NSF (0854551) tarafından finanse edilmiştir.

1. Ön-deneysel Hazırlama <ol><li> Aşağıdaki çözeltiler (kompozisyonlar için bakınız Tablo 1) hazırlanması: (a) 1 litre NFR (Normal Kurbağa Ringer), (b) ve 100 ml% 10 salin, (c) ve 100 ml CMF (Ca2 + / Mg2 +-serbest çözelti ), (d) 1 ml ITS (insülin transferin selenyum-, Sigma I1884), (e) ve 100 ml L-15 kültür ortamı, (f) 10 ml HCG (insan koryonik gonadotropin Sigma CG-10), (g) 100 ml K +-iç çözüm, çözüm 1 osmotik gücü bir buhar basıncı Osmometer (örn. Wescor model # 5100C) kullanarak yaklaşık 260 mOsm olduğundan emin olmak için kontrol edilmelidir Amfoterisin B (h) 100 ml K +-iç çözüm . </li><li> (B) çözümleri sterilize filtreleyin ve (c). Solutions (a) (b) ve (c) 4 ° C&#39;de üç aya kadar saklanabilir Bir 25 gauge iğne şırınga ve şırınga filtresi üzerinden liyofilize toz içeren şişe 1 ml deiyonize H2O ilave edilerek çözelti (d) hazırlayın. Bu nihai çözüm cBir 30 gün boyunca 4 ° C&#39;de saklanabilir. </li><li> Bir kabı veya şişesi tüm bileşenleri bir araya getirerek bir laminer akış kaputu çözeltisi (e) hazırlayın. Nihai çözüm sterilize ve steril santrifüj tüpleri içine 10 ml alikotları aktarmak filtreleyin. -20 ° C&#39;de saklayın </li><li> 25 gauge şırınga, iğne ve şırınga filtresi ile liyofilize toz içeren flakon H 2 0 deiyonize 10 ml ekleyerek HCG (çözüm (f)) hazırlayın. Bu, nihai çözelti 30 gün boyunca 4 ° C &#39;de muhafaza edilebilir. </li><li> Tutkallama siyanoakrilat yapıştırıcı ile cam bir Pasteur pipeti ucuna bir Minutien pim (26002-10, İnce Science araçları) ile en az iki mikrodisseksiyon araç imal. Pipet yaklaşık 0.5 cm sonunun uzatmak piminin sivri ucunu ver. </li><li> Hazırlama kültürleri İki gün önce, aşağıdaki prosedür ile üreme neden. Düzlemsel su üzerinde kadın ve koyu pigmentasyon göze çarpan, kırmızımsı kloaka gözlemleyerek Xenopus bir üreme hazır çifti tanımlayınerkek ön pençeleri rface. Kaçamayacağım böylece bir zaman, net her kurbağa Tek ve net bir lavabo üzerine ventral tarafında basılı tutun. Dorsal lenf keseler net ve deri altından içine biri ile çözeltinin 1 mL&#39;si (f) enjekte edilir. </li><li> Su ve bir kapalı on galon tank içinde bir arada üreme çifti yerleştirin. Hayvanların yeni koydu ve döllenmiş yumurta trample olmayacak emin olmak için, yaklaşık bir örgü boyutu ½ tankın alt (Şekil 1A) Yukarıdaki &quot;takılı yaklaşık 1-2 inç ekranlı bir zemin yüklemek. </li><li> Hayvanlar amplexus olan ve döllenmiş yumurta ekranının altındaki kat (Şekil 1B) gözlemlendi kadar 12-48 saat boyunca bozulmadan kurbağaları bırakın. </li><li> Tankından hayvanlar çıkarın, ama daha uzun en azından 24 saat için yumurta rahatsız bırakın. Kurbağa bu çifti altı hafta sonra yeniden yetiştirilen