Ringraziamo Maila Reh, Tharsana Tharmalingam e specialmente Altina Klein per un&#39;ottima assistenza tecnica. Questo lavoro è stato sostenuto dalla Fondazione di Ricerca Tedesca (DFG) (SFB1078 B2, FOR1279 SPP1665 a PH) e il Cluster of Excellence Unifying Concepts in Catalysis, UniCat, BIG-NSE (JV) e E4 (PH).

<ol>
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The authors have nothing to disclose.

Determinazione dei potenziali di inversione a condizioni ioniche e elettriche definite fornisce informazioni sulle specie ioniche trasportate dopo l&#39;attivazione luminosa dei ChRs. Se solo una specie di ioni varia in un complesso complesso fisiologico e il potenziale di inversione ottenuto si sposta in base al potenziale teorico di Nernst, questa specie ione è l&#39;unico trasportato. Tuttavia, per i ChRs, i cambi potenziali di inversione sono generalmente meno pronunciati di quelli previsti dall&#39;equazione di Nernst dovuti alla permeabilità alle diverse specie ioniche e alla concorrenza tra gli ioni condotti. In questo caso, la situazione diventa più complessa e tutti gli ioni potenzialmente condotti devono essere scambiati separatamente richiedendo una varietà di soluzioni di misura. Inoltre, la maggior parte dei CCR e dei primi ACR artificiali 9 , 10 , 21 sono conduttivi per i protoni, ma la concentrazione protonica variaL&#39;analisi richiede un&#39;analisi particolarmente approfondita, in quanto la concentrazione di protoni non solo può spostare le potenzialità di inversione, ma può anche influenzare la conducibilità ionica 9 , le cinetiche fotocorrenti 21 , 36 e la sensibilità spettrale di luce 11 , 37 , a causa del cambiamento di idrogeno, Singoli residui e cambiamenti della struttura secondaria. La conducibilità del protone può essere affrontata riducendo le concentrazioni di tutti gli altri ioni potenzialmente condotti come Na + , Ca 2+ o Cl - mantenendo costante il pH al fine di evitare variazioni proteiche menzionate sopra. I sostituti non condotti sono solitamente molecole ingombranti e cariche come NMG + per cationi 12 e gluconato, 2- ( N- morfolino) etansolfonato (MES) o aspartato per anioni. Confrontando i potenziali di inversione in differenti iLe soluzioni oniche consentono quindi di calcolare le relative permeabilità di ioni in condizioni di equilibrio dall&#39;equazione di tensione Goldman-Hodgkin-Katz. Quantificando l&#39;esatto contributo di ioni diversi alle fotocorri nette considerando la concorrenza ionica al di là delle condizioni di equilibrio, tuttavia, richiede complessi modelli matematici 12 , 38 . Conoscere la composizione degli ioni trasportati da ChR aiuta a chiarire eventuali effetti collaterali derivanti dall&#39;applicazione di ChR in particolare in un contesto neurofisiologico. Ad esempio, l&#39;attivazione prolungata di ChR può causare l&#39;acidificazione intracellulare 39 o condotta-Ca 2+ può innescare il rilascio sinaptico e il secondo percorso messaggero 40 . Poiché le cellule sono di solito imballate strettamente all&#39;interno dei tessuti, l&#39;attivazione delle rodopinte microbiche non può solo influenzare la composizione intracellulare di ioni, ma anche il lumen extracellulare, influenzando così nCellule ebbrezie 41 . Nonostante la selettività di ioni, altre caratteristiche biofisiche di ChR come ampiezze di fotocolore, inattivazione, sensibilità spettrale o leggera e proprietà cinetiche sono spesso di interesse per condurre, progettare e interpretare esperimenti che coinvolgono tecniche ottogenetiche. Alcune di queste proprietà possono anche essere determinate dal protocollo sopra descritto come descritto altrove 18 , 37 , 42 . Per studiare la sensibilità alla luce della cella espressa da ChR, l&#39;intensità della luce può essere variata regolando la potenza della sorgente luminosa o attenuando la luce di attivazione con filtri di densità neutra. Per ottenere la sensibilità spettrale, è necessaria una sorgente luminosa poligromatica e le intensità devono essere regolate a flusso di foton equivalente e larghezza di banda per tutte le lunghezze d&#39;onda. Gli spettri ad alta intensità assomigliano solo agli spettri di assorbimento del fotorecettore in caso di impulsi di luce corta oVengono utilizzati lampi laser, altrimenti possono verificarsi fenomeni di adattamento e reazioni fotochemiche. Il recupero da parte inattivato in modo completamente attivo (tempo totale di rotazione del fotociclo totale) può essere verificato applicando un protocollo a doppio impulso con incrementi di intervalli scuri tra i due impulsi luminosi. Le proprietà biofisiche dei ChRs discussi in precedenza sono importanti per la scelta di un appropriato ChR corrispondente alle esigenze sperimentali 40 . Derivare queste proprietà biofisiche con tecniche elettrofisiologiche si associa direttamente alla funzione proteica che è appena coperta da metodi spettroscopici. Dopo aver stabilito con successo le misurazioni della tensione-tensione, la tecnica può essere combinata con approcci di ingegneria proteica come la mutagenesi localizzata 9 , 11 , 43 , 44 , 37 , <supClass = &quot;xref&quot;&gt; 45, introduzione di sequenze di targeting 46 o fusione con altre proteine 41 , 47 . Ciò ha aumentato la conoscenza molecolare dei ChRs e del loro modo di operare e ha creato un&#39;elevata diversità di varianti ChR con alterata cinetica, conducibilità, sensibilità spettrale e selettività ionica 40 .

