Abstract
With the aid of a voltage-sensitive oxonol dye, flow cytometry was used to measure relative changes in resting membrane potential (Vm) and forward angle light scatter (FALS) profiles of a differentiating/differentiated murine neuroblastoma cell line (N1E-115). Electrophysiological differentiation was characterized by Vm establishment. The (Vm)-time profile was found to be seed cell concentration-dependent for cell densities of less than 2 × 104 cells/cm2. At higher initial cell densities, under differentiating culture conditions, Vm development commenced on day 2 and reached a steady-state on day 12. The relative distribution of differentiated cells between low and high FALS has been proposed as a potential culture electrophysiological differentiation state index. These experiments offer a general methodology to characterize cultured excitable cells of nervous system origin, with respect to electrophysiological differentiation. This information is valuable in studies employing neuroblastoma cells as in vitro screening models for safety/hazard evaluation and/or risk assessment of therapeutical and industrial chemicals under development.
Keywords: electrophysiological differentiation, flow cytometry, membrane, potential, N1E-115 neuroblastoma cells
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References
- Amano T, Richelson E, Nirenberg M. Neurotransmitter synthesis by neuroblastoma clones. Proc. Natl. Acad. Sci. 1972;69:258–263. doi: 10.1073/pnas.69.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Augusti-Tocco G, Sato G. Establishment of functional clonal lines of neuroblastoma clones. Proc Natl. Acad. Sci. 1969;64:311–315. doi: 10.1073/pnas.64.1.311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bashford CL, Alder GM, Gray MA, Micklem KJ, Taylor CC, Turek T, Pasternak CA. Oxonol dyes as monitors of membrane potential of animal cells in monolayer culture and in suspension. J. Cell Physiol. 1985;123:326–336. doi: 10.1002/jcp.1041230306. [DOI] [PubMed] [Google Scholar]
- Baumgold J, Spector I. Development of sodium channel protein during chemically induced differentiation of neuroblastoma cells. J. Neurochem. 1987;48:1264–1269. doi: 10.1111/j.1471-4159.1987.tb05656.x. [DOI] [PubMed] [Google Scholar]
- Bräuner T, Hülser TF, Straser RJ. Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes. Biochim. Biophys. Acta. 1984;771:208–216. doi: 10.1016/0005-2736(84)90535-2. [DOI] [PubMed] [Google Scholar]
- Chalazonitis A, Green LA. Enhancement in excitability properties of mouse neuroblastoma cells in the presence of dibutyl cyclic AMP. Brai Res. 1974;72:340–345. doi: 10.1016/0006-8993(74)90878-6. [DOI] [PubMed] [Google Scholar]
- Cosgrove C, Cobbett P. Induction of temporally dissociated morphological and physiological differentiation of N1E-115 cells. Brain Res. Bull. 1991;27:53–58. doi: 10.1016/0361-9230(91)90280-W. [DOI] [PubMed] [Google Scholar]
- Couly GF, Coltey PM, Le Douarin NM. The triple origin of skull in higher vertebrates — A study in quail-chick chimeras. Development. 1993;117:409–429. doi: 10.1242/dev.117.2.409. [DOI] [PubMed] [Google Scholar]
- Dwyer TM, Cuchens M. Membrane potential measurements by flow cytometry. J. Electrophysiol. Tech. 1987;14:43–57. [Google Scholar]
- EPA EPA's research strategies for the 1990s. Vet. Human Toxicol. 1991;33:69–70. [PubMed] [Google Scholar]
- Fishman MC, Spector I. Potassium currents suppression by quinidine reveals additional calcium currents in neuroblastoma cells. Proc. Natl. Acad. Sci. 1981;78:5245–5249. doi: 10.1073/pnas.78.8.5245. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gilbert JA, Knodel EL, Stenstrom SD, Richelson E. Function and regulation of methionine5-encephalon and its receptors in murine neuroblastoma cells. J. Biol. Chem. 1982;257:1274–1281. [PubMed] [Google Scholar]
