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

Etik onayı - kurumsal hayvan bakımı tarafından onaylanmış ve Baylor College of Medicine komitesini kullanmak gibi hayvan denekleri prosedürleri, kuralları ve araştırma hayvanlar için Sağlık kılavuzların National Institutes düzenlemelerine uygun olarak yapılmıştır. 1. Harici ve Dahili Çözümler <ol><li> retina diseksiyonu sırasında ve sonraki elektrofizyolojik kayıt harici bir çözüm olarak memeli Ringer çözeltisi kullanın. Kayıt günü (kalsiyum olmadan) 10x stok çözeltisinden, memeli Ringer çözeltisi hazırlayın ve carbogenation 15 dakika (% 95 O2 ve% 5 CO2) sonrasında CaCl2 damla damla ilave edin. Nihai 1x solüsyonu (mM cinsinden) şunları içerir: NaHCO 3, 0.8 Na 2 HPO 4, 0.1 NaH 2 PO 4, 2 CaCl2, 1 MgSO 4 ile 10 D-glikoz 120 NaCl, 5 KCl, 25. </li><li> SAC salınımların karakterize etmek için iki dahili çözümleri kullanın. TestYetişkin fare RGCs üzerinde daha uzun süreli tüm hücre yama kelepçe kayıtları hazırlanan çözeltiler yararlılığı ve kullanılana kadar -20 ° C&#39;de 500 ul alikolar içinde belirlenmiş gruplar saklayın. <ol><li> Membran potansiyeli salınım kaydetmek için, (mM olarak) ihtiva eden bir potasyum bazlı içi çözelti kullanımı: 125 K-glukonat, 8 NaCI, 4 ATP mg, 0.5 Li-GTP, 5 EGTA, 10 HEPES ve% 0.2 biocytin (ağırlık / h). KOH ile 7.3 pH ayarlayın. </li><li> (100 CS-metansülfonat, 8 NaCI, 4 ATP mg, 0.5 Li-GTP, 5 EGTA, 10 HEPES ve% 0.2 biocytin: eksitatör inhibe edici postsinaptik akımlar kaydetmek için, (mM olarak) ihtiva eden bir sezyum bir dahili solüsyon kullanmak ağırlık / hacim). CsOH ile 7.3 pH ayarlayın. </li></ol></li></ol> Kayıt Günü için 2. Hazırlık <ol><li> açık delikli bir nitroselüloz membran hazırlamak için, el ile körleşmiş 16 G şırınga iğnesi yapılmış özel bir zımba ile membran yumruk ve iki temiz bir cam levha arasına membran dümdüz. Bir toptan için, Retina montaj çapı merkezi delik 0.2 mm yapmak ve çapı 1-1,2 mm 4 büyük delikler tarafından onu çevreleyen. </li><li> elektrotlar içine iç çözümü yüklenmesi için özelleştirilmiş bir dolgu iplik yapmak için, ortada bir 10 ul pipet eritmek ve soğur ve sertleşir kadar yavaşça erimiş kısmı uzatmak. Hemen önce, kullanmak biraz daha uzun yama pipetler daha oluncaya kadar filamanın kesmek için temiz bir jilet kullanmak için. </li><li> bir iç filament ile borosilikat cam tüp programlanabilir çektirmenin yama pipetler çekin. Deney günü mikropipetler imalatı ve bunları hemen kullanın. NOT: Isı: kayıt BİLSEM&#39;ler için, şu çektirmenin ayarını kullanın 484; çekin: 0; hız: 25; gecikme süresi: 1; Basınç: 400 ve 462. borosilikat tüplerin OD ve kimliği bir rampa değeri sırasıyla 1.65 mm ve 1.0 mm vardır. pipetler direnç 10-14 MQ olduğunu. </li><li> Bir saat doku hasattan önce, Şeki iç çözümün bir tüp çözülme&gt; 30 dakika boyunca bir girdap üzerinde ng. </li></ol> 3. Retina Diseksiyon <ol><li> Hayvan bir ayak tutam yanıt kaybedene kadar oksijen izofluran% 4 ile hayvan anestezisi. servikal dislokasyon ile hayvan kurban. </li><li> uzakta düz sivri iris makası ile optik sinir başları 1-2 mm optik sinirlerin kapalı hem gözbebekleri kesin. </li><li> kan kaldırmak ve daha sonra carbogenated memeli Ringer solüsyonu sokmaktan temiz bir kağıt havlu üzerine gözbebekleri yuvarlayın. 