bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantification of Allosteric Interactions at G Protein Coupled Receptors Using Radioligand Binding Assays
Authors: Christopoulos Arthur
Published: June 2004

Allosteric interactions are non‐competitive in nature and involve the simultaneous binding of two ligands to the same receptor. These interactions are also commonly referred to as being cooperative. The effect of an allosteric modulator is to cause a conformational change in the receptor protein that yields a decrease or increase in the equilibrium binding affinity of a classical (orthosteric) agent, such as an agonist or competitive antagonist, for its binding site on the receptor, and vice versa. Because of the complex nature of allosteric phenomena, their detection and quantification relies on the use of both equilibrium and non‐equilibrium binding assays, otherwise the phenomenon may be misinterpreted. This unit outlines the most common experimental approaches to measuring allosterism at G protein‐coupled receptors, and also discusses the analysis of experimental data derived from such assays.
Cyclooxygenase Assays
Authors: Gierse James K. , Koboldt Carol M.
Published: June 2004

Cyclooxygenase (COX, also known as prostaglandin H2 synthase, PGH2) is one of the most significant enzymes in pharmacology since COX inhibition is the mechanism of action of most nonsteroidal anti‐inflammatory drugs (NSAIDs). There are two forms: COX‐1 is a constitutive form of the enzyme that has been linked to the production of physiologically important prostaglandins that may play a role in homeostasis (gastric, renal, etc.). COX‐2 is inducible by cytokines and growth factors, and induction of COX‐2 is linked to inflammatory cell types and tissues, so COX‐2 is believed to be the target for the anti‐inflammatory actions of NSAIDs. COX‐2 also exists in a constitutive form in kidney and brain. Thus, there is an ongoing effort to identify compounds that will inhibit COX‐2 in preference to COX‐1 since such an agent may be a safer, and perhaps more efficacious, NSAID. Three assays are provided in this unit for measuring COX activity: one is to measure the final product of the reaction, PGE2, by ELISA; another is to measure directly the oxygen uptake that occurs during the first step of the reaction using an oxygen sensor; and the third is to measure the second reaction (peroxidase function) spectrophotometrically.
Characterization of Matrix Metalloproteinase Inhibitors: Enzymatic Assays
Authors: Marcotte Patrick A. , Davidsen Steven K.
Published: June 2004

The matrix metalloproteinases (MMPs) are a family of tightly regulated proteases that are involved in the catabolic aspect of remodeling and maintenance of normal tissue, and more than 20 human MMPs have been identified thus far. The MMPs collectively degrade a broad range of protein components of the extracellular matrix. While some substrate overlap exists, individual MMPs have been shown to process certain substrates more efficiently than others. These differences raise the critical issue of whether broad‐spectrum inhibitors, active against all MMPs, or selective inhibitors, targeted to a subset of enzymes, represent the optimal therapeutic strategy for a given disease. This suggests the need to assess the inhibition potency of test compounds across a range of MMP family members. Described in this unit is a method for the in vitro characterization of MMP inhibitors. The is used to determine the potency of test compounds as inhibitors of 8 representative MMPs through the measurement of their inhibition of cleavage of a fluorogenic substrate. Since this substrate is efficiently hydrolyzed by all MMPs in the screening assays presented here, the method is convenient for assessing the selectivity of inhibitors against multiple enzymes. A describes the activation of MMP zymogens.
Cytochrome P450 Assays
Authors: Delaporte Enock , Rodrigues A. David
Published: June 2004

Cytochrome P450s (CYPs) play a major role in drug detoxification, and inhibition of CYP‐mediated metabolism may lead to accumulation of toxic drug levels in the plasma. To prevent adverse drug‐drug interactions, new drug candidates are routinely tested for their ability to inhibit these enzymes. This unit describes a variety of protocols for evaluating new chemical entities as inhibitors of CYP activity. An example protocol illustrates a high‐throughput screening format using a fluorogenic probe, and a method for evaluating a test compound as a time‐dependent inhibitor of CYP is also described.
Histamine Receptor Assays
Authors: Shankley Nigel P. , Morton Magda F. , Watt Gillian F.
Published: June 2004

This unit describes three standard in vitro bioassays for studying histamine H1, H2 and H3 receptors in isolated intact tissues removed from the guinea pig. Both the H1 and H3 receptor assays are based on preparations of the ileum, whereas the spontaneously beating right atrium assay is used for the H2‐receptor.

This unit describes three standard in vitro bioassays for studying histamine H1, H2 and H3 receptors in isolated intact tissue.
Models of Cardiovascular Disease: Measurement of Arrhythmogenic/Antiarrhythmic Activity in the Guinea Pig and Antihypertensive Activity in the Conscious Rat
Authors: Lacroix Pierre , Guillaume Philippe
Published: June 2004

