Voltage Clamp and Internal Perfusion With Suction-Pipette Method
The method discussed in this chapter fits between perfusion methods applied to very large cells such as the squid axon (1, 21) and diffusion methods applied to very small cells such as chromaffin cells (9). The suction-pipette method has been applied to spherical cells such as snail neurons between 50 and 100 µm in diameter (19) and to cylindrical cells such as mammalian ventricular myocytes with diameters, ≥10 µm and lengths ≤200 μm (1). There are several similar techniques available for the study of cells with these dimensions. Kostyuk et al. (15) originally introduced a partition method to isolate single neurons and have subsequently extended the method to a form of suction pipette (16) Takahashi and Yoshii (22) and Byerly and Hagiwara (5) also used modified suction-pipette methods to achieve the same results. In squid axon true perfusion is performed; in chromaffin cells exchange with intracellular components is by diffusion from a micropipette. In intermediate-sized cells, such as snail neurons in which the suction pipette is useful, control of the milieu interieur is achieved mainly by dialysis, although some bulk flow may occur. A true flow-through system based on simultaneous use of two suction pipettes has also been described (2, 18). In this chapter the adequacy of the exchange and the limitations of the voltage-clamp system are emphasized, beginning with an account of the design and fabrication.
KeywordsAgar Polyethylene Rubber Fluoride Trypsin
Baker, P. F., A. L. Hodgkin, and T. I. Shaw. Replacement of the protoplasm of a giant nerve fibre with artificial solutions. Nature London
274: 379–382, 1961.Google Scholar
Brown, A. M., K. S. Lee, and T. Powell. Voltage clamp and internal perfusion of single rat heart muscle cells. J. Physiol. London
318: 455–478, 1981.PubMedGoogle Scholar
Brown, A. M., K. Morimoto, Y. Tsuda, and D. L. Wilson. Calcium current-dependent inactivation of calcium channels in Helix aspersa. J. Physiol. London
320: 193–218, 1981.Google Scholar
Brown, A. M., Y. Tsuda, and D. L. Wilson. A description of activation and conduction in calcium channels based on tail and turn-on current measurements. J. Physiol. London
344: 549–584, 1983.PubMedGoogle Scholar
Byerly, L., and S. Hagiwara. Calcium currents in internally perfused nerve cell bodies of Limnea stagnalis. J. Physiol. London
322: 503–528, 1982.Google Scholar
Byerly, L., and W. J. Moody. Intracellular Ca’ and Ca currents in internally perfused snail neurons (Abstract). Biophys. J.
41: 292a, 1983.Google Scholar
Cole, K. S. Electrical properties of the squid axon sheath. Biophys. J.
16: 137–142, 1976.PubMedCrossRefGoogle Scholar
Eisenberg, R. S., and E. Engel. The spatial variation of membrane potential near a small source of current in a spherical cell. J. Gen. Physiol.
55: 736–757, 1970.PubMedCrossRefGoogle Scholar
Fenwick, E. M., A. Marty, and E. Neher. Sodium and calcium channels in bovine chromaffm cells. J. Physiol. London
331: 559–636, 1982.Google Scholar
Fishman, H. M. Techniques in Cellular Physiology
. Amsterdam: Elsevier North-Holland, 1982, p. 1–50.Google Scholar
Fishman, H. M., L. E. Moore, and D. Poussart. The Biophysical Approach to Excitable Membranes
. New York: Plenum, 1981, p. 65–87.CrossRefGoogle Scholar
Fishman, H. M., D. Poussart, and L. E. Moore. Complex admittance of Na conduction in squid axon. J. Membr. Biol.
50: 43–63, 1979.PubMedCrossRefGoogle Scholar
Hammil, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pfluegers Arch
. 391: 85–100, 1981.CrossRefGoogle Scholar
Katz, G. M., and T. L. Schwartz. Temporal control of voltage clamped membranes: an examination of principles. J. Membr. Biol.
17: 275–291, 1974.PubMedCrossRefGoogle Scholar
Kostyuk, P. G., O. A. Krishtal, and V. I. Pidoplichko. Intracellular dialysis of nerve cells: effect of intracellular fluoride and phosphate on inward current. Nature London
257: 691–693, 1975.PubMedCrossRefGoogle Scholar
Kostyuk, P. G., O. A. Krishtal, and V. I. Pidoplichko: Calcium inward current and related charge movements in the membrane of snail neurons. J. Physiol. London
310: 403–421, 1981.PubMedGoogle Scholar
Kostyuk, P. G., N. S. Veselonsky, and A. Y. Tsyndrenko. Ionic currents in the somatic membrane of rat dorsal root ganglion neurons. II. Calcium currents. Neuroscience
6: 2431–2438, 1981.PubMedCrossRefGoogle Scholar
Krishtal, O. A., V. I. Pidoplichko, and Y. A. Shakhovalov. Conductance of the calcium channel in the membrane of snail neurones. J. Physiol. London
310: 423–434, 1981.PubMedGoogle Scholar
Lee, K. S., N. Akaike, and A. M. Brown. Properties of internally perfused, voltage-clamped isolated nerve cell bodies. J. Gen. Physiol.
71: 489–507, 1978.PubMedCrossRefGoogle Scholar
Lee, K. S., N. Akaike, and A. M. Brown. The suction pipette method for internal perfusion and voltage clamp of small excitable cells. J. Neurosci. Methods
2: 51–78, 1980.PubMedCrossRefGoogle Scholar
Oikawa, T., G. S. Spyropoulos, I. Tasaki, and T. Teorell. Methods for perfusion of giant axon of Liligo pealii. Acta Physiol. Scand
. 51: 195–293, 1961.CrossRefGoogle Scholar
Takahashi, K., and M. Yoshii. Effects of internal free calcium upon the sodium and calcium channels in the tunicate egg analyzed by the internal perfusion technique. J. Physiol. London
279: 519–549, 1978.PubMedGoogle Scholar
© American Physiological Society 1985