23 G iğne ile limbus üzerinde bir delik ve mikro-makas ile limbusta boyunca keserek gözbebekleri ikiye bölmek. Ince forseps kullanılarak kornea ve lensi çıkarın. <ol><li> retina tam montaj için hafifçe ince forseps ile pigmente epitel kapalı tüm retina soyma ve optik sinir başı doğru kenarından dört 1.5-2 mm dik kesim yapmak. </li></ol></li><li> çanak içine bir delikli nitroselüloz membran daldırın ve hafifçe ile bitti retina sürükleyinGCL taraf yukarı. bir bütün montaj retina hazırlarken orta deliğe optik sinir başını yerleştirin. başka bir temiz kabına retina ile membran aktarın. Yavaşça bir Malta haçı gibi bakmak ve deliklerin üzerine dört kenarı koymak için iyi bir fırça ile retina dümdüz. <ol><li> retina kısmı bir seferde incelenecek olursa, pigment epitel bağlı kaldığı bir jilet ile 3-4 parçaya Aşama 3.3 ile üretilen her Göz yuvası kesti. carbogenated dış çözelti içinde ışıktan kullanılmayan parçalarını koruyun ve 12 saat içinde bunları kullanmak. </li></ol></li><li> Kuru filtre kağıt parçası ile nitroselüloz membran kurulayın ve forseps ile vitreus ve iç sınırlayıcı membran ve bir boya fırçası kaldırın. </li><li> tüm retina kenarları tamamen altta kapalı bir cam kapak kayma ile kayıt odasına aksamını aktarmadan önce zara bağlı olduğundan emin olun. Kapak kayma ve rehydrat vakum gres ile membran sabitleyinDış çözümü ile retina e. montaj altında herhangi bir hava kabarcıkları tuzak dikkatli olun. </li><li> dik bir mikroskop sahneye odasına ayarlayın. dakika başına ~ 3 ml bir oranda sıcak (34-35 ° C) carbogenated dış çözelti ile bölme serpmek. </li><li> diferansiyel girişim kontrast (DIC) ve / veya Epifloresans altında GCL ve INL nöronlar görmek için 60x su daldırma lens kullanabilirsiniz sonra ilk 10x objektif mercek altında retina incelemek ve. tdTomato salgılayan BİLSEM&#39;lere görselleştirmek için, beyaz bir 554 nm&#39;lik bir uyarılma filtreli ve 581 nm&#39;lik bir emisyon filtresi ile birlikte LED ışık kaynağı kullanır. </li></ol> Düz montaj Retina 4. Tüm hücre yama Kelepçe Kayıt <ol><li> Özel bir dolgu filaman bir şırınga filtre ile iç çözelti filtre. </li><li> taze çekilmiş mikropipet içine filamanın yerleştirin ve çözüm&gt; için gümüş elektrot teli kapsayan kadar ucuna yakın iç çözüm dağıtmak5 mm. iterek veya tüp bağlı 10 mi plastik şırınga pistonu çekerek ayarlanabilir elektrodun içinde bu basınç ile bir emme kutuplu bir elektrot tutucu üzerine mikropipet sabitleyin. </li><li> objektif altında pipet bulun ve retina üzerinde ~ 100 um aşağı getirmek. akım-takipçi modu (I = 0) kapsamında, DC ayakta DC gerilim sinyali sıfıra ofset kullanın. O banyoda iken pipet aracılığıyla sabit genlik kare dalga