Questo lavoro è stato sostenuto da NIH sovvenzioni EY019312 e EY021126 (vjk), EY002687 al Dipartimento di Oftalmologia e Scienze Visive presso la Washington University, e da Research per prevenire la cecità.

Tutti i protocolli sperimentali sono stati in conformità con la guida per la cura e l&#39;uso di animali da laboratorio e sono stati approvati dal Comitato istituzionale Animal Studies presso la Washington University. 1. Impostazione perfusione e Campione di laboratorio <ol><li> Preparare la soluzione per perfusione retina fresco il giorno dell&#39;esperimento. Utilizzare acqua distillata e deionizzata. Utilizzare una delle seguenti tre soluzioni. <ol><li> Preparare Bicarbonato contenenti &#39;soluzione (1 L): 1 bottiglia di Ames&#39; Ames media e 1,9 g di NaHCO 3, </li><li> Preparare la soluzione di Locke (in mM): 112.5 NaCl, 3.6 KCl, 2,4 MgCl 2, 1.2 CaCl 2, 10.0 HEPES, 20.0 NaHCO 3, 3.0 Na succinato, 0,5 Na glutammato, 0,02 EDTA, e 10,0 di glucosio, 0,1% vitamine MEM e ammino Acidi </li><li> Preparare soluzione tamponata HEPES Ringer (in mM): NaCl 133,3, KCl 3,3, 2,0 MgCl 2, 1.0 CaCl 2, 10.0 glucosio, 0,01 EDTA, 12.0 HEPES, pHregolato a 7,5 con ~ 5,8 ml di 1 M NaOH, aggiungere 0,72 g / L Leibovitz mezzo di coltura L-15. Usare 20 - 50 mM DL-AP4 e 50 - 100 pM BaCl 2 per isolare la risposta fotorecettore con qualsiasi mezzo di perfusione. </li></ol></li><li> Preparare la soluzione per elettrodi 28 (in mM): NaCl 140,0, KCl 3.6, 2.4 MgCl 2, 1.2 CaCl 2, 3, HEPES 0,01 EDTA e aggiustare il pH a 7,4-7,5 con NaOH. Soluzione elettrodo può essere conservata a temperatura ambiente per diversi mesi. </li><li> Preparare e testare il supporto del campione. <ol><li> Incollare il nero / carta grigia filtro in cima alle cupole della parte inferiore del titolare del campione (vedi Figura 1A). Distribuire accuratamente bicomponente 5 min colla epossidica intorno ai bordi delle cime piane delle cupole. Se necessario, farlo al microscopio dissezione. </li><li> Attendere che la colla è quasi asciutto (circa 4 min) e premere la carta filtro cupole utilizzando un elemento piatta. Carta da filtro colla almeno un giorno prima dell&#39;esperimento. Ilcarta da filtro può essere utilizzato più volte, ma deve essere sostituito dopo un mese di registrazioni. Usa il 70% di etanolo per pulire accuratamente cupole da eventuali residui di colla prima di installare la carta sostitutiva. </li><li> Riempire i canali degli elettrodi con soluzione elettrodo cercando di evitare le bolle d&#39;aria e vite elettrodi pellet chiusa con un adattatore filettato nei canali elettrodi (vedi figure 1 A e B). Collegare i superiore e inferiore pezzi del supporto del campione con quattro viti e riempire le linee di perfusione con soluzione elettrodi. </li><li> Misurare la resistenza e la tensione tra i conduttori di ciascuna coppia di elettrodi con un multimetro (Figura 1B). La resistenza deve essere inferiore a 100 Ωk e la tensione sotto 10 mV se i canali sono senza bolle e gli elettrodi sono in buone condizioni. </li></ol></li><li> Versare 400-700 ml di supporti perfusione nella bottiglia. Indipendente altri 300 ml da utilizzare nella dissezione di retine e sstrappò in un frigorifero. Impostare il tubo di perfusione nel blocco scambiatore di calore e posizionare il blocco preriscaldato sulla piastra di riscaldamento (vedi Figura 1D). </li><li> Racchiudere la bottiglia con un tappo che ha collegamenti di CO 2 / O 2 gas (se si usa Ames &#39;o Locke) e per le tubazioni perfusione (Figura 1D). Preriscaldare la bottiglia con il supporto a 37 o C e posizionarlo sulla piastra di riscaldamento o sulla parte superiore dell&#39;unità stimolazione luminosa in bagnomaria a 37-39 ° C 17. <ol><li> Prime linee di perfusione di riempimento d&#39;supporti perfusione di avviare il flusso di gravità-driven. <ol><li> Nel sistema LKC, posizionare la bottiglia sulla parte superiore dell&#39;unità stimolatore 17 per fornire un flusso gravitazionale significativo che viene poi regolata da regolatori di portata senza essere influenzato dal livello di riduzione della