Bu araştırma UT Güneybatı başlangıç ​​fonları (SK) tarafından desteklenmiştir.

<ol><li>Neher, E., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-channel+currents+recorded+from+membrane+of+denervated+frog+muscle+fibres%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-channel currents recorded from membrane of denervated frog muscle fibres.</a> Nature. 260 (5554), 799-802 (1976).</li>
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<li>Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correction+for+liquid+junction+potentials+in+patch+clamp+experiments%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Correction for liquid junction potentials in patch clamp experiments.</a> Methods Enzymol. 207, 123-131 (1992).</li>
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<li>Molleman, A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMolleman%2C+A+%22Patch+Clamping%3A+An+Introductory+Guide+To+Patch+Clamp+Electrophysiology%22">Patch Clamping: An Introductory Guide To Patch Clamp Electrophysiology</a>. , John Wiley & Sons, Ltd. (2003).</li>
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<li>Richerson, G. B., Messer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+composition+of+experimental+solutions+on+neuronal+survival+during+rat+brain+slicing%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Effect of composition of experimental solutions on neuronal survival during rat brain slicing.</a> Exp Neurol. 131 (1), 133-143 (1995).</li>
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</ol>

yazarların hiçbiri rakip çıkarları veya çakışan çıkarları var.

Bu protokol beyin dilimleri nöronlarda tüm hücre yama-kelepçe deneyler temel işlemi anlatılır. Bununla birlikte, bu tekniğin karmaşık, potansiyel ve hassasiyet tam olarak bu makalede açıklanan edilemez. Burada, en temel adımları tasvir ve başarılı ve titiz bütün hücre kayıtları elde etmek için kontrol edilmelidir önemli parametreleri altını çalıştık. Daha fazla teorik öğrenme, birçok kitap ve makale beyin dilimleri 3,21-24 ve hücre canlılığı geliştirmek amacıyla 25...

