NIH K08 EB003996

1. Poli-L-laktik asit (PLLA Çözüm İplik) <ol><li> 9 mL kloroform 0.4 g PLLA kısık ateşte karıştırarak eritin. </li><li> DMF: 09:01 (v / v) kloroform% 4 PLLA (w / v) çözümün nihai konsantrasyonu getirerek, çözüm için 1 ml dimetilformamid ekleyin. </li><li> Künt 23ga metal ucu ile bir polipropilen ya da cam şırınga çözüm yerleştirin. </li></ol> 2. Yüzey Hazırlığı 1 İplik <ol><li> 85:15 PLGA% 8 (w / v) solüsyonu (poli-laktik-ko-glikolik asit), kısık ateşte karıştırarak tarafından kloroform olun. </li><li> PLGA çözümü ile her kapak kayma yüzeyi kaplayan tarafından PLGA Coat temiz cam kapak fişleri. PLGA ince bir film (yaklaşık 30 dakika) kurumasını bekleyin. </li></ol> 3. Elektrospinning 2 <ol><li> Güvenli PLGA kaplı cam kapak, iletken karbon bant ile kolektör sığar. Hizalanmış lifleri için, toplayıcı, motorlu tekerlek. Kollektör rastgele lifleri için, sabit bir plaka. </li><li> Yeri şırınga ucu 20 cm toplayıcı tekerlek ile pompa. Yaklaşık 2 mL / saat pompa seti ve bir tekerleğini kullanarak, 300-400 RPM motor ayarlarsanız. Mümkünse, -2 kV DC kollektör önyargı ve 15 kV metal uç geçerlidir. Iki kutuplu bir güç kaynağına erişim yokluğunda, kollektör zemin. </li><li> Elyaf şırınga ucu jet ve dönen bir tekerlek üzerinde toplamak. Liflerin istenen yoğunluk elde edilinceye kadar iplik devam edin. Swipe bir kağıt havlu ile metal ucu olmayan bir iletken çubuğun ucunda periyodik tıkanmasını önlemek için yapıştırılmış. </li></ol> 4. 1 Moating <ol><li> Ardından iplik, kapak kayma çevresinde PLGA bir çizgi pipetleme hendek electrospun örnekleri. PLGA kurumasını bekleyin. Bu kültür sırasında lifler uyum koruyacaktır. </li></ol> 5. Protein Kaplama <ol><li> Electrospun cam elyaf ve kontrolleri hücre kültürü öncesinde protein kaplı olması gerekir. En az bir saat için bir protein çözüm substratlar Kapak ve daha sonra aşırı bir çözüm aspirat ve aspire substratlar kuruyana kadar devam edin. Olası protein çözümler şunlardır: PLL (poli-L-lisin) (100 mg / ml), laminin (25 mcg / ml) veya fibronektin (50 mg / ml). PLL kullanılması durumunda, substratlar steril su ile durulanır ve ilk kaplama aşağıdaki iki kez kuru olmalıdır. </li></ol> 6. E15 Rat Embriyolar Edinme 3 * Bütün deneyler NIH Hayvanların Kullanımı ve Bakımı, Michigan Komitesi Üniversitesi tarafından onaylanan Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu doğrultusunda yapıldı. <ol><li> Hazırlanışı: hayvanın gebe olup olmadığını kontrol edin. 3 ml tripsin, 3 mL FBS ve L15 sıcak bir şişe çözülme. Yangın bir Pasteur pipeti lehçe. 30 dakika süreyle% 70 etanol bütün cerrahi aletler dezenfekte edin. </li><li> Kaplama ortamı hazırlayın ve sıcak (bkz. Tablo 1). </li><li> Ötenazi: ketamin / xylazine bir IP enjeksiyon gerçekleştirin. 10-15 dakika bekleyin ve kornea eksikliği olup olmadığını kontrol edin ve geri çekilme refleksleri pedal. Refleksleri sonra, pentobarbital (FatalPlus) bir IC enjeksiyon gerçekleştirin. </li><li> Çadır karın cilt. Karın boşluğu ortaya çıkarmak için, kas ve deri katmanları yoluyla kesin. Bulun rahim ve rahim ağzı ve yumurtalıklar ayırmak. </li><li> Makas ile embriyolar rahim çıkarın ve hemen sıcak L15 içine koyun. (Diseksiyon mikroskobu ve embriyolar altında aşağıdaki işlemler tamamlandı ve disseke kablolarını her zaman sıcak L15 batık olmalıdır.), Kapatma ve çene etrafında forseps aşağıdaki oksipital kemik, kafatası embriyolar başını kesmek. </li></ol> 7. Diseksiyon 3 <ol><li> Bir tarafta embriyo yerleştirin. Bacaklarda ve karın içi organların şerit diğer seti kullanılarak bir dizi forseps ile omurilik koruyun. </li><li> Embriyo açın ve bir set forseps ile sabit tutun. Tüm kablosu maruz kadar, boyundan kuyruk uzunluğu boyunca embriyo Snip. </li><li> Kablosunun sonunda dikkatli bir şekilde tutun ve kaldırmak için kuyruk karşı kendini üzerinden çekin. Kablosu membran ve dorsal kök gangliyon (DRG) bağlı çeker. </li><li> DRG eksplant veya ayrışmış bir duyusal nöron kültürü yapılacak ise, kablo kapalı DRG snip ve L15 sıcak taze yemek aktarabilirsiniz. <ol><li> Eksplant kültür DRG için, 10.2 adıma geçin </li><li> Ayrışmış duyusal nöron kültürü için, 9. adıma geçin. </li><li> Motor nöron kültürü için, DRG adım 7.5 &#39;de membran ile silinecektir </li></ol></li><li> Uzun çorap çıkarmadan benzer kablosunun üst ucunda açgözlü ve aşağı soyma omurilik zarı, çıkarın. Tüm embriyolar için tekrarlayın ve sonra küçük parçalar halinde (2-3 mm) kordonlar doğrayın. </li></ol><pclass = &quot;jove_title&quot;&gt; 8. Motor Nöron (MN) İşleme 5,6,7 <ol><li> 2-3 dakika süreyle adet pelet 1000 RPM konik ve spin içine doğranmış kablolarını ve L15 dökün. </li><li> Süpernatantı dökün ve tripsin 3 mL pelet kabloları ekleyin. 