Name: one-channel cell-attached patch-clamp recording
Uploaded: 2014-06-09T14:45:00
Description: Watch this Scientific Journal Video about One-channel Cell-attached Patch-clamp Recording at JoVE.com

Detta arbete stöddes av F31NS086765 (KAC), F31NS076235 (MAP), och R01 NS052669 (GKP) och EIA9100012. Författarna tackar Eileen Kasperek för expertis och hjälp med molekylärbiologi och vävnadskultur; och Jason Myers för att dela data från tidiga prefrontala kortikala neuroner.

1. Cellodling och Protein Expression <ol><li> Bibehåll HEK293-celler (ATCC-nummer CRL-1573) mellan passagerna 22 och 40, som omfattar passagerna utförda av ATCC, i monolagerkultur i DMEM kompletterat med 10% fetalt bovint serum (FBS) och 1% penicillin / streptomycin blandningen vid 5% CO2 och 37 ° C. Mellan experiment, passage celler i T25-kolvar vid 5-20 faldiga späd i en slutlig volym på 10 ml. Anm: Med hjälp av celler under dessa passager garanterar gynnsam cell hälsa som gör det möjligt för op...

<ol>
	<li>Neher, E., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+currents+recorded+from+membrane+of+denervated+frog+muscle+fibres.">Single-channel currents recorded from membrane of denervated frog muscle fibres.</a> Nature. 260, 799-802 (1976).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pflugers Arch. 391, 85-100 (1981).</li><li>Piccolino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+electricity+and+the+birth+of+electrophysiology:+the+legacy+of+Luigi+Galvani.">Animal electricity and the birth of electrophysiology: the legacy of Luigi Galvani.</a> Brain Research Bulletin. 46, 381-407 (1998).</li><li>Albright, T. D., Jessell, T. M., Kandel, E. R., Posner, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Science:+A+Century+of+Progress+and+the+Mysteries+that+Remain.">Neural Science: A Century of Progress and the Mysteries that Remain.</a> Neuron. 25, (2000).</li><li>Popescu, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+glutamate+receptor+gating.">Modes of glutamate receptor gating.</a> The Journal of Physiology. 590, 73-91 (2012).</li><li>Morimoto-Tomita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoinactivation+of+Neuronal+AMPA+Receptors+via+Glutamate-Regulated+TARP+Interaction.">Autoinactivation of Neuronal AMPA Receptors via Glutamate-Regulated TARP Interaction.</a> Neuron. 61, 101-112 (2009).</li><li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> Journal of Pharmacological and Toxicological Methods. 51, 187-200 (2005).</li><li>Huang, Z., Li, G., Pei, W., Sosa, L. A., Niu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+protein+expression+in+single+HEK+293+cells.">Enhancing protein expression in single HEK 293 cells.</a> Journal of Neuroscience Methods. 142, 159-166 (2005).</li><li>Raymond, L. A., Moshaver, A., Tingley, W. G., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor+ion+channel+properties+predict+vulnerability+to+cytotoxicity+in+a+transfected+nonneuronal+cell+line.">Glutamate receptor ion channel properties predict vulnerability to cytotoxicity in a transfected nonneuronal cell line.</a> Mol Cell Neurosci. 7, 102-115 (1996).</li><li>Suchyna, T. M., Markin, V. S., Sachs, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+and+Structure+of+the+Patch+and+the+Gigaseal.">Biophysics and Structure of the Patch and the Gigaseal.</a> Biophysical Journal. 97, 738-747 (2009).</li><li>Qin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+single-channel+currents+using+the+segmental+k-means+method+based+on+hidden+Markov+modeling.">Restoration of single-channel currents using the segmental k-means method based on hidden Markov modeling.</a> Biophys J. 86, 1488-1501 (2004).</li><li>Colquhoun, D., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> chapter in Single-channel recording. 2nd edn eds B. Sakmann and E Neher. , (1995).</li><li>Popescu, G., Auerbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modal+gating+of+NMDA+receptors+and+the+shape+of+their+synaptic+response.">Modal gating of NMDA receptors and the shape of their synaptic response.</a> Nat Neurosci. 6, 476-483 (2003).