1. Instrumentation 
<ol>
<li>To measure the response of olfactory sensory neurons to photolysis of caged compounds we use a flash lamp in combination with a typical patch-clamp recording system including: a patch-clamp amplifier, a recording electrode and a reference electrode connected to the head-stage of a patch-clamp amplifier, a digitizer, a computer, software for data acquisition, micro-manipulators, an epifluorescence microscope, a perfusion system, an anti-vibration table and a Faraday cage (Figure 2).</li>
<li...

<ol>
	<li>Ellis-Davies, G. C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caged+compounds:+photorelease+technology+for+control+of+cellular+chemistry+and+physiology.">Caged compounds: photorelease technology for control of cellular chemistry and physiology.</a> Nat. Methods. 4, 619-628 (2007).</li><li>Pifferi, S., Boccaccio, A., Menini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+nucleotide-gated+ion+channels+in+sensory+transduction.">Cyclic nucleotide-gated ion channels in sensory transduction.</a> FEBS Lett. 580, 2853-2859 (2006).</li><li>Bozza, T. C., Kauer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+response+properties+of+convergent+olfactory+receptor+neurons.">Odorant response properties of convergent olfactory receptor neurons.</a> J. Neurosci. 18, 4560-4569 (1998).</li><li>Hagen, V., Bendig, J., Frings, S., Eckardt, T., Helm, S., Reuter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Efficient+and+Ultrafast+Phototriggers+for+cAMP+and+cGMP+by+Using+Long-Wavelength+UV/Vis-Activation.">Highly Efficient and Ultrafast Phototriggers for cAMP and cGMP by Using Long-Wavelength UV/Vis-Activation.</a> Angew. Chem. Int. Ed. Engl. 40, 1045-1048 (2001).</li><li>Kaplan, J. H., Ellis-Davies, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolabile+chelators+for+the+rapid+photorelease+of+divalent+cations.">Photolabile chelators for the rapid photorelease of divalent cations.</a> Proc. Natl. Acad. Sci. 85, 6571-6575 (1988).</li><li>Rapp, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+lamp-based+irradiation+of+caged+compounds.">Flash lamp-based irradiation of caged compounds.</a> Methods. Enzymol. 291, 202-222 (1998).</li><li>Gurney, A. M., Montenegro, I., Queiros, M. A., Daschbach, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+photolysis+of+caged+compounds.">Flash photolysis of caged compounds.</a> Microelectrodes: Theory and Applications. , (1991).</li><li>Lagostena, L., Menini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+recordings+and+photolysis+of+caged+compounds+in+olfactory+sensory+neurons+isolated+from+the+mouse.">Whole-cell recordings and photolysis of caged compounds in olfactory sensory neurons isolated from the mouse.</a> Chem. Senses. 28, 705-716 (2003).</li><li>Boccaccio, A., Lagostena, L., Hagen, V., Menini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+adaptation+in+mouse+olfactory+sensory+neurons+does+not+require+the+activity+of+phosphodiesterase.">Fast adaptation in mouse olfactory sensory neurons does not require the activity of phosphodiesterase.</a> J. Gen. Physiol. 128, 171-184 (2006).</li><li>Boccaccio, A., Menini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+development+of+cyclic+nucleotide-gated+and+Ca2++-activated+Cl-+currents+in+isolated+mouse+olfactory+sensory+neurons.">Temporal development of cyclic nucleotide-gated and Ca2+ -activated Cl- currents in isolated mouse olfactory sensory neurons.</a> J. Neurophysiol. 98, 153-160 (2007).</li><li>Sagheddu, C., Boccaccio, A., Dibattista, M., Montani, G., Tirindelli, R., Menini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+concentration+jumps+reveal+dynamic+ion+selectivity+of+calcium-activated+chloride+currents+in+mouse+olfactory+sensory+neurons+and+TMEM16B-transfected+HEK+293T+cells.">Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16B-transfected HEK 293T cells.</a> J. Physiol. 588, 4189-4204 (2010).</li><li>Balana, B., Taylor, N., Slesinger, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+and+Functional+Analysis+of+Ion+Channels+Heterologously+Expressed+in+Mammalian+Cells.">Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells.</a> J. Vis. Exp. (44), e2189-e2189 (2010).</li><li>Cygnar, K. D., Stephan, A. B., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+Responses+of+Mouse+Olfactory+Sensory+Neurons+Using+the+Air-phase+Electroolfactogram+Recording.">Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording.</a> J. Vis. Exp. (37), e1850-e1850 (2010).</li><li>Bernardinelli, Y., Haeberli, C., Chatton, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+photolysis+using+a+light+emitting+diode:+an+efficient,+compact,+and+affordable+solution.">Flash photolysis using a light emitting diode: an efficient, compact, and affordable solution.</a> Cell. Calcium. 37, 565-572 (2005).</li></ol>

