Long Duration Responses Obtained from Internally Perfused Axons

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Abstract
Methods Isolated and cleaned squid giant axons were internally perfused by a modifi- cation of the method of Adelman and Gilbert (1964) and Adelman and Fok (1964). A chemically driven syringe (Sage Instruments) was directly con- nected to the axon interior through a small cut in one end by means of a 200,/ diameter glass cannula. The internal axoplasm was eroded away upon continuous internal fluid flow of about 10 l/min. A carefully platinized axial platinum wire, 100u in diameter, was inserted through a large outflow hole at the other end of the axon. Both the outflow and inflow holes were electrically insulated from the external physiological salt solutions bathing the central 15 mm length of the axon by external flowing dextrose guards. The axial wire was used to pass current to external guard and current measuring electrodes either to excite the axon electrically to produce mem- brane responses or to control the membrane voltage in the voltage clamp. A glass micropipette electrode (3 M KC1 bridge to a calomel half-cell) was inserted just through the membrane for potential recording with respect to an external Ag, AgCl reference electrode. The point-control voltage clamp system was used (Cole and Moore, 1960), and membrane currents were measured over a 2 mm central length of axon. The microelectrode was located at the midpoint of the 2 mm long region. Potential differences between the external reference electrode and the tip electrode were balanced to zero for each of the external solutions. Osmolarities of all perfusion solutions were adjusted to that of artificial sea water by making measurements of osmolarity with a vapor pressure os- mometer. All solutions were adjusted to a pH of 7.4.