Rodent Sensory Neuron Culture and Analysis

Alison K. Hall1

1 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.19
DOI:  10.1002/0471142301.ns0319s36
Online Posting Date:  August, 2006
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Abstract

Sensory neurons have proven very useful for analysis of neuronal differentiation in vivo and in vitro. Their utility for in vitro work is based on the fact that sensory neurons are relatively easy to isolate in large numbers and are amenable to manipulations in culture. Lumbar ganglia are usually used because their location in the caudal nervous system means they are the least differentiated at any developmental stage, allowing the analysis of relatively undifferentiated cells. Rodent sensory ganglia from embryonic to adult stages can be dissected effectively and maintained in serumā€free medium or in coculture with other cells or factors. This unit describes generation of embryonic rat lumbar dorsal root ganglia (DRG) cultures, which form an important model system for investigating the cellular and molecular mechanisms that regulate neuronal differentiation. Adult DRG can also be successfully cultured, with a few modifications of the general protocol.

Keywords: sensory neuron; differentiation; dorsal root ganglion

     
 
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Table of Contents

  • Basic Protocol 1: Embryonic Rat Sensory Neuron Culture
  • Alternate Protocol 1: Modifications in Embryonic Rat Sensory Neuron Culture for Specific Assays
  • Alternate Protocol 2: Adult Rat DRG Culture
  • Support Protocol 1: Preplating or Cytotoxic Killing to Remove Non‐Neuronal Cells
  • Support Protocol 2: FudR Treatment to Remove Non‐Neuronal Cells from Adult DRG Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Embryonic Rat Sensory Neuron Culture

  Materials
  • 70% ethanol
  • Hanks' Balanced Salt Solution (HBSS; Invitrogen)
  • E14.5 timed pregnant Sprague‐Dawley rats (Zivic‐Miller, Charles River Laboratories, or Harlan Bioproducts for Science; order multiparous animals, as the dam is more likely to be pregnant and to have a large litter)
  • 5 mg/ml dispase (Roche Applied Science) in HBSS (store in 1‐ml aliquots in 15‐ml conical polypropylene centrifuge tubes at −20°C)
  • 0.1% bovine serum albumin (BSA) in HBSS (store in 10‐ml aliquots in 15‐ml conical polypropylene centrifuge tubes at −20°C)
  • Serum‐free neurobasal medium (see recipe)
  • Horizontal laminar‐flow tissue culture hood
  • Surgical instruments for fine dissection of embryos including:
    • Small scissors
    • Small toothed forceps
    • No. 5 dissecting forceps (four sets, at least two of which are very fine)
  • Surgical instruments for gross dissection to remove uteri, including large scissors and large toothed forceps
  • Plastic biohazard disposal bags (or equivalent thick plastic bags)
  • 100‐mm sterile polystyrene petri plates
  • Dissecting microscope
  • 15‐ml conical polypropylene centrifuge tubes
  • Cotton‐plugged sterile Pasteur pipets
  • Tabletop centrifuge (e.g., IEC Clinical)
  • Unpolished cotton‐stuffed pipets
  • Vacuum aspirator with tubing to accommodate glass pipets
  • Poly‐L‐lysine‐ and laminin‐coated 96‐ or 24‐well plates (see recipe)
  • 37°C, 5% CO 2 incubator
  • Additional reagents and equipment for euthanasia of the rat ( appendix 4H) and counting cells ( appendix 3B)
NOTE: To maintain sterility in the tissue culture room, the dam should be euthanized and the uterus removed in a room other than the tissue culture room. The embryos (still in the uterus) are transported in a clean petri plate to the tissue culture room, and all subsequent steps are performed in a laminar‐flow tissue culture hood under a dissecting microscope.

Alternate Protocol 1: Modifications in Embryonic Rat Sensory Neuron Culture for Specific Assays

  • Adult rat, preferably 8‐ to 10‐weeks old and not pregnant
  • HBSS (Invitrogen) containing 2.5 U/ml dispase and 200 U/ml collagenase (store at –20°C in sterile 1‐ml aliquots in 15‐ml microcentrifuge tubes)
  • Instruments for dissection:
    • Medium scissors (or rongeur)
    • Small (spring) scissors
    • Small toothed forceps
    • Dissection forceps: two coarse, two medium, two fine, one or two very fine (no. 55)
  • Hot‐bead dry sterilizer
  • Platform rotator
NOTE: To maintain sterility in the tissue culture room, euthanasia and gross dissections should performed in a room other than the tissue culture room. Once isolated, the DRG are transported in a clean petri plate to the tissue culture room, and all subsequent steps are performed in a laminar‐flow tissue culture hood under a dissecting microscope.

Alternate Protocol 2: Adult Rat DRG Culture

  Materials
  • Adult neuronal cell suspension ( protocol 3)
  • Growth medium, e.g., serum‐free neurobasal medium (see recipe)
  • 100‐mm polystyrene petri plates
  • 15‐ml conical polypropylene centrifuge tubes
  • Tabletop centrifuge
  • Additional reagents and equipment for counting cells ( appendix 3B) and plating/growing adult DRG neurons ( protocol 3)

Support Protocol 1: Preplating or Cytotoxic Killing to Remove Non‐Neuronal Cells

  Materials
  • Adult DRG neurons growing in 96‐ or 24‐well plates ( protocol 3)
  • 1000× (5 to 10 mM) 5‐fluoro‐2′‐deoxyuridine (FudR) in HBSS (Invitrogen), store in 500‐µl aliquots at –20°C
  • Additional reagents and equipment for plating/growing adult DRG neurons ( protocol 3)
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Figures

