SBC COBRE 1P20GM121310-01 NIH grant tarafından desteklenen.

Tüm yöntem tanımlamak burada kurumsal hayvan bakım ve kullanım Komitesi (IACUC), Wyoming Üniversitesi tarafından onaylanmıştır. Elektrofizyolojik kayıtlar da dahil olmak üzere tüm yordamları oda sıcaklığında yaklaşık 23 ° c yapılmaktadır Tüm yöntem tanımlamak burada tectal Kurbağa yavrularını gelişimsel sahne 42 ve (Neiuwkoop ve Faber15göre sahnelenen) 49 arasında neurons kaydetmek için optimize edilmiştir.
1. Vivo içinde hazırlık
<ol><li>İribaş anestezi.<ol>
<li>İribaş % 0,01 ile Steinberg'ın solüsyon içeren küçük bir Petri kabına yerleştirin MS-222 yaklaşık 5 min için. Not: Ortak bir balık ve amfibi anestezik MS-222 aka "Tricaine" dır. Kurbağa yavrularını mM Steinberg'ın çözümde yetiştirilir: 0.067 KCl, 0,034 Ca (NO3)2•4H2O, 0,083 MgSO4•7H2O, 5.8 NaCl, 4.9 HEPES.</li>
		<li>Adım 2 için devam etmeden önce derin anaesthetized (non-yanıt veren ve artık yüzme) iribaş olduğundan emin olun.</li>
	</ol>
</li>
	<li>Anatomi/kayıt çanak yere batık silikon bloğu için imzalat iribaş güvenli.<ol>
<li>Harici kayıt solüsyon içeren bir diseksiyon/kayıt yemek anaesthetized kurbağa taşımak için tek kullanımlık transfer pipet kullanın (115 mM NaCl, KCl, 2 mM CaCl23 mM, 3 mM MgCl2, HEPES, glikoz; 1 mM 5 mM ayarlamak 7,25 kullanarak pH 10 N NaOH, Osmolarite 255 mOsm).<ol>
<li>Spontan kas en aza indirmek için twitches, asetilkolin reseptör engelleyici, tubocurarine (100 µM) harici çözüm için ekleyin. (Genellikle, bu bir 10 mM tubocurarine stokunun 100 µL harici çözüm 10 mL için eklemek için 200 µL pipet kullanarak yapılır).</li>
		</ol>
</li>
		<li>Böcek iğne kullanarak, kurbağa, dorsal tarafta, silikon elastomer batık bir blok için güvenli (Sylgard 184 gibi Malzemeler tablo Ayrıntılar için bakınız), hangi-var yapıştırılmış diseksiyon çanak yere. Not: Pimleri yerleşimini önemlidir: Proje göz beyin, afferent RGC akson impaling önlemek için yeterince Kaudal her iki tarafında yer onları ve optik tectum (şekil 1A) anterior beyin girin.</li>
	</ol>
</li>
	<li>Orta çizgi boyunca beyin fileto.<ol>
<li>Beyin net görebilmek için steril 25-G iğne kullanarak orta çizgi boyunca yüzeysel bir kesi yaparak beyni örten cilt kaldırın.</li>
		<li>İğne Nöral tüp içine yerleştirip hafifçe yukarı doğru (dorsally) öyle ki tüp dorsal kısmı temiz bir şekilde yere plaka sağlam (şekil 1B) bırakarak (parçalanmış) kesilir çekerek aynı orta çizgi ekseni boyunca beyin fileto. Not: Afferent duyusal girdiler tectum kat plaka girin çünkü Nöral tüp zemin plakası olduğu gibi bırakılması önemlidir.</li>
	</ol>
</li>
	<li>Tectal nöronlar kapakları şeffaf ventrikül membran kaldırın.<ol>
<li>Kayıt çanak Elektrofizyoloji teçhizat taşımak ve bir kırık cam pipet ucu tectal nöron hücre organları örten ventrikül membran kaldırmak için kullanın. Ucunu hafifçe narin görev silecek arasında sürükleyerek kır.</li>
		<li>Pipet pipet yuvasına vida ve optik tectum micromanipulator üzerinden aşağı indir.</li>
		<li>Tectum uzak ventrikül membran soyma için kırık pipet kullanın. Not: Membran tüm tectum kaldırmak için gerekli değildir. küçük bir pencere yeterli ve nöronlar bol erişim sağlar.</li>
