Wir danken Maila Reh, Tharsana Tharmalingam und vor allem Altina Klein für hervorragende technische Unterstützung. Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft (DFG) (SFB1078 B2, FOR1279 SPP1665 bis PH) und dem Exzellenzcluster Vereinigungskonzepte in der Katalyse, UniCat, BIG-NSE (JV) und E4 (PH) unterstützt.

<ol>
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The authors have nothing to disclose.

Die Bestimmung von Umkehrpotentialen bei definierten ionischen und elektrischen Bedingungen liefert Informationen über die nach der Lichtaktivierung von ChRs transportierten Ionenspezies. Wenn nur eine Ionenspezies in einem komplexen physiologischen Medium variiert wird und die erhaltenen Umkehrpotentialverschiebungen nach dem theoretischen Nernstpotential sind, ist diese Ionensorte die einzige transportierte. Für ChRs sind jedoch Umkehrpotentialverschiebungen in der Regel weniger ausgeprägt als erwartet aus der Nernst-Gleichung aufgrund der Permeabilität gegenüber verschiedenen ionischen Spezies sowie der Konkurrenz unter den geführten Ionen. In diesem Fall wird die Situation komplexer und alle potentiell geführten Ionen müssen separat ausgetauscht werden, um eine Vielzahl von Messlösungen anzufordern. Darüber hinaus sind die meisten CCRs und die ersten künstlichen ACRs 9 , 10 , 21 für Protonen leitend, aber variierende ProtonenkonzentrationErfordert eine besonders gründliche Analyse, da die Protonenkonzentration nicht nur Umkehrpotentiale verschieben kann, sondern auch die Ionenleitfähigkeit 9 , die Photoströme Kinetik 21 , 36 und die spektrale Lichtempfindlichkeit 11 , 37 aufgrund einer Veränderung der Wasserstoffbindungsnetzwerke, des Protonierungszustandes von Individuelle Rückstände und sekundäre Strukturänderungen. Die Protonenleitfähigkeit kann durch Verringerung der Konzentrationen aller anderen potentiell durchgeführten Ionen wie Na &amp; spplus ; , Ca &amp; sub2; &amp; spplus ; oder Cl &amp; rgr; adressiert werden, während der pH-Wert konstant gehalten wird, um die oben erwähnten Proteinveränderungen zu vermeiden. Nicht durchgeführte Substitute sind meist sperrige, geladene Moleküle wie NMG + für Kationen 12 und Gluconat, 2- ( N- Morpholino) ethansulfonat (MES-) oder Aspartat für Anionen. Vergleich von Umkehrpotentialen in verschiedenen iOnic-Lösungen erlaubt dann die Berechnung der relativen Ionenpermeabilitäten bei Gleichgewichtsbedingungen durch die Goldman-Hodgkin-Katz-Spannungsgleichung. Die Quantifizierung des exakten Beitrags verschiedener Ionen zu den Netto-Photostraten unter Berücksichtigung des Ionenkonzers über Gleichgewichtsbedingungen erfordert jedoch komplexe mathematische Modelle 12 , 38 . Die Kenntnis der Zusammensetzung von Ionen, die von ChRs transportiert werden, hilft, mögliche Nebenwirkungen, die sich aus der ChR-Anwendung ergeben, besonders in einem neurophysiologischen Kontext zu erhellen. Zum Beispiel kann eine verlängerte ChR-Aktivierung eine intrazelluläre Säuerung verursachen 39 oder durchgeführt-Ca 2+ kann die synaptische Freisetzung und die zweiten Messengerwege auslösen 40 . Da die Zellen in der Regel fest in Gewebe gepackt sind, kann die Aktivierung von mikrobiellen Rhodopsinen nicht nur die intrazelluläre Ionenzusammensetzung beeinflussen, sondern auch das extrazelluläre Lumen, wodurch nBenachbarten Zellen 41 . Trotz Ionenselektivität sind andere biophysikalische Merkmale von ChRs wie Photostromamplituden, Inaktivierung, Spektral- oder Lichtempfindlichkeit und kinetische Eigenschaften oft von Interesse, um Experimente mit optogenetischen Techniken durchzuführen, zu entwerfen und zu interpretieren. Einige dieser Eigenschaften können auch aus dem oben beschriebenen Protokoll , wie an anderer Stelle 18 , 37 , 42 beschrieben , bestimmt werden . Um die Lichtempfindlichkeit der ChR-exprimierenden Zelle zu untersuchen, kann die Lichtintensität durch Einstellen der Leistung der Lichtquelle oder durch Dämpfen des Aktivierungslichts mit Neutral-Dichte-Filtern variiert werden. Um die spektrale Empfindlichkeit zu erhalten, wird eine polychromatische Lichtquelle benötigt und die Intensitäten müssen auf den gleichen Photonenfluss und die Bandbreite für alle Wellenlängen eingestellt werden. Hochintensitätsspektren ähneln nur Absorptionsspektren des Photorezeptors, wenn kurze Lichtimpulse oderLaser-Blitze werden verwendet, ansonsten können Anpassungsphänomene und photochemische Rückreaktionen auftreten. Die Erholung von teilweise inaktiviertem bis voll aktivem ChR (Gesamt-Photocycle-Umschlagszeit) kann durch Anlegen eines Doppelpuls-Protokolls mit zunehmenden Dunkelintervallen zwischen den beiden Lichtpulsen untersucht werden. Die oben diskutierten biophysikalischen Eigenschaften von ChRs sind wichtig für die Auswahl eines geeigneten ChR, der den experimentellen Bedürfnissen entspricht 40 . Die Ableitung dieser biophysikalischen Eigenschaften durch elektrophysiologische Techniken ähnelt direkt der Proteinfunktion, die kaum durch spektroskopische Methoden abgedeckt ist. Nach erfolgreicher Etablierung von Spannungsklemmmessungen kann die Technik mit Protein-Engineering-Ansätzen wie ortsgerichtete Mutagenese 9 , 11 , 43 , 44 , Helix-Shuffling 37 , <supClass = &quot;xref&quot;&gt; 45, Einführung von Targeting Sequenzen 46 oder Fusion mit anderen Proteinen 41 , 47 . Dies hat das molekulare Wissen über ChRs und ihre Funktionsweise erhöht und eine hohe Diversität von ChR-Varianten mit veränderter Kinetik, Leitfähigkeit, spektraler Empfindlichkeit und Ionenselektivität 40 erzeugt .

