Article Figures & Data
Figures
- Figure 1.
Electron tomography reveals robust ultrastructural differences between axonal mitochondria of fast-spiking and regular-spiking basket cells. A, Confocal images show that perisomatic synaptic boutons are either PV+ or CB1R+ in the hippocampal CA1 region. Pyramidal cell nuclei are labeled with DAPI (blue), vGAT-immunoreactive puncta represent GABAergic vesicle pools (cyan), PV labeling is green and CB1R labeling is magenta, white arrowheads mark GABAergic release sites. B, Transmission electron micrograph shows that CB1R-immunogold labeling reliably differentiates the two perisomatic bouton populations. Black granules are the silver-intensified CB1R-immunogold particles, white arrowheads mark a synapse. Pseudocolors: CB1R+ bouton, magenta; PV bouton, green; pyramidal cell cytoplasm, yellow; and nucleus, blue. C, CB1R-immunogold labeling was absent in CB1R-KO animals (tested membrane length: 38 µm in two WT, and 74.8 µm in two CB1R-KO mice). D, F, Representative 3D models of serial EM-reconstructed segments from a synaptic CB1R+ bouton (D), and a synaptic PV bouton. Bouton membrane is magenta for CB1R bouton, green for PV bouton, synapses are vivid green, mitochondria are blue, and red belts represent those sections of the organelles that were reconstructed through electron tomography. E, 1.5-nm-thick electron tomographic section and 3D model of the reconstructed mitochondrion from the bouton depicted in D (mitochondrial outer membrane is red, inner boundary membrane is cyan, CM is green). G, 1.5-nm-thick electron tomographic section and 3D model of the reconstructed mitochondrion from the bouton depicted in F. H, CM density is significantly higher in mitochondria of PV boutons than in those from CB1R+ boutons (Mann–Whitney U test, p = 0.0002, n = 20 mitochondria from two mice). I, Cristae are significantly more lamellar in mitochondria of PV boutons than in those from CB1R+ boutons, as verified by the higher crista shape factor values (Mann–Whitney U test, p = 0.0090, n = 20 mitochondria from two mice). H, I, Blue dots represent values from individual mitochondria, magenta rectangles represent interquartile ranges, and deep-magenta lines mark median values. Scale bars: 6 µm (A), 500 nm (B), and 100 nm (E, G). The 3D models in E, G are not displayed on the same scale. See also Extended Data Figures 1-1, 1-2.
- Figure 2.
Axonal mitochondria of fast-spiking basket cells express higher levels of CytC than those of regular-spiking basket cells. A, STORM super-resolution imaging confirms a near-complete overlap between TOM20 and CytC-labeled areas. B, Scatterplot shows a strong correlation (R = 0.98, n = 48 mitochondria from two mice) between the measured areas in the two channels of individual mitochondria. Each dot corresponds to a single mitochondrion. C, Confocal image shows a CB1R+ (blue) and a PV+ (yellow) perisomatic bouton, both containing a mitochondrion, labeled for CytC (magenta). D, CB1R+ bouton enlarged from C with overlaid STORM CytC localization points (cyan). E, Confocal and STORM image of mitochondrion enlarged from D. F, PV+ bouton enlarged from C with overlaid STORM CytC localization points. G, Confocal and STORM image of mitochondrion enlarged from F. Red lines in E, G mark the 2D convex hulls generated around the localization points. H, Mitochondria in the PV+ boutons are larger than those in the CB1R+ boutons, as the 2D areas of the convex hulls are significantly larger in the former group (Mann–Whitney U test, p = 0.0085, n = 57 mitochondria from two mice). I, Mitochondria in the PV+ boutons contain CytC in a higher density than those in the CB1R+ boutons (Mann–Whitney U test, p = 0.0384, n = 57 mitochondria from two mice). Blue dots represent values from individual mitochondria, magenta rectangles represent interquartile ranges, and deep-magenta lines mark median values. Scale bars: 800 nm (A, upper-left mitochondrion), 400 nm (A, all others), 1 µm (C), 600 nm (D, F), and 170 nm (E, G).
- Figure 3.
Mitochondrial ultrastructure is coupled to synaptic performance in a cell type-independent manner at glutamatergic synapses. A, 3D model of a serial EM reconstructed segment of a LP glutamatergic bouton from the dentate gyrus. Bouton membranes are semitransparent cyan, postsynaptic profiles are semitransparent magenta, synapses are yellow, mitochondria are blue, and red belts mark those sections of the organelles that were reconstructed through electron tomography. B, 1.5-nm-thick electron tomographic section and 3D model of the reconstructed mitochondrion from the bouton depicted in A (mitochondrial outer membrane is red, inner boundary membrane is cyan, CM is green). C, 3D model of a serial EM reconstructed segment of a HP glutamatergic bouton from the dentate gyrus (colors same as in A). D, 1.5-nm-thick electron tomographic section and 3D model of the reconstructed mitochondrion from the bouton depicted in C. E, Mitochondria are significantly larger in HP boutons than in LP boutons (Mann–Whitney U test, p = 0.0199, n = 19 mitochondria from two mice). F, CM density is significantly higher in mitochondria of HP boutons than in those from LP boutons (Mann–Whitney U test, p = 0.0009, n = 19 mitochondria from two mice). G, Cristae are significantly more lamellar in mitochondria of HP boutons than in those from LP boutons, as verified by the higher crista shape factor values (Mann–Whitney U test, p = 0.0080, n = 19 mitochondria from two mice). H–J, The volume of individual presynaptic mitochondria correlates with active zone area in the human hippocampus. H, Transmission electron micrographs of two presynaptic mitochondria from human samples, and their 3D reconstructions from serial images. I, 3D model of an axonal segment from human tissue, giving two synapses, each with an associated presynaptic mitochondrion. Axons are red, mitochondria green, spines blue, and active zones yellow. J, The volume of presynaptic mitochondria shows a strong correlation with active zone area in human axons (R = 0.86, p < 0.0001, n = 31 mitochondria from two patients). E–G, J, Blue dots represent values from individual mitochondria, magenta rectangles represent interquartile ranges, deep-magenta lines mark median values. Scale bars: 110 nm (B, D), 200 nm (H). The 3D models in B, D, H are not displayed on the same scale. See also Extended Data Figures 3-1, 3-2.
