Isolation and culture of adult neurons and neurospheres

Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable growth factors, adult neuron regeneration of axons and dendrites in culture proceeds over 1–3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in 1 week as a source of neuroprogenitors ethically preferred over embryonic or fetal sources. This protocol emphasizes two differences between neuron differentiation and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.

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References

  1. Banker, G.A. & Cowan, W.M. Rat hippocampal neurons in dispersed cell culture. Brain Res.126, 397–425 (1977). ArticleCASGoogle Scholar
  2. Brewer, G.J., Torricelli, J.R., Evege, E.K. & Price, P.J. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res.35, 567–576 (1993). ArticleCASGoogle Scholar
  3. Ahlemeyer, B. & Baumgart-Vogt, E. Optimized protocols for the simultaneous preparation of primary neuronal cultures of the neocortex, hippocampus and cerebellum from individual newborn (P0.5) C57Bl/6J mice. J. Neurosci. Methods149, 110–120 (2005). ArticleCASGoogle Scholar
  4. Brewer, G.J. Isolation and culture of adult rat hippocampal neurons. J. Neurosci. Methods71, 143–155 (1997). ArticleCASGoogle Scholar
  5. Brewer, G.J. Age-related toxicity to lactate, glutamate, and beta-amyloid in cultured adult neurons. Neurobiol. Aging19, 561–568 (1998). ArticleCASGoogle Scholar
  6. Brewer, G.J., Reichensperger, J.D. & Brinton, R.D. Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons. Neurobiol. Aging27, 306–317 (2006). ArticleCASGoogle Scholar
  7. Nathan, B.P. et al. Apolipoprotein E4 inhibits, and apolipoprotein E3 promotes neurite outgrowth in cultured adult mouse cortical neurons through the low-density lipoprotein receptor-related protein. Brain Res.928, 96–105 (2002). ArticleCASGoogle Scholar
  8. Parihar, M.S. & Brewer, G.J. Simultaneous age-related depolarization of mitochondrial membrane potential and increased mitochondrial ROS production correlate with age-related glutamate excitotoxicity in rat hippocampal neurons. J Neurosci. Res.85, 1018–1032 (2007). ArticleCASGoogle Scholar
  9. Struble, R.G., Nathan, B.P., Cady, C., Cheng, X.Y. & McAsey, M. Estradiol regulation of astroglia and apolipoprotein E: an important role in neuronal regeneration. Exp. Gerontol.42, 54–63 (2007). ArticleCASGoogle Scholar
  10. Zhou, L.P. et al. Neuroprotection by estradiol: a role of aromatase against spine synapse loss after blockade of GABA(A) receptors. Exp. Neurol.203, 72–81 (2007). ArticleCASGoogle Scholar
  11. Yip, P.K. et al. Lentiviral vector expressing retinoic acid receptor beta 2 promotes recovery of function after corticospinal tract injury in the adult rat spinal cord. Hum. Mol. Genet.15, 3107–3118 (2006). ArticleCASGoogle Scholar
  12. Tyler, W.J. et al. BDNF increases release probability and the size of a rapidly recycling vesicle pool within rat hippocampal excitatory synapses. J. Physiol.574, 787–803 (2006). ArticleCASGoogle Scholar
  13. Hurst, R.S. et al. A novel positive allosteric modulator of the alpha 7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J. Neurosci.25, 4396–4405 (2005). ArticleCASGoogle Scholar
  14. Brewer, G.J. & Price, P.J. Viable cultured neurons in ambient carbon dioxide and hibernation storage for a month. NeuroReport7, 1509–1512 (1996). ArticleCASGoogle Scholar
  15. Brewer, G.J. et al. Culture and regeneration of human neurons after brain surgery. J. Neurosci. Methods107, 15–23 (2001). ArticleCASGoogle Scholar
  16. Xie, C., Markesbery, W.R. & Lovell, M.A. Survival of hippocampal and cortical neurons in a mixture of MEM+ and B27-supplemented neurobasal medium. Free Radic. Biol. Med.28, 665–672 (2000). ArticleCASGoogle Scholar
  17. Viel, J.J., McManus, D.Q., Smith, S.S. & Brewer, G.J. Age- and concentration-dependent neuroprotection and toxicity by TNF in cortical neurons from beta-amyloid. J. Neurosci. Res.64, 454–465 (2001). ArticleCASGoogle Scholar
  18. Eide, L. & McMurray, C.T. Culture of adult mouse neurons. BioTechniques38, 99–104 (2005). ArticleCASGoogle Scholar
  19. Kivell, B.M., McDonald, F.J. & Miller, J.H. Serum-free culture of rat post-natal and fetal brainstem neurons. Brain Res. Dev. Brain Res.120, 199–210 (2000). ArticleCASGoogle Scholar
  20. Zhang, W., Hu, Y., Newman, E.A. & Mulholland, M.W. Serum-free culture of rat postnatal neurons derived from the dorsal motor nucleus of the vagus. J. Neurosci. Methods150, 1–7 (2006). ArticleCASGoogle Scholar
  21. Price, P.J. & Brewer, G.J. Protocols for Neural Cell Culture, 3rd edn. (eds. Fedoroff, S. & Richardson, A.) 255–264 (Humana Press, Inc., Totowa, New Jersey, 2000). Google Scholar
