bluuebunny/crossref_metadata_2025 huggingface dataset:
Overview of Pharmacogenetics
Authors: Katz David A.
Published: June 2004

Pharmacogenetics is the study of relationships between genetic variation and inter‐individual differences of drug response. As the field has matured over the past 15 years, a remarkable diversity of pathways, variation types, and mechanisms have been found to be relevant pharmacogenetic factors. Today, pharmacogenetics is becoming more important in pharmacology for target validation, lead optimization, and understanding of idiosyncratic toxicity. This unit provides an overview of the history of pharmacogenetics and current research applications in drug discovery, as well as a discussion of research quality issues relevant for human subjects research in the pharmacogenetics laboratory.
Estimating Intestinal Mucosal Permeation of Compounds Using Caco‐2 Cell Monolayers
Authors: Gao Jinnian , Hugger Erin D. , Beck‐Westermeyer Melissa S. , Borchardt Ronald T.
Published: June 2004

Step‐by‐step protocols are provided in this unit for the measurement of apparent permeability coefficients of compounds using Caco‐2 cell monolayers as an in vitro model of the intestinal mucosa. Procedures for culturing the cells and transmonolayer transport studies are also included. Critical issues for successfully estimating intestinal mucosal permeation of drugs are discussed. Step‐by‐step protocols are provided in this unit for the measurement of apparent permeability coefficients of compounds using
Overview of Drug Product Development
Authors: Lee Kathleen M.
Published: June 2004

Drug product development, also called formulation chemistry, is the process of developing formulations for use in toxicology and clinical studies and for commercial supply. In this overview, the development process is described, including dosage forms for various delivery routes, toxicology formulation development, ingredient selection and manufacturing process development.
Uptake Studies for Evaluating Activity of Efflux Transporters in a Cell Line Representative of the Blood‐Brain Barrier
Authors: Silverstein Peter S. , Karunaratne D. Nedra , Audus Kenneth L.
Published: June 2004

In this unit, protocols are presented for performing uptake studies with bovine brain microvessel endothelial cells (BBMECs) using either radiolabeled compounds, or rhodamine 123 as a marker for a P‐glycoprotein substrate. The protocols can easily be modified for the investigation of substrate uptake in other cell lines.
In Vitro Autoradiography
Authors: Kuhar Michael J.
Published: June 2004

The light‐microscopic localization of receptors and other molecules by autoradiography of radioligand binding in tissue sections provides a high degree of anatomical resolution and the ability to associate even small quantities of binding sites with anatomical structures. The basic procedure in this unit involves incubating slide‐mounted tissue sections with radioligands, washing and drying of the sections with specifically bound ligands under conditions that preserve ligand binding, and visualizing and quantifying the binding sites in the tissues. While the procedures were developed for drug and neurotransmitter receptors, any binding site can be visualized with a sufficiently selective, high‐affinity radioligand. If optimal conditions for radioligand binding to sites in slide‐mounted tissue sections are already known, the basic protocol in this unit can be used to create autoradiograms. However, if a radioligand is being studied for the first time, guidelines are provided on optimizing experimental parameters for binding, washing, and drying. The light‐microscopic localization of receptors and other molecules by autoradiography of radioligand binding in tissue sectio.
Cell‐Based Assays Using the Fluorometric Imaging Plate Reader (FLIPR)
Authors: Whiteaker Kristi L. , Sullivan James P. , Gopalakrishnan Murali
Published: June 2004

A critical component of the drug discovery and development process is the identification of novel pharmacophores. Such discovery efforts are, in general, facilitated by rapid and high‐throughput cell‐based assays of receptor/ion channel‐mediated signaling processes to screen diverse chemical libraries of compounds possessing activator or inhibitor activities at the desired target. The availability of the Flurometric Image Plate Reader (FLIPR) has made rapid assays of cellular signaling processes feasible by simultaneous kinetics measurement of cell‐based fluorescence changes in a 96‐ or 384‐well format. This unit describes the application of the FLIPR in cell‐based kinetic assays for measuring membrane potential changes and intracellular calcium dynamics.
Overview of Combinatorial Chemistry
Authors: Crooks Stephen L. , Charles Leslie J.
Published: June 2004

This unit provides an overview of combinatorial chemistry, including the design and construction of combinatorial libraries for drug discovery. Strategies and resources for library synthesis are summarized, and examples of combinatorial libraries are given. This unit provides an overview of combinatorial chemistry, including the design and construction of combinatorial libraries fo.
Overview of Microarrays in Drug Discovery and Development
Authors: Waring Jeffrey F.
Published: June 2004

With the sequencing of the human genome, new tools and technologies have been developed to identify and quantify global gene‐expression changes occurring in the cell. One of the main tools being utilized is microarray technology, which allows one to quantitate expression changes of thousands of genes in a single experiment. Microarrays allow researchers to gain an unprecedented understanding of the function and regulation of genes, and are transforming virtually all areas of biological research. In the drug‐discovery process, microarrays have the potential to play a role in all stages, from new target discovery through compound profiling and safety assessment. This overview highlights some of these studies and discusses how this technology is transforming the field of drug discovery and development.
Models of Inflammation: Measuring Gastrointestinal Ulceration in the Rat
Authors: Whiteley Phyllis E. , Dalrymple Stacie A.
Published: June 2004

