Supportato da NEI concedere EY014850.

1. Preparazione di piatti Sylgard coperto, camere sperimentali, e lame di rasoio <ol><li> Falcon 35 millimetri Petri rivestito con elastomero Sylgard sono necessari per il corretto taglio di una retina isolata per ottenere cellule fotorecettori singolo. L&#39;elastomero è preparato secondo le istruzioni del fornitore e una piccola quantità si riversa in ogni piatto per coprire il fondo con uno strato. Sostituire il piatto dopo il rivestimento copre e li memorizza. In pochi giorni &#39;l&#39;elastomero indurisce ei piatti sono pronti. </li><li> Fotorecettori isolato necessità di attenersi al fondo della camera sperimentale, in modo che siano immobilizzati durante il corso di un esperimento di imaging. Questo risultato è ottenuto rivestendo la fondali delle camere con poli-L-lisina o poli-L-ornitina. Aggiungere 200 ml di soluzione 0,01% di uno dei due per ogni camera, e coprire le camere con un tovagliolo di carta per proteggerli dalla polvere. Dopo che la soluzione si è asciugata, lavare le camere con acqua distillata e conservare in una scatola chiusa. Usare entro 2 settimane. </li><li> Per la pulizia delle camere alla fine di un esperimento, lavarli con un 100% di etanolo per rimuovere l&#39;olio dal petrolio-immersion lente e detriti cellulari. Per rimuovere i detriti cellulari, utilizzare punte di cotone e con attenzione macchia il fondo della camera. In seguito, lavare con acqua distillata e lasciare asciugare le camere prima di ri-verniciatura. </li><li> Taglio a doppio taglio lame di rasoio in piccoli pezzi (8 da una lama) con una fresa in metallo. </li></ol> 2. Preparazione delle soluzioni <ol><li> La composizione delle soluzioni dipende dalla specie. Per gli anfibi, il Ringer ha (in mmol / L): 110 NaCl, 2.5 KCl, 1.6 MgCl 2, 1 CaCl 2, 5 HEPES, pH = 7.55. Il pH deve essere regolato al valore finale con NaOH. Per i mammiferi, il Ringer ha (in mmol / L): 130 NaCl, 5 KCl, 0,5 MgCl 2, 2 CaCl 2, 25 hemisodium-HEPES, pH = 7.40. La soluzione di Ringer può essere tenuto ben chiuso a temperatura ambiente per un paio di mesi. </li><li> Soluzione di glucosio, 1 mol / L, conservato a -20 ° C per evitare la crescita batterica. </li><li> Il giorno dell&#39;esperimento, aggiungere il glucosio per la soluzione di Ringer ad una concentrazione finale di 5 mmol / L. Alla fine della giornata, scartare il Ringer contenente glucosio, perché possa crescere batteri. </li></ol> 3. Isolamento di retine <ol><li> Per la corretta asportazione di una retina, è importante che l&#39;animale sia adattata al buio per almeno 2-3 ore prima del sacrificio. Gli animali devono essere adattata al buio in un contenitore adatto ventilata in camera oscura. </li><li> Riempire due piastre di Petri 35 millimetri a metà strada con la soluzione di Ringer. </li><li> Sacrificare l&#39;animale sotto fioca luce rossa e rimuovere gli occhi. Successivamente, tutte le procedure sono effettuate sotto la luce a infrarossi utilizzando un microscopio da dissezione o una macchina fotografica con un monitor video. </li><li> Rimuovere eventuali residui di tessuto dalla superficie esterna dell&#39;occhio. Tagliare e rimuovere la parte anteriore, poi trasferire l&#39;oculare in uno dei piatti pieni di Petri Ringer. </li><li> Rimuovere il vitreo e la retina con attenzione separato dal resto della conchiglia oculare pizzicando off o tagliando gli eventuali allegati. Sollevare delicatamente la retina e separarlo pienamente il paraocchi. </li><li> Trasferire la retina per il secondo piatto di Petri con una pipetta di trasferimento di plastica. Tenere il piatto contenente la retina in una scatola a tenuta di luce. </li></ol> 4. Isolamento dei fotorecettori singolo <ol><li> Tutte le procedure sono effettuate sotto la luce a infrarossi. Tagliare un piccolo pezzo di retina e trasferire con una pipetta di plastica ad un Sylgard coperto piatto. Il volume della soluzione contenente il pezzo retina dovrebbe essere di circa 250 microlitri. </li><li> Prendete un piccolo pezzo di lama di un rasoio con il supporto della lama - il filo della lama deve essere di circa 45 ° angolo al titolare. Appiattire il pezzo di retina sullo strato Sylgard e usando la lama tagliare il pezzo di retina finemente mantenendolo attaccato allo strato Sylgard. </li><li> Trasferire 200 microlitri della soluzione contenente le cellule ad una camera sperimentale - lasciare alcun pezzo rimanente della retina nella Sylgard coperta di piatto. Tenere