akımları enjekte edilerek mevcut kelepçe (I Kelepçe) modu altında pipet direncini ölçün ve Raccess düğmeyi çevirerek farkı nötralize. Ohm kanunu ile pipet direnci hesaplamak için Raccess topuzu üzerinde okuma kullanın. </li><li> Yavaş yavaş 10 um Retina üzerinde elektroduna ~ getir. elektrot pozitif basınç uygulayın. o retina yaklaştıkça pipet yakın yansıma değişimini izleyin. Çabuk fakat nazikçe GCL içine pipet zorlamak ve pozitif pressu azaltmakhemen yeniden. </li><li> etiketli nöron doğru pipet hareket ettirin. diğer nöronlar, kan damarları ve Muller hücreleri endfeet temastan kaçının. elektrot tıkanmasını önlemek için gerekirse daha pozitif basınç uygulayın. </li><li> Bir çukur görünür olana kadar bir etiketli nöron orta hat yakınında pipet yerleştirin. pozitif basınç serbest bırakın ve plazma zarı pipet üzerine geri sıçrama sağlar. </li><li> Bir Giga-ohm mühür oluşumunu yardım etmek pipet 20 120 pA negatif akımları uygulayın. pipet içine plazma zarı çekmek için gerekirse yumuşak bir emme uygulayın. hücre zarı yırtılmasına mühür oluşumundan sonra 5 dakika bekleyin. Bu dökülen iç çözüm süperfüzyomu tarafından temizlenmesini sağlar. hafif bir emme ile membran rüptürü. </li><li> membran rüptürü sonra ve mevcut kelepçe modunda iken, köprü dengesi üzerinde geçiş ve Raccess düğmesini kullanarak ayarlayın. NOT: Varsa SAC gibi küçük bir hücre için, sadece hafif ayarlama gereklidir. Alternonları bir arada, kayıt uyarıcı ve sırasıyla (0 mV civarında) (-75 mV civarında) klorür ters potansiyelleri hücreyi ve glutamat tutarak gerilim kelepçe modunda (V Kelepçe) inhibitör postsinaptik akımlar. Kontrol ve ara sıra retina ve / veya mikromanipülatör sürüklenme karşılamak için gerekirse pipet konumunu ayarlayın. </li><li> Taze (örn, CNKX, AP5, pikrotoksin, tubokurarin, vs.) ve kanal modülatörleri (ör flupirtin, dopamin, meklofenamik asit, vs.), sinaptik blokerler hazırlanması sırasında veya ilaç tedavisi öncesi, membran potansiyel ya da mevcut bir değişiklik kaydetmek için dondurulmuş stoktan deney günü. carbogenated Ringer solüsyonu ile uyuşturucu seyreltiniz. perfüzyon yoluyla bir toplu şekilde onları uygulayın. </li><li> Kayıttan sonra, yavaşça soma gelen pipet kaldırmak. Temiz bir çanak odasından derleme aktarın. Ayır ve delikli hol olmadan başka basık nitroselüloz zar üzerine retinaya yeniden takmakes fiksasyon sırasında retina düzgünlüğü sağlamak. </li><li> Oda sıcaklığında 30 dakika süreyle% 4 paraformaldehid içinde daldırarak retina sabitleyin. nitroselüloz membran retina çıkarın ve 1x PBS ile yoğun durulayın. 4 ° C, 14% 0.4, Triton X-100 ile 1 x PBS içinde seyreltilmiş boya-konjüge streptavidin ile O / N boyama ile kaydedilen hücre morfolojisini ortaya koyuyor. antifade ortamda retina monte edin ve bir tarama konfokal mikroskop kullanılarak kaydedilen hücreleri gözlemleyin. Not: uçucu ve toksik olan paraformaldehid içeren prosedürler, güvenlik için bir taslak odası içinde yapılmalıdır. </li></ol>