- Figure 4.
CytC content of presynaptic mitochondria scales with synaptic performance at glutamatergic synapses. A, Confocal image shows a glutamatergic bouton (VG1, blue), containing a mitochondrion (Cyt-C, red), and its synapse (Homer1a, green). B, The 3D model of the bouton from A was reconstructed from the confocal stack. C, Confocal and STORM image of mitochondrion enlarged from the same bouton (Cyt-C confocal in magenta, Cyt-C localization points in cyan, 2D convex hull in red). D–F, Another bouton depicted as in A–C. Mitochondria-containing synaptic boutons were divided into LP and HP groups, based on the corresponding Homer volumes. Bouton in A–C represents a LP, bouton in D–F represents a HP bouton. G, Mitochondria in the HP boutons are larger than those in the LP boutons, as the CytC-volumes are significantly larger in the former group (Mann–Whitney U test, p = 0.00032, n = 42 mitochondria from two mice). H, Mitochondria in the HP boutons contain CytC in a higher density than those in the LP boutons (Mann–Whitney U test, p = 0.00016, n = 42 mitochondria from two mice). Blue dots represent values from individual mitochondria, magenta rectangles represent interquartile ranges, deep-magenta lines mark median values. Scale bars: 800 nm (A, D), 200 nm (C, F). The 3D models on B, E are not displayed on the same scale. See also Extended Data Figure 3-1.
Tables
Data structure Comparison Test Unknown Normality test Shapiro–Wilks W Not normal distribution Two independent groups Mann–Whitney U Not normal distribution Correlation Spearman’s correlation Normal distribution Correlation Pearson’s correlation The applied tests, the n-numbers and exact p-values for each comparison are presented in the Results section and in the figure legends.
Extended Data Figure 1-1
Staining with two anti-CB1R antibodies is overlapping in WT, but totally absent from CB1RKO mice; rate of tissue shrinkage due to electron beam irradiation. A, Triple-color confocal images show CB1R staining in the hippocampal CA1 region of a WT mouse with a rabbit (green) and a goat (magenta) antibody. Cell nuclei are stained with DAPI (blue). B, Staining with both antibodies are completely absent in CB1R-KO mice. Scale bar: 100 µm (upper panels) and 30 µm (lower panels). C, Rates of tissue shrinkage on 100-nm-thick (left) and 200-nm-thick (right) sections under 15 min of electron beam irradiation. Black bars represent interquartile ranges, black dots mark median values. Download Figure 1-1, TIF file.
Extended Data Figure 1-2
3D models of the reconstructed mitochondrial volumes from the hippocampal CA1 region. Top three rows show presynaptic mitochondria from regular spiking basket cell boutons, while the bottom three rows from fast-spiking basket-cell boutons. Mitochondrial outer membrane is red, inner boundary membrane is turquoise, CM is green. CM/MV, CM area/mitochondrial volume; CSF, crista shape factor. The models are not displayed on the same magnification. Download Figure 1-2, TIF file.
Extended Data Figure 3-1
Distribution of active zone sizes of the glutamatergic boutons in the tomographic and STORM measurements, and correlations of performance-determining mitochondrial features with synapse size. A, Active zone area distribution of the glutamatergic boutons examined with serial EM and electron tomography. B, The volume, CM density, and crista shape factor of presynaptic mitochondria are all strongly correlated to synapse size (R = 0.71, p = 0.0005; R = 0.77, p = 0.0001; and R = 0.65, p = 0.0026, respectively). Blue dots represent values from individual mitochondria, regression lines are red. C, Active zone area distribution of the glutamatergic boutons examined with CLSM and STORM super-resolution microscopy. All syn., all examined synaptic boutons; Mito. syn., synaptic boutons with mitochondria; LP syn., LP synaptic boutons; HP syn., HP synaptic boutons. Blue dots represent values from individual boutons, magenta rectangles represent interquartile ranges, deep-magenta lines mark median values. Red lines show values dividing HP and LP synaptic boutons. D The CytC-labeled volume and SLP density are both strongly correlated to synapse size (R = 0.77, p < 0.00001; R = 0.60, p = 0.00002, respectively). Blue dots represent values from individual mitochondria, regression lines are red. Download Figure 3-1, TIF file.
Extended Data Figure 3-2
3D models of the reconstructed mitochondrial volumes from the dentate gyrus. Top three rows show presynaptic mitochondria from LP glutamatergic boutons, while the bottom three rows from HP ones. Mitochondrial outer membrane is red, inner boundary membrane is turquoise, CM is green. CM/MV, CM area/mitochondrial volume; CSF, crista shape factor. The models are not displayed on the same magnification. Download Figure 3-2, TIF file.
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