We thank Joung Jang and Xin Guan for technical assistance. We thank Dr. Rory McQuiston of Virginia Commonwealth University for setting up our first patch clamp rig and advices on experimental procedures. We thank Dr. Samuel Wu for suggestions on voltage clamp recording. The work is supported by NIH grants EY013811, EY022228 and a vision core grant EY002520. C-KC is the Alice R. McPherson Retina Research Foundation Endowed Chair at the Baylor College of Medicine.

l&#39;approvazione etica - procedure che coinvolgono soggetti animali sono state condotte in conformità con le norme ei regolamenti del National Institutes of linee guida sanitarie per gli animali di ricerca, come approvato dalla cura degli animali istituzionale e usare comitato di Baylor College of Medicine. 1. esterno e delle soluzioni interne <ol><li> Utilizzare la soluzione di Ringer mammiferi durante la dissezione retina e la soluzione esterna nella successiva registrazione elettrofisiologica. Preparare la soluzione di Ringer mammiferi da 10x soluzione madre (senza calcio) il giorno della registrazione, e aggiungere CaCl 2 goccia a goccia dopo 15 min di carbogenation (95% O 2 e il 5% di CO 2). La soluzione finale 1x contiene (in mM): 120 NaCl, KCl 5, 25 NaHCO 3, 0.8 Na 2 HPO 4, 0,1 NaH 2 PO 4, 2 CaCl 2, 1 MgSO 4 e 10 D-glucosio. </li><li> Utilizzare due soluzioni interne per caratterizzare oscillazioni SAC. Testl&#39;utilità di soluzioni preparate in cellule intere registrazioni di patch clamp prolungati sulla RGCs topo adulto e memorizzare i lotti verificati in 500 microlitri aliquote a -20 ° C fino al momento dell&#39;uso. <ol><li> Per la registrazione potenziale di membrana di oscillazione, utilizzare una soluzione interna a base di potassio contenente (in mM): 125 K-gluconato, 8 NaCl, 4 ATP-Mg, 0,5 Li-GTP, 5 EGTA, 10 HEPES e 0,2% biocitina (w / v). Regolare il pH a 7,3 con KOH. </li><li> Per la registrazione eccitatori e correnti postsinaptiche inibitorie, utilizzare una soluzione interna cesio base contenente (in mM): 100 Cs-metansolfonato, 8 NaCl, 4 ATP-Mg, 0,5 Li-GTP, 5 EGTA, 10 HEPES e 0,2% biocitina ( w / v). Regolare il pH a 7,3 con CsOH. </li></ol></li></ol> 2. Preparazione per il giorno di registrazione <ol><li> Per preparare una membrana di nitrocellulosa con fori aperti, punch manualmente la membrana con un perforatore personalizzato fatto da un smussata 16 G ago della siringa e appiattire la membrana tra due lastre di vetro pulite. Per un all&#39;in- grossomontare retina, fare un foro centrale di 0,2 mm di diametro e circondarla da 4 fori più grandi che sono 1-1,2 mm di diametro. </li><li> Per fare un filamento di riempimento personalizzato per caricare soluzione interna in elettrodi, sciogliere un puntale 10 microlitri nel mezzo e allungare delicatamente la parte fusa fino a quando si raffredda e si indurisce. Appena prima dell&#39;uso, utilizzare una lama di rasoio pulito per tagliare il filamento finché è leggermente più lungo le pipette di patch. </li><li> Tirare le pipette di patch in un estrattore programmabile da tubo di vetro borosilicato con un filamento interno. Fabbricazione le micropipette il giorno dell&#39;esperimento e utilizzarli immediatamente. NOTA: per ZSC registrazione, usiamo la seguente impostazione puller: calore: 484; tirare: 0; Velocità: 25; tempo di ritardo: 1; pressione: 400 e un valore di rampa 462. L&#39;OD e ID dei tubi borosilicato sono rispettivamente di 1,65 millimetri e 1,0 mm. La resistenza delle pipette è 10-14 MW. </li><li> Una h prima della raccolta dei tessuti, scongelare un tubo di soluzione interna per shaking su un vortex per&gt; 30 