soluzione di perfusione nella bottiglia durante l&#39;esperimento. </li><li> Nel sistema di Ocuscience, posizionare il pbottiglia erfusion all&#39;interno della gabbia di Faraday per minimizzare il rumore e posizionare linee di uscita lungo perfusione del portacampioni (vedi passo 2.8) ben al di sotto del livello del portacampioni (e bottiglia perfusione) per aumentare l&#39;azionamento gravitazionale della soluzione. Schermare le linee di uscita e collegare lo schermo a terra dell&#39;amplificatore per evitare l&#39;accoppiamento del rumore elettromagnetico al segnale ERG. </li></ol></li></ol></li><li> Regolare la perfusione di 3 - 5 ml / min utilizzando regolatori di portata. Collegare 5% di CO 2/95% O 2 gas da una bombola con una corretta regolazione e regolare la portata per garantire gorgoglio costante dei mezzi di comunicazione nella bottiglia attraverso una pietra d&#39;aria. </li></ol> Preparazione 2. campione <ol><li> Montare gli strumenti di dissezione pulite e nitide compreso microscissors straight-lame, uno o due 45 o pinzette, lama di rasoio e un pezzo rettangolare di carta da filtro. </li><li> Versare circa 200 ml di perfu freddosoluzione sione in un grande piatto Petri in modo che tutta la parte inferiore del supporto del campione (comprese le cupole) può essere immerso nella soluzione. Questo passaggio diventa importante quando si monta la retina sui cupole (vedi punto 2.6). Sebbene alcune soluzioni sono progettate per essere saturo di CarboGen (5% CO 2/95% O 2), questo non è essenziale ai fini di dissezione e non è stato fatto negli esperimenti qui descritti. </li><li> Per un tipico esperimento, tenere gli animali in 12/12 ore scuro ciclo / luce e buio si adattano per 6-12 ore prima delle registrazioni. Euthanize l&#39;animale da CO 2 inalazione seguita da dislocazione cervicale sotto fioca luce rossa e fare tutte le seguenti procedure previste fioca luce rossa o IR (usare, ad esempio, il filtro rosso davanti alla sorgente luminosa del microscopio). <ol><li> Estrarre gli occhi con l&#39;ausilio di una pinzetta e metterli nei media. Inserire un occhio per volta su un piccolo pezzo di carta (ad esempio, carta da filtro regolare) e fare comeilluminato approssimativamente il livello di ora serrata tenendo l&#39;occhio con una pinzetta (questo è fatto di fuori della soluzione qui). </li></ol></li><li> Tagliare lungo la serrata (o più vicino all&#39;equatore dell&#39;occhio) con microscissors e rimuovere la cornea e la lente. Posizionare l&#39;oculare nei media freddo nella grande capsula di Petri e ripetere la stessa procedura con l&#39;altro occhio. </li><li> Tagliare una piccola incisione dall&#39;alto della tazza occhio verso il nervo ottico, mantenendo la forbice tra la retina e sclera per mantenere la retina intatto possibile. Grip sclera da entrambi i lati dell&#39;incisione utilizzando due pinzette e tirare le pinzette distanti per staccare la retina. <ol><li> Tagliare il nervo ottico e tentare di isolare la retina con una quantità minima di contatto fisico alla superficie distale. RPE sarà principalmente staccare automaticamente dalla retina durante il processo di dissezione. È più importante evitare disturbo meccanico della retina che per eseguire ladissezione rapidamente, generalmente retine può essere incubato almeno 30 min in soluzione senza effetti significativi sulle proprietà di risposta. </li></ol></li><li> Montare le retine sulle cupole del supporto del campione (Figura 1C). Immergere la parte inferiore del supporto del campione nella scatola di Petri con la retina sezionato. Scorrere la retina, fotorecettori lato (la superficie convessa della retina isolata) verso l&#39;alto, sopra la cupola e sollevare il supporto del campione in modo che la retina attacca sulla carta da filtro. Ripetere la procedura per l&#39;altro retina. </li><li> Asciugare con cura la piastra porta per evitare interferenze elettriche tra la retina, così come il rumore e il segnale shunt. Portacampione anelli O-ring intorno cupole parte inferiore per impedire soluzione fuoriuscita tra il fondo e le