3D Ultrastructural Study of Synapses in the Human Entorhinal Cortex

Abstract

The entorhinal cortex (EC) is a brain region that has been shown to be essential for memory functions and spatial navigation. However, detailed three-dimensional (3D) synaptic morphology analysis and identification of postsynaptic targets at the ultrastructural level have not been performed before in the human EC. In the present study, we used Focused Ion Beam/Scanning Electron Microscopy to perform a 3D analysis of the synapses in the neuropil of medial EC in layers II and III from human brain autopsies. Specifically, we studied synaptic structural parameters of 3561 synapses, which were fully reconstructed in 3D. We analyzed the synaptic density, 3D spatial distribution, and type (excitatory and inhibitory), as well as the shape and size of each synaptic junction. Moreover, the postsynaptic targets of synapses could be clearly determined. The present work constitutes a detailed description of the synaptic organization of the human EC, which is a necessary step to better understand the functional organization of this region in both health and disease.

Keywords: FIB-SEM; cerebral cortex; electron microscopy; neuropil; synaptology.

Figure 1

Coronal sections of the human hippocampal formation. (A, B) Low-power photographs showing the human EC (boxed areas). (C, D) Higher magnification of the boxed areas in A and B, to show the laminar pattern of EC (layers I to VI are indicated). Sections are stained for Nissl (A, C) and immunostained for anti-NeuN (B, D). WM, white matter. Scale bar (in D): 3 mm in panels A and B; 600 μm in panels C and D.

Figure 2

Correlative light/electron microscopy analysis of layer II and III of the EC. The delimitation of layers is based on the staining pattern of 1 μm-thick semithin section, stained with toluidine blue (A), which is adjacent to the block for FIB/SEM imaging (B). (B) SEM image to illustrate the block surface with trenches made in the neuropil (three per layer). Asterisks in A and B point to the same blood vessel, showing that the exact location of the region of interest was accurately determined. (C) Serial image obtained by FIB/SEM from layer II showing the neuropil, with two synapses indicated as examples of AS (green arrow) and SS (red arrow). Synapse classification was based on the examination of the full sequence of serial images; an SS can be visualized in D–H, and an AS in I–M. Scale bar (in M): 40 μm in A; 60 μm in B; 1000 nm in C; 1300 nm in D–M.

Figure 3

Screenshot 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). The 3D windows (B–D) show the three orthogonal planes and the 3D reconstruction of AS (green) and SS (red) segmented synapses (B), the reconstructed synapses (C), and the computed SAS for each reconstructed synapse (in yellow; D). Scale bar (in B): 7 μm in B–D.

Figure 4

Graph showing the mean AS SAS area (A), and the frequency distribution plots of AS SAS area (B), and perimeter (C), in layers II and III of the EC. A different color corresponds to each analyzed case, as denoted in the key (A). Statistical comparisons between layers showed differences in the size of the AS area (A) (asterisk; MW, P = 0.03), as well as in the frequency distribution of the area (B) and perimeter (C) (KS, P < 0.0001; indicated with an asterisk).

Figure 5

Proportions of the different synaptic shapes in layers II (A, B) and III (C, D) of the EC. (A) Shape proportions of the AS in layer II. (B) Proportions of AS and SS belonging to each shape type in layer II. (C) Shape proportions of the AS in layer III. A significantly fewer number of macular AS and a significantly higher number of perforated AS were found compared with layer II (asterisks; χ², P < 0.0001). (D) Proportions of AS and SS belonging to each shape type in layer III. In both layer II and layer III, the horseshoe-shaped synapses were significantly more frequent among SS than AS (asterisks; χ², P < 0.0001).

Figure 6

Analysis of the postsynaptic target distribution in layers II (A, B) and III (C, D) of the EC. (A) Graph showing the proportions of AS and SS corresponding to each postsynaptic target in layer II. (B) Schematic representation of the distribution of AS and SS on different postsynaptic targets in layers II. (C) Proportions of AS and SS corresponding to each postsynaptic target in layer III. In both layer II and layer III, the AS showed a preference for spine heads, whereas the SS showed a preference for dendritic shafts (asterisks; χ², P < 0.0001). (D) Schematic representation of the distribution of AS and SS on different postsynaptic targets in layer III. (B, D) Percentages of postsynaptic targets are indicated, showing —from left to right— the most frequent type (AS on dendritic shafts) to the least frequent type (SS on spine necks). (A–D) Synapses on spines have been sub-classified into those that are established on the spine head and those established on the neck.

Figure 7

Schematic representation of single and multiple synapses on dendritic spine heads in layers II and III of the EC. Percentages of each type are indicated. Synapses on the necks and other combinations that were rarely found (less than 1%) have not been included. AS have been represented in green and SS in red.