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Beyond counts and shapes: studying pathology of dendritic spines in the context of the surrounding neuropil through serial section electron microscopy - PubMed

  • ️Tue Jan 01 2013

Review

Beyond counts and shapes: studying pathology of dendritic spines in the context of the surrounding neuropil through serial section electron microscopy

M Kuwajima et al. Neuroscience. 2013.

Abstract

Because dendritic spines are the sites of excitatory synapses, pathological changes in spine morphology should be considered as part of pathological changes in neuronal circuitry in the forms of synaptic connections and connectivity strength. In the past, spine pathology has usually been measured by changes in their number or shape. A more complete understanding of spine pathology requires visualization at the nanometer level to analyze how the changes in number and size affect their presynaptic partners and associated astrocytic processes, as well as organelles and other intracellular structures. Currently, serial section electron microscopy (ssEM) offers the best approach to address this issue because of its ability to image the volume of brain tissue at the nanometer resolution. Renewed interest in ssEM has led to recent technological advances in imaging techniques and improvements in computational tools indispensable for three-dimensional analyses of brain tissue volumes. Here we consider the small but growing literature that has used ssEM analysis to unravel ultrastructural changes in neuropil including dendritic spines. These findings have implications in altered synaptic connectivity and cell biological processes involved in neuropathology, and serve as anatomical substrates for understanding changes in network activity that may underlie clinical symptoms.

Keywords: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 3D; 3D reconstruction; AD; ATLUM; Alzheimer’s disease; Aβ; CCD; DNMS; DS; Down’s syndrome; EM; FIB-SEM; GABA; MPTP; MSB; MSNs; MTLE; NSB; PSD; SA; SBFSEM; SEM; STEM; TEM; TEM camera array; TEMCA; amyloid β; automatic tape-collecting lathe ultramicrotome; charge-coupled device; connectome; delayed nonmatching-to-sample; electron microscopy; focused ion beam-SEM; medium spiny neurons; mesial temporal lobe epilepsy; multi-synaptic bouton; neuropil; non-synaptic bouton; pathology; postsynaptic density; scanning electron microscopy; scanning transmission electron microscopy; serial block face SEM; serial section electron microscopy; spine apparatus; ssEM; three-dimensional; transmission electron microscopy; ultrastructure; vGluT; vesicular glutamate transporter; γ-aminobutylic acid.

Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

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Figures

Figure 1
Figure 1

An example of the rat hippocampal dentate gyrus tissue embedded into a “Chien mold” block for accurate targeting of the region of interest for serial EM imaging. (Figures from Kuwajima et al., 2012) A: A parasagittal slice (70 μm thickness) of the hippocampal formation from a perfusionfixed rat. Area CA1 and the dentate gyrus (DG) are indicated. A’: The same slice as in A, with a superimposed image of the dentate tissue embedded in the epoxy block shown in B. B: The “Chien mold” was used to embed the dentate tissue dissected from the slice shown in A and A’ into epoxy resin. Note that the tissue can be cut from two faces of the block (Faces 1 and 2). A paper label identifying the block (“LE108RL1”) was embedded into resin along with the tissue. C: Left – A diagram indicating the orientation of the tissue in the block shown in B. The “Face 1” is perpendicular to the granule cell layer (gray spheres) and the “Face 2”, and dendrites of these cells (gray curved lines in the molecular layer) are cut longitudinally in this plane. Test-thin sections taken from Face 1 are used to measure the distance from the cell layer. The “Face 2” is parallel to the cell layer, and therefore the dendrites are cut in cross-sections. Right – The same block after trimming the final block face (gray trapezoid area) for serial sectioning. In this example, serial sections are cut from the Face 2 at 125 μm from the top of the granule cell layer to target the middle molecular layer. D: A low-magnification SEM image of a segment of serial sections on a single Pioloformcoated grid. Large capillaries are evident in each section. E: For serial EM imaging, our laboratory uses a field emission scanning EM (FE SEM) that is equipped with a secondary electron detector (s), an in-lens detector (i), and a retractable detector for transmitted electrons (STEM detector; t). The column (g) contains the gun assembly and objective lenses. The specimen chamber door (c) slides open outward with the stage. The SEM is controlled through the SEM interface and console (keyboard and joysticks can be seen in front of the monitors), or the integrated large-field image acquisition interface. Inset – TV camera view of the specimen chamber showing the arrangement of the final lens, STEM detector, and sample holder clipped onto the stage. The distance between the final lens and the specimen is 4-5 mm.