olabilir. </li><li> Tankın dibinden embriyolar gevşetin ve dört ya da beş 60X15 mm kült aktarabilirsiniz% 10 tuzlu su (çözüm b) içeren ure yemekleri. Niewkoop ve Faber (ref. 20) şemasına göre evre tarafından embriyolar sıralayın. Faydalı embriyo aşamasında 22-24 az olanlar olacaktır. Şekil 2B (yaklaşık aşamada 28) yararlı olduğu gelişme boyunca çok uzak bir embriyo gösterirken Şekil 2A yaklaşık aşamada 22 embriyo gösterir. Bu sağlıklı embriyolar seçmek önemlidir: açık kahverengi ve beyaz beneklenme ile görünüşte düzgün olanları idealdir. Büyük siyah ya da beyaz lekeler var Embriyolar genellikle sağlıksız ve kullanışsız. </li></ol> 2. Xenopus Embryolarının Mikrodisseksiyon <ol><li> Bir laminer akış kaputu, etiket içinde ve yaklaşık yarım üç 60X15 mm steril kültür% 10 tuzlu yemekleri ile doldurun ve CMF ile bir. </li><li> % 10 tuzlu su içeren yemekler birine steril, cam Pasteur pipeti transferi 09:55 aşamada 22-24 embriyo kullanma. Bir stereo zoom yardımıyla teşrih ilekaput içinde ing mikroskobu (10 x oküler ile 0.6-5x), steril # 5 forseps (11251-30, Güzel Bilim Araçları) iki çift kullanarak her embriyodan jöle ceket ve vitellin membran kaldırmak. (Şekil 3A ve 3B, bakınız.) </li><li> % 10 salin kalan iki yemekleri ile ve nihayet CMF içeren yemeğin içine, bir defada bir ileterek çıplak embriyolar yıkayın. Her transfer için yeni bir steril pipet kullanın ve çanak çanak transfer çözeltisinin hacmi en aza indirmek. </li><li> Buna karşılık, adım 1.5 sitemliyiz mikrodiseksiyon aracını kullanarak, forseps bir çift ile nazikçe ama sıkıca her embriyonun tutun ve nöral tüp ve hayvanın en dorsalinde yer almaktadır ilişkili Miyotom kaldırın. Üç dilim, nöral tüp konumu ve (Şekil 4A) için üçüncü sadece ventral iki ucundan birini yaparak bunu yapın. Uzaklıkta sarısı granül gelen çanak temiz bir parçası her disseke nöral tüp / myotome Taşıes ve diğer enkaz (Şekil 4B). Dorsal-ventral eksen, ve nöral tüp ve miyotomlarında ve konumları Şekil 4 içinde gösterilmektedir. </li><li> Bu çözüm yaklaşık on beş dakika sonra (CMF) kullanımı forseps disseke doku serbest pigmente deri kaldırın ve atmak. Birbirlerine (Şekil 4C) ayrışmış hale olarak bir ilave 30-60 dakika sonra, hücreler, bir &quot;kum yığını&quot; oluşturacak. </li></ol> 3. Sinir-kas Co-kültürlerin hazırlanması <ol><li> Kültür ortamı bir 10 ml tüp çözülme ve aseptik 70 ul ekleyin ITS ve 35 ng / ml &#39;BDNF (Sigma B3795). </li><li> Etiket ve steril 35 mm kültür kaplarına (FD35-100 Dünya Hassas Aletler) yaklaşık yarım L-15 kültür ortamı (çözüm (e)), disseke her bir embriyo için biri ile doldurun. </li><li> Bir alev üzerinde şevli kısım tutulurken, bir cam Pasteur pipeti her iki ucunda tutarak bir kaplama pipet