Arrhythmias arise as the result of either disorders of cardiac impulse formation or disorders of impulse conduction, or a combination of both. Screening methods have been developed to produce experimental arrhythmias and to select compounds on the basis of their ability to prevent or to reverse these arrhythmias. In this unit, cardiac glycosides are used produce arrhythmias (premature ventricular contractions, then sustained ventricular tachycardia and ventricular fibrillation) and can be used to detect the arrhythmogenic/antiarrhythmic potential of compounds in the guinea pig. The antihypertensive effects of compounds may be assessed using relatively simple in vivo techniques which are adapted to primary screening. The spontaneously hypertensive rat (SHR) model requires no surgery or pharmacological agent to induce hypertension, and it is sensitive to most of the clinically active antihypertensive drugs. The SHR method described in this unit can be used to detect the antihypertensive potential of compounds
Models of Affective Illness: Chronic Mild Stress
Authors: Papp Mariusz
Published: June 2004

Antidepressants can elevate the mood of depressed patients, but have no effect in normal (i.e., non‐depressed) individuals. This characteristic makes predicting the clinical action of an antidepressant, based on the effects it has in normal laboratory animals, difficult. The best strategy for studying the psychobiological causes and treatment of depression involves the use of appropriate animal models. Of the few models that meet the criteria required for a valid animal model of depression, the chronic mild stress paradigm is the most valid and the most extensively investigated. In addition to the protocol describing generation of the model, data supporting the model's validation are presented along with a discussion of technical aspects of the procedure.
Models of Anxiety: Ultrasonic Vocalizations of Isolated Rat Pups
Authors: van Oorschot Ruud , Mos Jan , Olivier Berend
Published: June 2004

When rat pups of 9 to 11 days are separated from their mother and littermates, they emit ultrasonic calls in the range of 30 to 50 kHz. This ultrasonic vocalization can be used as a measure of distress or anxiety. Measurement of the number of vocalizations during a short testing period (5 min) can be used to quantify the amount of distress (anxiety). While the ultrasonic cries are sensitive to drugs with anxiolytic properties, the paradigm also distinguishes other drug classes. Described in this unit are the basic methods for measurement and quantification of ultrasonic calls in rat pups under two different stress conditions: a cold plate (18°C) and a warm plate (37°C) condition. The utility of this protocol in psychopharmacologic testing and a method for determining drug side‐effects is described.
Models for Environmentally Induced Eating Disorders: Dietary Hyperphagia and Anorexia Nervosa
Authors: Pelleymounter Mary Ann , Kant R.H. , Aravich Paul
Published: June 2004

The two protocols in this unit provide suggestions for constructing models of eating disorders that are at the opposite ends of the spectrum: dietary hyperphagia and anorexia nervosa. The greatest degree of dietary hyperphagia is induced by giving rats or mice access to a daily choice of highly palatable foods (e.g., chocolate or bread) in addition to their regular chow. Like humans, rats overeat and actually develop physiological requirements for these foods. This model can be used to test the effects of putative anorectic agents on both acute and chronic administration regimens. The second protocol describes a model of compulsive behavior that results in profound weight loss, which is produced by moderate food deprivation along with continuous access to exercise wheels.
Models of Renal Insufficiency: The Anti‐Thy‐1.1 Model of Acute Proliferative Glomerulonephritis
Authors: Reinhart Glenn A. , Cox Bryan F.
Published: June 2004

This unit describes the rat anti‐Thy‐1.1 model of acute proliferative glomerulonephritis for the study of chronic renal insufficiency. A procedure is detailed for the induction of glomerulonephritis in rats as well as measurement of daily urinary excretion of protein, which is a convenient, primary screening tool. The unit also provides methods for assessment of glomerular filtration rate and effective renal plasma flow in anesthetized rats with anti‐Thy‐1.1‐induced renal insufficiency.
Models of Cardiac Ischemia‐Reperfusion Injury in Dogs and Rats
Authors: Gross Garrett J.
Published: June 2004

A marked reduction in coronary blood flow produces a reduction in myocardial function, electrocardiographic abnormalities and eventually a myocardial infarction if the ischemic episode were to persist for more than 20 to 30 min. There have been numerous studies in animal models in search of a magic bullet or drug that can ameliorate these symptoms and result in a reduction in infarct size, improvement in the recovery of contractile function, and abrogation of malignant ventricular arrhythmias in humans. This unit describes two animal models of myocardial ischemia/reperfusion injury which are used to evaluate pharmacological agents that may eventually demonstrate cardioprotective activity in a clinical setting.
Tissue Expression Profiling Using Real‐Time PCR
Authors: Strum Jay C. , Carrick Kevin M. , Stuart Joan S. , Martensen Shelby A.
Published: June 2004

The recent development of real‐time PCR allows for the rapid and accurate quantitation of gene expression in cells and tissues. Real‐time PCR instrumentation is designed for the simultaneous quantitation of gene expression from a few samples up to 384 samples. The normal tissue expression profile of a gene can provide useful insights into its potential role in normal physiological processes. When combined with the tissue expression profile of the gene in diseased tissues, information concerning the potential role in pathological processes can be determined. This unit describes a protocol to determine the relative abundance of mRNA in a panel of human tissues using real‐time PCR.
In Vivo Measurement of Blood‐Brain Barrier Permeability
Authors: Boje Kathleen M.K.
Published: June 2004

This unit describes various protocols for the in vivo quantitation of drug permeability across the rodent blood‐brain barrier. Methods for the measurement of drug influx and efflux are described, and support protocols are provided for determining intravascular capillary volume and cerebral perfusion flow. An in situ perfusion technique is also provided for assessing whether transport of a test compound occurs by carrier‐mediated or saturable transport.
Determination of the Stability of Drugs in Plasma
Authors: Hartman David A.
Published: June 2004