  •   FigureFigure 3.19.1 Embryonic day 14.5 (E14.5) rat, as staged according to Christie (). (A) For most immature sensory neurons, embryos are bisected at the “waist” (1), but to obtain the largest yield regardless of differentiation, embryos are bisected at “underarm” (2). (B) The desired vertebral tissue in the embryonic rat is shown in gray in the whole animal (bottom), and in isolation (middle) once pinched free (right) from the remainder of the embryo (top). The tissue should be placed on its side, as shown, and forceps used to pinch off the ventral 1/4 of the column all along its length, which breaks open the cartilaginous vertebral column that encases the spinal cord at this stage. The tissue should then be placed so that dorsal is up, so that the skin and the dorsal, weakly cartilaginous vertebral structures can be gently reflected and removed. This leaves a neural tube with associated, attached grape‐like DRGs (Fig. ). The DRGs may then be plucked off with forceps and placed in a pile.
  •   FigureFigure 3.19.2 Neural tube with attached DRG. (A) After removal of cartilaginous vertebrae and overlying skin, DRG look like grapes attached to the neural tube (arrows point to several DRGs, NT is neural tube). Carefully pinch each DRG off using forceps (examples, black lines). (B) Photograph of neural tube and DRG as seen under the microscope during dissection.
  •   FigureFigure 3.19.3 Illustration of transverse section through adult spinal column, with cuts through bone indicated by thick black bars. Heavy scissors or rongeurs are needed to cut apart a portion of the bony vertebra over the spinal cord on both sides. Care must be taken not to damage the spinal cord (shaded) and associated dorsal roots that connect to DRG on each side. Once these cuts have been made through several consecutive vertebrae, the DRG become easily identifiable when the spinal cord is gently moved laterally within the vertebral column.

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Literature Cited

Literature Cited
   Ai, X., MacPhedran, S.E., and Hall, A.K. 1998. Effects of depolarization on calcitonin gene‐related peptide expression in embryonic sensory ganglion cells in vitro. J. Neurosci. 18:9294‐9302.
   Ai, X., Cappuzzello, J., and Hall, A.K. 1999. Activin and bone morphogenetic proteins induce calcitonin gene‐related peptide in embryonic sensory neurons in vitro. Mol. Cell. Neurosci. 14:506‐518.
   Bayer, S.A. and Altman, J. 1995. Neurogenesis and neuronal migration. In The Rat Nervous System (G. Paxinos, ed.) pp. 1041‐1078. Academic Press, San Diego, Calif.
   Christie, G.A. 1964. Developmental stages in somite and post‐somite rat embryos, based on external appearance, and including some features of the macroscopic development of the oral cavity. J. Morphol. 114:263‐286.
   Cruise, B.A., Xu, P., and Hall, A.K. 2004. Wounds increase activin in skin and a vasoactive neuropeptide in sensory ganglia. Dev. Biol. 271:1‐10.
   Davies, S.J.A., Fitch, M.T., Memberg, S.P., Hall, A.K., Raisman, G., and Silver, J. 1997. Regeneration of adult axons in white matter tracts of the central nervous system. Nature 390:680‐683.
   Hall, A., Ai, X., Hickman, G., MacPhedran, S., Nduaguda, C., and Robertson, C. 1997. The generation of neuronal heterogeneity in a rat sensory ganglion. J. Neurosci. 17:2775‐2784.
   Hall, A.K., Dinsio, K.J., and Cappuzzello, J. 2001. Skin cell induction of calcitonin gene related peptide in embryonic sensory neurons in vitro involves activin. Dev. Biol. 229:263‐270.
   Hawrot, E. and Patterson, P.H. 1979. Long‐term culture of dissociated sympathetic neurons. Methods Enzymol. 58:574–584.
   Kaufman, M.H. 1992. Atlas of Mouse Development. Academic Press, London.
   Lawson, S.N., Caddy, K.W.T., and Biscoe, T.J. 1974. Development of rat dorsal root ganglion neurones: Studies of cell birthdays and changes in mean cell diameter. Cell Tissue Res. 153:399‐413.
   Lindsay, R.M. 1988. Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons. J. Neurosci. 8:2394‐2405.
   Lindsay, R.M. and Harmar, A.J. 1989. Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons. Nature 337:362‐364.
   Memberg, S.P. and Hall, A.K. 1995. Proliferation, differentiation and survival of rat sensory neuron precursors in vitro are selectively regulated by trophic factors. Mol. Cell. Neurosci. 6:323‐336.
   Mirnics, K. and Koerber, H.R. 1995. Prenatal development of rat primary afferent fibers: I. Peripheral projections. J. Comp. Neurol. 355:589‐600.
   Mu, X., Silos‐Santiago, I., Carroll, S.L., and Snider, W.D. 1993. Neurotrophin receptor genes are expressed in distinct patterns in developing dorsal root ganglia. J. Neurosci. 13:4029‐4041.
   Otten, U. and Lorez, H.P. 1982. Nerve growth factor increases substance P, cholecystokinin and vasoactive intestinal polypeptide immunoreactivities in primary sensory neurones of newborn rats. Neurosci. Lett. 34:153‐158.
   Woodruff, T.K., Sluss, P., Wang, E., Janssen, I., and Mersol‐Barg, M.S. 1997. Activin A and follistatin are dynamically regulated during human pregnancy. J. Endocrinol. 152:167‐174.
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