Neuromethods – serie

Techniques in the neurosciences are evolving rapidly. There are currently very few volumes dedicated to the methodology - ployed by neuroscientists, and those that are available often seem either out of date or limited in scope. This series IS about the methods most widely used by modern-day neuroscientists and is written by their colleagues who are practicing experts. Volume 1 will be useful to all neuroscientists since it concerns those procedures used routmely across the widest range of s- disciplines. Collecting these general techniques together in a s- gle volume strikes us not only as a service, but will no doubt prove of exceptional utilitarian value as well. Volumes 2 and 3 - scribe current procedures for the analyses of amines and their - tabolites and of amino acids, respectively. These collections will clearly be of value to all neuroscientists working in or contempl- ing research in these fields.Similar reasons exist for Volume 4 on receptor binding techniques since experimental details are p- vided for many types of ligand-receptor binding, including ch- ters on general principles, drug discovery and development, and a most useful appendix on computer programs for Scatchard, nonlinear and competitive displacement analyses. Volume 5 p- vides procedures for the assessment of enzymes involved in biogenic amine synthesis and catabolism. Volumes in the NEUROMETHODS series will be useful to neuro-chemists, -pharmacologists, -physiologists, -anatomists, psychopharmacologists, psychiatrists, neurologists, and chemists (organic, analytical, pharmaceutical, medicinal); in fact, everyone involved in the neurosciences, both basic and clinical.

Techniques u-r the neurosciences are evolving rapidly. There are currently very few volumes dedicated to the methodology - ployed by neuroscrentists, and those that are available often seem either out of date or limited in scope This series is about the methods most widely used by modern-day neuroscrentrsts and 1s written by their colleagues who are practicing experts Volume 1 will be useful to all neuroscientists since it concerns those procedures used routinely across the widest range of subdiscrplines Collectmg these general techmques together in a single volume strikes us not only as a service, but will no doubt prove of exceptronal utilrtarian value as well Volumes 2 and 3 - scribe all current procedures for the analyses of ammes and their metabolrtes and of ammo acids, respectively. These collectrons will clearly be of value to all neuroscrentrsts working m or contemplating research in those fields.Similar reasons exist for Volume 4 on receptor bmdmg techniques since experimental - tails are provided for all types of lrgand-receptor binding, including chapters on general principles, drug discovery and - velopment, and a most useful appendix on computer programs for Scatchard, nonlinear, and competitrve displacement analyses. Volume 5 provides procedures for the assessment of enzymes- volved m brogemc amme synthesis and catabolrsm. Volumes in the NEUROMETHODS series will be useful to neurochemists, -pharmacologists, -physrologrsts, -anatomrsts, psychopharmacologrsts, psychratrrsts, neurologrsts, and chemists (organic, analytrcal, pharmaceutrcal, medicinal), m fact, everyone involved m the neurosciences, both basic and clinical.

Techniques in the neurosciences are evolving rapidly. There are currently very few volumes dedicated to the methodology - ployed by neurosclentlsts, and those that are available often seem either out of date or limited m scope. This series is about the methods most widely used by modern-day neurosclentists and 1s wrltten by their colleagues who are practicing experts. Volume 1 will be useful to all neurosclentists since It concerns those procedures used routinely across the widest range of subdisciplines. Collecting these general techniques together m a single volume strikes us not only as a service, but will no doubt prove of exceptIona utilitarian value as well. Volumes 2 and 3 - scribe all current procedures for the analyses of amines and their metabolltes and of ammo aads, respectively. These collections will clearly be of value to all neuroscientlsts working m or contemplating research m these fields.Similar reasons exist for Volume 4 on receptor binding techniques since experimental - tails are provided for all types of llgand-receptor bmdmg, including chapters on general prmciples, drug discovery and - velopment, and a most useful appendix on computer programs for Scatchard, nonlinear, and competitive displacement analyses. Volume 5 provides procedures for the assessment of enzymes - volved m biogenic amme synthesis and catabolism. Volumes in the NELJROMETHODS series will be useful to neurochemists, -pharmacologists, -physiologists, -anatomlsts, psychopharmacologists, psychlatnsts, neurologists, and chemists (organic, analytical, pharmaceutical, medicinal), in fact, everyone involved m the neurosclences, both basic and clinical.