Patch-kelepçe tekniği, 1970&#39;lerin sonlarında 1,2 geliştirilmiştir bir elektrofizyolojik teknik canlı dokuda tekli veya çoklu iyon kanal işlevleri çalışmak için birincil araçtır. elde edilebilir, farklı bant konfigürasyonu arasında, bütün hücre patch-clamp kayıtları nöron önemli bir bölümünün, elektrik davranış çalışma sağlar. Klasik olarak, bu teknik, ya in vitro beyin kesitleri, yeni ayrışmış nöronlar üzerinde veya hücre kültürü modelleri 3 gerçekleştirilir. Beyin dilimleri nöronlar üzerinde gerçekleştirilen, bu teknik birçok avantaj sunar. Özellikle: (I) nöronları olduğu bir dereceye kadar nispeten korunmuş beyin devrelerinde kaydedilir ve hücre kültürü preparatlara kıyasla, 3 fizyolojik olarak uygun bir ortam sağlar. Bu, akut Pharmacolog her türlü tarafından tetiklenen hücresel ve moleküler olayları erken yakalayan, hatta gerçek zamanlı olarak izleme veriyornik manipülasyonlar - in vivo koşullarında, klasik kullanılarak elde edilemez bir zamansal çözünürlüğü; görsel beyin dilimleri beyin bölgelerini tespit etmek, (ii) yeteneği, hem de floresan belirteçler zaman beyin bölgesi okudu ve özel nöronlar için yüksek bölgesel özgüllüğü 3 sağlar; (iii) (hücre içi kayıtları için keskin bir mikropipet ile membran delme aksine) plazma zarının önemli bir bölümünü açarak hücrenin hücre içi alana erişim 4. Buna karşılık, bu iç çözüm oluşturan belirli iyonların içeriği veya konsantrasyonu o kadar moleküler hedeflerin değiştirilmesi veya hücresel mekanizmaları farklı koşullar altında ele alınabilir sağlar. Örneğin, tam-hücre konfigürasyonu, herhangi bir farmakolojik madde (örneğin, antagonistler) kurulması sırasında bir kayıt mikropipet (Yama pipet) çözeltisi doğrudan sitoplazma içine nüfuz ve putat hareket edecek ekle kiKomşu hücrelerde hedef fonksiyonu değiştirmeden hücre içi hedefleri ive. Ayrıca, keskin mikropipet kayda göre, yama kelepçe elektrodun ucundaki büyük açılış alt direnci, daha az rekabet halindeki gürültü ve hücrenin 4 içine böylece daha iyi elektrik erişim sağlar. Ancak, pipet ucunda büyük açılış hücre diyaliz yol ve çalışma 5,6 altında biyolojik olayların ifadesi için kritik olabilecek hücre içi moleküler makine, böylece kaybı unutmayın. Bu durumda, sert elektrot kayıtları daha uygun olabilir. Kayıtların Bu tür ve böylece hücre içi alanı ve iç pipet çözeltisi arasında iyon değişiminin çoğu önlenmesi, tüm hücre kayıtları için kullanılan daha küçük olan bir gözenek olan mikropipetler gerektirir. (Akut veya kronik) deneyimi herhangi bir şekilde dahil olmak üzere kötüye 11,1 ilaçlara, pozlama 7-10 öğrenme2, vb stres 13,14, spesifik beyin bölgelerinde nöronal fonksiyonu çeşitli yönlerini değiştirebilir. Bu değişiklikler genellikle (gün saat) geliştirmek için zaman gerektirdiğinden, belirli bir deneyim geçirmiş hayvanların beyin dilimleri tam hücre kayıtları araştırmacılar bu değişiklikleri tanımlamak için izin verir. Temelde, (hepsi değilse de) nöronal fonksiyonlar (örneğin, ligand-aktive iyon kanalları, voltaj kapılı iyon kanalları, nörotransmitter taşıyıcılar) ve böylece beyin devre aktivitesi ve davranış, deneyim