Nanophysiology approach reveals diversity in calcium microdomains across zebrafish retinal bipolar ribbon synapses

Rapid and high local calcium (Ca²⁺) signals are essential for triggering neurotransmitter release from presynaptic terminals. In specialized bipolar ribbon synapses of the retina, these local Ca²⁺ signals control multiple processes, including the priming, docking, and translocation of vesicles on the ribbon before exocytosis, endocytosis, and the replenishment of release-ready vesicles to the fusion sites for sustained neurotransmission. However, our knowledge about Ca²⁺ signals along the axis of the ribbon active zone is limited. Here, we used fast confocal quantitative dual-color ratiometric line-scan imaging of a fluorescently labeled ribbon binding peptide and Ca²⁺ indicators to monitor the spatial and temporal aspects of Ca²⁺ transients of individual ribbon active zones in zebrafish retinal rod bipolar cells (RBCs). We observed that a Ca²⁺ transient elicited a much greater fluorescence amplitude when the Ca²⁺ indicator was conjugated to a ribeye-binding peptide than when using a soluble Ca²⁺ indicator, and the estimated Ca²⁺ levels at the ribbon active zone exceeded 26 μM in response to a 10 millisecond stimulus, as measured by a ribbon-bound low-affinity Ca²⁺ indicator. Our quantitative modeling of Ca²⁺ diffusion and buffering is consistent with this estimate and provides a detailed view of the spatiotemporal [Ca²⁺] dynamics near the ribbon. Importantly, our data demonstrates that the local Ca²⁺ levels may vary between ribbons of different RBCs and within the same cells. The variation in local Ca²⁺ signals is found to correlate with ribbon size and active zone extent. Our serial electron microscopy results provide new information about the heterogeneity in ribbon size, shape, and area of the ribbon in contact with the plasma membrane.