This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Articles: Cellular/Molecular Differentiation and characterization of excitatory and inhibitory synapses by cryo-electron tomography and correlative microscopy Chang-Lu Tao1, Yun-Tao Liu1, Rong Sun1, Bin Zhang2, Lei Qi2, Sakar Shivakoti1, Chong-Li Tian2, Peijun Zhang3, Pak-Ming Lau2, Z. Hong Zhou1,4,5 and Guo-Qiang Bi1,6 1National Laboratory for Physical Sciences at the Microscale, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China 2CAS Key Laboratory of Brain Function and Disease, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China 3Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive Oxford OX37BN, UK 4The California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA 5Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA 6CAS 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. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 Differentiation and characterization of excitatory and inhibitory synapses by 2 cryo-electron tomography and correlative microscopy 3 4 Abbreviated title: Visualizing synapse types in 3D by cryoCLEM 5 6 Chang-Lu Tao1,*, Yun-Tao Liu1,*, Rong Sun1, Bin Zhang2, Lei Qi2, Sakar Shivakoti1, 7 Chong-Li Tian2, Peijun Zhang3, Pak-Ming Lau2, Z. Hong Zhou1,4,5,#, Guo-Qiang Bi1,6,# 8 1National Laboratory for Physical Sciences at the Microscale, and School of Life Sciences, 9 University of Science and Technology of China, Hefei, Anhui 230026, China; 2CAS Key 10 Laboratory of Brain Function and Disease, and School of Life Sciences, University of 11 Science and Technology of China, Hefei, Anhui 230026, China; 3Division of Structural 12 Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt 13 Drive Oxford OX37BN, UK; 4The California NanoSystems Institute, University of 14 California, Los Angeles, CA 90095, USA; 5Department of Microbiology, Immunology and 15 Molecular Genetics, University of California, Los Angeles, CA 90095, USA; 6CAS Center 16 for Excellence in Brain Science and Intelligence Technology, and Innovation Center for 17 Cell Signaling Network, University of Science and Technology of China, Hefei, Anhui 18 230026, China 19 *These authors contributed equally to this work. 20 #Correspondence: Guo-Qiang Bi, School of Life Science, USTC, 443 Huangshan Rd, 21 Hefei, China, 230027. E-mail: [email protected] and Z. Hong Zhou, The California 22 NanoSystems Institute, UCLA, 570 Westwood Plaza, CA 90095, USA. E-mail: 23 [email protected]. 24 Number of text pages: 51; Number of figures: 9; Number of movies: 5; 1 25 Number of words: Abstract (215); Significance Statement (118); Introduction (640); 26 Discussion (1207). 27 28 Conflict of Interest: The authors declare no competing financial interests. 29 30 Acknowledgements: This work was supported in part by grants from the CAS 31 (XDB02050000 to GQB), NSFC (30725017 and 91232722 to GQB; 31070935 to PML), 32 MOST (2009CB941300 to GQB), and NIH (GM071940 to ZHZ). We acknowledge use of 33 instruments at the Center for Integrative Imaging of Hefei National Laboratory for Physical 34 Sciences at the Microscale and those at the Electron Imaging Center for Nanomachines 35 of UCLA supported by NIH (S10RR23057 and S10OD018111) and NSF (DBI-133813). 36 We thank Xiaokang Zhang and Peng Ge for technical advice on imaging and data 37 processing, Jay He for help with design and construction of the cryoCLEM platform, 38 Weidong Yao and Ann Marie Craig for sharing the PSD-95 and mCherrry-gephyrin 39 plasmids, Xiaobing Chen for insightful discussions, Chunhong Qiu for help with 40 illustrations, and the anonymous reviewers for their insightful comments and constructive 41 suggestions to improve the paper. 42 43 2 44 Abstract 45 As key functional units in neural circuits, different types of neuronal synapses play distinct 46 roles in brain information processing, learning and memory. Synaptic abnormalities are 47 believed to underlie various neurological and psychiatric disorders. Here, by combining 48 cryo-electron tomography and cryo-correlative light and electron microscopy, we 49 distinguished intact excitatory and inhibitory synapses of cultured hippocampal neurons, 50 and visualized the in situ three-dimensional organization of synaptic organelles and 51 macromolecules in their native state. Quantitative analyses of over a hundred synaptic 52 tomograms reveal that excitatory synapses contain a mesh-like postsynaptic density 53 (PSD) with thickness ranging from 20-50 nm. In contrast, the PSD in inhibitory synapses 54 assumes a thin sheet-like structure ~12 nm from the postsynaptic membrane. On the 55 presynaptic side, spherical synaptic vesicles (SVs) of 25-60 nm diameter and 56 discus-shaped ellipsoidal SVs of various sizes coexist in both synaptic types, with more 57 ellipsoidal ones in inhibitory synapses. High-resolution tomograms obtained using a Volta 58 phase plate and electron filtering and counting reveal glutamate receptor-like and GABAA 59 receptor-like structures that interact with putative scaffolding and adhesion molecules, 60 reflecting details of receptor anchoring and PSD organization. These results provide an 61 updated view of the ultrastructure of excitatory and inhibitory synapses, and demonstrate 62 the potential of our approach to gain insight into the organizational principles of cellular 63 architecture underlying distinct synaptic functions. 64 3 65 Significance Statement 66 To understand functional properties of neuronal synapses, it is desirable to analyze their 67 structure at molecular resolution. We have developed an integrative approach combining 68 cryoET and correlative fluorescence microscopy to visualize 3D ultrastructural features of 69 intact excitatory and inhibitory synapses in their native state. Our approach shows that 70 inhibitory synapses contain uniform thin sheet-like PSDs, while excitatory synapses 71 contain previously known mesh-like PSDs. We discovered “discus-shaped” ellipsoidal 72 synaptic vesicles, and their distributions along with regular spherical vesicles in synaptic 73 types are characterized. High-resolution tomograms further allowed identification of 74 putative neurotransmitter receptors and their heterogeneous interaction with synaptic 75 scaffolding proteins. The specificity and resolution of our approach enables precise in situ 76 analysis of ultrastructural organization underlying distinct synaptic functions. 77 4 78 Introduction 79 Chemical synapses are basic functional units in neural circuits for information 80 transmission, processing and storage (Eccles, 1964; Sudhof and Malenka, 2008; Mayford 81 et al., 2012). From the enormous number of synapses in the brain, the plasticity of each 82 synapse, and the molecular and functional diversity across these synapses, arise the 83 brain’s remarkable computational power and cognitive capacity (Milner et al., 1998; Bi 84 and Poo, 2001). Glutamatergic and GABAergic synapses, the two main types of central 85 synapses, play opposite roles in excitation and inhibition. They have been shown by 86 biochemical and electrophysiological