min. </li></ol> 3. Retina Dissection <ol><li> Anestetizzare l&#39;animale del 4% isoflurano in ossigeno fino a quando l&#39;animale perde la reattività di un pizzico dito del piede. Sacrificare l&#39;animale da dislocazione cervicale. </li><li> Tagliare due bulbi oculari fuori i nervi ottici a distanza dalle teste del nervo ottico con Iris rette punte forbici 1-2 mm. </li><li> Lancia i bulbi oculari su un tovagliolo di carta pulito per rimuovere il sangue e poi immergerli in una soluzione carbogenated mammiferi di Ringer. Effettuare un foro sul limbus con un ago G 23 e bisect bulbi oculari tagliando lungo il limbus con micro-forbici. Rimuovere la cornea e la lente utilizzando una pinza sottile. <ol><li> Per retina tutto il montaggio, rimuovere delicatamente tutta la retina fuori dell&#39;epitelio pigmentato con una pinza sottile e fare tagli quattro 1,5-2 mm ortogonali dal bordo verso la testa del nervo ottico. </li></ol></li><li> Immergere una membrana di nitrocellulosa perforato nel piatto e trascinare delicatamente la retina su di esso conil GCL verso l&#39;alto. Mettere la testa del nervo ottico nel foro centrale quando si prepara un tutto il montaggio della retina. Trasferire la membrana della retina in un altro piatto pulito. appiattire delicatamente la retina con un pennello fine per assomigliare ad una croce di Malta e porre i quattro bordi sui fori. <ol><li> Se una frazione della retina è da esaminare alla volta, tagliare ogni eyecup preparati dal punto 3.3 in 3-4 pezzi con una lama di rasoio con l&#39;epitelio pigmentato rimane attaccato. Proteggere i pezzi inutilizzati dalla luce in soluzione esterna carbogenated e utilizzarli entro 12 ore. </li></ol></li><li> Tamponare la membrana di nitrocellulosa con un pezzo di carta da filtro asciutta e rimuovere la membrana vitreo e interna limitativo con una pinza e un pennello. </li><li> Assicurarsi che tutti i bordi retiniche sono completamente attaccati alla membrana prima di trasferire il gruppo nella camera di registrazione con una copertura in vetro sigillato antiscivolo sul fondo. Fissare la membrana con grasso vuoto per la polizza di copertura e rehydratvia e la retina con la soluzione esterna. Non essere attenti a intrappolare eventuali bolle d&#39;aria sotto l&#39;assemblaggio. </li><li> Impostare la camera sulla scena di un microscopio verticale. Profumato la camera con acqua calda (34-35 ° C) soluzione esterna carbogenated ad una velocità di ~ 3 ml al minuto. </li><li> Esaminare la retina sotto una lente obiettivo 10X prima, e poi utilizzare un obiettivo 60x acqua-immersion per vedere i neuroni GCL e INL sotto contrasto interferenziale differenziale (DIC) e / o epifluorescenza. Per visualizzare ZSC tdTomato esprimenti, utilizzare una sorgente di luce bianca in combinazione LED con un filtro di eccitazione di 554 nm e un filtro di emissione di 581 nm. </li></ol> 4. patch clamp whole-cell da Flat-mount Retina <ol><li> Filtrare la soluzione attraverso un filtro interno siringa nel filamento riempimento personalizzato. </li><li> Inserire il filamento in una micropipetta appena spillata e dispensare la soluzione interna vicino alla punta fino a che la soluzione copre il filo elettrodo d&#39;argento per&gt;5 mm. Fissare la micropipetta su un portaelettrodo con un palo di aspirazione, attraverso il quale la pressione all&#39;interno dell&#39;elettrodo può essere regolata premendo o tirando lo stantuffo di una siringa di plastica da 10 ml tubo collegato. </li><li> Trova la pipetta sotto l&#39;obiettivo e portarlo fino a ~ 100 micron sopra la retina. Sotto la modalità corrente-seguace (I = 0), utilizzare