In rats, nonsteroidal anti‐inflammatory drugs (NSAIDs) such as indomethacin induce gastrointestinal ulcerations. This unit describes a procedure to elicit and measure indomethacin‐induced gastrointestinal lesions. This approach can be used to test for gastrointestinal side effects of potential anti‐inflammatory and other agents.
Toxicology in the Drug Discovery and Development Process
Authors: Dorato Michael A. , Buckley Lorrene A.
Published: June 2004

The primary objective of toxicology studies in the drug development process is to evaluate the safety of potential drug candidates using relevant animal models and validated procedures. The ultimate goal is to interpret the animal data to assess risk for human subjects/patients. Therefore, the toxicologist must be aware of the international guidelines for safety evaluation in conjunction with the use of traditional and nontraditional models such as transgenic animals. This overview discusses the components of a typical toxicology profile including safety pharmacology, genetic toxicology, acute and subchronic toxicology, chronic toxicology, absorption, distribution, metabolism and excretion (ADME) studies, reproductive and developmental toxicology, and an evaluation of carcinogenic potential. (Due to the wide variety of therapeutic targets, toxicology profiles may differ from drug to drug.) Also, regulatory guidelines for toxicology testing are explained in relation to the drug discovery and development process.
Place Preference Test in Rodents
Authors: Roux Sylvain , Froger Christelle , Porsolt Roger D. , Valverde Olga , Maldonado Rafael
Published: June 2004

Conditioned place preference is a behavioral model currently used to measure the rewarding properties induced by the administration of a drug. In this paradigm, the rewarding properties of a compound are associated with the particular characteristics of a given environment. Advantages of this test are the absence of any instrumental learning and the possibility of evaluating reinforcing effects in the absence of the test substance. Due to the recent availability of multiple lines of genetically modified mice, this unit has been updated to include procedures for testing place preference in both rats and mice. Details are also provided for evaluating drug effects using this procedure.
Models of Neurological Disease (Substance Abuse): Self‐Administration in Monkeys
Authors: Carey Galen , Spealman Roger D.
Published: June 2004

Drug self‐administration is a procedure in which a subject performs a response, called an operant, that results in the delivery of a drug injection. This procedure is viewed as a relevant model for the study of human drug‐taking behavior. Drug self‐administration in primates has several characteristics that resemble drug‐taking behavior in humans, and drugs that are commonly abused by humans also typically maintain self‐administration behavior in monkeys. Drug self‐administration procedures allow for the study of a variety of drug properties. For instance, they are used to investigate the abuse potential of new compounds and to study the effects of candidate medications for the treatment of drug addiction. These procedures also can be used to study the process of drug reinforcement. This unit describes intravenous drug self‐administration in large primates, such as rhesus macaques, and smaller primates, such as squirrel monkeys.
Patch‐Clamp Studies of Human Cardiac Ion Channels in the Evaluation of Cardiac Electrophysiological Effects of Compounds
Authors: Crumb William J. , Cavero Icilio
Published: June 2004

Drugs prolonging the QT interval appear to consistently inhibit the outward, rapid delayed rectifier K+ current (IKr) conveyed by the HERG channel. Hence, for determining whether a new drug candidate blocks the latter channel, this unit presents a basic electrophysiology protocol to conduct patch clamp studies in single cell preparations expressing heterologously cloned HERG channels. An additional protocol details the isolation of myocytes from specimens of human atria which are used in the study of native cardiac ion currents (INa, ICa, Ito, Isus, IK1). The results of these tests are useful for determining whether drug candidates have the desired cardiac safety profile for human use.
Respiratory Function Assessment in Safety Pharmacology
Authors: Murphy Dennis J.
Published: June 2004

The known effects of drugs from a variety of pharmacological/therapeutic classes on the respiratory system, the life‐threatening consequences of respiratory dysfunction, and world‐wide regulatory safety guidelines all support the need for conducting respiratory evaluations in safety pharmacology. This unit provides an overview of the functional disorders of the respiratory system and presents the techniques and strategies considered to be most appropriate for detecting and characterizing drug‐induced respiratory disorders in safety pharmacology studies.
Electrophysiological Analysis of Heterologously Expressed Kv and SK/IK Potassium Channels
Authors: Castle Neil A. , Wickenden Alan D. , Zou Anruo
Published: June 2004

This unit describes protocols to aid investigators in determining the electrophysiological and pharmacological profile of heterologously expressed voltage or calcium‐activated potassium channels belonging to the Kv1.x and SK/IK gene families. Protocols for data acquisition as well as analysis are provided.
Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells
Authors: Abmayr Susan M. , Carrozza Michael J. , Workman Jerry L.
Published: June 2004