</li><li>Colquhoun, D., Hawkes, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+properties+of+ion+channel+openings+and+bursts+in+a+membrane+patch+that+contains+two+channels:+evidence+concerning+the+number+of+channels+present+when+a+record+containing+only+single+openings+is+observed.">Stochastic properties of ion channel openings and bursts in a membrane patch that contains two channels: evidence concerning the number of channels present when a record containing only single openings is observed.</a> Proc R Soc Lond B Biol Sci. 240, 453-477 (1990).</li><li>Kussius, C. L., Kaur, N., Popescu, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnanolone+Sulfate+Promotes+Desensitization+of+Activated+NMDA+Receptors.">Pregnanolone Sulfate Promotes Desensitization of Activated NMDA Receptors.</a> J. Neurosci. 29, 6819-6827 (2009).</li><li>Amico-Ruvio, S., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stationary+gating+of+GluN1/GluN2B+receptors+in+intact+membrane+patches.">Stationary gating of GluN1/GluN2B receptors in intact membrane patches.</a> Biophysical Journal. 98, 1160-1169 (2010).</li><li>Borschel, W. F., 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=Gating+reaction+mechanism+of+neuronal+NMDA+receptors.">Gating reaction mechanism of neuronal NMDA receptors.</a> J Neurophysiol. 108, 3105-3115 (2012).</li><li>Colquhoun, D., Hatton, C. J., Hawkes, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quality+of+maximum+likelihood+estimates+of+ion+channel+rate+constants.">The quality of maximum likelihood estimates of ion channel rate constants.</a> The Journal of Physiology. 547, 699-728 (2003).</li><li>Kussius, C. L., Kaur, N., Popescu, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnanolone+Sulfate+Promotes+Desensitization+of+Activated+NMDA+Receptors.">Pregnanolone Sulfate Promotes Desensitization of Activated NMDA Receptors.</a> The Journal of Neuroscience. 29, 6819-6827 (2009).</li><li>Popescu, G., Auerbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modal+gating+of+NMDA+receptors+and+the+shape+of+their+synaptic+response.">Modal gating of NMDA receptors and the shape of their synaptic response.</a> Nat Neurosci. 6, 476-483 (2003).</li><li>Popescu, G., Robert, A., Howe, J. R., Auerbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+mechanism+determines+NMDA+receptor+response+to+repetitive+stimulation.">Reaction mechanism determines NMDA receptor response to repetitive stimulation.</a> Nature. 430, 790-793 (2004).</li><li>Prieto, M. L., Wollmuth, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+Modes+in+AMPA+Receptors.">Gating Modes in AMPA Receptors.</a> The Journal of Neuroscience. 30, 4449-4459 (2010).</li><li>Poon, K., Nowak, L. M., Oswald, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+Single-Channel+Behavior+of+GluA3+Receptors.">Characterizing Single-Channel Behavior of GluA3 Receptors.</a> Biophysical Journal. 99, 1437-1446 (2010).</li><li>Smith, T. C., Wang, L. -. Y., Howe, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+Conductance+Levels+of+Native+AMPA+Receptors.">Heterogeneous Conductance Levels of Native AMPA Receptors.</a> The Journal of Neuroscience. 20, 2073-2085 (2000).</li><li>Coste, B., 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=Piezo1+and+Piezo2+Are+Essential+Components+of+Distinct+Mechanically+Activated+Cation+Channels.">Piezo1 and Piezo2 Are Essential Components of Distinct Mechanically Activated Cation Channels.</a> Science. 330, 55-60 (2010).</li><li>Coste, B., 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=Piezo+proteins+are+pore-forming+subunits+of+mechanically+activated+channels.">Piezo proteins are pore-forming subunits of mechanically activated channels.</a> Nature. 483, 176-181 (2012).</li><li>Benndorf, 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> chapter in Single-channel recording. 2nd edn eds B. Sakmann and E Neher. , (1995).</li></ol>

I jonkanalen fältet, är ett viktigt forskningsområde tillägnad förstå händelseförlopp som leder till kanalöppningen eller kanalens slussmekanismen. För de flesta kanaler, är processen komplex och involverar flera kinetiska steg som inte kan härledas från en makroskopisk flerkanalig signal. Däremot kan experiment utformas där observera sekvensen av öppna / stängda händelser i en kanal rekord kan producera mer detaljerad information om slussmekanismer. I de metoder som beskrivs här, gör det externt bel...