değiştirilebilir (deneyim bağımlı katılan bileşenler çok plastisite) 10,15-17. Nöronal düzeyde, beyin devresi etkinlik sinaptik arasında sürekli etkileşim (örn glutamat iletim) ve içsel hücresel heyecanlanma faktörleri (örneğin, axosomato-dendritik iyon kanalları çıkar: potasyum, K +, sodyum Na + ve kalsiyum, Ca 2+ ). Whol kullanarak belirli koşullar altındaE-hücre patch-kenetli elektrofizyolojik teknikler, sinaptik genel içsel uyarılabilirliğinin değişikliklerden özellikle kaynaklanan sinyal değişiklik izole edilebilir. Çoğu durumda, sinaptik uyarılma tüm hücre voltaj-kelepçe tekniği kullanılarak değerlendirilir. Bu kayıt modu α-amino-3-hidroksi-5-metil-4-isoxazolepropionic asit reseptörleri tarafından aracılık edilen, örneğin iyon akımlarının [ölçümünü (karşılaştırın AMPA reseptörleri) ve nöronal plazma zarından, N-metil-D-aspartik asit reseptörleri (NMDA reseptörleri)] bir dizi gerilim zar potansiyeli tutarak. Burada, deneyci sezyum ihtiva iç mikropipet çözümleri (Cs +), K + kanallarının geniş bir engelleyici (kilit iç uyarılma faktörler) kullanın. Tüm hücre konfigürasyonunun oluşturulmasından sonra, hücre içi boşlukta Cs + `nin difüzyon K + kanallarını bloke eder ve böylece, nispeten etkin bir alanı kelepçesi ve ön hem de izin verirdiğer ölçümler üzerinde içsel uyarılabilirlik faktörlerin etkisini havalandırın. Düzensiz şekilli hücreler (örneğin, nöronlar) ve 18,19 çardak geniş ve karmaşık dendritik özellikle nöronlar kaydederken Uzay kelepçe sorunları, yani gerilim-kelepçe zorluk tüm hücre, ortaya çıkar. somatik gerilim kelepçe kötü kontroller nöronların dendritik ağacında gerilimi nedeniyle, çalışma kapsamında dendritik elektrik sinyallerine çeşitli yönleri bir dendritik mesafe bağımlı bir şekilde bozulmuş. (Yapay beyin-omurilik sıvısı, ACSF) hücre-dışı çözelti içinde çözüldü bu pikrotoksin (y-aminobütirik asit, GABA A reseptör antagonisti) ya da kynurenic asit (glutamat reseptörlerinin birçok engelleyici) gibi farmakolojik araçlar ile birlikte, bu teknik, glutamat ölçümünü sağlar reseptör ve GABA A sırasıyla akımları R-aracılı. Buna karşılık, iç uyarılma genellikle akım kelepçe kayıt modunda değerlendirilir.voltaj-kelepçe kayıtları aksine, bu kayıt modu nöronal plazma zarı içinden akan iyon akımlarının neden membran potansiyelleri varyasyonlarının ölçümü. Nöronlar Na + ve K + kanalları gerektiren hem aksiyon potansiyelleri oluşturmak için Tipik olarak, içsel eksitabilite değişiklik yeteneği değişiklikler ile belirlenir. Akım kelepçe kayıtları yaparken, bu nedenle, mikropipetler K + yerine Cs + içeren bir iç çözelti ile doldurulur. Glutamat ve GABA ACSF içinde çözülür A reseptör-aracılı akımları bloke farmakolojik ajanlar ile kombine, bu deney tasarımı sinaptik eksitabilite olası değişiklikleri ile kontamine olmadan nöronal ateşleme içsel faktörler (örneğin, K + kanalları) katkısı ölçülmesini sağlar faktörler. Bu makale t gerekli temel usul adımları anlatacağızo (i) sağlıklı beyin dilimleri hazırlamak; (Ii) tüm hücre konfigürasyonu elde, ve (iii) sinaptik ve içsel heyecanlanma değerlendirmek için temel parametreleri izlemek.

