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Research Articles: Cellular/Molecular
Differentiation and characterization of excitatory and inhibitory synapses by cryo-electron tomography and correlative microscopy 1
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Chang-Lu Tao , Yun-Tao Liu , Rong Sun , Bin Zhang , Lei Qi , Sakar Shivakoti , Chong-Li Tian , Peijun 3
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1,4,5
Zhang , Pak-Ming Lau , Z. Hong Zhou
and Guo-Qiang Bi
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National Laboratory for Physical Sciences at the Microscale, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China 2
CAS Key Laboratory of Brain Function and Disease, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China 3
Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive Oxford OX37BN, UK 4
The California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
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Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
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CAS Center for Excellence in Brain Science and Intelligence Technology, and Innovation Center for Cell Signaling Network, University of Science and Technology of China, Hefei, Anhui 230026, China DOI: 10.1523/JNEUROSCI.1548-17.2017 Received: 2 June 2017 Revised: 17 December 2017 Accepted: 24 December 2017 Published: 8 January 2018
Author contributions: C.-L. Tao, Y.-T.L., P.Z., P.-M.L., Z.H.Z., and G.-Q.B. designed research; C.-L. Tao, Y.-T.L., R.S., B.Z., and L.Q. performed research; C.-L. Tao, Y.-T.L., R.S., S.S., C.-L. Tian, and G.-Q.B. analyzed data; C.-L. Tao, Y.-T.L., S.S., P.Z., P.-M.L., Z.H.Z., and G.-Q.B. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was supported in part by grants from the CAS (XDB02050000 to GQB), NSFC (30725017 and 91232722 to GQB; 31070935 to PML), MOST (2009CB941300 to GQB), and NIH (GM071940 to ZHZ). We acknowledge use of instruments at the Center for Integrative Imaging of Hefei National Laboratory for Physical Sciences at the Microscale and those at the Electron Imaging Center for Nanomachines of UCLA supported by NIH (S10RR23057 and S10OD018111) and NSF (DBI-133813). We thank Xiaokang Zhang and Peng Ge for technical advice on imaging and data processing, Jay He for help with design and construction of the cryoCLEM platform, Weidong Yao and Ann Marie Craig for sharing the PSD-95 and mCherrry-gephyrin plasmids, Xiaobing Chen for insightful discussions, Chunhong Qiu for help with illustrations, and the anonymous reviewers for their insightful comments and constructive suggestions to improve the paper. Correspondence: Guo-Qiang Bi, School of Life Science, USTC, 443 Huangshan Rd, Hefei, China, 230027. E-mail:
[email protected] and Z. Hong Zhou, The California NanoSystems Institute, UCLA, 570 Westwood Plaza, CA 90095, USA. E-mail:
[email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1548-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published.
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Differentiation and characterization of excitatory and inhibitory synapses by
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cryo-electron tomography and correlative microscopy
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Abbreviated title: Visualizing synapse types in 3D by cryoCLEM
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Chang-Lu Tao1,*, Yun-Tao Liu1,*, Rong Sun1, Bin Zhang2, Lei Qi2, Sakar Shivakoti1,
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Chong-Li Tian2, Peijun Zhang3, Pak-Ming Lau2, Z. Hong Zhou1,4,5,#, Guo-Qiang Bi1,6,#
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1
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University of Science and Technology of China, Hefei, Anhui 230026, China; 2CAS Key
National Laboratory for Physical Sciences at the Microscale, and School of Life Sciences,
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Laboratory of Brain Function and Disease, and School of Life Sciences, University of
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Science and Technology of China, Hefei, Anhui 230026, China; 3Division of Structural
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Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt
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Drive Oxford OX37BN, UK; 4The California NanoSystems Institute, University of
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California, Los Angeles, CA 90095, USA; 5Department of Microbiology, Immunology and
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Molecular Genetics, University of California, Los Angeles, CA 90095, USA; 6CAS Center
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for Excellence in Brain Science and Intelligence Technology, and Innovation Center for
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Cell Signaling Network, University of Science and Technology of China, Hefei, Anhui
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230026, China
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*These authors contributed equally to this work.
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#
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Hefei, China, 230027. E-mail:
[email protected] and Z. Hong Zhou, The California
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NanoSystems Institute, UCLA, 570 Westwood Plaza, CA 90095, USA. E-mail:
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[email protected].
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Number of text pages: 51; Number of figures: 9; Number of movies: 5;
Correspondence: Guo-Qiang Bi, School of Life Science, USTC, 443 Huangshan Rd,
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Number of words: Abstract (215); Significance Statement (118); Introduction (640);
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Discussion (1207).
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Conflict of Interest: The authors declare no competing financial interests.
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Acknowledgements: This work was supported in part by grants from the CAS
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(XDB02050000 to GQB), NSFC (30725017 and 91232722 to GQB; 31070935 to PML),
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MOST (2009CB941300 to GQB), and NIH (GM071940 to ZHZ). We acknowledge use of
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instruments at the Center for Integrative Imaging of Hefei National Laboratory for Physical
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Sciences at the Microscale and those at the Electron Imaging Center for Nanomachines
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of UCLA supported by NIH (S10RR23057 and S10OD018111) and NSF (DBI-133813).
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We thank Xiaokang Zhang and Peng Ge for technical advice on imaging and data
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processing, Jay He for help with design and construction of the cryoCLEM platform,
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Weidong Yao and Ann Marie Craig for sharing the PSD-95 and mCherrry-gephyrin
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plasmids, Xiaobing Chen for insightful discussions, Chunhong Qiu for help with
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illustrations, and the anonymous reviewers for their insightful comments and constructive
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suggestions to improve the paper.
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Abstract
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As key functional units in neural circuits, different types of neuronal synapses play distinct
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roles in brain information processing, learning and memory. Synaptic abnormalities are
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believed to underlie various neurological and psychiatric disorders. Here, by combining
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cryo-electron tomography and cryo-correlative light and electron microscopy, we
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distinguished intact excitatory and inhibitory synapses of cultured hippocampal neurons,
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and visualized the in situ three-dimensional organization of synaptic organelles and
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macromolecules in their native state. Quantitative analyses of over a hundred synaptic
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tomograms reveal that excitatory synapses contain a mesh-like postsynaptic density
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(PSD) with thickness ranging from 20-50 nm. In contrast, the PSD in inhibitory synapses
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assumes a thin sheet-like structure ~12 nm from the postsynaptic membrane. On the
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presynaptic side, spherical synaptic vesicles (SVs) of 25-60 nm diameter and
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discus-shaped ellipsoidal SVs of various sizes coexist in both synaptic types, with more
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ellipsoidal ones in inhibitory synapses. High-resolution tomograms obtained using a Volta
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phase plate and electron filtering and counting reveal glutamate receptor-like and GABAA
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receptor-like structures that interact with putative scaffolding and adhesion molecules,
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reflecting details of receptor anchoring and PSD organization. These results provide an
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updated view of the ultrastructure of excitatory and inhibitory synapses, and demonstrate
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the potential of our approach to gain insight into the organizational principles of cellular
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architecture underlying distinct synaptic functions.
