The neuron-glia ratio is of prime importance for maintaining the physiological homeostasis of neuronal and glial cells, and especially crucial for dopaminergic neurons because a reduction in glial density has been reported in postmortem reports of brains affected by Parkinson's disease. We thus aimed at developing an in vitro midbrain culture which would replicate a similar neuron-glia ratio to that in in vivo adult midbrain while containing a similar number of dopaminergic neurons. A sequential culture technique was adopted to achieve this. Neural progenitors (NPs) were generated by the hanging-drop method and propagated as 3D neurospheres followed by the derivation of outgrowth from these neurospheres on a chosen extracellular matrix. The highest proliferation was observed in neurospheres from day in vitro (DIV) 5 through MTT and FACS analysis of Ki67 expression. FACS analysis using annexin/propidium iodide showed an increase in the apoptotic population from DIV 8. DIV 5 neurospheres were therefore selected for deriving the differentiated outgrowth of midbrain on a poly-L-lysine-coated surface. Quantitative RT-PCR showed comparable gene expressions of the mature neuronal marker β-tubulin III, glial marker GFAP and dopaminergic marker tyrosine hydroxylase (TH) as compared to in vivo adult rat midbrain. The FACS analysis showed a similar neuron-glia ratio obtained by the sequential culture in comparison to adult rat midbrain. The yield of β-tubulin III and TH was distinctly higher in the sequential culture in comparison to 2D culture, which showed a higher yield of GFAP immunopositive cells. Functional characterization indicated that both the constitutive and inducible (KCl and ATP) release of dopamine was distinctly higher in the sequential culture than the 2D culture. Thus, the sequential culture technique succeeded in the initial enrichment of NPs in 3D neurospheres, which in turn resulted in an optimal attainment of the neuron-glia ratio on outgrowth culture from these neurospheres.

Keywords:
Hanging-drop method, Neural progenitors, Neurospheres, Dopaminergic neurons, Proliferation and apoptosis, Dopamine release
1.
Babu, H., J.H. Claasen, S. Kannan, A. Rünker, E. Palmer, G. Kempermann (2011) A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus. Front Neurosci 5: 89.
2.
Brito, C., D. Simão, I. Costa, R. Malpique, C.I. Pereira, P. Fernandes, M. Serra, S.C. Schwarz, J. Schwarz, E.J. Kremer, P.M. Alves (2012) 3D cultures of human neural progenitor cells: dopaminergic differentiation and genetic modification. Methods 56: 452-460.
3.
Brouard, A., D. Pelaprat, C. Dana, M. Vial, A.M. Lhiaubet, W. Rostène (1992) Mesencephalic dopaminergic neurons in primary cultures express functional neurotensin receptors. J Neurosci 12: 1409-1415.
4.
Brzozowski, M.J., Jenner P., Rose S. (2015) Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP+-induced neuronal toxicity. J Neural Transm (Vienna) 122: 779-788.
5.
Chung, I.Y., E.N. Benveniste (1990) Tumor necrosis factor-alpha production by astrocytes: induction by lipopolysaccharide, IFN-gamma, and IL-1 beta. J Immunol 144: 2999-3007.
6.
Collier, T.J., C.D. Sladek, M.J. Gallagher, R.W. Gereau IV, J.E. Springer (1990) Diffusible factor(s) from adult rat sciatic nerve increases cell number and neurite outgrowth of cultured embryonic ventral mesencephalic tyrosine hydroxylase-positive neurons. J Neurosci Res 27:394-399.
7.
Costa, M.R., F. Ortega., M.S. Brill, R. Beckervordersandforth, C. Petrone, T. Schroeder, M. Götz, B. Berninger (2011) Continuous live imaging of adult neural stem cell division and lineage progression in vitro. Development 138: 1057-1068.
8.
Cukierman, E., R. Pankov, K.M. Yamada (2002) Cell interactions with three-dimensional matrices. Curr Opin Cell Biol 14: 633-639.
9.
Damier, P., E.C. Hirsch, F. Javoy-Agid, P. Zhang, Y. Agid (1993) Protective role of glutathione peroxidase against neuronal death in Parkinson's disease. Neurosci 52: 1-6.
10.
Desbaillets, I., U. Ziegler, P. Groscurth, M. Gassmann (2000) Embryoid bodies: an in vitro model of mouse embryogenesis. Exp Physiol 85: 645-651.
11.
Doetsch, F. (2003) The glial identity of neural stem cells. Nat Neurosci 6: 1127-1134.
12.
Fasano, C., D. Thibault, L.E. Trudeau (2008) Culture of Postnatal Mesencephalic Dopamine Neurons on an Astrocyte Monolayer. Curr Protoc Neurosci DOI: 10.1002/0471142301.ns0321s44.