la camera con le cellule isolate in una scatola a tenuta di luce. </li><li> Attendere 10 minuti per le cellule di risolvere, quindi aggiungere 2-3 ml di Ringer. In questa fase, le cellule isolate può essere caricata con un particolare colorante fluorescente (per esempio Fura-2) secondo il protocollo di carico colorante. </li><li> Le cellule possono ora essere adottate per la fase di microscopio per gli esperimenti. </li></ol> 5. Fluorescenza di imaging <ol><li> Trasferimento della camera sul palco del microscopio in epifluorescenza. Regolare perfusione soluzione, sonde di temperatura, illuminazione a infrarossi, ecc </li><li> Chiudere le tende e iniziare a sperimentare. Accendere la luce a infrarossi all&#39;interno della gabbia microscopio, si concentrano sul fondo della camera sperimentale, e spostare il palco in cerca di cellule. </li></ol> 6. Rappresentante dei risultatis: Fig. 1 mostra la morfologia del sano asta isolati e fotorecettori coni ottenuti con questo protocollo da un (Ambystoma tigrinum) retina salamandra. Cellule salamandra sono stati ampiamente utilizzati per gli studi di imaging singola cellula fluorescenza a causa delle loro grandi dimensioni e la loro capacità di sopravvivere per parecchie ore dopo l&#39;isolamento dalla retina. Inoltre, da una retina salamandra si può ottenere sia regolarmente bastone e fotorecettori cono. Un criterio importante per la salute delle cellule è la presenza di un ellissoide intatta (Fig. 1), la parte della cellula dove sono concentrati i mitocondri. Quando le cellule sono visti sotto l&#39;ottica DAPI, questa concentrazione di mitocondri dà un forte segnale di fluorescenza (Fig. 2) a causa della presenza di NADH. Mancanza di un ellissoide intatto è il segno di una cellula danneggiata, generalmente inadatti per esperimento. Fig. 3 mostra una danneggiata salamandra asta fotorecettore, con un corpo cellulare gonfio e un nucleo condensato. Tali cellule schermo molto più bassi di fluorescenza in ottica DAPI, ma visto sotto l&#39;ottica FITC mostrano un segnale forte FAD nella regione ellissoide (provenienti da nucleotidi flavina ossidato e flavoproteine). Un altro criterio per la salute dei fotorecettori isolato è la loro capacità di generare all-trans retinolo (vitamina A) nei loro segmenti esterni alla stimolazione dalla luce. La generazione di vitamina A richiede notevoli quantità di NADPH, che dipende da un macchinario metabolico intatto. Figure 4 e 5 mostrano la formazione di vitamina A nei segmenti esterni di rana intatta e bastoncelli topo rispettivamente. <img src="/files/ftp_upload/2789/2789fig1.jpg" alt="Figura 1"/> Figura 1. Sano fotorecettori singola asta e cono. Le cellule sono state isolate da una retina salamandra tigre. Fototrasduzione avviene nel segmento esterno e l&#39;ellissoide è densamente ricco di mitocondri. Aste sono responsabili per dim visione della luce, i coni per la visione di luce intensa. <img src="/files/ftp_upload/2789/2789fig2.jpg" alt="Figura 2"/> Figura 2. Fluorescenza di vivere salamandra asta e cono. Questi sono adattata al buio cellule, che mostra una forte fluorescenza NADH nei loro rispettivi ellissoidi e nessuna vitamina significativo un fluorescenza nei loro segmenti esterni. La cattura delle immagini a fluorescenza rappresenta la loro prima esposizione alla luce visibile dopo il periodo di buio-adattamento. <img src="/files/ftp_upload/2789/2789fig3.jpg" alt="Figura 3"/> Figura 3. Danneggiato salamandra asta dei fotorecettori. Il corpo cellulare gonfio e il nucleo condensato sono indicativi di danno. La cella viene ossidato e non vi è minimo segnale di NADH (ottica DAPI), ma molto più forte segnale di FAD (FITC ottica). <img src="/files/ftp_upload/2789/2789fig4.jpg" alt="Figura 4"/> Figura 4. Rana con asta NADH e retinolo. Questo è un sano fotorecettore asta rana che mostra una forte fluorescenza NADH nella regione ellissoide. Prima di esposizione alla luce c&#39;è fluorescenza minima nel segmento esterno. A seguito di esposizione alla luce, vi è un significativo aumento della fluorescenza segmento esterno a causa della formazione di vitamina A. <img src="/files/ftp_upload/2789/2789fig5.jpg" alt="Figura 5"/> Figura 5. Topo asta con retinolo. Questo è un sano fotorecettore asta del mouse mostrando significativi fluorescenza esterno segmento dopo l&#39;esposizione alla luce a causa della formazione di vitamina A. Le regioni ellissoide di bastoncelli topo non mostrano un forte segnale di fluorescenza.

