Methods in Molecular Biology 1538 Alexandros Poulopoulos Editor Synapse Development Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB , UK For further volumes: http://www.springer.com/series/7651 Synapse Development Methods and Protocols Edited by Alexandros Poulopoulos Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Editor Alexandros Poulopoulos Department of Stem Cell and Regenerative Biology Harvard University Cambridge, MA, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6686-8 ISBN 978-1-4939-6688-2 (eBook) DOI 10.1007/978-1-4939-6688-2 Library of Congress Control Number: 2016956874 © Springer Science+Business Media LLC 2 017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Cover Illustration: Cover image design by Massimiliano Stagi Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A. Prefa ce This book is a collection of protocols for experimentation into how neurons connect to produce the extraordinary functionalities of the nervous system. The complexity underly- ing the development of synapses allows and warrants an assortment of approaches to probe the many layers of structure, mechanism, and emergent function. Appropriately, neurosci- ence is populated by researchers across disciplines, and with them comes a range of tech- nologies and methodologies that make the neuroscience toolkit one of characteristic wealth. The experimental diversity converging onto the synapse has made it one of the most intensely studied and intricately described biological systems. Observing in real-time how a single protein fl ips between conformational states to regulate ion fl ows, following how thin cellular protrusions traverse the body to form networks of the highest complexity seen in nature, measuring how circuits interact and morph throughout the span of life to docu- ment its story, our understanding of synapse development is arguably unparalleled in its pluralism of perspective. Yet it seems the more detailed our descriptions, the more expan- sive their heterogeneity and the less inclusive the models. Depending on the system in ques- tion, synaptogenic behaviors range anywhere from the preordained pairings of Sperrian chemoaffi nity to the play-by-ear modes of promiscuous exuberance and subsequent refi ne- ment. Plasticity in turn occurs up or down, fl eeting or perduring, and is stirred on by mol- ecules, voltages, and timing. While thankfully all these serve to construct a system able to produce both hiccups and quantum theory, they also do away with comfy notions of a generalizable synapse awaiting full description. The challenge is on, and the design of clever new experimentation ever more opportune. In putting together this volume, we’ve had the aim of compiling a laboratory guide that covers a broad spectrum of applicable methods (largely excluding electrophysiology , which has merited its own dedicated volumes in this series), with the benefi t of a narrower focus of implementation to the study of synapse development. I’ve asked experts to present the current state of the art of classic protocols, giving their insider tweaks, tips, and tricks for optimal application to the synapse and its particularities. I’ve also asked pioneers of highly specialized approaches to contribute detailed guides to methods utilizing sophisti- cated instrumentation that is often institutionally available to labs but for which method- ological expertise is diffi cult to come by. These 23 chapters by 63 investigators are presented in fi ve parts that deal with synapses: (1) in culture, (2) their constituents, (3) their struc- tures, (4) their dynamics, and (5) the assemblies they form. Our hope is that these guides will minimize the barrier to entry for the integration of new approaches with existing exper- tise, producing syntheses that will foster novel perspectives on the many ways in which synapses form, transform, and transmit. Cambridge, MA, USA Alexandros Poulopoulos v Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x PART I FORMING SYNAPSES IN A DISH 1 A Cell Culture System to Investigate the Presynaptic Control of Subsynaptic Membrane Differentiation at the Neuromuscular Junction. . . . 