13.
Fawcett, J.W., R.A. Barker, S.B. Dunnett (1995) Dopaminergic neuronal survival and the effects of bFGF in explant, three dimensional and monolayer cultures of embryonic rat ventral mesencephalon. Exp Brain Res 106: 275-282.
14.
Fu, H.Q., T. Yang, W. Xiao, L. Fan, Y. Wu, N. Terrando (2014) Prolonged neuroinflammation after lipopolysaccharide exposure in aged rats. PLoS One 9: e106331.
15.
Gaven, F., P. Marin, S. Claeysen (2014) Primary culture of mouse dopaminergic neurons. J Vis Exp 91: e51751.
16.
Gomes, P., J. Chevalier, W. Boesmans, L. Roosen, V. van den Abbeel, M. Neunlist, J. Tack, P. Vanden Berghe (2009) ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice. Neurogastroenterol Motil 21: 870-e62.
17.
Goslin, K., G. Banker (1998) Rat hippocampal neurons in low-density culture. Cult Nerve Cell 2: 339-370.
18.
Hatten, M.E., W.Q. Gao., M.E. Morriso, C.A. Mason (1998) The cerebellum: purification and coculture of identified cell populations. Cult Nerve Cell 1: 419-459.
19.
Haycock, J.W. (2011) 3D cell culture: a review of current approaches and techniques. Methods Mol Biol 695: 1-15.
20.
Hellstrom, I.C., M. Danik, G.N. Luheshi, S. Williams (2005) Chronic LPS exposure produces changes in intrinsic membrane properties and a sustained IL-β-dependent increase in GABAergic inhibition in hippocampal CA1 pyramidal neurons. Hippocampus 15: 656-664.
21.
Herculano-Houzel, S., R. Lent (2005) Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain. J Neurosci 25: 2518-2521.
22.
Hyman, C., M. Hofer, Y.A. Barde, M. Juhasz, G.D. Yancopoulos, S.P. Squinto, R.M. Lindsay (1991) BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 350: 230-232.
23.
Justice, B.A, N.A. Badr, R.A. Felder (2009) 3D cell culture opens new dimensions in cell-based assays. Drug Discov Today 14: 102-107.
24.
Kanafi, M., D. Majumdar, R. Bhonde, P. Gupta, I. Datta (2014) Midbrain cues dictate differentiation of human dental pulp stem cells towards functional dopaminergic neurons. J Cell Physiol 229: 1369-1377.
25.
Lee, J., M.J. Cuddihy., N. Kotov (2008) Three-dimensional cell culture matrices: state of the art. Tissue Eng Part B Rev 14: 61-86.
26.
Lu, H., K. Seare, Y. Liu, T. Parker (2012) The effect of dimensionality on growth and differentiation of neural progenitors from different regions of fetal rat brain in vitro: 3-dimensional spheroid versus 2-dimensional monolayer culture. Cells Tissues Organs 196: 48-55.
27.
Majumdar, D., R. Bhonde, I. Datta (2013) Influence of ischemic microenvironment on human Wharton's Jelly mesenchymal stromal cells. Placenta 34: 642-649.
28.
Marthiens, V., I. Kazanis, L. Moss, K. Long, C. French-Constant (2010) Adhesion molecules in the stem cell niche - more than just staying in shape? J Cell Sci 123: 1613-1622.
29.
McGeer, P.L., E.G. McGeer (2008) Glial reactions in Parkinson's disease. Mov Disord 23: 474-483.
30.
McNaught, K.S., P. Jenner (1999) Altered glial function causes neuronal death and increases neuronal susceptibility to 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced toxicity in astrocytic/ventral mesencephalic co-cultures. J Neurochem 73: 2469-2476.
31.
Medana, I.M., N.H. Hunt., G. Chaudhri (1997) Tumor necrosis factor-α expression in the brain during fatal murine cerebral malaria: evidence for production by microglia and astrocytes. Am J Pathol 150: 1473-1486.
32.
Mosharov, E.V., K.E. Larsen, E. Kanter, K.A. Phillips, K. Wilson, Y. Schmitz, D.E. Krantz, K. Kobayashi, R.H. Edwards, D. Sulzer (2009) Interplay between cytosolic dopamine, calcium, and α-synuclein causes selective death of substantia nigra neurons. Neuron 62: 218-229.
33.
Nedergaard, M., B. Ransom, S.A. Goldman (2003) New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci 26: 523-530.
34.
Olude, M.A., O.A. Mustapha, O.A. Aderounmu, J.O. Olopade, A.O. Ihunwo (2015) Astrocyte morphology, heterogeneity, and density in the developing African giant rat (Cricetomys, gambianus). Front Neuroanat 26: 67.
