bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gene Expression in Transgenic Mice Using Neural Promoters
Authors: Okabe Shigeo 
Published: June 2004

AbstractIn 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

AbstractOf 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

AbstractThis 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

AbstractThere 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

AbstractHerpes 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

AbstractThe 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

AbstractAdeno‐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.
Production of High‐Titer Lentiviral Vectors
Authors: Zufferey Romain , Trono Didier 
Published: June 2004

AbstractConventional 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

AbstractSemliki 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

AbstractNuclease 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

AbstractThe 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

AbstractThis 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

AbstractThis 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

AbstractThis 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

AbstractOrganotypic 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

AbstractXenopus 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

AbstractSynaptic 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

AbstractAmperometry 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

AbstractMicrodialysis 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

AbstractThe 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.
Measurement of Second Messengers in Signal Transduction: cAMP and Inositol Phosphates
Authors: Gusovsky Fabian 
Published: June 2004

AbstractcAMP acts as an intracellular mediator of hormone action and the importance of accurate quantitative determination of cAMP levels in cells and tissues is widely recognized. The most utilized procedures for the determination of adenylate cyclase activity in membranes are described here for measuring the conversion of [alpha‐32P]ATP into [32P]cAMP after a two‐step chromatographic separation. Also critical in signal tranduction is phosphoinositide turnover, which is linked to receptor activation resulting from changes in cytosolic calcium concentrations. Phosphoinositide turnover can be measured as described in this unit by labeling phospholipid pools with [3H]‐inositol and then analyzing for tritiated inositol phosphates.
Measurement of Nitric Oxide in Single Cells and Tissue Using a Porphyrinic Microsensor
Authors: Malinski Tadeusz , Huk Igor 
Published: June 2004

AbstractThis unit describes the preparation and applications of porphyrinic sensors for quantitative measurement of nitric oxide (NO) in single cells and in tissues. The determination of NO is based on the electrochemical oxidation of NO on a carbon fiber electrode covered with a thin layer of a conducting polymeric metalloporphyrin catalyst, overlaid with another thin film of Nafion, a cation exchange material. The electric current generated during NO oxidation on the surface of the polymeric porphyrin is linearly proportional to the concentration of NO, so this current is used as an analytical signal which can be measured in either the amperometric or the voltammetric mode. Both methods provide a quantitative signal. This unit describes the electrochemical setup for measurement of NO in single cells and tissue. Support protocols describe porphyrin synthesis, sensor preparation, and sensor calibration.
Locomotor Behavior
Authors: Pierce R. Christopher , Kalivas Peter 
Published: June 2004

AbstractLocomotor activity can be used to elucidate basic scientific questions, as the neural circuitry mediating the behavioral effects of different drug classes can be determined by direct pharmacological stimulation of specific brain nuclei. Locomotor activity is also used to assess the neural bases of ethological and biological rhythms. Methods for evaluating locomotor behavior in rodents fall into two general categories: direct observational techniques and automated monitoring of behavior. The first technique described in this unit consists of direct observation using interval and ordinal scales. The second approach is based on the measurement of locomotor activity with photocell‐based automated monitoring systems. Other methods used to quantify locomotor activity include video‐based systems, rotometers, and running wheels. The protocols in this unit are designed for rat subjects, although they can easily be adapted for use with mice and other rodents.
Social Learning of Food Preferences in Rodents: Rapid Appetitive Learning
Authors: Galef Bennett G. 
Published: June 2004

AbstractA procedure is described for quantitating the transfer of information about inducing increased intake of distinctively‐flavored foods or fluids among common laboratory rodents. The method provides a simple, efficient, non‐invasive way to produce robust, long‐lasting changes in appetitive behaviors of laboratory rodents that can be used in studies of the neuroanatomical, neurochemical or genetic substrates of learning and memory.