐ornithine (Sigma) diluted in borate buffer
  • Tissue culture–grade water
  • Laminin (BD Biosciences)
  • HBSS without Ca2+ and Mg2+ (CMF‐HBSS; HyClone)
  • Silicone high‐vacuum grease, Dow Corning (VWR, cat. no. 59344‐055)
  • 70% ethanol
  • Neuronal medium containing 1% (v/v) methocel (see recipe)
  • Neuronal medium (see recipe)
  • Teflon trichambers (Tyler Research; see recipe)
  • Pin rake (homemade or Tyler Research)
  • 35‐mm plastic tissue culture dishes
  • 15‐cm non‐tissue culture‐treated dishes (optional)
  • Disposable 3‐ml syringe
  • Machine‐sawed 18‐G hypodermic needle or truncated 200‐µl pipet tip
  • Autoclave
  • Hemostat (e.g., Roboz RS‐7293)
  • Additional reagents and equipment for cleaning the Teflon chambers ( protocol 3)

Basic Protocol 2: Plating and Maintaining Dissociated Neurons in the Trichamber System

  Materials
  • Pregnant mouse, E14 or E15
  • HBSS (HyClone)
  • HBSS without Ca2+ and Mg2+ (CMF‐HBSS; HyClone)
  • 2.5 mg/ml trypsin in HBSS (see recipe)
  • 1 mg/ml DNase in neurobasal medium (see recipe)
  • 2 mg/ml soybean trypsin inhibitor (SBTI) in neurobasal medium (see recipe)
  • Neuronal medium (see recipe)
  • Cytosine β‐D‐arabinofuranoside hydrochloride (AraC; see recipe)
  • 15‐ml conical tubes
  • Water bath at 37°C
  • Flame‐polished glass Pasteur pipet
  • Hemacytometer
  • Teflon trichambers (Tyler Research; see recipe)
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.1)

Support Protocol 1: Disassembling and Cleaning the Trichambers: Routine Cleaning

  Materials
  • Distilled water
  • 200‐proof (absolute) ethanol
  • Trichambers that need cleaning
  • Blunt forceps
  • Paper towels
  • Kimwipes
  • Autoclave
  • 10‐cm glass petri dish (optional)

Support Protocol 2: Disassembling and Cleaning the Trichambers: Occasional Cleaning

  • Concentrated sulfuric acid

Basic Protocol 3: Studying the Spread of Viral Infection: Anterograde Transport and Neuron‐To‐Cell Transmission of Infection

  Materials
  • Two‐week‐old neuron cultures in the trichamber system ( protocol 2)
  • A confluent monolayer of tissue culture cells (e.g., PK15 cells)
  • Trypsin (Invitrogen)
  • Dulbecco's Modified Eagle Medium (DMEM; Invitrogen)
  • Fetal bovine serum (HyClone)
  • Neuronal medium containing 1% (v/v) methocel (see recipe)
  • Viral stock to be studied (e.g., 107 to 108 PFU/ml PRV)
  • Disposable protein gel‐loading tip (Rainin)
  • −80°C freezer

Alternate Protocol 1: Studying the Spread of Viral Infection: Retrograde Transport and Axon‐Mediated Infection of Neurons

  Materials
  • Two‐week‐old neuron cultures in the trichamber system
  • Neuronal medium containing 1% methocel (see recipe)
  • Viral stock to be studied (e.g., PRV)
  • Disposable protein gel‐loading pipet tip (Rainin)
  • −80°C freezer

Basic Protocol 4: Visualizing Infection in the Trichamber System: Immunofluorescence

