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RECENTLY PUBLISHED:
UNIT 4.28 Gene Expression Analysis Using cDNA Microarrays (Stanislav L. Karsten and Daniel H. Geschwind, University of California-Los Angeles School of Medicine, Los Angeles, California). This unit focuses on the application of cDNA microarrays to large-scale gene expression studies in the nervous system. Specifically, the steps of probe preparation and microarray hybridization are described. Four methods are presented for labeling and detection of cDNA on microarray slides: direct labeling (DL) 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.
UNIT 9.13 Models of Amyotrophic Lateral Sclerosis (Mandy Jackson, Raquelli Ganel, and Jeffrey D. Rothstein, Johns Hopkins University, Baltimore, MD). Amyotrophic lateral sclerosis is an adult-onset chronic neuromuscular disease characterized pathologically by the relatively selective progressive degeneration of cortical motor neurons (upper motor neurons) and motor neurons in the brain stem and spinal cord (lower motor neurons). Two experimental models that can be used in screening putative therapeutic agents against neurodegeneration are described in this unit: organotypic cultures of spinal cord and transgenic mice expressing a human mutant SOD1 gene.
UNIT 2.5 Loading Neurons with Dextran-Conjugated Calcium Indicators in Intact Nervous Tissue (Kerry R. Delaney, Simon Fraser University, British Columbia, Canada). This unit describes methods for filling populations of neurons and their processes, including presynaptic terminals, with dextran-conjugated calcium indicators in central nervous tissue of mammals and lower vertebrates. Techniques for filling neurons in vivo for subsequent analysis either in vivo, or in brain slices or en bloc preparations, are described. These methods are also suitable for staining neurons in acute and organotypic brain slices.
UNIT 6.15 Recording with Multielectrode Arrays from Organotypic Cultures (Veronica C. Karpiak and Dietmar Plenz, National Institutes of Mental Health, NIH, Bethesda, Maryland). Recording from neuronal cultures with multielectrode arrays (MEAs) provides a powerful tool for studying neuronal activity in vitro with many neurons simultaneously. This unit describes the detailed steps necessary for growing organotypic cultures on MEAs and the typical neuronal activity that is obtained with this methodology.
UNIT 9.11 Intravenous Self-Administration of Ethanol in the Mouse (Nicholas J. Grahame, Indiana University - Purdue University at Indianapolis, Indiana; and Christopher L. Cunningham, Oregon Health Sciences University, Portland, Oregon). A more complete understanding of alcohol’s 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.
UNIT 9.12 Preclinical Models to Evaluate Potential Pharmacotherapeutic Agents in Treating Alcoholism and Studying the Neuropharmacological Bases of Alcohol-Seeking Behaviors in Rats (Harry L. June, Indiana University - Purdue University at Indianapolis, Indiana). The 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.
UNIT 8.11B Fear-Potentiated Startle in Mice (William A. Falls, The University of Vermont, Burlington, Vermont). Pavlovian fear conditioning procedures are frequently used to assess the behavioral, physiological, genetic, and molecular correlates of learning and memory. In the typical Pavlovian conditioned fear procedure, a neutral stimulus, such as a tone, is paired with a mildly aversive stimulus such as a foot shock. After a few of these pairings, the tone conditioned stimulus (CS) comes to elicit a variety of behaviors that are indicative of learned fear. One of the more prominent of these behaviors is a potentiated acoustic startle response. In this fear-potentiated startle effect, conditioned fear is operationally defined as elevated startle amplitude in the presence versus the absence of the CS. While fear-potentiated startle in mice is qualitatively similar to fear-potentiated startle in rats, it is now quite evident that the stimulus parameters and procedures for producing optimum fear-potentiated startle in mice differ considerably from those that produce optimum fear-potentiated startle in rats. Procedures outlined in this unit include initial assessment of startle, fear conditioning, and fear-potentiated startle testing. Special attention is paid to the parameters that affect the magnitude of fear-potentiated startle, and procedures designed to systematically examine these parameters are included.
UNIT 2.4 Electronic Imaging in Neuroscience (Kenneth R. Spring, National Heart, Lung and Blood Institute, NIH, Bethesda, Maryland). This unit is intended to aid the neuroscientist in understanding the basics of image detectors and selecting a suitable camera for various neuroscience research applications. A procedure is described for evaluating cameras in the laboratory.
UNIT 5.18 Phage Display in Neurobiology (Andrew Bradbury, Los Alamos National Laboratory, Los Alamos, New Mexico and the International School for Advanced Studies (SISSA), Trieste, Italy; Daniele Sblattero, the International School for Advanced Studies (SISSA), Trieste, Italy; Roberto Marzari, Universita' di Trieste, Trieste, Italy; Louise Rem and Hennie Hoogenboom, Department Pathologie, Academisch Ziekenhuis Maastricht and TargetQuest, Maastricht, The Netherlands). In this unit, some of the basic protocols involved in the manipulation of phage display libraries are described, including the rescue and amplification of such libraries, selection and screening from them, and testing of derived clones.
FORTHCOMING:
UNIT 6.16 Chronic Recording of Extracellular Neuronal Activity in Behaving Animals (Ronald Szymusiak, Veterans Administration Greater Los Angeles Healthcare System, North Hills, California and University of California, Los Angeles, California; and Douglas Nitz, Neurosciences Institute, La Jolla, California). Two 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 parardigms.
UNIT 8.5D Social Learning of Food Preference in Rodents: Rapid Appetitive Learning (Bennett G. Galef, Jr., McMaster University, Hamilton, Ontario, Canada). This unit describes a procedure for inducing increased intake of distinctively flavored foods or fluids in common laboratory rodents. The method provides a simple, efficient, non-invasive way to produce robust, long-lasting changes in appetitive behaviors of laboratory rodents that can be used in studies of the neuroanatomical, neurochemical, or genetic substrates of learning and memory.