offset DC a zero il segnale di tensione continua in piedi. Misurare la resistenza pipetta sotto il morsetto modalità corrente (I morsetto) iniettando corrente ad onda quadra di ampiezza fissa tramite la pipetta mentre è nel bagno e neutralizzare la differenza ruotando la manopola raccess. Utilizzare la lettura sulla manopola raccess per calcolare la resistenza pipetta dalla legge di Ohm. </li><li> Portare lentamente l&#39;elettrodo a ~ 10 micron sopra la retina. Applicare pressione positiva all&#39;elettrodo. Guarda il cambiamento riflessione vicino alla punta della pipetta mentre si avvicina la retina. Rapidamente, ma delicatamente forzare la pipetta nel GCL e ridurre il PRESSU positivori immediatamente. </li><li> Spostare la pipetta verso un neurone etichettati. Evitare il contatto con altri neuroni, vasi sanguigni e delle cellule endfeet Muller. Applicare una pressione più positivo, se necessario, per evitare l&#39;intasamento degli elettrodi. </li><li> Posizionare la punta della pipetta vicino alla linea mediana di un neurone marcato fino a quando una fossetta è visibile. Rilasciare la pressione positiva e lasciare la membrana plasmatica di riprendersi sulla punta della pipetta. </li><li> Applicare 20 a 120 pA correnti negative alla pipetta per aiutare la formazione di una tenuta giga ohm. Applicare una delicata aspirazione se necessario tirare la membrana plasmatica nella pipetta. Attendere 5 min dopo la formazione della guarnizione di rottura della membrana cellulare. In questo modo la soluzione interna versato viene controllata da superfusione. Rompere la membrana da una delicata aspirazione. </li><li> Dopo la rottura della membrana e in modalità morsetto corrente, accendere l&#39;equilibrio ponte e regolarlo con la manopola raccess. NOTA: Per una piccola cella come il SAC, è necessaria solo leggero aggiustamento, se presente. Alterntivamente, registrare eccitatorio e le correnti post-sinaptiche inibitorie in modalità morsetto tensione (V morsetto) tenendo il cellulare a potenziali inversione di cloruro (circa -75 mV) e il glutammato (circa 0 mV), rispettivamente. Controllare e regolare la posizione della pipetta, se necessario, per ospitare retina occasionale e / o micromanipolatore deriva. </li><li> Per registrare potenziale di membrana o variazione di corrente prima, durante e dopo il trattamento farmacologico, preparare bloccanti sinaptiche (ad esempio, CNQX, AP5, picrotoxin, tubocurarina, etc.) e modulatori dei canali (ad esempio, flupirtina, dopamina, acido meclofenamico, ecc) fresco su il giorno dell&#39;esperimento dalle scorte congelati. Diluire i farmaci con la soluzione carbogenated di Ringer. applicarle in maniera lotto attraverso la perfusione. </li><li> Dopo la registrazione, rimuovere delicatamente la pipetta dal soma. Trasferire il gruppo dalla camera di un piatto pulito. Staccare e ricollegare la retina su un altro membrana di nitrocellulosa appiattita senza Hol perforatoES per garantire la planarità della retina durante la fissazione. </li><li> Fissare la retina immergendolo in paraformaldeide al 4% per 30 minuti a RT. Rimuovere la retina dalla membrana di nitrocellulosa e risciacquare a lungo con PBS 1x. Rivela la morfologia delle cellule registrate da O / N colorazione con streptavidina colorante coniugato diluito in PBS 1x con 0,4% Triton X-100 a 4 ° C 14. Montare la retina in media antifade e osservare le cellule registrati utilizzando un microscopio confocale a scansione. NOTA: Le procedure che coinvolgono paraformaldeide, che è volatile e tossica, dovrebbero essere condotte in un progetto da camera per la sicurezza. </li></ol>

