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. 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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. 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Flash fotolisi di composti in gabbia in combinazione con registrazioni patch-clamp è una tecnica utile per ottenere salti rapidi e locali della concentrazione di molecole attive fisiologicamente sia all&#39;interno che all&#39;esterno delle cellule. Diversi tipi di compounds1 in gabbia sono stati sintetizzati, e questa tecnica può essere applicata a diversi tipi di cellule, comprese le cellule in coltura esprimono canali ionici che possono essere attivate o modulata da fotolisi di alcuni dei composti disponibili in ga...

<table border="1"><tbody><tr><th> Attrezzatura </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Adattatore modulo flash della lampada al microscopio </td><td> Rapp optoelettronici </td><td> FlashCube 70 </td><td></td></tr><tr><td> Air tavolo </td><td> TMC </td><td> MICRO-g 63-534 </td><td></td></tr><tr><td> Digitalizzatore </td><td> Axon Instruments </td><td> Digidata 1322A </td><td></td></tr><tr><td> Acquisizione Dati Software </td><td> Axon Instruments </td><td> pClamp 8 </td><td></td></tr><tr><td> Software per l&#39;analisi dei dati </td><td> WaveMetrics </td><td> Igor </td><td></td></tr><tr><td> Specchio per il modulo adattatore </td><td> Rapp optoelettronici </td><td> M70/100 </td><td></td></tr><tr><td> Elettrodo titolare </td><td> Axon Instruments </td><td> 1-HL-U </td><td></td></tr><tr><td> Gabbia di Faraday </td><td> Una soluzione su misura </td><td></td><td></td></tr><tr><td> Cubo filtro </td><td> Olimpo </td><td> U-ULU </td><td> Filtro di eccitazione rimosso </td></tr><tr><td> Flash lampada </td><td> Rapp optoelettronici </td><td> JML-C2 </td><td></td></tr><tr><td> Pinza Dumont # 55 </td><td> Mondo Strumenti di precisione </td><td> 14099 </td><td></td></tr><tr><td> Vetro capillari </td><td> Mondo Strumenti di precisione </td><td> PG10165-4 </td><td></td></tr><tr><td> Con fondo di vetro piatto </td><td> Mondo Strumenti di precisione </td><td> FD35-100 </td><td></td></tr><tr><td> Illuminatore </td><td> Olimpo </td><td> Evidenziare 3100 </td><td></td></tr><tr><td> Microscopio invertito </td><td> Olimpo </td><td> IX70 </td><td></td></tr><tr><td> Micromanipolatori </td><td> Luigs &amp; Neumann </td><td> SM Mi </td><td></td></tr><tr><td> Micropipetta Puller </td><td> Narishige </td><td> PP-830 </td><td></td></tr><tr><td> Controllare </td><td> HesaVision </td><td> MTB-01 </td><td></td></tr><tr><td> I filtri a densità neutra </td><td> Omega Optical </td><td> varia </td><td></td></tr><tr><td> Obiettivo 100X </td><td> Zeiss </td><td> Fluar 440285 </td><td> Sia Zeiss o Olympus </td></tr><tr><td> Obiettivo 100X </td><td> Olimpo </td><td> UPLFLN 100XOI2 </td><td> Sia Zeiss o Olympus </td></tr><tr><td> Ottica UV shortpass filtro </td><td> Rapp optoelettronici </td><td> SP400 </td><td></td></tr><tr><td> Patch-clamp amplificatore </td><td> Axon Instruments </td><td> Axopatch 200B </td><td></td></tr><tr><td> Photo Diode Assembly </td><td> Rapp optoelettronici </td><td> PDA </td><td></td></tr><tr><td> Quarzo luce guida </td><td> Rapp optoelettronici </td><td> varia </td><td> Usiamo 600 micron di diametro </td></tr><tr><td> In filo d&#39;argento </td><td> Mondo Strumenti di precisione </td><td> AGT1025 </td><td></td></tr><tr><td> Argento terreno pellet </td><td> Warner strumenti </td><td> 64-1309 </td><td></td></tr><tr><td> Lampada ad arco allo xeno </td><td> Rapp optoelettronici </td><td> XBL-JML </td><td></td></tr></tbody></table> <table border="1"><tbody><tr><th> Reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> BCMCM-gabbia cAMP </td><td> Biolog </td><td> B016 </td></tr><tr><td> Sieroalbumina bovina (BSA) </td><td> Sigma </td><td> A8806 </td></tr><tr><td> CaCl2 soluzione standard di 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> Cisteina </td><td> Sigma </td><td> C9768 </td></tr><tr><td> Concanavalina Un tipo 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> DNAsi I </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> Glucosio </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> Leupeptina </td><td> Sigma </td><td> L0649 </td></tr><tr><td> MgCl2 </td><td> Fluka </td><td> 63020 </td></tr><tr><td> Papaina </td><td> Sigma </td><td> P3125 </td></tr><tr><td> Poli-L-lisina </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

Fotolisi di composti in gabbia permette la produzione di un rapido aumento e localizzato della concentrazione di vari composti fisiologicamente attive 1. Composti in gabbia sono molecole fatte fisiologicamente inattivo da una gabbia chimico che può essere interrotta da un lampo di luce ultravioletta. Qui mostriamo come ottenere patch-clamp registrazioni combinate con fotolisi di composti in gabbia per lo studio della trasduzione olfattiva in dissociato topo neuroni sensoriali olfattivi. Il processo di trasduzione olfattiva (Figura 1) si svolge nel ciglia di neuroni sensoriali olfattivi, dove odoranti legandosi ai recettori porta all&#39;aumento di cAMP che si apre nucleotidi ciclici (CNG) canali 2. Ingresso di Ca attraverso canali CNG attiva Ca-attivati ​​i canali Cl. Mostriamo come dissociare i neuroni dell&#39;epitelio olfattivo del mouse 3 e come attivare i canali CNG o Ca-attivati ​​i canali Cl da fotolisi di cAMP in gabbia in gabbia Ca 4 o 5 &lt;/ Sup&gt;. Noi usiamo una lampada flash 6,7 per applicare lampeggia ultravioletti alla regione ciliare di uncage cAMP o Ca mentre patch-clamp registrazioni sono prese per misurare la corrente in tutta la cellula-voltage-clamp configurazione 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

Fotolisi di composti in gabbia permette la produzione di un rapido aumento e localizzato della concentrazione di vari composti fisiologicamente attivi. Qui mostriamo come ottenere patch-clamp registrazioni combinate con fotolisi di cAMP in gabbia o in gabbia Ca per lo studio della trasduzione olfattiva in dissociato topo neuroni sensoriali olfattivi.

Fotolisi lampo di composti Caged nel Cilia di neuroni sensoriali olfattivi

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

Research

JoVE Journal

Neuroscience

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

Analizzando le risposte di topo neuroni olfattivi sensoriali che utilizzano l&#39;aria in fase di registrazione Electroolfactogram

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

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Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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