<ol>
	<li>Burns, M. E., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Counting+Photons:+Trials+and+Trends+in+Vertebrate+Visual+Transduction.">Beyond Counting Photons: Trials and Trends in Vertebrate Visual Transduction.</a> Neuron. 48, 387-401 (2005).</li><li>Ebrey, T., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+Photoreceptors.">Vertebrate Photoreceptors.</a> Prog Retin Eye Res. 20, 49-94 (2001).</li><li>Fain, G. L., Matthews, H. R., Cornwall, M. C., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+in+Vertebrate+Photoreceptors.">Adaptation in Vertebrate Photoreceptors.</a> Physiol Rev. 81, 117-151 (2001).</li><li>Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+Protein-Coupled+Receptor+Rhodopsin.">G Protein-Coupled Receptor Rhodopsin.</a> Annu Rev Biochem. 75, 743-767 (2006).</li><li>Saari, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+Visual+Pigment+Regeneration:+The+Friedenwald+Lecture.">Biochemistry of Visual Pigment Regeneration: The Friedenwald Lecture.</a> Invest Ophthalmol Vis Sci. 41, 337-348 (2000).</li><li>Lamb, T. D., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dark+Adaptation+and+the+Retinoid+Cycle+of+Vision.">Dark Adaptation and the Retinoid Cycle of Vision.</a> Prog Retin Eye Res. 23, 307-380 (2004).</li><li>Imanishi, Y., Lodowski, K. H., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Photon+Microscopy:+Shedding+Light+on+the+Chemistry+of+Vision.">Two-Photon Microscopy: Shedding Light on the Chemistry of Vision.</a> Biochemistry. 46, 9674-9684 (2007).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleached+Pigment+Produces+a+Maintained+Decrease+in+Outer+Segment+Ca2++in+Salamander+Rods.">Bleached Pigment Produces a Maintained Decrease in Outer Segment Ca2+ in Salamander Rods.</a> J Gen Physiol. 111, 53-64 (1998).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., Bandarchi, J., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Dependent+Changes+in+Outer+Segment+Free-Ca2++Concentration+in+Salamander+Cone+Photoreceptors.">Light-Dependent Changes in Outer Segment Free-Ca2+ Concentration in Salamander Cone Photoreceptors.</a> J Gen Physiol. 113, 267-277 (1999).</li><li>Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Cytoplasmic+Calcium+Concentration+in+the+Rods+of+Wild-Type+and+Transducin+Knock-out+Mice.">Measurement of Cytoplasmic Calcium Concentration in the Rods of Wild-Type and Transducin Knock-out Mice.</a> J Physiol. 542, 843-854 (2002).</li><li>Leung, Y. T., Fain, G. L., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Measurement+of+Current+and+Calcium+in+the+Ultraviolet-Sensitive+Cones+of+Zebrafish.">Simultaneous Measurement of Current and Calcium in the Ultraviolet-Sensitive Cones of Zebrafish.</a> J Physiol. 579, 15-27 (2007).</li><li>Matthews, H. R., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Spot+Confocal+Technique+to+Measure+Cytoplasmic+Calcium+Concentration+in+Photoreceptors.">Laser Spot Confocal Technique to Measure Cytoplasmic Calcium Concentration in Photoreceptors.</a> Methods Enzymol. 316, 146-163 (2000).</li><li>Szikra, T., Cusato, K., Thoreson, W. B., Barabas, P., Bartoletti, T. M., Krizaj, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+Calcium+Stores+Regulates+Calcium+Influx+and+Signal+Transmission+in+Rod+Photoreceptors.">Depletion of Calcium Stores Regulates Calcium Influx and Signal Transmission in Rod Photoreceptors.</a> J Physiol. 586, 4859-4875 (2008).</li><li>Krizaj, D., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+Calcium+Extrusion+Mechanisms+in+the+Outer+and+Inner+Segments+of+Photoreceptors.">Compartmentalization of Calcium Extrusion Mechanisms in the Outer and Inner Segments of Photoreceptors.</a> Neuron. 21, 249-256 (1998).</li><li>Chen, C., Nakatani, K., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+Magnesium+Concentration+in+Salamander+Photoreceptor+Outer+Segments.">Free Magnesium Concentration in Salamander Photoreceptor Outer Segments.</a> J Physiol. 553, 125-135 (2003).</li><li>Chen, C., Jiang, Y., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Behavior+of+Rod+Photoreceptor+Disks.">Dynamic Behavior of Rod Photoreceptor Disks.</a> Biophys J. 83, 1403-1412 (2002).</li><li>Tsina, E., Chen, C., Koutalos, Y., Ala-Laurila, P., Tsacopoulos, M., Wiggert, B., Crouch, R. K., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+Microfluorometric+Studies+of+Reduction+and+Clearance+of+Retinal+in+Bleached+Rod+Photoreceptors.">Physiological and Microfluorometric Studies of Reduction and Clearance of Retinal in Bleached Rod Photoreceptors.</a> J Gen Physiol. 124, 429-443 (2004).</li><li>Ala-Laurila, P., Kolesnikov, A. V., Crouch, R. K., Tsina, E., Shukolyukov, S. A., Govardovskii, V. I., Koutalos, Y., Wiggert, B., Estevez, M. E., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle:+Dependence+of+Retinol+Production+and+Removal+on+Photoproduct+Decay+and+Cell+Morphology.">Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology.</a> J Gen Physiol. 128, 153-169 (2006).</li><li>Koutalos, Y., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluorometric+Measurement+of+the+Formation+of+All-Trans-Retinol+in+the+Outer+Segments+of+Single+Isolated+Vertebrate+Photoreceptors.">Microfluorometric Measurement of the Formation of All-Trans-Retinol in the Outer Segments of Single Isolated Vertebrate Photoreceptors.</a> Methods Mol Biol. 652, 129-147 (2010).</li><li>Wu, Q., Blakeley, L. R., Cornwall, M. C., Crouch, R. K., Wiggert, B. N., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interphotoreceptor+Retinoid-Binding+Protein+Is+the+Physiologically+Relevant+Carrier+That+Removes+Retinol+from+Rod+Photoreceptor+Outer+Segments.">Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments.</a> Biochemistry. 46, 8669-8679 (2007).</li><li>Kolesnikov, A. V., Ala-Laurila, P., Shukolyukov, S. A., Crouch, R. K., Wiggert, B., Estevez, M. E., Govardovskii, V. I., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle+and+Its+Metabolic+Support+in+Gecko+Photoreceptors.">Visual Cycle and Its Metabolic Support in Gecko Photoreceptors.</a> Vision Res. 47, 363-374 (2007).</li><li>Chen, C., Blakeley, L. R., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+All-Trans+Retinol+after+Visual+Pigment+Bleaching+in+Mouse+Photoreceptors.">Formation of All-Trans Retinol after Visual Pigment Bleaching in Mouse Photoreceptors.</a> Invest Ophthalmol Vis Sci. 50, 3589-3595 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Se sano cellule isolate non si ottengono, il problema è sia con l&#39;isolamento o la salute della retina o con il suo taglio. Di solito, dopo aver rimosso la parte anteriore dell&#39;occhio e il vitreo, la retina ascensori prontamente spento l&#39;epitelio pigmentato. Se non lo fa, cerca di non staccare a partire dalla periferia del oculare. Se è ancora difficile separare, una possibilità probabile è che l&#39;animale non è stata adattata al buio per un adeguato periodo di tempo, o la luce rossa è troppo luminoso...

