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Neuromethods 106 Gabriela K. Popescu Editor Ionotropic Glutamate Receptor Technologies N EUROMETHODS Series Editor Wolfgang Walz University of Saskatchewan Saskatoon, SK, Canada For further volumes: h ttp://www.springer.com/series/7657 Ionotropic Glutamate Receptor Technologies Edited by Gabriela K. Popescu Department of Biochemistry, University at Buffalo, Buffalo, NY, USA Editor Gabriela K. P opescu Department of Biochemistry University at Buffalo Buffalo, NY , U SA ISSN 0893-2336 ISSN 1940-6045 (electronic) Neuromethods ISBN 978-1-4939-2811-8 ISBN 978-1-4939-2812-5 (eBook) DOI 10.1007/978-1-4939-2812-5 Library of Congress Control Number: 2015944063 Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2 016 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. Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com) Series P reface Experimental life sciences have two basic foundations: concepts and tools. The N euromethods series focuses on the tools and techniques unique to the investigation of the nervous system and excitable cells. It will not, however, shortchange the concept side of things as care has been taken to integrate these tools within the context of the concepts and questions under investigation. In this way, the series is unique in that it not only collects protocols but also includes theoretical background information and critiques which led to the methods and their development. Thus it gives the reader a better understanding of the origin of the techniques and their potential future development. The N euromethods publishing program strikes a balance between recent and exciting developments like those concerning new ani- mal models of disease, imaging, in vivo methods, and more established techniques, including, for example, immunocytochemistry and electrophysiological technologies. New trainees in neurosciences still need a sound footing in these older methods in order to apply a critical approach to their results. Under the guidance of its founders, Alan Boulton and Glen Baker, the N euromethods series has been a success since its fi rst volume published through Humana Press in 1985. The series continues to fl ourish through many changes over the years. It is now published under the umbrella of Springer Protocols. While methods involving brain research have changed a lot since the series started, the publishing environment and technology have changed even more radically. N euromethods has the distinct layout and style of the Springer Protocols program, designed specifi cally for readability and ease of reference in a laboratory setting. The careful application of methods is potentially the most important step in the process of scientifi c inquiry. In the past, new methodologies led the way in developing new disciplines in the biological and medical sciences. For example, Physiology emerged out of Anatomy in the nineteenth century by harnessing new methods based on the newly discovered phenomenon of electricity. Nowadays, the relationships between disciplines and methods are more complex. Methods are now widely shared between disciplines and research areas. New developments in electronic publishing make it possible for scientists that encounter new methods to quickly fi nd sources of information electronically. The design of individual volumes and chapters in this series takes this new access technology into account. Springer Protocols makes it possible to download single protocols separately. In addition, Springer makes its print-on-demand technology available globally. A print copy can therefore be acquired quickly and for a competitive price anywhere in the world. Wolfgang Walz v Foreword Ionotropic glutamate receptors (iGluRs) are glutamate-gated ion channels that mediate fast excitatory neurotransmission in the central nervous system (CNS). They mediate the nor- mal development and function of the CNS and play critical roles in numerous neurologic and psychiatric disorders. The already high and increasing burden of neurodegenerative diseases across the world has prompted national interest to better understand the structure and function of the nervous system. This ambitious desideratum will require more advanced knowledge of the structure, function, and biological activities of iGluRs. The fi eld of ligand-gated ion channels has its roots in the pharmacologic investigations of excitable tissues in the second part of the nineteenth century. The observations that drugs and poisons (atropine, nicotine, morphine, etc.) have dramatic effects on the electrical properties of tissues and organs (heartbeat, muscle twitch, etc.) were most parsimoni- ously explained by postulating the existence of chemically receptive proteins that form transmembrane pores. Since then, the evolution of theories and concepts in the fi eld of ligand-activated channels has followed closely the development of technologies to apply and withdraw ligands (perfusion techniques), to record and interpret electrical signals (electrophysiology), and to access increasingly diverse biological preparations (tissue cul- ture, molecular biology). Many of these concepts and theories were developed in the fi rst part of the twentieth century, in advance of the fi rst direct observation of a single-channel current and before the cells of the mammalian central nervous system were amenable to direct experimental investigation. In fact, these concepts evolved primarily from observations of the more easily accessible nerve-muscle synapse; because the ion channel respon- sible for the endplate current is the muscle acetylcholine receptor, to this day, in many textbooks and reviews, the term ligand-gated ion channel often refers to this founding receptor and its family of pentameric ligand-gated ion channels, despite the growing diver- sity of proteins in this class. After the Second World War, two simultaneous technologic advances propelled the glutamate-gated ion channels to the experimentally accessible range. First, increased knowledge on how to prepare and maintain explants of central nervous tissue made it progressively feasible to investigate their molecular and cellular properties. In parallel, methods to record and measure electrical currents across biological membranes—fi rst with sharp intracellular electrodes and then with patch-clamp microelectrodes—made it possible to observe and record the electrical properties of much smaller cells in the manner already used for the neuromuscular junction. With what was at the time the state of the art in electrophysiology, it was repeatedly demonstrated that glutamate and related amino acids excited central neurons. In fact, kainate, a glutamate analogue, had been a well-known neurotoxin and was used by neuroanatomists to trace neuronal circuits by staining degenerating axons. Still, up until the early 1980s, the pervasive thought was that glutamate is too important a metabolite, and its concentration in brain too high, for it to possibly be a