Channelrhodopsine (ChR) sind lichtgesteuerte Ionenkanäle, die im Augenfleck von beweglichen Grünalgen vorkommen und als primäre Photosensoren für Phototaxis und phobische Reaktionen dienen 1 . Seit ihrer ersten Beschreibung im Jahr 2002 haben die ChRs den Weg für das entstehende Feld der Optogenetik geebnet und können in einer Vielzahl von erregbaren Zellen angewendet werden, z. B. in Skelettmuskeln, im Herzen oder im Gehirn 3 , 4 , 5 . Die Expression von ChRs in Zielzellen führt zu einer lichtsteuerbaren Ionenpermeabilität der jeweiligen Zelle. In einem neuronalen Kontext ermöglicht dies die Aktivierung 6 , 7 , 8 oder die Hemmung 9 , 10 des Aktionspotentials (AP), die - abhängig vom geführten Ion - mit dem räumlichen und zeitlichen abfeuertPräzision des Lichts, die hervorhebt, wie die Ionenselektivität einer ChR-Variante ihre optogenetische Anwendung bestimmt. Die ersten entdeckten ChRs von Chlamydomonas reinhardtii und Volvox carteri sind für Protonen durchlässig, aber auch für monovalente Kationen wie Natrium, Kalium und in geringerem Maße zu zweiwertigen Kationen wie Calcium und Magnesium 11 , 12 , 13 . Heute sind mehr als 70 natürliche kationenleitende Kanalrhodopsine (CCRs) 14 , 15 , 16 , 17 und mehrere konstruierte Varianten 18 , 19 , 20 mit unterschiedlichen Eigenschaften wie Photostromgröße, spektrale Empfindlichkeit, Kinetik und Kationenselektivität stehen zur Verfügung. Während in der Neurowissenschaften, CCRs aRe verwendet, um Zellen zu aktivieren und APs auszulösen, waren lichtgetriebene mikrobielle Pumpen die einzigen verfügbaren Antagonisten für die Stummschaltung von Neuronen seit Jahren. Im Jahr 2014 zeigten zwei Gruppen gleichzeitig, dass CCRs durch Veränderung der Polarität entlang der putativen Ionen leitenden Pore über molekulare Technik 9 , 21 in anion-leitende Kanalrhodopsine (ACRs) umgewandelt werden können . Anschließend wurden natürliche ACRs in mehreren Kryptophytenalgen 22 , 23 , 24 identifiziert. Am wichtigsten ist, dass die Lichtaktivierung von ACRs Chloridströme in erwachsenen Neuronen vermittelt, was eine Hemmung der neuronalen Aktivität bei viel geringeren Lichtintensitäten ermöglicht als mikrobielle Pumpen, die nur einzelne Ladungen pro absorbiertem Photon transportieren. Die ChR-Aktivität kann direkt durch elektrophysiologische Patch-Clamp-Aufnahmen von lichtinduzierten Strömen in HEK293-Zellen adressiert werden. Die Patch-ClampTechnik wurde ursprünglich in den späten 1970er Jahren entwickelt und weiter verbessert von Hamill et al. , Wodurch die Aufzeichnung der Entität von Strömen aus einer kleinen Zelle (Ganzzellenmodus) mit hoher Stromauflösung und direkter Steuerung der Membranspannung 26 ermöglicht wird. In der Zellkultur angewendet, bietet diese Technik eine genaue Kontrolle sowohl der ionischen als auch der elektrischen Aufzeichnungsbedingungen und ermöglicht die Untersuchung der Ionenselektivität zusammen mit dem relativen Beitrag der Ionen zum Gesamtstrom. Hier veranschaulichen wir die Untersuchung der Ionenselektivität für das anion-leitende Kanalrhodopsin von Proteomonas sulcata ( Ps ACR1) 22 , 23 über die Aufzeichnung von Strom-Spannungs-Beziehungen unter verschiedenen extrazellulären Chlorid-Konzentrationen, um eine hohe Chlorid-Leitfähigkeit zu beweisen.

