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

Bu çalışma F31NS086765 (KAC) tarafından desteklenen, F31NS076235 (MAP), ve R01 NS052669 (GKP) ve EIA9100012. Yazarlar uzmanlık ve moleküler biyoloji ve doku kültürü ile yardım için Eileen Kasperek teşekkür ediyorum; ve Jason Myers erken prefrontal kortikal nöronların elde edilen veri paylaşımı için.

1.. Hücre Kültürü ve Protein Expression <ol><li> HEK293 hücreleri,% 10 Fetal Sığır Serumu (FBS) ile takviye edilmiş DMEM içinde tek tabakalı kültürü, ATCC tarafından gerçekleştirilen parçaları içerir geçitleri 22 ve 40 arasında (ATCC numarası CRL-1573) ve% 5 CO2,% 1 penisilin / streptomisin karışımı tutun ve 37 ° C. Deneyler arasında, 10 ml&#39;lik bir final hacmi içinde, 5-20 kat seyreltmelerde T25 şişeleri içine geçiş hücreleri. Not: Bu geçişleri sırasında hücreler...

<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. 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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>

Iyon kanalı alanında, araştırma önemli bir alan açılmasını veya kanalın yolluk mekanizmasını kanal açan olaylar dizisini anlamak için adamıştır. En çok kanal için, bu işlem karmaşıktır ve gözle görülebilir çok kanallı sinyalden çıkarılabilir edilemez kinetik çeşitli adımlar içerir. Buna karşılık, deneyler tek kanallı kayıt, açık / kapalı olaylar dizisini gözlemleyerek mekanizmaları yolluk hakkında daha detaylı bilgi üretebilir nerede dizayn edilebilir. Minimal invaziv patc...

