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PSY2jan 29 psy29090 January 29.docx

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University of Toronto St. George
Junchul Kim

PSY290 January 29, 2013 Methods - how study biological neuroscientist in general - All materials centered around experimental procedure - 5 appraoches and all serve different goals - Staining revealed structure of nerve system - Imaging gross structure and function and activity - Recording – the fine property happening inside neuron - Want to develop hypothesis and test it by manipulating system - Observe what is happening in undisturbed system - To test hypothesis you manipulate system - And after manipulation want to see what type of consequences you generate can do in human and animal Staining - What strcuutre look like - And the layers and nuclei what do all the strucutures look like and how components connected with each other - Without staining see big mess not tell you anything of fine strcuutrue need to prepare tissue with stain and key to understanding the strucuture of neurons and brain and gernal is how you treat specimen Number of methods - Use to visualize cells and connectivity in different levels - Layers of neurons use nissl stain - Other meothod for other things Nissl - Method by german neurologists francis nissl in late 1980s and basically a die use blue or violet and incubate tissue in die solution penetrate ans stain cell body - Nissl stains cell structure contain ribosomes bc so many in they can visualize nuclei and what they look like and layers of neurons – used to look at cluster or layers of neurons and this is cross section of monkey brain and tell cerebral corte made of layer of neurons - Also look in middle see cluster in middle - Different from nissl stain and golgi Golgi - Is structure they lable - Golgi by neuranatomist staining method two chem. Compound and mix them get silver chromate penetrates cells not penetrate all cells only small number of cells work to great advantage bc stain small number help examine them in detail and stain cell and ciusalize it all process in neurons - See cell body and draw 3d digram of neurons - Stains limited number very small Fluroscent protein - Most popular gfp isolated from jelly fish and shine with blue light emit green fluroscent light - Derivative blue colour some yellow based on protein sequence - Naturally occruing some sea coral express gflurose with neon colour different emiision peaks need different wv of light to make them grow need to give energy to emit light - Different from firefly - Need to apply certain wv Gene for gfp - Alter chem. Propert and alter fluroscnt property - Many are modified gfp - And known collectiy as xfp - All diffent ecitation light need and emission light emut - Take some protein and inject in cell and label cell like golgi staining - Do simila thing Xfp introduce - Fill entire extent of neurons – genetically stain neuron at similar level - Similar details of golgi stain - Bc protein genetically encodable engineer genes into organisms the genome wusing transgenic approach – inject into egg and injected dna intergrated to genome and produce this protein Brainbow - Lable different neurons with different colour – indi neurons labeled have different amount of fluorescent protein some high levels some low levels – mixing paint - Allow random mixing of genetic protein - Advantage track connection – if all same colour axons intermingle not distinguish labeled different colour follow different neurons - All axon bundle on gfp – huge benefit labeling neurons with fluroscent protein learn about connectivity Tracing - Studying connection between neurons - Work by communicating with each other - Circuit is neuron and their connections - Circuits contribute to a system that serve a finction – heiracchail organization - Understand structure and connection before finction - Most bio systems structure determines function - Anterograde and retrograde Tracing - Identify projection and terminal field - Were axons end up which brain regions receive inervation of neuron of interest - Red neuron know cell body located in layer of cerebral cortex - Where psot synaptic partners are located inject molecule anterograde tracer molecule red die - Uptaken by cell body and uptaken molecules transported along axon and label all aonal process - Know where axon project - What ant tracers do label axons and axon terminals - Know If axon or axon terminal they look different – know tracer signal is labeling axon terminal - Pha and wga both plant protein and popular ones – transported along axons and label synaptic terminals Retro - Opp thing - Identify neurons that give rise to projection to target of interest - Receives input – want to know which neurons send axonons sends input to the target area - Tracers up by cell terminal not by cell bidy taken to cell body or soma from terminal - Then prepare thin section - Retro – toxin b chain most pop and flurogold - Ant visualize axon cell body intake - Rret neuron terminal and back to cell soma and lable cell body reveals connectivity between brain regions Not neuron or structure but detecting protein and gene Genes – mrna Immu - Use molecule antibody to detect protein - Anti body y shaped protein - Light and heavy chain small is light long heavy - Constant region shared between antibodies amino acids and variable different amino acids and