Channelrhodopsins (ChR) sono canali ionici leggeri che si verificano nel punto di occhio delle alghe verdi motile e servono come fotosensori primari per fototassi e risposte fobiche 1 . Dalla loro prima descrizione nel 2002 2 , i ChRs hanno aperto la strada per il campo emergente dell&#39;ottogenetica e possono essere applicati in una varietà di cellule eccitabili, ad es. Nei muscoli scheletrici, nel cuore o nel cervello 3 , 4 , 5 . L&#39;espressione di ChRs nelle cellule bersaglio produce una permeabilità ionica controllabile dalla luce della rispettiva cellula. In un contesto neuronale, questo consente l&#39;attivazione 6 , 7 , 8 o inibizione 9 , 10 di cottura del potenziale d&#39;azione (AP) - a seconda dello ione condotto - con lo spazio e temporaliPrecisione della luce sottolineando come la selettività ionica di una variante ChR determina l&#39;applicazione ottogenetica. I primi ChRs scoperti da Chlamydomonas reinhardtii e Volvox carteri sono permeabili a protoni, ma anche a cationi monovalenti come il sodio, il potassio e, in misura minore, a cationi bivalenti come il calcio e il magnesio 11 , 12 , 13 . Oggi, più di 70 cantipolisini naturali (CCR) 14 , 15 , 16 , 17 e varie varianti progettate 18 , 19 , 20 con proprietà diverse come La dimensione fotocirrente, la sensibilità spettrale, la cinetica e la selettività del cation sono disponibili. Mentre nella neuroscienza, CCRs aUtilizzati per attivare le cellule e attivare AP, le pompe microbiche a luce guidata sono stati gli unici antagonisti disponibili per annientare i neuroni. Nel 2014, due gruppi hanno dimostrato simultaneamente che i CCRs possono essere convertiti in channelrhodopsins (ACR) di anion-conducting mediante alterazione della polarità lungo il poro conducente ionico attraverso l&#39;engineering molecolare 9 , 21 . Successivamente, ACR naturali sono stati identificati in diverse alghe di crittophyte 22 , 23 , 24 . Più importante, l&#39;attivazione leggera di ACR mediata dalle correnti di cloruro nei neuroni adulti permette di inibire l&#39;attività neuronale a intensità luminosa molto minori rispetto alle pompe microbiche che trasportano solo singole cariche per fotone assorbito. L&#39;attività di ChR può essere direttamente affrontata mediante registrazioni elettrofisiologiche di patch-clamp di correnti indotte da luce nelle cellule HEK293. La pinzettaLa tecnica è stata originariamente sviluppata alla fine degli anni &#39;70 25 e ulteriormente migliorata da Hamill et al. , Consentendo la registrazione dell&#39;entità delle correnti da una piccola cella (modalità di intera cellula) con alta risoluzione di corrente e controllo diretto della tensione della membrana 26 . Applicata nella coltura cellulare, questa tecnica fornisce un preciso controllo delle condizioni di registrazione ioniche e elettriche e consente di studiare la selettività degli ioni insieme al relativo contributo degli ioni alla corrente totale. Qui esemplifica l&#39;esame della selettività ionica per il channelrhodopsin di Anion -conducting di Proteomonas sulcata ( Ps ACR1) 22 , 23 attraverso la registrazione di relazioni di tensione-corrente in varie concentrazioni di cloruro extracellulare per dimostrare un&#39;elevata conduttanza di cloruro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >HEK293 cells</td>
			<td >Sigma Aldrich</td>
			<td >85120602</td>
			<td >Human embryonic kidney cells</td>
		</tr>
		<tr >
			<td >Retinal</td>
			<td >Sigma Aldrich</td>
			<td>R2500</td>
			<td>all-trans retinal</td>
		</tr>
		<tr >
			<td >FuGENE HD</td>
			<td >Promega</td>
			<td>E2312</td>
			<td>Transfection reagent</td>
		</tr>
		<tr >
			<td >DMEM</td>
			<td >Biochrome</td>