20 dakika süreyle 37 ° C su banyosunda inkübe edin. </li><li> Konik ve 2-3 dakika süreyle yeniden pelet 1000 RPM dönüş 3 mL FBS ekleyin. </li><li> Süpernatantı ve kat FBS ile bir yangın cilalı Pasteur pipeti dökün. </li><li> Konik bir Pasteur, L15, pipet dolu ve çözüm görünür kümeleri ile homojen olana kadar yavaşça çiğnemek. Kabarcıkları kaçının. </li><li> L15 Optiprep% 9 çözüm hazırlayın. 3 mL Optiprep çözüm her iki embriyo (embriyo bir tek sayı varsa, kadar yuvarlak) bir tüp hazırlayın. </li><li> Tüm tüpler arasında eşit olarak bölünerek homojenize çözüm, Optiprep çözüm üzerine bırakın. Tüpleri 15 dakika için 2000 RPM Spin. </li><li> Üst 2 ml, 50 ml konik her tüp ve havuz dikkatlice toplamak. Optiprep hücreleri durulamak için 5 dakika için 1000 RPM hızında, L15 ve spin konik doldurun. </li><li> Süpernatantı dökün ve kaplama medya (250-500 ul) küçük bir miktar hücrelerin tekrar süspansiyon. Hücreler, canlı hücreleri belirlemek için tripan mavisi dışlama kullanarak güvenin. 10.1 adıma geç </li></ol> 9 - Duyusal Nöron (SN) İşleme 7 <ol><li> 2-3 dk pelet adet 1000 RPM konik bir tüp ve spin omurilik ve L15 kaldırılır DRG dökün. </li><li> Süpernatantı dökün ve tripsin 3 mL pelet DRG ekleyin. 10 dakika süreyle 37 ° C su banyosunda inkübe edin. </li><li> Konik ve 2-3 dakika süreyle yeniden pelet 1000 RPM dönüş 3 mL FBS ekleyin. </li><li> Süpernatantı ve kat FBS ile bir yangın cilalı Pasteur pipeti dökün. </li><li> Konik bir Pasteur, L15, pipet dolu ve çözüm görünür kümeleri ile homojen olana kadar yavaşça çiğnemek. Kabarcıkları kaçının. </li><li> 5 dakika için 1000 RPM Homojenat Spin. Süpernatantı dökün ve SN kaplama medya (250-500 ul) küçük bir miktar hücrelerin tekrar süspansiyon. Hücreler, canlı hücreleri belirlemek için tripan mavisi dışlama kullanarak güvenin. 10.1 adım devam edin </li></ol> 10 Kaplama ve Kültür <ol><li> : Ayrışmış SN veya MN kültür için <ol><li> Sonra birkaç damla medya substratlar Kapak uygun bir hacimli hücre süspansiyonu ekleyin. Disosiye hücreleri düşük yoğunluklu (25-50 hücre / mm 2) kaplama olmalıdır. Hücrelerin en az bir saat önce, medya ile kuyu sel uyması izin verin. </li></ol></li><li> Eksplant kültür DRG için: <ol><li> Medya birkaç damla substratlar Kapak sonra medyaya DRG ekleyin. Substrat (22x22 mm kapak astar üzerine yaklaşık 4 DRG uyacak) eşit dağılmış, böylece DRG düzenleyin. DRG medya ile kuyuların sel önce en az 4 saat boyunca uyması izin verin. </li></ol></li><li> Kültürler medya değişiklik olmadan 4 gün öncesine kadar devam edilebilir. Besleme medya ile uzun kültürler için, yarım medya değişiklikler yapılmalıdır. Besleme medya kaplama medya eksi L-glutamin aynı kompozisyon. </li></ol> 11. İmmünositokimya 1,5,8 <ol><li> Tespit hücreleri: aspire medya ve 30 dakika süreyle sıcak% 4 PFA (paraformaldehid) ile değiştirin. Sonra 5 dakika 1x PBS 3X yıkar gerçekleştirin. </li><li> Permeabilization Engelleme / blok / perma solüsyonu (% 1,25 1X PBS albümin sığır serumu,% 0.05 Triton X-100 ve% 2,0 &#39;normal keçi serumu) hazırlayın. Aspire PBS ve 30 dakika için örnek blok / perma çözümü uygulamak. Sonra 1X 5 dakika 1x PBS yıkar gerçekleştirin. </li><li> Primer antikor: primer antikor çalışma çözeltisi hazırlayın (% 10 normal keçi serumu,% 1.25 sığır serum albumin,% 0.1 Na azid, 1x PBS ile% 0.05 Triton X-100 ve 10 ml). (Bkz. Tablo 2) primer antikor uygun miktarda ekleyin. Parafilm doğrultusunda birkaç yemekler. Her bir yüzey için parafilm birincil antikor çözüm 200μl boncuk yerleştirin. Substratları haricindekileri, gece primer antikor çalışma çözeltisi hücre tarafı aşağı kayan (oda klimalı ya da özellikle kuru ise, suya doymuş kağıt bir parça çanak çalışma çözeltisi buharlaşma önlemek için bir köşesinde eklenebilir.) . </li><li> İkincil antikor: birincil (çalışma solüsyonu ° 4 saklanan, C toplanan ve yeniden olabilir) substratları çıkarın ve 2X 5 dakika 1x PBS yıkar gerçekleştirmek. 