Regulation of presynaptic function by sodium permeable ion channels at the calyx of Held synapse
Description
Previous work has revealed a presynaptic cytosolic Na+-dependent regulation on vesicular glutamate content and mEPSC amplitude via activating vacuolar Na+/H+ exchangers (NHEs) expressed on the synaptic vesicles, suggesting a presynaptic determinant of quantal size for synaptic strength. However, it remains unknown how spike activities control intracellular Na+ at the axon terminals and how the fluctuation of presynaptic Na+ during activities modulates quantal content and contributes to synaptic strength. I studied these questions using the calyx of Held, a giant glutamatergic synapse. With two-photon Na+ imaging, I found that presynaptic Na+ substantially accumulated during spike firing in a frequency and duration-dependent manner. This spike-induced elevation of presynaptic Na+ gradually increased EPSC amplitude by solely affecting vesicular glutamate filling, which was further confirmed as increased amplitude of asynchronous released vesicles, but without affecting the size of readily releasable pool or neurotransmitter release probability. This Na+-dependent modulation of EPSC amplitude resulted in a change of the reliability of transferring presynaptic spike to postsynaptic firing. Finally, blockade of NHEs reduced both EPSC amplitude and reliability of synaptic signaling, suggesting that NHEs are required for presynaptic Na+ regulation of synaptic transmission. Recent studies demonstrated that a non-inactivation cation channel NALCN (Na+ leak channel, non-selective), characterized as a major Na+ leak channel, is widely expressed in the central nervous system. Immunostaining revealed the expression of NALCN channel at the calyceal terminals. In line with a role of NALCN in controlling the cell excitability, calyces with conditional knockout (cKO) of NALCN exhibited a more hyperpolarized resting membrane potential compared with the wildtype (WT) calyces. Blockade of NALCN with a non-specific blocker gadolinium (Gd3+) induced a reduction of basal Na+ level and mEPSC amplitude in the WT but not in cKO group, suggesting the involvement of presynaptic NALCN channels in regulating the vesicular glutamate content. More importantly, two-photon Ca2+ imaging showed that NALCN channels were permeable to Ca2+, and Gd3+ decreased the basal Ca2+ level in WT but not cKO calyces. The Ca2+ permeability was further confirmed by reduced sensitivity of mEPSC frequency in response to increased extracellular Ca2+ concentration in cKO and reduced initial release probability in response to application of Gd3+ to block NALCN channels in WT group. Finally, Gd3+ induced a stronger reduction of EPSC amplitude in WT group compared to cKO group. Overall, these data indicate that NALCN channels regulate glutamate transmission through modulation of both quantal size and initial release probability.