Fig. 3.

Ca²⁺ hot spots co-localize with synaptic ribbons. Cone loaded via a patch pipette with the low-affinity Ca²⁺ indicator dye OGB-6F and the ribbon-specific peptide, Rh-RBP. Fluorescent images were taken at −70 mV (A) and during depolarization to −10 mV (B). The voltage clamp stimulus and times at which images A and B were obtained are illustrated below the images. Difference image (C) shows regions at the base of the terminal exhibiting increased fluorescence (green pixels), indicating a higher Ca²⁺ concentration. Note the three Ca²⁺ “hot spots.” OGB-6F was imaged with 488-nm excitation and 525-nm emission filters. Duration of each frame during Ca²⁺ imaging was 48 ms. (D) The graph shows changes in Ca²⁺ within the hot spot and soma over time. ΔF/F was converted to [Ca²⁺] using eqn (1) and a K_d of 3 μM for OGB-6F. (E) Binding of Rh-RBP to synaptic ribbons in the same cone was visualized by exciting the dye with 568-nm excitation and 607-nm emission filters. Note the three spots of bright labeling (red pixels), presumably from three distinct synaptic ribbons. (F) Overlap of the change in OGB-6F fluorescence and Rh-RBP binding (yellow pixels), showing co-localization of Ca²⁺ hot spots and synaptic ribbons (panel C + panel E). (G) Magnification of the region containing Ca²⁺ hot spots. Scale bar =10 μm.