Techniques in the neurosciences are evolving rapidly. There are currently very few volumes dedicated to the methodology - ployed by neuroscrentists, and those that are available often seem either out of date or limited in scope. This series is about the methods most widely used by modern-day neuroscientists and 1s written by their colleagues who are practicing experts. Volume 1 will be useful to all neuroscientists since it concerns those procedures used routinely across the widest range of s- drsciplines. Collecting these general techniques together in a single volume stnkes us not only as a service, but will no doubt prove of exceptional utilitarian value as well. Volumes 2 and 3 describe all current procedures for the analyses of ammes and theirmetabolites and of amino acrds, respectively. These collections will clearly be of value to all neuroscientists working in or contemplating research in these fields.Similar reasons exist for Volume 4 on receptor binding techniques since experimental details are provided for many types of ligand-receptor binding, including chapters on general prin- ples, drug discovery and development, and a most useful app- dix on computer programs for Scatchard, nonlinear, and compe- tive displacement analyses. Volume 5 provides procedures for the assessment of enzymes involved in biogenic amine synthesis and catabolism. Volumes in the NEUROMETHODS series will be useful to neurochemists, -pharmacologists, -physrologists, -anatomists, psychopharmacologists, psychiatrists, neurologists, and chemists (organic, analytical, pharmaceutical, medicinal); in fact, everyone involved in the neurosciences, both basic and clinical.

Techniques in the neurosciences are evolving rapidly. There are currently very few volumes dedicated to the methodology - ployed by neuroscrentists, and those that are available often seem either out of date or limited in scope. This series is about the methods most widely used by modern-day neuroscientrsts and is written by their colleagues who are practicing experts. Volume 1 will be useful to all neuroscientists since it concerns those procedures used routinely across the wrdest range of s- disciplines. Collecting these general techniques together in a single volume strikes us not only as a service, but will no doubt prove of exceptional utilitarian value as well. Volumes 2 and 3 describe all current procedures for the analyses of amines and their metabolites and of amino acids, respectively. These collections will clearly be of value to all neuroscientists working in or contemplating research in these fields. Similar reasons exist for Volume 4 on receptor binding techniques since experimental details are provided for all types of ligand-receptor binding, including chapters on general principles, drug discovery and development, and a most useful-appendix on computer programs for Scatchard, nonlinear, and competitive d- placement analyses. Volume 5 provides procedures for the asse- ment of enzymes involved in biogenic amme synthesis and catabolrsm. Volumes in the NEUROMETHODS series will be useful to neurochemists, -pharmacologists, -physiologists, -anatomists, psychopharmacologists, psychiatrists, neurologists, and chemists (organic, analyncal, pharmaceutical, medicinal); in fact, everyone involved in the neurosciences, both basic and clinical.

Techniques in the neurosciences are evolving rapidly. There are currently very few volumes dedicated to the methodology - ployed by neuroscrentists, and those that are available often seem either out of date or limited in scope. This series is about the methods most widely used by modern-day neuroscientists and is written by their colleagues who are practicing experts. Volume 1 will be useful to all neuroscientists since rt concerns those procedures used routmely across the wrdest range of s- disciplines. Collecting these general techniques together in a single volume strikes us not only as a service, but will no doubt prove of exceptronal utrlitarran value as well. Volumes 2 and 3 describe current procedures for the analyses of amines and their metabolites and of amino acids, respectively. These collections will clearly be of value to all neuroscrentists working m or contemplating research m these fields.Similar reasons exist for Volume 4 on receptor binding techniques, since experimental details are provided for many types of hgand-receptor binding, including chapters on general prin- ples, drug discovery and development, and a most useful app- dix on computer programs for Scatchard, nonlinear, and compe- tive displacement analyses. Volume 5 provides procedures for the assessment of enzymes involved in neurotransmitter synthesis and catabolism. Volumes in the NEUROMETHODS series will be useful to neurochemists, -pharmacologists, -physiologrsts, -anatomrsts, psychopharmacologists, psychiatrists, neurologists, and chemists (organic, analytical, pharmaceutical, medicinal); in fact, everyone involved m the neuroscrences, both basic and clmical.