<ol>
	<li>Choi, H., 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=Intrinsic+bursting+of+AII+amacrine+cells+underlies+oscillations+in+the+rd1+mouse+retina.">Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina.</a> J Neurophysiol. 112 (6), 1491-1504 (2014).</li><li>Gregg, R. G., 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=Nyctalopin+expression+in+retinal+bipolar+cells+restores+visual+function+in+a+mouse+model+of+complete+X-linked+congenital+stationary+night+blindness.">Nyctalopin expression in retinal bipolar cells restores visual function in a mouse model of complete X-linked congenital stationary night blindness.</a> J Neurophysiol. 98 (5), 3023-3033 (2007).</li><li>Hellmer, C. B., Ichinose, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+light-evoked+postsynaptic+responses+in+neurons+in+dark-adapted,+mouse+retinal+slice+preparations+using+patch+clamp+techniques.">Recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques.</a> J Vis Exp. (96), (2015).</li><li>Tu, H. Y., Bang, A., McQuiston, A. R., Chiao, C. C., Chen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+dendritic+branching+in+direction+selective+retinal+ganglion+cells+in+nob1+mice.">Increased dendritic branching in direction selective retinal ganglion cells in nob1 mice.</a> Invest Ophthalmol Vis Sci. 55 (13), (2014).</li><li>Tu, H. Y., Chen, Y. J., Chiao, C. C., McQuiston, A. R., Chen, C. K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+membrane+potential+fluctuations+of+cholinergic+amacrine+cells+in+mice+lacking+ERG+b-waves.">Rhythmic membrane potential fluctuations of cholinergic amacrine cells in mice lacking ERG b-waves.</a> Invest Ophthalmol Vis Sci. 56 (7), (2015).</li><li>Demas, J., 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=Failure+to+maintain+eye-specific+segregation+in+nob,+a+mutant+with+abnormally+patterned+retinal+activity.">Failure to maintain eye-specific segregation in nob, a mutant with abnormally patterned retinal activity.</a> Neuron. 50 (2), 247-259 (2006).</li><li>Tu, H. Y., Chen, Y. J., McQuiston, A. R., Chiao, C. C., Chen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Retinal+Oscillation+Mechanism+in+an+Autosomal+Dominant+Photoreceptor+Degeneration+Mouse+Model.">A Novel Retinal Oscillation Mechanism in an Autosomal Dominant Photoreceptor Degeneration Mouse Model.</a> Front Cell Neurosci. 9, 513 (2015).</li><li>Borowska, J., Trenholm, S., Awatramani, 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=An+intrinsic+neural+oscillator+in+the+degenerating+mouse+retina.">An intrinsic neural oscillator in the degenerating mouse retina.</a> J Neurosci. 31 (13), 5000-5012 (2011).</li><li>Margolis, D. J., Detwiler, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+origin+of+spontaneous+ganglion+cell+spike+activity+in+animal+models+of+retinitis+pigmentosa.">Cellular origin of spontaneous ganglion cell spike activity in animal models of retinitis pigmentosa.</a> J Ophthalmol. , (2011).</li><li>Stasheff, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+sustained+spontaneous+hyperactivity+and+temporary+preservation+of+OFF+responses+in+ganglion+cells+of+the+retinal+degeneration+(rd1)+mouse.">Emergence of sustained spontaneous hyperactivity and temporary preservation of OFF responses in ganglion cells of the retinal degeneration (rd1) mouse.</a> J Neurophysiol. 99 (3), 1408-1421 (2008).</li><li>Trenholm, S., Awatramani, 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=Origins+of+spontaneous+activity+in+the+degenerating+retina.">Origins of spontaneous activity in the degenerating retina.</a> Front Cell Neurosci. 9, 277 (2015).</li><li>Trenholm, S., 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=Intrinsic+oscillatory+activity+arising+within+the+electrically+coupled+AII+amacrine-ON+cone+bipolar+cell+network+is+driven+by+voltage-gated+Na++channels.">Intrinsic oscillatory activity arising within the electrically coupled AII amacrine-ON cone bipolar cell network is driven by voltage-gated Na+ channels.</a> J Physiol. 590 (Pt 10), 2501-2517 (2012).</li><li>Chen, C. K. J., 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=Cell-autonomous+changes+in+displaced+cholinergic+amacrine+cells+lacking+Gbeta5.">Cell-autonomous changes in displaced cholinergic amacrine cells lacking Gbeta5.</a> Invest Ophthalmol Vis Sci. 56 (7), (2015).</li><li>O'Brien, B. J., Isayama, T., Richardson, R., Berson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+physiological+properties+of+cat+retinal+ganglion+cells.">Intrinsic physiological properties of cat retinal ganglion cells.</a> J Physiol. 538 (Pt 3), 787-802 (2002).</li><li>Lee, S., 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=An+Unconventional+Glutamatergic+Circuit+in+the+Retina+Formed+by+vGluT3+Amacrine+Cells.">An Unconventional Glutamatergic Circuit in the Retina Formed by vGluT3 Amacrine Cells.</a> Neuron. 84 (4), 708-715 (2014).</li><li>Hoggarth, A., 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=Specific+wiring+of+distinct+amacrine+cells+in+the+directionally+selective+retinal+circuit+permits+independent+coding+of+direction+and+size.">Specific wiring of distinct amacrine cells in the directionally selective retinal circuit permits independent coding of direction and size.</a> Neuron. 86 (1), 276-291 (2015).</li><li>Margolis, D. J., Gartland, A. J., Singer, J. H., Detwiler, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+oscillations+drive+correlated+spiking+of+ON+and+OFF+ganglion+cells+in+the+rd1+mouse+model+of+retinal+degeneration.">Network oscillations drive correlated spiking of ON and OFF ganglion cells in the rd1 mouse model of retinal degeneration.</a> PLoS One. 9 (1), e86253 (2014).</li><li>Schmidt, T. M., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isolated+retinal+preparation+to+record+light+response+from+genetically+labeled+retinal+ganglion+cells.">An isolated retinal preparation to record light response from genetically labeled retinal ganglion cells.</a> J Vis Exp. (47), (2011).</li><li>Ivanova, E., Yee, C. W., Sagdullaev, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+phosphorylation+of+Cx36+gap+junctions+in+the+AII+amacrine+cells+of+RD+retina.">Increased phosphorylation of Cx36 gap junctions in the AII amacrine cells of RD retina.</a> Front Cell Neurosci. 9, 390 (2015).</li><li>Enoki, R., Koizumi, 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+method+of+horizontally+sliced+preparation+of+the+retina.">A method of horizontally sliced preparation of the retina.</a> Methods Mol Biol. 935, 201-205 (2013).</li><li>Akrouh, A., Kerschensteiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+function+of+three+VIP-expressing+amacrine+cell+types+in+the+mouse+retina.">Morphology and function of three VIP-expressing amacrine cell types in the mouse retina.</a> J Neurophysiol. 114 (4), 2431-2438 (2015).</li><li>Wei, W., Elstrott, J., Feller, M. B. <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+targeted+recording+of+GFP-expressing+neurons+for+light+responses+and+live-cell+imaging+in+the+mouse+retina.">Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina.</a> Nat Protoc. 5 (7), 1347-1352 (2010).</li></ol>