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The authors have nothing to disclose.

Birçok laboratuvar hazırlık 15-18 montaj düz GCL nöronlardan kaydettik, ama bizim prosedürler INL nöronlardan kayıt izin verir. Biz burada başarılı rutin kayıtlar için kritik olan birkaç adım vurgulamak. retina tazelik ve düzlük bir kayıt pipet ile nüfuz için önemlidir. Bu bağlamda, delme nitroselüloz zara retinanın sağlam bağlanma çok önemlidir ve zamanında tekrar nemlendirme (Aşama 3.4-3.6) ve ardından çözelti geçici emme ile elde edilir. Bu kı...

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

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

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

Memeli retina birden nöronal tip oluşan tabakalı bir dokudur. onun karmaşık sinaptik ağ içinde nasıl işlendiğini görsel sinyaller anlamak için, elektrofizyolojik kayıtlar sık ​​sık bireysel nöronlar arasındaki bağlantıları incelemek için kullanılır. Biz fare retina GCL (ganglion hücre tabakası) ve INL hem de genetik olarak işaretlenmiş nöronların yama kelepçe kayıt (iç nükleer tabaka) için düz montaj hazırlık optimize ettik. dikey ve yanal hem de bağlantıları eski yapılandırmasında korunur, çünkü düz bağlar kayıt INL nöronlar incelenecek büyük yanal bileşenleri ile retina devreleri izin dilimleri üzerinde tercih edilmektedir. Biz kolinerjik dolup taşan amacrine hücreleri (keseler) olarak retina ayna ortaklı nöronların cevapları karşılaştırmak için bu yordamı kullandık.

Içi ve bir deafferentated fare retina kapalı tip BİLSEM&#39;lerin Örnek kayıtları, Şekil 1 &#39;de gösterilmiştir. GCL ve INL hem de kolinerjik hücre güvenilir tdTomato floresan ile tanımlanmış ve DIC altında bütün hücre yama kelepçe kayıt için hedef olabilir (Şekil 1 A) onların membran potansiyelleri (üst izleri) ve (alt izleri, Şekil 1B) o sürücü sinaptik akımların salınımını ortaya çıkarmak için. I...

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

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

Bu protokol, düz monte hazırlık retina nöronlar üzerinde tüm hücre yama kelepçe kayıt yapmak gösterilmiştir.

Deafferentated Fare Retina bir düz montaj Hazırlık patlaması amacrine hücreleri Yama Kelepçe Kaydı

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

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

Research

JoVE Journal

Neuroscience

8.1K Görüntüleme.  Baylor College of Medicine.  Bu protokol, düz monte hazırlık retina nöronlar üzerinde tüm hücre yama kelepçe kayıt yapmak gösterilmiştir. 

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

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

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

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

Patch Kelepçe bir Karanlık adapte Dilim Hazırlık Fare Retina Nöronlar Kayıtlar

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

Nörobilim

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

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

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

Hücreler NRCC Patch-klemp Chips kültür ve Elektrofizyoloji

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

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

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

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

İç Kulak Saç Hücreleri Patch Clamp Kayıtlar İzole Zebrafish gelen

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

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

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

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

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

<sub&gt; 1</sub&gt; F<sub&gt; O</subSubmitochondrial veziküllü Membranların Patch Kelepçe Kayıtlar Sahne&gt; ATPaz veziküllü Hazırlık ve Tekniği

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

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

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

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

Kızılötesi Sinir Stimülasyon en mekanizmaları incelenmesi için tüm Hücre Patch Kelepçe

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

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

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

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

Embriyonik Zebra balığı Mauthner Hücreler Patch Kelepçe Kayıtlar

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

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

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

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

Kayıt Patch Kelepçe Teknikleri Kullanarak Koyu adapte Fare Retina Dilim Hazırlıkları Nöronlar içinde postsinaptik Yanıtları Işık-uyarılmış

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

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

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

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

Bozulmamış neuroepithelium Fare Koku Duyu Nöronlar Delikli Patch-kelepçe Kayıt: Bir Belirlenen Odorant Reseptör ifade Nöronlar Fonksiyonel Analiz

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

Whole-cell Patch-clamp Recordings in Brain Slices

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

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

Beyin Dilimleri tüm hücre Patch-kelepçe Kayıtlar

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

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

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

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

Elektrofizyolojik Çalışmalar Yetişkin Fare Retina Yatay Dilimleri hazırlanması

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