Isolation of Cerebellar Granule Cells from Neonatal Rats

Jan Oberdoerster1

1 Aventis CropScience, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.7
DOI:  10.1002/0471140856.tx1207s09
Online Posting Date:  November, 2001
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Abstract

Cerebellar Granule Cells in Neurotoxicology (Jan Oberdoerster, Aventis Corporation, Research Triangle Park, North Carolina). Cultured neurons allow the researcher to investigate mechanisms of toxicity on a relatively uniform population of cells. Primary cultures of cerebellar granule cells are post‐mitotic neurons that are readily isolated and may be used for experimental procedures including electrophysiology, neuronal maturation, and various biochemical and molecular analyses.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Preparation of Cerebral Granule Cells
  • Support Protocol 1: Preparation of the Culture Substrate of Cell Culture Plates
  • Support Protocol 2: Determining Cell Number with a Hemacytometer
  • Support Protocol 3: Heat‐Inactivation of Fetal Bovine Serum
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Tables
     
 
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Materials

Basic Protocol: Preparation of Cerebral Granule Cells

 Materials
  • 8‐day‐old postnatal Sprague‐Dawley rats
  • 70% ethanol
  • Antibiotic DMEM (see recipe)
  • 2.5% (w/v) trypsin solution (e.g., Life Technologies), 4°C
  • 100× DNase solution(see recipe)
  • Complete DMEM supplemented with 10% (v/v) heat‐inactivated fetal bovine serum (see recipe for DMEM; see Support Protocol 3 for heat‐inactivated fetal bovine serum)
  • Crystal violet solution (see recipe)
  • 500× AraC solution (see recipe)
  • Surgical gloves
  • 165‐mm large curved operating scissors
  • Laboratory pad (VWR)
  • Animal disposal plastic bags
  • Curved forceps (114‐mm length and 0.8‐mm tip width)
  • 115‐mm delicate dissecting scissors
  • Rounded weighing spatula (e.g., VWR)
  • 100‐mm an 60‐mm sterile tissue culture plastic dishes with and without treatment with poly‐d‐lysine (e.g., Falcon; see Support Protocol 1)
  • Dumont forceps
  • 115‐mm angled delicate dissecting scissors
  • 50‐ml and 15‐ml sterile conical tubes (e.g., Falcon)
  • 10‐ml and 25‐ml sterile serological pipets
  • 37°C water bath
  • 10‐ml syringe and 20‐G needle
  • 40‐, 70‐, and 100‐µm sterile nylon mesh cell strainers (e.g., Falcon), sterile
  • 75‐cm2 tissue culture flasks treated with poly‐d‐lysine (e.g., Falcon; see Support Protocol 1)
  • Inverted compound microscope
  • Hemacytometer (see Support Protocol 2)
  • Glass coverslips

NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic techniques should be used accordingly.

NOTE: All cultured incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

Support Protocol 1: Preparation of the Culture Substrate of Cell Culture Plates

 Materials
  • 100× poly‐d‐lysine stock solution (see recipe)
  • Phosphate‐buffered saline, pH 7.4, store at room temperature (PBS; appendix 2A)
  • 75‐cm2 tissue culture flasks, sterile
  • 10‐ml pipets, sterile
  • 36‐, 60‐, and 100‐mm tissue culture dishes, sterile

Support Protocol 3: Heat‐Inactivation of Fetal Bovine Serum

 Materials
  • Fetal bovine serum, qualified (Life Technologies)
  • 56°C water bath
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Figures

