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.
KeywordsVoltage Clamp Tail Current Access Resistance Spatial Control Squid Axon
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