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3
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Significance Statement
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To understand functional properties of neuronal synapses, it is desirable to analyze their
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structure at molecular resolution. We have developed an integrative approach combining
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cryoET and correlative fluorescence microscopy to visualize 3D ultrastructural features of
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intact excitatory and inhibitory synapses in their native state. Our approach shows that
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inhibitory synapses contain uniform thin sheet-like PSDs, while excitatory synapses
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contain previously known mesh-like PSDs. We discovered “discus-shaped” ellipsoidal
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synaptic vesicles, and their distributions along with regular spherical vesicles in synaptic
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types are characterized. High-resolution tomograms further allowed identification of
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putative neurotransmitter receptors and their heterogeneous interaction with synaptic
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scaffolding proteins. The specificity and resolution of our approach enables precise in situ
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analysis of ultrastructural organization underlying distinct synaptic functions.
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Introduction
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Chemical synapses are basic functional units in neural circuits for information
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transmission, processing and storage (Eccles, 1964; Sudhof and Malenka, 2008; Mayford
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et al., 2012). From the enormous number of synapses in the brain, the plasticity of each
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synapse, and the molecular and functional diversity across these synapses, arise the
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brain’s remarkable computational power and cognitive capacity (Milner et al., 1998; Bi
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and Poo, 2001). Glutamatergic and GABAergic synapses, the two main types of central
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synapses, play opposite roles in excitation and inhibition. They have been shown by
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biochemical and electrophysiological studies to contain different sets of molecular and
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cellular components and to exhibit distinct functional properties and plasticity rules (Craig
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and Boudin, 2001; Sudhof and Malenka, 2008; Vogels and Abbott, 2009;
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Sassoe-Pognetto et al., 2011). How are these different components organized into the
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intricate machinery to perform distinct synaptic functions? Electron microscopy (EM) has
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been a primary probe to address this question by visualizing the ultrastructure of various
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synapses (Siksou et al., 2007; Harris and Weinberg, 2012).
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Classical EM uses chemical fixation, dehydration and plastic embedding, followed by
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sectioning and heavy-metal staining to image brain tissues and cultured neurons. Such
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meticulous processing has allowed electron beams to image various biological
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specimens at high contrast. Indeed, classical EM observations have shaped much of our
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current knowledge about synaptic ultrastructure (Sorra and Harris, 2000; Harris and
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Weinberg, 2012). For example, prominent subcellular features such as the postsynaptic
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density (PSD) and synaptic vesicles (SVs) are well documented, especially for excitatory
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synapses (Gray, 1959; Colonnier, 1968; Schikorski and Stevens, 1997; Harris and 5
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Weinberg, 2012). The 3D resolving capability of electron tomography (ET) has yielded
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better views of synaptic ultrastructure (Harlow et al., 1998; Ress et al., 2004; Burette et
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al., 2012). The improved structural preservation provided by high-pressure freezing with
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freeze substitution (HPF-FS) (Tatsuoka and Reese, 1989) combined with ET (Rostaing et
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al., 2006) has allowed studies of the organization and dynamics of SVs (Siksou et al.,
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2007; Watanabe et al., 2013; Imig et al., 2014; Jung et al., 2016) and the 3D organization
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of macromolecular complexes in individual synapses, such as the PSD-95/glutamate
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receptor complex at the PSD (Chen et al., 2008; Chen et al., 2015).
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It remains challenging to characterize the structure and organization of cellular and
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molecular machinery of specific synaptic types at higher resolution (Hurbain and Sachse,
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2011; Tao et al., 2012), because damage or deformation from sample preparation
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procedures can complicate structural interpretation (Hurbain and Sachse, 2011).
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Cryo-electron tomography (cryoET), which aims to overcome this limitation, has been
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used to visualize distribution of SVs and other ultrastructural features in isolated
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synaptosomes and cryo-sections of brain tissues (Fernandez-Busnadiego et al., 2010;
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Shi et al., 2014; Wilhelm et al., 2014; Perez de Arce et al., 2015). However, cryoET alone
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cannot unambiguously identify synapse types due to a lack of specific labeling such as
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immunogold staining or photoconversion of diaminobenzidine for classical EM (Megias et
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al., 2001; Schikorski and Stevens, 2001; Rostaing et al., 2006; Harris and Weinberg,
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2012). One way to overcome this is to take advantage of the molecular specificity of
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fluorescence labeling and sample preservation of cryoET in cryo-correlative light and
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electron microscopy (cryoCLEM), as suggested earlier (Lucic et al., 2007), although the
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ability of this approach to distinguish different synapse types is yet to be realized. In the
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current study, we developed an efficient cryoCLEM platform to identify different types of 6
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synapses in cultured hippocampal neurons, and to define pre- and postsynaptic
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ultrastructural features of excitatory and inhibitory synapses in their native state. By
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high-resolution cryoET with cutting-edge direct electron detection (Li et al., 2013), Volta
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phase plate (VPP) (Danev et al., 2014; Fukuda et al., 2015), and electron energy filter
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(Verbeeck et al., 2004) technologies, we could also visualize putative glutamate and
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GABAA receptors and their organization at the postsynaptic membrane of excitatory and
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inhibitory synapses.
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Materials and Methods
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The overall workflow of experimental procedures is illustrated in Figure 1A. Primary
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neuronal cultures were grown on EM grids and then plunge-frozen for cryoET imaging
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followed by 3D reconstruction. For some cultures transfected with constructs of
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fluorescent protein-tagged synaptic proteins, cryo-fluorescence microscopy was
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performed before cryoET for correlative imaging. All animal procedures were performed
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following the guidelines of the Animal Experiments Committee at the University of
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Science and Technology of China.