Flash fotolyse av caged forbindelser kombinert med patch-clamp opptak er en nyttig teknikk for å oppnå raske og lokale hopper i konsentrasjonen av fysiologisk aktive molekyler både innenfor og utenfor cellene. Flere typer bur compounds1 har blitt syntetisert, og denne teknikken kan brukes til ulike typer celler, inkludert dyrkede celler uttrykker ionekanaler som kan aktiveres eller modulert av fotolyse av noen av de tilgjengelige bur forbindelser 11. Fotolyse av caged forbindel...

<table border="1"><tbody><tr><th> Utstyr </th><th> Firma </th><th> Katalognummer </th><th> Kommentarer </th></tr><tr><td> Adapter modul flash lampe til mikroskop </td><td> Rapp Optoelektronisk </td><td> FlashCube 70 </td><td></td></tr><tr><td> Air table </td><td> TMC </td><td> MICRO-g 63-534 </td><td></td></tr><tr><td> Digitizer </td><td> Axon Instrumenter </td><td> Digidata 1322A </td><td></td></tr><tr><td> Datainnsamling Software </td><td> Axon Instrumenter </td><td> pClamp 8 </td><td></td></tr><tr><td> Data Analysis Software </td><td> WaveMetrics </td><td> Igor </td><td></td></tr><tr><td> Speil for adapter modul </td><td> Rapp Optoelektronisk </td><td> M70/100 </td><td></td></tr><tr><td> Elektrode holder </td><td> Axon Instrumenter </td><td> 1-HL-U </td><td></td></tr><tr><td> Faradays bur </td><td> Skreddersydde </td><td></td><td></td></tr><tr><td> Filter kube </td><td> Olympus </td><td> U-MWU </td><td> Eksitasjon filter fjernet </td></tr><tr><td> Flash lampe </td><td> Rapp Optoelektronisk </td><td> JML-C2 </td><td></td></tr><tr><td> Forceps Dumont # 55 </td><td> Verden presisjonsinstrumenter </td><td> 14099 </td><td></td></tr><tr><td> Glass kapillærer </td><td> Verden presisjonsinstrumenter </td><td> PG10165-4 </td><td></td></tr><tr><td> Glassbunn rett </td><td> Verden presisjonsinstrumenter </td><td> FD35-100 </td><td></td></tr><tr><td> Illuminator </td><td> Olympus </td><td> Highlight 3100 </td><td></td></tr><tr><td> Invertert mikroskop </td><td> Olympus </td><td> IX70 </td><td></td></tr><tr><td> Micromanipulators </td><td> Luigs &amp; Neumann </td><td> SM jeg </td><td></td></tr><tr><td> Mikropipette Puller </td><td> Narishige </td><td> PP-830 </td><td></td></tr><tr><td> Monitor </td><td> HesaVision </td><td> MTB-01 </td><td></td></tr><tr><td> Neutral density filter </td><td> Omega Optisk </td><td> varierer </td><td></td></tr><tr><td> Mål 100X </td><td> Zeiss </td><td> Fluar 440285 </td><td> Enten Zeiss eller Olympuer </td></tr><tr><td> Mål 100X </td><td> Olympus </td><td> UPLFLN 100XOI2 </td><td> Enten Zeiss eller Olympus </td></tr><tr><td> Optisk UV shortpass filter </td><td> Rapp Optoelektronisk </td><td> SP400 </td><td></td></tr><tr><td> Patch-clamp forsterker </td><td> Axon Instrumenter </td><td> Axopatch 200B </td><td></td></tr><tr><td> Photo Diode Assembly </td><td> Rapp Optoelektronisk </td><td> PDA </td><td></td></tr><tr><td> Quartz lysleder </td><td> Rapp Optoelektronisk </td><td> varierer </td><td> Vi bruker 600 mikrometer diameter </td></tr><tr><td> Silver ledning </td><td> Verden presisjonsinstrumenter </td><td> AGT1025 </td><td></td></tr><tr><td> Silver bakken pellet </td><td> Warner instrumenter </td><td> 64-1309 </td><td></td></tr><tr><td> Xenon arc lampe </td><td> Rapp Optoelektronisk </td><td> XBL-JML </td><td></td></tr></tbody></table> <table border="1"><tbody><tr><th> Reagens </th><th> Firma </th><th> Katalognummer </th></tr><tr><td> BCMCM-caged cAMP </td><td> BioLog </td><td> B016 </td></tr><tr><td> Bovine serum albumin (BSA) </td><td> Sigma </td><td> A8806 </td></tr><tr><td> CaCl2 standardløsning 0,1 M </td><td> Fluka </td><td> 21059 </td></tr><tr><td> Caged Ca: DMNP-EDTA </td><td> Invitrogen </td><td> D6814 </td></tr><tr><td> Cystein </td><td> Sigma </td><td> C9768 </td></tr><tr><td> Concanavalin En type V (Cona) </td><td> Sigma </td><td> C7275 </td></tr><tr><td> CsCl </td><td> Sigma </td><td> C4036 </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D8418 </td></tr><tr><td> DNAse jeg </td><td> Sigma </td><td> D4527 </td></tr><tr><td> EDTA </td><td> Sigma </td><td> E9884 </td></tr><tr><td> EGTA </td><td> Sigma </td><td> E4378 </td></tr><tr><td> Glukose </td><td> Sigma </td><td> G5767 </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3375 </td></tr><tr><td> KCl </td><td> Sigma </td><td> P3911 </td></tr><tr><td> KOH </td><td> Sigma </td><td> P1767 </td></tr><tr><td> Leupeptin </td><td> Sigma </td><td> L0649 </td></tr><tr><td> MgCl2 </td><td> Fluka </td><td> 63020 </td></tr><tr><td> Papain </td><td> Sigma </td><td> P3125 </td></tr><tr><td> Poly-L-lysin </td><td> Sigma </td><td> P1274 </td></tr><tr><td> NaCl </td><td> Sigma </td><td> S9888 </td></tr><tr><td> NaOH </td><td> Sigma </td><td> S5881 </td></tr><tr><td> NaPyruvate </td><td> Sigma </td><td> P2256 </td></tr></tbody></table>