Extracts prepared from the isolated nuclei of cultured cells are functional and accurate in in vitro transcription and mRNA processing. Thus, such extracts can be used directly for functional studies and as the starting material for purification of the proteins involved in these processes. In this unit, a procedure for generating reproducible extracts from different preparations of nuclei is described. The conditions are optimized to produce transcriptionally active extracts from HeLa cells. The protocol can also be optimized to increase the yield of specific proteins or activities from different cells types. Preparation of the cytoplasmic (S‐100) fraction from the cytoplasmic extract produced in the main protocol is also described.Extracts prepared from the isolated nuclei of cultured cells are functional and accurate in in vitro transcription and mRNA.
Measurement of VLA‐4/CS‐1 and VLA‐4/VCAM Adhesion Inhibition
Authors: Mehlin Christopher
Published: June 2004

Cell adhesion, a critical early step in the inflammatory process, has increasingly become the target of drug discovery efforts. Described in this unit are techniques for measuring inhibitors of VLA‐4‐mediated adhesion to either VCAM or the connecting segment (CS‐1) of fibronectin.
Protein Analysis by SDS‐PAGE and Detection by Coomassie Blue or Silver Staining
Authors: Gallagher (electrophoresis, staining) Sean , Sasse (staining) Joachim
Published: June 2004

Electrophoresis is used to separate complex mixtures of proteins, to investigate subunit compositions and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, the combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit first presents an informative section about safe use of electricity and electrophoresis. Protocols are then provided for one‐dimensional gel electrophoresis under denaturing conditions and subsequent staining with either Coomassie blue or silver staining.Electrophoresis is used to separate complex mixtures of proteins, to investigate subunit compositions and to verify homogeneit
Purification and Concentration of DNA from Aqueous Solutions
Authors: Moore David
Published: June 2004

This unit presents procedures for manipulating solutions of single‐ or double‐ stranded DNA through purification and concentration steps. These techniques are useful when proteins or solute molecules need to be removed from aqueous solutions, or when DNA solutions need to be concentrated. The basic protocol, using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations lower than 1 mg/ml. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads and this is also presented. These protocols may also be used for purifying RNA. Two alternate protocols can be used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing low molecular weight oligonucleotides and triphosphates.
Cryosectioning
Authors: Watkins Simon
Published: June 2004

This unit describes sample preparation and sectioning methods for frozen tissue. Sections of this type are used in a variety of light microscopic procedures including in situ hybridization immunohistochemistry, and enzyme histochemistry.
Overview of Gene Delivery into Cells Using HSV‐1‐Based Vectors
Authors: Neve Rachael L. , Lim Filip
Published: June 2004

This overview describes the considerations involved in the preparation and use of herpes simplex virus type 1 (HSV‐1) as a vector for gene transfer into neurons. Strategies for gene delivery into neurons, either to study the molecular biology of brain function or for gene therapy, must utilize vectors that persist stably in postmitotic cells and that can be targeted both spatially and temporally in the nervous system in vivo. This unit describes the biology of HSV‐1 along with a discussion covering development of amplicon and genomic HSV‐1 vectors. Advantages and disadvantages of current HSV‐1 vectors are presented, and HSV‐1 vectors are compared with other vectors for gene transfer into neurons.
The Use of Phage Display in Neurobiology
Authors: Bradbury Andrew
Published: June 2004

Phage display is a technique that involves the coupling of phenotype to genotype in a selectable format. It has been extensively used in molecular biology to study protein‐protein interactions, receptor and antibody binding sites, and immune responses; to modify protein properties; and to select antibodies against a wide range of different antigens. In the format most often used, a polypeptide is displayed on the surface of a filamentous phage by genetic fusion to one of the coat proteins, creating a chimeric coat protein. As the gene encoding the chimeric coat protein is packaged within the phage, selection of the phage on the basis of the binding properties of the polypeptide displayed on the surface simultaneously results in the isolation of the gene encoding the polypeptide. This unit describes the background of the technique and illustrates how it has been applied to a number of different problems, each of which has its neurobiological counterparts.
Basic Neuroanatomical Methods
Authors: Gerfen Charles R.
Published: June 2004

This unit covers some of the basic procedures that are common to a wide range of neuroanatomical protocols. Procedures are provided for the preparation of unfixed, fresh brain tissue as well as for perfusion fixation of animals resulting in fixed neural tissue. A variety of methods for sectioning brains are described, including frozen sectioning in a cryostat, frozen sectioning with a microtome, and sectioning with a vibratome. The choice of sectioning method depends on how the brain has been prepared and what histochemical method is to be used. Three post‐sectioning procedures are provided: defatting of slide‐mounted sections, thionin staining of the sections, and coating of slides with photographic emulsion for autoradiography. Finally, a procedure is described for subbing slides with gelatin, which is necessary in some protocols in order for the sections to adhere to the slides.
Hybridization Histochemistry of Neural Transcripts
Authors: Young W. Scott , Mezey Éva
Published: June 2004