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Primary culture of hippocampal neurons
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Low-density cultures of dissociated embryonic rat hippocampal neurons were prepared
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as previously described (Bi and Poo, 1998) with modifications. Quantifoil R2/2 gold EM
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grids (200 mesh with holey carbon film of 2 μm hole size and 2 μm spacing) or Quantifoil
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R2/2 gold NH2 finder grids (100 mesh with holey carbon film of 2 μm hole size and 2 μm
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spacing) were plasma cleaned with H2 and O2 for 10 s using a plasma cleaning system
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(Gatan), and sterilized with UV light for 30 min. These grids were then coated with 7
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poly-L-lysine (Sigma) overnight, followed by washing with Hank’s balanced salt solution
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and ddH2O for about 12 hours each. Hippocampi were removed from embryonic day-18
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(E18) rats (without distinguishing sex differences) and were treated with trypsin for 15 min
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at 37 °C, followed by washing and gentle trituration. The dissociated cells were plated on
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the poly-L-lysine coated EM grids in 35-mm Petri dishes at a density of 40,000-60,000
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cells/ml, and maintained in incubators at 37 °C in 5% CO2. The culture medium was
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NeuroBasal (NB) (Invitrogen) supplemented with 5% heat-inactivated bovine calf serum
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(PAA) plus 5% heat-inactivated fetal bovine serum (HyClone), 1X Glutamax (Invitrogen)
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and 1X B27 (Invitrogen). Twenty-four hours after plating, half of the medium was replaced
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by serum-free culture medium. Subsequently, one third of the culture medium was
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replaced with fresh culture medium twice a week. For correlative microscopy, cultures
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were co-infected with lentiviruses encoding PSD-95-EGFP and mCherry-gephyrin
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constructs (see below) for 5-7 days in vitro before vitrification of the grid. Twelve hours
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after the infection, half of the culture medium was replaced by fresh medium.
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To prevent overgrowth of glial cells, the cultures were treated with cytosine
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arabinoside (Sigma) at various stages. Cultures were used for cryoEM imaging at 14-18
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days in vitro (DIV), when healthy, low-density cultures formed patches of monolayer
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neuronal processes (Figure 1B1). We judge whether the culture is healthy based on
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morphological criteria, e.g. smooth soma and dendrites with multiple branches viewed
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under phase-contrast light microscopy, and at least one probable synapse each few
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micrometers along the dendrites of transfected neurons, as viewed under fluorescence
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microscopy. According to our experience, such criteria predict retention of functional
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properties of synaptic transmission and plasticity evaluated with patch-clamp recording
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and calcium imaging. 8
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DNA constructs and lentivirus preparation
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The PSD-95 cDNA was amplified from GW-PSD-95-EGFP plasmid (a generous gift from
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Dr. Weidong Yao) and subcloned into pLenti-CaMKII-mKate2 vector to produce
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pLenti-CaMKII-PSD-95-mKate2. The GGGS-EGFP cDNA was amplified from the
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pEGFPN1 plasmid, and then subcloned into pLenti-CaMKII-PSD-95-mKate2 to produce
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the pLenti-CaMKII-PSD-95-EGFP plasmid. The mCherry-gephyrin lentiviral construct
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(Dobie and Craig, 2011) was a generous gift from Dr. Ann Marie Craig. Both
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PSD-95-EGFP and mCherry-gephyrin lentiviral constructs were packaged into lentivirus
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following a protocol from Dr. Karl Deisseroth’s lab (Zhang et al., 2010b).
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Frozen-hydrated sample preparation
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After being removed from the CO2 incubator, low-density neuronal cultures (DIV 14-18)
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on EM grids were first placed in extracellular solution (ECS, containing 150 mM NaCl, 3
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mM KCl, 3 mM CaCl2, 2 mM MgCl2, 10 mM HEPES and 5 mM glucose, pH 7.3), then
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mounted on a Vitrobot IV (FEI). Protein A-coated colloidal gold beads (15-nm size, CMC)
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were added to the grid (4 μl each, stock solution washed in ECS and diluted 10 times
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after centrifugation) as fiducial markers. The grids were then plunged into liquid ethane
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for rapid vitrification of the samples, which were then stored in liquid nitrogen until use.
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CryoET imaging
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CryoET data were collected with single-axis tilt using either a Tecnai F20 transmission
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electron microscope (Tecnai F20, FEI) equipped with an Eagle 4K×4K multiport CCD
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camera (FEI), or a Titan Krios (FEI) with a K2 Summit direct electron detector (K2 camera,
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Gatan). The Tecnai F20 was operated at an acceleration voltage of 200 KV. Tilt series 9
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were collected from -60° to +60° at of 2° intervals using FEI Xplore 3D software, with the
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defocus value set at -12 to -18 μm, and the total electron dosage of ~100 e-/Å2. The final
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pixel size was 0.755 nm. The Titan Krios were operated at an acceleration voltage of 300
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KV, with or without Volta phase plate (VPP) and Gatan image filter (GIF). In either
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configuration, images were collected by the K2 camera in counting mode. In the absence
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of VPP and GIF, tilt series were acquired from -64° to +64° at 2° intervals using Leginon
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(Suloway et al., 2005), with the defocus value maintained at -10 μm, and the total
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accumulated dose of ~120 e-/Å2. The final pixel size was 0.765 nm. When VPP and GIF
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were used, the energy filter slit was set at 20 eV, and VPP was conditioned by
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pre-irradiation for 60 s to achieve an initial phase shift of ~0.3π (Fukuda et al., 2015). Tilt
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series were acquired from -66° to +60° at an interval of 2° or 3° using SerialEM
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(Mastronarde, 2005) with the defocus value maintained at -1 μm and the total
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accumulated dose of ~150 e-/Å2. The final pixel size was 0.435 nm.
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For this study, we examined 78 grids, of which 12 were used for data collection. The
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rest were discarded because the grids were damaged during transfer or freezing, cultures
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were too dense and/or too thick, or cultures appeared not healthy with few or no
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synapses found. Usually 3-5 grid squares (each about 100×100 μm2) per grid were
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selected for imaging. To obtain high quality cryoET images, it is critical to choose thin
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culture areas with healthy yet relatively low-density dendrites (Figure 1B2). Generally,
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areas thicker than ~500 nm were ignored. At this thickness, subcellular structures such
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as mitochondria, microtubules and synaptic vesicles could not be distinguished in single
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projection images.
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Cryo-correlative light and electron microscopy imaging
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The hardware of our cryo-light microscope system includes a custom-built cryo-chamber
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with liquid nitrogen supply, a Gatan 626 EM cryo-holder, and an Olympus IX71 inverted
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fluorescence microscope (Figure 1C). The inside channel of the cryo-chamber was
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precooled to -190 °C by liquid nitrogen, and maintained below -180 °C, as monitored by a
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thermoelectric sensor. Nitrogen gas flowed through the objective lens and light source
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windows during the experiment to prevent frost accumulation. Then, an EM grid with
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frozen-hydrated sample was loaded onto an EM cryo-holder, which was subsequently
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inserted into the cryo-chamber.