	</ol>
</li>
	<li>Tüm cep telefonu yama kelepçe kayıtları elde etmek. Not: Bu aşamadan itibaren Segev, et al. açıklandığı gibi fare beyin dilimleri tüm cep telefonu yama kelepçe kayıtları taşımaya benzer bir yaklaşımdır 16 (Şekil 1C).</li>
	<li>Retina yansıtılan ışık tüm alanı parıltısı tectal nöron yanıtlarını kaydetmek.<ol>
<li>Tüm alan flaş ışık retina proje için bir optik fiber kurbağa'nın göz bitişik yerleştirin. Optik fiber diğer ucunda bir LED ışık parlaklığı denetlenmesini sağlar bir değişken direnç doğrultusunda olduğunu. Tetikleyici LED Dijital çıkış ile amplifikatör. Bu şekilde, tüm alanı değişen şiddetlerde ışık yanıp söner olabilir (şekil 4A) kaydedildi.</li>
	</ol>
</li>
</ol>2. tüm beyin hazırlık
<ol><li>1.1-1,3 adımları gerçekleştirin. Not: Bu hazırlık için dış çözümde tubocurarine gereği yok.</li>
	<li>Beyin izole et.<ol>
<li>25-G iğne (Şekil 2A) arka beyin sever için kullanın.</li>
		<li>İribaş bütün beyninden izole etmek için hafifçe beyinde tüm yanal ve ventral bag doku ve sinir lifleri koparmaya Kaudal-için-rostral bir yön altında iğne çalıştırın.</li>
	</ol>
</li>
	<li>Beyin silikon elastomer bloğu için güvenli.<ol>
<li>Tamamen serbest geldiğinde, silikon elastomer bloğu için beyin yanında duvar ilanı olfaktör ampuller biri aracılığıyla bir PIN ve arka beyin (Şekil 2B) aracılığıyla başka bir PIN güvenli. Bu kayıt tectal nöronlar için en uygun yapılandırmadır.</li>
	</ol>
</li>
	<li>Diseksiyon kapsamdan sabitlenmiş tüm beyin hazırlık Elektrofizyoloji teçhizat için içeren yemek taşımak. 1.4. adımda açıklandığı gibi bir kırık cam pipet kullanarak ventrikül membran kaldırın.</li>
	<li>İki kutuplu bir uyarıcı elektrot optik hemisferlerine (nerede her Eye'dan axon yolları orta çapraz) doğrudan RGC akson etkinleştirmek için yerleştirin.<ol>
<li>İki kutuplu uyarıcı elektrot rostral ve hemen hemen bitişiğinde, büyük orta ventrikül yerleştirin. Optik hemisferlerine hemen rostral büyük orta ventrikül (Şekil 2B) için yer alır.<ol>
<li>Böyle küçük bir göçük dokusunda oluşan yavaşça uyarıcı elektrot optik hemisferlerine indirin. İki kutuplu elektrot stimülasyon tam olarak kontrol edilebilir gücünü sağlayan bir darbe uyarıcı tarafından tahrik edilmektedir.</li>
		</ol>
</li>
	</ol>
</li>
	<li>Segev, et al. tarafından açıklandığı gibi bütün hücre yama kelepçe kayıt (Şekil 2C) gerçekleştirmek 16
</li>
</ol>3. yatay beyin dilim hazırlık
<ol><li>2.1-2,3 adımları gerçekleştirin.</li>
	<li>(Hangi vivo içinde karşılık gelen en dorsal dördüncü) en yanal dördüncü bir yüzünün bir optik tectum tüketim için jilet kullanın. # Bu parçayı şekil 3' teAgösterilen Kaudal rostral uçağa paralel yapılmıştır.</li>
	<li>Silikon elastomer ile dilimlenmiş tarafına beyin yeniden pin yukarı bakacak şekilde (somata ve neuropil doğrudan kayıt için erişilebilir böylece) ve silikon elastomer blok (şekil 3 uzak bakan beyin ventrikül yüzeyine yan B) böylece (İki kutuplu bir elektrot optik hemisferlerine üzerinde yerleştirilebilir).</li>
	<li>Segev, et al. açıklandığı gibi tüm cep telefonu yama kelepçe veya yerel dosyalanmış potansiyel kayıt (şekil 3C) yerine getirmek 16
</li>
</ol>