  Materials
  • Poly‐DL‐ornithine (Sigma)
  • Laminin (BD Biosciences)
  • Phosphate‐buffered saline (PBS; HyClone, cat. no. SH30028.03)
  • 3.2% (w/v) paraformaldehyde in PBS (store up to 2 weeks, in the dark, at 4°C)
  • PBS/BSA: 3% (w/v) bovine serum albumin (BSA) in PBS (HyClone store solution up to 6 months at 4°C)
  • PBS/BSA/SAP: PBS (HyClone)/3% (w/v) bovine serum albumin (BSA; Roche)/0.5% (v/v) saponin solution (Sigma; store solution up to 6 months at 4°C)
  • Primary antibody
  • Fluorophore‐conjugated secondary antibody
  • Tissue culture–grade water
  • Mounting medium (e.g., Aqua Poly/Mount from Polysciences)
  • Aclar (EM Sciences)
  • UV source for sterilization
  • Pin rake (Tyler Research or homemade)
  • 35‐mm tissue culture dish
  • Blunt forceps
  • Sharp forceps
  • Spatula
  • Dissection scissors, optional
  • Microscope slide
  • Microscope coverslip
  • Additional reagents and equipment for assembling the trichamber ( protocol 1) and plating dissociated neurons, maintaining the cultures, scoring axon penetration, and subsequent infection assays ( protocol 2 and 3 and protocol 6)

Alternate Protocol 2: Visualizing Infection in the Trichamber System: Electron Microscopy of Trichamber System Samples

  Materials
  • Two‐week‐old cultures in the trichamber system, set up on Aclar ( protocol 7)
  • Phosphate‐buffered saline (PBS; HyClone)
  • 2% (v/v) glutaraldehyde in sodium cacodylate buffer (see recipe)
  • 0.2 M sodium cacodylate buffer, pH 7.2 (see recipe)
  • 1% (w/v) osmium tetraoxide in sodium veronal buffer (see recipe)
  • Sodium veronal buffer (see recipe)
  • 0.25% (w/v) toluidine blue in sodium cacodylate buffer (see recipe)
  • 0.05 M sodium maleate buffer, pH 5.1 (see recipe)
  • 2% (w/v) uranyl acetate in sodium maleate buffer (see recipe)
  • Ethanol solutions of 30%, 50%, 70%, 90%, 95%, and 100%
  • Epon resin (Embed812; see recipe)
  • BEEM capsules
  • Fine‐tipped forceps
  • 60°C oven
  • Ultramicrotome (e.g., Leica UC6)
  • 200‐mesh hexagonal copper grid (EM Sciences)
  • Zeiss 912AB TEM with an Omega Energy Filter
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Figures