200 ul sekonder antikoru 1:200 sulandırılmış PBS (parafilm astarlı çanak ters) 4 saat süreyle uygulayın. </li><li> Sonra ikincil dan çıkarın 2X 5 dk 1x PBS yıkar gerçekleştirin. Dağı substratlar slaytlarla DAPI veya başka uygun bir nükleer sayaç içeren Altın leke uzatır. </li></ol> 12. Temsilcisi Sonuçlar: Hizalanmış ve rastgele El tipik morfolojiectrospun lifleri Şekil 1&#39;de gösterilmiştir. Biz genellikle bu protokol ile&gt;% 90 nöronlar 7,8 MN saflık elde etmek ve herhangi bir önemli fibroblast büyüme olmadan uzun bir hafta boyunca kültürlerin DRG korumuştur. Şekil 2, 24 saat sonra kültür ve immün cam ve elyaf tipik MN görünüm gösterir. Cam ve elyaf üç gün boyunca kültür immunohistokimyasal DRG Şekil 3&#39;te. <img src="/files/ftp_upload/2389/2389fig1.jpg" alt="Şekil 1" /> Şekil 1. Fiber hizalama, koleksiyoncu seçimi tarafından manipüle . A.)) dönen bir tekerlek toplayıcı ve B. üzerine bükülmüş liflerin Bağlantısızlar rasgele lifler sabit bir plaka toplayıcı üzerine döndü. Ölçek bar = 20 mm. <img src="/files/ftp_upload/2389/2389fig2.jpg" alt="Şekil 2" /> Şekil 2) PLL kaplı A. 24 saat sonra kültür TuJ1 (yeşil) ve DAPI (mavi) elyaf ve B. lekeli Temsilcisi motor nöronlar) cam. Ölçek bar = 20 mm. <img src="/files/ftp_upload/2389/2389fig3.jpg" alt="Şekil 3" /> Şekil 3 PLL kaplı A. 3 gün sonra kültür nörofilaman Temsilcisi DRG boyandı (yeşil)) cam ve B.) lifleri. Ölçek bar = 200 mm. <table border="1"><tbody><tr><td> Stok çözelti: </td><td> MN medya: </td><td> DRG / SN medya: </td></tr><tr><td> 10 mg mL -1 albümin </td><td> 12.5 ul </td><td> - </td></tr><tr><td> 10 mg mL -1 apo-transferrin </td><td> 50 ul </td><td> 40 ul </td></tr><tr><td> 50 mg mL -1 β-östradiol </td><td> 2.7 ul </td><td> 2.2 ul </td></tr><tr><td> 0.1 mg mL -1 biotin </td><td> 50 ul </td><td> - </td></tr><tr><td> 16 mg mL -1 katalaz </td><td> Ul 8 </td><td> - </td></tr><tr><td> 15 mg mL -1 D-galaktoz </td><td> 50 ul </td><td> - </td></tr><tr><td> 50 mg mL -1 hidrokortizon </td><td> 3.7 ul </td><td> 2.9 ul </td></tr><tr><td> 0.63 mg mL -1 progesteron </td><td> 0.5 ul </td><td> - </td></tr><tr><td> 16 mg mL -1 putrescine </td><td> 50 ul </td><td> - </td></tr><tr><td> 50 mg mL -1 selenyum </td><td> 3.4 ul </td><td> 4.1 ul </td></tr><tr><td> 2.5 mg mL -1 süperoksit dismutaz </td><td> 50 ul </td><td> - </td></tr><tr><td> * 500 ng mL -1 sinir büyüme faktörü </td><td> - </td><td> 1 ml </td></tr><tr><td> * 100X PSN </td><td> 0,5 mL </td><td> 0,5 mL </td></tr><tr><td> * B27 </td><td> 1 ml </td><td> 1 ml </td></tr><tr><td> * 2 mM L-glutamin </td><td> 35 ul </td><td> 35 ul </td></tr><tr><td> * Neurobasal </td><td> 50 ml </td><td> 50 ml </td></tr></tbody></table> Tablo 1: kullanımdan hemen önce medyaya (*) bileşenleri oynadı ekleyin. Geri kalan bileşenleri bir stok solüsyonu olarak hazırlanan ve 6,7 ihtiyaç duyulana kadar -20 ° C&#39;de muhafaza edilebilir. <table border="1"><tbody><tr><td> Primer (konsantrasyon) </td><td> Nöronlar: </td><td> Glia: </td><td> Fibroblastlar: </td></tr><tr><td> anti-β-tubulin (TuJ1) (1:1000) </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td> anti-nörofilaman (1:1000) </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td> anti-S-100 (1:250) </td><td> X </td><td> ✓ </td><td> ✓ </td></tr><tr><td> anti-NGFR P75 (1:500) </td><td> ✓ </td><td> ✓ </td><td> X </td></tr></tbody></table> Tablo 2. Primer antikor seçim araştırmacının hedeflerine bağlıdır. Yukarıda birkaç antikorlar ve laboratuvarda başarı ile kullanılan konsantrasyonları. S-100, Schwann hücreleri ve / veya fibroblastlar kirlenmesine kontrol tanımlamak, ancak glia ve fibroblasts4 ayırt etmek NGFR P75 ile birlikte kullanılmalıdır gerektiğini özellikle yararlıdır. Ayrıca nörofilaman veya TuJ1 amacı bireysel neurites görselleştirmek için ise mükemmel bir seçimdir NGFR P75 lekeleri çok hafifçe neurites fark var.