Techniques in the neurosciences are evolving rapidly. There are currently very few volumes dedicated to the methodology - ployed by neuroscientists, and those that are available often seem either out of date or limited in scope. This series is about the methods most widely used by modern-day neuroscientists and is written by their colleagues who are practicing experts. Volume 1 will be useful to all neuroscientists since it concerns those procedures used routmely across the widest range of s- disciplines. Collecting these general techniques together in a single volume strikes us not only as a service, but will no doubt prove of exceptional utilitarian value as well. Volumes 2 and 3 describe all current procedures for the analyses of amines and theirmetabolites and of amino acids, respectively. These collections will clearly be of value to all neurosclentists working m or contemplating research in these fields. Similar reasons exist for Volume 4 on receptor binding techniques, since experimental details are provided for all types of ligand-receptor binding, including chapters on general principles, drug discovery and development, and a most useful appendix on computer programs for Scatchard, nonlinear, and competitive d- placement analyses. Volume 5 provides procedures for the asse- ment of enzymes involved in biogenic amine synthesis and catabolism. Volumes in the NEUROMETHODS series will be useful to neurochemists, -pharmacologists, -physiologists, -anatomlsts, psychopharmacologists, psychiatrists, neurologists, and chemists (organic, analytical, pharmaceutical, medicinal); in fact, everyone involved in the neurosciences, both basic and clinical.

Recent years have seen remarkable advances in the devel- ment of techniques that have direct applications in neurological research. In consequence, the circulatory and metabolic status of the brain can be measured and correlated with changes m structure often noninvasively, m the same - and integrated function, perimental subIect This has stimulated an increased awareness of the complexity, under normal and pathological conditions, of the interdependence of these factors. Through the application of the methods described in this volume, however, these complexities can now be analyzed. The chapters m this volume present methodological - scriptions of some of the most powerful "physicochemical" methods for studymg the brain. Multidisciplmary teams are - quired to develop some of these methods, which are extremely expensive m terms of capital equipment costs and technological personnel support Thus, they will likely remain restricted to malor medical research centers Nevertheless, many recent concepts of brain responses to disease are a result of their application We have been fortunate m convincing active, leading sci- tists to contribute to this volume.The descrrptions of the basic prmciples of each method, and its applications and limitations, are derived primarily from their personal experiences. The first two chapters (Rowan, Auer) deal with methods for assessing brain hemodynamics. The two subsequent chapters (Greenberg; He- covitch) describe autoradiography and positron emission tomog- phy techniques, which provide quantitative measurements of brain metabolism as well as blood flow.

In this volume, we hope to cover the malor techniques that are presently being used to analyze the actions of drugs used in psyc- atry. The contributors of the chapters are active researchers who have considerable practical experience with the techniques they are describing, and the emphasis in the chapters is on three types of psychiatric drugs, namely antidepressants, antipsychotics, and anxiolytics. The first chapter, by Curry and Yu, discusses protein binding of psychotropic drugs, with special reference to equilibrium ana- sis as the method of assessment and to lipophilicity correlations. Since many of the drugs used in treating psychiatric disorders are bound extensively to protein, this aspect is of great importance with regard to their therapeutic actions and toxicity, Basic mat- matical models, techniques for the study of protein binding, molecular aspects of protein binding, binding in relation to lipophilicity, indirect approaches to measurement of the fraction of drug bound, the function of protein binding, and tissue binding are among the topics discussed. Chapter 2, by Norman and B- rows, deals with the principles of isotope derivative assays and their applications to antidepressants and antipsychotics; extensive protocols are provided. The third chapter, by Cooper, deals with analysis by gas-liquid chromatography. Sample collection and storage, extraction procedures, column selection, use of internal standards, types of detectors, techniques of application, and - plications to specific drugs are among the topics discussed.

