bluuebunny/crossref_metadata_2025 huggingface dataset:
Models of Schizophrenia: Phencyclidine Disruption of Prepulse Inhibition (PPI) of Startle in Rats
Authors: Kehne John H. , Hoffman Diane C. , Cassella James V.
Published: June 2004

Modern theories of schizophrenia implicate deficits in glutamatergic transmission in the etiology of this disorder. This unit describes an animal model in which a deficit in glutamatergic transmission is induced pharmacologically by the NMDA receptor ion channel blocker phencyclidine. Reversal of the resulting behavioral impairment, the reduction of sensory‐motor gating as measured by prepulse inhibition, can be used as a method for evaluating novel treatments for schizophrenia.
Models for Assessing Antipsychotics: Antagonism of Amphetamine‐Induced Hyperactivity and Stereotypies in Mice
Authors: Roux Sylvain , Hay Anne‐Marie , Porsolt Ruger D.
Published: June 2004

All classical antipsychotics, such as chlorpromazine and haloperidol, have potent dopamine receptor‐blocking properties

This unit describes the rat anti‐Thy‐1.1 model of acute proliferative glomerulonephritis for the study of chronic renal insufficiency. A procedure is detailed for the induction of glomerulonephritis in rats as well as measurement of daily urinary excretion of protein, which is a convenient, primary screening tool. The unit also provides methods for assessment of glomerular filtration rate and effective renal plasma flow in anesthetized rats with anti‐Thy‐1.1‐induced renal insufficiency.
Characterization of Matrix Metalloproteinase Inhibitors: Angiogenesis and Tumor Models
Authors: Albert Daniel H. , Davidsen Steven K.
Published: June 2004

Since the matrix metalloproteinases (MMPs) have an essential role in the process of tumor growth, invasion and metastasis, small molecule MMP inhibitors have the ability to modulate tumor progression in animals and the potential to be of therapeutic benefit to cancer patients. The antiangiogenic properties of MMP inhibitors can be assessed by the measurement of hemoglobin content of Matrigel plugs containing angiogenic growth factors introduced into the flanks of mice. A flank tumor growth model using B16 murine melanoma cells provides a useful means of determining the antitumor effects of MMP inhibitors as well as correlating efficacy with the concentration of drug in blood.
Overview of Animal Models of Asthma
Authors: Canning Brendan J.
Published: June 2004

Asthma is an inflammatory disease characterized by airways obstruction, airways hyperresponsiveness, excessive mucous secretion and cough. Guinea pig airways display many anatomical, physiological and pharmacological attributes of human airways, making this species ideal for modeling the asthmatic condition. This unit provides an overview of animal models of asthma, including definitions, descriptions of available animal models, and discussion of numerous critical issues to consider before designing a model to study this complex disease.
Models of Neuropathic Pain in the Rat
Authors: Bennett Gary J. , Chung Jin Mo , Honore (Table 5.32.2) Marie , Seltzer Ze'ev
Published: June 2004

Peripheral nerve injury due to trauma, disease, and certain toxins sometimes produces abnormal (neuropathic) pain syndromes that are chronic and refractory to standard analgesics. Knowledge of the mechanisms that produce neuropathic pain and the ability to search for new drugs to control it have been greatly advanced by the introduction of rat models of post‐traumatic painful peripheral neuropathy. There are currently three models of neuropathic pain in the rat that are widely used. The procedures to create these models and the behavioral assays used to quantify the resulting neuropathic pain symptoms are described in this unit: the chronic constriction injury (CCI) model, the partial sciatic ligation (PSL) model, and the spinal nerve ligation (SNL) model. Four kinds of abnormal pain sensations are commonly measured to assess the outcome: heat‐hyperalgesia, mechano‐hyperalgesia, mechano‐allodynia, and cold‐allodynia.
Electrical Amygdala Kindling
Authors: Dürmüller N. , Porsolt R.D.
Published: June 2004

This unit describes a method of electrical amygdala kindling in the rat. This procedure requires mastery of stereotaxic electrode implantation which is not covered in the current unit. Also, the investigator must have a sound knowledge of electronics and computing. The text gives instructions on how to render rats epileptic, how to determine the effects of compounds in kindled rats, and how to analyze the data. Results with three reference substances are illustrated. These substances are used in the clinic and give robust results in kindling.
A Rat Model of Postoperative Pain
Authors: Brennan Timothy J.
Published: June 2004