Biyolojik zarları arasında hızlı iletişim kanalları yaygın olarak adlandırılan oligomerik gözenek oluşturma membran proteinleri, neredeyse sadece dayanır. Bu proteinler aktivasyon sinyalleri, yolluk mekanizmaları ve iletkenlik özellikleri çok farklıdır. Gözenekleri iyonları seçici kanal proteinleri, iyon kanalları olarak sınıflandırılır; onların aktivasyon zarından iyonik akımları üretir ve bunların yanıtları elektrofizyolojik teknikler kullanarak gerçek zamanlı olarak yüksek çözünürlük ile kaydedilebilir. Aktivasyon sinyalleri konsantrasyon dereceleri, mekanik ve elektrik kuvvetleri, ve sıcaklık gibi kimyasal ve fiziksel girdilerin geniş bir dizi kapsar; Böylece, başka bir bağ içine iyon kanallarını sınıflandırmak, kapı mechanosensitive, voltaj kapılı veya ısıya duyarlı türleri. Bu makalede, protokoller patch-clamp tekniği kullanılarak, bir ligand kapılı kanal, NMDA reseptörü ve bir mechanosensitive kanal PIEZO1 gelen tek kanal aktivitesini kaydetmek için tarif edilmiştir.0; Patch-clamp elektrofizyoloji tek moleküllerin 1, 2 gözlem izin vermek için yeterince hassas ilk ve en yaygın olarak kullanılan bir deney yöntemidir. Bu zarif hassasiyete ek olarak, bu çok elektrofizyolojik kayıt için uygun biyolojik hazırlıkları genişletilmiş ve aynı zamanda sağlam zarlarında iyon kanallarının gözlem izin verdi. Gerilim ve akım sıkıştırma kaydı hem de aynı elektrot ile gerçekleştirilir, çünkü İlk olarak, bu küçük hücreler ya da zarlar üzerinde yamalar sinyalleri kaydetmek için kullanılabilir. Teknik iyon kanalları kurbağa kasları, yılan balığı electroplaques veya kalamar dev akson 3, 4, ancak bunun yerine transmembran sinyal mekanizmalarının her yerde demirbaş temsil eden ve tek veya her hücresel membran türlerine özgü olan bunun heyecanlı zarlarına sınırlı olmadığını ortaya koydu çok hücreli organizmalar ve aynı zamanda hücre içi zarlarına. Ithalatantly, sadece sağlam bir hücreye bir cam pipet takarak zar akımları kaydetmek için yeteneği kendi ana undisrupted zarlarında iyon kanalları aktivite kayıt için benzersiz bir fırsat sağladı. Böylece, bu protokol açıklanan hücre bağlı patch-clamp tekniği, on dakika ya da doğal bir ortamda uzun bir süre sürekli olarak iyon kanallarının etkinliği izleme izin verir. Normal termal dalgalanmalardan altında, iyon kanal proteinleri dahil tüm proteinler, yan zincir hareketleri ve bütünün yeniden konumlandırılması tarafından temsil çok daha yavaş, daha az sık değişikliklerden büyük olasılıkla temsil hızlı ve en sık düzenlenmeleri ile, geniş bir zaman ölçeğinde yapısal değişikliklere etki veya alt birimleri veya translasyon sonrası modifikasyon veya protein-protein etkileşimleri 5, 6, bazı durumlarda. Bir molekül tarafından oluşturulan faaliyet uzun süre gözlemleyerek fonk anlamak için yardımcı olabilirtional sağlam fizyolojik zarlarında iyon kanalları dinamikleri ve gözlenen molekülün işletme mekanizması hakkında değerli bilgiler sağlar. Hücre tipleri ve gelişim aşamaları boyunca iyon kanalları, doğal membran iyon kanallarının moleküler bileşimi hakkında bilgi çeşitliliğinin artan anlaşılması aksine hala sınırlıdır. Tüm iyon kanalları multimerik proteinleri ve doğal iyon kanallarının çoğunluğu genellikle çeşitli iletkenlik ve perdeleme özellikleri ile birlikte olan, geniş moleküler çeşitlilik, proteinleri üretme alt birimlerinin çeşitli türlerinden monte edin. Bu nedenle, tanımlanan terkibin moleküler iyon kanalları heterolog sistemlerde tanımlanması üzerine çalışılmıştır. Özellikle, ölümsüzleştirilmiş insan embriyonik böbrek hücreleri 7 klonal çizgi HEK293 hücreleri, yeniden birleştirici iyon kanallarının heterolog ekspresyonu için tercih edilen bir sistem olarak yaygın bir kabul gördü. Adam arasındaiyon kanal elektrofizyoloji için seçim sistemi olarak HEK293 hücreleri yükselmiş y avantajları kolaylığı ve Kültüre uygun fiyatta ve uzun ömürlü istikrarlı kültürlerini koruyarak, post-translasyon katlama, işleme ve memeli proteinlerinin ticaretini yürütmek için kendi yetenek ve birçok durumda , düşük düzeyde veya ilgi duyulan 7, 8, kanalın endojen ekspresyonunun da olmaması. Rekombinant ifade iyon kanallarını ve HEK293 hücrelerinde fonksiyonel özelliklerini inceleyerek yapı-işlev iyon kanalları özellikleri yanı sıra, bir iyon kanalı izoformları ve doğal doku içinde rolleri spesifik özellikleri hakkında bilgi elde etmek için bir değerli bir yaklaşım olmaya devam etmektedir. Bu makalede açıklanan protokoller HEK293 hücrelerinde eksprese edilen rekombinant iyon kanalları ve yerel iyon kanalları için eşit ölçüde iyi uygulanabilir. Özetle, onun eşi görülmemiş kapasitesi ile yama kelepçe tekniği, sinyal çözmek içinbir molekülden s, bugüne kadar, tek moleküllerin davranışını gözlemlemek için en doğrudan yöntem olmaya devam etmektedir. Kendi hücre-bağlı modunda, patch-clamp kayıt bir molekül için tamamlandığı zaman, iyon kanallarının devreye olağanüstü bilgi sağlar, uzun gözlem süreleri sağlar. Aşağıda, bir iyon kanalı protein ihtiva eden hücre bağlı yamalar yüksek çözünürlüklü akım kayıtları elde etmek için bir protokol sunulmuştur.

