Single‐Vesicle Electrochemical Profiling Unveils Chemical Gating of Delayed and Sustained Exocytosis by Vitamin D

Roles of vitamin D in modulating brain function and mitigating cognitive impairment have garnered increasing attention, yet the mechanisms underlying its actions on neuronal activity, especially neurotransmission, remain unclear. Here, using single‐vesicle electrochemical analysis, we find that a rapid stimulus (15 s) by vitamin D 3 in its hormonally active form, 1,25‐(OH) 2 D 3 , directly triggers vesicular exocytosis, inducing delayed (> 30 s) and ultralong (minutes) neurotransmitter release that displays a concentration‐thresholded releasing behavior. By using immunofluorescence, live‐cell calcium imaging, and protein knockdown, we find that the membrane vitamin D receptor critically mediates 1,25‐(OH) 2 D 3 ‐induced synapsin phosphorylation to elicit delayed release, and that 1,25‐(OH) 2 D 3 can drive internal calcium store release to sustain a gradual cytosolic calcium rise, thus facilitating a long‐lasting exocytosis. We further demonstrate that locally administered 1,25‐(OH) 2 D 3 directly activates persistent firing in the rat hippocampus in vivo. This study provides the first vesicle‐level, time‐resolved, quantitative perspective of nongenomic actions of vitamin D on neuronal functions, unveiling its chemical gating of neurotransmission with concentration and spatiotemporal precision.