Bu çalışma, Ulusal Sağlık Hibe RO1DC002756 Enstitüleri ve Purdue Araştırma Vakfı tarafından finanse edildi. Doris Wu ve Wiese Chang Biz deney ve figürleri ile yardım için Rodney McPhail tavsiye için teşekkür ederim.

Tarifler Chick Ringers <table border="1"><tbody><tr><td> 7.2 g </td><td> NaCl </td></tr><tr><td> 0.23 g </td><td> CaCl 2 + 2H 2 O </td></tr><tr><td> 0.37 g </td><td> KCl </td></tr><tr><td> 0,115 g </td><td> Na 2 HPO 4 </td></tr><tr><td> 900 ml </td><td> Su (Doku kültürü notu) </td></tr></tbody></table> Not: pH 7.4 &#39;e ayarlayın. 1000 ml su ile son hacim kazandırın. 10X PBS <table border="1"><tbody><tr><td> 40 g </td><td> NaCl </td></tr><tr><td> 1 g </td><td> KCl </td></tr><tr><td> 7 gr </td><td> Na 2 HPO 4 </td></tr><tr><td> 1.2 g </td><td> KH 2 PO 4 </td></tr><tr><td> 450 ml </td><td> DEPC su </td></tr></tbody></table> Not: pH 7.4 &#39;e ayarlayın. Son getirin500 ml su ile hacim. Çalışma konsantrasyonu (1X), 18 M cm iletkenlik suyu üretmek için bir Millipore UV ışınlama sistemi ile elde edilen gibi 9 parça, doku kültürü sınıf su ile 1 parçası 10X stok seyreltilmesi ile yapılır. Eksplant tutarak orta SAG <ul><li> DMEM/F12 L-glutamin, 10 mM HEPES </li><li> % 10 İnsülin, aktarma, Selenyum (DA 1) </li><li> % 1 pen-strep </li></ul> Eksplant kültür ortamı <ul><li> Eksplant tutarak orta SAG </li><li> 10 ng / ml nörotrofin-3 (NT-3) </li><li> 10 ng / ml siliyer nörotrofik faktör (CNTF) </li></ul> Omurilik diseksiyonu ve holding orta <ul><li> L-15 </li><li> % 10 fetal buzağı serumu </li><li> % 1 pen-strep </li></ul> 37-38 inkübe yumurta ° C % 70 etanol araçları ve Sylgard diseksiyon çanak diseksiyon sterilize edin. Diseksiyon yemekleri ve araçlar da UV gecede sterilize edilmelidir. <ol><li> 1 ml steril PBS, mix, ve tüp altına yerleşmek için boncuk beklemek bir mikrotüp yerleştirin boncuklar. </li><li> Boncuk boncuk yerleşti ve PBS içinde tekrar süspansiyon sonra süpernatantı kaldırarak birkaç kez yıkayın. </li><li> Boncuk saflaştırılmış protein (PBS içinde seyreltilmiş) veya PBS tek başına (Kontrol) oda sıcaklığında 1 saat inkübe. </li><li> Kollajen yerleştirilir kadar 1ml PBS ile 24 plaka PBS ve mağaza boncuk durulayın. </li></ol> 2. Statoacoustic ganglion diseksiyonu <ol><li> E4, yumurta, embriyo kaldırmak ve embriyonik membranlar kaldırmak için civciv Ringer solüsyonu ile bir tabak koyun. </li><li> Soğuk HBSS ve yukarı bakacak şekilde otik vezikül yan pozisyonda embriyo ile dolu bir Sylgard © çizgili petri embriyo yerleştirin. Diseksiyon pimleri kullanarak çanak embriyo Pin. </li><li> Iki diseksiyon pimleri kullanarak, deri yoluyla yatay bir kesi yapmak hemen ventralkanal torbalar, yaklaşık koklear kanal birleştiği yerde. Dikey bir kesim anterior ve posterior SAG olun. Üç kesimler tarafından sınırlanmıştır cilt kapağı kaldırın ve çıkarmak için forseps veya bir diseksiyon iğnesi kullanın. Not: SAG aydınlık temel aydınlatma ile kolayca görülebilir sırt çantası ve ventromedially projeleri ve faringeal kemerler gizlenmiş ventral koklear kanal arasında yarı yolda, yaklaşık otocyst anteroventral kenarında yer almaktadır. Koklear kanal uzar dorsoventral boyut böylece giderek Hamburger-Hamilton aşamaları 23-25 ​​Eylül arasında E4 artacaktır. </li><li> SAG ayrı bir posterior yönde otocyst çekin. SAG diseksiyonu iğneler ile çevresindeki doku çekilin ve SAG # 55 forseps kullanarak embriyo dikkatlice çıkarın. Mezenkimal doku ve sinir demetleri SAG çıkıntılı büyük boyutta çıkarın. </li><li> Geniş ağızlı pipetlemeyin uç kullanarak, bir eksplantOrta tutarak 0.5 ml SAG eksplant ile 24 plaka. 