3 Nadine Schmidt , S reya Basu , S tephan Kröger , and Hans Rudolf Brenner 2 C o-culture Synaptogenic Assay: A New Look at Fluorescence Reporters and Technological Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 Karen Perez de Arce and Massimiliano Stagi 3 S ynaptogenic Assays Using Neurons Cultured on Micropatterned Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 9 Katalin Czöndör and Olivier Thoumine 4 M onitoring Synapses Via Trans-Synaptic GFP Complementation. . . . . . . . . . . 4 5 Theodoros Tsetsenis 5 G eneration of Spinal Motor Neurons from Human Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 David P. Santos and Evangelos Kiskinis PART II ANALYZING SYNAPTIC CONSTITUENTS 6 B iochemical Purification of Binding Partners of Synaptic Scaffold Proteins . . . 6 9 Bekir Altas , O laf Jahn , and Hiroshi Kawabe 7 I n Situ Protein Binding Assay Using Fc-Fusion Proteins . . . . . . . . . . . . . . . . . 8 3 Nirmala Padmanabhan and Tabrez J. Siddiqui 8 R econstitution of Synaptic SNAREs into Large Liposomes with Reduced Curvature Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 Javier M. Hernandez 9 I solation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Mary L. Michaelis , L ei Jiang , and Elias K. Michaelis 10 P urification of Synaptosome Populations Using Fluorescence-Activated Synaptosome Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Elisa Luquet , C hristoph Biesemann , A nnie Munier , and Etienne Herzog PART III OBSERVING SYNAPTIC STRUCTURE AND ULTRASTRUCTURE 11 O ptimized Protocol for Imaging Cleared Neural Tissues Using Light Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 37 Yoh Isogai , D ouglas S. Richardson , C atherine Dulac , and Joseph Bergan vii viii Contents 12 S tructured Illumination Microscopy for the Investigation of Synaptic Structure and Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Soyon Hong , D aniel K. Wilton , B eth Stevens , and Douglas S. Richardson 13 3 D d STORM Imaging of Fixed Brain Tissue . . . . . . . . . . . . . . . . . . . . . . . . . 1 69 Frank Herrmannsdörfer , B enjamin Flottmann , S iddarth Nanguneri , Varun Venkataramani , H einz Horstmann , T homas Kuner , and Mike Heilemann 14 P hotomarking Relocalization Technique for Correlated Two-Photon and Electron Microcopy Imaging of Single Stimulated Synapses . . . . . . . . . . . 1 85 Miquel Bosch , J orge Castro , M riganka Sur , and Yasunori Hayashi 15 3 D Analysis of Synaptic Ultrastructure in Organotypic Hippocampal Slice Culture by High-Pressure Freezing and Electron Tomography . . . . . . . . . . . . 215 Cordelia I mig and Benjamin H. Cooper PART IV FOLLOWING SYNAPSE DYNAMICS 16 A nalyzing Endosomal Docking, Fusion, Sorting, and Budding Mechanisms in Isolated Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Sina V. Barysch and Ioanna Bethani 17 C oncurrent Imaging of Receptor Trafficking and Calcium Dynamics by Spinning Disk Confocal Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 DeLaine D. Larsen , R egina Wai-Yan Choy , and Minjong Park 18 I maging Activity-Dependent Signaling Dynamics at the Neuronal Synapse Using FRET-Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 61 Zohreh Farsi and Andrew Woehler 19 A nalyzing Structural Plasticity of Dendritic Spines in Organotypic Slice Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Mathias De Roo and Adema Ribic PART V REVEALING PATTERNS OF SYNAPTIC CONNECTIVITY 20 U sing Fluorescent Markers to Estimate Synaptic Connectivity In Situ . . . . . . . 293 Mrinalini Hoon , R aunak Sinha , and Haruhisa Okawa 21 D ual Anterograde and Retrograde Viral Tracing of Reciprocal Connectivity . . . . 3 21 Matthias G. Haberl , M elanie Ginger , and Andreas Frick 22 M apping Synaptic Inputs of Developing Neurons Using Calcium Imaging . . . . . 3 41 Johan Winnubst and Christian Lohmann 23 M onosynaptic Tracing in Developing Circuits Using Modified Rabies Virus . . . . 