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 fotólisis de compuestos enjaulados en combinación con patch-clamp grabaciones es una técnica útil para obtener saltos rápidos y locales en la concentración de las moléculas fisiológicamente activas, tanto dentro como fuera de las células. Existen varios tipos de compounds1 enjaulados han sido sintetizados, y esta técnica se puede aplicar a diferentes tipos de células, incluyendo células cultivadas que expresan canales iónicos que pueden ser activadas o modulada por fotólisis de algunos de los compues...

<table border="1"><tbody><tr><th> Equipo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Adaptador de módulo de lámpara de flash a microscopio </td><td> Rapp optoelectrónica </td><td> FlashCube 70 </td><td></td></tr><tr><td> Aire mesa </td><td> TMC </td><td> MICRO-63-534 g </td><td></td></tr><tr><td> Digitalizador </td><td> Axon Instruments </td><td> Digidata 1322A </td><td></td></tr><tr><td> Software de Adquisición de Datos </td><td> Axon Instruments </td><td> pCLAMP 8 </td><td></td></tr><tr><td> Software de análisis de datos </td><td> WaveMetrics </td><td> Igor </td><td></td></tr><tr><td> Espejo para el módulo de adaptador </td><td> Rapp optoelectrónica </td><td> M70/100 </td><td></td></tr><tr><td> Portaelectrodos </td><td> Axon Instruments </td><td> 1-HL-U </td><td></td></tr><tr><td> Jaula de Faraday </td><td> Una medida que se </td><td></td><td></td></tr><tr><td> Cubo de filtro </td><td> Olimpo </td><td> U-UTH </td><td> Filtro de excitación eliminado </td></tr><tr><td> Lámpara de flash </td><td> Rapp optoelectrónica </td><td> JML-C2 </td><td></td></tr><tr><td> Pinzas Dumont # 55 </td><td> Mundial de Instrumentos de Precisión </td><td> 14099 </td><td></td></tr><tr><td> Capilares de vidrio </td><td> Mundial de Instrumentos de Precisión </td><td> PG10165-4 </td><td></td></tr><tr><td> Vidrio placa inferior </td><td> Mundial de Instrumentos de Precisión </td><td> FD35-100 </td><td></td></tr><tr><td> Iluminador </td><td> Olimpo </td><td> Resalte 3100 </td><td></td></tr><tr><td> Microscopio invertido </td><td> Olimpo </td><td> IX70 </td><td></td></tr><tr><td> Micromanipuladores </td><td> Luigs y Neumann </td><td> SM I </td><td></td></tr><tr><td> Micropipeta Extractor </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> Filtros de densidad neutra </td><td> Omega Optical </td><td> varía </td><td></td></tr><tr><td> Objetivo 100X </td><td> Zeiss </td><td> Fluar 440285 </td><td> Ya sea Zeiss o Olympus </td></tr><tr><td> Objetivo 100X </td><td> Olimpo </td><td> UPLFLN 100XOI2 </td><td> Ya sea Zeiss o Olympus </td></tr><tr><td> Óptica UV filtro paso corto </td><td> Rapp optoelectrónica </td><td> SP400 </td><td></td></tr><tr><td> Patch-clamp amplificador </td><td> Axon Instruments </td><td> Axopatch 200B </td><td></td></tr><tr><td> Foto Asamblea Diodo </td><td> Rapp optoelectrónica </td><td> PDA </td><td></td></tr><tr><td> Guía de luz de cuarzo </td><td> Rapp optoelectrónica </td><td> varía </td><td> Nosotros utilizamos 600 m de diámetro </td></tr><tr><td> Alambre de plata </td><td> Mundial de Instrumentos de Precisión </td><td> AGT1025 </td><td></td></tr><tr><td> Plata baja de pellets </td><td> Warner instrumentos </td><td> 64-1309 </td><td></td></tr><tr><td> Lámpara de arco de xenón </td><td> Rapp optoelectrónica </td><td> XBL-JML </td><td></td></tr></tbody></table> <table border="1"><tbody><tr><th> Reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> BCMCM-jaula AMPc </td><td> Biolog </td><td> B016 </td></tr><tr><td> Albúmina de suero bovino (BSA) </td><td> Sigma </td><td> A8806 </td></tr><tr><td> CaCl2 solución estándar de 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> Cisteína </td><td> Sigma </td><td> C9768 </td></tr><tr><td> Concanavalina A de 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> DNAsa 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> Glucosa </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> Papaína </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

