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Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy - PubMed

  • ️Mon Jan 01 2024

Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy

Nicolás Cano-Astorga et al. Front Neuroanat. 2024.

Abstract

The brain contains thousands of millions of synapses, exhibiting diverse structural, molecular, and functional characteristics. However, synapses can be classified into two primary morphological types: Gray's type I and type II, corresponding to Colonnier's asymmetric (AS) and symmetric (SS) synapses, respectively. AS and SS have a thick and thin postsynaptic density, respectively. In the cerebral cortex, since most AS are excitatory (glutamatergic), and SS are inhibitory (GABAergic), determining the distribution, size, density, and proportion of the two major cortical types of synapses is critical, not only to better understand synaptic organization in terms of connectivity, but also from a functional perspective. However, several technical challenges complicate the study of synapses. Potassium ferrocyanide has been utilized in recent volume electron microscope studies to enhance electron density in cellular membranes. However, identifying synaptic junctions, especially SS, becomes more challenging as the postsynaptic densities become thinner with increasing concentrations of potassium ferrocyanide. Here we describe a protocol employing Focused Ion Beam Milling and Scanning Electron Microscopy for studying brain tissue. The focus is on the unequivocal identification of AS and SS types. To validate SS observed using this protocol as GABAergic, experiments with immunocytochemistry for the vesicular GABA transporter were conducted on fixed mouse brain tissue sections. This material was processed with different concentrations of potassium ferrocyanide, aiming to determine its optimal concentration. We demonstrate that using a low concentration of potassium ferrocyanide (0.1%) improves membrane visualization while allowing unequivocal identification of synapses as AS or SS.

Keywords: 3D-electron microscopy; FIB-SEM; VGAT; cerebral cortex; excitatory and inhibitory synapses; potassium ferrocyanide; ultrastructure.

Copyright © 2024 Cano-Astorga, Plaza-Alonso, Turegano-Lopez, Rodrigo-Rodríguez, Merchan-Perez and DeFelipe.

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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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1

Images obtained by FIB/SEM showing the neuropil of the somatosensory cortex of mice. The sample was treated with 1% potassium ferrocyanide and not permeabilized with liquid nitrogen. (A) Low-magnification FIB/SEM image from a stack to illustrate the good quality of the EM image. (B–I) Various examples of synapses on different dendritic spines, which typically establish AS. However, in this material, AS are challenging to identify because the postsynaptic densities are relatively thin. Scale bar (in I) indicates 468 nm for (A), and 315 nm for (B–I).

Figure 2
Figure 2

Images obtained by FIB/SEM showing the neuropil of the somatosensory cortex of mice. The sample was treated with 0.1% potassium ferrocyanide and not permeabilized with liquid nitrogen. In (A), an example of a low-magnification FIB/SEM image from a stack highlights AS and SS synapses with green and red arrowheads, respectively. (B–I) Various serial sections at higher magnification of the same SS (red arrow). (J–M) Various serial sections of the same AS (green arrow) from the image stack. The section number is indicated in the top right-hand corner of each image. Scale bar (in M) indicates 468 nm for (A), and 315 nm for (B–M).

Figure 3
Figure 3

VGAT-positive axon terminals in a single SEM image from tissue treated with 0.1% potassium ferrocyanide and permeabilized with liquid nitrogen. (A) A viewer trench was excavated using FIB milling on the surface of a brain section. The asterisks indicate the interface between the embedding medium (Araldite) and the brain tissue. (B) Higher magnification of the boxed area in A, showing the neuropil. Axon terminals 1 and 2 establish synapses with a cell body that becomes more apparent through the serial sections (see Figure 5). The red arrow indicates a VGAT-positive terminal forming an SS (magnified in C), AT indicates another VGAT-positive terminal (magnified in D), and double asterisks indicate neuropil magnified in E. (C) Higher magnification of the VGAT-positive terminal forming an SS (red arrow), and the VGAT-positive terminal (1) forming an SS with the cell somata in further serial sections shown in B. (D) Example of a VGAT-positive terminal in which the intensity of the staining decreases as the distance to the surface of the section increases. (E) Example of a VGAT-negative terminal forming an AS. Scale bar (in E) indicates 5 μm for (A), 800 nm for (B) and 370 nm for (C–E).

Figure 4
Figure 4

AS (A) and SS (B) identification from FIB/SEM images in VGAT-immunostained tissue permeabilized with liquid nitrogen and treated with 0.1% potassium ferrocyanide. Sequence of FIB-SEM serial images of an AS (C–J) and an SS (K–R). Numbers on the top right of each panel indicate the number of each section from the stack of FIB/SEM images. Synapse classification was performed based on the thickness of the PSD and the VGAT-positive labeling of the presynaptic terminal through the examination of full sequences of serial images. Green arrows indicate the beginning (C) and the end (J) of the AS. Red arrows indicate the beginning (K) and the end (R) of the SS. Note the VGAT-positive presynaptic staining on the SS. Scale bar (in R) indicates 250 nm for (A,B), and 500 nm for (C–R).

Figure 5
Figure 5

Identification of SS on neuronal soma from FIB/SEM images in VGAT-immunostained tissue permeabilized with liquid nitrogen and treated with 0.1% potassium ferrocyanide. White asterisk indicates the neuronal soma. (B–I) sequence of FIB-SEM serial images of an SS established on the neuronal soma. Numbers on the top right of each panel indicate the number of each section from the stack of FIB/SEM images. Red arrows indicate the beginning (B) and the end (I) of the SS. Scale bar (in I) indicates 520 nm for (A), and 500 nm for (B–I).

Figure 6
Figure 6

Identification and segmentation of synapses. (A–D) Screenshots of the EspINA software user interface. (A) In the main window, the sections are viewed through the xy plane (as obtained by FIB/SEM microscopy). The other two orthogonal planes, yz and xz, are also shown in adjacent windows (on the right). (B) 3D view showing the three orthogonal planes and the 3D reconstruction of AS (green) and SS (red) segmented synaptic junctions. (C) 3D reconstructed synaptic junctions of both AS and SS, displayed using the same colors as in B. (D) 3D reconstructed synaptic junctions of SS. Scale bar (in D) indicates 2 μm for (B–D).

Figure 7
Figure 7

AS (A) and SS (B) identification from FIB/SEM images in VGAT-immunostained tissue permeabilized with liquid nitrogen and not treated with potassium ferrocyanide. FIB-SEM serial images of an AS (C–J) and an SS (K–R) are shown. Numbers on the top right of each panel indicate the number of each section from the stack of FIB/SEM images. Synapse classification was performed based on the thickness of the PSD and the VGAT-positive labeling of the presynaptic terminal through the examination of full sequences of serial images. Green arrows indicate the beginning (C) and the end (J) of the AS. Red arrows indicate the beginning (K) and the end (R) of the SS. Note the VGAT-positive presynaptic staining on the SS. Scale bar (in R) indicates 230 nm for (A), 270 nm for (B), 253 nm for (C–J), and 540 nm for (K–R). Taken from unpublished material from Turégano-López et al. (2021).

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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the following Grants: PID2021-127924NB-I00 funded by MCIN/AEI/10.13039/501100011033; CSIC Interdisciplinary Thematic Platform - Cajal Blue Brain (PTI-BLUEBRAIN; Spain); and CIBERNED, ISCIII, CB06/05/0066. Research Fellowships funded by MCIN/AEI/10.13039/501100011033 for NC-A. (PRE2019-089228) and SP-A. (FPU19/00007).