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	<li>Corey, J. M., Gertz, C. C., Wang, B. S., Birrell, L. K., Johnson, S. L., Martin, D. C., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+electrospun+PLLA+nanofiber+scaffolds+compatible+with+serum-free+growth+of+primary+motor+and+sensory+neurons.">The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons.</a> Acta. Biomater. 4, 863-875 (2008).</li><li>Lin, D. Y., Johnson, M. A., Vohden, R. A., Chen, D., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailored+nanofiber+morphologies+using+modulated+electrospinning+for+biomedical+applications.">Tailored nanofiber morphologies using modulated electrospinning for biomedical applications.</a> Mat. Res. Soc. Symp. Proc. 736, D3.8.1-D3.8.6 (2003).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1998).</li><li>Kameda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+glial+progenitor+markers+p75NTR+and+S100+protein+in+the+developing+mouse+parathyroid+gland.">Expression of glial progenitor markers p75NTR and S100 protein in the developing mouse parathyroid gland.</a> Cell Tissue Res. 327, 15-23 (2007).</li><li>Corey, J. M., Lin, D. Y., Mycek, K. B., Chen, Q., Samuel, S., Feldman, E. L., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+electrospun+nanofibers+specify+the+direction+of+dorsal+root+ganglia+neurite+growth.">Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth.</a> J. Biomed. Mater. Res. A. 83, 636-645 (2007).</li><li>Vincent, A. M., Mobley, B. C., Hiller, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-I+prevents+glutamate-induced+motor+neuron+programmed+cell+death.">IGF-I prevents glutamate-induced motor neuron programmed cell death.</a> Neurobiol. Dis. 16, 407-416 (2004).</li><li>Russell, J. W., Windebank, A. J., Schenone, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor-I+prevents+apoptosis+in+neurons+after+nerve+growth+factor+withdrawal.">Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal.</a> J. Neurobiol. 36, 455-467 (1998).</li><li>Gertz, C. C., Leach, M. K., Birrel, L. K., Martin, D. C., Feldman, E. L., Corey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+neuritogenesis+and+maturation+of+primary+spinal+motor+neurons+in+response+to+nanofibers.">Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers.</a> Dev. Neurobiol. 70, 589-603 (2010).</li><li>Tan, S. -. H., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+parameter+study+for+ultra-fine+fiber+fabrication+via+electrospinning+process.">Systematic parameter study for ultra-fine fiber fabrication via electrospinning process.</a> Polymer. 46, 6128-6134 (2005).</li><li>Wang, H. B., Mullins, M. E., Cregg, J. M., Hurtado, A., Oudega, M., Trombley, M. T., Gilbert, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+highly+aligned+electrospun+poly-L-lactic+acid+fibers+for+nerve+regeneration+applications.">Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications.</a> J. Neural Eng. 6, (2009).</li></ol>