<table><tbody><tr><td>Isolated pulse stimulus generator</td><td>A.M.P.I</td><td>Master-8</td><td></td></tr><tr><td>Isolation unit (ISO-Flex)</td><td>A.M.P.I</td><td>ISO-Flex</td><td></td></tr><tr><td>Computer controlled Amplifier&amp;nbsp;</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Digital Acquisition system</td><td>Molecular Devices</td><td>Digidata 1500</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX-51</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>Chamber and in-line Heater</td><td>Warner Instruments</td><td>TC-344B</td><td></td></tr><tr><td>Vibratome Slicer</td><td>Leica&amp;nbsp;</td><td>VT1000 S</td><td></td></tr><tr><td>Micropipette Puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Imaging Camera</td><td>Q Imaging</td><td>QIClick-F-M-12</td><td></td></tr><tr><td>Narishige pipette puller PC-10</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Glass capillaries</td><td>WPI</td><td>TW150F-3</td><td></td></tr><tr><td>Slice hold-down (harp)</td><td>Warner Instruments</td><td>64-0255</td><td></td></tr><tr><td>Slice Chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Nonmetallic syringe needle</td><td>World Precision Instruments</td><td>MF28G67-5</td><td></td></tr><tr><td>Syringe filters</td><td>Nalgene</td><td>176-0045</td><td></td></tr><tr><td>Glue Gun</td><td>Home Depot</td><td>various</td><td></td></tr><tr><td>Gas dispersion tube</td><td>Ace Glass Inc.</td><td>various</td><td></td></tr><tr><td>Decapitation scissors</td><td>Home Depot</td><td>100649198</td><td></td></tr><tr><td>Scalpel Handle #3</td><td>World Precision Instruments</td><td>500236</td><td></td></tr><tr><td>Small straight sharp tips scissors</td><td>World Precision Instruments</td><td>14218</td><td></td></tr><tr><td>Vessel canulation forceps&amp;nbsp;</td><td>World Precision Instruments</td><td>500453</td><td></td></tr><tr><td>Curved hemostatic forceps</td><td>World Precision Instruments</td><td>501288</td><td></td></tr><tr><td>Economy Tweezers #3</td><td>World Precision Instruments</td><td>501976-6</td><td></td></tr><tr><td>Spatula</td><td>Fisher Scientific</td><td>14357Q</td><td></td></tr><tr><td>Scooping spatula</td><td>Fisher Scientific</td><td>14-357Q</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>08-747B</td><td></td></tr><tr><td>Filter paper</td><td>Lab Depot</td><td>CFP1-110</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Solutions&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Cs-Gluconate internal solution (pH 7.2&amp;ndash;7.3, 280&amp;ndash;290 mOsm)&lt;/strong&gt;</td></tr><tr><td>D-gluconic acid 50%&amp;nbsp;</td><td>Sigma Aldrich/various</td><td>G1951</td><td></td></tr><tr><td>Cesium-OH (CsOH) 50%&amp;nbsp;</td><td>Sigma Aldrich/various</td><td>232041</td><td></td></tr><tr><td>NaCl, 2.8 mM</td><td>Sigma Aldrich/various</td><td>S7653</td><td></td></tr><tr><td>HEPES, 20 mM</td><td>Sigma Aldrich/various</td><td>H3375</td><td></td></tr><tr><td>EGTA, 0.4 mM</td><td>Sigma Aldrich/various</td><td>E4378</td><td></td></tr><tr><td>tetraethylammonium-Cl, 5 mM</td><td>Sigma Aldrich/various</td><td>T2265</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;GTP, 0.3 mM</td><td>Sigma Aldrich/various</td><td>G8877</td><td></td></tr><tr><td>MgATP, 2 mM</td><td>Sigma Aldrich/various</td><td>A9187</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;K-Gluconate internal solution (pH 7.2&amp;ndash;7.3, 280&amp;ndash;290 mOsm)&lt;/strong&gt;</td></tr><tr><td>K D-gluconate, 120 mM</td><td>Sigma Aldrich/various</td><td>G4500</td><td></td></tr><tr><td>KCl, 20 mM</td><td>Sigma Aldrich/various</td><td>P3911</td><td></td></tr><tr><td>HEPES, 10 mM</td><td>Sigma Aldrich/various</td><td>H3375</td><td></td></tr><tr><td>EGTA, 0.2 mM</td><td>Sigma Aldrich/various</td><td>E4378</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich/various</td><td>M8266</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;GTP, 0.3 mM</td><td>Sigma Aldrich/various</td><td>G8877</td><td></td></tr><tr><td>MgATP, 2 mM</td><td>Sigma Aldrich/various</td><td>A9187</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Standard artificial cerebrospinal fluid (ACSF, osmolarity &amp;asymp; 300-310 mOsm)&lt;/strong&gt;</td></tr><tr><td>KCl, 2.5 mM</td><td>Sigma Aldrich/various</td><td>P3911</td><td></td></tr><tr><td>NaCl, 119 mM</td><td>Sigma Aldrich/various</td><td>S7653</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;H&lt;sub&gt;2&lt;/sub&gt;O, 1 mM</td><td>Sigma Aldrich/various</td><td>S9638</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3,&lt;/sub&gt; 26.2 mM</td><td>Sigma Aldrich/various</td><td>S8875</td><td></td></tr><tr><td>Glucose, 11 mM</td><td>Sigma Aldrich/various</td><td>G8270</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;-7H&lt;sub&gt;2&lt;/sub&gt;O, 1.3 mM</td><td>Sigma Aldrich/various</td><td>230391</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O, 2.5 mM</td><td>Sigma Aldrich/various</td><td>C3881</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Additional compounds used for solutions preparation&lt;/strong&gt;</td></tr><tr><td>KOH</td><td>various</td><td></td><td></td></tr><tr><td>Kynurenic acid</td><td>Sigma Aldrich/various</td><td>K3375</td><td></td></tr></tbody></table>