Research on pain resulting from an incision is crucial for developing therapies for postoperative pain, a condition for which effective and inexpensive treatments are not yet available. Because of the gap between preclinical models of persistent pain (e.g., inflammatory, neuropathic) and acute postsurgical pain, efforts have been undertaken to develop methods for studying pain caused by incisions. To this end, a rat model for postoperative pain has been developed. The model uses a plantar incision in the hindpaw and is characterized by persistent, reduced withdrawal thresholds to mechanical stimuli, as is the case in patients after surgery. The pain behaviors are greatest immediately after recovery from anesthesia. The enhanced responsiveness remains remarkable for several days and then gradually decreases. The model is useful for screening test compounds that may be effective in reducing postoperative pain and for understanding pain mechanisms associated with incisions.
Isotopic Assays for Reporter Gene Activity
Authors: Kingston Robert E. , Sheen Jen , Moore David
Published: June 2004

This unit describes two widely used reporter systems that are based on radioactive detection assays. The first assay uses chloramphenicol acetyltransferase (CAT) activity as a measure of the level of expression of a transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group from acetyl CoA to chloramphenicol. Transfected cells are harvested and lysed, and then acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and modified derivatives of the antibiotic are separated from the starting material. The second reporter system uses a kit to perform a simple two‐site radioimmunoassay to quantitate the amount of human growth hormone (hGH) secreted into culture medium by transfected cells. Medium is incubated with 125I‐labeled antibody specific for hGH, and immune complexes are collected by an avidin‐coated bead. The quantity of hormone is determined based on comparison with a standard curve.
Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics In Vivo
Authors: Kahana Jason A. , Silver Pam A.
Published: June 2004

Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.
Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Authors: Kramer Martha F. , Coen Donald M.
Published: June 2004

This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield. Hot‐start methods are described which can greatly improve specificity, sensitivity, and yield. This protocol suggests some relatively inexpensive methods to achieve hot start, and lists several commercial hot‐start options which may be more convenient, but of course more expensive. The unit has recently been updated to include new information on reagents to enhance the reaction, better cycling parameters, and innovations in robotics and high‐performance thermocyclers.
Mobility Shift DNA‐Binding Assay Using Gel Electrophoresis
Authors: Buratowski Stephen , Chodosh Lewis A.
Published: June 2004

DNA‐binding assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence‐specific DNA‐binding proteins. Proteins that bind specifically to an end‐labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein‐DNA complexes. The assay described in this unit can be used to test binding of purified proteins or of uncharacterized factors found in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA‐binding proteins. Three additional protocols describe a competition assay using unlabeled competitor DNA, an antibody supershift assay, and multicomponent gel shift assays.
Overview of Pharmacokinetics
Authors: Weber Steven J.
Published: June 2004

Pharmacokinetics is the study of the absorption, distribution, metabolism (biotransformation), and excretion (collectively referred to as ADME) of a compound following its administration. The physical/chemical state (e.g., molecular size and configuration, lipophilicity, and solubility) of a compound will greatly affect all these parameters. The aim of pharmacokinetic studies is to determine what dose and dosing schedule are sufficient to elicit and maintain a desired clinical response. This unit presents detailed procedures for dosing a rat via intravenous (i.v.), oral (p.o.), subcutaneous (s.c.), intraperitoneal (i.p.), and intramuscular (i.m.) routes. Protocols for blood collection, excreta collection, tissue collection, sample processing and calculation of data are also provided.
Determination of Compound Binding to Plasma Proteins
Authors: Dow Natasha
Published: June 2004

The pharmocokinetic and pharmacodynamic properties of a compound are profoundly affected by the extent of its binding to plasma proteins. Consequently, the determination of the plasma protein binding of a compound is essential during drug development and is increasingly required during lead prioritization. This unit describes the use of ultrafiltration and equilibrium dialysis techniques for determination of plasma protein binding. The pharmocokinetic and pharmacodynamic properties of a compound are profoundly affected by the extent of its binding to plasm
In Vitro Drug Metabolism Using Liver Microsomes
Authors: Hill Jeanette R.
Published: June 2004

Metabolic biotransformation of a drug can increase the rate of elimination from the body and have a significant effect on efficacy and safety. Drug candidates are screened early in the discovery process for metabolic stability using liver microsomes. Methods for microsome isolation and characterization and the determination of metabolic stability are presented.
Autoradiographic Localization of Growth Factor Receptors in Neuronal Tissues
Authors: Doré Sylvain , Kar Satyabrata , Auld Daniel , Chabot Jean‐Guy , Quirion Remi
Published: June 2004