Expression of genes is manifested by the production of RNA transcripts within cells. Hybridization histochemistry (or in situ hybridization) permits localization of these transcripts with cellular resolution or better. Furthermore, the relative amounts of transcripts detected within different tissues or the same tissues in different states (e.g., physiological or developmental) may be quantified. This unit describes hybridization histochemical techniques using either oligonucleotide probes or RNA probes (riboprobes). Also presented is the use of probes labeled with digoxigenin for colorimetric detection of RNA transcripts and a technique to detect the Y chromosome using either mouse or human riboprobes. Finally, a procedure is presented for the autoradiographic detection of radiolabeled probes. Methods are provided for labeling oligodeoxynucleotide and RNA probes and performing northern analyses using these probes.
Metabolic Mapping
Authors: McCasland James S. , Graczyk Ginny M.
Published: June 2004

This unit describes a high‐resolution [3H]2‐deoxyglucose (2DG)/immunohistochemistry double labeling protocol which enables simultaneous visualization of metabolic and immunohistochemical markers (e.g., neurotransmitter‐specific epitopes) in the same tissue section. This approach generates single‐cell metabolic measures from large (many thousands) samples of neurons, and allows the assignment of putative neurotransmitter types to large samples of functionally assayed single neurons. Thus, it provides a unique opportunity to assay the metabolic activities of histochemically identified neural elements throughout an entire pathway. [3H]2DG injection and perfusion fixation of hamsters or mice is described first, followed by immunohistochemical staining of brain slices for a monoclonal antibody against glutamic acid decarboxylase‐6 (GAD‐6). Included are the steps for dipping and developing slides for autoradiography. An alternate protocol gives details of staining for cytochrome oxidase (CO).
Electronic Imaging in Neuroscience
Authors: Spring Kenneth R.
Published: June 2004

This unit is intended to aid the neuroscientist in understanding the basics of image detectors and selecting a suitable camera for various neuroscience research applications. A procedure is described for evaluating cameras in the laboratory.
Loading Neurons with Dextran‐Conjugated Calcium Indicators in Intact Nervous Tissue
Authors: Delaney Kerry R.
Published: June 2004

This unit describes methods for filling populations of neurons and their processes, including presynaptic terminals, with dextran‐conjugated calcium indicators in central nervous tissue of mammals and lower vertebrates. Techniques for filling neurons in vivo for subsequent analysis either in vivo or in brain slices or en bloc preparations are described. These methods are also suitable for staining neurons in acute and organotypic brain slices.
Culture of Neuroepithelial Stem Cells
Authors: Hazel Thomas , Müller Thomas
Published: June 2004

In vitro culture of multipotent neural precursors allows researchers to study mechanisms regulating such processes as proliferation and lineage commitment within the developing central nervous system (CNS). The protocols presented in this unit describe the isolation and maintenance of stem cells from both fetal and adult rodent neural tissue. A procedure for treating tissue culture dishes with poly‐L‐ornithine and fibronectin, which is necessary before the dishes are used for neuroepithelial stem cell culture, is also included.
Long‐Term Culture of Hippocampal Neurons
Authors: Vicario‐Abejón Carlos
Published: June 2004

In culture, hippocampal cells can develop to express neuronal antigens and acquire mature neuronal morphologies, including axons, complex dendritic trees, and synapses that are electrophysiologically active. This system is suitable for studying neuronal differentiation and other events, such as synaptogenesis. It is also a valuable model for investigating synaptic plasticity and exploring the mechanisms of neuronal degeneration. This unit provides a protocol for culturing neurons prepared from embryonic (E‐18) rat or mouse hippocampus, but could also be used to grow neurons from embryonic cortex, olfactory bulb, striatum, or spinal cord. A second method is included for preparing neuronal cultures from embryos with different genotypes, such as those from transgenic mice. Also described is the preparation of polyornithine‐ and fibronectin‐coated coverslips, which are highly adhesive and promote neurite outgrowth, for use in the culture protocols.
Isolation and Purification of Primary Oligodendrocyte Precursors
Authors: Bögler Oliver
Published: June 2004

Oligodendrocytes are the myelinating cells of the central nervous system, and are avidly studied by investigators interested in neural cell lineages, myelination, multiple sclerosis, and oligodendroglioma. This unit presents a strategy for manipulating the division and differentiation of oligodendrocyte progenitors by isolating O‐2A progenitors from optic nerve and then purifying and expanding them by culture in a defined medium supplemented with platelet‐derived growth factor (PDGF) plus basic fibroblast growth factor (bFGF). Cells are then allowed to differentiate through removal of the growth factors. A protocol is also provided for monitoring the cell type composition of the cultures by immunohistochemistry. In addition to making it possible to generate large numbers of cells, this approach allows access to intermediate differentiation stages. Cells can thus be studied as they differentiate into oligodendrocytes, making the actual process of differentiation open to examination.
Experimental Transplantation in the Embryonic, Neonatal, and Adult Mammalian Brain
Authors: Brüstle Oliver , Cunningham Miles G. , Tabar Vivian , Studer Lorenz
Published: June 2004