Videos

Literature Cited

Literature Cited
   Bi, J., Tsai, N.P, Lin, Y.P., Loh, H.H., and Wei, L.N. 2006. Axonal mRNA transport and localized translational regulation of kappa‐opioid receptor in primary neurons of dorsal root ganglia. Proc. Natl. Acad. Sci. U.S.A. 103:19919‐19924.
   Brewer, G.J., Torricelli, J.R., Evege, E.K., and Price, P.J. 1993. Optimized survival of hippocampal neurons in B27‐supplemented Neurobasal, a new serum‐free medium combination. J. Neurosci. Res. 35:567‐576.
   Campenot, R.B. 1977. Local control of neurite development by nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 74:4516‐4519.
   Campenot, R.B. 1992. Compartmented culture analysis of nerve growth. In Cell‐Cell Interactions: A Practical Approach (R.B. Stevenson, Gallin, W.J., and Paul, D.L., ed.) pp. 275‐298. IRL Press, Oxford.
   Ch'ng, T.H., Flood, E.A., and Enquist, L.W. 2005. Culturing primary and transformed neuronal cells for studying pseudorabies virus infection. Methods Molec. Biol. 292:299‐316.
   Ch'ng, T.H., Spear, P.G., Struyf, F., and Enquist, L.W. 2007. Glycoprotein D‐independent spread of pseudorabies virus infection in cultured peripheral nervous system neurons in a compartmented system. J. Virol. 81:10742‐10757.
   Curanović, D., Lyman, M., Bisher, M., and Enquist, L.W. Mutations in UL21 affect the efficiency of retrograde transneuronal infection by PRV‐Bartha. Manuscript in preparation.
   DiCicco‐Bloom, E. and Black, I.B. 1988. Insulin growth factors regulate the mitotic cycle in cultured rat sympathetic neuroblasts. Proc. Natl. Acad. Sci. U.S.A. 85:4066‐4070.
   DiCicco‐Bloom, E., Townes‐Anderson, E., and Black, I.B. 1990. Neuroblast mitosis in dissociated culture: regulation and relationship to differentiation. J. Cell Biol. 110:2073‐2086.
   Feierbach, B., Bisher, M., Goodhouse, J., and Enquist, L.W. 2007. In vitro analysis of transneuronal spread of an alpha herpesvirus infection in peripheral nervous system neurons. J. Virol. 81:6846‐6857.
   Hayashi, H., Campenot, R.B., Vance, D.E., and Vance, J.E. 2004. Glial lipoproteins stimulate axon growth of central nervous system neurons in compartmented cultures. J. Biol. Chem. 279:14009‐14015.
   Hayat, M.A. 2000. Principles and Techniques of Electron Microscopy: Biological Applications. Cambridge University Press.
   Ivins, K.J., Bui, E.T., and Cotman, C.W. 1998. Beta‐amyloid induces local neurite degeneration in cultured hippocampal neurons: evidence for neuritic apoptosis. Neurobiol. Dis. 5:365‐378.
   Johnson, M.I. 2001. Primary cultures of sympathetic ganglia. Humana Press Inc., Totowa, N.J.
   Karten, B., MacInnis, B.L., Eng, H., Azumaya, Y., Martin, G., Lund, K., Watts, R.C., Vance, J.E., Vance, D.E., and Campenot, R.B. 2002. Analytical approaches for investigating apoptosis and other biochemical events in compartmented cultures of sympathetic neurons. In Apoptosis Techniques and Protocols Vol. 37. pp. 163‐175. Humana Press.
   Karten, B., Hayashi, H., Campenot, R.B., Vance, D.E., and Vance, J.E. 2005. Neuronal models for studying lipid metabolism and transport. Methods. 36:117‐128.
   Kimpinski, K., Campenot, R.B., and Mearow, K. 1997. Effects of the neurotrophins nerve growth factor, neurotrophin‐3, and brain‐derived neurotrophic factor (BDNF) on neurite growth from adult sensory neurons in compartmented cultures. J. Neurobiol. 33:395‐410.
   Kingsley, R.E. and Cole, N.L. 1988. Preparation of cultured mammalian cells for transmission and scanning electron microscopy using Aclar film. J. Electron Microsc. Tech. 10:77‐85.
   Lycke, E., Kristensson, K., Svennerholm, B., Vahlne, A., and Ziegler, R. 1984. Uptake and transport of herpes simplex virus in neurites of rat dorsal root ganglia cells in culture. J. Gen. Virol. 65:55‐64.
   Manning, P.T., Johnson, E.M., Jr., Wilcox, C.L., Palmatier, M.A., and Russell, J.H. 1987. MHC‐specific cytotoxic T lymphocyte killing of dissociated sympathetic neuronal cultures. Am. J. Pathol. 128:395‐409.
   Mikloska, Z. and Cunningham, A.L. 2001. Alpha and gamma interferons inhibit herpes simplex virus type 1 infection and spread in epidermal cells after axonal transmission. J. Virol. 75:11821‐11826.
   Penfold, M.E., Armati, P., and Cunningham, A.L. 1994. Axonal transport of herpes simplex virions to epidermal cells: Evidence for a specialized mode of virus transport and assembly. Proc. Natl. Acad. Sci. U.S.A. 91:6529‐6533.
   Samuel, M.A., Wang, H., Siddharthan, V., Morrey, J.D., and Diamond, M.S. 2007. Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis. Proc. Natl. Acad. Sci. U.S.A. 104:17140‐17145.
   Ziegler, R.J. and Herman, R.E. 1980. Peripheral infection in culture of rat sensory neurons by herpes simplex virus. Infect. Immun. 28:620‐623.
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