Serendipitous discovery has played an important role m the identification of physiological mechanisms in central and peri- eral neurotransmission. In many cases, the subsequent followup of these discoveries has been substantially aided by previouslyiden- fied natural substances that interfered with these mechanisms. The site of neuromuscular blockade produced by curare extracts, for example, was shown in the 1850s by Claude Bernard, and Sir Henry H. Dale m 1906 discovered the reversal of the pressor effects of adrenal extracts by ergot alkaloids. These findings were the first clues that led eventually to the definition of mcotinic cholinergic receptors and the present classification of adrenergic receptors. In the 1950s many discoveries, including the identification of the catecholamines and 5-hydroxytryptamine in the central n- vous system, followed by the development of specific sensitive biochemical and histochemical methods and the introduction of modern psychotropic drugs led to our increased understanding of chemical transmission and the formulation of theories that attempt to explain the causes of some mental disorders.The finding that hydroxylated derivatives of noradrenaline and- hydroxytryptamine are readily taken up by catecholaminergic or serotonergic neurons, and that this is followed by poisoning and death of the cell, encouraged the use of these compounds for the mapping of central and peripheral catecholamine or- hydroxytryptamine systems, and the mvestigation of the re- vance of these pathways to receptor regulation or behavioral - sponses.

Lipids m the nervous system are major components of the m- branes. The presence of glycolipids in high concentrations is unique for the nervous system. Recent discoverres of the functronal and pathological importance of lipids such as diacylglycerols, polyphosphoinositides, prostaglandins, leukotrienes, docosahexaenoic acid, platelet activating factor, and gangliosides have markedly increased the number of publications on nervous system lipids. Many new methods have been developed. Thus, there IS a need for this volume that is dedicated to that methodo- gy. This volume places all of the methods for lipids into perspective with recommendations concerning the selection of a method for a specific purpose. Lloyd A. Horrocks vii Contents ...Preface to the Series vi Preface...*...xix List of Contributors Lipid Extraction Norman S. Radin 1 1. Introduction ...5 2. Properties of Solvents 2.1. Ethers ...6 8 2.2. Alcohols ...9 2.3. Halocarbons ...9 2.4. Hydrocarbons ...2.5. Miscellaneous Solvents ...10 ...10 3. Storage of Lipid Extracts 14 4. Homogenizers ...15 5. Separating the Extract from the Tissue Residue 15 5.1. Use of Filtration ...16 5.2. Centrifugation ...17 6.Removing Nonlipids from Lipid Extracts 6.1. Removal of Nonlipids by Evaporation and Reextraction ...17 ...17 6.2. Prewashing the Tissue Sample. 6.3. Removal of Nonlipids by Precipitating the Lipids with the Proteins ...18 ...19 6.4. Liquid/Liquid Partitioning ...19 6.5. Removal of Nonlipids by Dialysis 6.6. Separation from Nonlipids by Changing a 21 Partition Constant ...

The study of drug effects on behavior is truly a confluence of streams of neuroscience research. The material in this volume covers a broad range of methodology representative of practically the entire scope of psychopharmacology for conscious animals. The contributions in this book represent careful evaluations by experts who were invited to participate because of their excellent practical application of research design and methodology. In each case, a particularly useful and well-written contribution has been forthcoming, and should serve either as a valuable introduction to developing researchers or as a practical reference to those active in the field. The material is arranged in an intuitive hierarchy. This arrangement is based on what are ostensibly increasing levels of integrated activity of the nervous system and, therefore, increasing levels of behavioral integration. Nevertheless, as all of the areas covered are rather detailed in their levels of intrinsic complexity, this arrangement of chapters does not imply successively more intricate problems. The reader will find some overlap between chapters, but little interdependence, i.e., each may be read as a separate or as an integral part of the volume.