parti superiori del titolare, nonché per aiutare l&#39;isolamento elettrico dei fotorecettori e cellule gangliari lati dei singoli retine. Fissare la parte superiore del supporto con le quattro viti (Figura 1B) e riempire i canali di perfusione con soluzione di perfusione utilizzando una siringa e un ago. </li><li> Trasferire il titolare del campione accanto al blocco scambiatore di calore e collegare le linee di ingresso e di uscita di perfusione al titolare del campione (Figura 1D). Collegare gli elettrodi all&#39;amplificatore ERG (top elettrodi collegano alla terra / meno pin nel amplificatore) e collegare l&#39;unità stimolatore / di controllo sul supporto del campione utilizzando un adattatore o far scorrere la piastra elettrica nell&#39;unità stimolatore a seconda di quale in vivo Sistema ERG è utilizzato. </li></ol> 3. Registrazioni <ol><li> Configurare le impostazioni dell&#39;amplificatore e di stimolo utilizzando il software del sistema in vivo. Impostare la frequenza di acquisizione di un valore compreso tra 1 e 10 kHz e filtrazione passabasso a 300 Hz. Non utilizzare il filtro passa-alto. Utilizzare 60 Hz (o 50 Hz in Europa) segnare il filtro, se necessario. </li><li> Record di base senza stimolazione luminosa e lo spiritoh stimolazione dim (ad es., la luce verde di -35 dB o 0.3 mCd sm -2) per testare per buon collegamento elettrico tra gli elettrodi e il campione retina. Attendere 10 - 20 min prima di iniziare la raccolta di dati in modo che la temperatura e la funzione retinica raggiungere uno stato stabile. <ol><li> Scegli i parametri di stimolo secondo qualsiasi esperimento specifico e avviare la raccolta dei dati. Ad esempio, utilizzare la luce verde da -40 a 0 dB o da 0,1 fino a 1.000 mCd sm -2 per una famiglia risposta scotopic. Usa circa 2 a 3 log-unità più luminoso lampeggia registrazioni fotopiche dove aste devono essere soppressi dalla luce di fondo (3-30 Cdm -2) o un&#39;unità flash sonda (circa 1,3 Cd sm -2 = 1 dB, vedi figura 3). </li><li> Tuttavia, ricordare che gli esatti valori di intensità della luce potrebbe essere un po &#39;dipende dal sistema di calibrazione e condizioni sperimentali. Come regola generale, le intensità di scala in calo di 5 - 10 volte rispetto a in vivo </em&gt; registrazioni quando si utilizza la luce verde 17. Un coperchio nero con aperture sopra la retina (inclusi nel sistema adattatore commerciale) può essere utilizzato per ridurre la dispersione della luce nel portacampioni e facilitare omogenea e stimolo uguale dei due retine dalla sfera Ganzfeld dei sistemi commerciali in vivo. NOTA: Per le registrazioni di retina adattata al buio, le intensità dei lampi di prova utilizzati in un tipico esperimento di candeggina solo una frazione trascurabile del pigmento in modo che la mancanza di rigenerazione RPE-driven non è un problema. Inoltre, è stato precedentemente dimostrato che la rigenerazione pigmento cono può ancora avvenire nella retina isolata mediante la cella Müller ciclo visivo 20. </li></ol></li><li> Come esperimenti durano in genere da 30 minuti fino a diverse ore, monitorare la deriva della linea di base, il livello di rumore e la stabilità di risposta durante l&#39;esperimento come movimenti di questi potrebbero indicare problemi tecnici. NOTA: L&#39;aumento o la diminuzione del rumoreampiezze d risposta possono indicare bolle d&#39;aria nei canali di perfusione o elettrodi, troppo bassa o alta temperatura, la soluzione di perdita / fuoriuscita o spostamento della retina. </li></ol> 4. Pulizia <ol><li> Staccare il supporto del campione dalle linee di perfusione, aprirlo e lavare la retina dalla carta da filtro. Rimuovere gli elettrodi e sciacquare con acqua distillata (non utilizzare etanolo). Pulire il supporto del campione (compresi i canali di perfusione) con etanolo e / o di acqua distillata. Tubazione perfusione Lavare accuratamente con&gt; 70% di etanolo. </li><li> Sciacquare la bottiglia di perfusione con acqua distillata (non utilizzare detergenti). L&#39;etanolo può essere utilizzata anche per pulire la bottiglia. </li></ol>