<table border="1"><tbody><tr><th> Nome </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Stanza buia (100-150 ft 2) </td><td></td><td></td><td></td></tr><tr><td> Luci rosse 19 </td><td></td><td></td><td> Negozi on-line </td></tr><tr><td> Fonti di luce a infrarossi e visualizzatori di immagini a raggi infrarossi </td><td> FJW Optical Systems, Inc. </td><td></td><td></td></tr><tr><td> Dissezione microscopio 19 </td><td></td><td></td><td> Dotato di visori a infrarossi </td></tr><tr><td> Microscopio a epifluorescenza racchiuso in una gabbia a tenuta di luce 19 </td><td></td><td></td><td></td></tr><tr><td> Strumenti di dissezione (forbici, pinze, porta lama) </td><td> Roboz o Strumenti Scienze Arti </td><td></td><td></td></tr><tr><td> Sylgard elastomero </td><td> Essex (Charlotte) </td><td> Sylgard 184 elastomero kit </td><td></td></tr><tr><td> Poli-L-ornitina (0,01%) </td><td> Sigma-Aldrich </td><td> P4957 </td><td></td></tr><tr><td> Poli-L-lisina (0,1%) </td><td> Sigma-Aldrich </td><td> P8920 </td><td> Diluire a 0,01% </td></tr><tr><td> Camere sperimentale </td><td> Warner Instruments (Hamden, CT) </td><td> D3512P </td><td></td></tr><tr><td> Piastre di Petri, pipette di plastica </td><td> Fisher Scientific </td><td></td><td></td></tr></tbody></table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

