Differentiation of Mouse Neuro 2A Cells Into Dopamine Neurons

J Neurosci Methods. 2010 Jan 30;186(1):60-7. doi: 10.1016/j.jneumeth.2009.11.004. Epub 2009 Nov 10.

Abstract

Neuro 2A (N2a) is a mouse neural crest-derived cell line that has been extensively used to study neuronal differentiation, axonal growth and signaling pathways. A convenient characteristic of these cells is their ability to differentiate into neurons within a few days. However, most differentiation methods reported for N2a cells do not provide information about the neuronal types obtained after each treatment. In this study, we evaluated the generation of N2a dopamine neurons following treatment with a number of factors known to induce neuronal differentiation. Our results showed that N2a cells express Nurr-related factor 1 (Nurr1) and produce low levels of tyrosine hydroxylase (TH) and dopamine. Both TH and dopamine levels were significantly enhanced in the presence of dibutyryl cyclic adenosine monophosphate (dbcAMP), as evidenced by Western blot, immunocytochemistry and high performance liquid chromatography (HPLC). In contrast to dbcAMP, other factors such as transforming growth factor beta1 (TGF beta 1), bone morphogenetic protein 4 (BMP4), glial cell-derived neurotrophic factor (GDNF) and retinoic acid (RA) did not increase TH expression. Further investigation confirmed that the effect of dbcAMP on production of TH-positive neurons was mediated through cyclic AMP (cAMP) responsive element binding protein (CREB) and it was antagonized by RA. Thus, although various treatments can be used to generate N2a neurons, only dbcAMP significantly enhanced the formation of dopamine neurons. Taken together, this study provided a simple and reliable method to generate dopamine neurons for rapid and efficient physiological and pharmacological assays.

MeSH terms

  • Animals
  • Bucladesine / pharmacology
  • Cell Culture Techniques
  • Cell Differentiation / drug effects
  • Cell Differentiation / physiology*
  • Cell Line
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • Dopamine / metabolism*
  • Mice
  • Nerve Growth Factors / pharmacology
  • Neurogenesis / drug effects
  • Neurogenesis / physiology*
  • Neurons / cytology
  • Neurons / metabolism*
  • Nuclear Receptor Subfamily 4, Group A, Member 2 / metabolism
  • Stem Cells / cytology
  • Stem Cells / drug effects
  • Stem Cells / metabolism*
  • Tretinoin / metabolism
  • Tretinoin / pharmacology
  • Tyrosine 3-Monooxygenase / metabolism

Substances

  • Creb1 protein, mouse
  • Cyclic AMP Response Element-Binding Protein
  • NR4A2 protein, human
  • Nerve Growth Factors
  • Nuclear Receptor Subfamily 4, Group A, Member 2
  • Tretinoin
  • Bucladesine
  • Tyrosine 3-Monooxygenase
  • Dopamine