102
Brain Research, 530 (1990) 102-104 Elsevier
BRES 24316
Whole-cell voltage-clamp study of sodium current in neuroblastoma cells: effects of inhibition of neurite outgrowth by colchicine L.M. Partlow, R. Karler and S.A. Turkanis Department of Pharmacology, School of Medicine, University of Utah, Salt Lake City, UT 84132 (U.S.A.)
(Accepted 26 June 1990) Key words: Colchicine; Neuroblastoma N1E-115; Whole-cell voltage clamp; Sodium current; Cell differentiation; Cell culture
A method is described for the production of large numbers of neurite-free neuroblastoma cells that are especially suitable for studies involving whole-cell voltage clamp. Differentiation in the presence of colchicine yielded cells having abundant sodium channels, highly reproducible peak currents and no space-clamp problems. Treatment with this drug did not alter the electrophysiologicalproperties of the cells. Colchicine might be similarly advantageous in voltage-clamp studies of different ion channels and other types of cultured neurons.
Many voltage-clamp studies have utilized neuroblastoma cells differentiated by exposure to dimethylsulfoxide (DMSO, 2%) 2-4'6. Typical cells in such cultures possess both nerve fibers and abundant ion channels 1"2. Differentiated cells lacking neurites are ideal for wholecell voltage-clamp studies 3'4'6, but these cells o c c u r infrequently (Fig. 1B) t. While the fraction of neurite-free cells in differentiated neuroblastoma cultures can be increased by use of a higher concentration of DMSO (4%), the resulting cells exhibit an unacceptably low level of electrical excitability 1. The fraction of neurite-free neuroblastoma cells can also be markedly increased by use of colchicine, a drug which inhibits nerve-fiber outgrowth by binding to microtubules 7. In the present study, neurite-free cells were prepared using colchicine and their electrophysiological properties were investigated by a whole-cell voltage-clamp technique. A neuroblastoma cell line (clone N1E-115) was grown at 35.5 °C in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 20 mM HEPES, 80 pg/ml gentamicin, and 4 mM glutamine. Neuronal differentiation occurred in growth medium modified to contain 2.5% fetal calf serum and 2% DMSO ]'s. Cultures were incubated for 4 days in growth medium or in differentiation medium with or without colchicine (10 ng/ml). Cells differentiated in the presence of colchicine were both morphologically and electrophysiologically stable for at least several weeks. The absence of discernable toxicity was probably due to the fact that
DMSO blocks cell division 1'5, making the cells less sensitive to the toxic effects of colchicine. The external bathing solution contained (mM): NaCl 130, CsCl 5.5, MgCl 2 0.8, CaCl 2 1.8, LaCl 3 0.01, glucose 25, HEPES 20. The internal electrode solution contained (mM): CsCl 80, sodium glutamate 20, NaCl 26, E G T A 20, HEPES 20. The osmolarity and pH of both solutions were 290-300 mosM and 7.3. Replacement of KC1 with CsCI prevented potassium currents; cold temperature (near 13 °C) and LaC13 blocked calcium currents 3'4. Conventional procedures for whole-cell voltage clamp were used 4. Average tip diameter and resistance of the glass microelectrodes were 3 p m and 3 M ~ ; seal resistances ranged from 3 to 10 GQ. Current was recorded using an Axopatch 1B patch clamp and an Axolab-1 interface; version 5.5 of pClamp (Axon Instruments) was employed for data acquisition. All cells examined electrophysiologically were round, neurite-free, and had diameters - 0.10, Student's t-test). A t a m e m b r a n e potential of 0 mV,
means and standard errors in cells differentiated in the presence and absence of colchicine were as follows (ms): latency 1.2 + 0.1 vs. 1.4 + 0.2; time to p e a k 1.6 + 0.1 vs. 1.6 + 0.1; tau 1.4 + 0.1 vs. 1.5 + 0.1. Thus, colchicine did not alter either activation or inactivation of the sodium channel. In summary, n e u r o b l a s t o m a cultures differentiated in the presence colchicine contained m a n y neurite-free cells that could be readily v o l t a g e - c l a m p e d without spaceclamp difficulties. Colchicine did not alter any electrophysiological p a r a m e t e r associated with the sodium current; in addition, it increased the reproducibility of the amplitude of the current. This technique may be useful in investigations of different ion channels and of other types of cultured neurons.