35.
Oorschot, D.E., D.G. Jones (1986) Non-neuronal cell proliferation in tissue culture: implications for axonal regeneration in the central nervous system. Brain Res 368: 49-61.
36.
Pampaloni, F., E.G. Reynaud., E.H. Stelzer (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8: 839-845.
37.
Pardo, B., P. Honegger (2000) Differentiation of rat striatal embryonic stem cells in vitro: monolayer culture vs. three-dimensional coculture with differentiated brain cells. J Neurosci Res 59: 504-512.
38.
Parmar, M.S., J.D. Jaumotte, S.L. Wyrostek, M.J. Zigmond, J.E. Cavanaugh (2014) The role of ERK1, 2, and 5 in dopamine neuron survival during aging. Neurobiol Aging 35: 669-679.
39.
Pastrana, E., V. Silva-Vargas, F. Doetsch (2011) Eyes wide open: a critical review of sphere formation as an assay for stem cells. Cell Stem Cell 8: 486-498.
40.
Pinteaux, E., L.C. Parker, N.C. Rothwell, G.N. Luheshi (2002) Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells. J Neurochem 83: 754-763.
41.
Raisman, G. (1991) Glia, neurons, and plasticity. Ann NY Acad Sci 633: 209-213.
42.
Robinson, L.J., I.B. Black, C.F. Dreyfus (1993) Hippocampal regulation of the survival and morphological development of locus coeruleus neurons in dissociated cell culture. J Comp Neurol 333: 567-577.
43.
Savchenko, V.L., J.A. McKanna, I.R. Nikonenko, G.G. Skibo (2000) Microglia and astrocytes in the adult rat brain: comparative immunocytochemical analysis demonstrates the efficacy of lipocortin 1 immunoreactivity. Neuroscience 96: 195-203.
44.
Shimoda, K., Y. Sauve, A. Marini, J.P. Schwartz, J.W. Commissiong (1992) A high percentage yield of tyrosine hydroxylase-positive cells from rat E14 mesencephalic cell culture. Brain Res 586: 319-331.
45.
Sommer, L., M. Rao (2002) Neural stem cells and regulation of cell number. Prog Neurobiol 66: 1-18.
46.
Song, T.T., Y.H. Bi, R. Huang, K. Hao, G. Xu, J.W. Tang, Z.Q. Ma, F.P. Kong, J.H. Coote, X.Q. Chen, J.Z. Du (2016) Systemic pro-inflammatory response facilitates the development of cerebral edema during short hypoxia. J Neuroinflammation 13: 63.
47.
Spijker, S. (2011) Dissection of rodent brain regions. Neuroproteomics Neuromethods 57: 13-26.
48.
Studer, L., M. Csete, S.H. Lee, N. Kabbani, J. Walikonis, B. Wold, R. McKay (2000) Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J Neurosci 20: 7377-7383.
49.
Svenningsen, A.F., W.S. Shan, D.R. Colman, L. Pedraza (2003) Rapid method for culturing embryonic neuron-glial cell cocultures. J Neurosci Res 72: 565-573.
50.
Tolosa, A., X. Zhou, B. Spittau, K. Krieglstein (2013) Establishment of a survival and toxic cellular model for Parkinson's disease from chicken mesencephalon. Neurotox Res 24: 119-129.
51.
Walker, T.L., G. Kempermann (2014) One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice. J Vis Exp 84: e51225.
52.
Walker, T.L., A. Wierick, A.M. Sykes, B. Waldau, D. Corbeil, P. Carmeliet, G. Kempermann (2013) Prominin-1 allows prospective isolation of neural stem cells from the adult murine hippocampus. J Neurosci 33: 3010-3024.
53.
Wang, C.N., L. Lin, Z.F. Duan, F. Zhong, D.Y. Zuo, Y.L. Wu (2013) Primary co-culture of cortical neurons and astrocytes of new-born SD rats (in Chinese). Yao Xue Xue Bao 48: 1729-1732.
54.
Weinert, M., T. Selvakumar, T.S. Tierney, K.N. Alavian (2015) Isolation, culture and long term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains. J Vis Exp 96: e52475.
55.
Yasuhara, T., N. Matsukawa, K. Hara, G. Yu, L. Xu, M. Maki, S.U. Kim, C.V. Borlongan (2006) Transplantation of human neural stem cells exerts neuroprotection in a rat model of Parkinson's disease. J Neurosci 26: 12497-12511.
56.
Zheng, K., T.Q. Liu, M.S. Dai, D. Ge, X.Q. Li, X.H. Ma, Z.F. Cui (2006) Comparison of different culture modes for long-term expansion of neural stem cells. Cytotechnology 52: 209-218.
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