All experiments were performed with a single electrode (filamented, GC120F - 10, Clark Electromedical, UK) pulled on a Kopf electrode puller (David Kopf, Tujunga California). Once backfilled with 3 M KCl only those with resistances falling between 30 and 50 M c were used. The bath electrode consisted of a silver - chloride pellet (E200, Clark Electromedical) fixed to a 1 ml syringe containing ASW with the circuit completed by a glass capillary tube filled with ASW.
After being stuck down to glass slides coated with poly-D-lysine, (20 pg/ml) current
the eggs were impaled. Before impalement zero was established after compensating the negative capacitance to replace the current being drawn by the microelectrode. After impalement the negative capacitance was adjusted to give the correct waveform (see Halliwell and Whitaker, 1987) during current clamp in the chopping mode at 5 kHz. Using a Narishige hydraulic micromanipulator the electrode was advanced towards the egg and placed against the surface of the egg indenting it slightly. Impalement was achieved by overcompensating the negative capacitance briefly. The electrode could be seen entering the egg on occasion. A holding current of 1 nA sometimes maintained the impalement while the egg recovered. During the next 10 minutes the electrode was typically moved deeper into the egg improving the seal as reflected by the reduction in holding current required. The egg membrane resistance was constantly monitored by passing 0.1 nA hyperpolarizing current pulses for 1 second. Eggs typically reached resting membrane potentials of either -10 mV or -70 mV. Only those eggs which reached the lower physiological membrane potential of -70 mV (Jaffe and Robinson, 1978;
Chambers and de Armendi, 1979; David et al., 1988) were used. Once at around -70mV the holding current was slowly removed, sometimes almost completely. If required at this point the egg was then microinjected. During this injection the membrane potential falls rapidly to zero, but as the microinjection pipette is removed the egg quickly regains a resting membrane potential equal to its preinjection potential if the injection was not too damaging. After this my criteria then was to use only those eggs which maintained a -70 mV resting membrane potential using < 500 pA holding current and which fired regenerative action potentials upon either removal of the holding current or injection of a depolarizing current. No compensation was done for leakage currents since all measurements here report voltage dependent currents, while leakage currents will display a linear current voltage relationship. Having satisfied the criteria the egg was sometimes voltage clamped. To assess the voltage clamp a voltage command step from -80 mV to -30 mV lasting 100-500 ms was applied while both increasing the gain and altering the phase to give the squarest wave possible with the least voltage ringing. This voltage step should result in the opening of the voltage dependent Ca'*"'' channels (David et al., 1988) which are clearly identifiable by their kinetics. This procedure also gives some indication of the accuracy of the voltage command step (since the eggs Ca"*"^ channels will respond only if the command step is in the correct range). The accuracy of the command step was previously shown by Swann (1987, thesis) by insertion of a second electrode (which will introduce a further leakage current and so reduce the efficacy of the clamp) to be within 10% of the command voltage. At the end of each experiment the electrode was removed from the egg and the holding current removed to measure the true zero voltage, which was invariably very close to the preinsemination value (within 5 mV either way) and the record corrected.
The recording electrode was mounted on a preamplifier based on the design of Purves (Purves, 1981). Recordings were achieved by coupling the electrode to a switching single - electrode voltage-clamp amplifier (Halliwell and Whitaker, 1987) based on the design of Wilson and Goldner (1975). This amplifier can either inject constant current or pulses of current. For voltage
clamp recordings pulses of current are injected with a frequency of 5 kHz. Because the time constant of the egg is large, about 100 ms (tau = membrane resistance x membrane capacitance) compared to the time constant of the microelectrode, about 50 ps (measured, where tau = microelectrode resistance x total stray capacitance), when the pulsing frequency is set at 5 kHz (1 pulse every 200 ps) only the egg will store charge while the current flowing through the microelectrode will decay. Furthermore, by using negative capacitance, much of the current being drawn from the microelectrode is replaced. The sample and hold set up passes current for 2/3 of the time and samples 1/3 of the time, therefore all currents measured are 1/3 their true value. This is compensated for during calibration which is achieved using a 100 M ohm resistor and a 1 nA current pulse to read 100 mV. Leakage currents are set to 10 pA or less. Recordings were made jointly via a Sony digital pulse code modulator (model 701- ES) onto Sony video 8 tape (MP90) on a video 8 recorder (model EV-C3E), directly as files using the UMANS software (Chester Reagan), and directly on a Gould chart recorder (model BS-272). One oscilloscope (Crotech, model 2021) was set at 50 ps / division to monitor the pulsing frequency and to apply the negative capacitance properly. Another oscilloscope (Tektronix, model 2221) was used to monitor the slower changes accompanying intracellular recordings. Driving the system was the software of Chester Reagan loaded onto a Ness PC (386 - 16 - SX series).