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	<li>Armington, 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=.">.</a> The Electroretinogram. , (1974).</li><li>Einthoven, W., Jolly, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+form+and+magnitude+of+the+electrical+response+of+the+eye+to+stimulation+by+light+at+various+intensities.">The form and magnitude of the electrical response of the eye to stimulation by light at various intensities.</a> Q J Exp Physiol. 1, 43 (1908).</li><li>Granit, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+components+of+the+retinal+action+potential+in+mammals+and+their+relation+to+the+discharge+in+the+optic+nerve.">The components of the retinal action potential in mammals and their relation to the discharge in the optic nerve.</a> J. Physiol. 77, 207-239 (1933).</li><li>Penn, R. D., Hagins, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transmission+along+retinal+rods+and+the+origin+of+the+electroretinographic+a-wave.">Signal transmission along retinal rods and the origin of the electroretinographic a-wave.</a> Nature. 223, 201-204 (1969).</li><li>Stockton, R. A., Slaughter, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-wave+of+the+electroretinogram.+A+reflection+of+ON+bipolar+cell+activity.">B-wave of the electroretinogram. A reflection of ON bipolar cell activity.</a> J. Gen. Physiol. 93, 101-122 (1989).</li><li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+linearity+and+kinetics+of+the+cat+retina:+the+bipolar+cell+component+of+the+dark-adapted+electroretinogram.">Response linearity and kinetics of the cat retina: the bipolar cell component of the dark-adapted electroretinogram.</a> Vis. Neurosci. 12, 837-850 (1995).</li><li>Green, D. G., Kapousta-Bruneau, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dissection+of+the+electroretinogram+from+the+isolated+rat+retina+with+microelectrodes+and+drugs.">A dissection of the electroretinogram from the isolated rat retina with microelectrodes and drugs.</a> Vis. Neurosci. 16, 727-741 (1999).</li><li>Steinberg, R. H., Schmidt, R., Brown, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+responses+to+light+from+cat+pigment+epithelium:+origin+of+the+electroretinogram+c-wave.">Intracellular responses to light from cat pigment epithelium: origin of the electroretinogram c-wave.</a> Nature. 227, 728-730 (1970).</li><li>Wilson, W. S., Shahidullah, M., Millar, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bovine+arterially-perfused+eye:+an+in+vitro+method+for+the+study+of+drug+mechanisms+on+IOP,+aqueous+humour+formation+and+uveal+vasculature.">The bovine arterially-perfused eye: an in vitro method for the study of drug mechanisms on IOP, aqueous humour formation and uveal vasculature.</a> Curr. Eye Res. 12, 609-620 (1993).</li><li>Frank, R. N., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+photoproducts:+effects+on+electroretinogram+sensitivity+in+isolated+perfused+rat+retina.">Rhodopsin photoproducts: effects on electroretinogram sensitivity in isolated perfused rat retina.</a> Science. 161, 487-489 (1968).</li><li>Donner, K., Hemila, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependence+of+rod+photoresponses+from+the+aspartate-treated+retina+of+the+frog+(Rana+temporaria).">Temperature-dependence of rod photoresponses from the aspartate-treated retina of the frog (Rana temporaria).</a> Acta Physiol. Scand. 134, 535-541 (1988).</li><li>Green, D. G., Kapousta-Bruneau, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+properties+of+a+new+isolated+rat+retina+preparation.">Electrophysiological properties of a new isolated rat retina preparation.</a> Vision Res. 39, 2165-2177 (1999).</li><li>Hamasaki, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sodium+ion+concentration+on+the+electroretinogram+of+the+isolated+retina+of+the+frog.">The effect of sodium ion concentration on the electroretinogram of the isolated retina of the frog.