Figure 2
Figure 2

A and B: Multisynaptic spines in the severe case of mesial temporal lobe epilepsy. A – Electron micrograph of a multisynaptic spine (yellow) with four synapses (red) and seven axons (green) that eventually synapse with this spine visible on this one section. A’ – 3D reconstruction of the same multisynaptic spine (yellow) showing all nine of its presynaptic axonal boutons (multicolored) and seven of its PSDs (eight and nine are hidden). B – In the severe case, the astroglial processes (turquoise) were loosely associated around the periphery of many presynaptic axons (multicolored) making synapses on a single multisynaptic spine (yellow). From Witcher et al. (2010). C: 3D reconstruction of extracellular deposits of amyloid β (Aβ) in the hippocampal stratum radiatum of a transgenic mouse model of Alzheimer’s disease. Aβ deposits (dots representing gold particles) form continuous bundles of fibrils varying in size and direction, some of which are associated with apical dendrites (ap1-ap4). The reconstruction was made from ~100 serial sections with post-embedding immunogold labeling for Aβ. Scale bar = 0.5 μm. Reprinted from Journal of Alzheimer’s Disease, Nuntagij P et al. (2009), Copyright 2009, with permission from IOS Press. D: Electron micrograph of a neurite diverticulum, containing a long, curved mitochondrion (arrows) located adjacent to a curved microtubule. D’ – 3D reconstruction of mitochondria in the same neurite demonstrates that the mitochondrion in the main axis (blue) has a normal cylindrical shape while the mitochondrion in the diverticulum (red) is convoluted and branching. Scale bar = 0.5 μm. Figures were reprinted from Fiala et al. (2007), Copyright 2007, with permission from Springer (pending). E: 3D reconstruction of multi-synaptic boutons (MSB; blue) with the spines (yellow) and PSDs (red) in the outer molecular layer of the monkey hippocampal dentate gyrus. Both MSBs form a synapse with one small spine with macular PSD, and another with a large mushroom spine with perforated PSD. Perforated PSD can be in a single piece with perforation (left), or completely segmented (right). Reprinted from The Journal of Neuroscience, Hara et al. (2011), Copyright 2011, with permission from the Society for Neuroscience. F: Representative 3D reconstructions of two giant mossy fiber boutons (GB; green and blue) contacting a thorny excrescence (gray) with multiple PSDs (red) in the mouse hippocampal area CA3. Scale bar = 1 μm. Reprinted from The Journal of Comparative Neurology, Popov et al. (2011), Copyright 2011, with permission from John Wiley & Sons. G-I: Afferent specific changes in axospinous synapses in the striatum of parkinsonian monkeys. G – Electron micrograph of a vGluT2-positive bouton (T) forming synapses with a dendritic shaft (D) and a spine (Sp). G’ – 3D reconstruction of the same synapses, viewed from two angles, illustrating the spatial relationship of the bouton (T) with its postsynaptic partners (D and Sp), as well as the synapses (PSD1 and PSD2) that are perforated. H and I – Electron micrographs (H and I) and 3D reconstructions (H’ and I’) of representative axospinous synapses receiving inputs from vGluT1-positive boutons (T) in control (H and H’) and parkinsonian (I and I’) animals. The reconstructions illustrate the spatial arrangements of the spine apparatus (SA; green) and PSD (red) in the spine head (H) and neck (N). Scale bar = 1 μm in E, F, and G. Reprinted from The Journal of Comparative Neurology, Villalba and Smith (2011), Copyright 2011, with permission from John Wiley & Sons.

Figure 3
Figure 3

A and B: Electron micrographs demonstrating changes in the spine apparatus morphology in peritumorous neocortical tissue from human patients. A – A large spine (Sp) forming a perforated asymmetric synapse with a bouton (B) contains a hypertrophic spine apparatus (asterisk). B – Another spine (Sp) containing a spine apparatus with dilated cisterns of smooth endoplasmic reticulum (asterisks). This spine forms an asymmetric synapse with a bouton (B) containing vesicles that appear to be disrupted. In both A and B, the dense plate of the spine apparatus appears to give rise to filamentous material (arrows) that contacts the base of PSD. Scale bars = 0.2 μm in A and B. Electron micrograph in A is from Spacek (1987).

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