bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vitro Autoradiography
Authors: Kuhar Michael J. 
Published: June 2004

AbstractThe 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

AbstractA 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 Microarrays in Drug Discovery and Development
Authors: Waring Jeffrey F. 
Published: June 2004

AbstractWith 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

AbstractIn 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

AbstractThe 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

AbstractConditioned 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

AbstractDrug 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

AbstractDrugs 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

AbstractThe 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

AbstractThis 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

AbstractExtracts 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

AbstractCell 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

AbstractElectrophoresis 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

AbstractThis 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.
Overview of Gene Delivery into Cells Using HSV‐1‐Based Vectors
Authors: Neve Rachael L. , Lim Filip 
Published: June 2004

AbstractThis 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

AbstractPhage 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

AbstractThis 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

AbstractExpression 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

AbstractThis 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).
Loading Neurons with Dextran‐Conjugated Calcium Indicators in Intact Nervous Tissue
Authors: Delaney Kerry R. 
Published: June 2004

AbstractThis 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

AbstractIn 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

AbstractIn 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

AbstractOligodendrocytes 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

AbstractThis 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

AbstractIn 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

AbstractThis 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.