neurotransmitter, and many scientists speculated that its excitatory effects had no physiologic meaning. More than two decades of careful pharmacologic vii viii Foreword and neurophysiologic investigations were necessary to slowly bring into unanimous acceptance that in the CNS excitatory postsynaptic currents are largely the result of iGluR activations. The molecular biology revolution of the late twentieth century allowed for the fi rst time the preparation and thus the functional investigation of proteins of defi ned molecular identity. It also exposed an unanticipated multiplicity of ligand-gated channels, which are organized not only as the prototypical acetylcholine receptor as pentamers but also as tri-, tetra-, or hexameric proteins. Furthermore, it became clear that ligand-gated channels are not only expressed at synapses and on the surface of cells but also embedded in virtually every biological membrane. For iGluRs, as well, this has been a particularly exciting era. It was demonstrated during this time that functional iGluRs assemble as homo- or hetero- tetramers of homologous subunits, and the subsequent decoding of several genomes has established that mammalian iGluRs assemble from a family of 18 homologous subunits. These cluster further into three classes or types, corresponding to the already established pharmacologically based nomenclature as AMPA, kainate, and NMDA receptors. The current volume compiles methods that have afforded important conceptual advances in the iGluR fi eld within the past decade. Among these recent developments per- haps the most spectacular are several atomic structures for functional AMPA and NMDA receptors, and parts of kainate receptors. Along with these new structural benchmarks, modern means of identifying and sorting intramolecular motions seek to associate conformational changes with state lifetimes and ultimately with functional output. Thus, a com- mon goal is to organize observed structural changes into a coherent chronologic sequence that narrates the molecular trajectories that produce function. Zooming out from the atomic to the molecular and cellular levels, methodologic advances described in this volume expose mechanisms that control receptor assembly, oligomerization, expression, and traffi cking, and provide approaches to identify or count molecular assemblies expressed on cells or at specifi c synapses. Lastly, the volume would have been incomplete without giving a modern account of classic electrophysiologic approaches that evaluate receptor function following mutagenesis, pharmacologic treatment, and a variety of stimulation protocols, whether for recombinant or native receptors. Taken together the chapters in this volume outline the contemporary landscape of iGluR technologies. They highlight exciting advances in the fi eld in a manner designed to facilitate additional investigations along these newly forged tracks. Necessarily, they also illustrate that progress has been uneven across the three classes of iGluRs, most likely due to the specifi c experimental challenges associated with each receptor type. By assisting new and established investigators to adopt these technologies, the present volume may expedite the development of the next generation of approaches and techniques to produce a com- prehensive understanding of how iGluRs work to fulfi ll their essential biologic functions in the CNS. Buffalo, NY, USA Gabriela K. Popescu Prefa ce Glutamate is the principal excitatory neurotransmitter in the brain and spinal cord and its rapid action at more than 90 % of central synapses occurs through membrane receptors of the ionotropic glutamate receptor (iGluR) family. Since their molecular cloning in the early 1990s, the number of PubMed indexed articles focusing on these receptors’ structure, function, and role in health and disease has exploded in the 1990s and has held steady during the past 15 years at ~2,500 publications per year, with no sign of a decline. This large number of publications refl ects the constant and substantial advances in our collective understanding of these receptors but also the development of new technologies that allow scientists to address gaps in knowledge in this area. Increasingly, scientifi c jour- nals that report primary research have moved to enforcing page limits for the articles they are willing to review and publish. This fact has resulted in deliberate abbreviation of the Methods section, usually accomplished by extensive referencing of previous literature or by relegating a major part of this section to Supplementary Material. This practice has made it cumbersome to follow the technical procedures and quite diffi cult to implement these in a lab with no previous experience with the particular technique. The chapters in this volume of N euromethods describe techniques, methods, and approaches that are either specifi c to iGluRs or have advanced the fi eld signifi cantly in recent years. They are intended as detailed practical guides that will facilitate the implementation of these technologies in new or established laboratories. Despite the critical roles of iGluRs in health and disease, much remains unknown about the operation, modulation, and the biological functions of iGluRs. The development, maintenance, and experience-dependent plasticity of excitatory CNS synapses depend criti- cally on the activity of iGluRs; and iGluRs participate in fundamental aspects of development and behavior including learning and memory, information processing, and cognition. In addition, iGluRs mediate glutamate neurotoxicity, a key component of pathology in a number of neurodegenerative conditions. Newly delineated atomic models of functional iGluRs have galvanized the fi eld with new information that had been previously diffi cult to obtain and have formulated new questions in iGluR research. In addition, recent national initiatives into the structure and function of the brain are sure to increase the demand for accessible techniques to evaluate the structure, function, and physiologic contributions of iGluRs. Helping investigators to implement successfully iGluR-specifi c methods will accel- erate the pace of discovery in this important scientifi c area. This volume compiles practical guides, organized as chapters, to technologies that are used currently to investigate iGluR structure, function, and physiology. Chapters focus on a particular approach that has been proven successful in revealing fundamental aspects of iGluRs’ involvement with health and disease. The fi rst section includes methods that can help illuminate the assembly, traffi cking, molecular composition, and subcellular location of iGluRs. The second section describes approaches used to understand the atomic organiza- tion of iGluRs and the intramolecular motions associated with function. The last section provides techniques to monitor receptor activity in real time, whether from single m olecules or receptor populations, and approaches to assemble a storyboard of conformational ix