Facilitation of presynaptic calcium currents in the rat brainstem

Abstract

1. To study use-dependent changes in the presynaptic Ca2+ influx and their contribution to transmitter release, we made simultaneous voltage clamp recordings from presynaptic terminals (the calyces of Held) and postsynaptic cells (the principal cells of the medial nucleus of the trapezoid body) in slices of the rat auditory brainstem. 2. Following a short (2 ms) prepulse to 0 mV, calcium channels opened faster during steps to negative test potentials. During trains of action potential waveforms the Ca2+ influx per action potential increased. At the same time, however, the amplitude of the EPSCs decreased. 3. The facilitation of the calcium currents appeared to depend on a build-up of intracellular Ca2+, since its magnitude was proportional to the Ca2+ influx and it was reduced in the presence of 10 mM BAPTA. 4. Facilitation of the presynaptic calcium currents may contribute to short-term facilitation of transmitter release, observed when quantal output is low. Alternatively, it may counteract processes that contribute to synaptic depression.

Figure 1. Facilitation of presynaptic calcium currents

A, from top to bottom: presynaptic command voltage (V_pre), presynaptic calcium current (I_pre) and postsynaptic currents (I_post). A 2 ms prepulse to 0 mV (thin line) resulted in a facilitation of the calcium current during a 5 ms step to −20 mV compared with the current in the absence of a prepulse (thick line). A somewhat smaller effect on the presynaptic current was observed after a prepulse to +60 mV (dotted line). The vertical arrow indicates where the isochronal current-voltage (I–V) relationship was evaluated. The horizontal arrows point to the peak amplitude of the tail currents with (lower arrow) and without (upper arrow) prepulse. B, no effect of the prepulse on the current during the test pulse was observed when the test pulse was to +20 mV. C, isochronal I-V relationship. Amplitudes of the currents during the test pulse were measured 1.5 ms after the start of the depolarizing step. The I-V relationship for the prepulse to 0 mV (□, thin line) was shifted to the left for negative voltages compared with the I-V relationship in the absence of a prepulse (•, thick line). An intermediate effect was observed after the prepulse to +60 mV (▴, dotted line). D, changes in volume-averaged Ca²⁺ concentration ([Ca²⁺]_i) resulting from the prepulse to 0 mV (thin line) or to +60 mV (dotted line). The thick line shows [Ca²⁺]_i in the absence of a voltage step. Different experiment from the one shown in A–C.

Figure 2. Facilitation of presynaptic calcium currents during a train of action potential waveforms

A, increase of the Ca²⁺ influx per action potential during a 100 Hz train of identical action potential waveforms. Simultaneous pre- and postsynaptic voltage clamp recording. From top to bottom, the panels show: presynaptic voltage command, presynaptic calcium currents, EPSCs and volume-averaged calcium concentration. Presynaptic pipette solution contained 50 μm fura-2. B, lack of a clear increase in the Ca²⁺ influx per action potential during a 100 Hz train of identical action potential waveforms in the presence of 10 mm BAPTA in the presynaptic pipette solution. Two-electrode voltage clamp recording of the calyx. Top panel, presynaptic voltage command; bottom panel, presynaptic calcium currents. Same scale applies as to the two top panels of A. C, average increase in the peak amplitudes of the calcium currents evoked by an action potential waveform in the presence of 10 mm BAPTA (○, n = 5) or 50 μm fura-2 (•, n = 6). Amplitudes were normalized to the first one. Following the second stimulus, normalized amplitudes in the presence of low and high Ca²⁺ buffer concentrations were significantly different (P < 0.05, unpaired t test for unequal variances).

Figure 3. Hodgkin-Huxley simulation of calcium currents

A, to mimic the effects of a 2 ms prepulse to +60 mV or to 0 mV on the calcium currents, the voltage dependence of the rate constant (α_m) for channel opening was shifted by −2.2 and −4.0 mV, respectively (see Methods). As a result, the simulated isochronal I-V relationship was shifted by −1.6 mV (dotted line) and −2.8 mV (thin line) at around −20 mV compared with control (thick trace) and the current evoked by a step to 0 mV increased by 4.4 % and 7.7 %, respectively, similar to that observed experimentally. B, simulation of calcium currents during an action potential using the same three conditions as in A. Top panel, voltage template. Middle panel, calculated open probability (P_o) of the calcium channels, displayed as a fraction of the maximum open probability. Lower panel, simulated calcium currents.