In order for a drug to be effective, enough of the active form must reach the target to elicit the desired effect. The first‐pass effect for drugs taken orally is well known but the metabolism of drugs in plasma must also be considered during the drug discovery and development process. For instance, hydrolysis of a compound by plasma enzymes can significantly alter the bioavailability of the active compound. This unit describes methods for the determination of the stability of compounds in plasma.
Screening in Constitutively Active Receptor Systems
Authors: Kenakin Terry
Published: June 2004

This unit discusses the use of constitutive G protein‐coupled receptor (GPCR) activity for the screening of new drug entities. It begins with an introduction to constitutive GPCR activity and then centers on the three basic components of a constitutive screening system, namely the receptor, the receptor coupling components (G protein) and the response reporting system. Then, the influence of ligands and particular application of such systems to drug screening, with reference to optimal conditions, is discussed. Finally, the relative advantages and disadvantages of constitutive screening are considered. This unit discusses the use of constitutive G protein‐coupled receptor (GPCR) activity for the screening of new drug entities
Overview of Safety Pharmacology
Authors: Porsolt Roger D.
Published: June 2004

This overview reviews some of the definitions, concepts, problems and regulatory requirements of safety pharmacology that are encountered by investigators in the field of drug discovery and development.
The Action Potential of the Purkinje Fiber: An In Vitro Model for Evaluation of the Proarrhythmic Potential of Cardiac and Noncardiac Drugs
Authors: Rouet René , Ducroq Joffrey , Picard Sandra
Published: June 2004

The proarrhythmic potential of new drugs can be investigated using in vitro electrophysiological techniques measuring the cardiac action potential in isolated Purkinje fibers. Different types of arrhythmias may occur, as early after‐depolarizations (EADs), which are favored by action potential duration lengthening and bradycardia, or delayed after‐depolarizations (DADs), which are facilitated by tachycardia. The effects of a test compound on the occurrence of these arrhythmias, known to induce in vivo torsades de pointes (or triggered activities), can be studied following the experimental protocols described in this unit.
Electrophysiological Analysis of ATP‐Sensitive Potassium Channels in Mammalian Cells and Xenopus Oocytes
Authors: Shieh Char‐Chang , Gopalakrishnan Murali
Published: June 2004

This unit describes general methodologies for the characterization of ATP‐sensitive K+ channels and the study of ligand‐channel interactions in native tissues and clonal cell lines by electrophysiological techniques. Detailed protocols on how to establish patch‐clamp single‐channel and whole‐cell current recording are presented. Two‐electrode voltage clamp techniques for studying ATP‐sensitive K+ channels expressed in Xenopus oocytes are also included.
Overview of Electrophysiological Characterization of Neuronal Nicotinic Acetylcholine Receptors
Authors: Bertrand Sonia , Bertrand Daniel
Published: June 2004

Electrophysiology is one of the best tools to characterize ligand‐gated channels such as neuronal nicotinic acetylcholine receptors. In this unit, the properties of these receptors are discussed along with approaches for how they can be characterized. Special emphasis is given to agonist and antagonist profiles as well as allosteric effectors that offer alternative possibilities in drug discovery.
Chondrocyte Culture and Assay
Authors: Liebman Jeffrey , Goldberg Ronald L.
Published: June 2004

Chondrocytes constitute the sole cell type found within cartilage, and control the formation and composition of cartilage. Cellular, biochemical and pharmacological studies of arthritis and other cartilage disorders have increasingly focused on chondrocyte function. Three methods are presented in this unit for culturing chondrocytes, and two assays are described that characterize proteoglycan synthesis, a key measure of chondrocyte function.Chondrocytes constitute the sole cell type found within cartilage, and control the formation and composition of cartilage
Immunohistochemical Localization of Proteins in the Nervous System
Authors: Volpicelli‐Daley Laura A. , Levey Allan
Published: June 2004

The immunohistological methods described in this unit can be used to determine the precise localization of neurochemicals, receptors, and proteins throughout the nervous system. Determining the localization of a protein within defined brain nuclei and neuronal cell populations can provide important clues regarding its potential function. Immunoperoxidase reactions and light microscopy are commonly used to visualize the distribution of a single primary antibody directed to an antigen of interest. Double‐labeling immunofluorescence and confocal microscopy techniques detect the localization of one protein relative to another protein and allow analysis of colocalization at a cellular and subcellular level. The colocalization of two proteins can also be quantified, allowing analysis of the extent of overlap between two labeled markers and measurements of changes in the localization of one protein relative to another following drug treatment or in animals that have been genetically modified. The theoretical limit of resolution of confocal microscopy is 0.1 to 0.2 µm.
Transneuronal Circuit Analysis With Pseudorabies Viruses
Authors: Card J. Patrick , Enquist Lynn W.
Published: June 2004