The authors have nothing to disclose.

Molti laboratori hanno registrato dai neuroni GCL nella preparazione 15-18 TV a monte, ma le nostre procedure consentono la registrazione da neuroni INL. Con la presente sottolineare diversi passaggi che sono fondamentali per le registrazioni di routine di successo. La freschezza e planarità della retina sono importanti per penetrare con una pipetta di registrazione. A questo proposito, il fermo attaccamento della retina alla membrana di nitrocellulosa perforato è fondamentale e si ottiene l&#39;assorbimento transiente della soluzione seguito da reidratazione tempestiva (Passo 3,4-3,6). Durante questo breve periodo, di solito meno di 30 secondi, il vitreo si comporta come una gelatina allentata e può essere staccata. La nostra tecnica è più efficiente rispetto alle diverse procedure pubblicate, dove il vitreo viene rimosso manualmente in soluzione 17,19 o da azioni enzimatiche 18,20. Un altro metodo utilizzato di frequente è quello di strappare la membrana limitante interna off utilizzando un pipetta di patch vuoto per ogni cellada registrare 21,22. Non abbiamo perseguire il metodo strappo per paura di smontaggio della retina. Tuttavia, peeling off vitrea in qualche modo trasparente e gelatinosa può a volte staccare la retina dalla membrana. Si tratta quindi di un passo che richiede pratica la conservazione della retina sulla membrana di nitrocellulosa è essenziale per la registrazione neuroni INL. Una buona pratica è quella di utilizzare la membrana di nitrocellulosa ben appiattita e completamente idratata. Un punto degno di nota qui è l&#39;apparente compromesso tra l&#39;area registrabile (cioè, la dimensione di un foro punzonato), la planarità della retina, e la fermezza di attacco alla membrana. Una finestra di registrazione più grande è preferibile ma un foro punzonato grande significa che c&#39;è area della membrana inferiore per la retina da allegare al. Analogamente, il montaggio della retina sopra un foro più piccolo assicura allegato aperta planarità può essere ridotto, e quindi è area registrabile. Per inserire un elettrodo attraverso il vitreo inil GCL e INL (punto 4.4), si applica una pressione positiva per evitare inceppamenti elettrodo. La riduzione immediata di questa pressione sulla penetrazione nella retina è essenziale anche per ridurre la colorazione di fondo e per preservare oscillazione. Un importante punto di controllo è quello di abbreviare il tempo tra penetrazione e ottenere la tenuta, preferibilmente minore di 30 secondi per i neuroni GCL ed entro 60 secondi per i neuroni INL. Un altro aspetto importante è il periodo di attesa 5 min dopo la formazione del sigillo giga ohm e prima delle rottura della membrana. Questo periodo di attesa assicura la compensazione della soluzione interna versato nel percorso dell&#39;elettrodo ed è particolarmente rilevante quando una soluzione interna base di potassio viene utilizzato perché l&#39;alto contenuto di potassio può depolarizzare temporaneamente neuroni vicini e disturbare oscillazione. Infine, una caratteristica un po &#39;non convenzionale del nostro approccio (Step 4.7) è che ci avviciniamo a un cellulare, formano un sigillo giga ohm, e quindi ottenere l&#39;accesso a cellula intera sotto il mo pinza correntede, come consigliato dal Dr. Rory McQuiston della Virginia Commonwealth University, che ci ha aiutato con le nostre registrazioni elettrofisiologiche iniziali. Questo metodo permette la variazione di resistenza rapido su break-in per essere catturato da cambiamenti di tensione (visibili attraverso un oscilloscopio) e protegge la cellula registrata dalla improvvisa oscillazione del potenziale di membrana a livello di tutta la cellula. Un&#39;altra caratteristica utile del nostro metodo è la corrente negativa applicata alla punta dell&#39;elettrodo, che aiuta ad attirare fosfolipidi di membrana carica positiva e facilita la formazione di tenuta. Per quanto riguarda la conservazione circuiti retinici laterali, una preparazione fette orizzontale della retina 20 è stato suggerito. Mentre questo metodo espone efficacemente i dendriti espansione orizzontale e connessioni sinaptiche nel IPL per la registrazione e l&#39;imaging, il percorso verticale è purtroppo interrotto e quindi questo metodo può essere utilizzato solo per scopi limitati. Infine, le raffinatezze che implementano inprocedure di preparazione dei tessuti e di registrazione consentono di routine mirata registrazioni di neuroni INL, come OFF-ZSC 7. Incorporando l&#39;imaging a due fotoni e contrasto migliorando la microscopia, mira le cellule bipolari e le cellule orizzontali nei pressi dello strato plessiforme esterno per la registrazione in condizioni di illuminazione patch clamp è ora considerato fattibile in un preparato flat-mount.

