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UNIT 7.18 Assessment of Cell Viability in Primary Neuronal Cultures (Howard S. Ying, Frank J. Gottron and Dennis W. Choi, Washington University School of Medicine). Four commonly used methods for the assessment of neuronal (or glial) cell viability are described in this unit. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (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.

UNIT 4.21 Gene Transfer Using Lentiviral Vectors (Romain Zufferey and Didier Trono, University of Geneva). Conventional retroviral vectors are of limited usefulness for neuroscience applications because they are derived from oncoretroviruses such as the Mouse Leukemia Virus (MLV), and, as a consequence, cannot transduce nondividing cells. Most cells in the nervous system proliferate very little, if at all. In contrast to oncoretroviruses, lentiviruses such as the human immunodeficiency virus (HIV) are a subfamily of retroviruses that can infect both growth-arrested and dividing cells. Accordingly, lentiviral vectors efficiently transduce targets such as neurons and glial cells, both in tissue culture and in vivo. This unit describes the production of high-titer lentiviral vectors by transient transfection of 293T cells and concentration of the particles by centrifugation. Guidelines for the titration of vector stocks are also given.

UNIT 4.22 Alphavirus-Mediated Gene Transfer into Neurons (Markus U. Ehrengruber, Brain Research Institute, University of Zurich, and Kenneth Lundstrom, F. Hoffmann-La Roche, Basel, Switzerland). Semliki Forest virus and Sindbis virus belong to the alphavirus genus of the togaviruses and have recently been shown to efficiently infect neurons in vitro and in vivo, i.e., under conditions where glial cells are present. This unit describes the preparation and application of recombinant, neurotrophic replicons derived from Semliki Forest virus and Sindbis virus for gene transfer into hippocampal neurons.

UNIT 4.23 Gene Transfer into Neural Cells In Vitro Using Adenoviral Vectors (Thomas D. Southgate, Paul A. Kingston, and Maria G. Castro, University of Manchester). 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; they are also 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.

UNIT 4.24 Gene Transfer into the Brain Using Adenoviral Vectors (Clare E. Thomas, Evelyn Abordo-Adesida, Tricia C. Maleniak, Daniel Stone, Christian A. Gerdes and Pedro R. Lowenstein, University of Manchester). Recombinant 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, and vector-mediated cytotoxicity). Finally, critical parameters for obtaining optimal gene transfer with minimum side effects are presented.

UNIT 9.5 Rodent Models of Global Cerebral Ischemia and UNIT 9.6 Rodent Models of Focal Cerebral Ischemia (Michael J. O’Neill and James A. Clemens, Eli Lilly and Co.). Brain damage after stroke and head injury remains a huge clinical problem. In stroke, the initial cause of the damage is a blockage in a blood vessel (often the middle cerebral artery) and this sets off several pathways that ultimately lead to cell death. Recent studies have demonstrated that several new mechanisms are involved in neuronal death, and this has led to an increase in research into novel molecules that might prevent brain damage or improve recuperation post-stroke. There are several models of global cerebral ischemia. Two of the most widely used models are discussed in detail in UNIT 9.5: the gerbil bilateral carotid artery occlusion (BCAO) model and rat 4-vessel occlusion (4-VO) model. Additionally, several models of focal cerebral ischemia have been developed to mimic the effects of human stroke. UNIT 9.6 presents models that are used both 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.

UNIT 1.6 Metabolic Activity in Antigentically Identified Neurons: A Double-Labeling Method for High-Resolution 2-Deoxyglucose Detection and Immunohistochemistry (James S. McCasland, SUNY Health Science Center at Syracuse, N.Y.). The 2-deoxyglucose (2DG) histochemical method of Sokoloff has been widely used to map metabolic activity in the brain, but lacks sufficient resolution to view individual neurons. Modifications of the original method have achieved cellular resolution with emulsion autoradiography and tissue apposed directly to the autoradiogram, but the ability to discriminate clearly between populations of neurons identified by antigenic markers has been lacking. A method is presented which makes it possible, for the first time, to study the metabolic activity of every immunohistochemically labeled neuron in a neural system of interest. The procedure combines a high-resolution 2DG technique and immunostaining for glutamate decarboxylase (GAD) or other antigens. The method stabilizes the label during immunohistochemical processing by the addition of glycogen to all solutions that come into contact with tissue after sectioning. The postulated effect of the added glycogen is to limit diffusion or net efflux of unfixed macromolecules that contain the 2DG label. When used with a GAD antibody to label putative GABAergic inhibitory interneurons, the procedure allows a direct assessment of the extent of inhibition in the circuitry of cerebral cortex.  A detailed protocol for this double-labeling procedure is presented, along with examples of the labeling patterns obtained in the whisker-related barrel field of rodent somatosensory cortex.
 

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UNIT 8.12 Motor Coordination and Balance in Rodents (Rebecca J. Carter, A. Jennifer Morton and Stephen B. Dunnett, University of Cambridge). Measurement 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 knockout 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.

UNIT 1.7 Targeted Toxins (Ronald G. Wiley, Vanderbilt University, Nashville, TN and Douglas A. Lappi, Advanced Targeting Systems, San Diego, CA). The use of targeted toxins to make highly selective neural lesions can serve as a bridge between molecular neuroscience and systems neuroscience. The “molecular neurosurgery” strategy presented in this unit consists of coupling a cytotoxic moiety such as saporin, a plant protein that irreversibly inhibits protein synthesis by catalytically inactivating ribosomes, to a targeting vector, such as an anti-neuronal antibody or neuropeptide, which will be selectively internalized by neurons that express a particular target surface molecule such as growth factor or neuropeptide receptors.  Animals with such neuron type–selective lesions can thus be studied to determine the impact of losing a precisely defined population of neurons.