Snabb kommunikation över biologiska membran bygger nästan uteslutande på oligomeric por bildar membranproteiner, som vanligtvis kallas kanaler. Dessa proteiner skiljer sig mycket i aktiveringssignaler, grindmekanismer och konduktans egenskaper. Kanal proteiner vars porer är selektiva för joner klassificeras som jonkanaler; deras aktivering producerar jonströmmar över membranet, och deras svar kan spelas in med hög upplösning i realtid med hjälp av elektrofysiologiska metoder. De aktiveringssignaler spänner över ett brett spektrum av kemiska och fysiska ingångar inklusive koncentrationsgradienter, mekaniska och elektriska krafter, och temperatur; alltså, ytterligare klassificera jonkanaler i ligand gated, mechanosensitive, spänning gated, eller värmekänsliga typer. I den här artikeln, är protokoll beskrivs för att spela in en kanal aktivitet från en ligand gated kanal, NMDA-receptorn, och en mechanosensitive kanal, PIEZO1, med hjälp av patch-clamp teknik.0; Patch-clamp-elektrofysiologi är det första och mest använda experimentella metoden tillräckligt känsligt för att medge observation av enstaka molekyler 1, 2. Förutom denna utsökta känslighet finns det omfattande utökad de biologiska preparat som lämpar sig för elektrofysiologisk registrering och även har tillåtit observationen av jonkanaler i intakta membran. Först, eftersom både spänning fastspänning och ström inspelning åstadkoms med samma elektrod, kan den användas för att spela in signaler över små celler eller membran patchar. Tekniken visade att jonkanaler inte är begränsade till hetsiga membran av groda muskler, ål electroplaques eller bläckfisk jätte axoner 3, 4, utan snarare att de representerar ubiquitous fixturer för transsignaleringsmekanismer och är inneboende i alla cellulära membran typer av uni-eller multicellulära organismer, och även till intracellulära membran. Importoch art, möjlighet att spela in trans strömmar genom att helt enkelt ansluta ett glas pipett till en intakt cell gett helt nya möjligheter att spela in aktivitet från jonkanaler i deras infödda störningsfri membran. Således cellen bifogade patch-clamp teknik, som beskrivs i detta protokoll, tillåter övervakning av aktiviteten av jonkanaler kontinuerligt under tiotals minuter eller längre i sin ursprungliga miljö. Under normala termiska variationer, alla proteiner, inklusive jonkanalsproteiner, genomgå strukturella förändringar över en bred tidsskala, med de snabbaste och mest frekventa omdisponeringar representerade troligen vid sida-kedja rörelser och mycket långsammare, mindre frekventa förändringar som representeras av ompositionering av hela domäner eller subenheter, eller i vissa fall av posttranslationella modifieringar eller protein-proteininteraktioner 5, 6. Observation långa perioder av verksamhet som genereras av en molekyl kan hjälpa till att förstå funktionentionella dynamiken i jonkanaler i intakta fysiologiska membran och ger värdefull information om det operativa system av molekylen observeras. I motsats till den ökande förståelsen av mångfalden av jonkanaler över celltyper och utvecklingsstadier, kunskap om den molekylära sammansättningen av jonkanaler i infödda membran är fortfarande begränsad. Alla jonkanaler är multimera proteiner och majoriteten av inhemska jonkanaler ihop från flera typer av subenheter som producerar proteiner med breda molekylära mångfalden, som ofta åtföljs med olika konduktans och gating egenskaper. Av denna anledning är jonkanaler med definierad molekylär sammansättning studerades vid expression i heterologa system. I synnerhet HEK293-celler, som är en klonal linje av immortaliserade humana embryonala njurceller 7, vunnit bred acceptans som det föredragna systemet för heterolog expression av rekombinanta jonkanaler. Bland manneny fördelar som förhöjda HEK293 celler som valet system för jonkanal elektrofysiologi är den lätthet och överkomliga priser på odling och bibehålla långlivade stabila kulturer, deras förmåga att utföra post-translationell vikning, förädling och handel med däggdjursproteiner, och i många fall , deras låga eller till och med frånvaro av endogen expression för kanal av intresse 7, 8. Att uttrycka rekombinanta jonkanaler och studera deras funktionella egenskaper i HEK293 celler fortsätter att vara en värdefull metod för att få information om struktur-funktionsegenskaper jonkanaler samt specifika egenskaper jonkanal isoformer och deras roller i infödda vävnad. De protokoll som beskrivs i denna artikel kan tillämpas lika väl på rekombinanta jonkanaler som uttrycks i HEK293 celler och infödda jonkanaler. Sammanfattningsvis, för att patch-clamp teknik, genom sin exempellösa förmåga att lösa signals från en molekyl förblir hittills den mest direkta metoden för att observera beteendet hos enskilda molekyler. I sin cell-anslutna läge, gör patch-clamp inspelning långa observationsperioder som, när det görs för en molekyl, kan ge exceptionell insikt i driften av jonkanaler. Nedan presenteras ett protokoll för att få hög upplösning nuvarande inspelningar från cell bifogade plåster innehållande en jonkanal protein.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:189px;">Chemicals</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:128px;">Various</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosillicate Glass</td>
			<td>Sutter</td>
			<td>BF-150-86-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bright field inverted microscope</td>
			<td>Olympus</td>
			<td>1x51</td>
			<td>Nikon also has similar microscopes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluroescent box</td>
			<td>X-cite</td>
			<td>Series 120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Light Guide</td>
			<td>X-cite</td>
			<td>OEX-LG15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS1001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amplifier</td>
			<td>Molecular Devices</td>
			<td colspan="2">Axon Axopatch 200B&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table</td>
			<td>TMC</td>
			<td align="right">63561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NIDAQ card</td>
			<td>National Instruments</td>
			<td>776844-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polisher</td>
			<td>Narishige</td>
			<td>Microforge MF-830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Faraday Cage</td>
			<td>TMC</td>
			<td align="right">8133306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Speed Pressure Clamp</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA HSPC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressue/Vaccuum Pump</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA PV-PUMP</td>
			<td>For HSPC-1</td>
		</tr>
	</tbody>
</table>