As an easily accessible part of the central nervous system, the retina has for decades been a useful model in neuroscience studies. Genetic marking of neurons has allowed detailed characterization of synaptic connections in the retina. With many methodologies available to examine function and morphology of retinal neurons, the patch clamp recording technique has been instrumental in our current understanding of vertically transmitted signals in the retina. These signals are originated from photon absorption in photoreceptors and sent to brain visual centers through spiking of retinal ganglion cells (RGCs). Despite a large body of knowledge accumulated thus far, neural diversity in vascularized mammalian retina remains unsolved and obstructs the full appreciation of retinal circuits that subserve normal vision. This is in part because most recordings were performed on retinal slices to trade lateral circuit integrity for access to more proximal retinal neurons1-3. To gain a comprehensive picture on how retina computes visual signals, it is thus desirable to record neurons in flat-mounts wherein lateral connections, large and small, may be better preserved.
When synaptic transmission from photoreceptors to bipolar cells is interrupted due to a defective metabotropic glutamate receptor 6 (mGluR6) signaling pathway in depolarizing bipolar cells4-6 or simply as the result of photoreceptor loss in degenerated retinas7-10, many RGCs exhibit oscillatory activities. These oscillations originate from multiple sources, however the one involving gap junction coupling between AII amacrine cells (AII-ACs) and depolarizing cone bipolar cells (DCBCs) has received the most attention and hence is best understood1,7,11. We have found another source, which persists under pharmacological blockade of the aforementioned AII-AC/DCBC network and drives oscillation of OFF-type SACs in Rho&#916;CTA and Nob mice with deafferentated retinas7,8,12. Here we detail our protocol of preparing retinal flat-mounts for INL neuron recording. This approach uses commercial mouse lines (Jax stock no. 006410 and 007905) to mark cholinergic retinal neurons by fluorescent protein (tdTomato) expression that is identifiable under a fluorescent microscope equipped with contrast enhancing optics. Some experimental results acquired through this approach have been previously reported4,5,7,13.