</a> J. Physiol. 167, 156-168 (1963).</li><li>Luke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+perfused+bovine+retina--a+sensitive+tool+for+pharmacological+research+on+retinal+function.">The isolated perfused bovine retina--a sensitive tool for pharmacological research on retinal function.</a> Brain Res. Brain Res. Protoc. 16, 27-36 (2005).</li><li>Bastian, B. L., 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+adaptation+in+toad+rods:+requirement+for+an+internal+messenger+which+is+not+calcium.">Light adaptation in toad rods: requirement for an internal messenger which is not calcium.</a> J. Physiol. 297, 493-520 (1979).</li><li>Arden, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+gradients+across+the+receptor+layer+of+the+isolated+rat+retina.">Voltage gradients across the receptor layer of the isolated rat retina.</a> J. Physiol. 256, 333-360 (1976).</li><li>Vinberg, F., Kolesnikov, A. V., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+ERG+analysis+of+photoreceptors+using+an+in+vivo+ERG+system.">Ex vivo ERG analysis of photoreceptors using an in vivo ERG system.</a> Vision Res. 101, 108-117 (2014).</li><li>Nymark, S., Heikkinen, H., Haldin, C., Donner, K., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+responses+and+light+adaptation+in+rat+retinal+rods+at+different+temperatures.">Light responses and light adaptation in rat retinal rods at different temperatures.</a> J. Physiol. 567, 923-938 (2005).</li><li>Heikkinen, H., Nymark, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cone+photoresponses+obtained+with+electroretinogram+from+the+isolated+retina.">Mouse cone photoresponses obtained with electroretinogram from the isolated retina.</a> Vision Res. 48, 264-272 (2008).</li><li>Wang, J. S., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+pathway+mediates+the+mouse+and+human+cone+visual+cycle.">An alternative pathway mediates the mouse and human cone visual cycle.</a> Curr. Biol. 19, 1665-1669 (2009).</li><li>Bolnick, D. A., Walter, A. E., Sillman, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barium+suppresses+slow+PIII+in+perfused+bullfrog+retina.">Barium suppresses slow PIII in perfused bullfrog retina.</a> Vision Res. 19, 1117-1119 (1979).</li><li>Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+conductance+block+by+barium+in+amphibian+Muller+cells.">Potassium conductance block by barium in amphibian Muller cells.</a> Brain Res. 498, 308-314 (1989).</li><li>Oakley, B., Katz, B. J., Xu, Z., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+buffering+of+extracellular+potassium+by+Muller+(glial)+cells+in+the+toad+retina.">Spatial buffering of extracellular potassium by Muller (glial) cells in the toad retina.</a> Exp. Eye Res. 55, 539-550 (1992).</li><li>Nymark, S., Haldin, C., Tenhu, H., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+measuring+free+drug+concentration:+retinal+tissue+as+a+biosensor.">A new method for measuring free drug concentration: retinal tissue as a biosensor.</a> Invest. Ophthalmol. Vis. Sci. 47, 2583-2588 (2006).</li><li>Walter, P., Luke, C., Sickel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotics+and+light+responses+in+superfused+bovine+retina.">Antibiotics and light responses in superfused bovine retina.</a> Cell. Mol. Neurobiol. 19, 87-92 (1999).</li><li>Luke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+safety+profile+of+alkylphosphocholines+in+the+model+of+the+isolated+perfused+vertebrate+retina.">The safety profile of alkylphosphocholines in the model of the isolated perfused vertebrate retina.</a> Graefes Arch. Clin. Exp. Ophthalmol. 248, 511-518 (2010).