4 saate kadar buz üzerinde veya oda sıcaklığında eksplantlar tutun. </li></ol> 3. Omurilik diseksiyonu <ol><li> E6 embriyo yumurta çıkarın ve civciv Ringer çözeltisi içeren bir çanak koyun. Baş ve embriyonik zarlar çıkarın. </li><li> Bir Sylgard vücudun © soğuk omurilik diseksiyon orta içeren diseksiyon çanak yerleştirin. Pozisyon ve deneyi karşı karşıya embriyo &quot;ventral aşağı&quot; ve kaudal pin. Her uzuv ile ön sonuna kadar diseksiyon pimleri yerleştirin. </li><li> Forseps ve / veya diseksiyon pimleri, omurilik dorsal yüzeyinde görünür oluncaya kadar dikkatle, ventral yönde hareket, deri ve doku çıkarmak. </li><li> Tüm uzunluğu boyunca, spinal kord, anterior yönüne posterior diseksiyon bir iğne ile omurilik dorsal orta hat kesin. Bu omurilik ayrı sol ve sağ tarafında bir &quot;açık kitap&quot; oluşturur. </li><li&gt;, Yanal, çevre dokuya çıkarın omurilik izole etmek ve dorsal kök gangliyon (DRG) maruz bırakmayın. DRG ve meninksler omurilik ve DRG arasında bir diseksiyon iğnesi ya da forseps ovuşturarak çıkarın. </li><li> Forseps ile omuriliğin en kaudal ucunu tutarak ve deneyci kadar uzak kaldırma vücut omurilik çıkarın. DRG ve meninksler kalan çıkarın. </li><li> Forseps (ya da çanak pin) ile omuriliğin bir ucundan tutun ve omurilik ikiye bölmek için, ventral orta hat boyunca kesmek için Vannas makas kullanın. </li><li> Doku hareketsiz ve doku uzunluğu boyunca küçük eksplantlar (100-500 um uzunluğunda) kesmek için Vannas makas kullanmak için forseps ile eksplant bir ucundan tutun. Taban plakası ile bir eksplant kenarı boyunca net bir doku kalınlaşması olarak kabul edilebilir. </li><li> 0.5 ml soğuk diseksiyon orta ile 24 plaka eksplantlar aktarmak için geniş bir ağız pipetlemeyin ucu kullanın. , F doku canlılığını korumak için buz üzerinde eksplantlar tutunveya 4 saate kadar. </li></ol> 4. Neurite yanıt test etmek için saflaştırılmış proteinlerin kültür Eksplant <ol><li> Üreticinin talimatlarına göre, buz, 15 ml konik bir tüp içinde bir 1.5 mg / ml kollajen çözüm hazırlayın. </li><li> Kollajen çözüm 7-7.4 kullanarak pH indikatör kağıdı pH olduğundan emin olun. 1 ul hacimleri NaOH ekleyerek pH ayarlayın. </li><li> Geniş bir ağız pipetlemeyin ucu ve aspirat aşırı sıvı kullanarak 24 plaka eksplantlar aktarın. Çoklu eksplantlar iyi bir kültür olabilir, ama birbirinden ayrı en az 500 mikron konulmalıdır. </li><li> Iyi eksplant içeren 0.5 ml kollajen ekleyin. Kuyunun alt yüzeyinde eksplant yerleştirin. Kollajen, oda sıcaklığında polimerize başlayacak, çünkü her kültürün sonraki iyi geçmeden önce tamamen emin olun. </li><li> Kollajen polimerize ° C slayt 30-45 dakika süreyle sıcak bir 37 kültür plakası yerleştirin. Kollajen polymebir jel benzer rized. </li><li> Saflaştırılmış proteinlerin (PBS içinde seyreltilmiş) veya PBS (kontrol) ile desteklenmiş 0.5 ml sıcak eksplant kültür ortamı ve 37 inkübe ° C,% 5 CO 2. Neurite akıbet Üzerindeki Etkileri 24 saat görünür olmalıdır. NOT: Biz 42 saat kültür SAG eksplantlar aynı protokolü