Properties of internally perfused, voltage-clamped, isolated nerve cell bodies

Abstract

The membrane properties of isolated neurons from Helix aspersa were examined by using a new suction pipette method. The method combines internal perfusion with voltage clamp of nerve cell bodies separated from their axons. Pretreatment with enzymes such as trypsin that alter membrane function is not required. A platinized platinum wire which ruptures the soma membrane allows low resistance access directly to the cell's interior improving the time resolution under voltage clamp by two orders of magnitude. The shunt resistance of the suction pipette was 10-50 times the neuronal membrane resistance, and the series resistance of the system, which was largely due to the tip diameter, was about 10(5) omega. However, the peak clamp currents were only about 20 nA for a 60-mV voltage step so that measurements of membrane voltage were accurate to within at least 3%. Spatial control of voltage was achieved only after somal separation, and nerve cell bodies isolated in this way do not generate all-or-none action potentials. Measurements of membrane potential, membrane resistance, and membrane time constant are equivalent to those obtained using intracellular micropipettes, the customary method. With the axon attached, comparable all-or-none action potentials were also measured by either method. Complete exchange of Cs+ for K+ was accomplished by internal perfusion and allowed K+ currents to be blocked. Na+ currents could then be blocked by TTX or suppressed by Tris-substituted snail Ringer solution. Ca2+ currents could be blocked using Ni2+ and other divalent cations as well as organic Ca2+ blockers. The most favorable intracellular anion was aspartate-, and the sequence of favorability was inverted from that found in squid axon.