3 53 Laura Cocas and Gloria Fernandez Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 67 Contributors BEKIR ALTAS • Department of Molecular Neurobiology, M ax Planck Institute of Experimental Medicine , G öttingen, G ermany SINA V. BARYSCH • Zentrum für Molekulare Biologie Heidelberg (ZMBH), DKFZ-Z MBH Alliance , H eidelberg, G ermany SREYA BASU • Department of Cell Biology and Genetics, E rasmus MC , R otterdam , The Netherlands JOSEPH BERGAN • Department of Psychological and Brain Sciences, U niversity of Massachusetts , A mherst , M A, U SA IOANNA BETHANI • Department of Neurobiology, M ax Planck Institute for Biophysical Chemistry , G öttingen, G ermany CHRISTOPH BIESEMANN • Department of Molecular Neurobiology, M ax Planck Institute of Experimental Medicine , G öttingen, G ermany MIQUEL BOSCH • Institute for Bioengineering of Catalonia (IBEC) , B arcelona, S pain HANS RUDOLF BRENNER • Department of Biomedicine, U niversity of Basel , B asel, Switzerland JORGE CASTRO • Department of Brain and Cognitive Sciences, The Picower Institute for Learning and Memory , M assachusetts Institute of Technology , C ambridge, M A, U SA REGINA WAI-YAN CHOY • Department of Psychiatry, School of Medicine, U niversity of California , S an Francisco, C A , U SA LAURA COCAS • Department of Neurology, University of California , San Francisco, CA , USA BENJAMIN H. COOPER • Department of Molecular Neurobiology, M ax Planck Institute of Experimental Medicine , G öttingen, G ermany KATALIN CZÖNDÖR • Interdisciplinary Institute for Neuroscience , U niversity of Bordeaux and CNRS , B ordeaux, F rance ; Interdisciplinary Institute for Neuroscience, CNRS, UMR 5297, Bordeaux, France MATHIAS DE ROO • Department of Neuroscience, School of Medicine , U niversity of Geneva , Geneva, S witzerland CATHERINE DULAC • Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, H arvard University , C ambridge, M A, U SA ZOHREH FARSI • Department of Neurobiology, M ax Planck Institute for Biophysical Chemistry , G öttingen, G ermany GLORIA FERNANDEZ • Department of Biological Sciences, C alifornia State University , Los Angeles, C A , U SA BENJAMIN FLOTTMANN • Institute of Physical and Theoretical Chemistry , G oethe-U niversity Frankfurt , F rankfurt, G ermany ANDREAS FRICK • Neurocentre Magendie, I NSERM , B ordeaux, F rance ; U niversity of Bordeaux , Bordeaux, F rance MELANIE GINGER • Neurocentre Magendie , I NSERM , B ordeaux, F rance ; U niversity of Bordeaux , B ordeaux, F rance ix x Contributors MATTHIAS G. HABERL • Neurocentre Magendie , I NSERM , B ordeaux, F rance ; U niversity of Bordeaux , B ordeaux, F rance YASUNORI HAYASHI • Brain Science Institute, RIKEN , W ako , J apan MIKE HEILEMANN • Institute of Physical and Theoretical Chemistry , G oethe-University Frankfurt , F rankfurt, G ermany JAVIER M. HERNANDEZ • Department of Structural Biochemistry, Max Planck Institute for Molecular Physiology, Dortmund, Germany FRANK HERRMANNSDÖRFER • Department of Functional Neuroanatomy, H eidelberg University , H eidelberg, G ermany ETIENNE HERZOG • Interdisciplinary Institute for Neuroscience , U niversity of Bordeaux , Bordeaux, F rance ; I nterdisciplinary Institute for NeuroScience, CNRS , B ordeaux, France SOYON HONG • Department of Neurology, F.M. Kirby Neurobiology Center , B oston Children’s Hospital, Harvard Medical School , B oston , M A, U SA ; Program in Neuroscience, Harvard Medical School, Boston, MA, USA MRINALINI HOON • Department of Biological Structure, U niversity of Washington , S eattle , WA, U SA HEINZ HORSTMANN • Department of Functional Neuroanatomy, H eidelberg University , Heidelberg, G ermany CORDELIA IMIG • Department of Molecular Neurobiology, M ax Planck Institute of Experimental Medicine , G öttingen, G ermany YOH ISOGAI • Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA; Sainsbury Wellcome Centre for Neural Circuits and Behaviour , U niversity College London , L ondon , U K OLAF JAHN • Proteomics Group , M ax Planck Institute of Experimental Medicine , Göttingen, G ermany LEI JIANG • Higuchi Biosciences Center, U niversity of Kansas , L awrence, K S , U SA HIROSHI KAWABE • Department of Molecular Neurobiology, M ax Planck Institute of Experimental Medicine , G öttingen, G ermany EVANGELOS KISKINIS • The Ken & Ruth Davee Department of Neurology & Clinical Neurological Sciences, Department of Physiology, Feinberg School of Medicine, N orthwestern University , C hicago, I L, U SA STEPHAN KÖGER • Department of Physiological Genomics, L udwig Maximilians University , Munich, G ermany THOMAS KUNER • Department of Functional Neuroanatomy, H eidelberg University , Heidelberg, G ermany DELAINE D. LARSEN • Department of Biochemistry and Biophysics, School of Medicine, University of California , S an Francisco, C A , U SA CHRISTIAN LOHMANN • Department of Synapse and Network Development, N etherlands Institute for Neuroscience , A msterdam, T he Netherlands ELISA LUQUET • Interdisciplinary Institute for Neuroscience , U niversity of Bordeaux , Bordeaux, F rance ; I nterdisciplinary Institute for NeuroScience, CNRS , B ordeaux, France ELIAS K. MICHAELIS • Higuchi Biosciences Center, University of Kansas , Lawrence, KS , USA MARY L. MICHAELIS • Higuchi Biosciences Center, University of Kansas , Lawrence, KS , USA ANNIE MUNIER • Institut de Biologie Paris Seine, U niversity Pierre et Marie Curie , P aris , France SIDDARTH NANGUNERI • Department of Functional Neuroanatomy, H eidelberg University , Heidelberg, G ermany Contributors xi HARUHISA OKAWA • Department of Biological Structure, U niversity of Washington , S eattle , WA, U SA NIRMALA PADMANABHAN • Department of Physiology and Pathophysiology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg , MB, C anada ; N euroscience Research Program, Kleysen Institute for Advanced Medicine, Health Sciences Centre, University of Manitoba , W innipeg, M B, C anada MINJONG PARK • Department of Psychiatry, School of Medicine, U niversity of California , San Francisco, C A , U SA KAREN PEREZ DE ARCE • Department of Neuroscience, School of Medicine , T ufts University , Boston , M A, U SA ADEMA RIBIC • Department of Neuroscience, School of Medicine , T ufts University , B oston , MA, U SA DOUGLAS S. RICHARDSON • Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA; Harvard Center for Biological Imaging, Harvard University, Cambridge, MA, USA DAVID P. SANTOS • The Ken & Ruth Davee Department of Neurology & Clinical Neurological Sciences, Department of Physiology, Feinberg School of Medicine, Northwestern University , C hicago, I L, U SA NADINE SCHMIDT • Department of Physiology II, A lbert-Ludwigs University , F reiburg, Germany TABREZ J. SIDDIQUI • Department of Physiology and Pathophysiology, Max Rady College of Medicine, Rady Faculty of Health Sciences ; U niversity of Manitoba , W innipeg, M B, Canada ; Neuroscience Research Program, Kleysen Institute for Advanced Medicine, Health Sciences Centre, University of Manitoba, Winnipeg, MB RAUNAK SINHA • Department of Physiology and Biophysics, U niversity of Washington , Seattle , W A, U SA MASSIMILIANO STAGI • Department of Cellular and Molecular Physiology, Institute of Translational Medicine, U niversity of Liverpool , L iverpool, U K ; University of Liverpool, Crown Street, Liverpool, UK BETH STEVENS • Department of Neurology, F.M. Kirby Neurobiology Center , Boston Children’s Hospital, Harvard Medical School , B oston , M A, U SA ; Program in Neuroscience, Harvard Medical School, Boston, MA, USA MRIGANKA SUR • Department of Brain and Cognitive Sciences, The Picower Institute for Learning and Memory , M assachusetts Institute of Technology , C ambridge, M A, U SA OLIVIER THOUMINE • Interdisciplinary Institute for Neuroscience , U niversity of Bordeaux , Bordeaux, F rance ; C NRS , B ordeaux, F rance ; Interdisciplinary Institute for Neuroscience, CNRS, UMR 5297, Bordeaux, France THEODOROS TSETSENIS • Department of Neuroscience, Mahoney Institute for Neurosciences, Perelman School for Medicine, U niversity of Pennsylvania , P hiladelphia, P A, U SA VARUN VENKATARAMANI • Department of Functional Neuroanatomy, Heidelberg University , Heidelberg, G ermany DANIEL K. WILTON • Department of Neurology, F.M. Kirby Neurobiology Center , B oston Children’s Hospital, Harvard Medical School , B oston , M A, U SA ; Program in Neuroscience, Harvard Medical School, Boston, MA, USA JOHAN WINNUBST • Janelia Research Campus, H oward Hughes Medical Institute , A shburn, VA, U SA ANDREW WOEHLER • Berlin Institute for Medical Systems Biology, M ax Delbrueck Center for Molecular Medicine in the Helmholtz Association , B erlin-Buch, G ermany