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.

<table><tbody><tr><td>Fixed-stage fluorescent microscope with DIC</td><td>Olympus</td><td>BX51-WI</td><td></td></tr><tr><td>Micromanipulators</td><td>Sutter</td><td>MP-225</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>A-M System</td><td>AM2400</td><td></td></tr><tr><td>AD converter</td><td>National Instrument</td><td>NI-USB-6221</td><td></td></tr><tr><td>Heater controller</td><td>Warner Instrument</td><td>TC-324B</td><td></td></tr><tr><td>Inline heater</td><td>Warner Instrument</td><td>SC-20</td><td></td></tr><tr><td>Peristaltic pump</td><td>Rainin</td><td>Dynamax</td><td></td></tr><tr><td>pipette puller</td><td>Sutter Instrument</td><td>P-1000</td><td></td></tr><tr><td>Glass tube with filament</td><td>King Precision Glass</td><td>Customized</td><td></td></tr><tr><td>Stimulator</td><td>A.M.P.I.</td><td>Master-8</td><td></td></tr><tr><td>Biocytin</td><td>Sigma</td><td>B4261</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S6191</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher</td><td>BP328-1</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S0876</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S5011</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C5670</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M1880</td><td></td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G6152</td><td></td></tr><tr><td>K-gluconate</td><td>Sigma</td><td>G4500</td><td></td></tr><tr><td>ATP-Mg</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>Li-GTP</td><td>Sigma</td><td>G5884</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E0396</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>KOH</td><td>Sigma</td><td>P5958</td><td></td></tr><tr><td>Cs-methanesulfonate</td><td>Sigma</td><td>C1426</td><td></td></tr><tr><td>CsOH</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>Syringer filter</td><td>Nalgene</td><td>171</td><td></td></tr><tr><td>1 ml syring</td><td>Rainin</td><td>17013002</td><td></td></tr><tr><td>10 &amp;mu;l pipette tip</td><td>Genesee Scientific</td><td>24-130RL</td><td></td></tr><tr><td>Streptavidin-488</td><td>ThermoFisher</td><td>S-11223</td><td></td></tr><tr><td>10x PBS</td><td>Lonza</td><td>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

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Genetik Etiketli Retina Ganglion Hücreleri Işık Tepkisi Record İzole Retina Hazırlık

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

Işık tam hücreli Kayıtlar Retina Dilim Uyarıcı Synaptic Akımlar Uyarılmış

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Biyoloji

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-kelepçe Kapasitans Ölçümler ve Ca 2 + Retina Dilimleri Tek Sinir Terminalleri Görüntüleme

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

Fare Retina gelen Transretinal ERG Kayıtlar: Rod ve Koni Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Bozulmamış Retina Elektrofizyolojik Karakterizasyonu GFP-ifade eden hücre popülasyonlarının

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Fare Retina Ganglion Hücreleri Synaptic ve Yapay iletkenlikleri Dinamik Kelepçe Uygulama

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

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. ...

Murin bölünmüş retinada bipolar ve yatay nöronal hücrelerin incelenmesi

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

Yazar Spotlight: Gelişmiş Erişim ve Hızlandırılmış Deneyler için Bölünmüş Retina Tekniğini Kullanma 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

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

Özet

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