Name: spectral confocal imaging of fluorescently tagged nicotinic receptors in knock-in mice with chronic nicotine administration
Uploaded: 2012-02-10T12:00:00
Description: Watch this Scientific Journal Video about Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration at JoVE.com

Anthony Renda blev støttet af en University of Victoria Graduate Fellowship Award. Denne forskning blev støttet af en Natural Sciences and Engineering Research Council of Canada Discovery Grant, en NARSAD Young Investigator Award (til RN), en Victoria Foundation - Myre og Winifred Sim Fund, en canadisk Institut for Innovation tilskud, en British Columbia Viden Fond og en naturlig Sciences and Engineering Research Council of Canada Research Tools og Instrumentering Grant. Vi takker Jillian McKay, Christina Barnes, Ariel Sullivan, Jennifer MacDonald og Daniel Morgado for fremragende mus dyrehold.

1. Pumpe implantation <ol><li> Før pumpen implantation, fylde og forberede Alzet mini-osmotiske pumper (Alzet, model 2002, Cupertino, USA) er forsigtig med ikke at indføre luftbobler. Denne model af mini-osmotisk pumpe leverer opløsning ved en hastighed på 0,5 gl / time i 14 dage. Sikre sterile forhold. Vejes tomme og fyldte pumper. Ved afslutningen af ​​forsøget (10 dage efter implantation), kan den resterende væske i pumpen fjernes med en sprøjte og nål og vejet til beregning af den pumpes. </li><li> Pum...

