bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct, Continuous Behavioral Analysis of Drug Action on Feeding
Authors: Halford Jason , Blundell John 
Published: June 2004

AbstractAnalysis of rodent feeding behavior during the study of anorectic drugs reveals that contextual variables such as type of food, drug, environment, and physiological state influence food intake. Temporal changes in behavior can signal the onset of a feeding modulation process, as is the case with anorectic drugs acting on distinct neurochemical mechanisms that change temporal behavioral profiles. Comparing the effects of anorectic agents in controlled animal assays can identify potential therapeutic drugs that may influence human eating behavior. This unit specifies the materials and procedures required for continuously monitoring the Behavioral Satiety Sequence, as well as a device for continuous, automatic measurement of the mass of food consumption by the animal during behavioral monitoring called the RUEMA (Rat Universal Eating Monitor Apparatus).
Conditioned Flavor Preferences: Evaluating Postingestive Reinforcement by Nutrients
Authors: Ackroff Karen , Sclafani Anthony 
Published: June 2004

AbstractThe acceptance of food and the expression of preferences for particular foods have both innate and learned components. To determine the mechanism(s) responsible for the acquisition of learned preferences and the enhancement of inborn taste preferences, it is important to separate the component stimuli: the oral flavor cues and the postingestive nutrient cues that are associated with the flavors. Unambiguous analysis of postingestive reinforcement of a nutrient requires post‐oral presentation of the nutrient during or after oral intake of the flavor stimulus. This protocol concentrates on controlling stimulus presentation in associative conditioning, pairing a flavor with positive postingestive effects.
Measurement of Rodent Stereotyped Behavior
Authors: Kelley Ann E. 
Published: June 2004

AbstractThis unit presents a quantitative, observational method for the assessment of rodent stereotyped behavior which consists of motor responses that are repetitive, invariant, and seemingly without purpose or goal. The most classic behavioral pattern that is characteristic of stereotypy is that elicited by high doses of stimulants, such as cocaine and amphetamine, in rats, although it can also occur in response to other drugs or neurotoxic treatments affecting the basal ganglia. An observational time‐sampling procedure is described in which animals are observed and rated by an experimenter, who is blind to treatment, at regular time points over the course of a behavioral testing period. The frequency of different behaviors is measured by scoring the presence or absence of a given behavior during predetermined time bins. The apparatus and test procedures are described, and a comprehensive list of commonly observed behaviors that may appear as stereotyped is provided. In addition to being ideally suited to the measurement of stereotypy, the protocol can be adapted to sampling many forms of spontaneous behaviors, including locomotion, rearing, grooming, eating, and drinking. Samples of behavioral checklists and scoring sheets are also provided.
Motor Coordination and Balance in Rodents
Authors: Carter Rebecca J. , Morton Jennifer , Dunnett Stephen B. 
Published: June 2004

AbstractMeasurement of motor coordination and balance can be used not only to assess the effect of drugs or other experimental manipulations on mice and rats, but also to characterize the motor phenotype of transgenic or knock‐out animals. Three well established and widely used protocols for measuring motor coordination and balance in mice and rats (rotarod, beam walking and footprint analysis) are described in this unit. The tests can be used equally well for rats and mice, and have been used both for the phenotypic characterization of transgenic mice and for evaluating the effects of lesions and aging in rats. The protocols are described in the primary context of testing mice, but modifications of the test apparatus or variations in the test parameters for assessment of rats are noted.
Parental Behaviors in Rats and Mice
Authors: Lonstein Joseph S. , Fleming Alison S. 
Published: June 2004

AbstractParental care in mammals is often critical for survival and development of the offspring. The multiple behaviors that comprise parental care in rats and mice are controlled by a complex array of factors. This unit describes the components of parental care in rats and mice and the frequently used methods to assess the performance of these behaviors, a subject's preference for pup‐related cues, as well as a subject's motivation to act parentally.
Assessment of Murine Startle Reactivity, Prepulse Inhibition, and Habituation
Authors: Geyer Mark A. , Dulawa Stephanie C. 
Published: June 2004

