L-type Ca²⁺ channels mediate regulation of glutamate release by subthreshold potential changes

Subthreshold depolarization enhances neurotransmitter release evoked by action potentials and plays a key role in modulating synaptic transmission by combining analog and digital signals. This process is known to be Ca²⁺ dependent. However, the underlying mechanism of how small changes in basal Ca²⁺ caused by subthreshold depolarization can regulate transmitter release triggered by a large increase in local Ca²⁺ is not well understood. This study aimed to investigate the source and signaling mechanisms of Ca²⁺ that couple subthreshold depolarization with the enhancement of glutamate release in hippocampal cultures and CA3 pyramidal neurons. Subthreshold depolarization increased presynaptic Ca²⁺ levels, the frequency of spontaneous release, and the amplitude of evoked release, all of which were abolished by blocking L-type Ca²⁺ channels. A high concentration of intracellular Ca²⁺ buffer or blockade of calmodulin abolished depolarization-induced increases in transmitter release. Estimation of the readily releasable pool size using hypertonic sucrose showed depolarization-induced increases in readily releasable pool size, and this increase was abolished by the blockade of calmodulin. Our results provide mechanistic insights into the modulation of transmitter release by subthreshold potential change and highlight the role of L-type Ca²⁺ channels in coupling subthreshold depolarization to the activation of Ca²⁺-dependent signaling molecules that regulate transmitter release.