This unit provides three basic experimental protocols for transplantation into the adult, neonatal, and embryonic rat or mouse brain. These approaches differ not only with respect to their technical peculiarities but also in the spectrum of biological questions they can address. Also included are procedures for the construction of a simple injection device used in delivery of the cell suspension to be transplanted into the embryonic brain; preparation of single‐cell suspensions, which are the preferred form of donor material for transplantations; and two procedures, technically similar to the transplantation procedures, for modeling neurodegenerative disease in rodents: 6‐hydroxydopamine (6‐OHDA) lesioning as a model of Parkinson's disease and ibotenic acid lesioning as a model for Huntington's disease.
Purification of Oligodendrocytes and Their Progenitors Using Immunomagnetic Separation and Percoll Gradient Centrifugation
Authors: Colello Raymond J. , Sato-Bigbee Carmen
Published: June 2004

In this unit, two techniques are described for the purification of oligodendrocytes and their progenitors from the developing mammalian central nervous system (CNS). The first method utilizes the technique of immunomagnetic separation to selectively isolate oligodendrocytes and their progenitor cells from the optic nerve of prenatal and early postnatal rats. This technique takes advantage of the surface antigens expressed on these cells. A paramagnetic bead is attached to the cells via an antibody bridge. Target cells that are coupled to magnetic beads can then be separated from a heterogeneous cell population using a magnetic field. The second method for isolating oligodendrocytes uses Percoll gradient centrifugation to separate oligodendrocytes from a heterogeneous cell population by virtue of their cell density and allows the direct isolation of oligodendrocytes from animals aged postnatal day 4 (P‐4) to adult. This method is particularly useful for assessing physiological systems present in development that may be lost as a result of growing purified neonatal cells in vitro in the absence of neuronal influence.
Neuronal Transfection Using Particle‐Mediated Gene Transfer
Authors: Lo Donald C.
Published: June 2004

This unit describes the use of particle‐mediated gene transfer (also known as biolistics) for the transfection of neuronal cell lines and brain slices. Like nuclear microinjection of DNA, biolistics results in the direct introduction of DNA into the nucleus; it is perhaps for this reason that biolistics works as well in mitotic cells as in postmitotic cells such as skeletal muscle, skin, liver, and neurons. The basic principle of biolistics is to accelerate micron‐sized gold particles coated with DNA towards target cells or tissue. Cells penetrated by these particles have a high likelihood of being transfected by the DNA thus introduced. The motive force for particle acceleration can come from a variety of sources, the most widely used is described in this unit and is a supersonic shock wave generated by the rupture of a kapton membrane induced by high‐pressure helium. Another option included in this unit is to propel the gold particles by gas jet entrainment.
Gene Expression in Transgenic Mice Using Neural Promoters
Authors: Okabe Shigeo
Published: June 2004

In the first part of this unit, major considerations for the analysis of neural promoters in transgenic mice are discussed. Detailed protocols on the production of transgenic mice are not described in this unit. Advantages and disadvantages of the transgenic approach for analysis of neural cis‐acting elements are also presented. The concept of transient transgenic mice is then introduced; this method compensates for some disadvantages associated with the conventional transgenic approach. Finally, major factors influencing the efficiency of transgenic mouse production are discussed. The second part of the unit presents detailed information on a variety of neural‐specific cis‐acting elements that have been characterized by a transgenic approach. This information is useful both as a guide for carrying out the analysis of cis‐acting elements and as a reference for selection of promoter/enhancer elements for designing an appropriate transgenic construct.
PCR Cloning of Neural Gene Products
Authors: Grandy David K. , Bunzow James R. , Dorit Robert L.
Published: June 2004

Of the many proteins that are known to be involved in neuronal signaling, one family of gene products, collectively referred to as the G protein‐coupled receptors (GPCRs), has received considerable attention. Within the transmembrane domains of GPCRs are clusters of amino acids that tend to be conserved among receptors that bind related ligands. Polymerase chain reaction (PCR)‐based approaches to cloning novel GPCRs typically begin with the identification of these well‐conserved amino acid motifs, which are then back‐translated into degenerate oligonucleotide primers. These pools of degenerate oligonucleotides are the most important variables in PCR cloning of GPCRs. Although GPCRs are used as the focus of this unit, the strategies and techniques described are applicable to the cloning of a wide variety of neuronal gene products. In the first procedure in this unit, either total or poly(A)+ purified RNA is reverse transcribed into first‐strand cDNA. In subsequent steps the cDNA product serves as the template for synthesis and amplification of target receptor sequences by PCR primed with degenerate oligodeoxynucleotides. The product is ready to be cloned and screened as described. Guidelines for database searching are provided to help identify the cloned gene from the known sequence. Typically, only a portion of the receptor coding region is cloned by the above approach. Rapid amplification of cDNA ends (RACE) or anchored PCR is described in this unit and is used to obtain a full‐length cDNA amenable for expression studies.
cDNA Expression Cloning in Mammalian Cells
Authors: Hoffman Beth J.
Published: June 2004