imal. Yaklaşık 1 ila uçlarında ayrı çek0 cm ve daha sonra yaklaşık 0.2 &#39;lik bir uç elde etmek için son kopar. </li><li> Çizilmiş çözüm miktarını minimize tek embriyo &quot;kum yığını&quot; yalakalık kaplama pipet kullanın. Bir kültür çanağı alt üzerine hücreleri Expel. Birkaç satır içine Levha hücreleri. Kaplama farklılaşmamış hücreler (Şekil 5A ve 5B) uyun. </li><li> Hücreler zaman çanak takmak için izin vermek için en az on beş dakika boyunca rahatsız edilmeden kaplı hücreler bulaşık bırakın. </li></ol> 4. Patch Sıkma Sinir-kas Sinapslar <ol><li> Kas hücreleri iğ şeklinde olur ve spinal nöronlar uzun süreçler (Şekil 6) uzatmak: kültüründe 12-24 saat sonra, kaplama hücreleri belirgin morfolojik özellikleri üzerine alır. Nörit varisler ve kas hücreleri arasındaki fonksiyonel sinaptik iletişim simultane eşleştirilmiş elektrofizyolojik kayıtlar ile teyit edilebilir. &quot;M&quot;, &quot;S&quot; ve &quot;V&quot; nöronal, kas hücresi sırasıyla bakınsoma ve presinaptik varisler. </li><li> Presinaptik varisler için ve kas hücresi için bir: Kayıt için iki yama elektrot hazırlayın. </li><li> Presinaptik elektrot için, aşağıdaki gibi bir amfoterisin B içeren çözeltinin hazırlanması: 5 mg amfoterisin B (Sigma A4888) ihtiva eden 1.5 ml mikrosantrifüj tüpüne 100 ul DMSO ekleyin. 10 sn vorteksleyin bu çözüm. Sonraki, Amfoterisin B (çözüm (h)) için +-iç çözüm 625 ul K içeren ikinci bir mikrosantrifüj tüpüne bu çözeltinin 10 ul ekleyin. Yukarıdaki gibi Vortex. </li><li> Amfoterisin B ihtiva eden K basamak 4.3 &#39;de hazırlanmış +-iç çözelti ile K +-iç çözeltisi (çözeltisi (g)) ile birlikte post-sinaptik elektrot ile pre-sinaptik elektrot doldurun. </li><li> NFR ile kültür çanak medya değiştirin ve faz kontrast optik ile donatılmış bir inverted mikroskop gittiğinizde. </li><li> Sadece kendi hedeflerinin üzerinde her iki elektrot yerleştirin. Tüm-hücre eş edininvarisler elektrot ile delikli yama yapılandırma önce kas hücresi elektrot ile nfiguration. </li><li> Sinaps işlevselliğini doğrulamak için, yazılım denetimi altında (örn. Axopatch 200B, Moleküler Cihazlar) bir yama kelepçe amplifikatör ile varisler depolarize (pCLAMP 9, Molecular Devices örneğin) ve eşzamanlı presinaptik ve postsinaptik akımları gözlemlemek. Şekil 7 görülen akımlar gösterilir +40 mV&#39;ye -30 mV 10 mV artışlarla presinaptik varis verilen gerilimi adımlar depolarize yanıt olarak. Presinaptik hücre görülen içe akımları Na + ve Ca 2 + ve K + ile dış akımlar tarafından yürütülmektedir. Postsinaptik akımlar hücre içinde kaydedilmiş varis serbest nörotransmitter yanıtları vardır ve nikotinik asetilkolin reseptörü kanallar vasıtasıyla Na + tarafından çoğunlukla taşınmaktadır. </li></ol>