  22. Gage, F.H. et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc. Natl. Acad. Sci. USA92, 11879–11883 (1995). ArticleCASGoogle Scholar
  23. Caldwell, M.A. et al. Growth factors regulate the survival and fate of cells derived from human neurospheres. Nat. Biotechnol.19, 475–479 (2001). ArticleCASGoogle Scholar
  24. Tatebayashi, Y., Iqbal, K. & Grundke-Iqbal, I. Dynamic regulation of expression and phosphorylation of tau by fibroblast growth factor-2 in neural progenitor cells from adult rat hippocampus. J. Neurosci.19, 5245–5254 (1999). ArticleCASGoogle Scholar
  25. Alexanian, A.R. & Nornes, H.O. Proliferation and regeneration of retrogradely labeled adult rat corticospinal neurons in culture. Exp. Neurol.170, 277–282 (2001). ArticleCASGoogle Scholar
  26. Palmer, T.D., Ray, J. & Gage, F.H. FGF-2—responsive neuronal progenitors reside in proliferation and quiescent regions of the adult rodent brain. Mol. Cell. Neurosci.6, 474–486 (1995). ArticleCASGoogle Scholar
  27. Zheng, T. et al. Transplantation of multipotent astrocytic stem cells into a rat model of neonatal hypoxic-ischemic encephalopathy. Brain Res.1112, 99–105 (2006). ArticleCASGoogle Scholar
  28. Shetty, A.K. & Turner, D.A. In vitro survival and differentiation of neurons derived from epidermal growth factor-responsive postnatal hippocampal stem cells: inducing effects of brain-derived neurotrophic factor. J. Neurobiol.35, 395–425 (1998). ArticleCASGoogle Scholar
  29. Gobbel, G.T., Choi, S.J., Beier, S. & Niranjan, A. Long-term cultivation of multipotential neural stem cells from adult rat subependyma. Brain Res.980, 221–232 (2003). ArticleCASGoogle Scholar
  30. Evans, J. et al. Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem. J. Neurophysiol.87, 1076–1085 (2002). ArticleGoogle Scholar
  31. Brewer, G.J. & Cotman, C.W. Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen. Brain Res.494, 65–74 (1989). ArticleCASGoogle Scholar
  32. Kaplan, F.S. et al. Enhanced survival of rat neonatal cerebral cortical neurons at subatmospheric oxygen tensions in vitro . Brain Res.384, 199–203 (1986). ArticleCASGoogle Scholar
  33. Studer, L. et al. Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J. Neurosci.20, 7377–7383 (2000). ArticleCASGoogle Scholar
  34. Haynes, L.W. (ed.) The Neuron in Tissue Culture (John Wiley & Sons, New York, 1999). Google Scholar
  35. Banker, G. & Goslin, K. (eds.) Culturing Nerve Cells 2nd edn. (The Bradford Book, MIT Press, Cambridge, Massachusetts, 1998). Google Scholar
  36. Meberg, P.J. & Miller, M.W. Culturing hippocampal and cortical neurons. Methods Cell Biol.71, 111–127 (2003). ArticleGoogle Scholar
  37. Brewer, G.J., Lim, A., Capps, N.G. & Torricelli, J.R. Age-related calcium changes, oxyradical damage, caspase activation and nuclear condensation in hippocampal neurons in response to glutamate and beta-amyloid. Exp. Gerontol.40, 426–437 (2005). ArticleCASGoogle Scholar
  38. Evans, M.S., Collings, M.A. & Brewer, G.J. Electrophysiology of embryonic, adult and aged rat hippocampal neurons in serum-free culture. J. Neurosci. Methods79, 37–46 (1998). ArticleCASGoogle Scholar
  39. Patel, J.R. & Brewer, G.J. Age-related changes in neuronal glucose uptake in response to glutamate and beta-amyloid. J. Neurosci. Res.72, 527–536 (2003). ArticleCASGoogle Scholar
  40. Oddo, S. et al. Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron39, 409–421 (2003). ArticleCASGoogle Scholar
  41. Davis, A.A. & Temple, S. A self-renewing multipotential stem cell in the embryonic rat cerebral cortex. Nature372, 263–266 (1994). ArticleCASGoogle Scholar
  42. Temple, S. & Alvarez-Buylla, A. Stem cells in the adult mammalian central nervous system. Curr. Opin. Neurobiol.9, 135–141 (1999). ArticleCASGoogle Scholar
  43. Ostenfeld, T. et al. Regional specification of rodent and human neurospheres. Brain Res. Dev. Brain Res.134, 43–55 (2002). ArticleCASGoogle Scholar
  44. Mayer-Proschel, M., Kalyani, A.J., Mujtaba, T. & Rao, M.S. Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron19, 773–785 (1997). ArticleCASGoogle Scholar
  45. Wohl, C.A. & Weiss, S. Retinoic acid enhances neuronal proliferation and astroglial differentiation in cultures of CNS stem cell-derived precursors. J. Neurobiol.37, 281–290 (1998). ArticleCASGoogle Scholar
  46. Takahashi, J., Palmer, T.D. & Gage, F.H. Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures. J. Neurobiol.38, 65–81 (1999). ArticleCASGoogle Scholar
  47. Laywell, E.D. et al. Neuron-to-astrocyte transition: Phenotypic fluidity and the formation of hybrid asterons in differentiating neurospheres. J. Comp. Neurol.493, 321–333 (2005). ArticleGoogle Scholar

Acknowledgements

This work was supported by a Temple Foundation award from the Alzheimer Association and the National Institute on Aging.