differntitrates antibody - Constant regions similar or same - Different variable region - Different antigen or molecule - Antibody specific to that protein Staining - Label antibody with some type of probe you can visualize chem. Labels added to heavy chain that can readily detect glow when apply light - Many chemical labels people use - Organic flur chem. Added to heavy chain - Thin brain section think contain protein of you interest and coapter of glass and overly solution containing anitobody and is labeled with chemical probe - Enough time ab in solution antibody find antigen and stay there even when wash away non specific binding then look at microcope and see protein of interest - Bc detect antigen by lableling antibody this is direct methods Direct method - Only dop neuron express certain enzyme - Find antibody specific to enzyme and use method to lable dopamingeric neuron use directly lableling primary antibody - Primary ab - Signal you gain Is weak bc signal intrnsity is correlated with amount of antigen in target cell Indirect method - Primary antibody sees antibody directly - Prim ab recognize antigen and secondary antibody recognize primary antibody - Direct one to one indirect more then one to one – ab to protein either directly by lableling primary or two different types one to ligand and one primary antibody Genes In situ - Detect molecule mrna - Need know mrna and why detect - Know proteins are made in cells How proteins are made in cells - Are genetic information is carried in dna in nucleotide sequence - First sequence to rna called transpction dna to rna – polymerase caal - Rna then to protein this is translation - Infor from dna to rna to protein is dogma of biology Double dna transcriped to mrna and then to amino acids Why detect mrna is bc presence garunttess prescen of protein but not always* - good measure of protein location Detecting mrna in cell tell those cells contain corresponding protein which neurons produce which protein Mrna through transcription and detect this is specific way Need to make strand of nuclei acid bind to mrna sequence through complimentary base pairs sequence Prepare comp sequence base pair to that strand Usually rna strand called riboprobe can locate mrna of interest Tag the rna probe with something you can readily detect and incubate with brain tissue and this riboprobe will find target rna sequence – Ribopobe – red signal rep neurons express endorphins – w/o using antibody still find where proteins found in brain Allen brain atlas - Coordinated utilze technique in situ - Riboprobe for every gene and develop database people share - See where genes expressed in brain - See strcuteure at differne levels - Clusters and layers by suing dies - And indi cells using dies and flur protein - And method connection brain region - Method detect protein and gene Brain imaging - Strucurer and function larger scale however - Study structure and function of living brain Xray - Simplest form of brain imaging - Shot spec with xray and record whatever xray pass through need device shot xray and another capture xray signal and records it on other side - Xray to object some will pass and absorb diff strcture in brain absorb different amounts of axray and reveal anatomical strcuutre of specime - Limitation xray technique cannot distinguish vertically – flattened image – 2d image not three d image Ct - Xray scanning much higher spatial resol - Recording device and xray emiting device rotate around specimen - In cyclinder and have xray sorce and detect and both rotate around specimen head and capturing xrays at different angles as result many angles and combine to reconstruct 3d image and ct can distinguish vertically at z plane - Use xray difference in machine collect data Mri - Higher spatial resolution - Mri use phenom h atoms of water - Apply strong magnetic force h act like magnet and aligh them apply strong magnetic field with mag force movement is random cant predict orientation string magnet align - When h align - Use aligned state to generate enrgy – apply energy wave to sample and uses radio frequency high energy hit aligned h molecule h tilt alittle bit whilst spinning when go back emit energy - Fmri detect small amount of energy emitted by h - Those signals all differ in differat areas of brain and tissue density major factor of signal internsity - Using difference can generate image of brain strcuutre - This is structure no function Pet and fmri for brain activity Pet - Radioactive substance - Use glucose derivative - Glusocse main form of energy – uptake glucose in blood stream get to cells receptor in cell recognize glucose and uptake them and broken down and make energy - Glucose main soruce cells utlize - Picky back system label glucose radioactive substance - Reach brain and uptaken by cells and label them - Trick active regions use more energy and take more glucose molecules – more activiated labeld more densly – detecting density of radio label glucose estimate activy of different brain regions - Big disadvatge not reveal strucute not fine resolution xray or mri scan provide Fmri - Idea that increased neural activity increase oxy blood flow to that area more active brain region use more oxygen - Technqiue utlize property of oxy blood - Oxy and dexoy blood show difference – apply magnetic field and see difference can estimate which brain regio
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