kullandık. </li></ol> 5. Yönlü neurite akıbet test etmek için protein kaplı boncuklar ile birlikte kültür Eksplant <ol><li> Eksplant kültür 4,1-4,4 adımları ile başlayın. </li><li> PBS kollajen çözüm ve eksplant içeren bir pipetlemeyin kullanarak, transferi 1-5 boncuk. </li><li> Eksplant kenarına boncuk 50-500 mikron konumlandırmak için forseps kullanın. </li><li> Kollajen polimerize ° C slayt 30-45 dakika süreyle sıcak bir 37 plaka koyun. Doku veya boncuk kollajen polimerize olarak yerinden olabilir. Ilk 5 dakika boyunca kültürler ve yeniden pozisyon doku ve boncuk kontrol edin. </li><li> 0,5 ml Her iyi medya SAG ve 24 saat inkübe edin. </li></ol> 6. Immünhistokimya neurite akıbet Görselleştirme <ol><li> PBS içinde kültürleri çalkalayın ve oda sıcaklığında 1 saat süreyle PBS içinde% 4 paraformaldehid düzeltmek. </li><li> Jellerin kenarında bir teflon mikro spatula yuvarlak ucu ile izleme kuyuların duvarlarında jeller bırakın. Kuyularında jeller immunostain gerçekleştirin. PBS içinde birkaç kez durulayın. </li><li> 4 geceleme 0.5 ml engelleme solüsyonu (% 10 buzağı serumu, PBS içinde% 0,1 Triton X-100,% 0,1 sodyum azid) ° C de inkübe </li><li> 4 gecede 0.5 ml primer antikor (anti-β-tubulin antikor 1:500 sulandırılmış çözümü engelleme) inkübe ° C </li><li> PBS içinde birkaç kez durulayın. 4 gecede 0.5 ml sekonder antikoru (Alexa Florlu 488 keçi anti-fare IgG 2b antikor 1:500 sulandırılmış) inkübe ° C Bu adım ileri, ışık duyarlılığı nedeniyle, folyo ile karanlık veya sarın tabak, tutmaksekonder antikorlar. </li><li> 4 ° C PBS içinde bir haftaya kadar, PBS ve mağaza birkaç kez durulayın. </li></ol> 7. Temsilcisi Sonuçlar <img alt="Şekil 1" src="/files/ftp_upload/3600/3600fig1.jpg" /> Şekil 1. Saflaştırılmış NT-3 ve NT-3-kaplı boncuklar E4 civciv SAG eksplantlar neurite destekleyici etkileri Temsilcisi sonuçlar. SAG nöron hücre gövdeleri ve neurites β-tubulin ile boyanmış ve konfokal mikroskobu 10x amacı ile görüntülenmiş. In vitro olarak 24 saat sonra, SAG eksplantlar denetimleri (1A) ile karşılaştırıldığında, 100 ng / ml saflaştırılmış insan NT-3 hücre kültür vasatı (1B) varlığında büyük neurite akıbet görüntülenir. SAG nöronlar, NT-3 bir nokta kaynak yerel neurite akıbet teşvik olduğunu göstermek için in100 ng / ml, NT-3 batırılmış boncuklar ile birlikte kültüre edildi. Eksplantlarındaki uzun görüntülenir ve yan yoğun neurite akıbetboncuk karşı tarafında (1D) ve PBS (1C) batırılmış boncuklar ile kültürler kontrol ile karşılaştırıldığında karşı karşıya eksplant. Ölçek çubuğu = 200 mm. <img alt="Şekil 2" src="/files/ftp_upload/3600/3600fig2.jpg" /> Şekil 2. E6 civciv omurilik saflaştırılmış Wnt5a ve Wnt5a kaplı boncuklar neurite destekleyici etkileri Temsilcisi sonuçlar. Omurilik eksplantlar β-tubulin ile boyanmış ve bir konfokal mikroskobu ile görüntülenmiş. 24 saat saflaştırılmış fare Wnt5a (400ng/ml), civciv, spinal kord eksplantlar neurite akıbet varlığı büyüme artış sonra (2B) (2A) Wnt5a olmadan kültür kontrollere kıyasla. 500 ng / ml Wnt5a batırılmış boncuklar, aynı zamanda kültürleri (2C) kontrolü ile karşılaştırıldığında neurite akıbet (2D) terfi 300-500 mikron omurilik eksplantlar kenarına yerleştirilir. Benzer ko-kültür sonuçları Wnt5a-ifade hücreleri 8 ile daha önce yayımlanmıştır . Ölçek çubuğu = 200mikron.