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Çıkar çatışması ilan etti.

Motonöronlar ve kas hücrelerinin Başarılı co-kültür Key adımları uygun Xenopus kurbağaların indüklenen üreme üretilen embriyolar sahnelenen kullanımı ve farklılaşmamış spinal nöronlar ve iskelet hücreleri dikkatli aseptik diseksiyonu vardır. Er sık ​​onların gelişimi onları durdurur hareket gibi onlar yaklaşık 10 ya da öylesine aşamasına ulaşıncaya kadar Döllenen yumurtalar, bozulmamış bırakılmalıdır. Sağlıklı embriyoların pürüzsüz bir görünüm ve kahverengi ve beyaz benekli boyama ile tanımlanır. Bu miyositlerin önemli ölçüde farklılaştığını sadece nöral tüp kapanır sonra ve öncesinde geliştirme sırasında noktasıdır, çünkü Evre 22-24 embriyoların çok yararlıdır. Buna ek olarak, daha yaşlı edilen hücrelerden CMF iyi ayrıştırmak için başarısız. Bu embriyo kuvvetle yapışır gibi vitellin membran sökerken dikkat edilmelidir. Embriyonun bozulmadan ortaya böylece membran keskin forseps kullanarak parçalandı edilmelidir. Bir başka önemli precaution kültür çanak (yerine onları oraya yerleşmek icar fazla) alt üzerine hücreler dikkatle yerleştirmektir. Bu hücrelerin kültür çanak uyulması yönündeki olasılığını artırır gibi bu yöntem tercih edilir. Sinir ve kas arasındaki Fonksiyonel nörotransmisyonu sadece postsinaptik kaydı ile tespit edilebilir ve sık sık innervasyon sonra spontan kas kasılması gözlem tarafından tespit edilebilir. Un-innerve kas hücreleri kültür içinde seğirme yoktur. Yalnız postsinaptik kayıt minyatür son plak akımları ya da potansiyelleri ancak uyarılmış boşalma ölçümleri öncesi ve postsinaptik akımların presinaptik uyarımı ve korelasyon patch-klemp çift gerektirir gerektirir kayıt için yararlıdır. Burada açıklanan paired-yama kayıt yöntemi yanısıra, bu hazırlık Bu sayede nöronal soma 5 üçüncü bir pipet tanıtma fırsatı sunan bir aksiyon potansiyeli tha nesilt presinaptik varisler yaymak ve nörotransmitter salınımına yol açabilir. Buna ek olarak, arabuluculuk düşünce veya sinaptik iletimi modüle edilir olası ajanlar sinaps iki tarafına da tanıttı olabilir: kas hücresi pipet aracılığıyla veya soma yerleştirilmiş üçüncü bir pipetle difüzyon yoluyla.

<table border="1"><tbody><tr><td> Ekipman / Malzemeleri </td><td> Satıcı </td><td> Katalog / Model Numarası </td><td></td></tr><tr><td> İnsülin-Transferrin-Selenyum </td><td> Sigma </td><td> I1884 </td><td></td></tr><tr><td> İnsan koryonik gonadotropin </td><td> Sigma </td><td> CG-10 </td><td></td></tr><tr><td> Buhar Basıncı Ozmometre </td><td> Wescor </td><td> 5100C </td><td></td></tr><tr><td> Minutien Pins </td><td> Güzel Bilim Araçları </td><td> 26002-10 </td><td></td></tr><tr><td> Amfoterisin B </td><td> Sigma </td><td> A4888 </td><td></td></tr><tr><td> Forseps </td><td> Güzel Bilim Araçları </td><td> 11251-30 </td><td></td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Çözüm Adı </td><td> Yemek tarifi </td></tr><tr><td> (A) NFR (Normal Kurbağa Ringer (mM) </td><td> 116 NaCl, 2 KCl, 1.8 CaCl2, 5 Na-HEPES, pH 7.35. </td></tr><tr><td> (B)% 10 salin </td><td> 10 ml NFR +90 ml H2O deiyonize </td></tr><tr><td> (C) CMF (Ca2 + / Mg2 +-serbest çözelti) </td><td> 125 NaCl, 2 KCl, 1.2 EDTA, 5 Na +-HEPES, pH 7.35. </td></tr><tr><td> (E) L-15 Kültür ortamı </td><td> 50 ml L-15 ortamı (Sigma L1518) + 50 ml NFR. </td></tr><tr><td> (G) K +-iç solüsyonu (mM cinsinden) </td><td> 116 KCl, 1 NaCl, 1 MgCl2, 10 EGTA, 5 HEPES, pH 7.3 </td></tr><tr><td> Amfoterisin B (h) K +-iç solüsyonu (mM cinsinden) </td><td> 52 K 2 SO 4, 38 KCl, 1 EGTA, 5 HEPES, pH 7.3 </td></tr></tbody></table></td></tr></tbody></table> Table 1.

simultaneous pre- and post-synaptic electrophysiological recording from xenopus nerve-muscle co-cultures