<ol>
	<li>Perry, D. C., Davila-Garcia, M. I., Stockmeier, C. A., Kellar, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+nicotinic+receptors+in+brains+from+smokers:+membrane+binding+and+autoradiography+studies.">Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies.</a> J. Pharmacol. Exp. Ther. 289, 1545-1552 (1999).</li><li>Schwartz, R. D., Kellar, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+cholinergic+receptor+binding+sites+in+the+brain:+regulation+in+vivo.">Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo.</a> Science. 220, 214-216 (1983).</li><li>Marks, M. J., Burch, J. B., Collins, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chronic+nicotine+infusion+on+tolerance+development+and+nicotinic+receptors.">Effects of chronic nicotine infusion on tolerance development and nicotinic receptors.</a> J. Pharmacol. Exp. Ther. 226, 817-8125 (1983).</li><li>Carlezon, W. A. J., Nestler, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+levels+of+GluR1+in+the+midbrain:+a+trigger+for+sensitization+to+drugs+of+abuse.">Elevated levels of GluR1 in the midbrain: a trigger for sensitization to drugs of abuse.</a> Trends Neurosci. 25, 610-615 (2002).</li><li>Fitzgerald, L. W., Ortiz, J., Hamedani, A. G., Nestler, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+of+abuse+and+stress+increase+the+expression+of+GluR1+and+NMDAR1+glutamate+receptor+subunits+in+the+rat+ventral+tegmental+area:+common+adaptations+among+cross-sensitizing+agents.">Drugs of abuse and stress increase the expression of GluR1 and NMDAR1 glutamate receptor subunits in the rat ventral tegmental area: common adaptations among cross-sensitizing agents.</a> J. Neurosci. 16, 274-2782 (1996).</li><li>Saal, D., Dong, Y., Bonci, A., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+of+abuse+and+stress+trigger+a+common+synaptic+adaptation+in+dopamine+neurons.">Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons.</a> Neuron. 37, 577-5782 (2003).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Feng, G., Mellor, R. H., Bernstein, M., Keller-Peck, C., Nguyen, Q. T., Wallace, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Livet, J., Weissman, T. A., Kang, H., Draft, R. W., Lu, J., Bennis, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450, 56-62 (2007).</li><li>Nashmi, R., Dickinson, M. E., McKinney, S., Jareb, M., Labarca, C., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+&#945;4&#946;2+nicotinic+acetylcholine+receptors+assessed+with+functional+fluorescently+labeled+subunits:+effects+of+localization,+trafficking,+and+nicotine-induced+upregulation+in+clonal+mammalian+cells+and+in+cultured+midbrain+neurons.">Assembly of &#945;4&#946;2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons.</a> J. Neurosci. 23, 11554-11567 (2003).</li><li>Nashmi, R., Xiao, C., Deshpande, P., McKinney, S., Grady, S. R., Whiteaker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+nicotine+cell+specifically+upregulates+functional+Î±4*+nicotinic+receptors:+basis+for+both+tolerance+in+midbrain+and+enhanced+long-term+potentiation+in+perforant+path.">Chronic nicotine cell specifically upregulates functional Î±4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path.</a> J. Neurosci. 27, 8202-8218 (2007).</li><li>Dickinson, M. E., Bearman, G., Tilie, S., Lansford, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-spectral+imaging+and+linear+unmixing+add+a+whole+new+dimension+to+laser+scanning+fluorescence+microscopy.">Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy.</a> BioTechniques. 31, 1272-1278 (2001).</li><li>Nashmi, R., Fraser, S. E., Lester, H., Dickinson, M. E., Periasamy, A., Day, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular imaging: fret microscopy and spectroscopy. , 180-192 (2005).</li><li>Zimmermann, T., Rietdorf, J., Girod, A., Georget, V., Pepperkok, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+imaging+and+linear+un-mixing+enables+improved+FRET+efficiency+with+a+novel+GFP2-YFP+FRET+pair.">Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair.</a> FEBS Lett. 531, 245-249 (2002).</li><li>Larson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nikon+C1si+combines+high+spectral+resolution,+high+sensitivity,+and+high+acquisition+speed.">The Nikon C1si combines high spectral resolution, high sensitivity, and high acquisition speed.</a> Cytometry A. 69, 825-8234 (2006).</li><li>Melvin, N. R., Sutherland, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+caveats+of+standard+immunohistochemical+procedures:+implications+for+optical+disector-based+designs.">Quantitative caveats of standard immunohistochemical procedures: implications for optical disector-based designs.</a> J. Histochem. Cytochem. 58, 577-5784 (2010).</li><li>Jones, I. W., Wonnacott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+doesn't+nicotinic+ACh+receptor+immunoreactivity+knock+out.">Why doesn't nicotinic ACh receptor immunoreactivity knock out.</a> Trends Neurosci. 28, 343-345 (2005).</li><li>Moser, N., Mechawar, N., Jones, I., Gochberg-Sarver, A., Orr-Urtreger, A., Plomann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+suitability+of+nicotinic+acetylcholine+receptor+antibodies+for+standard+immunodetection+procedures.">Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures.</a> J. Neurochem. , (2007).</li><li>Whiteaker, P., Cooper, J. F., Salminen, O., Marks, M. J., McClure-Begley, T. D., Brown, R. W., Collins, A. C., Lindstrom, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolabeling+demonstrates+the+interdependence+of+mouse+brain+a4+and+b2+nicotinic+acetylcholine+receptor+subunit+expression.">Immunolabeling demonstrates the interdependence of mouse brain a4 and b2 nicotinic acetylcholine receptor subunit expression.</a> The Journal of Comparative Neurology. 499, 1016-1038 (2006).</li><li>Marks, M. J., McClure-Begley, T. D., Whiteaker, P., Salminen, O., Brown, R. W. B., Cooper, J., Collins, A. C., Lindstrom, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+nicotinic+acetylcholine+receptor+protein+underlies+chronic+nicotine-induced+up-regulation+of+nicotinic+agonist+binding+sites+in+mouse+brain.">Increased nicotinic acetylcholine receptor protein underlies chronic nicotine-induced up-regulation of nicotinic agonist binding sites in mouse brain.</a> The Journal of Pharmacology and Experimental Therapeutics. , 337-3187 (2011).</li><li>Marks, M. J., Rowell, P. P., Cao, J. Z., Grady, S. R., McCallum, S. E., Collins, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsets+of+acetylcholine-stimulated+86[Rb]++efflux+and+125[I]-epibatidine+binding+sites+in+C57BL/6+mouse+brain+are+differentially+affected+by+chronic+nicotine+treatment.">Subsets of acetylcholine-stimulated 86[Rb]+ efflux and 125[I]-epibatidine binding sites in C57BL/6 mouse brain are differentially affected by chronic nicotine treatment.</a> Neuropharmacology. 46, 1141-1157 (2004).</li><li>King, S. L., Caldarone, B. J., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta2-subunit-containing+nicotinic+acetylcholine+receptors+are+critical+for+dopamine-dependent+locomotor+activation+following+repeated+nicotine+administration.">Beta2-subunit-containing nicotinic acetylcholine receptors are critical for dopamine-dependent locomotor activation following repeated nicotine administration.</a> Neuropharmacology. 47, 132-139 (2004).</li><li>Robinson, S. F., Marks, M. J., Collins, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbred+mouse+strains+vary+in+oral+self-selection+of+nicotine.">Inbred mouse strains vary in oral self-selection of nicotine.</a> Psychopharmacology (Berl). 124, 332-339 (1996).</li><li>Sparks, J. A., Pauly, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+continuous+oral+nicotine+administration+on+brain+nicotinic+receptors+and+responsiveness+to+nicotine+in+C57Bl/6+mice.">Effects of continuous oral nicotine administration on brain nicotinic receptors and responsiveness to nicotine in C57Bl/6 mice.</a> Psychopharmacology (Berl). , 141-145 (1999).</li><li>Rahman, S., Zhang, J., Engleman, E. A., Corrigall, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroadaptive+changes+in+the+mesoaccumbens+dopamine+system+after+chronic+nicotine+self-administration:+a+microdialysis+study.">Neuroadaptive changes in the mesoaccumbens dopamine system after chronic nicotine self-administration: a microdialysis study.</a> Neuroscience. 129, 415-4124 (2004).</li><li>Picciotto, M. R., Zoli, M., Rimondini, R., Lena, C., Marubio, L. M., Pich, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+containing+the+Î²2+subunit+are+involved+in+the+reinforcing+properties+of+nicotine.">Acetylcholine receptors containing the Î²2 subunit are involved in the reinforcing properties of nicotine.</a> Nature. 391, 173-177 (1998).</li><li>Fowler, C. D., Lu, Q., Johnson, P. M., Marks, M. J., Kenny, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habenular+&#945;5+nicotinic+receptor+subunit+signalling+controls+nicotine+intake.">Habenular &#945;5 nicotinic receptor subunit signalling controls nicotine intake.</a> Nature. 471, 597-601 (2011).</li><li>Maskos, U., Molles, B. E., Pons, S., Besson, M., Guiard, B. P., Guilloux, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+reinforcement+and+cognition+restored+by+targeted+expression+of+nicotinic+receptors.">Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors.</a> Nature. 436, 103-107 (2005).</li><li>Matta, S. G., Balfour, D. J., Benowitz, N. L., Boyd, R. T., Buccafusco, J. J., Caggiula, A. R., Craig, C. R., Collins, A. C., Damaj, M. I., Donny, E. C., Gardiner, P. S., Grady, S. R., Heberlein, U., Leonard, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+nicotine+dose+selection+for+in+vivo+research.">Guidelines on nicotine dose selection for in vivo research.</a> Psychopharmacology. 190, 269-319 (2007).</li><li>Lang, T., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+protein+clusters+at+nanoscale+resolution:+more+than+pretty+pictures.">Membrane protein clusters at nanoscale resolution: more than pretty pictures.</a> Physiology (Bethesda). 25, 116-1124 (2010).</li></ol>