Bu protokol, pek çok kritik adımlar vardır. Ilk electrospun fiber yüzeylerin uygun üretim içerir. PLLA lifler, PLGA film ve PLGA hendek sıvı medya, tek bir birim olarak cam kapak slip ayrı olacaktır. PLGA atlanırsa, PLLA lifleri, düz bir levha kalamaz kullanılamaz karmaşasının içine kıvrılıp. Böylece, PLGA filmi kültür sırasında fiber uyum sağlamak için uygun bir substrat olarak yer almaktadır. Hendek lifler güvenli PLGA film sabit ve filmin yapısal katılık eklemek sağlamak için her iki iplik sonra eklenir. Yüzeyler, montaj, boyama ve montaj sırasında manipüle hendek de yararlı bir kolu olarak hizmet vermektedir. Öğütme ikinci kritik bir adımdır. Kabarcık oluşumunu önlemek için her türlü çabayı yapılmalıdır. Ayrıca, bu adımı bir yangın cilalı pipet kullanıldığında çok daha yüksek verim elde edilmiştir. Lehçe yangın, bir Bunsen beki ışık ve pipet bir ampul takın. Sürekli sıkma ve ampul bırakırken alev ile 2-3 kez hızlı bir şekilde pipet ucu Pass - pipet ile hava akışı tamamen kapatarak ucu önlemeye yardımcı olur. Pipet ucu delik hala açık olduğundan emin olmak için inceleyin ve kullanmadan önce biraz yuvarlatılmış kenarları görünür. Elektrospinning çok yönlü bir süreçtir; elektrospinning parametreleri morfolojileri çeşitli lifler üretmek için modifiye edilebilir. , Tan ve ark. (2005) PLLA electrospun liflerin çapı etkileyen parametrelerin sistematik bir çalışma . Wang ve ark (2009), hizalama electrospun PLLA lifleri etkileyen parametreler detaylı bir çalışma sunar .