flash photolysis of caged compounds in the cilia of olfactory sensory  neurons

Fotolyse av caged forbindelser gjør at produksjonen av raske og lokalisert økning i konsentrasjonen av ulike fysiologisk aktive forbindelser en. Caged forbindelser er molekyler laget fysiologisk inaktive ved en kjemisk bur som kan brytes av en flash av ultrafiolett lys. Her viser vi hvordan du anskaffer patch-clamp opptak kombinert med fotolyse av caged forbindelser for studiet av olfactory transduksjon i dissosiert mus olfactory sensoriske nevroner. Prosessen med olfactory transduksjon (Figur 1) foregår i flimmerhårene av olfactory sensoriske nevroner, hvor odorant binding til reseptorer fører til økning av cAMP som åpner syklisk nukleotid-gated (CNG) kanaler 2. Ca oppføringen gjennom CNG kanaler aktiverer Ca-aktivert Cl kanaler. Vi viser hvordan man skal distansere neuroner fra musen olfactory epitelet 3 og hvordan du kan aktivere CNG kanaler eller Ca-aktivert Cl kanaler ved fotolyse av caged cAMP 4 eller bur Ca 5 &lt;/ Sup&gt;. Vi bruker en flash-lampe 6,7 til å søke ultrafiolett blinker til ciliary regionen for å uncage cAMP eller Ca mens patch-clamp opptakene er tatt for å måle strømmen i hele-cellers spenning-klemme konfigurasjon 8-11.

Watch this Scientific Journal Video about Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons at JoVE.com

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons (Video) | JoVE

Fotolyse av caged forbindelser gjør at produksjonen av raske og lokalisert økning i konsentrasjonen av ulike fysiologisk aktive forbindelser. Her viser vi hvordan du anskaffer patch-clamp opptak kombinert med fotolyse av caged cAMP eller bur Ca for studiet av olfactory transduksjon i dissosiert mus olfactory sensoriske nevroner.

Flash Fotolyse av Caged forbindelser i Cilia av olfactory sensoriske nevroner

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

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Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

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Hele Mount Merking av Cilia i hovedluktsystemet av Mus

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