Videos

Literature Cited

 Literature Cited
    Aloisi, F., Clotti, M.T., Levi, G. 1985. Characterization of GABAergic neurons in cerebellar primary cultures and selective neurotoxic effect of serum fractions. J. Neurosci. 5:2001‐2008.
    Balazs, R., Jørgensen, O., and Hack, N. 1988. N‐methyl‐d‐aspartate promotes the survival of cerebellar granule cells in culture. Neuroscience 27:437‐451.
    Burgoyne, R., Graham, M., and Cambray‐Deakin, M. 1993. Neurotrophic effects of NMDA receptor activation on developing cerebellar granule cells. J. Neurocytol. 22:689‐695.
    Copani, A., Bruno, V., Barresi, V., Battaglia, G., Condorelli, D., and Nicoletti, F. 1995. Activation of metabotropic glutamate receptors prevents neuronal apoptosis in culture. J. Neurochem. 64:101‐108.
    Cull‐Candy, S.G., Howe, J.R., and Ogden, D.C. 1988. Noise and single channels activated by excitatory amino acids in rat cerebellar granule neurones. J. Physiol. 400:189‐222.
    D'Mello, S., Anelli, R., and Calissano, P. 1994. Lithium induces apoptosis in immature cerebellar granule cells but promotes survival of mature neurons. Exp. Cell Res. 211:332‐338.
    Dessi, F., Pollard, H., Moreau, J., Ben‐Ari, Y., and Charriaut‐Marlangue, C. 1995. Cytosine arabinoside induces apoptosis in cerebellar neurons in culture. J. Neurochem. 64:1980‐1987.
    Fields, K.L., Currie, D.N., and Dutton, G.R. 1982. Development of THY‐1 antigen on cerebellar neurons in culture. J. Neurosci. 2:663‐673.
    Gallo, V., Kingsbury, A., Balazs, R., and Jorgensen, O.S. 1987. The role of depolarization in the survival and differentiation of cerebellar granule cells in culture. J. Neurosci. 7:2203‐2213.
    Levi, G., Aloisi, F., Ciotti, M.T., and Gallo, V. 1984. Autoradiographic localization and depolarization‐induced release of acidic amino acids in differentiating cerebellar granule cell cultures. Brain Res. 290:77‐86.
    Marini, A.M., Ueda, Y., and June, C.H. 1999. Intracellular survival pathways against glutamate receptor agonist excitotoxicity in cultured neurons. Intracellular calcium responses. Ann. N.Y.Acad. Sci. 890:421‐437.
    Oberdoerster, J. and Rabin, R.A. 1999. Enhancd caspase activity during ethanol‐induced apoptosis in rat cerebellar granule cells. Eur. J. Pharmacol. 385:273‐282.
    Oberdoerster, J., Guizzetti, M., and Costa, L.G. 2000. The effect of phenylalanine and its metabolites on the proliferation and viability of neuronal and astroglial cells: Possible relevance in maternal phenylketonuria. J. Pharmacol. Exp. Ther. 295:295‐301.
    Pantazis, N., Dohrman, D., Goodlett, C., Cook, R., and West, J. 1993. Vulnerability of cerebellar granule cells to alcohol‐induced cell death diminishes with time in culture. Alcohol Clin. Exp. Res. 17:1014‐1021.
    Saunders, P.A., Chalecka‐Franaszek, E., and Chuang, D.M. 1997. Subcellular distribution of glyceraldehyde‐3‐phosphate dehydrogenase in cerebellar granule cells undergoing cytosine arabinoside‐induced apoptosis. J. Neurochem. 69:1820‐1828.
    Schramm, M., Eimerl, S., Costa, E. 1990. Serum and depolarizing agents cause acute neurotoxicity in cultured cerebellar granule cells: Role of the glutamate receptor responsive to N‐methyl‐d‐aspartate. Proc. Natl. Acad. Sci. U.S.A. 87:1193‐1197.
    Wyllie, D.J. and Cull‐Candy, S.G. 1994. A comparison of non‐NMDA receptor channels in type‐2 astrocytes and granule cells from rat cerebellum. J. Physiol. 475:95‐114.
 Key References
    Reberl, G., Haynes, L., and Lelong, I.H. 1999. Nerve cell culture methodology: The medium environment. In The Neuron in Tissue Culture. (L.W. Haynes, ed.) pp. 323‐352, John Wiley & Sons, New York.

An excellent review of potential pitfalls and methodologies in both primary and clonal neuronal cell cultures.

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