Over the past decade there has been a dramatic increase in the use of viruses as transneuronal tracers of neuronal circuitry. The method exploits the propensity of neurotropic viruses to invade neurons and then produce infectious progeny that cross synapses to infect other neurons within a circuit. The protocols and commentaries included in this unit focus upon the use of the swine alpha herpesvirus known as pseudorabies virus (PRV) for polysynaptic analysis. Here, the aspects of experimental design that have the greatest import for successful use of viruses in circuit definition are presented. Accordingly, the protocols included in this unit can be applied in concert with methods in which the use of classical tract tracers has been detailed. A procedure for retrograde infection of CNS circuits in the rat CNS by peripheral injection of virus is detailed, while transneuronal analysis by intracerebral injection is also described. A variant of these procedures, transneuronal analysis with multiple recombinant strains, is also described along with methods for growing and titering viral stocks, and procedures for single and dual immunohistochemical localization of viral antigens in fixed brain tissue.
Counting Cells in Sectioned Material: A Suite of Techniques, Tools, and Tips
Authors: Williams Robert W. , von Bartheld Christopher S. , Rosen Glenn D.
Published: June 2004

This unit presents protocols to obtain accurate estimates of cell density and cell number in sectioned material by using a light microscope. The “optical disector” or “3‐D counting method” is described, followed by Abercrombie's less commonly used two‐section comparison (TSC) method. These basic protocols are accompanied by four support protocols: one for celloidin embedding, which renders superb morphology, one for point counting, which is important for volume measurements and is almost always used in conjunction with the disector or 3‐D counting, one for handling the potential problem of z‐axis distortion and the consequences that this error can have on density estimates and sampling tactics when using the disector, and finally, one that provides a guide for calibrating and verifying estimates obtained by counting methods.
Intracellular and Juxtacellular Staining with Biocytin
Authors: Wilson Charles J. , Sachdev R.N.S.
Published: June 2004

Many physiological studies require microscopic examination of the recorded neuron for identification. This unit describes how intracellular and extracellular recording can be combined with single‐neuron staining to enable sequential physiological and morphological studies.
Basic Confocal Microscopy
Authors: Smith Carolyn L.
Published: June 2004

Confocal microscopy produces sharp images of structures within relatively thick specimens (up to several hundred microns). It is particularly useful for examining fluorescent specimens. This overview intended to provide background and practical tips needed to get started with confocal microscopy. It begins with a description of the basis of optical sectioning, then discusses various types of confocal microscopes, and concludes with practical guidelines for sample preparation and optimizing image acquisition parameters.
Culture of Substantia Nigra Neurons
Authors: Studer Lorenz
Published: June 2004

Primary cultures of nigral tissue are widely used as a model system to assay effects of trophic and toxic agents on dopaminergic neurons. Cultured dopaminergic neurons have been successfully transplanted in animals and led to behavioral improvement in animal models of Parkinson's disease. Cell cultures have also been used to study the development of substantia nigra, allowing investigators to identify early inductive events important for nigral development and to study dopaminergic differentiation and target innervation. This unit provides simple and reliable culture protocols for these applications. The first approach presented is the preparation of dissociated nigral cell cultures, the later steps of which can be used as a simple and efficient assay for testing growth factors. A second approach is the preparation of free‐floating roller tube cultures, which may be used as a tool for neural transplantation and to study more complex developmental events. A third approach is the production of organotypic cultures using chicken plasma as a matrix. Organotypic cultures can maintain the in vivo cytoarchitecture of a host region in vitro.
Calcium Phosphate Transfection of DNA into Neurons in Primary Culture
Authors: Dudek Henryk , Ghosh Anirvan , Greenberg Michael E.
Published: June 2004

Calcium phosphate/DNA coprecipitation is a widely used method for the introduction of foreign DNA into cells. DNA and calcium phosphate are allowed to form a precipitate that is then added to cells in culture. The cells internalize the DNA, leading to the expression of the transfected genes in the cell. Despite the simplicity of this method, it has not been used very often for primary neurons because of its potential to cause neuronal toxicity. However, low toxicity and reasonably high transfection efficiency (0.5% to 5%) can be achieved by optimization of the transfection parameter, combined in some cases with the use of inhibitors of neuronal activity. This unit describes a very easy and inexpensive method for neuronal gene delivery that can be used with standard eukaryotic expression vectors for the gene of interest.
Overview of Neural Gene Expression
Authors: Neve Rachael L. , Neve Kim A.
Published: June 2004

This overview describes issues that must be considered before attempting to express neural cDNAs in mammalian cells, including the choice of expression vector and cell type. Considerations for introducing recombinant vectors into cells are discussed along with a comparison of achieving stable or transient expression. Finally, the appropriate promoter is crucial and must be chosen to fit the design of the expression system.
Protein Expression in the Drosophila Schneider 2 Cell System
Authors: Schetz John A.
Published: June 2004

The Schneider‐2 (S2) Drosophila cell line is well suited for the stable overexpression of recombinant proteins using plasmid‐based protein expression vectors. Following drug selection, a polyclonal S2 cell line can be induced to express on the order of 2 to 100 pmol/mg membrane protein for G‐coupled protein receptors, 4000 to 100,000 sites/cell for other membrane receptors and 3 to 35 mg/liter for soluble and secreted proteins.
Gene Transfer into Rat Brain Using Adenoviral Vectors
Authors: Thomas Clare E. , Abordo‐Adesida Evelyn , Maleniak Tricia C. , Stone Daniel , Gerdes Christian A. , Lowenstein Pedro R.
Published: June 2004