Nörotransmitter serbest bırakılması ile presinaptik iyonik akımların kaplin hakkında çok bilgi en önemlisi omurgasız hazırlıkları, kalamar dev sinaps 1 elde edilmiştir. Bununla birlikte, hazırlama hariç burada açıklandığı gibi, az omurgalı preparatlar bu nörotransmitter serbest ve presinaptik iyonik akımlarının eşzamanlı ölçümler yapmak mümkündür ki içinde bulunmaktadır. (Dek 12-24 saat içinde işlevsel sinaps oluşturacak ve birkaç gün boyunca sinir ve kas hücre gelişimi ve sinaptik etkileşimleri çalışmak için de kullanılabilir; Embriyonik Xenopus motor nöronlar ve kas hücreleri oda sıcaklığında basit bir kültür ortamı içinde yetiştirilen bir arada büyümesi) oluşur. Diğer omurgalı preparatlar içinde bu ko-kültürler bazı avantajları hazırlama kolaylığı, oda sıcaklığında kültürleri ve çalışma muhafaza etme özelliğine sahiptir, ve 2 oluşturduğu sinaps hazır erişilebilirlik-4. Hazırlık presinaptik iyon kanalları ve verici sürümü 5-8 yönetmeliğin biyofiziksel özelliklerini incelemek için yaygın olarak kullanılmaktadır. Buna ek olarak, hazırlık 9-12 akson uzantısı ve sinaptogenez çalışma, nörotransmitter salınımını 13-15, nöromodülasyon 16,17 içinde difüze haberciler rolünü moleküler mekanizmaları ve in vitro sinaptik plastisite 18 de dahil olmak üzere diğer kullanımlar için kendini vermiştir - 19.

Şekil 4B hemen 22 Xenopus embriyosunun. Şekil 4C olduğunu, Ca 2 +-Mg 2 + ücretsiz bir çözüm cilt kaldırma ve inkübasyon (CMF, çözümden sonra (gösteren bir sahne alanından çıkarılmasını sonrası kordon / myotome izole spinal dorsal görünümü gösterir c)), hücreleri bir &quot;kum yığını&quot; içine ayrışır ve kaplama için hazır. Kültür ortamı hücreleri içine kaplama biraz morfolojik değişkenliği (Şekil 5B) gösterirler ancak kültür yirmi dört saat sonra farklı şekiller almaktadırlar hemen sonra. Nöronlar uzanan neurites iken küresel kalırken kas hücreleri iğ şeklinde haline kas hücreleri ile sinaps (Şekil 6) de ayrıntılı variköz. Varis presinaptik ve postsinaptik kas hücresi (Şekil 7) aynı anda eşleştirilmiş yama kayıt nörotransmiterin salınışı ve resulta ile ilişkili presinaptik içe ve dışa doğru akımlar ortaya koymaktadırnt postsinaptik akımlar eksitatör. <img src="/files/ftp_upload/50253/50253fig1.jpg" alt="Şekil 1"/> Şekil 1 A:.. Ekranda tepesinde çiftleşme kova kurbağa Damızlık çifti B: döllenmiş yumurta ile amplexus kurbağalar. <img src="/files/ftp_upload/50253/50253fig2.jpg" alt="Şekil 2,"/> Şekil 2 A:.. &quot;Uygun&quot; aşaması 22 embriyo B: &quot;uygunsuz&quot; aşaması 28 embriyo. Ölçek çubuğu 1 mm temsil eder. <img src="/files/ftp_upload/50253/50253fig3.jpg" alt="Şekil 3"/> Şekil 3 A:. Aşama 22 embriyo vitellin membran ve jöle kat temizlenmeden önce. Ok jöle kat dış kenarına işaret. Vitellin membran embr yakından yapışıryo ve neredeyse görünmez B:. Bare embriyo. Ölçek çubuğu 1 mm temsil eder. <img src="/files/ftp_upload/50253/50253fig4.jpg" alt="Şekil 4,"/> Şekil 4 A: to-be disseke B kısmı gösteren kesikli çizgi ile Sahne 22 embriyo:.. Embriyonun Akut izole dorsal kısmı; &quot;d&quot; ve &quot;v&quot; embriyoların dorsal ve ventral yönlerini bakın, ise &quot;m&quot; ve &quot;n&quot; mytomes ve nöral tüp yaklaşık konumları belirtin C:. CMF 60 dk sonra hücrelerin &quot;Kum yığını&quot;. Ölçek çubuğu A için 1 mm ve B ve C için 0.5 mm temsil eder. <img src="/files/ftp_upload/50253/50253fig5.jpg" alt="Şekil 5,"/> Şekil 5 A:. Hemen sonra kaplama kültürdeki hücre 5x güç görünümü. B: 40x de aynı kültür. Ölçekçubuğu A için 40 mikron ve B için 5 mikron temsil <img src="/files/ftp_upload/50253/50253fig6.jpg" alt="Şekil 6"/> Şekil 6. Nöronal soma (S), presinaptik varisler (V) ve postsinaptik kas hücresi (M) kimlik ile kültür Nöromusküler kavşak. Ölçek çubuğu: 5 mm. <img src="/files/ftp_upload/50253/50253fig7.jpg" alt="Şekil 7" fo:content-width="5in" fo:src="/files/ftp_upload/50253/50253fig7highres.jpg"/> 10 mV artan voltaj uygulaması yanıt olarak görüldü Şekil 7. Presinaptik (üst) ve postsinaptik (alt) akımları -30 mV dan +40 mV presinaptik varisler sunulan adımları. Gerilim adımlar presinaptik izleri bazı yanında gösterilir. Hem hücreler için tutma potansiyeli -70 mV idi.