AbstractThe study of the startle response and its plasticity provides a powerful research paradigm for examining the neural control of behavior. The neural substrates underlying phenomena such as information‐processing functions, including sensorimotor gating, affective modulation, and habituation, can be studied using startle plasticity as an operational measure. The mouse is rapidly becoming the most powerful model system for the study of human CNS function and is highly amenable to both genetic and pharmacological study. This unit describes procedures for the assessment of the startle response to acoustic stimuli in mice; methods for assessing prepulse inhibition (PPI) of startle, with treatments including drug administration and genetic manipulation; and methods for testing habituation.
Dural Inflammation Model of Migraine Pain
Authors: Phebus Lee A. , Johnson Kirk W. 
Published: June 2004

AbstractThe cause of migraine pain is controversial. One recently proposed theory is that migraine pain may originate from inflammation of the meninges, particularly the dural membranes that surround the brain. This theory proposes that, during a migraine, there is an idiopathic activation of trigeminal sensory afferents, resulting in nociceptive transmission to the CNS as well as the release of pro‐inflammatory substances in the periphery, particularly the dura. Dural inflammation is thought to lower the nociceptive threshold of dural afferents and facilitate nociceptive transmission to the central nervous system. In the procedure described in this unit, trigeminal sensory afferents are activated by electrically stimulating the trigeminal ganglion. This stimulation causes trigeminal peripheral sensory afferents to depolarize, inflammatory substances to be released from these afferents, and dural inflammation to appear. Dural inflammation is quantified by measuring plasma protein extravasation. The basic protocol describes this model in rats, and the alternate protocol describes the analogous procedure in guinea pigs.
Rodent Models of Focal Cerebral Ischemia
Authors: O'Neill Michael J. , Clemens James A. 
Published: June 2004

AbstractThis unit presents models that are both used to study ischemic mechanisms and to test for neuroprotective agents or agents that enhance recovery from stroke. The Tamura model is one of the best characterized focal ischemia models in which the middle cerebral artery is occluded by electrocoagulation. Also described is the intraluminal monofilament model, the spontaneously hypertensive rat (SHR), and the newer endothelin‐1 model. The rationale behind the use of animal models, the various types of models and advantage and disadvantages of each model are presented.
A Murine Model of Retroviral Encephalopathy
Authors: Sei Yoshitatsu , Basile Anthony S. 
Published: June 2004

AbstractThis unit delineates the steps for production of a murine model of retroviral encephalopathy. The LP‐BM5 infected mouse develops a chronic inflammation of the brain secondary to profound immune deficiency. The model is robust, develops rapidly and does not require the use of human pathogens. In addition, the behavioral and neurochemical characteristics of this model is reviewed.
Intravenous Self‐Administration of Ethanol in Mice
Authors: Grahame Nicholas J. , Cunningham Christopher L. 
Published: June 2004

AbstractA more complete understanding of alcohols reinforcing actions is obtained when multiple behavioral procedures are used, some of which bypass taste factors. This unit describes a method for assessing the reinforcing effects of alcohol in mice using the most widely accepted procedure for assessing drug reward: intravenous self‐administration.
Preclinical Models to Evaluate Potential Pharmacotherapeutic Agents in Treating Alcoholism and Studying the Neuropharmacological Bases of Ethanol‐Seeking Behaviors in Rats
Authors: June Harry L. 
Published: June 2004

AbstractThe unit outlines four basic protocols designed to systematically evaluate the capacity of potential pharmacotherapeutic agents to effectively treat alcohol addiction and dependence in rats. Also included are procedures designed to study the neural mechanisms regulating alcohol‐seeking behaviors.
Measurement of Panic‐Like Responses Following Intravenous Infusion of Sodium Lactate in Panic‐Prone Rats
Authors: Sajdyk Tammy J. , Keim Stanley R. , Thielen Shelley R. , Fitz Stephanie D. , Shekhar Anantha 
Published: June 2004

AbstractThis unit describes a putative animal model for panic disorder. The basic premise is that pharmacological disruption of critical brain regions implicated in the circuitry of anxiety will lead to a condition similar to that of the human disorder. A clinically relevant test, the sodium lactate challenge, is utilized to assess parallels between the human condition and this rat model.
Patch‐Pipet Recording in Brain Slices
Authors: Stuart Greg 
Published: June 2004