The physiological and pharmacological effects of growth factors, like those of neurotransmitters and neuromodulators, are mediated by binding to specific receptor sites. Using radiolabeled molecules with high specific activity, such as the endogenous ligand itself, it is possible to measure receptor binding in membrane preparations. This technique provides a means to characterize the selectivity and specificity of the receptors for a particular growth factor. Additionally, ligand‐binding assays make it possible to study in detail the structure‐activity relationships and mechanisms of action of growth factors. As an example of this methodology, this unit describes the use of both film and emulsion autoradiography to study the pharmacological characteristics and cellular distribution profile of insulin‐like growth factor receptors in the central nervous system. For the sake of clarity, in vitro film autoradiography in tissue sections and liquid emulsion autoradiography in cultured neurons are discussed separately. The physiological and pharmacological effects of growth factors, like those of neurotransmitters and neuromodulators, are medi.
Vigilance‐Controlled Quantified EEG in Safety Pharmacology
Authors: Dürmüller N. , Porsolt R.D. , Scherschlicht R.
Published: June 2004

The vigilance‐controlled quantified EEG can be used either as a safety, or as a discovery pharmacology procedure. Put strategically into the preclinical development process of a drug, it can be useful for making the decision about the future research direction to be taken. The experimental approach described in this unit is based on the rat EEG. Even though there are considerable differences in function and structure between human and rat brain, the EEG response to psychoactive drugs and convulsants is similar in the two species. Thus, the rat EEG is generally a reliable predictor for human CNS drug effects.
An Overview of QT Interval Assessment in Safety Pharmacology
Authors: Lacroix Pierre
Published: June 2004

Medicinal products that prolong cardiac repolarization unintentionally, as assessed in terms of prolongation of the QT interval of the electrocardiogram, may trigger a potentially fatal arrhythmia called torsade de pointe (TDP). This lethal risk necessitates a robust preclinical evaluation before engaging in clinical trials. There are two different and complementary approaches to assess the potential of drugs to cause QT interval prolongation. The in vivo approach provides information on the potential of the compound to prolong the QT interval under near‐physiological conditions. It is mostly descriptive and not explanatory in terms of mechanisms of action. The in vitro approach provides much more mechanistic information, but is far removed from the clinical situation. Both approaches appear to possess reasonable predictive value, but the results obtained may largely depend on the experimental conditions used. This unit describes and presents an analytic strategy aimed at understanding the problems associated with this cardiovascular risk.
Overview of Electrophysiology
Authors: Bertrand Daniel
Published: June 2004

This unit provides an overview of the electrophysiological techniques used to determine the physiological and/or pharmacological properties of membrane ion channels at the single‐cell level. Only those techniques that measure potential changes at the transmembrane and membrane levels are discussed. The basics of current‐clamp recording, voltage‐clamp recording, and patch‐clamp procedures are presented along with their advantages and disadvantages. Additional topics are discussed with regard to their relation to electrophysiological measurements, such as membrane properties, ion selectivity, time resolution, ligand‐gated ion channels (LGICs), voltage‐dependent channels, and channel modulation and interactions. Also included is a discussion the electrophysiological properties of seven‐transmembrane‐domain receptors. This unit provides an overview of the electrophysiological techniques used to determine the physiological and/or pharmacologic of ion channels.
Electrophysiological Analysis of G Protein–Coupled Receptors in Mammalian Neurons
Authors: Proctor William R. , Dunwiddie Thomas V.
Published: June 2004

This unit describes general techniques that are useful for recording electrophysiological responses that are mediated via the activation of G‐protein coupled receptors (GPCRs). It includes a brief description of preparations, but focuses primarily on experiments using hippocampal brain slice preparations. Techniques for the preparation of brain slices, electrodes, filling solutions, and the recording protocols that are suitable for assessing the activity of GPCRs using electrophysiological techniques are summarized, and various protocols for the activation of these receptors are discussed.
Electrophysiological Analysis of Tetrodotoxin‐Resistant Sodium Channel Pharmacology
Authors: Dong Xiao‐Wei , Priestley Tony
Published: June 2004

This unit describes essential procedures to record the activity of and the effects of pharmacological manipulation upon tetrodotoxin‐resistant (TTXr) sodium channels. Readers are guided through the process of assembling a functional |patch‐clamp| electrophysiology apparatus and construction of software‐controlled voltage protocols that address various aspects of channel gating. Guidelines are also presented for analysis and display of the data typically generated by these experiments. For investigators wishing to study native channels in their appropriate cellular environment, protocols describing the isolation of viable cells from the main tissues that commonly express TTXr channels are also included.
Methods of Measuring Internalization of G Protein–Coupled Receptors
Authors: Evans Nicholas
Published: June 2004

