Short-Term Depression of Axonal Spikes at the Mouse Hippocampal Mossy Fibers and Sodium Channel-Dependent Modulation

Loose-patch clamp recordings of axonal spikes from the single mossy fiber boutons. A, Schematic drawing of experimental arrangement. Stimulating electrode was placed in the granule cell layer of the dentate gyrus, and the evoked responses were recorded from visually-identified single mossy fiber boutons. Surrounding region of the recording site was focally perfused with a continuous flow of perfusate through a flow-pipe. A photograph showing IR-DIC image of the recorded bouton (arrow). B, Representative traces of the axonal spikes recorded from the single mossy fiber boutons. C, All-or-none feature of the axonal spikes, which appear above the threshold stimulus intensity (0.2 mA in this recording). D, Effect of focal application of TTX at 0.5 μM. E, Comparison of the time course of simulated dV_m/dt and the recorded axonal spikes. Simulated membrane potential (V_m) during axonal action potential was calculated according to the latest model of action potentials at mossy fibers (see Materials and Methods). In the right panel, the recorded axonal spike in B was superimposed with the first derivative of simulated V_m (dV_m/dt, middle panel).

PPD of axonal spikes recorded from single mossy fiber boutons. A, At a 50-ms interval, the amplitude of the second spike (closed circle) was slightly reduced than the first spike (open circle). B, Superimposed traces of paired-pulse responses at 50-, 100-, 200-, and 500-ms intervals. C, Time course of PPD of axonal spikes recorded at 25 ± 1°C are shown in black circles (n = 9). Data of similar experiments recorded at 33 ± 1°C are also shown in red circles (n = 7). D, Time-integrated traces of axonal spikes recorded extracellularly by loose-patch clamp configuration, which are supposed to reflect intracellular membrane potential changes during action potentials, in response to the first (open circle) and second stimuli (closed circle, blue trace). Note that superimposed traces in the right panel show reduction of the peak amplitudes.

Minimal effects of Ca²⁺-containing [Ca²⁺(+)] ACSF on axonal spikes. A, Focal application of Ca²⁺(+) ACSF to the surrounding area of the recorded boutons (see Fig. 1A) exhibited no clear effects on the 1st (open circles) and 2nd (closed circles) axonal spikes. B, Time course of the amplitude of the first and second spikes during Ca²⁺(+) ACSF application. C, Summary data of the effect of Ca²⁺(+) ACSF on the first (open bar) and second spikes (closed bar, n = 8). D, Summary data of PPD of axonal spikes in control condition (open bar) and in Ca²⁺(+) ACSF. E, Superimposed traces of the first (open circle) and 2nd spike (closed circle) in the control condition and in Ca²⁺(+) ACSF.

Selective modulation of PPD of axonal spikes by veratridine, an inhibitor of inactivation of sodium channels. A, Focal application of 1 μM veratridine selectively suppressed the amplitude of the second spike (closed circle) with minimal effect on the first spike (open circle). B, Time course of the amplitude of the first and second spikes during veratridine application (n = 20). C, Summary data on the effect of veratridine on the first (open bar) and second spikes (closed bar, n = 20, ** P < 0.01). D, Summary data of PPD of axonal spikes in control condition (open bar) and in the presence of veratridine (closed bar). E, Superimposed traces of the first (open circle) and 2nd spike (closed circle) in control condition and in the presence of veratridine.

Veratridine-induced use-dependent modulation of axonal spikes during repetitive stimuli. A, B, Veratridine at 1 μM minimally affected the first spike (open circle), while selectively reduced the subsequent responses to 20 Hz (A) and 100 Hz (B) trains. C, D, Time course of the first, 10th or 100th spikes during veratridine application (n = 13 and 14, respectively). E, Superimposed traces of the first (open diamond) and 100th spike during 100-Hz train (closed diamond) in the control condition and in the presence of veratridine. F, G, Summary data of latency of the first and the last spikes during 20-Hz (F) and 100-Hz trains (G). The 100th spikes during 100-Hz trains in the presence of veratridine (G) were reduced in size substantially, and therefore were unable to measure the latency quantitatively.

Effect of low concentration of TTX on axonal spike. A, TTX at 50 nM partially suppressed both the first (open circle) and second spike (closed circle). B, Time course of the amplitude of the first and 2nd spikes during application of 20 nM and 50 nM TTX (n = 7 and 9, respectively) as shown by open and closed diamonds and circles, respectively. C, Summary data of the effect of 20 and 50 nM TTX on the first (open bar) and second spikes (closed bar). D, PPD was weakly restored by 50 nM TTX (closed bar, n = 9, *p < 0.05), while 20 nM TTX did not affect significantly (n = 7). E, Superimposed traces of the first (open circle) and second spike (closed circle) in control condition and in the presence of 50 nM TTX.

Veratridine potentiated ADP in the granule cell soma. A, Veratridine at 1 μM strongly enhanced ADP following action potentials elicited by brief current injection into the dentate granule cells, which originates the mossy fiber axons. Prolonged ADP during veratridine application was often accompanied by multiple spikes after current injection. B, Expanded time course of action potential and ADP. ADP amplitude was quantified at 10 ms after the peak of the action potential (arrowhead). C, Summary data on the effect of veratridine on the ADP amplitude (n = 7, *p < 0.05).