Mechanisms underlying presynaptic facilitatory effect of cyclothiazide at the calyx of Held of juvenile rats


Corresponding author T. Takahashi: Department of Neurophysiology, University of Tokyo, Faculty of Medicine, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Email:


  • Excitatory postsynaptic currents (EPSCs) were recorded using the whole-cell patch-clamp technique at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) in auditory brainstem slices from juvenile rats.

  • Bath application of cyclothiazide (CTZ, 100 μm) significantly increased the amplitude of EPSCs mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptors. Cyclothiazide increased the magnitude of paired-pulse depression of both AMPA-EPSCs (intervals, 50 and 500 ms) and NMDA-EPSCs (interval, 20 ms). In low Ca2+, high Mg2+ solution, CTZ decreased the number of failures and increased the mean amplitude of AMPA-EPSCs more than three-fold.

  • Presynaptic Ca2+ currents and K+ currents were directly recorded from the calyceal nerve terminals. These currents were attenuated by CTZ in a reversible manner. The magnitude of inhibition of presynaptic K+ currents by CTZ (100 μm) was comparable to that by 5 μm 4-aminopyridine (4-AP). Both CTZ and 4-AP slowed the repolarizing phase of presynaptic action potentials.

  • The inhibitory effects of CTZ on presynaptic ion channels were mimicked by a solution having reduced Ca2+ concentration and 5 μm 4-AP. This solution facilitated EPSCs, but the magnitude of facilitation was significantly less than that caused by CTZ.

  • In the presence of tetrodotoxin (TTX), CTZ increased the mean frequency of miniature EPSCs three-fold. CTZ prolonged their decay time but had no effect on their amplitude. The facilitatory effect of CTZ on the miniature frequency was neither blocked by a protein kinase C inhibitor nor occluded by phorbol ester, suggesting that a distinct mechanism underlies the effect of CTZ.

  • We conclude that CTZ facilitates transmitter release through suppression of presynaptic potassium conductance and stimulation of exocytotic machinery downstream of Ca2+ influx.