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Methods Lecture 3: Molecular Biological techniques Dr Bill Phillips, Dept of Physiology Rm N348 Anderson Stuart Bldg Uses of molecular biology techniques in neuroscience: • Study connections among neurons in the brain and spinal cord • Investigate the role of particular genes in the function of the nervous system • Analyse the molecular structure and function of neurons and glia • Identify changes in gene expression that occur in developmental, adaptive or disease states Tracers for tract tracing- Inject a “tracer” into one brain nucleus or ganglion, it travels along the nerve fibres to label full extent of the nerves • Retrograde tracers: eg HRP (active transport), rhodamine (diffusion) • Anterograde tracers eg radioactive amino acids • Functional pathway tracing- activation of immediate early genes (c-fos) when a nucleus is repetitively activated. • Recombinant viruses- transported retrogradely, trans-synaptically Poly-synaptic tract tracing with Herpes virus • Some strains of Herpes virus are transported anterogradely (fig) others retrogradely • Cross synapses to label 2nd and 3rd order neurons (pontine nucleus & cerebellum here) • Visualise on section by staining for antigen or by histochemical enzyme Kandel,Schwartz and Jessel 2000 Imaging individual living neurons in the brain • During development and during remodelling of synapses neurons change shape, grow and withdraw dendritic spines • Too many neurons packed together to be able to study individuals • Solution? Selectively label just a few using a recombinant virus and Green Fluorescent Protein (GFP) from jellyfish GFP Labelling studies combine molecular techniques with: • Confocal microscopy • High sensitivity low-light fluorescent imaging so cells are not damaged • Developmental and/or behavioural studies Investigate the role of particular genes in the function of the nervous system Genetics Identify Phenotype Reverse genetics Clone suspect gene (say poor motor control) Study Inheritance Inactivate gene by homologous recombination Clone gene responsible and try to relate its function to the pheno type Study Phenotype and try to relate known function of protein to the pheno type Genetic approaches to understanding the role of genes and proteins in the nervous system • identify neurological phenotypes (trait): behavioural, electrophysiological, neuroanatomical • clone the gene based upon the pattern of inheritance of the trait • sequence the gene, identify the protein • infer from the phenotype the function of this protein Examples of forward genetic studies in nervous system • human startle disease- identified as a mutation to the glycine receptor a-subunit • low IQ in boys with Duchenne Muscular Dystrophyhow does lack of dystrophin leads to this phenotype? • Spontaneous neurological mutants in mice such as stargazin. Stargazin mice are ataxic and prone to epilepsy. Stargazin protein expressed in cell lines showed that it is important for delivering and clustering AMPA receptors in the postsynaptic membrane • Chemically induced mutants in Drosophilla and C. elegans Reverse Genetic approaches: clone the gene for the receptor or other synaptic protein then: • Express the gene in cultured cells- to study the function of the protein in neuronal neuronal-like (PC12) or non-neuronal cells • Inactivate candidate gene (“gene knockout” in fly, worm or mouse) • ‘Overexpress’ the gene (transgenic mice, flies, worms etc) Studies of neuronal proteins in cultured cells • Introduce the cDNA encoding the protein into Xenopus oocyte (injection) or “cell line” (transfection) • Study its ion channel properties, protein interactions (adaptor proteins) or transporter function (eg amino acid transporters) • Infer the likely role of the protein in neurons Structure/function studies of ion channels with patch-clamp • • • • Clone channel Make specific mutations to particular amino acids Express wild-type cf mutant in Xenopus oocytes Measure changes in gating, conductance etc with patch clamp recordings of membrane currents Example: Properties of P2X receptor sub-types • New family of channels is cloned ~7 members• what are their channel properties? • how might their presence on a particular neuron modify its excitability • Can we find new drugs that will be selective • Channel permeability, agonist sensitivity, gating kinetics, inactivation Gene knockout in mice • Isolate (clone) “candidate gene” DNA • Mutate gene DNA so that it can’t function • Replace natural gene in chromosomes of embryonic stem cells by homologous recombination in cell culture • Derive mice by injecting modified stem cells into blastocyst stage of embryo • Mate progeny so that mutation becomes homozygous • Examine abnormalities in structure and function of nervous system and infer the normal role of the protein Gene knockout- recent examples • Synaptotagmin I - testing the hypothesis that synaptotagmin is the Ca2+ sensing trigger • Clarifying the role of P2X receptor subtypes • Role of GABAA receptor subunits (a6-, dsubunits) in cerebellar granule cellsinhibition. ‘Overexpress’ the gene in transgenic mice eg NMDA receptor NR2B • NMDA receptor subunit expressed in developing but not adult mice • Might NR2B help make developing brain more adaptable than adult brain? • Overexpressed NR2B using a ‘strong’ promoter • LTP response AND learning ability improved Tang et al. Nature 401, 63 - 69 (1999) © Macmillan Publishers Ltd. Basic molecular methods widely used in neuroscience: • Recombining DNA: PCR (amplify, modify), restriction enzymes (cut) and ligase (paste) DNA • Cloning DNA: E coli, lambda bacteriophage, yeast • Studying where and when mRNA is expressed: in situ hybridization, RT-PCR, Northern blots, Gene arrays • Transgenic mice • Homologous recombination Gene Arrays- flavour of the month • Genome sequencing projects have identified and sequenced most human genes • These sequences are like fingerprints and are easy to match to on Web databases (NCBI USA, ANGIS Australia etc) • Gene arrays/chips allow one to determine changes in expression of mRNA for a wide range of genes simultaneously. • IF properly controlled, may give clues to how expression of certain genes makes neurons behave differently in health and disease. Great care needed with controls.