Fotólisis de compuestos enjaulados permite la producción de aumentos rápidos y localizados en la concentración de diversos compuestos fisiológicamente activos 1. Compuestos enjaulados son moléculas hechas fisiológicamente inactivo por una jaula de sustancia química que puede ser rota por un destello de luz ultravioleta. Aquí, se muestra cómo obtener el patch-clamp grabaciones en combinación con la fotólisis de compuestos enjaulados para el estudio de la transducción olfativa en disociada del ratón neuronas sensoriales olfativas. El proceso de transducción olfativa (Figura 1) lleva a cabo en los cilios de las neuronas sensoriales olfativas, que une a los receptores odorantes conduce al aumento de AMPc que se abre de nucleótidos cíclicos-dependientes (GNC) 2 canales. Entrada de Ca a través de los canales de GNC activa Ca-Cl canales activados. Mostramos cómo disociar las neuronas del epitelio olfatorio de ratón 3 y la forma de activar los canales de GNC o Ca-Cl canales activados por fotólisis de AMPc jaula de 4 o 5 jaulas Ca &lt;/ Sup&gt;. Nosotros utilizamos una lámpara de flash 6,7 aplicar destellos ultravioleta a la región ciliar a desenjaular cAMP o Ca, mientras patch-clamp grabaciones se toman para medir la corriente en el conjunto de células voltaje-clamp configuración 08/11.

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

Fotólisis de compuestos enjaulados permite la producción de aumentos rápidos y localizados en la concentración de diversos compuestos fisiológicamente activos. Aquí, se muestra cómo obtener el patch-clamp grabaciones en combinación con la fotólisis de AMPc jaula o jaulas Ca para el estudio de la transducción olfativa en disociada del ratón neuronas sensoriales olfativas.

Flash fotólisis de compuestos enjaulados en los cilios de las neuronas sensoriales olfativas

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

Research

JoVE Journal

Neuroscience

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

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording (Video) | JoVE

Las respuestas de los análisis de las neuronas sensoriales olfativas ratón Utilizando la grabación Electroolfactogram Aire fase

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

Investigación

Neurociencias

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Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons (Video) | JoVE

En vivo de imágenes de la translocación de PKC en células Sf9 y en las neuronas sensoriales Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo (Video) | JoVE

Lentivirus mediada por la manipulación genética y la visualización de las neuronas sensoriales olfativas In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis (Video) | JoVE

Intracelular de grabación, Sensorial Asignación de campos, y el cultivo de neuronas identificadas en el Leech, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna (Video) | JoVE

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el<em&gt; Drosophila</em&gt; Antena

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

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice (Video) | JoVE

Todo el montaje de etiquetado de los cilios en el sistema olfativo principal de ratones

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor (Video) | JoVE

Perforado grabación de patch-clamp de ratón neuronas sensoriales olfativas en neuroepitelio Intacto: Análisis funcional de las neuronas que expresan un odorante del receptor Identificado

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain (Video) | JoVE

Imágenes en vivo de la ependimarios cilios en los ventrículos laterales del cerebro del ratón

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer (Video) | JoVE

Comportamientos olfativas ensayó mediante seguimiento informático de<em&gt; Drosophila</em&gt; En un olfatómetro de cuatro cuadrantes

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana (Video) | JoVE

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The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...