<ol>
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Yazarlar ifşa gerek yok.

Bu çalışmada açıklanan tüm yöntemleri tectal Kurbağa yavrularını gelişimsel sahne 42 ve (Neiuwkoop ve Faber15göre sahnelenen) 49 arasında neurons kaydetmek için optimize edilmiştir. Sahne 42 ile Kurbağa yavrularını yeterince büyük ve yeterince gelişmiş böylece böcek pimleri beyin vivo kayıtları için ve tüm beyin diseksiyon taşımak için her iki tarafında yerleştirilebilir. Daha önceki aşamalarında, Kurbağa yavrularını aslında iki boyutlu olduğunda (...

Retinotectal devre amfibi görsel sisteminin önemli bileşenidir. Hangi onların aksonlar optik tectum nerede postsinaptik tectal nöronlar ile sinaptik bağlantıları oluşturmak için proje RGCs gözünde, oluşur. Xenopus iribaş retinotectal devre nöral devre oluşumu ve işlev eğitim için popüler bir gelişim modelidir. Bu kurbağa'nın retinotectal devrenin bir güçlü deneysel model1,2,3render birçok özniteliği vardır. Bir büyük öznitelik ve odağı bu makalenin tüm cep telefonu yama kelepçe kayıtları tectal nöronlar, içinde VIVO veya tüm beyin hazırlık kullanarak yürütmek için yeteneği var. Gerilim ve akım kayıt modları kelepçe destekler bir amplifikatör ile donatılmış bir Elektrofizyoloji sondaj platformu ile tüm cep telefonu yama kelepçe kayıtları bir nöron'ın Elektrofizyoloji yüksek çözünürlükte karakterize edilebilir izin verir. Sonuç olarak, tüm cep telefonu yama kelepçe kayıtları tectal nöronlar retinotectal devre oluşumu temel aşamaları boyunca geliştirme detaylı ve kapsamlı bir anlayış ve plastisite içsel4,5 hazırladık , 6 , 7 ve sinaptik8,9,10,11 özellikleri. Tüm cep telefonu yama kelepçe tectal nöron kayıtları birleştirerek, genlerin veya morpholinos bu nöronların12ve kurulan görsel kaçınma testi13 ile görsel destekli davranışını değerlendirmek için bir yöntem ilgi ifade yeteneği teşvik molekülleri, devre fonksiyon ve davranışı arasındaki bağlantıları tanımlaması.
Tüm cep telefonu yama kelepçe kayıtları elde veri çünkü genetik kalsiyum göstergesi GCaMP6, gibi daha yeni görüntüleme yaklaşımları kullanarak mümkün değildir yüksek çözümleme türü bulunmakla birlikte kalsiyum göstergeler kullanarak görüntüleme verir unutmamak gerekir Kalsiyum dynamics büyük popülasyonlarda nöronların arasında aynı anda, orada hayır doğrudan veya özel elektrik parametreleri somata delta Floresans ölçerek elde edilebilir ve gerilim bir yolu yoktur belirgin şekilde kelepçe ölçmek için nöron akım-gerilim ilişkileri. Açıkça bu ayrı iki yaklaşım, Elektrofizyolojik kayıtlar ve kalsiyum görüntüleme, üst üste güçlü sahip ve farklı veri oluşturmak. Bu nedenle, en iyi yaklaşım belirli deneysel soru ele alınmaktadır bağlıdır.
Burada, biz tüm cep telefonu yama kelepçe kayıtları bir vivo içinde hazırlık, tüm beyin hazırlık, kullanarak iribaş optik tectum nöronlar edinme için bizim yöntem tanımlamak ve daha yeni bir14 bizim laboratuarımızda geliştirildi tüm beyin hazırlık modifiye . Temsilcisi sonuçları bölümünde her hazırlık ve elde edilebilir veri farklı türde deneysel avantajları göstermektedir. Sınırları ve güçlü ipuçları sorun giderme, yanı sıra farklı ürünleri bir tartışma bölümünde dahil edilir.