UNIT 1.8 Compartmental Analysis of Temporal Activity Using Fluorescent In Situ Hybridization (catFISH) (John F. Guzowski, University of Arizona, and Paul F. Worley, Johns Hopkins University School of Medicine). The subcellular location of IEG RNA molecules, detected by fluorescent in situ hybridization (FISH) and laser scanning confocal microscopy, can be used to infer the activity history of neurons at two instances before the sacrifice of the animal. In this unit, special procedures for FISH are provided to yield the high sensitivity required for successful catFISH experiments, including mounting sections, preparation of riboprobes, and tyramide signal amplification detection of the probes. Additionally, special microscopic considerations are presented so the reader can obtain images of appropriate resolution and sensitivity. Also detailed are image scoring methodologies.

UNIT 8.10A Behavioral Despair: Forced Swimming and Tail Suspension Tests in Rodents (Roger D. Porsolt, Geneviève Brossard and Sylvain Roux, Porsolt and Partners Pharmacology, France). Rodents forced to swim in a narrow space from which there is no escape will, after an initial period of vigorous activity, adopt a characteristic immobile posture, making only those movements necessary to keep their heads above the water. It was hypothesized that immobility reflected the animals' having learned that escape was impossible and their having given up hope. Immobility was therefore given the name "behavioral despair". Immobility was subsequently found to be reduced by a wide range of clinically active antidepressant drugs. This simple behavioral procedure has since become a useful test for screening novel antidepressants in rats and is presented in this unit. An equivalent procedure in the mouse is also described along with a “dry” version of the test where immobility is induced simply by suspending the mouse by the tail.

UNIT 8.10B The Use of a Triadic Design to Produce Behavioral Learned Helplessness in Rats (Robert C. Drugan, University of New Hampshire, Durham, NH). Certain types of human depression are precipitated by stressful life events, and vulnerable individuals experiencing these stressors may develop clinical depression. Understanding the neurobiology of stress vulnerability (depression) as well as stress resiliency (coping) is critical for guiding the development of novel pharmacotherapeutic agents for stress-related disorders such as depression in humans. The use of a triadic design (escapable shock, yoked-inescapable shock and restrained control) allows the investigator to examine the various sequellae of stress exposure, while manipulating and quantifying the impact of psychological dynamics of stress such as active behavioral coping (i.e., stress control). Both escape and yoked subjects are exposed to the identical amount, intensity, pattern, and duration of stress. The critical distinction between these two groups is that the escape group has the opportunity to terminate the shock stress by turning a wheel at the front of a chamber, while wheel-turning for the yoked subject is of no consequence. Any difference observed between the escape and yoked subjects is a result of the effects of coping, rather than stress exposure per se. The restrained group is included to control for the effects of  handling. Any differences between this group and the escape and yoked subjects reflects the impact of stress per se.

UNIT 8.10C Induction of a “Learned Helplessness Effect” in Mice (Hymie Anisman, Carleton University, Ottawa and Zul Merali, University of Ottawa). Uncontrollable stressors induce a variety of behavioral disturbances that are in many ways reminiscent of the symptoms that characterize clinical depression. These deficits are evident across a range of species, including mice. Given the increasing focus on genetic techniques involving mice to identify the mechanisms subserving these behavioral disturbances (e.g., recombinant, knockout, and transgenic strains), it is of particular interest to provide a detailed description of the method to induce behavioral deficits in response to uncontrollable stressors. This unit describes the procedure used to assess the effects of controllable and uncontrollable shock on subsequent shock-escape performance in mice using an escape-delay procedure.

UNIT 9.7 Experimental Autoimmune Encephalomyelitis (Michael K. Racke, University of Texas–Southwestern Medical Center at Dallas, Tex.). Experimental autoimmune encephalomyelitis (EAE) is the prime animal model for the human disease multiple sclerosis (MS). Many new experimental therapies for MS have initially been tested in the EAE model. With increasing evidence that EAE and MS result in damage to axons in the CNS, the EAE model will become increasingly valuable to neuroscientists as a means of testing new neuroprotective strategies for inflammatory demyelinating diseases.

UNIT 9.8 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.

APPENDIX 4A High-Precision Stereotaxic Surgery in Mice (Jaime Athos and Daniel R. Storm, University of Washington). This unit provides protocols for cannulation and site-specific central microinjection of mice using a recently developed high-precision stereotaxic frame. The construction of cannulae, wire plugs, and injection needles are also described.

UNIT 5.15 Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics In Vivo (Jason A. Kahana, University of California–San Diego, and Pamela A. Silver, Dana Farber Cancer Institute, Boston, MA). Fluorescent 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.

UNIT 5.16 GFP in the Study of Neuronal Signaling Pathways (Leslie Blair, Kendra Bence-Hanulec and John Marshall, Brown University, Providence, RI). In recent years, techniques have been established for transiently cotransfecting cells with cDNA of the jellyfish green fluoresent protein (GFP), a reporter gene that encodes a nontoxic 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.

UNIT 5.17 Analysis of RNA by Northern and Slot Blot Hybridization (Terry Brown, University of Manchester Institute of Science and Technology, United Kingdom, and Karol Mackey, Molecular Research Services, Cincinnati, OH). Specific 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 rehybridized with a different probe.

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