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Çıkar çatışması ilan etti.

Biz bir yöntem serum koşullar altında, civciv, kültür E4 SAG ve E6 omurilik eksplantlar teşrih ve sunar. Bu prosedür şu anda SAG nöron hayatta kalma ve neurite akıbet çeşitli salgılanan ligandlar etkilerinin incelendiği laboratuvarda kullanılır. Bu protokolün Roman yönlerini, kültür eksplantlar ve SAG neurite akıbet etkilerini incelemek için büyüme faktörleri batırılmış boncuk kullanımı için serum ücretsiz sistem kullanımını içerir. Civciv omurilik 10 bizimkine benzer bir ...

<table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Katalog numarası </td><td> Yorum </td></tr><tr><td> Ekipman </td><td></td><td></td><td></td></tr><tr><td> Diseksiyon mikroskobu </td><td></td><td></td><td> Biz aydınlık baz aydınlatma ile Leica M80 stereomikroskopta </td></tr><tr><td> Sıcak kaydırın. </td><td> CS &amp; E. </td><td></td><td> Kollajen polimerizasyon </td></tr><tr><td> Piliç kuluçka </td><td></td><td></td><td></td></tr><tr><td> 37 ° C /% 5 CO 2 nemlendirilmiş hücre kültürü inkübatör </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Diseksiyon Malzemeleri </td><td></td><td></td><td></td></tr><tr><td> Sylgard &amp;kopyası; kaplı petri </td><td></td><td></td><td></td></tr><tr><td> 24-kuyu hücre kültürü plaka </td><td> Corning </td><td> 3526 </td><td></td></tr><tr><td> # 5 Dumont forseps </td><td> Güzel Bilim Araçları </td><td> 11252-30 </td><td></td></tr><tr><td> # 55 Dumont forseps </td><td> Güzel Bilim Araçları </td><td> 11295-51 </td><td></td></tr><tr><td> Paslanmaz çelik diseksiyon pimleri </td><td> Güzel Bilim Araçları </td><td> 26002-10 </td><td> Embriyo çivileme, ince diseksiyon </td></tr><tr><td> Moria delikli kaşık </td><td> Güzel Bilim Araçları </td><td> 10370-17 </td><td> Embriyo hasat / transfer </td></tr><tr><td> 200 ul geniş ağız pipetlemeyin ipuçları </td><td> Nokta Scientific Inc </td><td> 118-96R </td><td> Eksplant transferi </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> E4 ve E6 tavuk yumurtası </td><td></td><td></td><td> &amp; Nbsp; </td></tr><tr><td> Chick Ringer solüsyonu </td><td></td><td></td><td> Embriyo hasat </td></tr><tr><td> HBSS </td><td> Sigma </td><td> H8264 </td><td> Diseksiyonu SAG </td></tr><tr><td> L15 orta </td><td> Sigma </td><td> L5520 </td><td> Omurilik diseksiyon orta </td></tr><tr><td> Fetal buzağı serumu </td><td> Atlanta Biyolojik </td><td> S11150 </td><td> Omurilik diseksiyon orta </td></tr><tr><td> Vannas Makas </td><td> Dünya Hassas Aletler </td><td> 501778 </td><td> Omurilik