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For cryoCLEM imaging, fluorescence images were taken using a 40X air objective
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lens (Olympus LUCPLFLN 40X, NA 0.6) and an ANDOR NEO sCMOS camera (Andor)
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attached to the fluorescence microscope. For each field of view, three images were
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collected, one in bright-field, another in the EGFP channel (Ex:470/40, DM: 495, Em:
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525/50; Chroma, 49002) and the third in the mCherry channel (Ex: 562/40, DM: 593, Em:
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641/75; Semrock, mCherry-B-000). Typically about 10 sets of images were sufficient to
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cover all good areas (about 40 squares) on each grid, which took about 20 min to
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complete; a "good area" was defined as a grid square (~100×100 μm2) of appropriate
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sample thickness that displayed multiple dendritic branches, usually containing dozens of
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PSD-95-EGFP puncta or multiple mCherry-gephyrin puncta under fluorescence
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microscopy (Figure 1D1).
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Immediately after the light microscopy (LM) imaging, the EM cryo-holder with grid
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was directly transferred into a Tecnai F20 scope. Areas of the sample imaged in cryo-light
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microscope were identified in the EM using the indexes of the finder grids (Figure 1D1,
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D2). Low magnification (330X) EM images were collected and roughly aligned with
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bright-field LM images using Midas program in IMOD package (RRID: SCR_003297) 11
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(Kremer et al., 1996). After rough alignment, a set of holes on the carbon layer of the grid
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were picked from both the low magnification EM images and their corresponding
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fluorescence images using 3dmod in IMOD package. Transformation functions between
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the EM and LM images was calculated based on the selected positions by minimizing the
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mean squared error.
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When the low magnification EM image and LM image were optimally aligned (Figure
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1D3), ~15 holes on carbon (in one square) in the low magnification EM image were
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selected, with their pixel positions recorded. The same holes were identified at 5,000X
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magnification and the mechanical coordinates were recorded. Afterwards, the
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transformation function from the pixel positions to EM mechanical coordinates was
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determined using similar linear regression methods. With the transformation functions,
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positions of selected fluorescent puncta (putative excitatory or inhibitory synapses) were
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converted into corresponding EM mechanical coordinates, which were used to guide EM
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image acquisition. Tilt series were collected on the area with selected fluorescent signals.
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Finally, tomographic slices were fine-aligned and merged with the fluorescence images to
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identify each synapse (Figure 1D4) using Midas and ImageJ (RRID: SCR_003070).
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Python scripts (RRID: SCR_008394) to integrate the correlation procedures are available
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to interested readers upon request.
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3D Reconstruction and rendering
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Tilt series were aligned and reconstructed using IMOD. Gold beads added to the sample
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before plunge freezing were used as fiducial markers to align the tilt series.
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Reconstruction was performed using a simultaneous iterative reconstruction technique
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with 5 or 15 iterations. A fraction of the data collected were discarded during 12
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reconstruction for technical reasons: for Tecnai F20, ~50% of the data were discarded
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because of issues such as stage drift, beam blockade at high tilt angles, and occasional
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auto-focus failure; for Titan Krios, 20 nm) PSD structures 21
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right next to the plasma membrane were easily identifiable in 13 out of the 14 excitatory
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synapses (Figure 3A) and spanned nearly the entire area of the uniform synaptic cleft
471
(Figure 3A1). The existence of such thick PSDs is consistent with the common belief that
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excitatory synapses are “asymmetric”, with dense molecular scaffolds on the
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postsynaptic side (Colonnier, 1968; Peters and Palay, 1996). In contrast, nearly all (7 out
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of the 8) inhibitory synapses identified by cryoCLEM (Figure 3B) had distinct thin
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sheet-like PSD (thin PSD for short), positioned in parallel and close to the postsynaptic
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membrane (Figure 3B1). Thus, the previously-termed “symmetric” inhibitory synapses
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are in fact asymmetric under cryoET.
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Quantitative analyses of PSD structures in excitatory and inhibitory synapses.
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We quantified the PSD profiles of the above 20 identified synapses together with 90
480
additional synapses with visible PSDs from the 101 synapses obtained with cryoET but
481
not CLEM, by plotting the mean pixel density as a function of its distance to the
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postsynaptic membrane (Figure 4A, B). The curve contains two major peaks representing
483
the pre- and postsynaptic membranes. There are also two smaller peaks on the curve,
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one between the pre- and postsynaptic membrane, representing an electron-dense band
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within the synaptic cleft reported previously as an intermediate band (Gray, 1959), and
486
the other to the right of the postsynaptic membrane defined as “PSD peak,” presumably
487
indicating a postsynaptic proteinaceous layer. This quantitative approach allowed us to
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identify d1, the peak position of the PSD, and d2, which provides a measure of the
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thickness of the PSD (Figure 4A2, B2). Scatter plot of PSD thickness and PSD peak
490
positions of all synapses that contain visible PSDs shows two well-defined clusters, thick
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and thin, which overlap with the two distinct populations formed by the CLEM-identified
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excitatory and inhibitory synapses, respectively (Figure 4C). Thus, the distinct PSD 22
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patterns detected by cryoET can be used as hallmarks to distinguish the two types of
494
synapses.
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With the PSD pattern as an unequivocal criterion, we systematically characterized
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pre- and postsynaptic features in all 110 synapses with visible PSDs analyzed above,
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including 85 excitatory synapses and 25 inhibitory synapses. Their postsynaptic densities
498
exhibit distinct PSD peak positions (14.7±3.0 nm, n=85 for excitatory synapse; 9.1±1.1
499
nm, n=25 for inhibitory synapse) and thickness (32.7±7.7 nm, n=85 for excitatory synapse;
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12.3±1.8 nm, n=25 for inhibitory synapse). Compared to the uniformly thin PSDs of
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inhibitory synapses, the thick PSDs of excitatory synapses exhibit substantial variability
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(Figure 4C, D). By averaging all density profiles for excitatory and inhibitory synapses,
503
respectively, we found that the two types of synapses have similar cleft width (~26 nm)
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(Figure 4E), in contrast to the previous report of narrower cleft for inhibitory synapses
505
(Peters and Palay, 1996). Inside the cleft, distinctive band-like structures are visible in all
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synapses (Figure 4A, B), as reported previously (Gray, 1959; Zuber et al., 2005). We
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speculate that these structures are protein complexes involved in cell adhesion (Missler
508
et al., 2012). Intriguingly, the density profile around the presynaptic membrane peak is
509
asymmetric; the density values on the cytoplasmic side are slightly higher than that on the
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cleft side, especially in excitatory synapses. This presumably reflects extra protein on the
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cytoplasmic side of the presynaptic membrane, forming a weak version of the active zone
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commonly observed in conventional EM (Phillips et al., 2001; Sudhof, 2012). Additional
513
studies with better molecular identifications are needed to gain more insight into these
514
structures.