<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

In den letzten zehn Jahren wurden Channelrhodopsine in der neurowissenschaftlichen Forschung unentbehrlich, wo sie als Werkzeuge zur nicht-invasiven Manipulation elektrischer Prozesse in Zielzellen verwendet werden. In diesem Zusammenhang ist die Ionenselektivität eines Kanalrhodopsins von besonderer Bedeutung. Dieser Artikel beschreibt die Untersuchung der Chloridselektivität für ein kürzlich identifiziertes anion-leitendes Kanalrhodopsin von Proteomonas sulcata über elektrophysiologische Patch-Clamp-Aufnahmen auf HEK293-Zellen. Das experimentelle Verfahren zur Messung von lichtgesteuerten Photoströmen erfordert eine schnell umschaltbare - idealerweise monochromatische - Lichtquelle, die in das Mikroskop eines ansonsten konventionellen Patch-Clamp-Setups eingekoppelt ist. Vorbereitende Verfahren vor dem Experiment werden unter Berücksichtigung von gepufferten Lösungen, Überlegungen über Flüssigkeitsübergangspotentiale, Seeding und Transfektion von Zellen und Ziehen von Patchpipetten skizziert. Die tatsächliche Aufzeichnung der Strom-Spannungs-BeziehungS zur Bestimmung der Umkehrpotentiale für verschiedene Chloridkonzentrationen findet nach der Transfektion 24 h bis 48 h statt. Schließlich werden elektrophysiologische Daten in Bezug auf theoretische Überlegungen zur Chloridleitung analysiert.