This unit contains protocols for expression cloning in mammalian cells. Either calcium phosphate‐ or liposome‐mediated transfection of mammalian cells, or virus infection and liposome‐mediated transfection are used to screen pools derived from a cDNA library. cDNA pools are prepared for cloning from library‐transformed E. coli grown in liquid culture medium or on antibiotic‐containing selection plates. Results of screening assays for expression can be detected using autoradiography of dishes of cultured cells to identify clones, direct visualization of radiolabeled cells on emulsion‐coated and developed chamber slides, detection and quantification of gene activity by a functional (transport) assay with scintillation counting, or detection using a filter‐based assay for binding of radioligand to membranes or whole cells. The most critical step of any cDNA cloning project is the establishment of the screening protocol. Therefore, the bioassay for the gene product must be established prior to executing any of these protocols, including construction of the cDNA library.
Generation of High‐Titer Defective HSV‐1 Vectors
Authors: Lim Filip , Neve Rachael L.
Published: June 2004

There are two types of replication‐deficient herpes simplex virus type 1 (HSV‐1) vectors: those in which the foreign DNA of interest is cloned into the viral genome itself, and those that are comprised of a plasmid (amplicon) carrying minimal HSV‐1 sequences that allow it to be packaged into virus particles with the aid of a helper virus. This unit describes the generation of helper virus stocks. Preparation of recombinant amplicon vector particles by transfection of amplicon and superinfection of helper virus into cells, and harvesting of packaged particles, is also delineated. Thorough characterization of the viral vector stock involves measuring (1) the helper virus plaque‐forming units per ml (pfu/ml) on 2‐2 cells, (2) the wild‐type HSV‐1 pfu/ml on Vero cells, and (3) the amplicon stock infectious vector units per ml (ivu/ml) on PC12 cells. Support protocols detail methods for determining titers of helper virus and wild‐type virus by plaque assay, and of amplicon stocks by vector assay.
Gene Delivery Using Helper Virus–Free HSV‐1 Amplicon Vectors
Authors: Fraefel Cornel
Published: June 2004

Herpes simplex virus type 1 (HSV‐1)‐based amplicon vectors contain only a very small percentage of the 152‐kbp viral genome. Consequently, replication and packaging of amplicons depend on helper functions that are provided either by replication‐defective mutants of HSV‐1 or by replication‐competent, but packaging‐defective, HSV‐1 genomes. Sets of cosmids that overlap and represent the entire HSV‐1 genome can form, via homologous recombination, circular replication‐competent viral genomes, which give rise to infectious virus progeny. However, if the DNA cleavage/packaging signals are deleted, reconstituted virus genomes are not packageable, but still provide all the helper functions required for the packaging of cotransfected amplicon DNA. The resulting stocks of packaged amplicon vectors are essentially free of contaminating helper virus. This unit describes the cotransfection of amplicon and cosmid DNA into 2‐2 cells by cationic liposome‐mediated transfection using and the harvesting of packaged vector particles. Support protocols provide methods for preparing cosmid DNA and determining the titers of amplicon stocks.
The Substituted‐Cysteine Accessibility Method (SCAM) to Elucidate Membrane Protein Structure
Authors: Liapakis George , Simpson Merrill M. , Javitch Jonathan A.
Published: June 2004

The substituted‐cysteine accessibility method (SCAM) provides an approach to identifying the residues in the membrane‐spanning segments that line a channel, transporter, or binding‐site crevice. SCAM can also be used to determine differences in the structures of the membrane‐spanning segments in different functional states of the proteins, to map electrostatic potential in the membrane‐spanning domains, and to size a channel or binding‐site crevice. The protocol in this unit describes the use of SCAM to map the binding‐site crevice of a G‐protein coupled receptor (GPCR) which binds ligand within the transmembrane portion of the receptor.
Production of Recombinant Adeno‐Associated Viral Vectors and Use in In Vitro and In Vivo Administration
Authors: Haberman Rebecca A. , Kroner‐Lux Gabriele , McCown Thomas J. , Samulski Richard Jude
Published: June 2004

Adeno‐associated virus (AAV) vectors are able to transduce CNS cells in vitro and in vivo for extended periods of time without immune complications. The ability to efficiently deliver transgenes in vivo with recombinant AAV makes this an ideal vector for neuronal gene therapy. This unit describes the production of recombinant AAV vectors and presents a protocol for infusing these viruses into the brains of intact animals.
Calcium Flux Assay in Xenopus Oocytes
Authors: Murphy Philip M.
Published: June 2004

Many G protein‐coupled receptors of interest to neuroscientists induce transient increases in [Ca2+]i, which can be used as a convenient measure of receptor activation in a variety of applications. This unit describes a simple calcium flux assay applied to Xenopus oocytes
Production of High‐Titer Lentiviral Vectors
Authors: Zufferey Romain , Trono Didier
Published: June 2004