one-channel cell-attached patch-clamp recording

Jonkanalsproteiner är universella enheter för snabb kommunikation över biologiska membran. Den tidsmässiga underskrift joniska flux de genererar beror på egenskaperna inneboende i varje kanal protein samt den mekanism genom vilken den genereras och kontrolleras och utgör ett viktigt område av aktuell forskning. Information om de operativa dynamik jonkanalproteiner kan erhållas genom observation av långa sträckor av ström som produceras av en enda molekyl. Beskrivet här är ett protokoll för att erhålla en-kanaliga cell bifogade patch-clamp nuvarande inspelningar för en ligand jonkanal, NMDA-receptorn, uttryckt heterologt i HEK293-celler eller inbyggt i kortikala neuroner. Också är instruktioner om hur man ska anpassa metoden till andra jonkanaler av intresse genom att presentera exempel på mekano känsliga kanal PIEZO1. Denna metod kan ge uppgifter om kanalens konduktans egenskaper och den tidssekvens av OPEn-slutna konformationer som utgör kanalens aktiveringsmekanism, vilket bidrar till att förstå deras funktion vid hälsa och sjukdom.

Rekombinant NMDA-receptorer NMDA-receptorer binder och svara på den åtföljande handlingen av två co-agonister: glutamat och glycin. De monterar som heterotetramerer av två glycin bindande GluN1 subenheter och två glutamat bindande GluN2 subenheter. GluN2 subenheter kodas av fyra gener (AD) och av dessa de mest transkriberade former i hjärnan är GluN2A i vuxen och GluN2B hos unga djur. På grund av mångfalden av NMDA-receptor subtyper hos infödda beredningar, som ut...

Watch this Scientific Journal Video about One-channel Cell-attached Patch-clamp Recording at JoVE.com

One-channel Cell-attached Patch-clamp Recording

Beskrivs här är ett förfarande för att få långa sträckor av aktuell inspelning från en jonkanal med cell bifogade patch-clamp teknik. Denna metod gör det möjligt att observera, i realtid, mönstret av öppen-nära kanalkonformationer som ligger bakom den biologiska signalen. Dessa data informera om kanal fastigheter i ostörda biologiska membran.

En-kanals Cell-ansluten Patch-clamp inspelning

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

one-channel-cell-attached-patch-clamp-recording

Research

JoVE Journal

Biology

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Metoder för Kapacitans Patch Clamp inspelningar från Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Patch Clamp Registrering av jonkanaler Uttryckt i Xenopus oocyter

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

Tryck-polering Pipetter för förbättrad patch-clamp-inspelning

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dela upp och inspelning från The C. elegans neuromuskulära förbindelsen

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Elektrofysiologiska Inspelning i Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

Ambulatoriska EKG-registrering hos möss

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch Clamp och tekniker perfusion för att studera jonkanaler Uttryckt i Xenopus Oocyter

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Exploring arteriella glatta muskel KV7 Funktion kaliumkanal med Elektrofysiologi Patch Clamp och tryck Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

Jätte liposomberedningen för Imaging och Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Beredning av ett transmembranprotein, den spänningskänsliga jonkanaler, KvAP, in Giant enlamellvesiklar för mikroskopi och Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...