whole-cell patch-clamp recordings in brain slices

Tüm hücre yama kelepçe kayıt nöron önemli bir bölümünün elektriksel özelliklerinin çalışma sağlayan bir elektrofizyolojik bir tekniktir. Bu yapılandırmada, mikropipet, daha önce kullanılan hücre içi keskin elektrot kayıt yöntemine göre daha doğru bir iyonik akım ölçümleri mevcut sızmasını önler ve böylece içerir hücre membranı ile sıkı bir temas halindedir. Klasik olarak, tam-hücre kayıt hücre kültürü modelleri, ayrışmış nöronlar, beyin dilimleri nöronlar dahil preparasyonların çeşitli üzere nöronlar üzerinde yapıldı ve sağlam anestezi ya uyanık hayvanlarda edilebilir. Özetle, bu teknik son derece uyarılabilir hücrelerin pasif ve aktif biyofiziksel özelliklerinin anlaşılması için katkıda bulunmuştur. Bu tekniğin önemli bir avantajı da manipülasyonlar (örneğin, farmakolojik, deneyci kaynaklı plastisite) belirli nöronal fonksiyonlar veya c değiştirebilir nasıl özel bilgi sağlamasıdırgerçek zamanlı olarak hannels. Buna ek olarak, plazma zarının önemli bir açıklığı iç pipet çözeltisi serbest sokulması ilaçlar, örneğin, agonistleri veya belirli bir hücre içi proteinlerin antagonistleri için araçlar temin edilmesi ve komşu hücrelerde fonksiyonlarını değiştirmeden bu hedeflerin manipüle, sitoplazma içine nüfuz sağlar. Tam hücreli kayıt üzerinde durulacak Bu makale, beyin dilimleri nöronlar üzerinde bir fizyolojik ilgili bağlamda, yani nispeten iyi korunmuş beyin devreleri, nöronların kayıt avantajı vardır bir hazırlık yapıldı. Özel olarak, uygun bir farmakoloji ile birleştirildiğinde, bu yöntem, öğrenme, ilaç kötüye kullanımı maruz kalma ve stres gibi deneyimler, herhangi bir türü aşağıdaki Meydana gelen neuroadaptations belirlenmesini sağlayan güçlü bir araçtır. Özet olarak, beyin dilimleri tam hücre patch-clamp kayıtları ex vivo preparat uzun süreli değişiklikler ölçmek için araç sağlarnöronal işlevleri bu sağlam uyanık hayvanlarda gelişmiştir.

Sıcaklık, kolay deneyi tarafından kontrol edilen bir unsur, iyon kanalları ve reseptörleriyle biyofiziksel özelliklerini etkiler ve böylece sinaptik sonrası akımlar (PSC) (EPSC ve iPSCs) ve sivri ortaya çıkarmak için nöronların özelliği dalga biçimi. Şekil 3 ve Şekil 4, nöronal ateşleme üzerinde sıcaklığın etkisini sırasıyla uyarılmış EPSCs (eEPSCs) eğimini göstermektedir. Ateşleme paterni (Şekil 3) zam...

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings in Brain Slices at JoVE.com

Whole-cell Patch-clamp Recordings in Brain Slices

Bu protokol tüm hücre yama-kelepçe kayıtları gerçekleştirmek için temel usul adımları açıklar. Bu teknik nöronların elektriksel davranışının çalışma sağlar ve beyin dilimleri yapıldığında, hala nispeten iyi korunmuş beyin devreleri entegre edilmiştir nöronlardan farklı nöron işlevlerinin değerlendirilmesine olanak sağlamaktadır.

