Culture of Postnatal Mesencephalic Dopamine Neurons on an Astrocyte Monolayer

Caroline Fasano1, Dominic Thibault1, Louis‐Éric Trudeau1

1 Département de Pharmacologie, Faculté de Médecine, Université de Montréal, Québec, Canada
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.21
DOI:  10.1002/0471142301.ns0321s44
Online Posting Date:  July, 2008
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This unit presents a protocol for primary culture of postnatal mesencephalic dopamine neurons grown on an astrocyte monolayer, which can be used to investigate cellular and molecular mechanisms regulating dopamine neuron function. Using this in vitro approach, dopamine neurons survive for an extended period of time and establish functional axon terminals and dendrites that display properties similar to those observed in vivo and in brain slices. An alternate protocol is provided for a microculture system in which astrocytes are grown on a spatially limited surface where single or small groups of dopamine neurons develop. Under such conditions, isolated neurons establish synaptic contacts, or autapses, onto their own somatodendritic compartment, thus facilitating morphological and physiological experiments. Curr. Protoc. Neurosci. 44:3.21.1‐3.21.19. © 2008 by John Wiley & Sons, Inc.

Keywords: primary culture; microculture; dopamine neuron; astrocyte; mesencephalon; cortex; mouse

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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture of Mouse Postnatal Mesencephalic Dopamine Neurons on a Cortical Astrocyte Monolayer
  • Alternate Protocol 1: Culture of Mouse Postnatal Mesencephalic Dopamine Neurons on Astrocyte Microislands to form Autapses
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Culture of Mouse Postnatal Mesencephalic Dopamine Neurons on a Cortical Astrocyte Monolayer

  • 12 N HCl
  • Sterile‐filtered, cell culture‐tested water (Sigma)
  • 95% and 70% ethanol
  • Collagen solution 1 (see recipe)
  • Poly‐L‐lysine solution (see recipe)
  • MEM+ solution (see recipe)
  • Dissociation solution (see recipe)
  • Papain solution (see recipe)
  • Inhibitory solution (see recipe)
  • Mice aged from P0 to P5 (no specific requirement in terms of gender or strain)
  • Minimum Essential Medium (MEM, 1×; Invitrogen)
  • Versene solution (Invitrogen)
  • PBS ( appendix 2A)
  • 0.5% trypsin solution (see recipe)
  • 0.4% trypan blue stain (Invitrogen)
  • Conditioned MEM+ solution (see recipe)
  • FUDR solution (see recipe)
  • Centrifugation solution (see recipe)
  • Trituration solution (see recipe)
  • Neurobasal‐A+/MEM+ medium (see recipe)
  • Kynurenic acid solution (see recipe)
  • 1.5‐cm round glass coverslips (Ted Pella)
  • Dissection instruments (most surgical instruments can be obtained from suppliers such as Fine Science Tools):
    • 1 pair of microdissecting scissors
    • 1 pair of thin forceps (no. 5, Dumont)
    • 1 pair of curved forceps (no. 7, Dumont)
    • 1 scalpel handle (no. 3, Feather)
    • Scalpel blades (no. 10, Feather)
  • Alcohol burner to place in the sterile hood
  • 90‐mm Whatman paper filters
  • UV light
  • 100‐mm × 15‐mm sterile petri dishes (Fisher)
  • Airtight box large enough to contain the desired number of 100‐mm petri dishes
  • Soldering iron or flame‐heated screwdriver
  • 10‐ml syringes
  • 0.2‐µm RC 15‐mm diameter syringe filters (Corning)
  • 35 × 10–mm sterile tissue culture dishes (Falcon)
  • Autoclave
  • Two 5‐ml sterile serological glass pipets (Fisher) with flamed‐polished tips with ∼1.5‐ and 0.5‐mm i.d
  • Aluminum foil
  • Flame‐curved Pasteur pipet (5 ¾‐in.; Fisher, see Fig. )
  • Binocular inverted microscope
  • 10‐ml sterile serological glass pipets (Fisher)
  • 15‐ml sterile centrifuge tubes (Corning)
  • Submersible water bath with agitation system and temperature control
  • 25‐cm2 cell culture flasks, tissue culture treated (Corning)
  • 175‐cm2 cell culture flasks, tissue culture treated (Nunc)
  • Clinical centrifuge (for 15‐ml centrifuge tubes)
  • Additional reagents and equipment for cell counting using a hemacytometer and trypan blue ( appendix 3B)
NOTE: Autoclave all instruments before use and clean them as needed during the dissection procedure in 70% ethanol. During dissection, store all brain tissues in ice‐cold dissociation medium on ice.