Watch this Scientific Journal Video about Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures at JoVE.com

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Bu video embriyonik primer kültürlerinde büyümek için kullanılan prosedürleri gösterir<em&gt; Xenopus</em&gt; Sinir ve kas hücreleri ve eşzamanlı öncesi ve post-sinaptik yama kelepçe kayıt yapmak için bu hazırlık kullanışlılığı.

Eşzamanlı Öncesi ve Sonrası-sinaptik Elektrofizyolojik Recording<em&gt; Xenopus</em&gt; Sinir-kas Co-kültürlerin

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

simultaneous-pre-post-synaptic-electrophysiological-recording-from

Research

JoVE Journal

Neuroscience

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

10.9K Görüntüleme.  David Geffen School of Medicine at UCLA. Bu video embriyonik primer kültürlerinde büyümek için kullanılan prosedürleri gösterir Xenopus Sinir ve kas hücreleri ve eşzamanlı öncesi ve post-sinaptik yama kelepçe kayıt yapmak için bu hazırlık kullanışlılığı.

Video: Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Elektrot İmalat ve İmplantasyon Aplysia californica Bozulmamış, Serbestçe Behaving Hayvanlar Çok kanallı Sinir ve Kas Kayıtlar

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Nörobilim

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

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

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

Xenopus Oosit ifade İyon Kanalları Patch Kelepçe Kaydı

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

Biyoloji

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Drosophila Larva NMJ Synaptic Potansiyeller kayıt için Elektrofizyolojik Yöntemler

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

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

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

C. Elegans Nöromüsküler Kavşağı Diseksiyon ve kayıt

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

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

Erişkin Fare yılında Motor Ünite tarafından üretilen bir Lomber Motonöron ve Kuvvet Eşzamanlı intrasellüler Kaydı<em&gt; In vivo</em

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Electrophysiological Recording in the Drosophila Embryo

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

Electrophysiological Recording in the Drosophila Embryo

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

Elektrofizyolojik Kayıt Drosophila Embriyo

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

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila larva nöromüsküler kavşak sinaptik akımlardan Fokal Macropatch kayıtları

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Ürünleri ve tüm hücre yama kelepçe kayıt Xenopus laevis Tectal nöronlar için iletişim kuralları

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Recording Gap Junction Current from Xenopus Oocytes

