Principle of SV recycling monitoring using a pH-sensitive probe. Left and middle: the pHluorin placed in the SV lumen is quenched under resting conditions (Rest). Upon SV exocytosis, the probe is exposed to a neutral environment, leading to fluorescence de-quenching (Stim). Subsequent endocytosis and re-acidification reduce fluorescence back to baseline levels (After stim). At the end of recordings, NH4Cl solutions are applied to visualize all SEP probes at the presynaptic terminal. White arrows indicate typical SypHy-positive puncta that respond to electrical stimulation. Scale bar: 1 m. Right: changes in SypHy fluorescence intensity in selected regions of interest (ROIs). The traces were normalized to the fluorescence intensity at the time of NH4Cl application.