</li><li>Januschowski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+toxicity+testing+of+VEGF+Trap-Eye+in+an+isolated+perfused+vertebrate+retina+organ+culture+model.">Electrophysiological toxicity testing of VEGF Trap-Eye in an isolated perfused vertebrate retina organ culture model.</a> Acta Ophthalmol. 92, e305-e311 (2014).</li><li>Kolesnikov, A. V., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transretinal+ERG+recordings+from+mouse+retina:+rod+and+cone+photoresponses.">Transretinal ERG recordings from mouse retina: rod and cone photoresponses.</a> J Vis Exp. , (2012).</li><li>Koskelainen, A., Hemila, S., Donner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+sensitivities+of+short-+and+long-wavelength+sensitive+cone+mechanisms+in+the+frog+retina.">Spectral sensitivities of short- and long-wavelength sensitive cone mechanisms in the frog retina.</a> Acta Physiol. Scand. 152, 115-124 (1994).</li><li>Lyubarsky, A. L., Falsini, B., Pennesi, M. E., Valentini, P., 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=UV-+and+midwave-sensitive+cone-driven+retinal+responses+of+the+mouse:+a+possible+phenotype+for+coexpression+of+cone+photopigments.">UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments.</a> J. Neurosci. 19, 442-455 (1999).</li><li>Lyubarsky, A. L., Daniele, L. L., 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=From+candelas+to+photoisomerizations+in+the+mouse+eye+by+rhodopsin+bleaching+in+situ+and+the+light-rearing+dependence+of+the+major+components+of+the+mouse+ERG.">From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG.</a> Vision Res. 44, 3235-3251 (2004).</li><li>Azevedo, A. W., Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+protocols+alter+phototransduction:+the+implications+for+retinal+processing+at+visual+threshold.">Experimental protocols alter phototransduction: the implications for retinal processing at visual threshold.</a> J. Neurosci. 31, 3670-3682 (2011).</li><li>Carter-Dawson, L. D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rods+and+cones+in+the+mouse+retina.+I.+Structural+analysis+using+light+and+electron+microscopy.">Rods and cones in the mouse retina. I. Structural analysis using light and electron microscopy.</a> J. Comp. Neurol. 188, 245-262 (1979).</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>Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., 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=Light-driven+translocation+of+signaling+proteins+in+vertebrate+photoreceptors.">Light-driven translocation of signaling proteins in vertebrate photoreceptors.</a> Trends Cell Biol. 16, 560-568 (2006).</li><li>Schneeweis, D. M., Schnapf, 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=The+photovoltage+of+macaque+cone+photoreceptors:+adaptation,+noise,+and+kinetics.">The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics.</a> J. Neurosci. 19, 1203-1216 (1999).</li><li>Heikkinen, H., Vinberg, F., Nymark, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesopic+background+lights+enhance+dark-adapted+cone+ERG+flash+responses+in+the+intact+mouse+retina:+a+possible+role+for+gap+junctional+decoupling.">Mesopic background lights enhance dark-adapted cone ERG flash responses in the intact mouse retina: a possible role for gap junctional decoupling.</a> J. Neurophysiol. 105, 2309-2318 (2011).</li><li>Gouras, P., MacKay, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+in+amplitude+of+the+human+cone+electroretinogram+with+light+adaptation.">Growth in amplitude of the human cone electroretinogram with light adaptation.</a> Invest. Ophthalmol. Vis. Sci. 30, 625-630 (1989).</li><li>Peachey, N. S., Goto, Y., al-Ubaidi, M. R., Naash, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+mouse+cone-mediated+electroretinogram+during+light+adaptation.">Properties of the mouse cone-mediated electroretinogram during light adaptation.</a> Neurosci. Lett. 162, 9-11 (1993).</li></ol>