<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

Iyon kanal proteinleri biyolojik zarların üzerinden hızlı iletişim için evrensel cihazlardır. Ürettikleri iyonik akının zamansal imzası her kanal proteini bunun yanı sıra oluşturulur ve kontrol edildiği mekanizma içsel özelliklerine bağlıdır ve güncel araştırma önemli bir alanını temsil eder. Iyon kanalı proteinlerin işlevsel dinamikleri ile ilgili bilgiler, tek bir molekül tarafından üretilen akımın uzun uzanır gözlemleyerek elde edilebilir. Ligand kapılı iyon kanalı, NMDA reseptörü için bir kanalı, hücre bağlı patch-clamp kayıtları akım elde etmek için bir protokol, aşağıda tarif edilmiştir, kortikal nöronlarda HEK293 hücrelerinde doğal olarak ya da heterolog bir şekilde eksprese. Ayrıca mekano duyarlı kanal PIEZO1 örneğini sunarak ilgi diğer iyon kanallarına yöntemi nasıl adapte talimatları vardır sağladı. Bu yöntem, kanal iletkenlik özellikleri ve ope zamansal dizisi ile ilgili verileri sağlayabilirBöylece sağlık ve hastalık onların işlevlerini anlamak için yardımcı kanalın aktivasyon mekanizmasını oluşturan n-kapalı konformasyonlar.

Rekombinant NMDA Reseptör NMDA reseptörleri bağlayan ve iki eş agonistlerinin birlikte eyleme yanıt: glutamat ve glisin. Bunlar GluN1 alt birimleri ve GluN2 alt birimleri bağlayıcı iki glutamat bağlanması, iki glisin heterotetramers olarak monte edin. GluN2 alt birimleri beyindeki en yaygın formları transkribe çocuk ve yetişkin hayvanlarda GluN2B olarak GluN2A dört gen (MS) ile ve bunlardan kodlanır. Çünkü (Şekil 1A) alt birimi bileşimin...

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

One-channel Cell-attached Patch-clamp Recording

Burada tarif edilen hücre-bağlı patch-clamp tekniği ile, bir iyon kanalının güncel kayıt uzun uzanır elde edilmesi için bir işlemdir. Bu yöntem, gerçek zamanlı olarak, gözlemlemek için izin veren, biyolojik sinyali altında yer open-close kanal konformasyonlarının desen. Bu veriler rahatsız biyolojik membranların kanal özellikleri hakkında bilgilendirmek.

Tek kanallı bir hücre-bağlı Patch-clamp kayıt

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.

Kaliks Patch Kelepçe Kapasitans Kayıtlar Yöntemleri

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

Biyoloji

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.

Xenopus Oosit ifade İyon Kanalları Patch Kelepçe Kaydı

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.

Geliştirilmiş Patch-kelepçe kayıt için Basınç-parlatma Pipetler

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.

C. Elegans Nöromüsküler Kavşağı Diseksiyon ve kayıt

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.

Elektrofizyolojik Kayıt Drosophila Embriyo

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.

Farelerde ambulatuvar EKG Kaydı

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.

İyon Kanalları incelenmesi için yama Kelepçe ve Perfüzyon Teknikleri cinsinden ifade edilir Xenopus Oosit

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.

Patch Clamp Elektrofizyoloji ve Basınç kasılmaların kâğıda çizilmesi kullanarak arteriyel düz kas KV7 Potasyum Kanal Fonksiyon Exploring

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.

Görüntüleme ve Patch-Kelepçe Elektrofizyoloji için dev Liposome Hazırlık

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.

Bir transmembran proteinin yeniden oluşturulması, Mikroskop ve yama kelepçe Çalışmaları Dev tek-katmanlı keseler içine Voltaj kapılı iyon kanalı, KvAP,

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