Conventional retroviral vectors are of limited usefulness for neuroscience applications because they are derived from oncoretroviruses such as the Mouse Leukemia Virus (MLV) and, as a consequence, cannot transduce nondividing cells. Most cells in the nervous system proliferate very little, if at all. In contrast to oncoretroviruses, lentiviruses such as the human immunodeficiency virus (HIV) are a sub‐family of retroviruses that can infect both growth‐arrested and dividing cells. Accordingly, lentiviral vectors efficiently transduce targets such as neurons and glial cells, both in tissue culture and in vivo. This unit describes the production of high‐titer lentiviral vectors by transient transfection of 293T cells and concentration of the particles by centrifugation. Guidelines for the titration of vector stocks are also given.
Alphavirus‐Mediated Gene Transfer into Neurons
Authors: Ehrengruber Markus U. , Lundstrom Kenneth
Published: June 2004

Semliki Forest virus and Sindbis virus belong to the alphavirus genus of the togaviruses and have recently been shown to efficiently infect neurons in vitro and in vivo, i.e., under conditions where glial cells are present. This unit describes the preparation and application of recombinant, neurotrophic replicons derived from Semliki Forest virus and Sindbis virus for gene transfer into hippocampal neurons.
RNA Analysis by Nuclease Protection
Authors: Goldrick Marianna , Kessler Donald
Published: June 2004

Nuclease protection assays (S1 nuclease protection and RNase protection) are extremely sensitive procedures for detection and quantitation of mRNA species in complex mixtures of total cellular RNA. These assays are well suited for mapping positions of external and internal junctions in RNA, such as transcription initiation and termination sites and intron/exon boundaries, and to discriminate between closely related targets by using probes designed to span the regions where the related genes differ the most. Also, because the size of the probes used in nuclease protection assays is a variable chosen by the investigator, probes may be designed to protect fragments of different sizes. This feature permits the simultaneous analysis of several different mRNAs in the same total RNA sample. In this unit, a method is included for RNase protection of target mRNA sequences, including hybridization of the probe to the target sequence, details of the actual protection assay, and detection of reaction products. An alternative method is provided for performing the RNase protection assay on a microvolume scale, which is useful when there are many samples to be analyzed. Support protocols describe synthesis and gel purification of labeled RNA probes; preparation of RNase‐free yeast RNA, which acts as an aid in the quantitative precipitation of newly synthesized probe; and quantitation of target mRNA. A method describing S1 nuclease protection of target mRNA using either RNA or DNA probes is also included. Additional support protocols provide instructions for the preparation of radiolabeled DNA probes by primer‐extension of double‐stranded plasmid or PCR product using Klenow fragment of E. coli DNA polymerase I or Taq or Tth polymerase in a thermal cycler. Another radiolabeling method details 5' end labeling of oligodeoxynucleotides and oligoribonucleotides using T4 polynucleotide kinase. Additionally, a method is described for mapping transcription start sites using the S1 nuclease protection assay.
Overview of Membrane Protein Solubilization
Authors: Schimerlik Michael I.
Published: June 2004

The solubilization of membrane proteins (i.e., extraction of the protein from the membrane in a disperse state in aqueous solution) in a stable state that retains the native activity of the membrane‐bound form is more an empirical art than a protocol‐driven science. Although a large number of molecules have been invented that display detergent properties, selection of the proper solubilizing agent is largely a matter of trial and error that depends on the protein of interest and the particular goals of the investigator. The purpose of this overview is to describe the methodology for finding the optimal conditions for detergent solubilization and to relate the selection of a specific detergent to explicit experimental goals.
The Electrophysiology Setup
Authors: Finkel Alan , Bookman Richard
Published: June 2004

This unit describes components and considerations that are common to all electrophysiology setups dedicated to measurement of electrical activity in cells. First, the basic concerns of setting up the equipment in a functional and accessible manner are described. Then two typical setups are briefly described: one for in vitro extracellular recording, the other for single‐channel patch‐clamping. An electrophysiological setup has four main requirements: (1) environment: the means of keeping the preparation healthy; (2) optics: the means of visualizing the preparation; (3) mechanics: the means of stably positioning the microelectrode; and (4) electronics: the means of amplifying and recording the signal. This unit addresses primarily the mechanics and electronics of the electrophysiological laboratory setup.
Preparation of Hippocampal Brain Slices
Authors: Madison Daniel V. , Edson Eleanore B.
Published: June 2004

This unit presents a procedure for the preparation of acute mammalian hippocampal slices for electrophysiological recording. Although this protocol should not be taken as the only means of making brain slices, it is a widely‐used typical procedure. It is simple and straightforward. It can be used on a variety of mammalian species, though it is discussed here for use in rats.
Whole‐Cell Voltage Clamp Recording
Authors: Jackson Meyer B.
Published: June 2004

This unit describes the use of whole‐cell voltage clamping to study voltage‐gated channels. Stepwise changes in voltage produced by this technique cause channels to interconvert between different states, and these transitions are monitored as changes in membrane current. The time course of this redistribution of states contains a great deal of information about the mechanism of channel gating. Furthermore, the voltage clamp can be used to activate different populations of channels selectively. In this way, a specific channel targeted by biological or pharmacological manipulations can often be identified and studied in detail. This technique is also readily adapted to the study of ligand‐gated channels, synaptic potentials, and exocytosis.
Preparation and Maintenance of Organotypic Slice Cultures of CNS Tissue
Authors: Gähwiler Beat H. , Thompson Scott M. , Muller Dominique
Published: June 2004

