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. 2020 Mar 14;30(3):1887-1901.
doi: 10.1093/cercor/bhz211.

Size, Shape, and Distribution of Multivesicular Bodies in the Juvenile Rat Somatosensory Cortex: A 3D Electron Microscopy Study

M Turegano-Lopez  1 , A Santuy  1 , J DeFelipe  1   2   3 , A Merchan-Perez  1   3   4
Affiliations

Size, Shape, and Distribution of Multivesicular Bodies in the Juvenile Rat Somatosensory Cortex: A 3D Electron Microscopy Study

M Turegano-Lopez et al. Cereb Cortex. .

Abstract

Multivesicular bodies (MVBs) are membrane-bound organelles that belong to the endosomal pathway. They participate in the transport, sorting, storage, recycling, degradation, and release of multiple substances. They interchange cargo with other organelles and participate in their renovation and degradation. We have used focused ion beam milling and scanning electron microscopy (FIB-SEM) to obtain stacks of serial sections from the neuropil of the somatosensory cortex of the juvenile rat. Using dedicated software, we have 3D-reconstructed 1618 MVBs. The mean density of MVBs was 0.21 per cubic micron. They were unequally distributed between dendrites (39.14%), axons (18.16%), and nonsynaptic cell processes (42.70%). About one out of five MVBs (18.16%) were docked on mitochondria, representing the process by which the endosomal pathway participates in mitochondrial maintenance. Other features of MVBs, such as the presence of tubular protrusions (6.66%) or clathrin coats (19.74%) can also be interpreted in functional terms, since both are typical of early endosomes. The sizes of MVBs follow a lognormal distribution, with differences across cortical layers and cellular compartments. The mean volume of dendritic MVBs is more than twice as large as the volume of axonic MVBs. In layer I, they are smaller, on average, than in the other layers.

Keywords: 3D electron microscopy; FIB-SEM; cerebral cortex; endosomal pathway; multivesicular bodies.

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Figures

Figure 1
Figure 1
Schematic representation of the role of MVBs within the endosomal pathway. Endosomes originate by endocytosis, and intraluminal vesicles accumulate inside them as they mature. Cargo (red bars) transported by MVBs can come from the plasma membrane or from organelles such as mitochondria, as is the case in this example. The content of MVBs can be recycled, released to the extracellular space as exosomes, or degraded by fusion with lysosomes.
Figure 2
Figure 2
3D view of MVBs in a stack of images. MVBs are colored either by their size or by the compartment where they are located.
Figure 3
Figure 3
Examples of different types and locations of MVBs. A, B, C. Serial electron microscopy images showing a spheroidal MVB (arrows) that is isolated in the cytoplasm. Numbers in the top right-hand corner indicate the position of each micrograph in the series. Arrowhead in C points to a membrane thickening possibly representing a clathrin coat (see text for further explanation). D, E, F. Another series of images showing an MVB docked on a mitochondrion. The MVB (arrows) has several tubular expansions (t). Arrowhead in E shows the contact point between the mitochondrion and the MVB. G. Three-dimensional reconstruction of the same MVB and mitochondrion shown in D–F. H, I, J. MVBs located in a dendritic spine, an excitatory axon, and an inhibitory axon, respectively. Note that the postsynaptic density (PSD) is thick in excitatory synapses shown in H and I, while it is thin in the inhibitory synapse shown in J. Scale bar shown in J is 520 nm in A to H; 300 nm in I; 370 nm in J.
Figure 4
Figure 4
Density of MVBs in the neuropil. A Density of MVBs (MVBs/μm3) in the six cortical layers (mean + sem). B Scatterplot showing the lack of correlation between the density of MVBs and the density of synapses in the six cortical layers (R2 = 0.1429).
Figure 5
Figure 5
Distribution of MVBs across cortical layers and subcellular compartments. A Percentage of MVBs located in dendrites, axons, and nonsynaptic fibers across cortical layers. *In layer I, the proportions of dendritic and axonic MVBs were lower and higher, respectively, than in any other layer (χ2, P < 0.05). B Percentage of MVBs located in excitatory and inhibitory axons. C Ratio between the percentage of MVBs in each compartment (nonsynaptic cell processes, dendrites, excitatory, and inhibitory axons) and the volume fraction occupied by each compartment. Values over 1 indicate that MVBs are relatively more concentrated in that compartment, while values below 1 indicate that MVBs are less concentrated.
Figure 6
Figure 6
Distribution of MVBs docked on mitochondria and isolated in the cytoplasm. A. Distribution of MVBs docked on mitochondria in dendrites, axons, and nonsynaptic cell processes. B. Percentage of docked and isolated MVBs in the six cortical layers. *The number of MVBs docked on mitochondria in layer II was lower than in any other layer (χ2, P < 0.05). C, D, E. Proportions of MVBs docked on mitochondria and isolated in the cytoplasm within the three subcellular compartments.
Figure 7
Figure 7
Example of an MVB with tubular protrusions. A. Serial images of an MVB (lightly stained) that shows tubular protrusions. An invagination of its membrane is also visible (arrows). The number in the top-right corner indicates the position of each micrograph in the series. Section thickness was 20 nm. Scale bar = 280 nm. B. 3D reconstruction of the MVB shown in A, from three different points of view. The arrow indicates the invagination of the membrane.
Figure 8
Figure 8
Distribution of spheroidal MVBs and MVBs with tubular protrusions. A. Distribution of MVBs with tubules in dendrites, axons, and nonsynaptic fibers. B Percentage of MVBs with tubules and without tubules (spheroidal) in the six cortical layers. C, D, E. Proportion of MVBs with and without tubules (spheroidal) in dendrites, axons, and nonsynaptic cell processes.
Figure 9
Figure 9
Size of MVBs across cortical layers. A. Size of MVBs in the six cortical layers (mean and standard error of the mean). *The mean size of MVBs in layer I was smaller than in the other cortical layers (KW, P < 0.0001). B. Size of the different morphological types of MVBs. *Differences in size were statistically significant (KW, P < 0.05) except between the two groups of MVBs with tubules (docked on mitochondria and isolated). C. Frequency histogram of the size of MVBs and the corresponding best-fit log-normal distribution (μ = −5.1375; σ = 0.9173). D. Frequency distribution of the size of MVBs in each cortical layer.
Figure 10
Figure 10
Size of MVBs in different subcellular compartments and cortical layers. A. Size of MVBs in different compartments (dendrites, axons, and nonsynaptic cell processes). *The mean size of dendritic MVBs was larger than axonic and nonsynaptic MVBs (KW, P < 0.0001). B. Frequency histograms of the sizes of MVBs in the different compartments. C. Size of dendritic MVBs across cortical layers. *Dendritic MVBs were smaller in layer I than in other layers except layer IV (KW, P < 0.05). D. Size of axonic MVBs across layers. *Axonic MVBs in layer VI were larger than MVBs in layers I, II, and IV (KW, P < 0.05). E. Size of MVBs in nonsynaptic cell processes across the six cortical layers. Sizes are represented as mean volume (μm3) plus sem.
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