515 516
In addition to synapses with visible PSD, we also observed 13 structures that met our criteria for synapses but exhibited no visible PSD (Figure 5A). They differ from the more 23
517
frequently observed nonsynaptic boutons similar to those reported previously (Bourne et al.,
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2013), because they had distinct uniform synaptic cleft structures that were absent in the
519
latter. These “PSD-free synapses" could be either excitatory or inhibitory as evidenced
520
from the cryoCLEM data (Figure 5B, C). They might reflect a special transient stage, e.g.,
521
at an early phase of synaptogenesis or on the way towards elimination (Klemann and
522
Roubos, 2011). The variation in the existence and thickness of PSDs reflects potentially
523
diverse synaptic subtypes and associated signaling and structural mechanisms.
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Quantitative analyses of synaptic vesicles in excitatory and inhibitory synapses
525
On the presynaptic side, we found that in both excitatory and inhibitory synapses, the
526
majority of SVs are perfectly spherical (Figure 6A-C), with an average diameter of about
527
40 nm (40.9±5.0 nm, 16476 vesicles in excitatory synapses; 41.3±6.6 nm, 4766 vesicles
528
in inhibitory synapses, Figure 6D). This differs from the classical EM observation that
529
inhibitory vesicles tend to be smaller compared to excitatory ones (Peters and Palay,
530
1996), but is consistent with findings using HPF-FS (Tatsuoka and Reese, 1989; Korogod
531
et al., 2015), and with measurements from synaptosomes using cryoEM
532
(Fernandez-Busnadiego et al., 2010). Intriguingly, we also observed some apparently
533
ellipsoidal vesicles in both types of synapses (Figure 6A, B). To quantify the shape of
534
different vesicles, we first performed two-dimensional (2D) analysis, based on the
535
maximal cross-section of each SV in the x-y plane, of 19,056 SVs in both excitatory and
536
inhibitory synapses. This analysis identified a relatively small population of ellipsoidal
537
vesicles with major/minor axis ratio significantly greater than 1 (Figure 6E). Interestingly,
538
although ellipsoidal vesicles were found in both excitatory and inhibitory synapses, more
539
were found in the latter (8.5% ± 9%, n=35 in excitatory synapses and 16.9% ± 11%, n=15
540
in inhibitory synapses; p=0.006, two-sample Kolmogorov-Smirnov test). Indeed, 5 out of 24
541
15 inhibitory synapses analyzed (in contrast to 3 out of 35 excitatory synapses) had 20%
542
or more vesicles that were ellipsoidal (Figure 6F).
543
A limitation of 2D analysis is that the true 3D shape of the vesicles could not be
544
determined. For example, if the 2D cross-sections of a vesicle is circular, its 3D shape
545
can be either spherical, “discus-shaped” (i.e., oblate spheroid), or “olive-shaped” (i.e.,
546
prolate spheroid) (Figure 6G). Therefore, we measured the three principal axes of each
547
vesicle using a 3D fitting program (see Materials and Methods) similar to a method
548
described previously (Kukulski et al., 2012). Measurement of axes with this program on
549
70 ellipsoidal vesicles and 70 of their neighboring spherical vesicles in 5 excitatory and 5
550
inhibitory synapses (in high-resolution tomograms obtained with VPP, electron filtering
551
and counting) revealed that the majority of the ellipsoidal vesicles were discus-shaped
552
rather than olive-shaped for both excitatory and inhibitory synapses (Figure 6H). The
553
ellipticity of the ellipsoidal vesicles exhibit large variability (Figure 6H), which may reflect
554
their different compositions and functional roles in synaptic transmission.
555
Visualization of putative receptors and scaffolding proteins in individual synapses
556
New tools that allow cryoET to achieve higher resolution, including direct electron
557
detection, Volta phase plate, and electron energy filter have facilitated characterization of
558
molecular complexes such as proteasome in intact cultured neurons (Asano et al., 2015).
559
Using these tools, we obtained high-quality tilt series (Figure 7A, B and Movie 1) and
560
tomograms with high contrast and high resolution, permitting visualization of features
561
such as the two leaflets of the membrane bilayer, microtubule protofilaments, and
562
putative proteasomes (Figure 7C-E). Two high-quality synaptic tomograms obtained
563
using Volta phase plate, and electron filtering and counting, were selected for further 25
564
study. One is a spine excitatory synapse with thick PSD structure. The other is a shaft
565
inhibitory synapse with thin sheet-like PSD.
566
In the excitatory synapse (Figure 8A), large features such as membranous
567
organelles and ribosomes, as well as actin and microtubule filaments with ultrastructural
568
details were readily identified and segmented (Figure 8B and Movie 2). Furthermore,
569
numerous particles and filamentous structures of various sizes and shapes were
570
visualized within and across pre- and postsynaptic compartments (Figure 8C). These
571
structures were presumably individual protein molecules and complexes. Of special
572
interest were particles near the postsynaptic membrane, some of which have shapes
573
similar to that known for glutamate receptors, including N-methyl-D-aspartate receptors
574
(NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors
575
(AMPARs), which constitute a major fraction of the postsynaptic membrane proteins
576
(Valtschanoff and Weinberg, 2001; Chen et al., 2008; Dani et al., 2010; Jacob and
577
Weinberg, 2015). We thus visually classified these particles as putative “glutamate
578
receptors”, and defined the remaining particles visible on the cleft side of the postsynaptic
579
membrane as “non-receptor” particles (Figure 8C). Plotting the length and width of all
580
these particles reveals that the visually identified putative glutamate receptors form a
581
cluster, although not well separated from the “non-receptor” particles (Figure 8D). The
582
average length (12.1±1.4 nm, n=81) and width (8.6±1.4 nm, n=81) of particles in this
583
cluster is similar to that of the extracellular domain of AMPARs (length: 12.0±0.2 nm,
584
width: 10.5±2.4 nm) and NMDARs (length: 10.5±0.2 nm, width: 10.3±1.4 nm) based on
585
their crystal structures [see detailed calculation of averaged dimensions in Materials and
586
Methods] (Figure 8D-D2). In total, this synapse contained 81 putative glutamate
587
receptors, intermingled with other membrane proteins to occupy the surface of the 26
588
postsynaptic membrane area (Figure 8E, F and Movie 3). This number agrees with
589
estimates of the total number of AMPARs and NMDARs in a glutamatergic synapse
590
based on quantitative immunoEM (Nusser et al., 1998b; Takumi et al., 1999), quantitative
591
mass spectrometry (Sheng and Hoogenraad, 2007; Lowenthal et al., 2015) and visual
592
identification with ET after HPF-FS (Chen et al., 2008; Chen et al., 2015).