<p class="jove_content"&gt; Anvendelsen af ​​et fluorescerende receptor i en afsmittende i musemodel til at bestemme mængden og lokalisering af en specifik ion-kanal tilvejebringer et antal fordele. I modsætning til proteiner såsom actin, som er ubikvitært udtrykt i alle celler, er ionkanaler stede i meget færre antal og deres ekspression varierer mellem neuronale undertyper gør nøjagtig analyse via traditionelle immunohistokemiske teknikker udfordrende. Den α4YFP genproduktet udtrykkes på WT niveauer, under kontrol af de samme pro...

<table border="1"><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> mini-osmotiske pumper </td><td> Alzet </td><td> model 2002 </td><td></td></tr><tr><td> saltvand </td><td> Teknova </td><td> S5819 </td><td></td></tr><tr><td> (-)-Nikotin hydrogen tartratsalt </td><td> Sigma </td><td> N5260 </td><td></td></tr><tr><td> øjendråber </td><td> Novartis </td><td> Tear-Gel </td><td></td></tr><tr><td> Vetbond lim </td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td> heparin-natriumsalt </td><td> Sigma </td><td> H4784 </td><td></td></tr><tr><td> 10x PBS </td><td> Invitrogen </td><td> 70011 </td><td></td></tr><tr><td> ketamin </td><td> Wyeth Dyresundhed </td><td> 0856-4403-01 </td><td></td></tr><tr><td> medatomidine hydrochlorid </td><td> Pfizer </td><td> 1950673 </td><td></td></tr><tr><td> 23G butterfly nål </td><td> Becton Dickinson </td><td> 367253 </td><td></td></tr><tr><td> paraformaldehyd </td><td> Electron Microscopy Sciences </td><td> 15710 </td><td></td></tr><tr><td> plast indlejring skimmel </td><td> VWR </td><td> 18986-1 </td><td></td></tr><tr><td> Oktober Montering Forbindelse </td><td> Tissue-Tek </td><td> 4583 </td><td></td></tr><tr><td> Mowiol 4-88 </td><td> EMD-Calbiochem </td><td> 475904 </td><td> pH 8,5 </td></tr></table>

spectral confocal imaging of fluorescently tagged nicotinic receptors in knock-in mice with chronic nicotine administration