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions (GJs). However, this approach is technically challenging because it requires a differential voltage clamp of two opposed oocytes sharing a common ground. Although a small number of labs have succeeded in performing this technique, essentially all of them have used either homemade amplifiers or commercial amplifiers that were designed for single-oocyte recordings. It is often challenging for other labs to implement this technique. Although a high side current measuring mode has been incorporated into a commercial amplifier for dual oocyte voltage-clamp recordings, there had been no report for its application until our recent study. We have made the high side current measuring approach more practical and convenient by introducing several technical modifications, including the construction of a magnetically based recording platform that allows precise placement of oocytes and various electrodes, use of the bath solution as a conductor in voltage differential electrodes, adoption of a commercial low-leakage KCl electrode as the reference electrode, fabrication of current and voltage electrodes from thin-wall glass capillaries, and positioning of all the electrodes using magnetically based devices. The method described here allows convenient and robust recordings of junctional current (Ij) between two opposed Xenopus oocytes.

Recording Gap Junction Current from Xenopus Oocytes

Here we present a protocol to express gap junction proteins in Xenopus oocytes and record junctional current between two apposed oocytes using a commercial amplifier designed for dual oocyte voltage-clamp recordings in a high side current measuring mode.

Ksenopus Oositlerinden Boşluk Bağlantı Akımının Kaydedilmesi

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions ...

Electrophysiological Recordings For Assessing Neuronal Regenerations in Co-cultured Spinal Cord Slices

Put a Petri dish with a coated microelectrode array inside under a stereomicroscope. Bring the array into focus, and center a six-microliter droplet of chicken plasma on the electrode array. Using a small spatula, carefully slide two spinal cord sections with their ventral sides facing each other into the plasma droplet.
Next, add eight microliters of thrombin around the chicken plasma droplet. Then, use the pipette tip to carefully mix and spread the chicken plasma and thrombin mixture. Just before the chicken plasma and thrombin mixture becomes too stringy and starts to coagulate, aspirate the excess liquid. Then, cap the Petri dish and place it in a humidified chamber.
Place the chamber inside an incubator at 37 degrees Celsius for about an hour. After incubation, carefully add 10 microliters of nutrient medium to the sample. Cap the Petri dish and place it back into the incubator for an additional 45 minutes. Next, place each of the multi-electrode array culture assemblies into a roller tube and add three milliliters of nutrient medium. Close the lid tightly and place the roller tube into the roller drum. Rotate the drum at 1 to 2 RPM in the incubator at 37 degrees Celsius.
Using sterile rubber-tipped forceps, remove the multi-electrode array culture assemblies from the roller tube and place them into a Petri dish under a stereomicroscope. Bring the tissue into focus and verify that the two slices are fused. Next, hold the assembly steady and place a scalpel blade in the groove of the multielectrode array close to the tissue slices. Hold the scalpel rather horizontally and then lift the scalpel handle up, but let the scalpel blade stay in the groove of the array in such a way that the blade rolls from base to tip, cutting through the tissue, covering the groove.
Sever any residual tissue connections with a 25-gauge needle tip if necessary. Work only in the area within the groove, and do not touch the tender edges. Put the multi-electrode array culture assemblies back into the roller tube and add three milliliters of fresh nutrient medium to the cultures. Then, place the roller tube back onto the roller drum in the incubator at 37 degrees Celsius.
Mount a multi-electrode array culture assembly into a recording chamber and apply about 500 microliters of extracellular solution to the array. Then, mount the assembly on the microscope. Wait 10 minutes for the system to stabilize, and then, record basic spontaneous activity from each of the activity-detecting electrodes for 10 minutes. Repeat the recordings a total of two times.
To ensure stable extracellular conditions, exchange the extracellular solution after every recording session. If desired, disinhibit the network by applying extracellular solution containing one micromolar of strychnine and 10 micromolar of gabazine and wait for at least two minutes before recording the electrical activity.

Eşzamanlı Öncesi ve Sonrası-sinaptik Elektrofizyolojik Recording

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