Presynaptic membrane potential affects transmitter release in an identified neuron in Aplysia by modulating the Ca 2 + and K + currents ( ionic current / voltage clamp / synaptic transmission )

We have examined the relationships between the modulation of transmitter release and of specific ionic currents by membrane potential in the cholinergic interneuron LIO of the abdominal ganglion of Aplysia californica. The presynaptic cell body was voltage-clamped under various pharmacological conditions and transmitter release from the terminals was assayed simultaneously by recording the synaptic potentials in the stsynaptic cell. When cell L1O was voltageclamped from a holding potential of -60 mV in the presence of tetrodotoxin, graded transmitter release was evoked by depolarizing command pulses in the membrane voltage range (-35 mV to +10 mV) in which the Ca2+ current was also increasing. Depolarizing the holding potential of L1O results in increased transmitter output. Two ionic mechanisms contribute to this form of plasticity. First, depolarization inactivates some K+ channels so that depolarizing command pulses recruit a smaller K+ current. In unclamped cells the decreased K+ conductance causes spike-broadening and increased influx of Ca2+ during each spike. Second, small depolarizations around resting potential (-55 mV to -35 mV) activate a steady-state Ca2+ current that also contributes to the modulation of transmitter release, because, even with most presynaptic K+ currents blocked pharmacologically, depolarizing the holding potential still increases transmitter release. In contrast to the steady-state Ca2+ current, the transient inward Ca2+ current evoked by depolarizing clamp steps is relatively unchanged from various holding potentials. In some spike-generating neurons the presynaptic terminals controlling transmitter release are electrically sufficiently close to the cell body so that the release of transmitter from the terminals can be modified by injecting current into the cell body (1-4). Klein and Kandel (5) have recently found that changes in the Ca2+ current in the cell body of an Aplysia neuron parallel changes in transmitter release at the terminals. These two sets of observations suggested to us that we might be able to examine the relationships between transmitter release and specific ionic currents of the presynaptic membrane. Toward this end we have combined two separate techniques: (i) pharmacological separation and voltage-clamp analysis of the ionic currents in the cell body of the presynaptic neuron and (ii) assay of transmitter release obtained by means of intracellular recordings of the synaptic potential in the post-synaptic cells. We have found that this combined technique provides a powerful method for studying changes in specific ionic conductances associated with various presynaptic mechanisms for synaptic plasticity. For these studies we have used the identified cholinergic neuron L10 of the abdominal ganglion of Aplysia. We eliminated impulse conduction by blocking Na+ channels with tetrodotoxin (TTX) and found that graded depolarizing commands, applied to the membrane of the presynaptic cell body under voltage-clamp control, caused graded transmitter release from the terminals as determined by the graded changes in the synaptic potential in the postsynaptic cell. Although in many cases we probably lacked ideal voltage control of the terminals, this procedure nonetheless allowed sufficient control to study transmitter release while examining specific ionic currents in the soma of the presynaptic neuron. Voltage control can be further improved by axotomizing the presynaptic cell (6, 7) and by adding pharmacological agents that block each of the three known outward K+ currents (8) and thereby also lengthen the effective space constant of the neuron. By using these approaches, we have found that depolarizing the presynaptic membrane potential enhances transmitter release by two mechanisms: (i) by activation of a steady-state Ca2+ current and (ii) by decreasing the outward K+ currents activated by a step depolarization (9, 10). This decrease in the K+ currents increases the duration of the spike and, therefore, the transient Ca2+ current activated by the spike from depolarized levels of membrane potential. In a subsequent paper (11) we use the same voltage-clamp approach to examine presynaptic inhibition at the synapses made by L1O and find that this action results from a direct modulation of the transient Ca2+ current. These findings indicate that the amount of Ca2+ coming into the presynaptic terminals with each action potential is not fixed but can be modulated by membrane voltage and by chemical transmitters. Moreover, the existence of several independent ways for regulating the Ca2+ current suggests that Ca2+ current modulation may be a fairly common mechanism for the presynaptic regulation of transmitter release.