1 Kimhi, Y., Palfrey, C., Spector, I., Barak, Y. and Littauer, U.Z., Maturation of neuroblastoma cells in the presence of dimethylsulfoxide, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 462-466. 2 Moolenaar, W.H. and Spector, I., Ionic currents in cultured mouse neuroblastoma cells under voltage-clamp conditions, J. Physiol., 278 (1978) 265-286. 3 Narahashi, T., Tsunoo, A. and Yoshii, M., Characterization of two types of calcium channels in mouse neuroblastoma cells, J. Physiol., 383 (1987) 231-249. 4 Quandt, F.N. and Narahashi, T., Isolation and kinetic analysis of
inward currents in neuroblastoma cells, Neuroscience, 13 (1984) 249-262. 5 Schilling, K.L. and Pilgrim, C., Developmental effect of dimethylsulfoxide on hypothalamo-neurohypophysialneurons in vitro, J. Neurosci. Res., 19 (1988) 27-33. 6 Twombly, D.A., Yoshii, M. and Narahashi, T., Mechanisms of calcium channel block by phenytoin, J. Pharmacol. Exp. Ther., 246 (1988) 189-195. 7 Yamada, K.M., Spooner, B.S. and Wessells, N.K., Axon growth: roles of microfilaments and microtubules, Proc. Natl. Acad. Sci. U.S.A., 66 (1970) 1206-1212.
This work was supported by NIDA Research Grant DA-00346. We are especially grateful to Drs. Toshio Narahashi and Fred Quandt for their advice and support and to Dr. Marshall Nirenberg for supplying us with the N1E-115 neuroblastoma clone.
Brain Research, 530 (1990) 102-104 Elsevier
BRES 24316
Whole-cell voltage-clamp study of sodium current in neuroblastoma cells: effects of inhibition of neurite outgrowth by colchicine L.M. Partlow, R. Karler and S.A. Turkanis Department of Pharmacology, School of Medicine, University of Utah, Salt Lake City, UT 84132 (U.S.A.)
(Accepted 26 June 1990) Key words: Colchicine; Neuroblastoma N1E-115; Whole-cell voltage clamp; Sodium current; Cell differentiation; Cell culture
A method is described for the production of large numbers of neurite-free neuroblastoma cells that are especially suitable for studies involving whole-cell voltage clamp. Differentiation in the presence of colchicine yielded cells having abundant sodium channels, highly reproducible peak currents and no space-clamp problems. Treatment with this drug did not alter the electrophysiologicalproperties of the cells. Colchicine might be similarly advantageous in voltage-clamp studies of different ion channels and other types of cultured neurons.