If one envisages neuroscience as a pyramid, with the more mole- lar disciplines forming the base and the more integrative d- ciplines positioned above, then neuropsychology clearly would be near the tip. Neuropsychology seeks to find order in the ultimate product of all neural systems, namely behavior, and to relate that product to its neural substrate. Relationships between brain and behavior are sought, but reductionistic explanations are eschewed. Attempting to “explain” complex behaviors in terms of neuronal activity is no more satisfying than attempting to “explain” artificial intelligence in terms of voltages within a computer’s central pr- essing unit. If one is to comprehend the functioning of either the brain or the digital computer, one must know something about not only the structure and mechanics of the device, but also the prin- ples according to which components of the device are organized and the context in which the device is operating (e.g., environm- tal inputs and stored information).

It goes without saying that the principles and techniques of molecular biology are having and will continue to have a major impact on investigations into nervous system structure and func tion. It is becoming increasingly apparent to neuroscientists in all subdisciplines that a working knowledge of the language, approaches, and techniques of molecular biology is indispensable for their work. For these reasons, the editors have decided to devote this volume of Neuromethods to the techniques of molecular biology and their application to neural systems. There currently exist a number of excellent reference technical manuals that de scribe molecular neurobiological techniques in great detail, and many of these are cited within the chapters included in this volume. It was not the intention of the editors or authors of this volume to duplicate these efforts. Rather, our intention was to present to the neuroscientist who is relatively unfamiliar with these methodologies an understanding of how specific techniques are used to approach major molecular neurobiological problems as well as a set of techniques that work in the laboratories of the individuals writing the chapters. In some cases, there are duplica tions of techniques these have been retained to illustrate the range of variability of the technique and/or the flexibility of the method to study different types of problems. We hope that the chapters will provide the reader with an understanding of the methods and their applicability to neurobiological problems; and, perhaps, suggest new directions for the reader's research efforts. Anthony T.

The mammalian central nervous system depends almost - clusively on glucose as its major energy source. In addition, g- cose participates in other cerebral metabolic functions including the biosynthesis of neurotransmitters, such as acetylcholine and the amino acids. This volume of Neuromethods assembles currently available methods for the study of cerebral glucose and energy metabolism in vitro and in mm. In the first chapter, Lust et al. describe the various methods available for the appropriate fixation of brain tissue necessary for the study of cerebral energy metabolism. Different fixation methods are compared, and some concerns raised by the USDHHS in their guidelines for the care and use of laboratory animals are addressed. Specific fixation methods pertinent to the various measurements are also covered in other chapters. In vitro p- parations have, despite certain limitations, been found to be useful in the study of brain metabolism, since the biochemical envir- ment is amenable to rapid, controlled manipulation.The chapter by Lai and Clark describes methods for the isolation and characterization of metabolically active preparations of synaptic and non-synaptic mitochondria from brain, and studies of - zymes involved in glucose metabolism and glucose-derived neurotransmitter synthesis in these preparations are summarized. The chapter by Whittingham discusses methods of preparations of hippocampal slices for use in the study of energy metabolism. Measurement of glucose and of glycolytic and dicarboxylic acid cycle intermediates in neural tissues are described in the chapter by Bachelard.