<table border="0" cellpadding="0" cellspacing="0" width="984">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Fixed-stage fluorescent microscope with DIC</td>
			<td style="width:155px;">Olympus</td>
			<td style="width:219px;">BX51-WI</td>
			<td style="width:419px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Micromanipulators</td>
			<td style="width:155px;">Sutter</td>
			<td style="width:219px;">MP-225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Patch clamp amplifier</td>
			<td style="width:155px;">A-M System</td>
			<td style="width:219px;">AM2400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">AD converter</td>
			<td style="width:155px;">National Instrument</td>
			<td style="width:219px;">NI-USB-6221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Heater controller</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">TC-324B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Inline heater</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">SC-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Peristaltic pump</td>
			<td style="width:155px;">Rainin</td>
			<td style="width:219px;">Dynamax</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">pipette puller</td>
			<td style="width:155px;">Sutter Instrument</td>
			<td style="width:219px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Glass tube with filament</td>
			<td style="width:155px;">King Precision Glass</td>
			<td style="width:219px;">Customized</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Stimulator</td>
			<td style="width:155px;">A.M.P.I.</td>
			<td style="width:219px;">Master-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Biocytin</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">B4261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S6191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5405</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:219px;">BP328-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Na2HPO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaH2PO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S5011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C5670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgSO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">M1880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">D-glucose</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">K-gluconate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">ATP-Mg</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Li-GTP</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G5884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">EGTA</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">E0396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">HEPES</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KOH</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Cs-methanesulfonate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CsOH</td>
			<td style="width:155px;">Sigma</td>
			<td align="right" style="width:219px;">232041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Syringer filter</td>
			<td style="width:155px;">Nalgene</td>
			<td align="right" style="width:219px;">171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">1 ml syring</td>
			<td style="width:155px;">Rainin</td>
			<td align="right" style="width:219px;">17013002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10 ul pipette tip</td>
			<td style="width:155px;">Genesee Scientific</td>
			<td style="width:219px;">24-130RL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Streptavidin-488</td>
			<td style="width:155px;">ThermoFisher</td>
			<td style="width:219px;">S-11223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10X PBS</td>
			<td style="width:155px;">Lonza</td>
			<td style="width:219px;">17-517Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina

La retina dei mammiferi è un tessuto stratificato composto da più tipi di neuroni. Per capire come i segnali visivi vengono elaborati all&#39;interno della propria rete sinaptica intricata, registrazioni elettrofisiologiche sono spesso utilizzati per studiare le connessioni tra i singoli neuroni. Abbiamo ottimizzato una preparazione TV a monte per la registrazione patch clamp di neuroni geneticamente marcate sia in GCL (strato di cellule gangliari) e INL (strato nucleare interno) di retine di topo. Registrazione neuroni INL a Flat-monti è favorita rispetto fette perché le connessioni sia verticali e laterali sono conservati nella ex di configurazione, consentendo circuiti della retina con componenti di grandi dimensioni laterali da studiare. Abbiamo usato questa procedura per confrontare le risposte dei neuroni specchio-partnered in retine come i colinergici cellule amacrine starburst (ZSC).