This unit provides detailed protocols for measuring receptor internalization. The techniques are sufficiently generalized to be applicable to most receptors in a wide variety of cell types. Both radioactive and non‐radioactive techniques are described that may be used to quantify receptor internalization, and the differences between the two are highlighted. This unit discusses how quantification of internalization may be achieved, and the advantages and drawbacks of each technique. Low‐ and higher‐throughput methods are compared, and the technologies required to conduct the analyses are discussed.
Receptor Nomenclature Guidelines
Authors: Williams Michael
Published: June 2004

Receptors are typically characterized via two distinct approaches: (1) the identification and pharmacological characterization of a receptor‐mediated response using classical pharmacological and/or radioligand approaches in tissues and animal models using selective agonist and antagonist ligands, and; (2) the cloning and expression of proteins with structural homology to known receptors, the function of which is subsequently established by studying the structure activity relationship (SAR) of receptor‐mediated responses. An additional means to characterize receptors proceeded, and evolved, with the structural approach, namely classification in terms of signal transduction mechanisms. The International Union of Pharmacology (IUPHAR) created guidelines and selected working groups for each receptor family to establish a common nomenclature system. Reports from those groups that have reached some degree of consensus have been summarized in this appendix.
Assays for Total Protein
Authors: Lovrien Rex , Matulis Daumantas
Published: June 2004

This unit describes three copper‐based assays to quantitate total protein: the biuret method, a variation of the Lowry method (Hartree‐Lowry method), and the bicinchoninic acid (BCA) assay. Acid hydrolysis of a protein is coupled with ninhydrin detection to quantitate amino acid content of a sample. Ultraviolet spectrophotometry is used to measure total protein and evaluate samples for the presence of contaminants. The Coomassie dye binding, or Bradford, assay is a quite simple assay and frequently is quite sensitive, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acid pH. Finally, dry weight measurement is used to quantitate pure protein. Support protocols describe heat sealing glass tubes for acid hydrolysis, sample dialysis in polyacrylamide gel wells to remove low‐molecular‐weight contaminants, and TCA precipitation to precipitate and concentrate proteins and remove low‐molecular‐weight contaminants.
Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
Authors: Zeller Rolf
Published: June 2004

This unit describes selected methods for fixing and sectioning various forms of biological materials ranging from tissues to single cells. Sections prepared according to these protocols can then be used to examine cell and tissue morphology and in studies involving in situ hybridization, immunohistochemistry, and enzyme histochemistry. The basic protocol describes how tissues and embryos can be prepared for sectioning by fixing in paraformaldehyde followed by embedding in wax, while an alternate protocol describes fixation of suspended or cultured cells. Two support protocols cover preserving and fixing organs by perfusion of whole animals with paraformaldehyde and a procedure for sectioning wax blocks of fixed tissue, plus the subsequent mounting of sections onto prepared or “subbed” glass slides.
In Situ Hybridization to Cellular RNA
Authors: Zeller Rolf , Rogers Melissa , Haramis Anna G. , Carrasceo Andrés E.
Published: June 2004

In situ hybridization to cellular RNA is used to determine the cellular localization of specific messages within complex cell populations and tissues. Tissues may either be embedded in paraffin and sectioned on a microtome (seeHybridization Using Paraffin Sections and Cells), or frozen and sectioned in a cryostat (seeHybridization Using Cryosections). RNA contained in the specimens is hybridized to a specific radiolabeled probe (seeSynthesis of35S‐Labled RiboprobesandSynthesis of35S‐Labled Double‐Stranded DNA Probes), which is then detected using film autoradiography or emulsion autoradiography (Chapter 8).
Rapid Solution Application Methods
Authors: Tang Cha‐Min
Published: June 2004

Solution changes to deliver solutes of different compositions are required in virtually every cellular electrophysiological experiment. Also, in many neurobiology experiments, it is necessary to make rapid step changes in the concentration of a test compound in order to outpace receptor desensitization or to mimic the brief lifetime of a fast synaptic response. The goal of this unit is to aid the investigator in choosing the rapid solution application method that is most appropriate for the experimental situation and to describe the methods for fabricating two relatively simple devices for making rapid changes between different solutions.
cDNA Library Construction from Single Cells
Authors: Kramer Phillip R.
Published: June 2004

cDNA Library Construction from Single Cells (Phillip R. Kramer, National Institute of Neuronal Disorders and Stroke, NIH, Bethesda, Maryland). Analysis of gene expression or comparing gene expression patterns between cells and/or tissues facilitates the identification of molecules activated by a cellular program or by a particular physiological or pharmacological treatment. This unit presents a protocol for studying these molecules at the level of a single cell.
Targeted Toxins
Authors: Wiley Ronald G. , Lappi Douglas A.
Published: June 2004

