bluuebunny/crossref_metadata_2025_split huggingface dataset:
Counting Cells in Sectioned Material: A Suite of Techniques, Tools, and Tips
Authors: Williams Robert W. , von Bartheld Christopher S. , Rosen Glenn D. 
Published: June 2004

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

AbstractThere are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteination and recovery of DNA. The main differences between various approaches lie in the extent of deproteination and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.
Development of a Universal Nucleobase and Modified Nucleobases for Expanding the Genetic Code
Authors: Romesberg Floyd E. , Yu Chengzhi , Matsuda Shigeo , Henry Allison A. 
Published: June 2004

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

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

AbstractThis unit begins with a discussion of the advantages and disadvantages of oligonucleotide synthesis using solid supports. The physical and chemical properties of solid‐phase supports are discussed in terms of their suitability for oligonucleotide synthesis. In addition, the unit outlines the properties of linkers used for transient or permanent attachment of properly protected nucleosides to the derivatized support, as well as strategies for coupling nucleosides to linkers and conditions for the release of synthetic oligonucleotides from specific supports.