Figure S1.
Quantitative analysis of microglia-synapse contacts and high-resolution imaging of dendrodendritic synapses in the OB by SBF-SEM. (A) Representative SBF-SEM image of microglia (green) in the GCL of the adult OB (n = 22 cells from three mice, pooled from five independent experiments). Yellow arrows, green arrowheads, and yellow arrowheads indicate long endoplasmic reticulum, extracellular space, and lysosomes, respectively. 3D reconstruction image of a synapse in the boxed area is shown in D. (B) 3D reconstruction of microglia in the GCL (semi-transparent green) shown in A and its associated synapses. While spines on distal, proximal, and basal dendrites of granule cells in the GCL form synapses to receive excitatory inputs from axonal terminals of mitral/tufted (M/T) cells in the OB and projection neurons in higher olfactory centers, those on apical dendrites in the EPL form dendrodendritic synapses with secondary dendrites of M/T cells (see Figs. 1 and S1 G). Presynaptic structures of putative M/T or centrifugal axons and spines of granule cells in the GCL are indicated by yellow and blue, respectively. (C–E) Representative three-dimensional reconstruction of small (C) and large (D) microglia-synapse contacts and distribution of microglia-synapse contact areas in the GCL (E) and corresponding gaussian fitting curves (E, trimodal distribution, AIC = 169.9, also see Materials and methods). The trimodal distribution consists of two distributions for small contacts (light orange and orange) and one distribution for large contacts (blue) and shows the minimum Akaike’s information criterion (AIC) in fitting curve analysis (E, see Materials and Methods). Arrows in E under the x axis indicate the center value (Xcenter) of each fitting curve (small contact-1, light orange, Xcenter = 0.070 µm2; small contact-2, orange, Xcenter = 2.2 × 10−8 µm2; large contact, magenta, Xcenter = 0.81 µm2). Most of the contacts between microglia and synapses are classified as small contacts (C and E). At large microglia-synapse contacts, microglial processes contained lysosomes and were wrapping synaptic structures (A and D), unlike the small contact-forming microglial processes (C). Microglia, presynapses, spines, and contact area in B–D are shown in green, yellow, blue, and red, respectively. 22 cells from three mice (pooled from five independent experiments) are analyzed (E). (F) Experimental scheme of sample preparation and image acquisition of microglia in the adult OB for high-resolution SBF-SEM imaging. Also see Materials and methods. (G) Representative SBF-SEM images of dendrodendritic synapses between secondary dendrites of M/T (blue) cells and dendritic spines of granule cells (G, pink) in the EPL of the adult OB (n = 23 cells from three mice, pooled from eight independent experiments). Magnified image of boxed area (right) shows 40 nm synaptic vesicles in both M/T and granule cell spines and postsynaptic density in the granule cell spines (arrowheads). Scale bars: A and B, 2 µm; G, 400 nm.

Quantitative analysis of microglia-synapse contacts and high-resolution imaging of dendrodendritic synapses in the OB by SBF-SEM. (A) Representative SBF-SEM image of microglia (green) in the GCL of the adult OB (n = 22 cells from three mice, pooled from five independent experiments). Yellow arrows, green arrowheads, and yellow arrowheads indicate long endoplasmic reticulum, extracellular space, and lysosomes, respectively. 3D reconstruction image of a synapse in the boxed area is shown in D. (B) 3D reconstruction of microglia in the GCL (semi-transparent green) shown in A and its associated synapses. While spines on distal, proximal, and basal dendrites of granule cells in the GCL form synapses to receive excitatory inputs from axonal terminals of mitral/tufted (M/T) cells in the OB and projection neurons in higher olfactory centers, those on apical dendrites in the EPL form dendrodendritic synapses with secondary dendrites of M/T cells (see Figs. 1 and S1 G). Presynaptic structures of putative M/T or centrifugal axons and spines of granule cells in the GCL are indicated by yellow and blue, respectively. (C–E) Representative three-dimensional reconstruction of small (C) and large (D) microglia-synapse contacts and distribution of microglia-synapse contact areas in the GCL (E) and corresponding gaussian fitting curves (E, trimodal distribution, AIC = 169.9, also see Materials and methods). The trimodal distribution consists of two distributions for small contacts (light orange and orange) and one distribution for large contacts (blue) and shows the minimum Akaike’s information criterion (AIC) in fitting curve analysis (E, see Materials and Methods). Arrows in E under the x axis indicate the center value (Xcenter) of each fitting curve (small contact-1, light orange, Xcenter = 0.070 µm2; small contact-2, orange, Xcenter = 2.2 × 10−8 µm2; large contact, magenta, Xcenter = 0.81 µm2). Most of the contacts between microglia and synapses are classified as small contacts (C and E). At large microglia-synapse contacts, microglial processes contained lysosomes and were wrapping synaptic structures (A and D), unlike the small contact-forming microglial processes (C). Microglia, presynapses, spines, and contact area in B–D are shown in green, yellow, blue, and red, respectively. 22 cells from three mice (pooled from five independent experiments) are analyzed (E). (F) Experimental scheme of sample preparation and image acquisition of microglia in the adult OB for high-resolution SBF-SEM imaging. Also see Materials and methods. (G) Representative SBF-SEM images of dendrodendritic synapses between secondary dendrites of M/T (blue) cells and dendritic spines of granule cells (G, pink) in the EPL of the adult OB (n = 23 cells from three mice, pooled from eight independent experiments). Magnified image of boxed area (right) shows 40 nm synaptic vesicles in both M/T and granule cell spines and postsynaptic density in the granule cell spines (arrowheads). Scale bars: A and B, 2 µm; G, 400 nm.

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