- Givan AL. Flow Cytometry: First Principles. New York: Wiley; 1992. pp. 90–91. [Google Scholar]
- Gunderson RW, Berrett JN. Neuronal chemotaxis: chick dorsal-root axons turn toward high concentrations of nerve growth factor. Science. 1979;206:1079–1080. doi: 10.1126/science.493992. [DOI] [PubMed] [Google Scholar]
- Hogg RV, Craig AT. Introduction to Mathematical Statistics. New Jersey: Prentice-Hall; 1995. pp. 521–526. [Google Scholar]
- Ishikawa S. Differentiation of human neuroblastoma cells in vitro — morphological changes induced by dibutyryl cyclic AMP. Acta Path. Jap. 1977;27:697–711. doi: 10.1111/j.1440-1827.1977.tb00186.x. [DOI] [PubMed] [Google Scholar]
- Kato E, Narahashi T. Characteristics of the electrical response to dopamine in neuroblastoma cells. J. Physiol. 1982;333:213–226. doi: 10.1113/jphysiol.1982.sp014450. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigene M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell. 1994;78:425–435. doi: 10.1016/0092-8674(94)90421-9. [DOI] [PubMed] [Google Scholar]
- Kimhi Y, Palfrey C, Spector I, Barak Y, Littauer UZ. Maturation of neuroblastoma cells in the presence of dimethyl-sulfoxide. Proc. Nat. Acad. Sci. 1976;73:462–466. doi: 10.1073/pnas.73.2.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kisaalita WS, Bowen JM. Effect of culture age on the susceptibility of differentiating neuroblastoma cells to retinois cytotoxicity. Biotechnol. Bioeng. 1996;50:580–586. doi: 10.1002/(SICI)1097-0290(19960605)50:5<580::AID-BIT13>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
- Kisaalita WS, Lund RB and Evans MD (1996) Size changes in differentiating neuroblastoma cells. In Vitro accepted. [DOI] [PubMed]
- Kohen E, Hirschberg JA. Cell Structure and Function by Microspectrofluorometry. New York: Academic Press; 1989. [Google Scholar]
- Krieger C, Paul E, Kim SU. Development of resting membrane potentials of embryonic murine spinal cord cells evaluated by flow cytometry analysis. Dev. Neurosci. 1991;13:11–19. doi: 10.1159/000112136. [DOI] [PubMed] [Google Scholar]
- Lumsden A, Davies A. Earliest sensory nerve fibers are guided to peripheral targets by attractants other than nerve growth factor. Nature. 1983;306:786–788. doi: 10.1038/306786a0. [DOI] [PubMed] [Google Scholar]
- Miyake M, Kurihara K. Resting potential of the mouse neuroblastoma cells I: The presence of K+ channels activated at high K+ concentration but closed at low K+ concentration including the physiological concentration. Biochim. Biophys. Acta. 1983;762:248–255. doi: 10.1016/0167-4889(83)90078-2. [DOI] [PubMed] [Google Scholar]
- Moolenaar WH, Spector I. Membrane currents examined under voltage clamp in cultured neuroblastoma cells. Science. 1977;196:331–333. doi: 10.1126/science.557842. [DOI] [PubMed] [Google Scholar]
- Nummery CL, Van Den Brink CE, Van Der Saag PT, De Laat SW. A short-term screening test for teratogens using differentiating neuroblastoma cells. Teratology. 1984;29:271–279. doi: 10.1002/tera.1420290213. [DOI] [PubMed] [Google Scholar]
- Nelson PG, Ruffner BW, Nirenberg MW. Neuronal tumor cells with excitable membranes grown in vitro. Proc. Natl. Acad. Sci. 1969;64:1004–1010. doi: 10.1073/pnas.64.3.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Risk Assessment in the Federal Government: Managing the Process. Washington D.C.: National Academy Press; 1983. [PubMed] [Google Scholar]
- Orkland RK. Glial cells. In: Kandel ER, editor. Cellular Biology of Neurons. Bethesda, MD: American Physiological Society; 1977. pp. 855–875. [Google Scholar]
