(A) Three neighboring presynaptic boutons located along the same axon (translucent gray). Bouton 1 (b1) was an MSB with two postsynaptic partners (PSDs, red) that contained a mitochondrion (navy), 303 nondocked vesicles (green), and 12 docked vesicles (blue, inset). Nearest distances from each PSD to the mitochondrion were less than 1 µm (Dm1a, 1b). For b2, the distance from the edge of the PSD to the nearest mitochondrion was 1.4 µm (Dm2, blue line), and it contained 124 nondocked vesicles and nine docked vesicles. The b3 had 82 nondocked vesicles and two vesicles docked at the PSD which was 4.4 µm from the nearest mitochondrion (b4, nonsyn mito, Dm3, orange line) and 5.3 µm from the end of the axon reconstruction (D3end). Scale cube is 0.5 µm on each side. (B) Representative EMs from four regions along an axon illustrating boutons with PSDs (red arrows), nondocked vesicles (green arrows), and docked vesicles (blue arrows) and a nonsynaptic inter-bouton region containing a mitochondrion (nonsyn mito). Scale bar is 0.25 µm in b4 for all four micrographs. (C) Of all the axonal boutons, 76% were SSBs and 24% were MSBs. Absolute numbers of boutons are in corresponding bars that also indicate the presence (black) or absence of a mitochondrion (gray). (D) Overall, the PSD areas did not differ between synapses on MSBs and SSBs (n = 216, F1,215 = 0.026, p=0.87). However, PSD areas were larger on SSBs when mitochondria were present (n = 140, F1,138 = 15.4, p<0.001), but on MSBs this difference did not reach significance (n = 75, F1,74 = 2.791, p=0.10). (E) The number of docked vesicles per PSD did not differ between MSBs and SSBs with or without mitochondria (n = 177, F1,175 = 0.26, p=0.61); however, the number of docked vesicles per PSD was greater for both types of boutons with mitochondria than those without mitochondria (n = 177, F1,175 = 65.1, p<0.001). (F) Boutons with mitochondria had more nondocked vesicles whether they were SSBs or MSBs (n = 180, F1,178 = 16.2). Since the pool of nondocked vesicles could not be attributed to a specific PSD, the MSBs had more nondocked vesicles than SSBs, which reached significance for those boutons without mitochondria (gray line above the bars). (G) Axon segments containing mitochondria somewhere along their lengths were analyzed and the amount of axon volume with mitochondria was significantly less than the amount of axon volume without mitochondria (n = 81, ANOVA, F1,161 = 104.1, p<0.0001). (H) Of all axonal mitochondria in the perfusion fixed condition from the brick analyses (see Figure 2E), 51% of mitochondria resided in nonsynaptic regions (Nonsyn), while 49% resided in synaptic boutons (Syn). (I) Axonal and mitochondrial diameters were correlated and this relationship did not differ significantly between mitochondria located in synaptic or nonsynaptic regions of the axons from (H) (n = 75, ANCOVA, F1,72 = 1.91, p=0.17). Significant post hoc differences are indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.