UNIT 9.15 Models of Neuropathic Pain in the Rat (Gary J. Bennett, McGill University, Montreal, Quebec, Canada; Jin Mo Chung, University of Texas Medical Branch, Galveston, Texas; and Ze’ev Seltzer, Hebrew University, Jerusalem, Israel). There are now three models of neuropathic pain in the rat that are in widespread use: the chronic constriction injury, the partial sciatic ligation model, and the spinal nerve ligation model. The procedures to create these models and the behavioral assays used to quantify the resulting abnormal pain sensations are described in this unit.
UNIT 8.16 Mouse Social Recognition and Preference (James T. Winslow, Jennifer Ferguson and Kiran Rao, Emory Medical School, Atlanta, Georgia). Social 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.
UNIT 9.14 Conditioned Place Preference in Mice (Olga Valverde and Rafael Maldonado, Universitat Pompeu Fabra, Barcelona, Spain). Conditioned place preference is a behavioral model used to measure the rewarding properties induced by the administration of a drug. In this paradigm, the rewarding properties of a compound are associated with the particular characteristics of a given environment. This unit describes a procedure in which a mouse learns to associate one compartment of a two-compartment box with a drug and the other compartment with a vehicle control. The two compartments differ in texture and visual cues. After conditioning, the animal will prefer to spend more time in the compartment associated with the most rewarding effect.
UNIT 4.29 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
UNIT 4.30 Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout) (Richard Mortensen, University of Michigan Medical School, Ann Arbor, Michigan). Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. This unit provides a protocol in which the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker. A negative selectable marker is included in the construct outside the region of target gene homology in order to enrich for clones in which the target gene has undergone homologous recombination. The altered target gene is then expressed in embryonic stem cells. A support protocol describes a method for transient expression of Cre recombinase to remove sequences between lox sites, which can also be used as a selection method.
UNIT 8.5E Assessment of Sustained and Divided Attention in Animals (H. Moore Arnold, Sention, Providence, Rhode Island; John P. Bruno and Martin Sarter, The Ohio State University, Columbus, Ohio). Disruption of attentional processes has been implicated in a wide range of neuropsychiatric disorders, including schizophrenia and senile dementia. This unit describes two different operant tasks for use in rodents; one task assesses the animals’ ability to sustain attention, the other assesses the animals’ ability to divide attention.
UNIT 6.17 Imaging Nervous System Activity with Voltage-Sensitive Dyes (Dejan Zecevic1, Chun X. Falk1,2, Maja Djurisic1, Lawrence B. Cohen1,, Michal R. Zochowski1,3,4, Srdjan Antic1, and Matt Wachowiak1, 1Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut; 2RedShirtImaging, Fairfield, Connecticut; 3Center for Complex Systems, Warsaw School of Advanced Social Psychology, Warsaw, Poland). Optical recording with a voltage-sensitive dye is advantageous in situations where membrane potential must be recorded in many sites at once. Compared to other optical signals, the voltage-sensitive dye signal is fast (response time constant < 0.01 msec) and thus far the signals have been uniquely interpretable as changes in membrane potential. This unit describes methods for making voltage-sensitive dye measurements on three different preparations from which three very different kinds of information about the nervous system can be obtained. First, snail (invertebrate) ganglion is used to study how a neuron integrates its synaptic input into its action potential output by measuring membrane potential everywhere synaptic input occurs and at the places where spikes are initiated. Second, to understand how a nervous system generates a behavior, voltage-sensitive dye methods are used to measure the action potential activity of many (all) of the participating neurons in Aplysia abdominal ganglion. Third, responses to sensory stimuli and generation of motor output in the vertebrate brain are often accompanied by synchronous activation of many neurons in wide-spread brain areas, and the turtle olfactory bulb preparation is used to obtain simultaneous measurement of population signals from many areas. The general approach is three-pronged: (1) test dyes to find the dye with the largest signal-to-noise ratio; (2) reduce the extraneous sources of noise (vibrations, line frequency noise, preparation movement, etc.); and (3) maximize the number of photons measured to reduce the relative shot noise (noise arising from the statistical nature of photon emission and detection). A discussion of optical recording methods including the choice of dyes, light sources, optics, cameras, and minimizing noise is also provided.
UNIT 9.16 Models of Neuropathic Pain in the Rat (Gary J. Bennett, McGill University, Montreal, Canada; Jin Mo Chung, University of Texas Medical Branch, Galveston, Texas; and Ze’ev Seltzer, Hebrew University, Jerusalem, Israel). Three models of neuropathic pain in the rat are provided: the chronic constriction injury, the partial sciatic ligation model, and the spinal nerve ligation model. Both the procedures to create these models and the behavioral assays used to quantify the resulting abnormal pain sensations are described.
UNIT 9.17 Photochemical Cortical Lesion in Rodent Brain (Marcelle
Bergeron, Lilly Research Laboratories, Indianapolis, Indiana). In the rat
photochemical cortical lesion model described in this unit, an intravascular
photochemical reaction induces endothelial damage resulting in platelet aggregation,
thrombosis, thrombotic response (secretion of factors by the platelets) and
permanent cerebral vascular occlusion. Because thrombosis is produced in pial
vessels, the resulting cortical infarct is generally smaller and more reproducible
than in the models involving occlusion of the middle cerebral artery. The
surgical procedures involved are limited, making this model generally easier
to perform and less invasive than most other models of permanent focal ischemia
that involve mechanical occlusion of major cerebral arteries.