Molecular neurosurgery can be used to make selective neural lesions by targeting cytotoxins to specific populations of neurons based on their common expression of a particular surface molecule. The targeted toxins employed in this unit consist of a targeting moiety (vector) and an effector moiety (cytotoxin). In all cases discussed in this unit, the cytotoxic moiety is an enzyme that catalytically inactivates the large ribosomal subunit, irreversibly inhibiting protein synthesis and resulting in cell death. These toxins appear to kill in an all‐or‐none fashion, indicating that one molecule of free cytotoxin in the cytoplasm of a cell is sufficient to kill the cell. Three general molecular neurosurgery protocols are presented in this unit. The first describes suicide transport, which refers to the use of targeted toxins to make anatomically restricted lesions based on retrograde axonal transport of the toxin. The second involves immunolesioning and uses anti‐neuronal immunotoxins to make type‐selective and anatomically restricted lesions. The final protocol uses neuropeptide‐toxin conjugates to selectively destroy neurons expressing the receptor for the specific neuropeptide.
Cellular Compartment Analysis of Temporal Activity by Fluorescence In Situ Hybridization (catFISH)
Authors: Guzowski John F. , Worley Paul F.
Published: June 2004

This protocol describes a method (cellular compartment analysis of temporal activity by fluorescent in situ hybridization or catFISH) that uses fluorescent in situ hybridization to immediate‐early gene RNAs and confocal microscopy to identify neuronal populations activated at two distinct times. The combination of cellular and temporal resolution makes catFISH a valuable tool for investigating the dynamic interactions of neuronal populations associated with different behaviors or cognitive challenges.
Detection of DNA Damage in Tissue Sections by In Situ Nick Translation
Authors: Chesselet Mary‐Franciose , MacKenzie Larami , Hoang Tuan
Published: June 2004

The technique of in situ nick translation (ISNT) is used to detect DNA strand breaks in tissue sections at the cellular level with great sensitivity. In fact, ISNT can be used to detect DNA damage in a single cell, which is particularly useful to assess programmed cell death during development. One crucial advantage of ISNT is the anatomical resolution that permits a detailed topographical analysis of DNA damage. This can be useful to identify cells that are more vulnerable to an experimental insult. Furthermore, cells with DNA damage can be identified morphologically with this method. This can be of interest to determine whether cells that exhibit DNA damage already exhibit clear features of dying cells or are still relatively intact morphologically. This can be useful to identify the mode of cell death involved. This unit provides a protocol that describes tissue preparation, in situ nick translation and emulsion autoradiography.
Quantitative In Situ Hybridization for the Study of Gene Expression at the Regional and Cellular Levels
Authors: Le Moine Catherine
Published: June 2004

Quantitative in situ hybridization allows measurement of mRNA level modifications in a variety of experimental conditions. This analysis may be performed both at the regional anatomical and cellular levels by densitometry, neuronal counting and silver grain measurements.
Theory and Application of Fluorescence Microscopy
Authors: Coling Donald , Kachar Bechara
Published: June 2004

The growing importance in biology and especially in neurobiology of fluorescence microscopy is due to (1) the extraordinary development of new fluorescent molecular probes and (2) the development of improved low light level imaging systems and confocal microscopy techniques. This overview covers fluorescent molecular probes, filters and filter sets, multiband filters and multidye fluorescence, light sources, microscope objectives, image resolution and the point‐spread function, and general steps for immunolabeling.
Imaging Nervous System Activity
Authors: Fields R. Douglas , O'Donovan Michael J.
Published: June 2004

Optical imaging methods rely upon visualization of three types of signals: (1) intrinsic optical signals, including light scattering and reflectance, birefringence, and spectroscopic changes of intrinsic molecules, such as NADH or oxyhemoglobin; (2) changes in fluorescence or absorbance of voltage‐sensitive membrane dyes; and (3) changes in fluorescence or absorbance of calcium‐sensitive indicator dyes. Of these, the most widely used approach is fluorescent microscopy of calcium‐sensitive dyes. This unit describes protocols for the use of calcium‐sensitive dyes and voltage‐dependent dyes for studies of neuronal activity in culture, tissue slices, and en‐bloc preparations of the central nervous system.
Use of FM1‐43 and Other Derivatives to Investigate Neuronal Function
Authors: Cousin Mike
Published: June 2004

This unit describes how to use FM1‐43 and its derivatives to investigate the physiology of synaptic vesicle recycling in neurons. This includes basic protocols for visualizing synaptic vesicle exocytosis and endocytosis and information on more advanced uses of the probes to examine different aspects of the vesicle life cycle.
Identification of Neural Programmed Cell Death Through the Detection of DNA Fragmentation In Situ and by PCR
Authors: Chun Jerold , Blaschke Anne J.
Published: June 2004