<table border="0" cellpadding="0" cellspacing="0" style="width:1012px;" width="1011">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Stemi Stereo 508</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:148px;">495009-0006-000&#160;</td>
			<td style="width:299px;">Dissecting microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS-222 &#34;Tricane&#34;</td>
			<td>Finquel</td>
			<td>ARF5G</td>
			<td>Amphibian general anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Used to prepare Stienberg&#39;s solution and external solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Used to prepare Stienberg&#39;s solution and external solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-1KG</td>
			<td>Used to prepare Stienberg&#39;s solution and external solution</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Calcium nitrate tetrahyrate (Ca(NO3)&#8226;4H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>237124-500G</td>
			<td>Used to prepare Stienberg&#39;s solution&#160;&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Magnesium Sulfate (MgSO4)</td>
			<td>Mallinckrodt Chemicals</td>
			<td>6066-04</td>
			<td>Used to prepare Steinberg&#39;s solution</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Calcium Chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C5080-500G</td>
			<td>Used to prepare external recording solution</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Magnesium Chloride (MgCl2)</td>
			<td>J.T. Baker</td>
			<td>2444-01</td>
			<td>Used to prepare external recording solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose Anhydrous</td>
			<td>Mallinckrodt Chemicals</td>
			<td>6066-04</td>
			<td>Used to prepare external recording solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubocurarine hydrochloride pentahydrate</td>
			<td>Sigma</td>
			<td>T2379</td>
			<td>Nicotinic acetylcholine receptor antagonist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>0.1mm diameter stainless steel pins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>761028</td>
			<td>Preweighed monomer and curing agent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Polystyrene Petri Dish - 60x15mm</td>
			<td>Fisher Scientific</td>
			<td>AS4052</td>
			<td>Small petri dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrecisionGlide Needle 25Gx5/8 (.0.5mm X 16mm)</td>
			<td>BD</td>
			<td>305122</td>
			<td>Syringe needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1mL Slip Tip Tuberculin Syringe&#160;</td>
			<td>BD</td>
			<td>309659</td>
			<td>Disposable, sterile syringes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate pipette glass</td>
			<td>Sutter Instrument</td>
			<td>BF150-86-10HP</td>
			<td>Pulled to desired specifications using pipette pulling machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td>Fabricates micropipettes for electrophysiology recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipes Kimtech wipes</td>
			<td>Kimberly-Clark</td>
			<td>34120</td>
			<td>Delicate task lint-free wipers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axon Instruments MultiClamp 700B Headstage CV-7B</td>
			<td>Molecular Devices</td>
			<td>1-CV-7B</td>
			<td>Current clamp and voltage clamp headstage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MP-285 Motorized Manipulator with Tabletop Controller</td>
			<td>Sutter Instrument</td>
			<td>MP-285/T</td>
			<td>Control for headstage on electrophysiology rig</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiber-Coupled LED (Green)</td>
			<td>Thorlabs</td>
			<td>M530F2</td>
			<td>Fiber optic cable paired with green LED</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cluster Bipolar Electrode (25&#181;m diameter)</td>
			<td>FHC</td>
			<td>30207</td>
			<td>Bipolar stimulating electrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISO-Flex Stimulator</td>
			<td>A.M.P.I. (Israel)&#160;</td>
			<td>Contact manufacturer</td>
			<td>Flexible stimulus isolator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axon Instruments 700B Multipatch Amplifier</td>
			<td>Molecular Devices</td>
			<td>2500-0157</td>
			<td>Amplifier for voltage- and current-clamp recording&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digidata 1322A digitizer</td>
			<td>Molecular Devices</td>
			<td>2500-135</td>
			<td>Data acquisition system for electrophysiology recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axio Examiner.A1</td>
			<td>Zeiss</td>
			<td>491404-0001-000&#160;</td>
			<td>Microscope for electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-g Lab Table</td>
			<td>TMC</td>
			<td>63-533</td>
			<td>Air table for electrophysiology microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inspiron 620 Personal Desktop Computer with Windows 7 64-bit</td>
			<td>Dell</td>
			<td>D06D001</td>
			<td>Computer running electrophysiology software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">c2400 CCD camera</td>
			<td>Hamamatsu</td>
			<td>70826-5</td>
			<td>Charge-coupled device camera for electrophysiology imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7 O&#39;Clock Super Platinum Stainless Razorblades</td>
			<td>Gillette</td>
			<td>CMM01049</td>
			<td>Platinum-coated stainless razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td>Disposable Polyethylene transfer pipets</td>
		</tr>
	</tbody>
</table>

preparations and protocols for whole cell patch clamp recording of xenopus laevis tectal neurons