593
Among the 81 receptor-like structures in the excitatory synapse, 16 displayed a clear
594
globular density (~10 nm in diameter) on the cytoplasmic side (Figure 8G1). Such
595
globular densities are unlikely to belong to the MAGUK-family proteins, which have
596
filamentous shapes (Nakagawa et al., 2004; Chen et al., 2008; Chen et al., 2011). We
597
thus suspect that these 16 structures are likely NMDARs, known to have much larger
598
cytoplasmic domains than AMPARs (Chen et al., 2008). Of the remaining 65 receptor-like
599
structures, 11 had relatively low image quality and thus prevented classification based on
600
their cytoplasmic structures, whereas 54 structures could be visually identified as
601
AMPAR-like structures based on the lack of large globular density. Among them, 44 were
602
found to each link to one or two filamentous structure that might represent PSD-95 or
603
similar MAGUK-family proteins (Figure 8G2, G3). These putative scaffolding structures
604
appeared to contact the cytoplasmic side of AMPAR-like structures (Figure 8G2, G3),
605
reminiscent of PSD-95 anchoring AMPAR through its interaction with stargazin, which
606
binds to the side of the AMPAR (Meyer et al., 2004; Nakagawa et al., 2006). We also
607
observed 10 AMPAR-like structures not associated with any PSD-95-like structures
608
(Figure 8G4). The majority of these PSD-95-like structures linking to AMPAR-like
609
structures were in near-perpendicular orientation with respect to the postsynaptic
610
membrane (Figure 8G). Together with about two hundred similar filaments connecting
611
directly to the membrane, they form a set of “vertical pillars” to shape an overall core 27
612
structure of the PSD (Movie 3), as also seen by ET of samples prepared using HPF-FS
613
(Chen et al., 2008).
614
A high-resolution tomogram of an inhibitory synapse also revealed rich ultrastructural
615
details (Figure 9A-C and Movie 4). On the cleft side of the postsynaptic membrane, many
616
particles (Figure 9C) were found with shapes similar to that of the type-A γ-aminobutyric
617
acid receptors (GABAAR), the primary inhibitory transmitter receptor in these
618
hippocampal neurons (Bi and Poo, 1998; Nusser et al., 1998a). With visual inspection,
619
we provisionally identified about 143 particles as GABAAR, and about 109 other particles
620
visible on the extracellular side of the postsynaptic membrane as “non-receptor” particles
621
that likely represent other synaptic proteins such as adhesion molecules. Plotting the
622
length and width of all these particles revealed that the visually-identified putative
623
GABAARs formed a cluster, and that the sizes of these putative GABAARs (length:
624
7.1±0.9 nm, width: 5.9±0.9 nm, n=143) were similar to that of the extracellular domain of
625
GABAAR based on its crystal structure (length: 6.2±0.1 nm, width: 6.4±0.1 nm) (Figure
626
9D-D2). Note that the averaged putative GABAAR, similar to the averaged putative
627
glutamate receptor, is sounded by a “halo” (Figure 8D1, 9D1), which could be partially
628
due to fringes arising from uncorrected contrast transfer function. However, for individual
629
particles, such effects appeared to be minimal and did not affect the measurements of
630
particle sizes. The majority of the putative “non-receptor” particles were uniformly skinny
631
but with variable lengths (Figure 9D). Within this inhibitory synapse, the 143 putative
632
GABAARs lying amidst 109 “non-receptor” membrane protein molecules covered the
633
entire ~0.1 μm2 postsynaptic membrane (Figure 9E, F and Movie 5). The number and
634
density of GABAAR-like particles are consistent with a previous estimate of 30-200
635
GABAARs per GABAergic synapse (1,250 receptors/μm2) (Nusser et al., 1997). 28
636
The majority of the GABAAR-like particles were associated with one or two
637
“hammer-shaped” structures on the cytoplasmic side, each with a dense “head” and a
638
thin “neck” (Figure 9G). The heads of these hammer-shaped structures were consistently
639
located at ~12 nm from the postsynaptic membrane, and the necks bridged the
640
transmembrane domain between the GABAAR-like structure and the dense head (Figure
641
9G1-3). We speculate that these hammer-shaped structures are protein complexes
642
containing gephyrin molecules, the major postsynaptic scaffolding component of
643
inhibitory synapse (Tretter et al., 2012). Some of the GABAAR-like structures had only
644
thin necks on their cytoplasmic side, lacking the head density (Figure 9G4), suggesting
645
that the neck might be the cytosolic domain of GABAAR. These putative receptor-linked
646
gephyrin-like structures, together with similar particles not linked to receptor-like particles
647
but also lying ~12 nm from the postsynaptic membrane, appeared to form a cross-linked
648
matrix, which could provide anchoring sites and structural support for GABAARs, as
649
previously proposed (Tyagarajan and Fritschy, 2014). Interestingly, we also found many
650
GABAAR-like structures also linked to densities on the cleft side (Figure 9G3, G4). These
651
densities might represent the cell adhesion molecule neurexin, previously reported to
652
bind directly to GABAAR (Zhang et al., 2010a).
653
Discussion
654
The complex and highly organized molecular machinery inside neuronal synapses
655
provides the structural basis for synaptic transmission and plasticity. In this study, we
656
have developed an approach of cryo-correlative microscopy to distinguish excitatory and
657
inhibitory synapses in intact neurons in culture and to visualize their 3D structures in their
658
native state. By quantifying ultrastructural features of more than a hundred hippocampal 29
659
synapses, we have characterized the ultrastructural features across excitatory and
660
inhibitory central synapses. Because the neurons we used were from embryonic brain
661
and cultured for only a couple of weeks, their synapses may not be as mature as those in
662
the adult brain. Nonetheless, such cultured neurons has been shown to exhibit basic
663
physiological properties of synaptic transmission and plasticity similar to those in more
664
intact preparations such as brain slices. Thus, it is likely that the basic ultrastructural
665
features we observed also reflect synaptic architecture in the brain, at least during its
666
early development.