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>PLLA</td>
<td>&nbsp;</td>
<td>Boehringer Ingelheim</td>
<td>Resomer L210</td>
<td>&nbsp;</td>
<tr>
<td>PLGA 85:15</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43471</td>
<td>&nbsp;</td>
<tr>
<td>L15</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>11415</td>
<td>&nbsp;</td>
<tr>
<td>Albumin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2289 
</td>
<td>&nbsp;</td>
<tr>
<td>Apo-transferrin</td>
<td>&nbsp;</td>
<td>Akron </td>
<td>AK8227</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC 23009-0010</td>
<td>&nbsp;</td>
<tr>
<td>Galactose</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>G0625</td>
<td>&nbsp;</td>
<tr>
<td>Progesterone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7556</td>
<td>&nbsp;</td>
<tr>
<td>Putrescine </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5780</td>
<td>&nbsp;</td>
<tr>
<td>Selenium </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S9133</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-estradiol</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>E 1132 </td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>H0396</td>
<td>&nbsp;</td>
<tr>
<td>Catalase </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C40 </td>
<td>&nbsp;</td>
<tr>
<td>Superoxide dismutase</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S4636</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>PSN</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15640 </td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1680149</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1689149</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Optiprep</td>
<td>&nbsp;</td>
<td>Accurate </td>
<td>2011</td>
<td>&nbsp;</td>
<tr>
<td>PLL</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4832 </td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>F 4759 </td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>B27</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
<tr>
<td>Anti-TuJ1</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556321</td>
<td>&nbsp;</td>
<tr>
<td>Anti-neurofilament</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB1987</td>
<td>&nbsp;</td>
<tr>
<td>Anti-S100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2532</td>
<td>&nbsp;</td>
<tr>
<td>Anti-NGFR p75</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N3908</td>
<td>&nbsp;</td>
<tr>
<td>Normal goat serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G6767</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8032</td>
<td>&nbsp;</td>
<tr>
<td>Carbon tape</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13073-1</td>
<td>&nbsp;</td>
<tr>
<td>Prolong Gold +DAPI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P36931</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the culture of primary motor and sensory neurons in defined media on electrospun poly-l-lactide nanofiber scaffolds

