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>

No conflicts of interest declared.

Flash photolysis of caged compounds combined with patch-clamp recordings is a useful technique to obtain rapid and local jumps in the concentration of physiologically active molecules both inside and outside cells. Several types of caged compounds1 have been synthesized, and this technique can be applied to various types of cells, including cultured cells expressing ion channels that can be activated or modulated by photolysis of some of the available caged compounds11.
Photolysis o...

<table border="1">
		<tbody>
 <tr>
 <th>Equipment</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>Adapter module flash lamp to microscope</td>
 <td>Rapp OptoElectronic</td>
 <td>FlashCube 70</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Air table</td>
 <td>TMC </td>
 <td>MICRO-g 63-534</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digitizer</td>
 <td>Axon Instruments </td>
 <td>Digidata 1322A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Data Acquisition Software</td>
 <td>Axon Instruments</td>
 <td>pClamp 8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Data Analysis Software</td>
 <td>WaveMetrics</td>
 <td>Igor</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mirror for adapter module</td>
 <td>Rapp OptoElectronic</td>
 <td>M70/100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electrode holder</td>
 <td>Axon Instruments</td>
 <td>1-HL-U</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Faraday&#x2019;s cage</td>
 <td>Custom made</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Filter cube</td>
 <td>Olympus</td>
 <td>U-MWU</td>
 <td>Excitation filter removed</td>
 </tr>
 <tr>
 <td>Flash lamp</td>
 <td>Rapp OptoElectronic</td>
 <td>JML-C2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Forceps Dumont #55 </td>
 <td>World Precision Instruments</td>
 <td>14099</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass capillaries </td>
 <td>World Precision Instruments</td>
 <td>PG10165-4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass bottom dish</td>
 <td>World Precision Instruments</td>
 <td>FD35-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Illuminator</td>
 <td>Olympus</td>
 <td>Highlight 3100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td>Olympus</td>
 <td>IX70</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micromanipulators </td>
 <td>Luigs &amp; Neumann</td>
 <td>SM I</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micropipette Puller </td>
 <td>Narishige</td>
 <td>PP-830</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Monitor</td>
 <td>HesaVision</td>
 <td>MTB-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Neutral density filters</td>
 <td>Omega Optical</td>
 <td>varies</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Objective 100X</td>
 <td>Zeiss</td>
 <td>Fluar 440285</td>
 <td>Either Zeiss or Olympus </td>
 </tr>
 <tr>
 <td>Objective 100X</td>
 <td>Olympus</td>
 <td>UPLFLN 100XOI2</td>
 <td>Either Zeiss or Olympus</td>
 </tr>
 <tr>
 <td>Optical UV shortpass filter </td>
 <td>Rapp OptoElectronic</td>
 <td>SP400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Patch-clamp amplifier</td>
 <td>Axon Instruments</td>
 <td>Axopatch 200B</td>
 <td>&nbsp;</td>
 </tr>
					 <tr>
						<td>Photo Diode Assembly </td>
						<td>Rapp OptoElectronic</td>
						<td>PDA</td>
						<td>&nbsp;</td>
					 </tr>
 <tr>
 <td>Quartz light guide</td>
 <td>Rapp OptoElectronic</td>
 <td>varies</td>
 <td>We use 600 &mu;m diameter</td>
 </tr>
 <tr>
 <td>Silver wire</td>
 <td>World Precision Instruments</td>
 <td>AGT1025</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Silver ground pellet</td>
 <td>Warner instruments</td>
 <td>64-1309</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Xenon arc lamp</td>
 <td>Rapp OptoElectronic</td>
 <td>XBL-JML</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
 </table> 
 <table border="1">
 <tbody>
 <tr>
 <th>Reagent</th>
 <th>Company</th>
 <th>Catalogue number</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 standard solution 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>Cysteine</td>
 <td>Sigma </td>
 <td>C9768</td>
 </tr>
 <tr>
 <td>Concanavalin A 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 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>Glucose </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-lysine </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

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

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

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

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

flash-photolysis-caged-compounds-cilia-olfactory-sensory

Research

JoVE Journal

Neuroscience

12.7K vues.  International School for Advanced Studies. La photolyse de composés en cage permet la production d'une augmentation rapide et localisé dans la concentration de divers composés physiologiquement actifs. Ici, nous montrons comment obtenir des enregistrements de patch-clamp combinée avec la photolyse de l'AMPc en cage ou en cage Ca pour l'étude de la transduction olfactive chez la souris dissocié neurones sensoriels olfactifs.