667
Our approach allows for unequivocal differentiation of the ultrastructure of excitatory
668
and inhibitory synapses in hippocampal neurons. Our results show that excitatory
669
synapses have distinct thick PSDs, and inhibitory synapses have more uniformly thin
670
PSDs. These corroborate classic findings regarding the ultrastructure of excitatory and
671
inhibitory synapses (Colonnier, 1968; Gray, 1969), while providing an updated
672
description of different PSD types in their native state. Quantitative analysis reveals that
673
excitatory PSDs have a broad distribution of thickness. This is consistent with the idea
674
that the dynamically interacting PSD molecules may be in a mixed gel/liquid phase (Zeng
675
et al., 2016), and suggests the existence of multiple structural configurations, perhaps
676
reflecting different states of their activation (Dosemeci et al., 2001) and plasticity (Bi and
677
Poo, 1998; Montgomery and Madison, 2004). The existence of multiple functional and
678
plasticity states in excitatory synapses could be critical for optimal learning and memory
679
storage in neuronal circuits, as suggested by theoretical studies (Fusi et al., 2005; Fusi
680
and Abbott, 2007). In contrast, inhibitory PSDs are much more uniformly and regularly
681
organized, consistent with the meager evidence for structural plasticity in such synapses.
682
Intriguingly, there is a “gap” area with relatively lower electron density between the 30
683
postsynaptic membrane and the PSD peak in both excitatory and inhibitory synapses
684
(see the density profiles of Figure 4A2, B2). We suspect that much of this gap reflects the
685
relatively lower protein content here, compared to the PSD peak where more extensive
686
interactions between protein molecules may occur.
687
High-resolution cryoET has allowed visualization of the distinct molecular
688
organization underlying the different functional properties of excitatory and inhibitory
689
synapses. In the excitatory synapse, a set of PSD-95-like filamentous structures formed
690
“vertical pillars” immediately underneath the postsynaptic membrane to organize the thick
691
PSD meshwork. These filaments have not been observed in classic studies with
692
conventional EM (Gray, 1959; Colonnier, 1968; Peters and Palay, 1996), or later studies
693
by ET with brain slices prepared using HPF-FS (Rostaing et al., 2006; Siksou et al., 2007),
694
but is consistent with more recent observation using HPF-FS and ET in cultures (Chen et
695
al., 2008). The discrepancies could be due to differences in image resolution, or could
696
reflect differences between synapses in slice and culture. Consistent with previous
697
observations (Chen et al., 2008; Chen et al., 2015), we also found that some of the
698
vertical filaments were linked to putative NMDA and AMPA type glutamate receptors.
699
However, the cytoplasmic domains of the putative receptors we observed are much
700
smaller. We speculate that the previously-seen large cytoplasmic domains were due to
701
heavy metal staining.
702
In the inhibitory synapse, the thin sheet-like PSD is likely to represent putative
703
gephyrin complexes forming a layer of interacting “heads”, which connect to putative
704
GABAARs and the postsynaptic membrane through filamentous thin “necks”. This is
705
consistent with the hypothesis that gephyrin molecules form a single layer comprising a
706
hexagonal planar lattice that provides docking sites of GABAARs (Tretter et al., 2012; 31
707
Heine et al., 2013). Such interactions could provide a stable structural matrix to ensure
708
efficient synaptic transmission.
709
On the presynaptic side, fine details of synaptic vesicle organization were revealed,
710
including filamentous structures tethering vesicles to the presynaptic membrane, as
711
observed previously in slices using ET with HPF-FS (Siksou et al., 2007), as well as in
712
slices and isolated synaptosomes using cryoET (Fernandez-Busnadiego et al., 2010;
713
Fernandez-Busnadiego et al., 2013). Of considerable interest is the presence of
714
discus-shaped ellipsoidal vesicles in both excitatory and inhibitory synapses.
715
“Pleomorphic” vesicles have been observed since early EM studies of the synapse, and
716
were generally considered an indicator of inhibitory synapses (Uchizono, 1965). The
717
nature of such vesicles has been debated as more recent studies indicated that their
718
occurrence was associated with specific conditions of sample processing (Tatsuoka and
719
Reese, 1989; Peters and Palay, 1996; Korogod et al., 2015). Although synapses of
720
cultured hippocampal neurons may have different characteristics compared to mature
721
synapses in brain slices, our results suggest that ellipsoidal vesicles could exist in both
722
excitatory and inhibitory native synapses, but their existence may not be used as a
723
definitive criterion to classify synapse types.
724
A technological advantage of our approach was the ability to use cryoCLEM for
725
unambiguous identification of excitatory and inhibitory synapse types. This is potentially
726
extendable to broader applications especially in light of functional heterogeneity of
727
synapses in neuronal circuits (Dobrunz and Stevens, 1997; Bi and Poo, 2001; Craig and
728
Boudin, 2001; Vogels and Abbott, 2009; Letellier et al., 2016). Compared to immuno-EM
729
labeling, the use of fluorescent protein tagging ensured high label density, while avoiding
730
additional staining steps that can cause significant structural distortions and structural 32
731
artifacts. The platform we developed here uses the same EM cryo-holder for shuttling the
732
sample between the light and electron microscopes. Besides being more convenient for
733
reliable correlation between light microscopy and EM, this method also avoids repeated
734
grid transfers, thus protecting the sample from potential damage and contamination. This
735
together with our method for accurate correlation between light and electron microscopy
736
greatly improved the efficiency of our approach, and was key to the success of our
737
cryoCLEM experiments.
738
Another advantage of our approach is that direct plunge-freezing of neurons cultured
739
on EM grids at low density prevents unwanted disturbance to the synapses unavoidable
740
in the synaptosome preparations previously used for cryoET studies
741
(Fernandez-Busnadiego et al., 2010; Shi et al., 2014; Perez de Arce et al., 2015).
742
Therefore, the ultrastructure of intact synapses can be preserved near their physiological
743
state. However, plunge-freezing is limited to monolayer cultured neurons and synapses
744
that are no more than a few hundred nanometer thick. High-pressure freezing and
745
cryo-sectioning could provide an appropriate tool to extend cryoET to native circuits in
746
brain tissue (Zuber et al., 2005). By implementing the latest cryoET technologies
747
including VPP, electron energy filter and direct electron detection, which greatly improve
748
resolution and signal-to-noise ratio (Danev et al., 2014; Fukuda et al., 2015), we were
749
able to visualize synaptic ultrastructural features. Individual molecules in the synapses,
750
such as GABAA receptors that were previously not accessible, could be identified,
751
localized and counted, thus providing a straightforward way to study key synaptic proteins
752
in individual synapse. With further technical improvements along the lines outlined here,
753
future studies with 3D classification and sub-tomogram averaging could identify additional
33
754
synaptic proteins more confidently, and reveal finer structural details of protein
755
complexes in situ.