Recombinant adenovirus vectors are attractive vehicles to deliver genes into the brain for the purposes of neurobiological research and for gene therapy of neurological diseases. This unit provides a comprehensive set of protocols for adenovirus vector‐mediated gene transfer to the brain, including introduction of the vector into the brain by stereotaxic injection and preparation and processing of brain tissue for the evaluation of gene transfer. The potential side‐effects of administering adenovirus vectors to the brain are discussed in detail. The unit also provides protocols for evaluating these side‐effects (e.g., demyelination, inflammation, vector‐mediated cytotoxicity, etc.). Finally, critical parameters for obtaining optimal gene transfer with minimum side‐effects are presented.
Overview of Nucleic Acid Arrays
Authors: DeRisi Joseph
Published: June 2004

Nucleic acid array technology refers to the use and fabrication of arrays containing thousands of nucleic acid samples bound to solid substrates such as glass microscope slides or silicon wafers. Because the physical area occupied by each sample is usually 50 to 200 micrometers in diameter, it is possible to assay nucleic acid samples representing entire genomes, ranging in size from 3,000 to 32,000 genes, on a single slide. Microarrays are useful for analyzing gene expression patterns, genotyping and genetic mapping, comparative genomic hybridization, polysome analysis, and DNA‐protein interactions. This overview describes the technology and applications, and provides valuable web site listings for obtaining additional information.
Reducing Gene Expression in the Brain via Antisense Methods
Authors: Ouagazzal Abdel‐Mouttalib , Tepper James M. , Creese Ian
Published: June 2004

This unit presents protocols that employ antisense oligodeoxynucleotides to reduce expression of target proteins in the brain. These oligonucleotides are generally designed to inhibit synthesis of a specific protein by hybridization to its mRNA. Because oligonucleotides show very poor penetration into the central nervous system (CNS) after systemic administration, they are either injected into the cerebrospinal fluid (CSF) or infused directly into the brain parenchyma. In this unit, the procedure most commonly used for delivering oligonucleotides continuously into CSF is outlined. In addition, a procedure is described for continuous infusion of oligonucleotides into a specific brain region, using the substantia nigra as an example.
Epitope Tagging of Recombinant Proteins
Authors: Brizzard Bill , Chubet Richard
Published: June 2004

Epitope tagging is a method of expressing proteins whereby an epitope for a specific monoclonal antibody is fused to a target protein using recombinant DNA techniques. The fusion gene is cloned into an appropriate expression vector for the experimental cell type and host cells are transfected. The fusion protein can then be detected and/or purified using a monoclonal antibody specific for the epitope tag. This unit presents protocols for detection and purification of proteins tagged with a particular epitope, the FLAG tag, although the same general approach can be applied to other epitope tags. The protocols in this unit employ the anti‐FLAG M2 antibody to detect and purify FLAG‐tagged proteins. The methods presented are immunoprecipitation of FLAG fusion proteins from cells using an anti‐FLAG M2 affinity gel, detection of FLAG fusion proteins by western blotting, and purification of FLAG fusion proteins by anti‐FLAG M2 affinity chromatography.
Fluorescent In Situ Transcription in Cells and Tissues
Authors: Ariano Marjorie A.
Published: June 2004

This method assesses cellular mRNA transcripts in tissue sections and cell cultures using unique short anti‐sense primers directed against sequences in particular protein(s). The unlabeled synthetic cDNA oligonucleotide primers are extended complementary to a sense mRNA transcript using reverse transcriptase and labeled through incorporation of a fluorescent‐labeled dUTP nucleotide base. The new cDNA will be synthesized upstream from the point of primer hybridization, and has a specific activity of fluorescent labeling dependent upon the length of the template mRNA from the primer location to the 5'‐terminus. This procedure provides rapid detection of low abundance mRNA messages that can be related to other cellular protein components, labeled experimentally with alternative fluorochromes.
Recording and Analyzing Synaptic Currents and Synaptic Potentials
Authors: Trussell Laurence
Published: June 2004

Intracellular recording of synaptic currents (PSCs) under voltage clamp conditions provides the most accurate and direct means for measuring the earliest effects of neurotransmitters. With this tool, combined with pharmacological or ionic manipulations, one can obtain information about the type of transmitter used at a synapse, the dynamics of transmitter‐receptor interactions, the types and numbers of receptors activated, the effects of drugs on transmission, functional neural circuitry, and indications about the mechanisms of synaptic plasticity. Each synaptic current or potential is a reflection of many experimental variables: the ionic composition of the solutions, the temperature, the presence of pharmacological agents, the rate of synaptic stimulation, the history of stimulation, the variables of the recording system, as well as other factors unique to each preparation. Correct analysis of data requires all these parameters be considered. Both stimulus‐evoked and spontaneous synaptic events are covered in this unit since conclusions about synaptic and drug mechanisms are strongest when based upon recording of both types of activity. This unit outlines basic considerations for recording PSCs and PSPs in addition to guidelines for data analysis.
Overview of Microdialysis
Authors: Shippenberg Toni S. , Thompson Alexis C.
Published: June 2004

