Ca(2+) current facilitation determines short-term facilitation at inhibitory synapses between cerebellar Purkinje cells

Abstract

Key points: Short-term facilitation takes place at GABAergic synapses between cerebellar Purkinje cells (PCs). By directly patch clamp recording from a PC axon terminal, we studied the mechanism of short-term facilitation. We show that the Ca(2+) currents elicited by high-frequency action potentials were augmented in a [Ca(2+) ]i -dependent manner. The facilitation of synaptic transmission showed 4-5th power dependence on the Ca(2+) current facilitation, and was abolished when the Ca(2+) current amplitude was adjusted to be identical. Short-term facilitation of Ca(2+) currents predominantly mediates short-term facilitation at synapses between PCs.

Abstract: Short-term synaptic facilitation is critical for information processing of neuronal circuits. Several Ca(2+) -dependent positive regulations of transmitter release have been suggested as candidate mechanisms underlying facilitation. However, the small sizes of presynaptic terminals have hindered the biophysical study of short-term facilitation. In the present study, by directly recording from the axon terminal of a rat cerebellar Purkinje cell (PC) in culture, we demonstrate a crucial role of [Ca(2+) ]i -dependent facilitation of Ca(2+) currents in short-term facilitation at inhibitory PC-PC synapses. Voltage clamp recording was performed from a PC axon terminal visualized by enhanced green fluorescent protein, and the Ca(2+) currents elicited by the voltage command consisting of action potential waveforms were recorded. The amplitude of presynaptic Ca(2+) current was augmented upon high-frequency paired-pulse stimulation in a [Ca(2+) ]i -dependent manner, leading to paired-pulse facilitation of Ca(2+) currents. Paired recordings from a presynaptic PC axon terminal and a postsynaptic PC soma demonstrated that the paired-pulse facilitation of inhibitory synaptic transmission between PCs showed 4-5th power dependence on that of Ca(2+) currents, and was completely abolished when the Ca(2+) current amplitude was adjusted to be identical. Thus, short-term facilitation of Ca(2+) currents predominantly mediates short-term synaptic facilitation at synapses between PCs.

Figure 1. PPF of synaptic transmission at the cultured PC–PC synapse

A, fluorescence image of a synaptically‐connected PC–PC pair. Green: a presynaptic PC expressing EGFP. Red: a postsynaptic PC visualized by CF633 applied through a patch pipette (removed when the image acquisition). To show several synapses around the postsynaptic PC soma and proximal dendrites, the area surrounded by a white rectangle is presented as an enlarged image. B, representative traces of presynaptic Na⁺ and K⁺ currents and PSCs upon paired pulse stimulation at a 10 ms interval (depolarization pulses to 0 mV for 2 ms). Presynaptic currents through voltage‐dependent channels were isolated by subtracting the 7‐fold capacitive currents upon the 10 mV depolarization pulse. C, normalized PSC traces upon paired‐pulse stimulation at 5, 10, 20 or 50 ms intervals. D, averaged ratio of PSC amplitudes (PSC2/PSC1) is plotted against the interval of paired pulse stimulation (n = 6–7 pairs).

Figure 2. PC–PC synaptic transmission upon an AP voltage command at a terminal

A, representative image for paired recordings from the presynaptic PC terminal (highlighted by a white arrowhead) and the postsynaptic PC soma. Both pre‐ and postsynaptic PCs are EGFP‐positive in this case. B, AP waveform recorded from a PC terminal impinging on a PC (left) and the AP waveform used for voltage command in the present study that was recorded from a PC terminal on a DCN (right). C, left: representative traces of different amplitudes of AP commands (V _com), the Ca²⁺ currents (ICa²⁺) in a presynaptic PC terminal and the PSCs simultaneously recorded from the postsynaptic PC. Right: PSC amplitudes upon various amplitudes of AP commands were plotted against the ICa²⁺ amplitudes. The grey line represents the 4.5th power relationship between x‐ and y‐axis. D, left: a representative trace of PSC evoked by the 5 ms depolarization pulse to 0 mV in the presynaptic terminal. For hundreds of milliseconds after the large PSC, asynchronous events could be observed. These events were used for sampling mPSCs because this is the most reliable way of collecting miniature events from a given presynaptic PC terminal. Right: amplitude histogram of asynchronous PSCs. Pooled data from six cells are shown.