The development of neurophysiology, the study of the activity of living nervous tissue, has relied heavily on the techniques of electrophysiology. This emphasis is revealed in volumes 14 and 15 of this series, which show how electrophysiological techniques can be applied to research topics ranging from ion channels to human behavior. Kitai and Park show how cellular neurophysiology can be related to classical neuroanatomy, an important basis for any type of functional analysis. Wonderlin, French, Arispe, and Jones describe new (single channel) and more traditional (whole cell) techniques for studying the role of ion channels in cellular pr- esses, a field that is currently developing very rapidly. An exciting nontraditional approach to the study of cellular electrophysiology is discussed by Hopp, Wu, Xiao, Rioult, London, Zecevic, and Cohen in their paper on optic measurement of membrane pot- tials. Humphrey and Schmidt offer a thoughtful review of the uses and limitations of the technique of recording extracellular unit potentials in the brain. Hoffer presents an introduction to a field that is of great interest but is technically very difficult-the reco- ing from cells and axons in the spinal cord and peripheral nervous system in freely moving animals. An electrophysiological approach to the analysis of the neural mechanisms of normal behavior is presented by Halgren in a wide-ranging review of the field of evoked potentials in humans.

The two Animal Models in Psychiatry volumes are loosely organized by subject. The first volume contains a number of chapters concerned with schizophrenia, psyc- ses, neuroleptic-induced tardive dyskinesias, and other d- orders that may involve dopamine, such as attention deficit disorder and mania. Also included is a chapter describing a behavioral model for activity-induced anorexia. The second volume deals with affective and anxiety disorders, but also includes chapters on subjects not easily classified as either psychotic, affective, or anxiety-related, such as aggression, mental retardation, and memory disorders. Four chapters on animal models of schizophrenia or psychoses are included in Volume 18 because of the importance of these disorders in psychiatry. Likewise, three chapters in the present v- ume deal with affective disorders, with a fourth chapter on circadian rhythms that also contributes to methods for a- mal models in affective disorders. Following the first four chapters are two chapters dealing with models of anxiety and panic, two chapters on aggression, one on mental retardation, and a final chapter covering memory disorders. Many of the behaviorally-based models of affective disorders involve inducing stress in a- mals, usually on a chronic basis. The first chapter by Anisman, Zalcman, Shanks, and Zacharko describes some of the neurochemical effects that are associated with the chronic application of sensors.

The two Animal Models in Psychiatry volumes are loosely organized by subject. The first volume contains a number of chapters concerned with schizophrenia, psyc- ses, neuroleptic-induced tardive dyskinesias, and other d- orders that may involve dopamine, such as attention deficit disorder and mania. The second volume deals with affective and anxiety disorders, but also includes chapters on subjects not easily classified as either psychotic, or affective, or an- ety-related, such as aggression, mental retardation, and memory disorders. Four chapters on animal models of schizophrenia or psychoses are included in the present v- ume because of the importance of these disorders in p- chiatry. Likewise, three chapters in the subsequent volume deal with depression. The first of the two volumes begins with an introd- tion by Paul Willner reviewing the criteria for assessing the validity of animal models in psychiatry. He has written - tensively on this subject, and his thorough description of the issues of various forms of validity provides a framework in which to evaluate the subsequent chapters. As will be seen, the remaining chapters in both volumes will refer frequently to these issues. The second chapter, by Melvin Lyon, describes a large number of different procedures that have been p- posed as potential animal models of schizophrenia. This is a departure from the usual format, consisting of detailed - scriptions of specific models.

This timely new volume in the highly acclaimed Neuromethods series focuses on major new advances in understanding the signaling mechanisms employed by neural tissues. Chapters, contributed by experts in the field, provide thorough, up-to-date coverage of a variety of topics and techniques, including: single-cell imaging technology phosphorylation of target proteins analysis of phosphoinositides and inositol phosphates inositol trisphosphate and intracellular calcium guanine nucleotide-binding proteins ion channel gating measurement of intracellular calcium with fluorescent calcium indicators protein kinase C synthetic analogs of intracellular messengers cyclic nucleotides caged intracellular messengers intracellular regulators and components of the exocytotic pathway intracellular messengers in vertebrate and invertebrate photoreceptors. Neuromethods 20 Intracellular Messengers is an essential resource for neurobiologiests at all levels of research.