The technique of microdialysis enables the monitoring of neurotransmitters and other molecules in the extracellular environment. This method has undergone several modifications and is now widely used for sampling and quantitating neurotransmitters, neuropeptides, and hormones in the brain and periphery. This unit describes the principles of conventional and quantitative microdialysis as well as strategies in designing a dialysis experiment. It establishes the groundwork for the basic techniques of preparation, conduct, and analysis of dialysis experiments in rodents and subhuman primates. Although the methods described are those used for monitoring CNS function, they can be easily applied with minor modification to other organ systems.
Analyzing Radioligand Binding Data
Authors: Motulsky Harvey , Neubig Richard
Published: June 2004

Radioligand binding experiments are easy to perform, and provide useful data in many fields. They can be used to study receptor regulation, discover new drugs by screening for compounds that compete with high affinity for radioligand binding to a particular receptor, investigate receptor localization in different organs or regions using autoradiography, categorize receptor subtypes, and probe mechanisms of receptor signaling, via measurements of agonist binding and its regulation by ions, nucleotides, and other allosteric modulators. This unit reviews the theory of receptor binding and explains how to analyze experimental data. Since binding data are usually best analyzed using nonlinear regression, this unit also explains the principles of curve fitting with nonlinear regression.
Saturation Analysis of Ligand Binding Using a Centrifugation Procedure
Authors: Layer Richard T.
Published: June 2004

The use of filtration assays is often unsuitable for radioligands with rapid dissociation rates. Rapid dissociation may result in the loss of significant amounts of bound ligand during the separation of bound from free ligand associated with filtration. The primary advantage of the centrifugation assay for characterizing the binding of a radioligand is that the bound ligand is separated from free ligand without initiating significant ligand dissociation from its receptor. Nonetheless, the small volume of supernatant that remains trapped within the membrane pellet (even after a superficial rinsing of the pellet surface) often results in an increased nonspecific binding compared to filtration techniques. The unit presents a protocol for the binding of [3H]glycine to the glycine recognition site of the NMDA receptor complex. This assay is commonly employed because of the current lack of high‐affinity agonist ligands that interact with this recognition site. The use of other radioligands may require different tissue preparations, buffer systems, and assay conditions, but the basic steps involving sedimentation of the pellet, removal of supernatant, superficial washing of the pellet surface, and solubilization of the pellet will remain the same.
Ligand Characterization Using Microphysiometry
Authors: Pitchford Simon
Published: June 2004

This unit describes the use of a Cytosensor microphysiometer for functional characterization of an agonist and antagonist to a G protein‐coupled receptor, the muscarinic M1 receptor. Concentration‐response profiles are used to calculate values for the EC50 of the response of cells to the agonist and the pA2 value for the antagonist. Support protocols describe optimization of two aspects of this procedure: the duration of ligand exposure at a given concentration and the length of recovery time between the administration of two different concentrations of ligand to minimize the impact of desensitization. The Cytosensor microphysiometer allows the measurement of receptor activation in both adherent cells, such as the M1WT3 cells used here or in suspension cultures.
Peripheral Type Benzodiazepine Binding Sites as a Tool for the Detection and Quantification of CNS Injury
Authors: Benavides Jesús , Dubois Alain , Scatton Bernard
Published: June 2004

The concentration of peripheral type benzodiazepine binding sites (PTBS) in the brain parenchyma is greatly increased following brain lesions, reflecting the glial reaction and/or presence of hematogenous cells. Thus, PTBS density is a sensitive and reliable marker of brain injury in a large number of experimental models (ischemia, trauma, excitotoxic lesions, brain tumors) and equivalent human neuropathological conditions. PTBS density can be measured using specific radioligands and a conventional binding technique, or by quantitative autoradiography in tissue sections.
Assessment of Cell Viability in Primary Neuronal Cultures
Authors: Ying Howard S. , Gottron Frank J. , Choi Dennis W.
Published: June 2004

Four commonly used methods for the assessment of neuronal (or glial) cell viability are described in this unit. The MTT assay is sensitive to the function of labile mitochondrial enzymes, which typically lose activity early in the progression towards death. The lactate dehydrogenase (LDH) assay measures the appearance of this cytosolic enzyme in the bathing medium, providing a measure of plasma membrane integrity. Loss of plasma membrane integrity is also the basis of the trypan blue dye assay and the propidium iodide assay. Trypan blue staining is assessed by cell counts; propidium iodide labeling can be assessed either by cell counts, typically in conjunction with fluorescein diacetate counterstaining to identify intact cells containing adequate levels of functional esterases, or with a fluorescence plate reader.
Assessment of Spatial Memory Using the Radial Arm Maze and Morris Water Maze
Authors: Wenk Gary L.
Published: June 2004

Behavioral tasks must be evaluated in terms of the cognitive functions they require in order to be performed. All of the tasks described in this chapter can be used with each of four experimental manipulations: stimulation of a single brain region by drugs or small electrical current, impairment of normal function by production of a lesion or administration of appropriate pharmacological agents, recording of brain activity during the performance of a specific behavioral task, or behavioral phenotyping of transgenic and knockout mice for genes expressed in specific brain regions. This unit describes protocols for the radial arm maze task and the water maze task, both of which require intact spatial memory abilities.
Models of Nociception: Hot‐Plate, Tail‐Flick, and Formalin Tests in Rodents
Authors: Malmberg Annika B. , Bannon Anthony W.
Published: June 2004