Figure 3. Ca²⁺‐dependent facilitation of presynaptic Ca²⁺ currents at PC–PC synapses

A, image of direct patch clamp recording from an EGFP‐positive axon terminal on a proximal dendrite of another PC. B, pair of APs recorded from a PC terminal on a PC (top), representative traces of paired‐pulse voltage commands with an AP waveform (middle, V _com) and the resultant Ca²⁺ currents (bottom, ICa²⁺). C, normalized Ca²⁺ currents upon paired‐pulse AP commands at 3.33, 5, 10, 20 or 50 ms interval. The dotted line represents the average peak amplitude of the first Ca²⁺ current. D, representative traces of the paired‐pulse voltage command with an identical AP waveform without AHP and the resultant Ca²⁺ currents. E, averaged Ca²⁺ current facilitation upon various intervals of paired‐pulse AP waveforms in the presence of 0.5 or 5 mm EGTA or of 20 mm BAPTA_.The data obtained without AHP of the AP waveform in the presence of 0.5 mm EGTA are also shown (no AHP) [n = 4 (0.5 mm EGTA, 5 mm EGTA and 20 mm BAPTA); n = 5 (no AHP)].

Figure 4. Tight coupling of Ca²⁺ current facilitation and synaptic facilitation at PC–PC synapses

A, representative traces of ICa²⁺ (average of 20 traces) and PSCs upon paired pulse voltage commands consisting of an identical AP waveform. Black PSC trace is the average of 20 PSC traces shown in grey. B and C, averaged PPR of ICa²⁺ (B) and that of PSC (C) in the presence of intracellular 0.5 or 5 mm of EGTA are plotted against the interstimulus interval. Grey or black continuous lines in (C) are the 4.5th power of ICa²⁺ (shown as dotted lines, which are same as continuous lines in B) [n = 6 pairs (5 mm EGTA); n = 4 pairs) (0.5 mm EGTA)].

Figure 5. ICa²⁺ facilitation determines the synaptic facilitation

A and B, representative traces of voltage commands consisting of paired‐pulse AP waveforms (V _com) and the Ca²⁺ currents (ICa²⁺) in a presynaptic PC terminal (A) and of PSCs (B). The second AP amplitude was changed (1.05, 1, 0.95, 0.9 or 0.85 times that of the first) to alter the second Ca²⁺ current amplitude. The PSC amplitudes were normalized to the first one. C, the averaged PPR of PSCs was plotted against that of ICa²⁺. The data were obtained from (A) and (B) (n = 6 pairs).

Figure 6. Residual Ca²⁺ increase at PC terminals

A, representative fluorescence image of a PC loaded with 200 μm OGB‐1. At an axon varicosity (highlighted by an white arrow), fluorescence change was measured before and after the action potential evoked at the soma. B, time courses of normalized fluorescence intensity change of OGB‐1 or OGB‐6 recorded from an axon varicosity from a PC, an IN or a GC. Representative traces for each condition are also shown in grey. C, ΔF/F upon single AP in a GC (n = 6 cells for OGB‐1 and 8 for OGB‐6), IN (n = 5 cells for OGB‐1 and 6 for OGB‐6) or PC (n = 7 cells for OGB‐1 and 5 for OGB‐6). *P < 0.05.

Figure 7. Ca²⁺‐dependent PPF of Ca²⁺ currents and PSCs at PC–DCN synapses

A, representative image for paired recordings from a presynaptic PC terminal and a postsynaptic DCN neuron. B, representative traces of the paired‐pulse AP command (V _com) and the Ca²⁺ currents (ICa²⁺) in a presynaptic PC terminal (top) and the PSCs simultaneously recorded from a postsynaptic DCN neuron (bottom). Red trace is the averaged PSC from 20 PSC traces (grey). C and D, averaged PPR of ICa²⁺ (C) and that of PSC (D) in the presence of intracellular 0.5 or 5 mm of EGTA are plotted against the interstimulus interval. Black or grey lines in (D) are the 4.5th power of ICa²⁺ (shown as dotted lines, which is same as the continuous lines in C) (n = 5 and 6 for 5 and 0.5 mm EGTA, respectively).

Figure 8. Cm increases at a PC terminal on a PC or DCN neuron

Top: representative traces of presynaptic ICa²⁺ and Cm recorded from a PC terminal on a PC (A) or that on a DCN neuron (B). Bottom: Cm increases recorded with intracellular 0.5 or 5 mm EGTA were plotted against the depolarization pulse duration [n = 7 (0.5 mm EGTA) and n = 13 (5 mm EGTA) terminals on a PC; n = 7 (0.5 mm EGTA) and n = 5 (5 mm EGTA) terminals on a DCN neuron].