A universal feature in the development of multicellular organisms is a physiological form of cell death called programmed cell death (PCD). A subset of PCD is apoptosis, which is defined by characteristic morphological changes and genomic DNA fragmentation producing what are referred to as nucleosomal ladders. To understand how PCD operates in a developing tissue or in a tissue following an experimental procedure, dying cells must be identified in relation to their surviving neighbors. One way to accomplish this is to visualize fragmented DNA in situ, in conjunction with gel electrophoresis of isolated DNA to visualize the nucleosomal ladders associated with apoptosis. Two approaches are presented in this unit: in situ end‐labeling plus (ISEL+), a technique to identify dying cells in tissue sections or cell cultures of central nervous system (CNS) tissue (optimized for embryonic samples); and the use of ligation‐mediated polymerase chain reaction (LMPCR) to identify nucleosomal ladders from intact tissues. Also included are procedures for preparing thymocyte cell cultures for use as controls in the ISEL+ procedure and for isolating genomic DNA for LMPCR.
Quantitative Analysis of In Vivo Cell Proliferation
Authors: Cameron Heather A.
Published: June 2004

Two similar techniques, [3H]thymidine autoradiography and 5‐bromo‐2'‐deoxyuridine (BrdU) immunohistochemistry, have been developed to label dividing cells and identify them following survival times of up to several years, so that different aspects of their fate can be examined. Both techniques use markers that are incorporated into the DNA of proliferating cells as it is synthesized during the S phase of mitosis. The techniques differ in the markers used and the detection systems used to recognize them. In the first method described in this unit, tritiated thymidine is injected into animals and is incorporated into dividing cells. These cells can then be identified using emulsion autoradiography. In the second method presented, BrdU, a halogenated thymidine analog, is similarly injected and incorporated into cells in S phase, and then detected immunohistologically with an antibody specific for DNA containing BrdU.
Isolation and Generation of Oligodendrocytes by Immunopanning
Authors: Mayer‐Pröschel Margot
Published: June 2004

This unit presents a procedure for the purification of oligodendrocyte progenitor cells, their expansion in vitro, and differentiation of these cells to yield oligodendrocyte cultures. A variation is also presented, detailing the direct isolation of differentiated oligodendrocytes from postnatal brain. The purification of the target cell population is achieved by exploiting the differential binding of cells to tissue culture dishes coated with an antibody directed against a specific cell‐surface antigen. Cells expressing this surface antigen are retained on the dish and are thereby separated from the remaining cell population.
Generation and Utilization of Phosphorylation State–Specific Antibodies to Investigate Signaling Pathways
Authors: Alberta John A. , Segal Rosalind A.
Published: June 2004

Phosphorylation state‐specific antibodies can be of great use, for example, in studying individual steps within a given signal transduction pathway. This unit presents a general approach to the generation and purification of phosphorylation state‐specific antibodies. In addition to their ability to detect phosphorylation at a particular key site, these antibodies are often more sensitive for biochemical studies. Besides their application in immunoblotting procedures, activation state‐specific antibodies can be used as immunohistochemical reagents. Thus, critical changes in phosphorylation can be monitored as described on an individual cell basis or in fixed tissue sections. Such antibodies can be used to address fundamental questions about signal transduction pathways during physiologic events that cannot be resolved by more conventional methodologies.
Homology Cloning of cDNA or Genomic DNA
Authors: Albert Paul R.
Published: June 2004

The principle of cloning utilizes hybridization of single‐stranded DNA probes to denatured DNA fixed on membranes to detect the identical DNA molecules present in a large population of diverse DNAs. In homology or low‐stringency cloning, hybridization of oligonucleotide, cDNA, or genomic DNA probes to denatured DNA from plasmid or bacteriophage libraries is carried out under low‐stringency conditions that promote hybridization in the presence of sequence mismatch (i.e., mispaired nucleotides). The procedures used are identical to those for screening libraries at high stringency, with the exception that hybridization and washing conditions are altered to permit hybridization with mismatched sequence; salt concentration is increased and the hybridization and wash temperatures are decreased. This unit describes conditions that have been used to clone and identify novel genes and cDNA clones using low‐stringency hybridization of known probes to membranes that contain libraries of bacterial or bacteriophage DNA.
Expression Cloning of Neural Genes Using Xenopus laevis Oocytes
Authors: Boulter Jim , Boyer Christopher
Published: June 2004