Ligand-gatede ionkanaler i centralnervesystemet (CNS) er impliceret i talrige betingelser med alvorlige medicinske og sociale følger. For eksempel er afhængighed af nikotin via tobaksrygning en førende årsag til for tidlig død på verdensplan (World Health Organization), og er sandsynligvis forårsaget af en ændring af ion-kanal fordeling i hjernen 1. Kronisk eksponering for nikotin i både gnavere og mennesker resulterer i forøgede antal af nicotinacetylcholinreceptorer (nAChRs) i hjernevæv 1-3. Ligeledes har ændringer i glutamaterge GluN1 eller GluA1 kanaler blevet impliceret i at udløse sensibilisering andre vanedannende stoffer, såsom kokain, amfetamin og opiater 4-6. Følgelig evnen til at kortlægge og kvantificere distribution og ekspressionsmønstre af specifikke ionkanaler er kritisk vigtigt at forstå mekanismerne i afhængighed. Undersøgelsen af ​​hjerne region-specifikke efeffekter af individuelle lægemidler blev fremført ved fremkomsten af ​​teknikker såsom radioaktive ligander. Den lave rumlige opløsning af radioaktiv ligand binding forhindrer muligheden for at kvantificere ligand-gatede ionkanaler i specifikke undertyper af neuroner. Genetisk indkodede fluorescerende reportere, såsom grønt fluorescerende protein (GFP) og mange andre farver, har revolutioneret inden for biologi 7. Ved genetisk mærkning af en fluorescerende reporter til et endogent protein kan man forestille proteiner in vivo 7-10. En fordel ved fluorescens mærkning proteiner med en probe er elimineringen af ​​antistof brug, som har problemer med nonspecificity og tilgængelighed til målproteinet. Vi har brugt denne strategi til fluorescens-mærke nAChRs, som gjorde det muligt for studiet af receptoren samling bruge Förster Resonance Energy Transfer (FRET) i transficerede dyrkede celler 11. Nylig, har vi brugt knock-in tilgang til ingeniør mus med gult fluorescerende protein tagged a4 nAChR underenheder (α4YFP), hvilket muliggør præcis kvantificering af receptoren ex vivo ved submikrometer opløsning i CNS-neuroner via spektral konfokal mikroskopi 12. Den målrettede fluorescerende knock-in mutation er inkorporeret i det endogene locus og under kontrol af dens native promotor, der producerer normale niveauer af ekspression og regulering af receptoren sammenlignet med umærkede receptorer i vildtype-mus. Dette knock-i fremgangsmåde kan udvides til fluorescens mærke andre ionkanaler og giver en kraftig metode til at visualisere og kvantificere receptorer i CNS. I dette papir beskriver vi en metode til at kvantificere ændringer i nAChR udtryk i specifikke CNS-neuroner efter udsættelse for kronisk nikotin. Vores metoder omfatter mini-osmotisk pumpe implantation, intrakardial perfusion fiksering, billedbehandling og analyse af fluorescens mærket nikotinsyre receptor underenheder fra α4YFP knock-i mus (fig. 1). Vi har optimeret fiksering teknikken for at minimere autofluorescens fra fast hjerne tissue.We detaljeret beskrivelse vores billeddannelse metode under anvendelse af en spektral konfokalt mikroskop i forbindelse med en lineær spektral unmixing algoritme til at subtrahere autofluoresent signal for nøjagtigt at opnå α4YFP fluorescenssignal. Endelig viser vi resultatet af kronisk nikotin-fremkaldt opregulering af α4YFP receptorer i den mediale perforante sti i hippocampus.

Watch this Scientific Journal Video about Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration at JoVE.com

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Vi har udviklet en ny teknik til at kvantificere nicotinacetylcholinreceptorneurotransmission ændringer inden for subcellulære regioner af specifikke undertyper af CNS-neuroner til bedre at forstå mekanismerne i nikotintrangen ved hjælp af en kombination af metoder, herunder fluorescerende protein tagging af receptoren ved hjælp af knock-in tilgang og spektral konfokal billeddannelse.

Spektral konfokal afbildning af Fluorescerende mærkede nikotinreceptorer i Knock-i mus med kronisk Nikotin Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Research

JoVE Journal

Neuroscience

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Imaging Calcium respons i GFP-mærkede neuroner i hypothalamus musehjerne Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Inducerende plasticitet astrocytisk receptorer ved manipulation af neuronfyring priser

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Live-Imaging af Nikotin induceret Calcium Signaling og neurotransmitterfrigivelse Langs ventrale Hippocampale Axoner

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

High-Resolution Kvantitativ immunoguld Analyse af membranreceptorer på retinale Ribbon synapser

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Kvantificering af infra-langsom dynamik for spektral kraft og hjertefrekvens i sovende mus

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

Automatiseret ganganalyse hos mus med kronisk konstriktion skade

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

En In Vivo blod - hjerne barrieren permeabilitet Assay i mus ved hjælp af Fluorescently mærket røbestoffer

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Spectral Reflectometric mikroskopi på myelinerede axoner In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Kvantitative cellebiologi af Neurodegeneration i Drosophila gennem saglig analyse af Fluorescently mærket proteiner ved hjælp af ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...