to the Animal Models Volumes This and several other volumes in the Neuromethods series will describe a number of animal models of neu- psychiatric disorders. Because of increasing public concern over the ethical treatment of animals in research, we felt it incumbent upon us to include this general preface to these volumes in order to indicate why we think further research using animals is necessary and why animal models of psychi- ric and neurologic disorders, in particular, are so important. We recognize that animals should only be used when suitable alternatives are not available. We think it self-e- dent, however, that humans can only be experimented upon in severely proscribed circumstances and alternative pro- dures using cell or tissue culture are inadequate in any models requiring assessments of behavioral change or of complex in vivo processes. However, when the distress, discomfort, or pain to the animals outweighs the anticipated gains for human welfare, then the research is not ethical and should not be carried out.

to the Animal Models Volumes This and several other volumes in the Neuromethods series will describe a number of animal models of neuropsychiatric disorders. Because of increasing public concern over the ethical treatment of animals in research, we felt it incumbent upon us to include this general preface to these volumes in order to indicate why we think further - search using animals is necessary and why animal models of psychiatric disorders, in particular, are so important. We recognize that animals should only be used when suitable alternatives are not available. We think it self-e- dent, however, that humans can only be experimented upon in severely proscribed circumstances and alternative pro- dures using cell or tissue culture are inadequate in any models requiring assessments of behavioral change or of complex in vivo processes. However, when the distress, discomfort, or pain to the animals outweighs the anticipated gains for human welfare, then the research is not ethical and should not be carried out. It is imperative that each individual researcher examine his/her own research from a critical moral standpoint - fore engaging in it, taking into consideration the animals’ welfare as well as the anticipated gains. Furthermore, once a decision to proceed with research is made, it is the researcher’s responsibility to ensure that the animals’ w- fare is of prime concern in terms of appropriate housing, feeding, and maximum reduction of any uncomfortable or distressing effects of the experimental conditions, and that these conditions undergo frequent formalized monitoring.

Most cells will survive removal from the natural mic- environment of their in vivo tissue and placement into a sterile culture dish under optimal conditions. Not only do they survive, but they also multiply and express differen- ated properties in such a culture dish. A few cells do this in suspension, but most will need some kind of mechanical support substituting for their natural connections with other cells. The surface of a culture dish that might have to be coated is usually sufficient. The recent trend to standa- ization of conditions and the existence of commercial ent- prises with adequate funds and specializing in the needs of scientists were responsible for the tremendous proliferation of cell culture techniques in all fields of research in the last 20 years. No longer does a scientist have to concentrate all his/her efforts on that technology; the new trends make it feasible to employ cell culture techniques as only one of the many methods available in a small corner of a larger research laboratory. Some areas of research depend more heavily than others on cell culture techniques. Neuroscience is one of the areas that has developed hand in hand with the prol- eration of cell culture methodology. Molecular biological aspects, cell differentiation and development, neurophy- ological and neurochemical studies, as well as investigations into the nature of various diseases are now to a large extent dependent on the use of cell cultures.

A comprehensive guide to the use of animal models for understanding the many facets of drug and alcohol abuse - problems that are now worldwide health concerns. Expert investigators have contributed state-of-the-art chapters on the use of animal models for research on substance abuse.

Leading researchers present those techniques that have been crucial to propelling the neurotrophic factors field to its current state of high visibility. The techniques range from molecular biological methods used for cloning and production, to cell culture methods for assessing biological activities, to animal models of nervous system injury (necessary for the development of therapeutic agents from neurotrophic factors).

Patch-Clamp Applications and Protocols presents the broadest coverage of techniques for measuring and recording directly activity within a cell. The first part of the book offers modern developments associated with the technology of path-clamp electrodes, of cell-free ion-channel recording, and of the whole-cell patch clamp technique. Chapters on such recent offspring of the technique as the concentration clamp technique, the pressure clap method, and the perfusion of patch clamp electrodes are contributed by authors intimately involved with their development. Also covered is the loose patch-clamp technique, single-channel recording, the perforated patch-clamp technique, molecular biological aspects of the technique, and the use of the technique in brain slices. Every scientist working with path-clamp methods will find this protocols manual invaluable to their continued use and development of this powerful technique.

