In spite of this, the biggest bottleneck in the use of serial section TEM is that a great deal of skilled labor is required to cut ribbons of serial sections that can break, be missed, or distorted during sectioning and sample collection on the metal grids. Specialized, advanced equipment is not necessary for three-dimensional studies using serial section TEM.Ī principal advantage of serial ultrathin sections is that once the sections are obtained, they can be safely stored in a grid case for several years for later use and the same grid (or even the same neuronal profile) can be reimaged multiple times. Serial section TEM studies can be performed in any basic EM facility that is equipped with a regular conventional TEM. Once serial images are acquired, they are aligned, and the same neuronal profile is traced in each section to render a three-dimensional shape. Much of our knowledge of the three-dimensional structure of dendritic spines has been obtained from serial-section transmission EM (TEM) studies, in which resin-embedded brain sections are sliced into ribbons of serial ultrathin sections, typically 40–80 nm thick ( Wilson et al., 1983 Harris and Stevens, 1988, 1989 Harris et al., 1992 Ichikawa et al., 2002, 2016 Stewart et al., 2005, 2010 Medvedev et al., 2010, 2014 Mishchenko et al., 2010). Here, we review the subcellular structure of excitatory synapses, with a particular focus on dendritic spine structure obtained from volume EM studies. However, despite the technical developments in LM imaging tools volume electron microscopy (EM) techniques are indispensable for the visualization of high-resolution three-dimensional structures and the precise quantification of various morphological parameters that govern synaptic transmission. In recent years, super-resolution microscopes that break the diffraction barrier of conventional LM techniques are becoming increasingly popular and powerful tools to image the surface geometry of dendritic spines in live preparation ( Chereau et al., 2015 Kashiwagi et al., 2019). The peculiar bulbous morphology of individual dendritic spines can be readily identified in light microscopy (LM) preparations ( Okabe, 2020). Owing to their role in mediating neuronal excitability and biochemical signaling, dendritic spines have been intensely studied using multiple experimental approaches, including biochemical, electrophysiological, molecular biological, and imaging techniques. In many regions of the brain, excitatory synaptic contacts are formed on tiny dendritic protrusions known as dendritic spines ( Harris and Weinberg, 2012 Frotscher et al., 2014 Parajuli et al., 2017 Parajuli, 2018). Understanding the precise ultrastructure of synapses is essential to unravel the intricate neuronal circuitry and physiological functions of the brain. With a particular focus on dendritic spine synapses in the rodent brain, we discuss various key studies that have highlighted the structural diversity of spines, the principles of their organization in the dendrites, their presynaptic wiring patterns, and their activity-dependent structural remodeling. Here, we review studies that have been instrumental in determining the three-dimensional ultrastructure of synapses. The challenges of low throughput EM imaging have been addressed to an appreciable degree by the development of automated EM imaging tools that allow imaging and reconstruction of dendritic segments in a realistic time frame. While volumetric imaging of synapses can be routinely obtained from the transmission EM (TEM) imaging of ultrathin sections, it requires an unimaginable amount of effort and time to reconstruct very long segments of dendrites and their spines from the serial section TEM images. Furthermore, a complete three-dimensional reconstruction of an individual synaptic profile is required for the precise quantitation of different parameters that shape synaptic transmission. Although conventional light microscopic techniques have substantially contributed to our ever-increasing understanding of the morphological characteristics of the putative synaptic junctions, EM is the gold standard for systematic visualization of the synaptic morphology. A quantitative understanding of synaptic ultrastructure also serves as a basis to estimate the relative magnitude of synaptic transmission across individual circuits in the brain. ![]() ![]() 2Advanced Research Institute for Health Science, Juntendo University, Tokyo, JapanĮlectron microscopy (EM)-based synaptology is a fundamental discipline for achieving a complex wiring diagram of the brain.1Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo, Japan.Laxmi Kumar Parajuli 1* Masato Koike 1,2*
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