Expression cloning requires a representative cDNA or genomic DNA library and a host organism in which the cloned genes can be transcribed and/or translated. It likewise requires a method to detect the expressed protein using, for example, the inherent biological activity of the gene or antibodies specific for the gene product. Most successful expression cloning strategies have employed cDNA libraries constructed in plasmid or bacteriophage lambda vectors and Xenopus oocytes or cultured mammalian cells as hosts. This unit presents several protocols designed for expression cloning paradigms that rely on electrophysiological recordings from Xenopus laevis oocytes.
Mutagenesis Approaches for Elucidation of Protein Structure‐Function Relationships
Authors: Strader Catherine D.
Published: June 2004

Several mutagenesis strategies have been used to allow the identification of specific receptor‐ligand interactions to support the docking of ligands within various receptor models. The strengths and limitations of each strategy are discussed in this overview. Large‐scale mapping is described using deletion and chimeric receptors. Fine tuning with single residue replacements is also covered along with two‐dimensional approaches.
Oligonucleotide‐Directed Mutagenesis Without Phenotypic Selection
Authors: Kunkel Thomas A.
Published: June 2004

A DNA sequence can be specifically altered by synthesizing the desired sequence change within an oligonucleotide, and then converting this into a biologically active circular DNA strand by using the oligonucleotide to prime in vitro synthesis on a single‐stranded circular template. This unit presents a protocol which uses a DNA template containing a small number of uracil residues in place of thymine. Use of the uracil‐containing template allows rapid and efficient recovery of mutants; in principle this same template can be applied to most of the other mutagenesis protocols in use. The length of the oligonucleotide primer is highly variable and depends on the nature of the change being made.
Directed Mutagenesis Using the Polymerase Chain Reaction
Authors: Cormack Brendan
Published: June 2004

The polymerase chain reaction (PCR) is most often used for the enzymatic amplification and direct sequencing of small quantities of nucleic acids. This technology can also be used as a quick and efficient method for introducing any desired sequence change into the DNA of interest. This unit contains two basic protocols for introducing base changes into specific DNA sequences. The first describes the incorporation of a restriction site and the second details the generation of specific point mutations. An alternate protocol describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.
Overview of the Baculovirus Expression System
Authors: Murphy Cheryl Isaac , Piwnica‐Worms Helen
Published: June 2004

Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This unit gives an overview of the baculovirus expression system, including discussion of the baculovirus life cycle, and post‐translational modifications that occur in insect cells. In addition, the steps for overproducing proteins in the baculovirus systems are described along with recommendations for choosing an appropriate baculovirus vector and DNA, and reagents and equipment necessary for implementing the whole overexpression system.
Protein Expression in the Baculovirus System
Authors: Bernard Alain , Payton Mark , Radford Kathryn R.
Published: June 2004

Insect cell‐recombinant baculovirus co‐cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post‐translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large‐scale production of recombinant proteins in the baculovirus expression system. A method for large‐scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end‐point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small‐scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS‐PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed‐batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided
Gene Transfer into Neural Cells In Vitro Using Adenoviral Vectors
Authors: Southgate Thomas D. , Kingston Paul A. , Castro Maria G.
Published: June 2004

Adenoviruses (Ads) have become a very attractive and versatile vector system for delivering genes into brain cells in vitro and in vivo. One of the main attractions of Ads is that they can mediate gene transfer into post‐mitotic cells, i.e. neurons. Ads are easy to grow and manipulate, stable, and their biology is very well understood. This unit is designed to help newcomers into the field, to design, prepare and grow replication‐defective recombinant adenovirus vectors with the aim of transferring genes into neurons and glial cells in primary culture. It provides step‐by‐step methods describing the preparation of brain cell cultures, their infection using recombinant adenovirus vectors and also the assessment of transgene expression using a variety of techniques including fluorescence immunocytochemistry and fluorescence activated cell‐sorting (FACS) analysis. The methods described will be useful to scientists wishing to enter the adenovirus field to construct adenovirus vectors to be used for gene transfer into neural cells.
Preparation of mRNA for Expression Monitoring
Authors: Byrne Michael C. , Whitley Maryann Z. , Follettie Maximillian T.
Published: June 2004

The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A+) versus total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.
Gene Expression Analysis Using cDNA Microarrays
Authors: Karsten StanisLav L. , Geshwind Daniel H.
Published: June 2004

This unit focuses on cDNA microarrays, which are being increasingly applied to large‐scale gene expression studies in the nervous system, concentrating on the steps of probe preparation and microarray hybridization. Four protocols for the attachment of fluorophores to cDNA (labeling) and cDNA hybridization onto microarrays are presented: direct labeling using either Klenow fragment or reverse transcriptase, and two signal amplification techniques represented by the tyramide signal amplification method (TSA), and detection and labeling of cDNA using PCR amplification. Basic issues in experimental design, sources of variability and potential pitfalls in microarray experiments are described. Anticipated results and follow up of microarray experiments, including confirmation of the results and troubleshooting are also discussed.
Overview of Gene Targeting by Homologous Recombination
Authors: Mortensen Richard
Published: June 2004