Efforts to measure neurotransmitters and related s- cies in living brain tissue by faradic electrochemistry began in earnest in the early 1970s. During the ensuing years, s- eral monographs and various symposia on the subject have appeared. The present volume of Neuromethods, Voltammetric Methods in Brain Systems, can be considered different from previous offerings in several respects. The methodology of in vivo electrochemistry has now reached a stable level of maturity. It is no longer necessary to convince interested re- ers that the techniques can measure, with specified degrees of reliability, certain neurotransmitters and metabolites in the extracellular fluid space. Moreover, the basic approaches of electroanalytical measurements are more widely appre- ated by neuroscientists and one need not explain in tutorial fashion the details of oxidative electrochemistry. Nevert- less, in keeping with the nature of the Neurumethods series, this is a "how to" volume and readers will find ample detail on the preparation of electrodes, practical experimental details, and the interpretation of results. But the emphasis is strongly on the application of voltammetric methods.The first two chapters deal with fundamentals inherent in employing in vivo electrochemical measures. The first - ticle offers an authoritative account of our understanding of the surface states of carbon and carbon fiber electrodes. It provides a rationale for the various pretreatments that have allowed the "tuning" of electrode properties to obtain sen- tivity and fast response times.

Most cells will survive removal from the natural mic- environment of their in vivo tissue and placement into a sterile culture dish under optimal conditions. Not only do they survive, but they also multiply and express differen- ated properties in such a culture dish. A few cells do this in suspension, but most will need some kind of mechanical support substituting for their natural connections with other cells. The surface of a culture dish that might have to be coated is usually sufficient. The recent trend to standa- ization of conditions and the existence of commercial ent- prises with adequate funds and specializing in the needs of scientists were responsible for the tremendous proliferation of cell culture techniques in all fields of research in the last 20 years. No longer does a scientist have to concentrate all his/her efforts on that technology; the new trends make it feasible to employ cell culture techniques as only one of the many methods available in a small corner of a larger research laboratory. Some areas of research depend more heavily than others on cell culture techniques. Neuroscience is one of the areas that has developed hand in hand with the prol- eration of cell culture methodology. Molecular biological aspects, cell differentiation and development, neurophy- ological and neurochemical studies, as well as investigations into the nature of various diseases are now to a large extent dependent on the use of cell cultures.

Distinguished international experts offer critical reviews of the major categories of posttranslational protein modification in neurons, as well as a comprehensive collection of state-of-the-art techniques for their analysis. They provide detailed and specific methods for studying protein phosphorylation/dephospohorylation, methylation, long chain fatty acylation, ADP-ribosylation, glycosylation, and glycosylphosphatidylinositol anchors. Using both conventional analytical and novel molecular biological approaches, these experts present important techniques and reagents and cite numerous examples from the neuroscience literature in a way that allows investigators to determine the best experimental approaches for their own systems. The book offers the most complete review of protein kinase and phosphatase inhibitor applications available.

Two distinguished international experts offer a fully illustrated, thoroughly referenced guide to the use of microinjected Xenopus embryos to study the role and spatiotemporal regulation of nervous system proteins during vertebrate development. Drs. Seidman and Soreq include all the basic scientific background and protocols needed to consider and exploit the vast potential of this model system. They provide an outline of Xenopus development, detailed methods for in vitro fertilization and microinjection, and protocols and references for a variety of biochemical and histochemical techniques to analyze the results of a microinjection experiment. They also present extensive experimental data illustrating the use of enzyme activity assays, gel electrophoresis, density ultracentrifugation, EAIA, RT-PCR, cytochemical and immunocytochemical staining, and electron microscopy. Transgenic Xenopus demonstrates convincingly the special utility of Xenopus embryo microinjection as a sophisticated and versatile tool for studying nervous system proteins.