bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of mRNA Populations from Single Live and Fixed Cells of the Central Nervous System
Authors: Eberwine James , Crino Peter 
Published: June 2004

AbstractThis 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

AbstractAntibodies 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

AbstractThe 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

AbstractRecombinant 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

AbstractThis 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

AbstractPhosphorylation 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.
Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics In Vivo
Authors: Kahana Jason A. , Silver Pam A. 
Published: June 2004

AbstractFluorescent molecules serve as valuable tools for the detection of numerous biochemical phenomena and have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting and determination of chemical concentrations. However, the use of such techniques generally requires significant nonphysiological perturbations to the biological system being studied; therefore, they are not always appropriate for the observation of dynamic phenomena. Green fluorescent protein (GFP), cloned from jellyfish, has been used to overcome many of these problems. It is a small, extremely stable fluorescent protein that has been successfully expressed and detected in a wide variety of organisms, both in intact form and fused to other proteins. This overview unit describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena.
Green Fluorescent Protein in the Study of Neuronal Signaling Pathways
Authors: Blair Leslie , Bence‐Hanulec Kendra , Marshall John 
Published: June 2004

AbstractIn recent years, techniques have been established for transiently co‐transfecting cells with cDNA of the jellyfish green fluoresent protein (GFP), a reporter gene that encodes a non‐toxic marker. This approach can be applied to primary neurons where it has become especially useful for the study of neuronal second messenger pathways. This unit describes procedures for transfecting neurons in primary culture: transfection with GFP DNA, including co‐transfecting with separate GFP and gene‐of‐interest constructs, transfecting with a single construct containing the gene of interest fused to a GFP gene, and transfecting with a single construct containing separate gene‐of‐interest and GFP cassettes. Also included is a method for the rapid, large‐scale preparation of a nearly homogeneous population of neurons from rat cerebellum. The Commentary provides several examples of how this approach can be applied to specific biological questions on neuronal signaling pathways.
Analysis ofRNAby Northern and Slot‐Blot Hybridization
Authors: Brown Terry , Mackey Karol 
Published: June 2004

AbstractSpecific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting), while unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Included in this unit are detailed procedures for RNA denaturation, blotting and hybridization. Also described is a method for stripped hybridization probes from blots so the blots can be re‐hybridized with a different probe.
Principles of Signal Conditioning
Authors: Finkel Alan , Bookman Richard 
Published: June 2004

AbstractIt is rare for biological, physiological, chemical, electrical, or physical signals to be measured in the appropriate format for recording and interpretation. Usually, a signal must be conditioned to optimize it for both of these functions. This overview describes the fundamentals of signal filtering, how to prepare signals for A/D conversion, signal averaging to increase the signal‐to‐noise ratio, line frequency pickup (hum), peak‐to‐peak and rms noise measurements, blanking, audio monitoring, testing of electrodes and the common‐mode rejection ratio.
Fabrication of Patch Pipets
Authors: Rae J.L. , Levis R.A. 
Published: June 2004

AbstractPatch clamping refers to a wide range of electrophysiological measurements, all of which have in common the use of patch pipets and the formation of gigaohm seals. The purpose of this unit is to describe the fabrication of patch pipets. The aspects of the pipet geometry that are important to different applications and the different procedures that have been found to most reliably and simply achieve these results are described. Parameters for glass selection are detailed in the beginning of the unit. Pulling patch and whole‐cell pipets, elastomer coating, fire polishing, pipet filling, and pipet testing in an experimental setup are highlighted. Additional support protocols describe alternative ways to optimize pipet geometry and cleaning the glass before pulling. Considerations for noise and dynamic performance are emphasized as these two requirements for single‐channel and whole‐cell current measurements dictate how the pipets must be fabricated.
Acute Isolation of Neurons from the Mature Mammalian Central Nervous System
Authors: Kay Alan R. , Krupa David J. 
Published: June 2004