			<td>FG 0445</td>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
		</tr>
		<tr >
			<td >Agarose</td>
			<td >Roth</td>
			<td>3810</td>
			<td>Agar bridges</td>
		</tr>
		<tr >
			<td >CaCl2</td>
			<td >Roth</td>
			<td>5239</td>
			<td>CaCl2 2H2O</td>
		</tr>
		<tr >
			<td >CsCl</td>
			<td >Biomol</td>
			<td>2452</td>
			<td></td>
		</tr>
		<tr >
			<td >EGTA</td>
			<td >Roth</td>
			<td>3054</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS</td>
			<td >Biochrome</td>
			<td>S0615</td>
			<td>Cell culture</td>
		</tr>
		<tr >
			<td >Glucose</td>
			<td >Roth</td>
			<td>HN06</td>
			<td>D(+)-Glucose</td>
		</tr>
		<tr >
			<td >KCl</td>
			<td >Roth</td>
			<td>6781</td>
			<td></td>
		</tr>
		<tr >
			<td >MgCl2</td>
			<td >Roth</td>
			<td>2189</td>
			<td>MgCl2 6H2O</td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >Roth</td>
			<td>3957</td>
			<td></td>
		</tr>
		<tr >
			<td >NMG</td>
			<td >Sigma Aldrich</td>
			<td>M2004</td>
			<td>N-Methyl-D-glucamine</td>
		</tr>
		<tr >
			<td >Na-Aspartate</td>
			<td >Sigma Aldrich</td>
			<td>A6683</td>
			<td>L-Aspartic acid sodium salt monohydrate</td>
		</tr>
		<tr >
			<td >Citric acid</td>
			<td >Roth</td>
			<td>6490</td>
			<td></td>
		</tr>
		<tr >
			<td >AgeI</td>
			<td >ThermoFischerScientific</td>
			<td>ER1462</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr >
			<td >XhoI</td>
			<td >ThermoFischerScientific</td>
			<td>ER0695</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr >
			<td >NheI</td>
			<td >ThermoFischerScientific</td>
			<td>ER0975</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr >
			<td >XL1Blue E.coli/</td>
			<td >Agilent Technologies</td>
			<td>200249</td>
			<td>Chemocompetent E.coli</td>
		</tr>
		<tr >
			<td >Kanamycin</td>
			<td >Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr >
			<td >Lysogeny broth medium</td>
			<td >Roth</td>
			<td>X964</td>
			<td></td>
		</tr>
		<tr >
			<td >Agar-Agar</td>
			<td >Roth</td>
			<td>6494</td>
			<td>Agar plates</td>
		</tr>
		<tr >
			<td >Plasmid purification kit</td>
			<td >Marchery-Nagel</td>
			<td>740727.25</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicilin/Streptomycin</td>
			<td >Biochrome</td>
			<td>A 2213</td>
			<td>Cell culture</td>
		</tr>
		<tr >
			<td >Poly-D-lysine hydrobromide</td>
			<td >Sigma Aldrich</td>
			<td>P6407-5MG</td>
			<td>Cover slip coating</td>
		</tr>
		<tr >
			<td >Microforge</td>
			<td >Custom made</td>
			<td></td>
			<td>Fire polishing</td>
		</tr>
		<tr >
			<td >Serological pipettes</td>
			<td >TPP</td>
			<td></td>
			<td>Different sizes</td>
		</tr>
		<tr >
			<td >Clean bench</td>
			<td >Kojair</td>
			<td>Biowizard SL130</td>
			<td></td>
		</tr>
		<tr >
			<td >Stirrer</td>
			<td >IKA</td>
			<td>RCT classic</td>
			<td></td>
		</tr>
		<tr >
			<td >Silver wire</td>
			<td >Science Products</td>
			<td>AG-T25; AG-T10</td>
			<td>Electrodes, 0.64 mm (bath); 0.25 mm (electrode)</td>
		</tr>
		<tr >
			<td >pH-meter</td>
			<td >Knick</td>
			<td>765 Calimetric</td>
			<td></td>
		</tr>
		<tr >
			<td >Osmometer</td>
			<td >Vogel</td>
			<td>OM 815</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td >Carl Zeiss</td>
			<td>ID03</td>
			<td>Fire polishing</td>
		</tr>
		<tr >
			<td >CO2 incubator</td>
			<td >Binder</td>
			<td>CB150</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell culture dishes</td>
			<td >TPP</td>
			<td>93040</td>
			<td>34 mm internal diameter</td>
		</tr>
		<tr >
			<td >Cover slips</td>
			<td >Roth</td>
			<td>P232</td>
			<td>15 mm diameter</td>
		</tr>
		<tr >
			<td >Thermometer</td>
			<td >R&#246;ssel Messtechnik</td>
			<td>MTM12</td>
			<td></td>