AbstractPatch‐clamp recording in brain slices provides a powerful approach for investigating the intrinsic electrical properties of neurons and glia and analyzing synaptic interactions between neurons. There are two main methods presently in use to obtain patch‐clamp recordings from neuronal or glial cell bodies and their processes in brain slices, both described in this unit. In the blind technique described in this unit, the approach to a cell is performed blindly and recordings are obtained relying purely on electrical cues. Alternatively, the movement of the recording pipet through the brain slice and its placement onto the cell membrane can be performed as described under visual control; this method is usually performed with the aid of differential interference contrast (DIC) optics and is referred to as the DIC technique. Increased resolution of cells and their processes can be obtained by combining DIC optics with infrared illumination and video microscopy techniques. Using either of these methods it is possible to reliably apply the full power of the patch‐clamp technique to the study of neurons and glial cells in brain slices.
Optimization of Transfection
Authors: Rose John K. 
Published: June 2004

AbstractThe single most important factor in optimizing transfection efficiency is selecting the proper transfection protocol. This overview discusses the most commonly used procedures: calcium phosphate‐mediated gene transfer, DEAE‐dextran‐mediated gene transfer, electroporation, and liposome‐mediated transfection. Fusion techniques such as protoplast fusion and microinjection may also be considered.
Preparation of RNA from Tissues and Cells
Authors: Ribaudo Randall , Gilman Michael , Kingston Robert E. , Chomczynski Piotr , Sacchi Nicoletta 
Published: June 2004

AbstractMost procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA. The first basic protocol describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second basic protocol allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A+) mRNAs from total RNA.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C. 
Published: June 2004

AbstractThe first section of this unit discusses basic principles of sterile technique, while the second section discusses medium preparation. The basic protocol describes subculturing of a monolayer culture grown in petri plates or flasks. Support protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. An alternate protocol describes freezing suspension cells.
Syntheses of Specifically 15N‐Labeled Adenosine and Guanosine
Authors: Gaffney Barbara L. , Jones Roger A. 
Published: June 2004

AbstractThis unit describes the specific incorporation of 15N into the N7 and amino positions of adenosine, and conversion of the adenosine to guanosine labeled at the N1, N7, and amino positions. Two variations of the procedures are also presented that include either 12C or 13C at the C8 position of adenosine, and 13C at either the C8 or C2 position of guanosine. These 13C tags permit the incorporation of two 15N‐labeled nucleosides into an RNA strand while ensuring that their nuclear magnetic resonance (NMR) signals can be distinguished from each other by the presence or absence of C‐N coupling. While the major application of these specifically 15N‐labeled nucleosides is NMR, the additional mass makes them useful in mass spectrometry (MS) as well. The procedures can also be adapted to synthesize the labeled deoxynucleosides. A support protocol describes the synthesis of 7‐methylguanosine.
Synthesis of Protected 2′‐Deoxy‐2′‐fluoro‐β‐D‐arabinonucleosides
Authors: Elzagheid Mohamed I. , Viazovkina Ekaterina , Damha Masad J. 
Published: June 2004

AbstractThis unit describes in detail the preparation of protected 2'‐deoxy‐2'‐fluoroarabinonucleosides. These building blocks are required for the synthesis of 2'‐deoxy‐2'‐fluoroarabinonucleic acid (2'F‐ANA), an oligonucleotide analog exhibiting very promising antisense properties. The preparation of phosphoramidites from these building blocks and the synthesis of 2'F‐ANA are described elsewhere in the manual.
Synthesis of 1,5‐Anhydrohexitol Building Blocks for Oligonucleotide Synthesis
Authors: Lagoja Irene M. , Marchand Arnaud , Van Aerschot Arthur , Herdewijn Piet 
Published: June 2004

AbstractThis unit describes in detail, the optimized preparations of 1,5‐anhydrohexitol and the 1,5‐anhydrohexitol building blocks for oligonucleotide synthesis (hG, hA, hC, hT).
Protection of 5′‐Hydroxy Functions of Nucleosides
Authors: Seliger H. 
Published: June 2004