<html> Fig. 2 zeigt repräsentative Ergebnisse, die aus Messungen nach dem beschriebenen Protokoll erhalten wurden. Bei der Beleuchtung mit grünem Licht weist der Ps ACR1 einen schnellen Übergangsstrom auf, der schnell auf einen stationären Strompegel abfällt. Nach dem Ausschalten des Lichtes zerfallen die Photoströme innerhalb von Millisekunden auf Null (Abbildung 2A ). Der Austausch der extrazellulären Chloridkonzentration bewirkt eine Verschiebung des Umkehrpotentials, das direkt in den erworbenen Photostromspuren beobachtet werden kann. Die Auswertung von Umkehrpotentialen aus mehreren Messungen quantifiziert die dramatische Umkehrpotentialverschiebung, die durch die Variation der äußeren Chloridkonzentration verursacht wird (Abbildung 2B ). Auffallend entsprechen die gemessenen Umkehrpotentiale direkt den berechneten Nernstpotentialen für Chlorid (Abbildung 2C ), die die hohe Chloridselektivität von Ps ACR1 bestätigen. T &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figur 2" src="/files/ftp_upload/55497/55497fig2.jpg" /> Abbildung 2: Chloridselektivität von Ps ACR1 . ( A ) Repräsentative Stromspuren von Ps ACR1 in HEK293-Zellen. Die intrazelluläre Chloridkonzentration wurde bei 120 mM gehalten, während die extrazelluläre Konzentration von 150 mM (grün) bis 10 mM (lila) Chlorid durch teilweises Ersetzen von NaCl mit Na-Aspartat variierte. Das Haltungspotential wurde in 20 mV Schritten von -80 mV auf +40 mV (LJP korrigiert) erhöht. ( B ) Durchschnittliche Strom-Spannungs-Relationen (n = 6 entsprechend den Bedingungen in A). ( C ) Umkehrpotentiale (Mittellinien zeigen Medianen, Feldgrenzen zeigen die 25. und 75. Perzentile an, wobei die Whisker das 2-fache der Standardabweichung und die Kreise auf einzelne Datenpunkte hindeuten, die aus den Strom-Spannungs-Relationen extrahiert werden. Für die hohe Chlorid-Kon-(Grün) wurde der Wert zwischen -20 mV und 0 mV Haltepotential linear interpoliert, während der Wert linear über +40 mV (lila, gestrichelte Linie, Tafel B) extrapoliert wurde. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55497/55497fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</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.

Ganzzellige Patch-Clamp-Aufnahmen zur elektrophysiologischen Bestimmung der Ionenselektivität in Channelrhodopsinen

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 Aufrufe.  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.

Serie: 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.

Patch-Clamp-Aufnahmen im inneren Haarsinneszellen Isoliert von Zebrafisch

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  ...

Forschung

Neurowissenschaften

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.

Whole Cell Patch-Clamp für erforschen die Mechanismen der Infrarot 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 ...

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.

Isolation von sensorischen Neuronen von<em&gt; Aplysia californica</em&gt; Für Patch-Clamp-Recordings von Glutamaterge Currents

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.

Patch-Clamp-Aufnahmen aus embryonalen Zebrafisch-Mauthner-Zellen

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-Aufnahmen aus Morphologically- und neurochemisch identifizierten Hippocampus Inter

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.

Gepaart ganze Zelle Recordings in Organotypische Hippocampus

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.

Elektrophysiologische und morphologische Charakterisierung neuronaler Schaltkreise in akuten Hirnschnitten Mit Paired-Patch-Clamp-Aufnahmen

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.

Whole-Cell-Patch-Clamp-Aufnahmen in Hirnschnitten

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.

Elektrophysiologische Methode für Ganzzell-Spannungsklemmen Aufnahmen von<em&gt; Drosophila</em&gt; Fotorezeptoren

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.

Auditory Brainstem Response und äußeren Haarzellen Whole-Cell Patch Clamp Aufnahme bei postnatalen Ratten

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 ...