Experimental models of pain include tests of response thesholds to high intensity stimuli (acute pain tests) and changes in spontaneous or evoked behavioral responses in animals with peripheral injury or inflammation (persistent pain models). Acute thermal pain is modeled by the hot‐plate and tail‐flick test, while persistent pain can be modeled by the formalin test. This unit presents protocols for all three of these tests, including preparation of animals (rats or mice), administration of a compound being tested for its analgesic properties and data collection.
Mouse Social Recognition and Preference
Authors: Winslow James T.
Published: June 2004

Social recognition in mice is represented by a simple pattern of behavior that can be accurately and reliably quantified by trained observers. The paradigm presented in this unit takes advantage of an ethologically relevant phenomenon marked by a vigorous and species‐typical sequence of investigatory behaviors that occurs when conspecifics meet. Recognition is noted by decreased investigation of a previously encountered animal.
Assessment of Developmental Milestones in Rodents
Authors: Heyser Charles J.
Published: June 2004

Developmental tests are used to characterize early markers of behavior for investigation of the neurobiology of these behaviors, and to assess the impact of early prenatal or postnatal insult. These perturbations may include pharmacological, environmental, and genetic manipulations. At birth the rat is capable of some specific activities, but its movements are uncoordinated and seemingly random, its tactile sensitivity is not fully developed, and its ear canals and eyes remain closed until several days after birth. Postnatal development consists mainly of the continuation of processes begun earlier. This unit presents protocols for the most commonly used animal tests of developmental reflexology, including negative geotaxis, cliff avoidance, placing responses, tactile and acoustic startle responses, surface and air righting reflexes, crossed extensor reflex, rooting reflex, grasp reflex, bar holding, and horizontal and vertical screen tests.
Preclinical Models of Parkinson's Disease
Authors: Bankiewicz Krys S. , Sanchez‐Pernaute Rosario , Oiwa Yoshitsugu , Kohutnicka Malgorzata , Cummins Alex , Eberling Jamie
Published: June 2004

Hepatic encephalopathy is a multifactorial neuropsychiatric syndrome accompanying acute or chronic liver failure. Techniques for developing animal models of hepatic encephalopathy associated with acute or chronic liver failure, or vascular shunting are illustrated. In addition, the behavioral and biochemical characteristics of these models are described.
Models of Amyotrophic Lateral Sclerosis
Authors: Jackson Mandy , Ganel Raquelli , Rothstein Jeffrey D.
Published: June 2004

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder caused by degeneration of the motor neurons in cortex, brainstem and spinal cord. Two experimental models of ALS are described in this unit: organotypic cultures of spinal cord, and transgenic mice expressing a human mutant superoxide dismutase 1 (SOD1) gene. Appropriate animal and cell culture models of ALS can be used to help unravel the sequence of events in motor neuronal degeneration and test potential therapies.
Preparation of Genomic DNA from Mammalian Tissue
Authors: Strauss William M.
Published: June 2004

There are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteination and recovery of DNA. The main differences between various approaches lie in the extent of deproteination and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.
Mouse and Rat Anesthesia and Analgesia
Authors: Davis Judith A.
Published: June 2004

Many animal models used in neuroscience research must be surgically created. The purpose of this unit is to review the advantages and disadvantages of various anesthetic agents in rodents; to discuss state‐of‐the‐art methods for anesthesia; and to present suggestions for analgesic management of mice and rats.
Development of a Universal Nucleobase and Modified Nucleobases for Expanding the Genetic Code
Authors: Romesberg Floyd E. , Yu Chengzhi , Matsuda Shigeo , Henry Allison A.
Published: June 2004

This unit presents protocols for the synthesis and characterization of nucleosides with unnatural bases in order to develop bases for the expansion of the genetic alphabet or for nonselective pairing opposite natural bases. Protocols describe the design, synthesis, and characterization of unnatural base pairs involving 1‐β‐D‐2‐deoxyribosyl‐N‐ and ‐C‐nucleosides. Determination of the thermodynamic and kinetic parameters of unnatural nucleosides is accomplished by incorporation into oligonucleotides and subsequent evaluation as described herein.
A Base‐Labile Protecting Group (Fluorenylmethoxycarbonyl) for the 5′‐Hydroxy Function of Nucleosides
Authors: Gait Michael J. , Lehmann Christian
Published: June 2004

Many popular synthesis strategies look for appropriate 2′‐O‐protection methods to use in conjunction with 5′‐O‐trityl chemistry. In contrast, this unit describes the use of FMOC as a 5′‐protecting group in conjunction with a ketal‐type 2′‐O‐protecting group, 4‐methoxytetrahydropyran‐4‐yl (MTHP). The synthesis of all four 2′‐O‐MTHP‐5′‐O‐FMOC‐protected ribonucleosides and 5′‐O‐FMOC‐2′‐deoxythymidine is described, as is the preparation of the N‐protected, 2′‐O‐MTHP‐protected starting nucleosides.
Solid‐Phase Supports for Oligonucleotide Synthesis
Authors: Pon Richard T.
Published: June 2004