Elektrospinning nano ölçekli liflerin mikro üretmek için kullanılan bir tekniktir. Elyaf yüksek tamamen rasgele hizalanır, uyum değişen derecelerde ile electrospun olabilir. Buna ek olarak, lifler, poli-L-laktik asit (PLLA) gibi biyolojik olarak parçalanabilen polimerler dahil olmak üzere çeşitli malzemeler spun olabilir. Bu özellikler, doku mühendisliği iskele uygulamaları bir dizi için uygun electrospun elyaf olun. Bizim odak sinir rejenerasyonu için hizalanmış electrospun liflerin kullanımı ile ilgili. Biz daha önce hizalı electrospun PLLA lifleri in vitro hem de primer duyu ve motor nöronların akıbet doğrudan göstermiştir . Biz mümkün olduğunca yakın bir in vivo model gerçek nöronların biyomateryaller değerlendirirken birincil hücre kültürü sisteminin kullanılması gerekli olduğunu korur. Burada, fibröz iskeleleri ve kültür dorsal kök gangliyon eksplantlar, yanı sıra electrospun iskeleleri dissosiye duyu ve motor nöronlar, electrospin laboratuvarımızda kullanılan teknikler açıklanmaktadır. Ancak, elektrospinning ve / veya burada sunulan kültür teknikleri kolayca diğer uygulamalarda kullanılmak üzere uyarlanmıştır.

Watch this Scientific Journal Video about The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds at JoVE.com

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Bağlantısızlar electrospun lifler in vitro nöron büyüme doğrudan ve sinir rejenerasyonu iskeleleri potansiyel bir bileşenidir. Biz electrospun elyaf yüzeyleri ve birincil sıçan E15 duyusal (DRG) ve motor nöronların serum serbest kültür hazırlanması için bir prosedür açıklanmaktadır. Immünsitokimya tarafından nöronların Görselleştirme de dahildir.

Kültür İlköğretim Motor ve Electrospun Poli-L-lactide Nanofiber Yapı iskeleleri Tanımlı Medya Duyu Nöronlar

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

the-culture-primary-motor-sensory-neurons-defined-media-on

Research

JoVE Journal

Neuroscience

19.3K Görüntüleme.  University of Michigan. Bağlantısızlar electrospun lifler in vitro nöron büyüme doğrudan ve sinir rejenerasyonu iskeleleri potansiyel bir bileşenidir. Biz electrospun elyaf yüzeyleri ve birincil sıçan E15 duyusal (DRG) ve motor nöronların serum serbest kültür hazırlanması için bir prosedür açıklanmaktadır. Immünsitokimya tarafından nöronların Görselleştirme de dahildir.

Video: The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

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

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Embriyonik Fare Hipokampal ve Kortikal nöronlar primer Nucleofection ve Kültür

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Koku Duyu Nöronlar Cilia Kafesli Bileşiklerin Flash Fotoliz

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

İlköğretim Rat Kortikal Nöronlar ve Glia bilaminar Co-kültür

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Kortikal Nöronlar In Vivo İki foton Of Görüntüleme Deneyimi bağımlı Moleküler Değişiklikler

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

In Koku Reseptör Nöronlar Tüm Dağı ile immün<em&gt; Drosophila</em&gt; Anten

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Fare dopaminerjik nöronlar İlköğretim Kültür

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Civciv Embriyolar itibaren İzolasyon ve Disosiye Duyu Nöronlar Kültür

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebra balığı<em&gt; Situ</em&gt; Omurilik Omurilik duyusal ve motor nöronlar gelen Elektrofizyolojik Recordings Hazırlık

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Larva zebra balığı sonradan içini kaplamak saç hücrelerinin vivo kalsiyum görüntüleme

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...