- Peacock JH, Mina J, Nelson PG. The use of aminopterin in selecting electrically active neuroblastoma cells. Exp. Cell Res. 1972;73:367–377. doi: 10.1016/0014-4827(72)90060-2. [DOI] [Google Scholar]
- Plazek M, Tessier-Lavigne M, Yamada T, Dood J, Jessell TM. Guidance of developing axons by diffusible chemoattractants. Cold Springs Harbor Symp. Quant. Biol. 1990;55:279–289. doi: 10.1101/sqb.1990.055.01.030. [DOI] [PubMed] [Google Scholar]
- Quandt FN, Narahashi T. Isolation and kinetic analysis of inward currents in neuroblastoma cells. Neuroscience. 1994;13:249–262. doi: 10.1016/0306-4522(84)90275-6. [DOI] [PubMed] [Google Scholar]
- Ramsden JJ, Li S-Y, Prenosil JE, Heinzle E. Kinetics of adhesion and spreading of animal cells. Biotechnol. Bioeng. 1993;43:939–945. doi: 10.1002/bit.260431007. [DOI] [PubMed] [Google Scholar]
- Rouget P, Le Bert M, Borde, Evrard C. Generation of neural cell lines by transfer of viral oncogenes. In: Woods JN, editor. Neuronal Cell Lines: A Practical Approach. Oxford: Oxford University Press; 1992. pp. 27–54. [Google Scholar]
- Santone KS, Oakes GS, Taylor SR, Powis G. Anthracycline-induced inhibition of calcium action potential in differentiated murine neuroblastoma cells. Cancer Res. 1986;46:2659–2664. [PubMed] [Google Scholar]
- Seeds NW, Gilman AG, Amano T, Nirenberg MW. Regulation of axon formation by clonal lines. Proc. Nat. Acad. Sci. 1970;66:160–167. doi: 10.1073/pnas.66.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spector I. Electrophysiology of clonal nerve cell lines. In: Nelson PG, Liebermann M, editors. Excitable Cells in Tissue Culture. New York: Plenum; 1981. pp. 257–277. [Google Scholar]
- Tilson HA. Risk assessment for neurotoxicity: An introductory overview. In: Chang LW, Slikker W, editors. Neurotoxicology: Approaches and Methods. New York: Academic Press; 1995. pp. 767–770. [Google Scholar]
- Tosney KW. The segregation and early migration of cranial neural crest cells in avian embryo. Dev. Biol. 1982;89:13–24. doi: 10.1016/0012-1606(82)90289-5. [DOI] [PubMed] [Google Scholar]
- Tuttle JB, Richelson E. Ionic excitation of a clone of mouse neuroblastoma. Brain Res. 1975;84:128–135. doi: 10.1016/0006-8993(75)90806-9. [DOI] [PubMed] [Google Scholar]
- Veronesi B. The use of cell culture for evaluating neurotoxicity. In: Tilson H, Mitchel C, editors. Neurotoxicology. New York: Raven Press; 1992. pp. 21–49. [Google Scholar]
- Walum E, Ekblad-Sekund G, Nyberg E, Gustaffsson L. Cultured neuroblastoma cells as neurotoxicological models: Acrylamide induced neurite disintegration. In: Shaler A, Goldber AM, editors. Model Systems in Neurotoxicology: Alternative Approaches to Animal Testing. New York: Alan Liss; 1987. pp. 121–136. [PubMed] [Google Scholar]
- Walum E, Nordin M, Beckman M, Odland L. Cellular methods foridentification of neurotoxic chemicals and estimation of neurotoxicological risk. Toxic. in Vitro. 1993;7:321–326. doi: 10.1016/0887-2333(93)90022-W. [DOI] [PubMed] [Google Scholar]
- Williams SP, Davenport-Jones J, Egan C, O'Hare S, Cookson M, McClean R, Garle MJ, Pentreath V, Atterwill CH. Phase 1 of an in vitro neurotoxicological pre-validation trial. Toxic. in Vitro. 1994;8:799–802. doi: 10.1016/0887-2333(94)90071-X. [DOI] [PubMed] [Google Scholar]
- Wilson HA, Chused TM. Lymphocyte membrane potentials and Ca2+-sensitive potassium channels described by oxonol dye fluorescence measurements. J. Cell. Physiol. 1985;125:72–81. doi: 10.1002/jcp.1041250110. [DOI] [PubMed] [Google Scholar]
- Zwart R, Abraham D, Oortgiessen M, Vijverberg HPM. α4β2 subunits combination specific pharmacology of neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells. Brain Res. 1994;654:312–318. doi: 10.1016/0006-8993(94)90493-6. [DOI] [PubMed] [Google Scholar]