Organotypic slice cultures are the in vitro method of choice for applications requiring long‐term survival of the preparation and a high degree of cellular differentiation and organization resembling that of the original tissue. Long‐term survival is achieved by culturing slices at the air/liquid interface, either by continuously rotating the preparation (roller‐tube cultures) or by culturing them on semiporous membranes (stationary interface cultures). Both culture techniques yield nerve cells which are highly differentiated in terms of their morphological and physiological characteristics. Because most of these cultures are prepared from 1‐week‐old postnatal animals, in which the cellular and tissue organization is already relatively advanced, the original cytoarchitecture is often remarkably well maintained. Moreover, the presence of a full complement of glial and nerve cells is thought to provide a microenvironment facilitating differentiation of neurons. Slice culture also offers unique advantages for recording from pairs of cells, as a consequence of the high degree of connectivity between nerve cells. Recently, new applications have emerged such as the cultivation of slices from knock‐out animals with limited postnatal survival time or alteration of gene expression by viral vectors.
Voltage Clamp Recordings from Xenopus Oocytes
Authors: Dascal Nathan
Published: June 2004

Xenopus oocytes serve as a standard heterologous expression system for the study of cloned ion channels. The large size of these cells allows for relatively easy expression and recording of activity of exogenous ion channels (together with neurotransmitter receptors and/or various regulatory proteins) using the whole‐cell two‐electrode voltage clamp (TEVC) technique, as well as standard single‐channel patch clamp recordings. Although usually advantageous, the cell size also dictates certain limits on the accuracy of recordings and requires specific modifications of recording methods. However, combining the advantages of the system with available recording methods enables the use of Xenopus oocytes for sophisticated multidisciplinary studies of ion channels.
Synaptic Plasticity in the Hippocampal Slice Preparation
Authors: Bortolotto Zuner A. , Anderson William W. , Isaac John T.R. , Collingridge Graham L.
Published: June 2004

Synaptic plasticity is the process by which the brain alters the strength of its synaptic connections, a fundamental function of the brain that enables individuals to learn from experience. The study of synaptic plasticity often involves the application of standard in vitro electrophysiological techniques to hippocampal slice preparations. This unit discusses many of the special considerations that are applicable for the optimal study of synaptic plasticity in this system. Most of these principles also apply to the study of synaptic plasticity in other brain slice preparations.
Amperometry and Cyclic Voltammetry with Carbon Fiber Microelectrodes at Single Cells
Authors: Mundorf Michelle L. , Wightman R. Mark
Published: June 2004

Amperometry and cyclic voltammetry are two electrochemical techniques that enable the detection of electroactive neurotransmitters that are released from single cells. These techniques have provided the first chemical view of the events that occur during exocytosis. This unit describes the isolation of several cell types known for their exocytotic properties, the fabrication and calibration of carbon fiber microelectrodes, as well as some of the equipment and software requirements for obtaining electrochemically generated data.
Microdialysis in Rodents
Authors: Thompson Alexis C. , Shippenberg Toni S.
Published: June 2004

Microdialysis is an established sampling technique for the in vivo measurement of a variety of substances in both blood and tissue. These include neurotransmitters and neuropeptides, enzymes, and electrolytes, as well as various hormones and pharmaceutical agents. More recently, microdialysis has been used to infuse exogenous as well as endogenous substances into the brain and spinal cord. In microdialysis, a semipermeable dialysis membrane is introduced into the fluid or tissue compartment to be sampled and perfused with physiological fluid. As a result of passive diffusion, molecules migrate across the membrane along their concentration gradient. Molecules found in high concentrations within the tissue compartment migrate across the membrane into the dialysis tubing where they can be collected for subsequent quantification, whereas molecules found in high concentrations within the membrane diffuse outward into the surrounding tissue compartment. This unit describes construction of concentric and side‐by‐side microdialysis probes, which differ with respect to size and inflow/outflow tube compositions, as well as a modification of a horizontal probe. This unit also covers methods for surgically implanting microdialysis probes in rats and mice and for conducting microdialysis in vitro and in vivo in rodents. Finally, procedures are provided for carrying out quantitative microdialysis techniques.
Uptake and Release of Neurotransmitters
Authors: Janowsky Aaron , Neve Kim , Eshleman Amy J.
Published: June 2004

The availability of clonal cell lines for norepinephrine, dopamine, and serotonin transporters allows the characterization of drug interactions with transporter recognition sites using radioligands, as well as the characterization of drug effects on selective transporter‐mediated uptake and release of substrate. In addition to clonal cell lines, synaptosomes prepared from specific brain regions can be used to conduct these studies without interference by endogenous transporters or binding proteins that are present in other tissues. This unit presents protocols for uptake and release of tritiated substrates using intact cells (either detached or in suspension) or synaptosomes. An HPLC procedure for electrochemical detection of nonradiolabeled substrates is also provided. Time‐dependent release can also be measured in assays involving real‐time sampling.