Fig 2. Mitochondria-free presynaptic boutons have increased neurotransmitter release during repetitive stimulation.

(A) Presynaptic release properties at mitochondria-associated and -free boutons were monitored using synaptophysin-pHluorin and mito-mTagBFP in cultured neurons at 15–17 DIV. (B) Representative image of sypH peak value following 20 AP at 10 Hz. sypH images were visualized by ratio of the peak image normalized by Fmax signal. Fmax of sypH was obtained by NH₄Cl (50mM) incubation. White arrows indicate mitochondria-associated presynaptic site and orange arrows indicate mitochondria-free presynaptic site. (C and E) Graphs of exocytic response at presynaptic sites with or without mitochondria show increased release in mitochondria-free bouton following both 20 and 100 AP stimuli. (D and F) Quantification of normalized peaks from C and E. Presynaptic sites without mitochondria have significantly increased release. n = 26 for mitochondria-associated boutons and 15 for mitochondria-free boutons from 15 neurons in 20AP condition. n = 31 for mitochondria-associated boutons and 17 for mitochondria-free boutons from 15 neurons in 100AP condition. * p < 0.05, Mann-Whitney test. Scale bar = 10μm. Individual values are available in S1 Data.