AbstractThe acute dissociation procedure provides a simple means of isolating neurons from the mature mammalian central nervous system. The method was primarily devised to isolate neurons for patch‐clamp electrophysiology. It may also prove useful for single‐cell PCR, immunocytochemistry, sorting of fluorescently labeled cells, or long‐term tissue culture of mature neurons. Dissociation is brought about by a combination of proteolysis and an ionic environment that encourages breakdown of the tissue. The method allows the isolation of neurons free of glial ensheathments in as little as 45 min after the sacrifice of the animal. Neurons so isolated lose fine dendritic branches, although the structure proximal to the cell body is often maintained, allowing identification of the morphological type of the neuron. The preparation has the following advantages: (1) the neurons are fully differentiated; (2) the cells are electronically compact, which improves the fidelity of the voltage clamp; (3) the cells are removed from the influence of surrounding cells; and (4) neurons can be isolated from small, circumscribed loci within the adult central nervous system.
Chronic Recording of Extracellular Neuronal Activity in Behaving Animals
Authors: Szymusiak Ronald , Nitz Douglas 
Published: June 2004

AbstractTwo methods for recording extracellular neuronal activity in unanesthetized, unrestrained rats are described in this unit. Both use chronically‐implanted bundles of fine microwires to record electrophysiological activity. One method provides recordings of single and/or multiple unit activity from individual wires in a bundle (monotrode). Discrimination of individual neuronal potentials is based on action potential amplitude, or on a combination of action potential amplitude and shape. The second method uses a 2‐ to 4‐microwire array (stereotrode‐tetrode) to yield multiple unit recordings. Discrimination of individual neuronal potentials is based on action potential shapes and the relative amplitude of action potentials recorded simultaneously on the different wires in the array. These methods can provide stable, long‐term recording of neuronal activity during a variety of behavioral paradigms.
Recording in the Cerebellar Slice
Authors: Swanson Geoffrey T. , Contractor Anis 
Published: June 2004

AbstractThis unit describes techniques for the preparation of mouse cerebellar slices and electrophysiological recording from neurons in the slice. The cerebellum provides a model of motor learning that can be correlated with alterations in synaptic function. In addition, the architecture and well‐defined synaptic pathways in the cerebellar slice make this preparation a useful model for exploring general principles of synaptic transmission.
Microdialysis in Nonhuman Primates
Authors: Saunders Richard C. , Kolachana Bhaskar S. , Weinberger Daniel R. 
Published: June 2004

AbstractThis unit presents a technique that allows for routine repeated microdialysis experiments in the monkey using a sedated preparation or, with further minor modification, in the awake behaving animal. Protocols are provided for construction and in vitro calibration of dialysis probes, obtaining magnetic resonance imaging (MRI) scans of the monkey brain, surgically attaching a guide holder to the skull, and performing postoperative MRI scanning, in vivo dialysis sample collection, anatomical verification of the probes, and neurochemical analysis. An alternate protocol describes microdialysis in awake, behaving monkeys; this requires substantial preparatory work in training the monkeys to sit quietly in a restraining chair and/or to perform a series of behavioral tasks.
Detection and Quantification of Neurotransmitters in Dialysates
Authors: Pani (catecholamines/indoleamines and acetylcholine) A.K. , Shippenberg (catecholamines/indoleamines) Toni S. , Heidbreder (acetylcholine) Christian , Espey (amino acids) Michael Graham 
Published: June 2004

AbstractThis unit describes methods for the separation and quantification of neurotransmitters, peptides, and second messengers in microdialysis samples by high‐pressure liquid chromatography (HPLC) coupled to either electrochemical detection (EC) or fluorescence detection. The protocols describe commonly used procedures for the detection of catecholamines and indoleamines (HPLC‐EC detection; detection of acetylcholine (HPLC‐EC detection with enzyme reactor), and detection of amino acids (HPLC‐fluorescence detection). Each of these protocols affords detection limits in the low nanomolar range.
Saturation Assays of Radioligand Binding to Receptors and Their Allosteric Modulatory Sites
Authors: Basile Anthony 
Published: June 2004