Nella retina dei vertebrati, fototrasduzione, la conversione della luce in un segnale elettrico, è effettuata dal bastone e coni fotorecettori 1-4. Bastoncelli sono responsabili della visione in penombra, i coni di luce. Fototrasduzione avviene nel segmento esterno della cellula fotorecettore, un comparto specializzato che contiene un&#39;alta concentrazione di pigmento visivo, il rilevatore di luce principale. Il pigmento visivo è composto da un cromoforo, 11 - cis retinico, attaccato ad una proteina, opsina. Un fotone assorbito dal pigmento visivo isomerizza il cromoforo da 11 - cis a tutto-trans. Questo fotoisomerizzazione determina un cambiamento conformazionale nel pigmento visivo che avvia una cascata di reazioni che culmina in un cambiamento di potenziale di membrana, e determinando la trasduzione dello stimolo della luce in un segnale elettrico. Il recupero della cellula da stimolazione luminosa comporta la disattivazione degli intermedi attivati ​​dalla luce, e il ripristino del potenziale di membrana. Ca 2 + modula l&#39;attività dei diversi enzimi coinvolti nella fototrasduzione, e la sua concentrazione è ridotta alla stimolazione luminosa. In questo modo, Ca 2 + gioca un ruolo importante nel recupero della cellula da stimolazione luminosa e il suo adattamento alla luce di fondo. Un&#39;altra parte essenziale del processo di recupero è la rigenerazione del pigmento visivo che è stato distrutto durante la luce il rilevamento da parte dei suoi fotoisomerizzazione 11 - cromoforo cis a tutto-trans 5-7. Questa rigenerazione inizia con il rilascio di tutti-trans retinico dal pigmento fotoattivati, lasciandosi alle spalle l&#39;apo-proteina opsina. La rilasciato all-trans retinico è rapidamente ridotto in una reazione che utilizza NADPH a tutto-trans retinolo e opsina combina con fresca 11 - retina cis portato nel segmento esterno di riformare il pigmento visivo. All-trans retinolo viene poi trasferito al di fuori del segmento esterno e nelle celle attigue dalla proteina specializzata retinoidi Interphotoreceptor vettore Binding (IRBP). Imaging di fluorescenza di cellule fotorecettrici singolo può essere usato per studiare la loro fisiologia e biologia cellulare. Ca 2 +-sensitive coloranti fluorescenti possono essere utilizzate per esaminare in dettaglio l&#39;interazione tra il segmento esterno Ca 2 + modifiche e risposta alla luce 8-12 così come il ruolo del segmento Ca 2 + interna negozi a Ca 2 + omeostasi 13,14 . Coloranti fluorescenti possono essere utilizzati anche per misurare la concentrazione di Mg 2 + 15, pH, e come traccianti di acquosa e scomparti membrana 16. Infine, la fluorescenza intrinseca di all-trans retinolo (vitamina A) può essere utilizzato per monitorare la cinetica della sua formazione e la rimozione delle cellule fotorecettori singolo 17-19.

Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

Un metodo è descritto per la preparazione di singole cellule fotorecettori che vivono da diverse specie di vertebrati per l&#39;imaging di fluorescenza. Il metodo può essere utilizzato per l&#39;immagine della fluorescenza di fluorofori endogeni, come la NADH o vitamina A, o quella di esogena coloranti aggiunti fluorescenti sensibili a Ca<sup&gt; 2 +</sup&gt; O altri fattori.

Preparazione delle condizioni di vita isolata fotorecettori dei Vertebrati per l&#39;imaging di fluorescenza

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

preparation-living-isolated-vertebrate-photoreceptor-cells-for

Research

JoVE Journal

Neuroscience

12.2K Views.  Medical University of South Carolina. Un metodo è descritto per la preparazione di singole cellule fotorecettori che vivono da diverse specie di vertebrati per l'imaging di fluorescenza. Il metodo può essere utilizzato per l'immagine della fluorescenza di fluorofori endogeni, come la NADH o vitamina A, o quella di esogena coloranti aggiunti fluorescenti sensibili a Ca 2 + O altri fattori.

Video: Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Una preparazione isolati retina per registrare Risposta Luce da Geneticamente etichetta cellule gangliari della retina

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Ricerca

Neuroscienze

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Rilevamento di ipossia microregionale nella corteccia cerebrale di topo Imaging a due fotoni di fluorescenza endogena NADH

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparazione di acuta fette zona subventricolare for Imaging di calcio

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Visualizzazione del endosome Dynamics in Vivere terminazioni nervose con quattro-dimensionale imaging di fluorescenza

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

Vibratome sezionamento mouse Retina preparare Culture fotorecettori

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imaging Ca<sup&gt; 2+</sup&gt; Dinamica a cono fotorecettore Axon Terminali della retina di topo

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Sviluppo di analisi per la quantificazione alto contenuto di formazione aggregata di Sod1 mutante della proteina in cellule viventi

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Due metodi di peeling per l'isolamento dei compartimenti cellulari fotorecettori nella retina del topo per l'analisi delle proteine

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Spettrometria di massa a scambio idrogeno/deuterio al millisecondo per lo studio della dinamica strutturale dell'alfa-sinucleina in condizioni fisiologiche

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...