AbstractThe 5‐hydroxy group is the primary hydroxy group of nucleosides. It is mandatory to protect 5‐hydroxyls in all methods of oligonucleotide synthesis that require nucleoside synthons. This unit discusses a wide variety of acid‐labile and base‐labile protecting groups, as well as enzymatic methods for 5‐protection and deprotection.
Deoxyribo‐ and Ribonucleoside H‐Phosphonates
Authors: Stawinski Jacek , Strömberg Roger 
Published: June 2004

AbstractMost methods for preparing H‐phosphonate monoesters suffer from variable yields and are often incompatible with common protecting groups used in oligonucleotide synthesis. This unit describes four procedures that consistently give high yields of the desired products. Taken together, they provide an arsenal of phosphonylation prodecures that it compatible with most common protecting groups.
Regioselective 2′‐Silylation of Purine Ribonucleosides for Phosphoramidite RNA Synthesis
Authors: Gaffney Barbara L. , Jones Roger A. 
Published: June 2004

AbstractThis unit describes high‐yield procedures for protection of purine ribonucleosides based on a reaction that allows concomitant highly regioselective 2′‐silylation and 3′‐phosphitylation to give, in one step, monomers that are ready for H‐phosphonate synthesis. For preparation of phosphoramidites, the H‐phosphonate monoester is cleaved without silyl migration to give intermediates ready for phosphitylation by standard methods.
Preparation of 2′‐O‐[(Triisopropylsilyl)oxy]methyl‐protected Ribonucleosides
Authors: Pitsch Stefan , Weiss Patrick A. 
Published: June 2004

AbstractThe [(triisopropylsilyl)oxy]methyl (TOM) group is a useful protecting group for the 2′‐OH of ribonucleosides to be used for oligoribonucleotide synthesis by the phosphoramidite method. It is completely stable to all reaction conditions applied during assembly and the first deprotection step. It does not interfere with the coupling reaction and leads to very good coupling yields under DNA‐coupling conditions. The final cleavage occurs quantitatively without concomitant destruction of the RNA product. This unit describes the synthesis and characterization of 2′‐O‐TOM‐5′‐O‐dimethoxytrityl‐N‐acetyl ribonucleosides in full detail. The TOM‐group is introduced via a dibutyltin dichloride‐mediated reaction into N‐acetylated, 5′‐O‐dimethoxytritylated ribonucleosides. Support protocols describe the synthesis of N‐acetylated, 5′‐O‐dimethoxytritylated adenosine and guanosine, as well as synthesis of the starting reagent [(triisopropylsilyl)oxy]methyl chloride (TOM‐Cl). Preparation of the phosphoramidites and their use in solid‐phase oligonucleotide synthesis are described elsewhere in the series.
Attachment of Nucleosides to Solid‐Phase Supports
Authors: Pon Richard T. 
Published: June 2004

AbstractSpecific step‐by‐step instructions are given for coupling nucleosides to LCAA‐CPG supports (supports consisting of a long‐chain alkylamine linked to controlled‐pore glass). Protocols are given for a succinic acid linker and a hydroquinone‐O,O′‐diacetic acid linker. The former is the most widely used linker arm, and the starting materials are widely available. The latter offers greater compatibility with base‐sensitive sequence modifications and great synthetic throughput because it can be cleaved under milder and faster conditions. Additional guidelines are given for selecting a linker arm and coupling protocol. Almost any application requiring synthetic oligonucletodies can be satisfied using one of these linker arms.
Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribonucleotides According to the Phosphoramidite Method
Authors: Beaucage Serge L. , Caruthers Marvin H. 
Published: June 2004

AbstractThe phosphoramidite approach has had a major impact on the synthesis of oligonucleotides. This unit describes parameters that affect the performance of this method for preparing oligodeoxyribonucleotides, as well as a number of compatible strategies. Milestones that led to the discovery of the approach are chronologically reported. Alternate strategies are also described to underscore the versatility by which these synthons can be obtained. Mechanisms of deoxyribonucleoside phosphoramidite activation, factors affecting condensation, and deprotection strategies are discussed.
Strategies for Oligoribonucleotide Synthesis According to the Phosphoramidite Method
Authors: Wincott Francine E. 
Published: June 2004