Alternate Protocol 1: Culture of Mouse Postnatal Mesencephalic Dopamine Neurons on Astrocyte Microislands to form Autapses

  • Poly‐L‐ornithine solution (see recipe)
  • 0.15% agarose solution (see recipe)
  • Collagen solution 2 (see recipe)
  • TLC Reagent sprayer (Kimble/Kontes)
  • UV‐light
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Literature Cited

Literature Cited
   Bourque, M.‐J. and Trudeau, L.E. 1998. GDNF enhances the synaptic efficacy of dopaminergic neurons in culture. Eur. J. Neurosci. 12:3172‐3180.
   Cardozo, D.L. 1993. Midbrain dopaminergic neurons from postnatal rat in long‐term primary culture. Neuroscience 56:409‐421.
   Congar, P., Bergevin, A., and Trudeau, L.E. 2002. D2 receptors inhibit the secretory process downstream from calcium influx in dopaminergic neurons: Implication of K+ channels. J. Neurophysiol. 87:1046‐1056.
   Forget, C., Stewart, J., and Trudeau, L.E. 2006. Impact of basic FGF expression in astrocytes on dopamine neuron synaptic function and development. Eur. J. Neurosci. 23:608‐616.
   Fortin, G.D., Desrosiers, C.C., Yamaguchi, N., and Trudeau, L.E. 2006. Basal somatodendritic dopamine release requires snare proteins. J. Neurochem. 96:1740‐1749.
   Jomphe, C., Bourque, M.J., Fortin, G.D., St‐Gelais, F., Okano, H., Kobayashi, K., and Trudeau, L.E. 2005. Use of TH‐EGFP transgenic mice as a source of identified dopaminergic neurons for physiological studies in postnatal cell culture. J. Neurosci. Meth. 146:1‐12.
   Jomphe, C., Lemelin, P.L., Okano, H., Kobayashi, K., and Trudeau, L.E. 2006. Bidirectional regulation of dopamine D2 and neurotensin NTS1 receptors in dopamine neurons. Eur. J. Neurosci. 24:2789‐2800.
   Masuko, S., Nakajima, S., and Nakajima, Y. 1992. Dissociated high‐purity dopaminergic neuron cultures from the substantia nigra and the ventral tegmental area of the postnatal rat. Neuroscience 49:347‐364.
   Mytilineou, C. and Cohen, G. 1984. 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine destroys dopamine neurons in explants of rat embryo mesencephalon. Science 225:529‐531.
   Mytilineou, C., Cohen, G., and Heikkila, R.E. 1985. 1‐Methyl‐4‐phenylpyridine (MPP+) is toxic to mesencephalic dopamine neurons in culture. Neurosci. Lett. 57:19‐24.
   Schlumpf, M., Shoemaker, W.J., and Bloom, F.E. 1977. Explant cultures of catecholamine‐containing neurons from rat brain: Biochemical, histofluorescence, and electron microscopic studies. Proc. Natl. Acad. Sci. U.S.A. 74:4471‐4475.
   Shimoda, K., Sauve, Y., Marini, A., Schwartz, J.P., and Commissiong, J.W. 1992. A high percentage yield of tyrosine hydroxylase‐positive cells from rat E14 mesencephalic cell culture. Brain Res. 586:319‐331.
   Staal, R.G.W., Rayport, S., and Sulzer, D. 2006. Amperometric detection of dopamine exocytosis from synaptic terminals. In Electrochemical Methods in Neuroscience (A. Michael, ed.) in the series Methods and New Frontiers in Neuroscience (M. Nicolelis and S.A. Simon, eds.) CRC Press, Boca Raton, Fla.
   Sulzer, D., Joyce, M.P., Lin, L., Geldwert, D., Haber, S.N., Hattori, T., and Rayport, S. 1998. Dopamine neurons make glutamatergic synapses in vitro. J. Neurosci. 18:4588‐4602.
   Takeshima, T., Johnston, J.M., and Commissiong, J.W. 1994. Mesencephalic type I astrocytes rescue dopaminergic neurons from death induced by serum deprivation. J. Neurosci. 14:4769‐4779.
   Takeshima, T., Shimoda, K., Johnston, J.M., and Commissiong, J.W. 1996. Standardized methods to bioassay neurotrophic factors for dopaminergic neurons. J. Neurosci. Meth. 67:27‐41.
   Zhuang, X., Masson, J., Gingrich, J.A., Rayport, S., and Hen, R. 2005. Targeted gene expression in dopamine and serotonin neurons of the mouse brain. J. Neurosci. Meth. 143:27‐32.
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