This unit begins with a discussion of the advantages and disadvantages of oligonucleotide synthesis using solid supports. The physical and chemical properties of solid‐phase supports are discussed in terms of their suitability for oligonucleotide synthesis. In addition, the unit outlines the properties of linkers used for transient or permanent attachment of properly protected nucleosides to the derivatized support, as well as strategies for coupling nucleosides to linkers and conditions for the release of synthetic oligonucleotides from specific supports.
A Brief History, Status, and Perspective of Modified Oligonucleotides for Chemotherapeutic Applications
Authors: Cook P. Dan
Published: June 2004

The advent of rapid and efficient methods of oligonucleotide synthesis has allowed the design of modified oligonucleotides that are complementary to specific nucleotide sequences in mRNA targets. Such modified oligonucleotides can be used to disrupt the flow of genetic information from transcribed mRNAs to proteins. This antisense strategy has been used to develop therapeutic oligonucleotides against cancer and various infectious diseases in humans. This overview reports recent advances in the application of oligonucleotides as drug candidates, describes the relationship between oligonucleotide modifications and their therapeutic profiles, and provides general guidelines for enhancing oligonucleotide drug properties.
Modification of the 5′ Terminus of Oligonucleotides for Attachment of Reporter and Conjugate Groups
Authors: Thuong Nguyen T. , Asseline Ulysse
Published: June 2004

Reporter and conjugate groups can be added directly to the 5′ terminus of oligonucleotides by appropriate modification. Conjugate groups can be used to increase the affinity of complementary strands, induce irreversible modification of target sequences, or enable sequences to recognize and permeate target cell membranes. This overview discusses the 5′ modifications that can be used and strategies for the covalent attachment of ligands to the modified oligonucleotides. Step‐by‐step protocols for attachment of conjugate groups are given elsewhere in the series.
Synthesis and Characterization of Chimeric 2‐5A‐DNA Oligonucleotides
Authors: Player Mark R. , Torrence Paul F.
Published: June 2004

This unit provides protocols for the synthesis and characterization of 2‐5A‐antisense nucleic acids. These chimeric oligonucleotides consist of 2′,5′‐phosphodiester‐linked oligoadenylates ligated to 3′,5′‐deoxyribonucleotides and are readily prepared using phosphoramidite chemistry on CPG solid supports. The 3′,5′‐deoxyribonucleotide functions as the antisense domain to target a given mRNA sequence, while the 2′,5′‐phosphodiester‐linked oligoadenylate serves to locally activate 2‐5A‐dependent RNase L, causing the targeted sequence to be cleaved.
Synthesis and Purification of Peptide Nucleic Acids
Authors: Braasch Dwaine A. , Nulf Christopher J. , Corey David R.
Published: June 2004

Peptide nucleic acids (PNAs) are DNA analogs in which the normal phosphodiester backbone is replaced by 2‐aminoethyl glycine linkages. Hybridization of PNAs with RNA or DNA follows normal rules for Watson‐Crick base pairing and occurs with high affinity. Thus, PNAs are a promising choice for applications that benefit from high‐affinity hybridization. They are assembled using techniques adapted from peptide chemistry. Protocols are given for both automated and manual synthesis of PNAs as well as their purification. The advantages of each method are discussed, as are the different monomers and reagents that are required. Additionally, protocols are given for adding peptides to PNAs (which can enhance hybridization or cell uptake of the PNA) and for adding a biotin label.
Probing RNA Structure by Lead Cleavage
Authors: Pan Tao
Published: June 2004

Lead cleavage causes a transesterification reaction that breaks the 5′,3′‐phosphodiester backbone of RNA, leaving a 2′,3′‐cyclic phosphate and a 5′‐hydroxyl. Since the efficiency of the reaction at the 2′‐hydroxyl is related to steric and chemical constraints on particular 2′‐hydroxyls embedded in the RNA, this reaction can be used to examine the structure of individual nucleotides within an RNA molecule. It is a sensitive probe of tertiary RNA structure, provided that Pb2+‐binding sites are created in the tertiary structure.
Probing RNA Structures with Hydroxyl Radicals
Authors: Celander Daniel W.
Published: June 2004

Fe(II)‐EDTA can be used to conveniently generate hydroxyl radicals to promote cleavage of RNA at nucleotide resolution. Two procedures are described, involving the generation of free radicals from solvated molecular oxygen and from hydrogen peroxide added to the RNA solution. Unlike other footprinting reagents, hydroxyl radicals cleave the sugar‐phosphate backbone at every residue and thus provide uniform cleavage in a given RNA secondary structure. Because some positions become protected by tertiary folding, this reagent is useful for monitoring the global folding of RNA at equilibrium.
Chemical Reagents for Investigating the Major Groove of DNA
Authors: Rokita Steven E.
Published: June 2004

Chemical modification provides an inexpensive and rapid method for characterizing the structure of DNA and its association with drugs and proteins. Numerous conformation‐specific probes are available, but most investigations rely on only the most common and readily available of these. The major groove of DNA is typically characterized by reaction with dimethyl sulfate, diethyl pyrocarbonate, potassium permanganate, osmium tetroxide, and, quite recently, bromide with monoperoxysulfate. This commentary discusses the specificity of these reagents and their applications in protection, interference, and missing contact experiments.