		</tr>
		<tr >
			<td >Beamsplitter</td>
			<td >Chroma</td>
			<td>21011</td>
			<td>90/10 transmission</td>
		</tr>
		<tr >
			<td >Pipette holder</td>
			<td>ALA Scientific Instruments</td>
			<td>PPH-1P-AXU-0-1.5</td>
			<td></td>
		</tr>
		<tr >
			<td >Headstage</td>
			<td >Molecular Devices</td>
			<td>CV203BU</td>
			<td></td>
		</tr>
		<tr >
			<td >Amplifier</td>
			<td >Molecular Devices</td>
			<td >AxoPatch200B</td>
			<td></td>
		</tr>
		<tr >
			<td >Digitizer</td>
			<td >Molecular Devices</td>
			<td >DigiData1400</td>
			<td>Digital analog converter</td>
		</tr>
		<tr >
			<td >Lightsource</td>
			<td >TILL Photonics</td>
			<td >Polychrome V</td>
			<td>Set to 540 nm full intensity</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td >Carl Zeiss</td>
			<td >Axiovert 100</td>
			<td></td>
		</tr>
		<tr >
			<td >Shutter</td>
			<td >Vincent Associates</td>
			<td >VS25</td>
			<td></td>
		</tr>
		<tr >
			<td >Shutter driver</td>
			<td >Vincent Associates</td>
			<td >VCM-D1</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass capilarries</td>
			<td >Warner Instruments</td>
			<td>G150F-3</td>
			<td >Boresilicate capillaries with fire polished ends OD 1.5 mm ID&#160; 0.86 mm</td>
		</tr>
		<tr >
			<td >Micropipette puller</td>
			<td >Sutter Instruments</td>
			<td >P1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Bath handler</td>
			<td >Lorenz Messger&#228;tebau</td>
			<td >MPCU</td>
			<td></td>
		</tr>
		<tr >
			<td >Tripleband filterset</td>
			<td >Chroma</td>
			<td>69008</td>
			<td>Fluorescence filter&#160; ECFP/EYFP/mCherry&#160;</td>
		</tr>
		<tr >
			<td >CCD camera</td>
			<td >Watec</td>
			<td >Wat-221SCCD</td>
			<td></td>
		</tr>
		<tr >
			<td >Optometer</td>
			<td >Gigahertz Optik</td>
			<td >P9710</td>
			<td>Measure light intensities</td>
		</tr>
		<tr >
			<td >Objective</td>
			<td >Carl Zeiss</td>
			<td>421462-9900-000</td>
			<td >W Plan-Apochromat 40X/1.0 DIC</td>
		</tr>
		<tr >
			<td >Micromanipulator</td>
			<td >Scientifica</td>
			<td>PatchStar</td>
			<td></td>
		</tr>
		<tr >
			<td >Recording chamber</td>
			<td >Custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Power supply</td>
			<td >Manson</td>
			<td>HCS-3202</td>
			<td>Avoids electrical noise from microscope built-in power supply</td>
		</tr>
		<tr >
			<td >Vibration isolated table</td>
			<td >Newport</td>
			<td>M-VW-3636-OPT-01</td>
			<td></td>
		</tr>
		<tr >
			<td >Faraday cage</td>
			<td >Custom made or any commercial matching table</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Hoses</td>
			<td >Any comercial; e.g. Roth</td>
			<td></td>
			<td>Different sizes and materials for bath handling and application of pipette pressure; agar bridges</td>
		</tr>
		<tr >
			<td >Linear shaker</td>
			<td >Sunlab Instruments</td>
			<td>SU 1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Liquid junction potential calculator</td>
			<td >Molecular Devices or directly from Peter H. Barry</td>
			<td></td>
			<td>Program is included in the Clampex aquisition software or can be obtained from&#160; p.barry@unsw.edu.au</td>
		</tr>
		<tr >
			<td >Data acquisition software</td>
			<td >Molecular Devices</td>
			<td>Clampex 10.X</td>
			<td></td>
		</tr>
		<tr >
			<td >Data evaluation software</td>
			<td >Molecular Devices</td>
			<td>Clampfit 10.X</td>
			<td></td>
		</tr>
		<tr >
			<td >PsACR1</td>
			<td>GenBank or Addgene</td>
			<td >KF992074.1 or Addgene plasmid #85465</td>
			<td>Gene encoding for PsACR1</td>
		</tr>
		<tr >
			<td >Amplifier guide</td>
			<td >Molecular Devices</td>
			<td>The Axon Guide</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins

Negli ultimi dieci anni, i channelrhodopsins sono diventati indispensabili nella ricerca neuroscientificale, dove vengono utilizzati come strumenti per manipolare in modo non invasivo i processi elettrici nelle cellule bersaglio. In questo contesto, la selettività ionica di un channelrhodopsin è di particolare importanza. In questo articolo si descrive l&#39;analisi della selettività di cloruro per una scanografia di Proteomonas sulcata recentemente identificata da anion-conducting channel mediante protezioni elettrofisiologiche di patch-clamp sulle cellule HEK293. La procedura sperimentale per misurare le fotocorri luminose richiede una sorgente luminosa rapida - idealmente monocromatica - accoppiata nel microscopio di una configurazione di patch-clamps altrimenti convenzionale. Sono state descritte procedure preparative prima dell&#39;esperimento che prevedono la preparazione di soluzioni tamponate, considerazioni sui potenziali di giunzione liquidi, la semina e la trasfezione delle cellule e la trazione delle pipette patch. La registrazione effettiva della relazione di tensione-correnteS per determinare le potenzialità di inversione per diverse concentrazioni di cloruro si svolge 24 h a 48 h dopo la trasfezione. Infine, i dati elettrofisiologici vengono analizzati rispetto alle considerazioni teoriche della conduzione di cloruro.