AbstractThe protocols in this unit describe methods for measuring radioligand binding to sites on the GABA‐A receptor complex. This system was chosen because of the fundamental role of GABA‐A receptors in central nervous system function and the presence of multiple binding sites on one supramolecular complex. However, these basic techniques can be used to analyze ligand binding to a wide variety of intracellular and extracellular sites associated with ion channels, transporters, or G proteins with relatively simple modifications of the protocols (e.g., tissue preparation, buffers, assay termination techniques, incubation times, and temperatures). A saturation assay is presented to examine ligand binding to the benzodiazepine site on the GABA‐A receptor, while a second protocol uses a similar technique to examine the binding site for GABA in this complex.
Measurement of Chloride Movement in Neuronal Preparations
Authors: Schwartz‐Bloom Rochelle D. , Engblom A. Christine , Åkerman Karl E. O. , Inglefield Jon R. 
Published: June 2004

AbstractIn this unit, protocols are described for biochemical and optical techniques that have been used by investigators to measure ligand‐gated chloride movement in vesicular structures called synaptoneurosomes (also referred to as microsacs), in cultured neurons, and in the acute brain slice. These techniques can be applied to other ions as well. The measurement of uptake and efflux of radioisotopic chloride in synaptoneurosomes is used to study the responses of gamma‐aminobutyric acid (GABA) receptors, which are coupled to chloride channels. Similar chloride flux assays for primary neuronal cultures are also presented. Alternatively, the efflux of chloride from synaptoneurosomes and primary neuronal cultures can be studied using fluorescent dyes and photometry. Finally, the measurement of chloride uptake can be studied in individual neurons in brain slices using fluorescent dyes and optical imaging by nonconfocal and confocal microscopy. Several support protocols are provided as well, outlining the preparation of synaptoneurosomes from specific brain regions, and the preparation, loading, and calibration of chloride‐sensitive fluorescent dyes.
Measurement of Cation Movement in Primary Cultures Using Fluorescent Dyes
Authors: Reynolds Ian J. 
Published: June 2004

Abstract
Ca2+, Na+, K+, and Mg2+ have a central role in neuronal excitability. The concentration of these cations in the cytoplasm of neurons (generically termed [ion+]i) provides a marker of the excitation state of the neurons, and may also illuminate the activity of specific signaling mechanisms that involve Ca2+‐ or Mg2+‐activated enzymes. The measurement of [ion+]i in cultured neurons is achieved with the use of an ion‐sensitive fluorescent dye in combination with equipment designed to quantitatively measure fluorescence. Specificity is obtained by choosing dyes with the appropriate selectivity for the ion of interest. Measurements of steady state ion concentrations can be made, as well as measurements of the net difference between ion movement into the cytoplasm (in response to a stimulus) and the physiological buffering of that ion. The procedures in this unit for loading and recording from dyes are broadly similar for each ion when ratiometric dyes are used as described, and can readily be modified for use with single‐wavelength dyes. Support protocols are provided for calibration of individual dyes, which can be more problematic.
In Vivo Measurement of Blood‐Brain Barrier Permeability
Authors: Boje Kathleen M.K. 
Published: June 2004

AbstractThis unit describes various protocols for the in vivo quantitation of drug permeability across the rodent blood ‐ brain barrier. Methods for the measurement of drug influx or efflux are described, and support protocols are provided for determining intravascular capillary volume and cerebral perfusion flow. An in situ perfusion technique is also provided for assessing whether transport of a test compound occurs by carrier‐mediated or saturable transport.
Methods for Sample Preparation for Direct Immunoassay Measurement of Analytes in Tissue Homogenates: ELISA Assay of Amyloid β‐Peptides
Authors: Hyslop Paul A. , Bender Mark H. 
Published: June 2004