diseksiyonu </td></tr><tr><td>&nbsp;</td><td></td><td></td><td></td></tr><tr><td> Kollajen hazırlık </td><td></td><td></td><td></td></tr><tr><td> Rat-tail Kollajen Tip I </td><td> BD Biosciences </td><td> 354249 </td><td> Eksplant kültür </td></tr><tr><td> 10X PBS (Steril) </td><td></td><td></td><td> Neu içinkollajen tralize </td></tr><tr><td> 1N NaOH (Steril) </td><td></td><td></td><td> Kollajen nötralize </td></tr><tr><td> Damıtılmış su (Steril) </td><td></td><td></td><td> Kollajen nötralize </td></tr><tr><td> pH indikatör kağıtları (6,0-8,1) </td><td> Whatman </td><td> 2629-990 </td><td> Kollajen pH kontrol etmek için </td></tr><tr><td> 15 ml konik tüpler </td><td> Nokta Scientific Inc </td><td> 818-PG </td><td></td></tr><tr><td> 500 ul geniş ağız pipetlemeyin ipuçları </td><td> Nokta Scientific Inc </td><td> 119-R100 </td><td> Pipetlemeyin kollajen </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Eksplant kültür </td><td></td><td></td><td></td></tr><tr><td> DMEM/F12 orta </td><td> Sigma </td><td> D8437 </td><td> Eksplant kültür ortamı </td></tr><tr><td> ITS 1 </td><td> Sigma </td><td> I2521 </td><td> Orta ek </td&gt; </tr><tr><td> Penisilin-Streptomisin </td><td> Invitrogen </td><td> 15140-122 </td><td> Orta ek </td></tr><tr><td> CNTF (sıçan) </td><td> Sigma </td><td> C3835 </td><td> Orta ek </td></tr><tr><td> NT-3 (insan) </td><td> Sigma </td><td> N1905 </td><td> Orta ek </td></tr><tr><td> Saflaştırılmış fare Wnt5a </td><td> R &amp; D Systems </td><td> 645-WN-010 </td><td></td></tr><tr><td> Affi-gel Blue jel boncuklar </td><td> Bio-Rad </td><td> 153-7302 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> İmmünohistokimya </td><td></td><td></td><td></td></tr><tr><td> Paraformaldehit </td><td> Sigma </td><td> P6148 </td><td> Tespit </td></tr><tr><td> PBS </td><td></td><td></td><td> Duruluk </td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T9284 </td><td> Engelleme çözüm </td></tr><tr><td> Sodyumazid </td><td> Sigma </td><td> S8032 </td><td> Engelleme çözüm </td></tr><tr><td> Buzağı serumu </td><td> Invitrogen </td><td> 16170-078 </td><td> Engelleme çözüm </td></tr><tr><td> Fare monoklonal IgG2b anti-β tubulin 1 + II antikor </td><td> Sigma </td><td> T8535 </td><td> Etiketler hücre gövdeleri ve neurites </td></tr><tr><td> Alexa Florlu 488 keçi anti-fare IgG2 b antikor </td><td> Invitrogen </td><td> A21141 </td><td> Β tubulin antikor Algılar </td></tr><tr><td> Teflon mikro spatula </td><td> VWR </td><td> 57949-033 </td><td> Kuyudan Yayın kollajen jeller </td></tr></tbody></table>

dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays

Tavuk iç kulak duyu organları statoacoustic ganglion (SAG) nöronların periferik süreçleri tarafından innerve edilir. Duyu organı innervasyon akson rehberlik yörünge boyunca büyüyen aksonlar ve / veya duyu organı hedeflerine içinde bulunan ipuçları 1 ve hayatta kalma faktörleri 2 bir arada bağlıdır. Örneğin, klasik bir akson rehberlik sinyal yolağı, semaphorin-neuropilin ile fonksiyonel otik aksonlar 3 parazit misrouting oluşturulur. Ayrıca, çeşitli büyüme faktörleri nörotrofin-3 (NT-3) ve beyin türevli Nörotrofik Faktör (BDNF) de dahil olmak üzere, iç kulak duyu hedefler, ifade SAG aksonlar 4 misrouting yeniden lider, transgenik hayvanlar manipüle edilmiştir. Bu aynı molekülleri in vitro 5,6 hayatta kalma ve civciv SAG nöronların neurite akıbet teşvik. Burada, tarif ve in vitro yöntem şu anda u göstermekbilinen Wnts gibi morphogens yanı sıra SAG neurite akıbet ve nöron hayatta kalma teşvik için önemli olan büyüme faktörleri de dahil olmak üzere çözünür proteinler, civciv SAG neurites yanıt test için şarkı. Bu model sistemi kullanarak, salgılanan ligandlar SAG nöron hayatta kalma ve neurite akıbet uygulamayın ki etkileri hakkında sonuçlara varmak için umuyoruz. SAG eksplantlar, embriyonik gün 4 (E4) disseke ve 24 saat boyunca serum serbest koşullar altında, üç boyutlu kollajen jel kültür. İlk olarak, neurite yanıt verme, protein takviye orta kültür eksplantlar tarafından test edilir. Daha sonra, salgılanan ligandlar noktası kaynakları neurite akıbet yönlü etkilere sahip olup olmadığını sormak için, eksplantlar protein kaplı boncuklar ile co-kültürlü ve yerel teşvik etmek veya akıbet inhibe boncuk yeteneği test. Ayrıca diseksiyon gösteri (protokol 7 değiştirilmiş) ve E6, spinal kord eksplantlar kültürü içerir. Bizrutin proteinleri ve protein batırılmış boncuklar biyoaktivite onaylamak için omurilik eksplantlar kullanımı ve SAG eksplantlar aynı kültür koşulları altında, civciv dokusu ile türlerin çapraz reaktivite doğrulamak için. In vitro tayinlerde Bunlar hızlı bir şekilde özellikle in vivo çalışmalar gerçekleştirmeden önce, trofik (hayatta kalma) veya SAG nöronlar tropik (yönlü) etkilerini gösterirler molekülleri için tarama için uygundur. Dahası, bu yöntem yüksek nöron hayatta kalma 8, serum serbest şartlar altında bireysel moleküllerin test izin verir.

Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Biz civciv E4 statoacoustic ganglion ve E6 omurilik eksplantlar incelemek ve kültürüne nasıl gösterilmektedir. Eksplantlarındaki 24 saat için 3D kollajen jel serum serbest koşullar altında kültüre. Neurite yanıt büyüme faktörü desteği orta ve protein kaplı boncuklar ile test edilir.

Diseksiyon ve Chick Statoacoustic Ganglion ve Kollajen Jeller Omurilik eksplantlar Kültür Neurite akıbet Tahliller

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

dissection-culture-chick-statoacoustic-ganglion-spinal-cord-explants

Research

JoVE Journal

Neuroscience

12.0K Görüntüleme.  Purdue University. Biz civciv E4 statoacoustic ganglion ve E6 omurilik eksplantlar incelemek ve kültürüne nasıl gösterilmektedir. Eksplantlarındaki 24 saat için 3D kollajen jel serum serbest koşullar altında kültüre. Neurite yanıt büyüme faktörü desteği orta ve protein kaplı boncuklar ile test edilir.

Video: Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nörobilim

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Kültür Bulaşık A Fonksiyonel Motor Birim: Omurilik Eksplantlarından ve Kas Hücrelerinin Co-kültür

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Fare Dopaminerjik ve Üç Boyutlu Collagen Matrix Tahliller striatal Eksplantlarından diseksiyonu ve Kültür

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Tavuk embriyo Omurilik Dilim Kültür Protokolü

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

Diseksiyon ve Zebra balığı Embriyolar Yanal Montaj: Omurilik Kalkınma Analizi

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Sonradan wholemount için komple Omurilik Yaralanmalı ve Beyin Diseksiyon Protokolü<em&gt; Yerinde</em&gt; Larva Deniz taşemen içinde Hibritleşme

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

Electroporated Chick Embriyonik Omurilik Diferansiyel Gen İfade RNA Sıra Analizi

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Yenidoğan Faresinden İzole Edilen Spinal Kord Nöronları İzolasyonu ve Kültürü

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Nöral Kök Hücrelerin Sıçan ve Fare Beynine İntra-Arteriyel Teslimatı: Serebral İskemiye Uygulama

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...