AbstractAdvances in oligoribonucleotide synthesis have lagged behind those in oligodeoxyribonucleotide synthesis because of the difficulty in identifying orthogonal protecting groups for the 2′‐ and 5′‐hydroxyls. Adaptation of the phosphoramidite method for DNA synthesis to RNA synthesis has greatly improved our understanding of RNA. It allows site‐specific introduction of modified nucleosides to any position in an RNA molecule, as well as introduction of variations at multiple sites in the molecule. This overview discusses issues that are relevant to RNA synthesis by the phosphoramidite approach, including supports used, activation of the ribonucleoside phosphoramidites, and protection of the nucleobase, phosphate, and 2′‐ and 5′‐hydroxyls.
Direct Attachment of Conjugate Groups to the 5′ Terminus of Oligodeoxyribonucleotides
Authors: Asseline Ulysse , Thuong Nguyen T. 
Published: June 2004

AbstractThis unit gives protocols for the attachment of intercalating and photoreactive conjugate groups to oligodeoxyribonucleotides. Protocols are given for acridine‐ and psoralen‐conjugated oligonucleotides, and include attachment of the linker, preparation of the phosphoramidite, coupling to the oligonucleotide, deprotection, purification, and characterization.
Attachment of Reporter and Conjugate Groups to the 3′ Termini of Oligonucleotides
Authors: Greenberg Marc M. 
Published: June 2004

AbstractConjugation of oligonucleotides at the 3 terminus is less common because this site is used for covalent linkage to solid‐phase oligonucleotide synthesis supports. However, 3‐oligonucleotide conjugates have several valuable physicochemical properties, including their ability to stabilize nucleic acid hybridization complexes and to retard the activity of exonucleases. This unit discusses methods for preparing oligonucleotides conjugated at the 3 terminus.
Incorporation of Halogenoalkyl, 2‐Pyridyldithioalkyl, or Isothiocyanate Linkers into Ligands
Authors: Asseline Ulysee , Thuong Nguyen T. 
Published: June 2004

AbstractLigands can be introduced at the 5' terminus of an oligonucleotide by adding a linker to the ligand and modifying the 5' terminus of the oligonucleotide. These are then reacted to give the ligand‐oligonucleotide conjugate. The addition of appropriate linkers to ligands is described in this unit. 5'Modification of the oligonucleotide and the final reaction that produces the ligand‐conjugated oligonucleotide are described elsewhere in the series. This approach is particularly useful when there is a limited amount of ligand available, when the ligand is sensitive to chemical conditions required for oligonucleotide deprotection, or when the ligand is weakly soluble in solvents required for phosphoramidite‐ or H‐phosphonate‐mediated oligonucleotide synthesis.
Conjugation of 5′‐Functionalized Oligodeoxyribonucleotides with Properly Functionalized Ligands
Authors: Asseline Ulysse , Thuong Nguyen T. 
Published: June 2004

AbstractThis unit reports the synthesis of oligodeoxyribonucleotides covalently linked via their 5′ termini to various ligands such as intercalating agents, reactive groups, or labels. Methods for incorporation of halogenoalkyl, isothiocyanate, and 2‐pyridyldisulfide linkers onto ligands, and addition of amino, carboxyl, thiophosphate, phosphate, and masked thiol groups at the 5 terminus of an oligodeoxyribonucleotide are described elsewhere in the series. This unit reports procedures for coupling the ligands and oligonucleotides, as well as details for purification and characterization of the final products.
Solid‐Phase Synthesis of Branched Oligonucleotides
Authors: Carriero Sandra , Damha Masad J. 
Published: June 2004

AbstractBranched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single‐stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2′,5′‐ and 3′,5′‐phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre‐mRNA splicing. The protocols described herein allow for the facile solid‐phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion‐exchange HPLC and PAGE are also illustrated.