Washington University di St. Louis ha un accordo di licenza con Xenotec, Inc. e può ricevere una royalty dalla vendita dell&#39;adattatore ex vivo.

<html> Dimostriamo qui i passaggi critici per l&#39;ottenimento di alta qualità ex registrazioni vivo ERG contemporaneamente da due retine isolate del mouse utilizzando componenti in vivo del sistema ERG insieme ad un ex vivo adattatore ERG. In questo studio abbiamo perfusione entrambe le retine da animali con la stessa soluzione (sia Ames &#39;, Locke o Ringer), ma è anche possibile perfusione ogni retina con una soluzione diversa per esempio, a scopo di test di droga. I pa...

Elettroretinogramma (ERG) è una tecnica ben consolidata che può essere utilizzato per registrare l&#39;attività elettrica della retina innescata dalla luce. Il segnale ERG è generato principalmente dalle variazioni di tensione causate da correnti radiali (lungo l&#39;asse dei fotorecettori e cellule bipolari) scorre nello spazio extracellulare resistivo della retina. Il primo segnale ERG è stato registrato nel 1865 dal Holmgren dalla superficie di un occhio di pesce 1. Einthoven e Jolly 1908 2 diviso la risposta ERG all&#39;insorgenza di luce in tre differenti onde, denominato a-, b-, e c-onde, che sono ormai noti per riflettere principalmente l&#39;attività dei fotorecettori, cellule bipolari ON, e dell&#39;epitelio pigmentato cellule, rispettivamente 3-8. ERG può essere registrato dagli occhi di animali o esseri umani (in vivo) anestetizzati, dalla preparazione isolato occhio 9, attraverso isolato retina intatto (ex vivo) 3,10-15 o attraverso strati della retina specifici con i microelettrodi (localeERG) 4,16. Di questi, in vivo ERG è attualmente il metodo più usato per valutare la funzione della retina. Si tratta di una tecnica non invasiva che può essere usato per scopi diagnostici o per seguire la progressione di malattie retiniche negli animali o pazienti. Tuttavia, in vivo registrazioni ERG producono un segnale complicato con diversi componenti sovrapposti, spesso contaminato da rumore extraocular fisiologico (ad esempio, la respirazione e l&#39;attività cardiaca). ERG locale può essere utilizzato per registrare il segnale attraverso strati specifici della retina ma è il più invasivo e ha il segnale-rumore più basso (SNR) rispetto alle altre configurazioni di registrazione ERG. ERG locale è anche tecnicamente impegnativo e richiede costose attrezzature (ad esempio, microscopio e micromanipolatori). Transretinal ERG dal intatto, isolato retina (ex vivo ERG) offre un compromesso tra in vivo e metodi ERG locali permettendo stabile e high SNR registrazioni da retine intatte di animali o esseri umani 17. Recentemente, questo metodo è stato utilizzato con successo per studiare l&#39;asta e cono fotorecettori funzione in mammiferi, primati e umani retine 18-20. In aggiunta, a causa dell&#39;assenza di dell&#39;epitelio pigmentato in ex vivo retina, il componente c-onda positiva del segnale ERG viene rimosso e un componente importante negativo lento PIII è rivelato nelle ex vivo registrazioni. Il componente lenta PIII è stato dimostrato che provengono dalla attività delle cellule glia Müller nella retina 21-23. Così, ex vivo metodo ERG potrebbe essere utilizzato anche per studiare le cellule Müller nella retina intatto. Diversi studi hanno anche dimostrato che ex vivo registrazioni ERG potrebbero essere utilizzati per misurare la concentrazione di agenti farmacologici in giro per la retina 24 e testare la sicurezza e l&#39;efficacia dei farmaci 25-27. Commerciale multipla sistemi in vivo sono disponibili eutilizzato in molti laboratori che non hanno necessariamente vasta elettrofisiologia sfondo. Al contrario, ex vivo dispositivi non sono stati disponibili fino a poco 17 e come risultato solo pochi laboratori sono attualmente in corso vantaggio di questa tecnica potente. Sarebbe utile per effettuare registrazioni ex vivo ERG disponibile per più laboratori per far avanzare le nostre conoscenze sulla fisiologia della retina e la patologia, e di sviluppare nuove terapie per le malattie accecante. Dimostriamo qui un dispositivo semplice ed economico ex vivo ERG 17 e mostriamo come può essere utilizzato in combinazione con diversi sistemi disponibili in commercio in vivo ERG per registrare la segnalazione Rod- e cono-mediata (a- e b-onde) e la funzione di cellule Müller (lenta PIII) da intatte retine di topo wild-type.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>In vivo ERG system</td>
			<td>OcuScience</td>
			<td>HMsERG</td>
			<td><a href="http://www.ocuscience.us/id77.html">www.ocuscience.us/id77.html</a></td>
		</tr>
		<tr>
			<td>In vivo ERG system</td>
			<td>LKC Technologies</td>
			<td>UTAS-E 3000</td>
			<td><a href="http://www.lkc.com/products/UTAS/bigshot.html">www.lkc.com/products/UTAS/bigshot.html</a></td>
		</tr>
		<tr>
			<td>Ex vivo adapter</td>
			<td>OcuScience</td>
			<td>Ex VIVO ERG adapter</td>
