Neuroscience is one of the most dynamic disciplines in biology. The inherent interest of the nervous system is not sufficient to explain the explosion of work in this field. The last decade has seen considerable conceptual progress, but technical advances leading to the introduction of new methods in laboratories across the world have also played a major part in driving the field forward. The techniques for investigating the nervous system that have been developed require familiarity with electrophysiology, mouse genetics, optics, brain anatomy, and many other methods. More than most areas of biology, neuroscience is a multidisciplinary activity. The rapid transfer of technical information and experience is the partner of profound conceptual change. Although it is hard to be comprehensive in this diverse field, our goal in creating this manual is to provide a single source for core techniques in all branches of neuroscience, in the form of an accurate and accessible guide to researchers starting a new experiment. The methods are presented as protocols that can be readily implemented in the lab; in most cases, this book provides a direct statement of the experimental procedures. The basic methods of neuroanatomy and electrophysiology are described here, but they are presented in the context of molecular and cellular neurobiology. In some cases, for example in the chapter on imaging, the style differs. Here, the material is discussed to present a background for the design of an experiment rather than as a detailed procedure. In both cases, researchers will find these contributions are presented with a practical emphasis so that they can be directly followed in the lab. The information in this manual is designed to be useful to a wide range of scientists. It is presented in a style that should permit new investigators to use it to set up an experiment for the first time. However, the format and frequent updates of the Current Protocols series ensure that the material will contain the latest technical developments and be of continuing interest to experienced investigators who wish to review technical developments in a familiar area. Our goal is to make this series a useful source of experimental methods for both the novice and the expert. Subjects in this manual are organized by chapters, and protocols are contained in units. Protocol units, which constitute the bulk of the book, generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method. Other units present more general information in the form of explanatory text with no protocols. Overview units contain theoretical discussions that lay the foundation for subsequent protocols. Other discussion units present more general information; for example, Chapter 7 contains a unit on analysis and interpretation of radioligand binding data (including the underlying theory behind the practical equations that are used for this purpose), to which the reader may turn to gain a greater understanding of experimental results. Page numbering reflects the modular arrangement by unit; for example, page 1.2.3 refers to Chapter 1 (Neuroanatomical Methods), UNIT Unavailable (Immunohistochemical Localization of Neurochemicals), page 3 of that particular unit. Many reagents and procedures are employed repeatedly throughout the manual. Instead of duplicating this information, cross-references among units are used extensively. Cross-referencing helps to ensure that lengthy and complex protocols are not overburdened with steps describing auxiliary procedures needed to prepare raw materials and analyze results. Certain units that describe commonly used techniques and recipes (e.g., neural tissue sectioning and mounting, solutions for preparing coated tissue culture dishes) are cross-referenced in other units that describe their application. Thus, whenever it is necessary to prepare slide-mounted rat brain sections for microscopy, the appropriate unit in Chapter 1—describing various procedures for brain preparation, sectioning, and mounting—is cross-referenced (i.e., UNIT Unavailable). For some widely used molecular biological techniques, Current Protocols in Molecular Biology (CPMB) is cross-referenced throughout the entire manual, and all CPMB cross-references are compiled in a table in APPENDIX Unavailable. Because this publication is first and foremost a compilation of laboratory techniques in neuroscience, we have not provided extensive instructive material. We have, however, included explanatory information where required to help readers gain an intuitive grasp of the procedures. Chapter 1 provides an overview of strategies for neural tissue preparation and characterization, to help the reader plan for all aspects of an experimental study. Some chapters contain special overview units that describe the state of the art of the topic matter and provide a context for the procedures that follow. Chapter and unit introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters, troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key references to inform users of particularly useful background reading, original descriptions, or applications of a technique. Many units in the manual contain groups of protocols, each presented with a series of steps. One or more basic protocols are presented first in each unit and generally cover the recommended or most universally applicable approaches. Alternate protocols are provided where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the basic protocol. Support protocols describe additional steps that are required to perform the basic or alternate protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual, or because they are performed in a time frame separate from the basic protocol steps. Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, while others are solutions unique to a particular protocol. Recipes for the latter solutions are provided in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., SDS sample buffer) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used stock solutions and buffers are provided once in APPENDIX Unavailable. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. Throughout the manual, the authors have recommended commercial suppliers of chemicals, biological materials, and equipment. In some cases, the noted brand has been found to be of superior quality or it is the only suitable product available in the marketplace. In other cases, the experience of the author of that protocol is limited to that brand. In the latter situation, recommendations are offered as an aid to the novice neuroscientist in obtaining the tools of the trade. Experienced investigators are therefore encouraged to experiment with substituting their own favorite brands. Anyone carrying out these protocols may encounter the following hazardous or potentially hazardous materials: (1) radioactive substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, and (3) pathogenic and infectious biological agents. Check the guidelines of your particular institution with regard to use and disposal of these hazardous materials. Although cautionary statements are included in the appropriate units, we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials must be used in strict accordance with local and national regulations. Another source for use and disposal guidelines can be found in APPENDIX Unavailable and APPENDIX Unavailable of Current Protocols in Molecular Biology. Many protocols call for use of live animals (usually rats or mice) for experiments. Prior to conducting any laboratory procedures with live subjects, the experimental approach must be submitted in writing to the appropriate Institutional Animal Care and Use Committee (IACUC). Written approval from this committee is absolutely required prior to undertaking any live-animal studies. Some specific animal care and handling guidelines are provided in the protocols where live subjects are used, but check with your IACUC guidelines to obtain more extensive guidelines. Most of the protocols included in this manual are used routinely in our own laboratories. These protocols work for us; to make them work for you we have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers' observations and suggestions. We encourage readers to send their comments and questions to currentprotocols@wiley.com. Another valuable resource is the companion website http://www.currentprotocols.com. This site features tools, calculators, apps, troubleshooting tips, webinars, and videos to assist in the experiments presented in this manual. The individual article pages provide valuable summary information and easy access to the content. While the editorial board enlists contributors for most of the topics presented in this manual, we invite individuals to submit letters of intent describing new topics that they would like to publish in CPNS. The editorial board will carefully consider suggested topics and invite authors to submit full manuscripts in some cases. Letters of intent may be submitted by email to currentprotocols@wiley.com. This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the hard work by the Current Protocols editorial staff at John Wiley and Sons. We are extremely grateful for the critical contributions by Virginia Chanda and Gwen Taylor (Developmental Editors) who kept the editors and the contributors on track and played a key role in bringing the entire project to completion. Other skilled members of the Current Protocols staff who contributed to the project include Janet Blair, Michael Gates, Scott Holmes, Alice Ro, Joseph White, and Kathy Wisch. The extensive copyediting required to produce an accurate protocols manual was ably handled by Rebecca Barr, Mark Bowes, Lisa Christenson, Elizabeth Harkins, Monte Kendrick, Kathy Morgan, Connie Parks, Allen Ranz, and Kristine Templeman, and electronic illustrations were prepared by Gae Xavier Studios. Roles of hormones in reproductive behaviors, plus chapters on the role of hormones in feeding, stress, aggression, biological rhythms, and cognition. General overview of neurotransmitter pathways in the brain, with some discussion of the role of neurotransmitter systems in behavior.