756
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Figure/Movie Legends
965
Figure 1. Imaging primary rat hippocampal neurons with cryoET / cryoCLEM. (A)
966
Illustration of the workflow of cryoET/cryoCLEM imaging of neurons grown on gold EM
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grids. (B) Representative results from different stages of the workflow. (B1) Light
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microscopy image of cultured neurons (red arrows indicating cell bodies). (B2) CryoEM
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image of neuronal processes in one grid square. (B3) A single cryoEM projection image
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of the boxed area in (B2) showing a synapse-like structure with a presynaptic bouton
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(Bouton) containing a dense population of SVs (green circles), a postsynaptic spine
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(Spine), and a relatively uniform cleft (yellow arrow). Inset shows a zoomed-in view of
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the synaptic bouton area with a dense population of SVs. (B4) A tomographic slice
974
showing fine structure of the same synapse in (B3), which was identified as a spine
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synapse by following through the tomogram in 3D, with mitochondrion (Mit),
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microtubules (MT), and SVs (green circles) and superposed with segmented presynaptic
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membrane (green) and postsynaptic membrane (red). Inset shows a zoomed-in view of
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the synaptic cleft area with transcleft structures. (C) Schematics depicting main
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components of cryo-fluorescence light microscope with an EM cryo-holder. (D) Pipeline 41
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of imaging synapse with cryoCLEM. (D1) Merged cryo-fluorescence and cryo-bright-field
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light images. (D2) Low magnification cryoEM image including the same grid square. (D3)
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Merged images of boxed area in (D1) and (D2) after fine alignment. (D4) Tomographic
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slice of the boxed area in (D3) superimposed with aligned fluorescence image showing
984
the structure of a synapse with a green fluorescent punctum.
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Figure 2. Synapses of various sizes, shapes and ultrastructural details imaged
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with cryoET. (A-C) Three tomographic slices showing structures of different synapses.
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In the synapses, structures such as SVs and dense core vesicle in presynaptic boutons
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(Bouton), microtubules (MT) in boutons and dendritic shaft (Shaft), mitochondria (Mit) in
990
presynaptic bouton and postsynaptic spine (Spine) are clearly visible. (A1-C1)
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Zoomed-in views of corresponding boxed areas from (A-C) showing thick (dashed
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parallel lines in (A1 and B1)) and thin (dashed parallel lines in (C1)) PSDs, as well as
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SVs attached (cyan arrowheads in (A1)) or fused (pink arrowheads in (B1)) to the
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presynaptic membrane. (D and E) Two synapses sharing the same presynaptic axon
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(determined by following through their tomograms in 3D), both with thick PSDs (D1 and
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D2) or both with thin PSDs (E1 and E2), respectively. (F and G) Two synapses sharing
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the same postsynaptic spine, both with thick PSDs (F1 and F2), or one with thin PSD
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(G1) and the other with thick PSD (G2).
999 42
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Figure 3. Identification of excitatory and inhibitory synapses with cryoCLEM. (A
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and B) Tomographic slices of an excitatory (A) and inhibitory (B) synapse colocalized
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with PSD-95-EGFP and mCherry-gephyrin puncta, respectively. (A1 and B1) Zoomed-in
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views of the boxed area in (A) and (B) showing the synapse with thick and thin PSD,
1004
respectively. Red dashed lines indicate the range of the PSD.
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Figure 4. Quantitative and statistical characterization of excitatory and inhibitory
1007
PSDs. (A and B) Tomographic slices of two synapses with thick and thin PSD
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respectively. (A1 and B1) Zoomed-in views of the marked areas in (A) and (B). (A2 and
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B2) Normalized density profiles of the two synapses in (A) and (B) respectively with
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cross-sectional mean density plotted against distance to postsynaptic membrane (see
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Materials and Methods). On the x-axis of this plot, 0 was set to be the position of the
1012
postsynaptic membrane, and positive values are on the postsynaptic side. The density
1013
profiles were normalized against the density values at distance ranging from 100 to 200
1014
nm such that the average density value in this range is zero and their standard deviation
1015
is unity. d1: PSD peak position, d2 is the sum of d1 and the length constant obtained
1016
from the exponential fit of the profile from d1 to the flattened background, to provide a
1017
measure of the thickness of the PSD (see Materials and Methods). (C) Scatter plot of
1018
PSD peak position and PSD thickness of all synapses show two well-defined clusters. (D)
1019
Histogram shows the PSD thickness distribution of all excitatory and inhibitory synapses 43
1020
respectively. (E) Averaged density curve of all excitatory synapses and all inhibitory
1021
synapses respectively.
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Figure 5. Synapses without visible PSDs. (A) A 15 nm-thick tomographic slice of an
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unidentified synapse imaged by cryoET only. (B and C) A 15 nm-thick tomographic slice
1025
of an excitatory (B) and an inhibitory (C) synapse identified by cryoCLEM superposed
1026
with the fluorescence image of colocalized PSD-95-EGFP and mCherry-gephyrin,
1027
respectively. (A1-C1) Zoomed-in views of the boxed area in (A-C) respectively showing
1028
that no PSD structures are visible in these synapses.
1029 1030
Figure 6. Heterogeneity of synaptic vesicles in excitatory and inhibitory synapses.
1031
(A and B) Tomographic slices of an excitatory and an inhibitory synapse respectively.
1032
Insets are zoomed-in views showing thick and thin PSDs from (A) and (B) respectively.
1033
(C) Scatter plot showing the major and minor axes of SVs in the two synapses in (A and
1034
B) measured by 2D fitting. (D) Distribution of vesicle sizes in excitatory and inhibitory
1035
synapses (16,476 vesicles in 35 excitatory synapses and 4,766 vesicles in 15 inhibitory
1036
synapses). (E) Distribution of ellipticity of SVs (major to minor axis ratio) in excitatory and
1037
inhibitory synapses. A threshold (dashed line) was set at major/minor = 1.14, which is
1038
about twice the peak position (major/minor = 1.07) from perfect circle (major/minor = 1)
1039
to separate ellipsoidal from spherical vesicles. Coincidentally, this threshold is also close 44
1040
to the cross point of the two distribution curves. (F) Cumulative frequency of the fraction
1041
of ellipsoidal vesicles in excitatory and inhibitory synapses. (G) Tomographic slices of
1042
spherical (left column), discus-shaped (i.e., oblate spheroid, middle column) and
1043
olive-shaped (i.e., prolate spheroid, right column) SVs viewed in three orthogonal planes
1044
that were rotated so that each of the three principle axes (a, b, and c) of the vesicles can be
1045
measured horizontally in the corresponding plane. For the spherical vesicles, aҮbҮc; for
1046
discus-shaped ones, a