Overview of Gene Targeting by Homologous Recombination (Richard Mortensen, University of Michigan Medical School, Ann Arbor, Michigan).This overview describes the basic methodology and applications of homologous recombination. Both insertion and replacement constructs are discussed along with methods of selection. Gene mutation and inactivation to produce a knockout animal can be accomplished in several ways, and the Cre‐loxP system is described for this purpose in some detail.
Analysis of mRNA Populations from Single Live and Fixed Cells of the Central Nervous System
Authors: Eberwine James , Crino Peter
Published: June 2004

This unit presents a method for the amplification of poly(A)+ mRNA extracted from the cytoplasm of a single cell. After cDNA is synthesized from the mRNA, it is made double stranded, denatured, and reverse transcribed to yield antisense RNA (aRNA). Another round of amplification results in a relatively large amount of aRNAs in essentially the same proportion as in the starting mRNA population. RNA amplification protocols can be used for many purposes, including generation of disease expression profiles, making of cDNA libraries, and generation of diagnostics and therapeutics for disease. An alternate protocol is used to amplify RNAs from single neurons in fixed tissue specimens. Support protocols gives instructions for reverse northern analysis, which allows analysis of the presence or absence and relative levels of mRNA expression in selected cells, and a convenient method to assess the RNA content in fixed tissue sections using the fluorescent dye acridine orange (which binds single‐stranded nucleic acids).
Production of Polyclonal Antisera
Authors: Cooper Helen M. , Paterson Yvonne
Published: June 2004

Antibodies are serum immunoglobulins with binding specificity for particular antigens, and polyclonal antibodies are particularly valuable for immunoprecipitation and immunoblotting. In this unit, the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters is described. A support protocol is included for preparing serum from blood.
Production of Antisera Using Fusion Proteins
Authors: Gilmor Michelle , Heilman Craig , Nash Norman , Levey Allan
Published: June 2004

The use of fusion proteins for the production of antisera allows specific areas of proteins to be targeted as epitopes and facilitates the purification of the antisera. This unit first describes the use of standard molecular biological techniques to construct a fusion‐protein expression plasmid by inserting a region of cDNA into a pGEX vector. Next, E. coli are transformed with the plasmid and induced to generate fusion protein. Also described is the purification of the soluble fusion protein, which is necessary for immunization and other subsequent procedures. This is accomplished by taking advantage of the GST fusion tag's affinity for glutathione. The purified fusion protein is then used to immunize animals, and antisera from these animals are then purified using affinity columns. Support protocols describe the construction and calibration of affinity columns for purifying antibodies using soluble fusion proteins, the use of insoluble fusion proteins for animal immunization, and preparation of affinity columns for purifying antibodies using insoluble fusion proteins.
Metal‐Chelate Affinity Chromatography
Authors: Petty Kevin J.
Published: June 2004

Recombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxy terminus can be purified using a resin containing nickel ions (Ni2+) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique is know as metal‐chelate affinity chromatography and can be performed using either native or denatured protein. This unit presents protocols for expression of histidine‐tail fusion proteins and their purification in either native or denatured form (along with procedures for renaturation by either dialysis or solid‐phase renaturation). Also provided are procedures for analysis of the purified produce and regeneration of the NTA resin.
Immunoaffinity Chromatography
Authors: Springer Timothy A.
Published: June 2004

This technique involves elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. Antibodies are coupled to Sepharose and the cell lysate is passed over the column. After washing, the specific antigen is eluted from the column using one of a number of methods: brief exposure to either high or low pH, or using the detergent octyl beta‐D‐glucoside (easily removable by dialysis). All these methods are described in this unit along with a procedure for covalently linking an antibody to Sepharose using the cyanogen bromide activation method.
Detection of Protein Phosphorylation in Tissues and Cells
Authors: Roche Katherine W. , Huganir Richard L.
Published: June 2004

Phosphorylation is one of the principal regulatory mechanisms in the nervous system. Several different procedures used to characterize the phosphorylation state of neuronal proteins are described in this unit, including analysis of phosphorylation in situ, phosphoamino acid analysis, and phosphopeptide map analysis. In addition, there is a protocol describing in vitro phosphorylation of fusion proteins. These methods are often combined to provide a comprehensive evaluation of the phosphorylation state of a particular protein.