<html> La Figura 2 mostra i risultati rappresentativi ottenuti dalle misurazioni seguendo il protocollo descritto. Durante l&#39;illuminazione con luce verde, Ps ACR1 presenta una corrente transitoria veloce che decade rapidamente a un livello corrente fisso. Dopo la spegnimento della luce, le fotocorrienti decadono a zero entro millisecondi ( Figura 2A ). Scambiare la concentrazione di cloruro extracellulare provoca uno spostamento del potenziale di inversione che può essere visto direttamente nelle tracce acquisite di fotocirato. La valutazione dei potenziali di inversione da molteplici misurazioni quantifica il drammatico spostamento del potenziale di inversione causato dalla variazione della concentrazione di cloruro esterno ( Figura 2B ). Attentamente, i potenziali di inversione misurati corrispondono direttamente ai potenziali Nernst calcolati per il cloruro ( Figura 2C ) che confermano l&#39;alta selettività di cloruro di Ps ACR1. T &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="figura 2" src="/files/ftp_upload/55497/55497fig2.jpg" /> Figura 2: Selettività del cloruro di Ps ACR1 . ( A ) Tracce correnti rappresentative di Ps ACR1 nelle cellule HEK293. La concentrazione di cloruro intracellulare è stata mantenuta a 120 mM, mentre la concentrazione extracellulare varia da 150 mM (verde) a 10 mM (viola) cloruro per parziale sostituzione di NaCl con Na-aspartato. Il potenziale di mantenimento è aumentato a 20 mV da -80 mV a +40 mV (corretto LJP). ( B ) Rapporti di corrente-tensione mediati (n = 6 corrispondenti alle condizioni in A). ( C ) Potenziali di inversione (le linee centrali indicano i mediani, i limiti della casella indicano i percentili 25 e 75, i baffi estendono 2 volte la deviazione standard ei cerchi indicano punti dati singoli) estratti dalle relazioni di tensione di corrente. Per l&#39;alto cloruro con(Verde) il valore è stato interpolato linearmente tra potenziale di mantenimento -20 mV e 0 mV, mentre il valore è stato estrapolato linearmente superiore a +40 mV (viola, linea tratteggiata, pannello B). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55497/55497fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Registrazioni patch-clamp intere cellule per la determinazione elettrofisiologica della selettività ionica in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

whole-cell-patch-clamp-recordings-for-electrophysiological

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.0K Views.  Humboldt-Universität zu Berlin. This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Video: Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

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  ...

Ricerca

Neuroscienze

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 ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolamento dei neuroni sensoriali<em&gt; Aplysia californica</em&gt; Per Bloccare Registrazioni patch di correnti glutamatergico

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Whole-cell patch-clamp registrazioni da Morphologically- e neurochimico-identificati interneuroni dell&#39;ippocampo

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Associati a cellule intere registrazioni in organotipiche fettine di ippocampo

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Elettrofisiologici e morfologica Caratterizzazione neuronali Microcircuiti in Acute Fette cervello Utilizzando accoppiati di patch-clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

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 ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Metodo elettrofisiologico per le registrazioni a morsetto di tensione a cellule intere da<em&gt; Drosophila</em&gt; Fotorecettori

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

Risposta uditiva del tronco cerebrale ed esterna della cellula ciliata cellule intere Patch Clamp registrazione in ratti postnatali

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...