AbstractUse of low abundance analytes in whole tissue homogenates has been realized with the development of assays in which a specific analyte is captured and detected using immunological reagents. One of the many advantages of analyte immunoassay in crude homogenates is its relative simplicity, allowing high throughput analysis of samples. In this unit, some major key determinants in sample and standard preparation and handling are described that have been shown to improve the performance and reliability of these assay systems. The ELISA assay of amyloid peptides from brain tissue is described as an example, since the protocols for this analysis exemplify many of the techniques and problems that are encountered in the development of new assays.
Sexual and Reproductive Behaviors
Authors: Sisk Cheryl L. , Meek Leslie R. 
Published: June 2004

AbstractThe procedures described in this unit include testing procedures for male and female reproductive behaviors, gonadectomy, and hormonal treatments appropriate for inducing male and female reproductive behaviors. Because reproductive behaviors are social behaviors, and therefore require the presence of stimulus animals, the protocols in this unit also provide information on the preparation of stimulus animals. The protocols are written for use with laboratory rats, although a discussion of issues related to species differences in the study of reproductive behaviors is included.
Application of Experimental Stressors in Laboratory Rodents
Authors: Heinrichs Stephen C. , Koob George F. 
Published: June 2004

AbstractThis unit presents seven separate stressor protocols for laboratory rodents. Stress induction is a critical element in the study of neural and neuroendocrine mechanisms involved in establishing and maintaining a state of stress. The first three procedures, immobilization, footshock, and swimming, involve acute exposure to noxious stimuli. The next three procedures, social isolation, resident/intruder aggression, and maternal deprivation, induce social disruption by withdrawal from a group housing condition, attack within the unfamiliar territory of a dominant male, or segregation of a preweanling pup from its mother, respectively. The final procedure, sleep deprivation, involves passive denial of the opportunity to sleep. Support protocols are provided to address the need for environmental acclimation and calming procedures prior to any stress‐related studies (including, for rats, handling of the animals as a calming measure) and to detail a simple method of quantifying the response to a given stressor by direct measurement of levels of the stress hormones adrenocorticotrophic hormone (ACTH) and corticosterone.
Cued and Contextual Fear Conditioning in Mice
Authors: Wehner Jeanne M. , Radcliffe Richard A. 
Published: June 2004

AbstractContextual and cued fear conditioning is a robust form of learning in which an association is made between stimuli and their aversive consequences. Fear conditioning has been used in laboratory rodents in part because it is a highly conserved form of behavior that is exhibited in both laboratory situations and in normal environments. Training requires only a single trial and this makes it adaptable to genetic, pharmacological, and biochemical studies. Clinically, it is has relevance to human behavior in that fear conditioning can be produced in humans, and damage to the amygdala prevents fear conditioning.
Assessment of Sustained and Divided Attention in Rats
Authors: Arnold H. Moore , Bruno John P. , Sarter Martin 
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.
Overview of Methodological Approaches to the Study of Ingestive Behavior
Authors: Hoebel Bartley G. , Wellman Paul J. 
Published: June 2004

AbstractSome examples of procedures used by feeding researchers are discussed in this unit and include ablation of neural function; inhibition of behaviors by selective neurotoxins and antisense oligonucleotides; staining of sensory and motor mechanisms; electrical stimulation of the brain; local injection and microdialysis of nutrients, neurotransmitters, and drugs; autoradiography and in situ hybridization of neurotransmitters with their receptors; electrophysiological techniques for multi‐ and single‐unit recording of cells in the hypothalamus; and gene technology using inbred strains of genetically obese mice.
Basic Measures of Food Intake
Authors: Wellman Paul J. , McMahon Lance R. 
Published: June 2004

AbstractThis unit describes a method for measuring the consumption of a pellet diet during a 30‐min testing session conducted during the late portion of the day. The procedure generates reliable and stable baseline measures of feeding, and is appropriate for assessing experimental manipulations that either enhance (e.g., injecting a peptide into the brain) or suppress feeding (e.g., systemic injections of amphetamine).