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. 2021 Jan 28:14:573944.
doi: 10.3389/fncel.2020.573944. eCollection 2020.

Characterization of Subcellular Organelles in Cortical Perisynaptic Astrocytes

Amina Aboufares El Alaoui  1   2 , Molly Jackson  2 , Mara Fabri  1 , Luisa de Vivo  2 , Michele Bellesi  2
Affiliations

Characterization of Subcellular Organelles in Cortical Perisynaptic Astrocytes

Amina Aboufares El Alaoui et al. Front Cell Neurosci. .

Abstract

Perisynaptic astrocytic processes (PAPs) carry out several different functions, from metabolite clearing to control of neuronal excitability and synaptic plasticity. All these functions are likely orchestrated by complex cellular machinery that resides within the PAPs and relies on a fine interplay between multiple subcellular components. However, traditional transmission electron microscopy (EM) studies have found that PAPs are remarkably poor of intracellular organelles, failing to explain how such a variety of PAP functions are achieved in the absence of a proportional complex network of intracellular structures. Here, we use serial block-face scanning EM to reconstruct and describe in three dimensions PAPs and their intracellular organelles in two different mouse cortical regions. We described five distinct organelles, which included empty and full endosomes, phagosomes, mitochondria, and endoplasmic reticulum (ER) cisternae, distributed within three PAPs categories (branches, branchlets, and leaflets). The majority of PAPs belonged to the leaflets category (~60%), with branchlets representing a minority (~37%). Branches were rarely in contact with synapses (<3%). Branches had a higher density of mitochondria and ER cisternae than branchlets and leaflets. Also, branches and branchlets displayed organelles more frequently than leaflets. Endosomes and phagosomes, which accounted for more than 60% of all the organelles detected, were often associated with the same PAP. Likewise, mitochondria and ER cisternae, representing ~40% of all organelles were usually associated. No differences were noted between the organelle distribution of the somatosensory and the anterior cingulate cortex. Finally, the organelle distribution in PAPs did not largely depend on the presence of a spine apparatus or a pre-synaptic mitochondrion in the synapse that PAPs were enwrapping, with some exceptions regarding the presence of phagosomes and ER cisternae, which were slightly more represented around synapses lacking a spine apparatus and a presynaptic mitochondrion, respectively. Thus, PAPs contain several subcellular organelles that could underlie the diverse astrocytic functions carried out at central synapses.

Keywords: astrocyte; electron microscopy; mouse; perisynaptic astrocytic processes; synapse.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A) Example of region of interest (ROI) showing an axo-spinous synapse with perisynaptic astrocytic process (PAP; blue), axon terminal (AxT), spine head (SpH), axon-spine interface (ASI, red), and relative 3D reconstruction. Scale bar: 400 nm. (B,C) Average ROI and PAP volumes, respectively. (D) Ratio between PAP and ROI volumes. (E) Average ASI size. (F) Percentage of synapses contacting the spine (spine+) and the synaptic cleft (ASI+). (G) Synaptic coverage as measured by the size of the interfacing surface between the PAP and the spine head. In panels (B–G) AC and SS refer to anterior cingulate and somatosensory cortex (paired t-test, n = 4).
Figure 2
Figure 2
(A) Top: PAP composition in our dataset (n = 863, four animals). Bottom: examples of the leaflet, branchlet, and branch depicted in blue and surrounding an axo-spinous synapse. The scale bar is 350 nm for all panels. (B) Average surface-to-volume ratio values in AC and SS (mixed-effects analysis, n = 4). Lines indicate significant pairwise comparisons (p < 0.05, Bonferroni corrected).
Figure 3
Figure 3
(A–D) Examples of empty endosomes (EE), full endosomes (FE), mitochondria (MT), ER-cisternae (ER). (E) Axon (a) being engulfed by a PAP caught in several consecutive z-steps showing the formation of the phagosome (PH). Scale bar is 300 nm for all panels.
Figure 4
Figure 4
(A,B) Quantification of PAP subcellular organelle distribution (A) and percentage of perisynaptic astrocytic processes (PAPS) displaying at least one organelle (B). All data are segregated for leaflets, branchlets, and branches (n = 4). Empty endosomes (EE), full endosomes (FE), phagosome (PH), mitochondria (MT), and ER cisternae (ER) for all panels. *Indicates significant pairwise comparisons (mixed-effects analysis, p < 0.05, Bonferroni corrected) relative to leaflets, whereas, the #indicates significant pairwise comparisons (mixed-effects analysis, p < 0.05, Bonferroni corrected) relative to branchlets.
Figure 5
Figure 5
(A) Correlation matrix indicating the strength of the correlation between all the described organelles in leaflets, branchlets, and branches separately. (B) Correlation matrix indicating strength (left) and significance (right) of the correlation between all the described organelles in all the PAPs pooled together. Only significant correlations after Bonferroni’s correction for multiple comparisons are displayed. Empty endosomes (EE), full endosomes (FE), phagosome (PH), mitochondria (MT), and ER cisternae (ER) for all panels.
Figure 6
Figure 6
(A) Subcellular organelle distribution in PAPs surrounding synapses with (left) or without (middle) spine apparatus and relative statistical analysis (right, rmANOVA followed by post hoc Bonferroni tests, n = 4). (B) Subcellular organelle distribution in PAPs surrounding synapses with (left) or without (middle) presynaptic mitochondrion and relative statistical analysis (right, rmANOVA followed by post hoc Bonferroni tests n = 4). Empty endosomes (EE), full endosomes (FE), phagosome (PH), mitochondria (MT), and ER cisternae (ER) for all panels.
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