Registrazioni rappresentativi di on e off-tipo ZSC da una retina deafferentated del mouse sono mostrati in figura 1. Le cellule colinergici sia in GCL e INL può essere attendibilmente identificate da tdTomato fluorescenza e mirati per la cellula intera registrazione patch clamp sotto DIC (Figura 1A) a rivelare l&#39;oscillazione dei loro potenziali di membrana (in alto tracce) e le correnti sinaptiche che la determinano (tracce di fondo, figura 1b). Correnti sinaptiche inibitorie ed eccitatorie sono rivelati tenendo le cellule a 0 mV e -75 mV, rispettivamente in condizioni di clamp di tensione (Figura 1B, tracce di fondo). Tipici dendritiche livelli arborization e stratificazione SAC nel IPL (Figura 1C) possono essere visualizzati dal post-hoc streptavidina colorazione per la biocitina dializzata internamente. La ritmicità e la frequenza del potenziale di membrana fluttuazioni e cambiamenti in corso post-sinaptici puòessere quantificato calcolando la densità spettrale di potenza. Blocco farmacologico, o la sua mancanza, possono essere utilizzati per distinguere diversi meccanismi sinaptici sottostanti potenziale di membrana di oscillazione 7. <img alt="Figura 1" src="/files/ftp_upload/54608/54608fig1.jpg" /> Figura 1. Morfologia e membrane potenziali di bilancio e fuori starburst cellule amacrine nella retina di topo Nob. (A) sia on-ZSC a GCL e OFF-ZSC a INL sono stati facilmente identificati da espressioni tdTomato Cre-driven e mirati per la registrazione sotto DIC. bar scala uguale 20 micron come indicato. (B) Le tracce superiori sono membrane potenziali variazioni registrate dalla cassa e fuori ZSC come indicato. Le tracce inferiori sono rappresentativi corrente post-sinaptica eccitatoria ritmica (EPSC) che guidano l&#39;oscillazione del potenziale di membrana e la corrente post-sinaptica inibitoria aritmico (IPSC). (C) Post hoc istologica caratterizzazione delle cellule registrate indica le tipiche dendritiche livelli morfologia e stratificazione SAC in IPL. Bar scala uguale a 50 micron, come indicato. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54608/54608fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina at JoVE.com

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Questo protocollo dimostra come eseguire cellula intera registrazione patch clamp su neuroni della retina da una preparazione TV a montare.

Patch clamp di celle Starburst amacrine in un appartamento di montaggio Preparazione di Deafferentated mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

patch-clamp-recording-starburst-amacrine-cells-flat-mount-preparation

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.1K Views.  Baylor College of Medicine.  Questo protocollo dimostra come eseguire cellula intera registrazione patch clamp su neuroni della retina da una preparazione TV a montare. 

Video: Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Bloccare Registrazioni patch da neuroni della retina del mouse in un Dark-adattato Preparazione Slice

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Ricerca

Neuroscienze

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Coltura ed Elettrofisiologia di Celle NRCC patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Registrazioni patch clamp in cellule interne dell&#39;orecchio Capelli Isolato da Zebrafish

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; ATPasi Vesicle Preparazione e tecnica per permettere Bloccare Registrazioni patch di membrane delle vescicole submitocondriali

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

Cellula intera Patch Clamp per lo studio dei meccanismi di Infrared Stimolazione Neurale

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Bloccare Registrazioni patch da cellule embrionali Zebrafish Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Registrazione Light-evocato Risposte postsinaptica nei neuroni in Dark-adattati, i preparativi della retina del mouse Slice Utilizzando tecniche di patch clamp

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforato Patch-clamp registrazione di topo neuroni sensoriali olfattivi In neuroepithelium intatto: Analisi funzionale di neuroni Esprimendo un Identificato odorizzante Receptor

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

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Registrazioni Whole-cell patch-clamp nel cervello fette

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Preparazione di orizzontali Fette di topo adulto Retina per elettrofisiologiche Studi

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...