Many voltage-clamp studies have utilized neuroblastoma cells differentiated by exposure to dimethylsulfoxide (DMSO, 2%) 2-4'6. Typical cells in such cultures possess both nerve fibers and abundant ion channels 1"2. Differentiated cells lacking neurites are ideal for wholecell voltage-clamp studies 3'4'6, but these cells o c c u r infrequently (Fig. 1B) t. While the fraction of neurite-free cells in differentiated neuroblastoma cultures can be increased by use of a higher concentration of DMSO (4%), the resulting cells exhibit an unacceptably low level of electrical excitability 1. The fraction of neurite-free neuroblastoma cells can also be markedly increased by use of colchicine, a drug which inhibits nerve-fiber outgrowth by binding to microtubules 7. In the present study, neurite-free cells were prepared using colchicine and their electrophysiological properties were investigated by a whole-cell voltage-clamp technique. A neuroblastoma cell line (clone N1E-115) was grown at 35.5 °C in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 20 mM HEPES, 80 pg/ml gentamicin, and 4 mM glutamine. Neuronal differentiation occurred in growth medium modified to contain 2.5% fetal calf serum and 2% DMSO ]'s. Cultures were incubated for 4 days in growth medium or in differentiation medium with or without colchicine (10 ng/ml). Cells differentiated in the presence of colchicine were both morphologically and electrophysiologically stable for at least several weeks. The absence of discernable toxicity was probably due to the fact that
DMSO blocks cell division 1'5, making the cells less sensitive to the toxic effects of colchicine. The external bathing solution contained (mM): NaCl 130, CsCl 5.5, MgCl 2 0.8, CaCl 2 1.8, LaCl 3 0.01, glucose 25, HEPES 20. The internal electrode solution contained (mM): CsCl 80, sodium glutamate 20, NaCl 26, E G T A 20, HEPES 20. The osmolarity and pH of both solutions were 290-300 mosM and 7.3. Replacement of KC1 with CsCI prevented potassium currents; cold temperature (near 13 °C) and LaC13 blocked calcium currents 3'4. Conventional procedures for whole-cell voltage clamp were used 4. Average tip diameter and resistance of the glass microelectrodes were 3 p m and 3 M ~ ; seal resistances ranged from 3 to 10 GQ. Current was recorded using an Axopatch 1B patch clamp and an Axolab-1 interface; version 5.5 of pClamp (Axon Instruments) was employed for data acquisition. All cells examined electrophysiologically were round, neurite-free, and had diameters - 0.10, Student's t-test). A t a m e m b r a n e potential of 0 mV,
means and standard errors in cells differentiated in the presence and absence of colchicine were as follows (ms): latency 1.2 + 0.1 vs. 1.4 + 0.2; time to p e a k 1.6 + 0.1 vs. 1.6 + 0.1; tau 1.4 + 0.1 vs. 1.5 + 0.1. Thus, colchicine did not alter either activation or inactivation of the sodium channel. In summary, n e u r o b l a s t o m a cultures differentiated in the presence colchicine contained m a n y neurite-free cells that could be readily v o l t a g e - c l a m p e d without spaceclamp difficulties. Colchicine did not alter any electrophysiological p a r a m e t e r associated with the sodium current; in addition, it increased the reproducibility of the amplitude of the current. This technique may be useful in investigations of different ion channels and of other types of cultured neurons.
1 Kimhi, Y., Palfrey, C., Spector, I., Barak, Y. and Littauer, U.Z., Maturation of neuroblastoma cells in the presence of dimethylsulfoxide, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 462-466. 2 Moolenaar, W.H. and Spector, I., Ionic currents in cultured mouse neuroblastoma cells under voltage-clamp conditions, J. Physiol., 278 (1978) 265-286. 3 Narahashi, T., Tsunoo, A. and Yoshii, M., Characterization of two types of calcium channels in mouse neuroblastoma cells, J. Physiol., 383 (1987) 231-249. 4 Quandt, F.N. and Narahashi, T., Isolation and kinetic analysis of
inward currents in neuroblastoma cells, Neuroscience, 13 (1984) 249-262. 5 Schilling, K.L. and Pilgrim, C., Developmental effect of dimethylsulfoxide on hypothalamo-neurohypophysialneurons in vitro, J. Neurosci. Res., 19 (1988) 27-33. 6 Twombly, D.A., Yoshii, M. and Narahashi, T., Mechanisms of calcium channel block by phenytoin, J. Pharmacol. Exp. Ther., 246 (1988) 189-195. 7 Yamada, K.M., Spooner, B.S. and Wessells, N.K., Axon growth: roles of microfilaments and microtubules, Proc. Natl. Acad. Sci. U.S.A., 66 (1970) 1206-1212.
This work was supported by NIDA Research Grant DA-00346. We are especially grateful to Drs. Toshio Narahashi and Fred Quandt for their advice and support and to Dr. Marshall Nirenberg for supplying us with the N1E-115 neuroblastoma clone.