			<td><a href="http://www.ocuscience.us/id107.html">www.ocuscience.us/id107.html</a></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>North Central Instruments</td>
			<td>Leica M80</td>
			<td><a href="http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;idCategory=27">May use any brand</a></td>
		</tr>
		<tr>
			<td>IR emitter</td>
			<td>Opto Diode Corp.</td>
			<td>OD-50L</td>
			<td><a href="http://www.optodiode.com/">www.optodiode.com</a></td>
		</tr>
		<tr>
			<td>Prowler Night Vision Scopes</td>
			<td>B.E. Meyers Electro Optics</td>
			<td>D4300-I</td>
			<td>Military grade product.</td>
		</tr>
		<tr>
			<td>Red filter</td>
			<td>Rosco Laboratories</td>
			<td>Roscolux #27 Medium Red</td>
			<td>May be used instead of IR system</td>
		</tr>
		<tr>
			<td>Red head light</td>
			<td>OcuScience</td>
			<td>ERGX011</td>
			<td><a href="http://www.ocuscience.us/catalog/i29.html">www.ocuscience.us/catalog/i29.html</a></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>WPI, Inc.</td>
			<td>500086</td>
			<td><a href="http://www.wpiinc.com/">www.wpiinc.com/</a></td>
		</tr>
		<tr>
			<td>Dumont tweezers #5</td>
			<td>WPI, Inc.</td>
			<td>14101</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com</a></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Metler Toledo</td>
			<td>AB54-S/FACT</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>pH meter and electrode</td>
			<td>Beckman Coulter</td>
			<td>pHI 350</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>60129</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>63020</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>21114</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>431788</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Ames medium</td>
			<td>Sigma-Aldrich</td>
			<td>A1420</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>B0750</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>DL-AP4</td>
			<td>Tocris Bioscience</td>
			<td>101</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Succinic acid disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>224731</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>L-Glutamic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G2834</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Leibovitz culture medium L-15</td>
			<td>Sigma-Aldrich</td>
			<td>L4386</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>MEM vitamins</td>
			<td>Sigma-Aldrich</td>
			<td>M6895</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M5550</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Airgas</td>
			<td>UN3156</td>
			<td>5% CO2</td>
		</tr>
	</tbody>
</table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Abbiamo registrato risposte flash di tipo selvaggio adattata al buio (WT) C57BL / 6 retine topo seguendo i protocolli sperimentali sopra descritte ed illustrate in figura 1 utilizzando diverse soluzioni standard di perfusione (Figura 2). Le forme d&#39;onda di risposta e la cinetica e sensibilità bastoncelli apparsi simili in Ames &#39;e mezzi di Locke (Figura 2A e B). D&#39;altra parte, in soluzione di Ringer HEPES-buffered (senza bicarbonato o 5% CO ...

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultaneo<em&gt; Ex vivo</em&gt; Test funzionale di due retine per<em&gt; In vivo</em&gt; Sistema Elettroretinogramma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

simultaneous-ex-vivo-functional-testing-two-retinas-vivo

Research

JoVE Journal

Neuroscience

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

9.1K Views.  Washington University in St. Louis. Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Video: Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Ricerca

Neuroscienze

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimaging funzionale con l&#39;impiego dell&#39;ecografia emato-encefalica e la barriera di Turbativa del Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo Imaging in diretta di divisioni cellulari singoli in mouse neuroepitelio

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

In Vivo microscopia a due fotoni di Endings singolo nervo in pelle

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

In vivo Interrogatorio del Sistema Nervoso Centrale Translatome da Polyribosome Frazionamento

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Ex Vivo preparati della lampadina vomeronasale Organo e accessori olfattivo intatto

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultanea a due fotoni<em&gt; In Vivo</em&gt; Imaging di Synaptic ingressi e degli obiettivi postsinaptici nella corteccia mouse retrosplenial

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo optogenetic dissezione di circuiti paura nel cervello fette

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

In Vivo Due-fotone Imaging dei neuroni corticali in topi neonatali

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

Un sistema impiantabile per l'elettromiografia cronica in vivo

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...