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 ...

whole-cell-patch-clamp-recordings-in-brain-slices

Research

JoVE Journal

Neuroscience

59.7K Görüntüleme.  University of Texas Southwestern Medical Center.  Bu protokol tüm hücre yama-kelepçe kayıtları gerçekleştirmek için temel usul adımları açıklar. Bu teknik nöronların elektriksel davranışının çalışma sağlar ve beyin dilimleri yapıldığında, hala nispeten iyi korunmuş beyin devreleri entegre edilmiştir nöronlardan farklı nöron işlevlerinin değerlendirilmesine olanak sağlamaktadır. 

Video: Whole-cell Patch-clamp Recordings in Brain Slices

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Fizyolojik Sıcaklık Akut Yetişkin Beyin Dilimleme

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Nörobilim

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Akut Beyin Dilimleri nöronal Mikro şemalar Elektrofizyolojik ve Morfolojik Karakterizasyonu Eşleştirilmiş Patch-Kelepçe Kayıtlar Kullanma

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

En iyi duruma getirilmiş bir Nkullanarak akut beyin dilimleri hazırlanması-metil-D-glucamine koruyucu kurtarma yöntemi

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

Dilim yama klemp tekniği öğrenme kaynaklı plastisite analiz etmek için

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

Sinaptik çeşitlilik bütün hücreli yama-kelepçe Elektrofizyoloji kullanarak değerlendirilmesi

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.

Here, we present a protocol to perform a whole-cell patch-clamp on brain slices containing kisspeptin neurons, the primary modulator of gonadotrophin-releasing hormone (GnRH) cells. By adding knowledge about kisspeptin neuron activity, this electrophysiological tool has served as the basis for significant advancements in the neuroendocrinology field over the last 20 years.

Tüm hücre yama-kelepçe kayıtları için bir hedef olarak hipotalamik kisspeptin nöronları

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in ...

Establishing a Whole-Cell Configuration through the Patch Clamp Method

Source: Segev, A. et al., <a href="https://app.jove.com/v/54024">Whole-cell Patch-clamp Recordings in Brain Slices.</a>&#160;J. Vis. Exp.&#160;(2016)
This video demonstrates the whole-cell patch clamp technique in a mouse brain slice. This technique establishes a stable connection between micropipette and neuron membrane, which is further disrupted to establish a whole-cell configuration to study the neuron&#39;s health and physiological responses.

Yama Kelepçesi Yöntemi ile Tüm Hücre Konfigürasyonunun Oluşturulması

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Slice ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Patch-Clamp Recordings from the Dendrite of a Dopaminergic Neuron in a Brain Slice

Source: Engel, D. <a href="https://app.jove.com/v/54601">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for patch-clamp recording from the dendritic membrane surface. This helps to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels in dopaminergic neurons.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Establishing a Whole-Cell Voltage-Clamp Configuration for Electrophysiological Recordings in Brain Slices

Source: Sunstrum, J. K., et al. <a href="https://app.jove.com/v/59461">Evaluation of synaptic multiplicity using whole-cell patch-clamp electrophysiology.</a> J. Vis. Exp. (2019)
This video demonstrates positioning a fine-pointed pipette near brain slices, forming a seal with a neuronal cell. Suction breaks the cell membrane, thereby creating a whole-cell patch clamp that enables current measurements for electrophysiology experiments.

Beyin Dilimlerinde Elektrofizyolojik Kayıtlar için Tüm Hücre Voltaj-Kelepçe Konfigürasyonunun Oluşturulması

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review ...

Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices

Source: Wu, Q., et al. <a href="https://app.jove.com/v/56010">Application of automated image-guided patch clamp for the study of neurons in brain slices.</a> J. Vis. Exp. (2017).
In this video, brain slices are immobilized in a recording chamber, and cells of interest are located and focused on using a computer-controlled microscope. The automated image processing system then precisely positions a patch pipette to seal the cell membrane and monitor electrical signals.

Beyin dilimlerindeki nöronları incelemek için otomatik görüntü kılavuzlu yama kelepçesi tekniği

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. System Setup

	Construct ...