Hangi formu synapses doğrudan üzerine nöronlarda optik tectum göz retina ganglion hücrelerinin (RGCs) oluşan Xenopus iribaş retinotectal devre nasıl sinir devreleri çalışma popüler modelidir kendini topla. Bütün taşıma yeteneği yama kelepçe kayıtları tectal nöronlar ve RGC uyarılmış yanıt, ya da içinde vivo hücre veya tüm beyin hazırlık kullanarak büyük bir vücut, normal yatan mekanizmaları yüksek çözünürlüklü verilerin üretti ve anormal, devre oluşumu ve işlev. Burada nasıl vivo içinde hazırlık, özgün tüm beyin hazırlık gerçekleştirileceğini açıklar ve daha yakın zamanda yatay beyin dilim hazırlık tectal neurons tüm cep telefonu yama kelepçe kayıtları elde etmek için geliştirilmiştir. Her hazırlık benzersiz deneysel avantajları vardır. Vivo hazırlık tectal nöronlar doğrudan yanıt göz yansıtılan görsel uyaranlara karşı kayıt sağlayan. Tüm beyin hazırlık için son derece kontrollü bir şekilde var olmak harekete geçirmek RGC akson, ve yatay beyin dilim hazırlık kaydından tectum tüm katmanları arasında sağlar.

Tüm alan flaş ışık ışık uyarılmış yanıt-e doğru kaydetmek için elde edilen yanıt bireysel tectal neurons (şekil 4A) kaydedilir iken retina tahmin edilmektedir. Bu belirli iletişim kuralı her iki nöron (yanıt "On") açma ve 15 off dönüm ışığa tepki ölçmek için tasarlanmıştır daha sonra "Off yanıt." ölçmek için s Tectal nöronlar genellikle sağlam ve yanıt kapalı sergi (nöron gösterilen burada içinde k...

Watch this Scientific Journal Video about Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons at JoVE.com

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

Bu yazıda, Xenopus laevis Kurbağa yavrularını retinotectal devre eğitim için tüm cep telefonu yama kelepçe kaydetmek için kullanılan üç beyin hazırlıkları görüşmek. Kendi belirli avantajları ile her hazırlık Xenopus iribaş deneysel tractability nöral devre işlevi incelemek için bir model olarak katkıda bulunmaktadır.

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 ...

preparations-protocols-for-whole-cell-patch-clamp-recording-xenopus

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

9.4K Görüntüleme.  University of Wyoming. Bu yazıda, Xenopus laevis Kurbağa yavrularını retinotectal devre eğitim için tüm cep telefonu yama kelepçe kaydetmek için kullanılan üç beyin hazırlıkları görüşmek. Kendi belirli avantajları ile her hazırlık Xenopus iribaş deneysel tractability nöral devre işlevi incelemek için bir model olarak katkıda bulunmaktadır.

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

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Dan Sinir Eksplantasyon Kültürleri<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Nörobilim

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Diseksiyon, Kültür ve Analizi<em&gt; Xenopus laevis</em&gt; Embriyonik Retina Doku

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

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

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

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

Kızılötesi Sinir Stimülasyon en mekanizmaları incelenmesi için tüm Hücre Patch Kelepçe

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

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Duyusal nöronlar izolasyonu<em&gt; Aplysia californica</em&gt; Glutamaterjik Akımlar Patch Kelepçe Kayıtlar için

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- ve nörokimyasal tanımlanan hipokampal internöronlardan gelen tüm hücre Patch-kelepçe Kayıtlar

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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

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

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

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

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

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

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

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

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

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

Whole-cell Patch-clamp Recordings in Brain Slices

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

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

Beyin Dilimleri tüm hücre Patch-kelepçe Kayıtlar

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

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

koku ampul kalsiyum Değişiklikleri İzleme yoluyla Sıcaklık kaynaklı Nöronal Aktivite Kayıt<em&gt; Xenopus laevis</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

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

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Dan Sinir Eksplantasyon Kültürleri

Diseksiyon, Kültür ve Analizi

Kızılötesi Sinir Stimülasyon en mekanizmaları incelenmesi için tüm Hücre Patch Kelepçe

Duyusal nöronlar izolasyonu

Morphologically- ve nörokimyasal tanımlanan hipokampal internöronlardan gelen tüm hücre Patch-kelepçe Kayıtlar

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

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

Beyin Dilimleri tüm hücre Patch-kelepçe Kayıtlar

koku ampul kalsiyum Değişiklikleri İzleme yoluyla Sıcaklık kaynaklı Nöronal Aktivite Kayıt

Channelrhodopsins'teki İyon Seçiciliğinin Elektrofizyolojik Olarak Belirlenmesi için Tam Hücreli Yama Kelepçesi Kayıtları