Yazarlar Dr Alistair Garratt (Max Delbrück Merkezi, Berlin) faydalı yorumları için teşekkür etmek istiyorum. Bu çalışma Alman Araştırma Kurumu (DFG) ortak araştırma merkezi (SFB665) tarafından desteklenmiştir.

1. Diseksiyon işlemi Not: farelerde deneysel laboratuvar hayvanlarının bakımı ve kullanımı için kurallar resmen onaylanmış takip etmelidir. <ol><li> Hazırlanmadan önce, diseksiyon mikroskobu kurmak ve forseps (iç cilalanmış ipuçları var ikisi) diseksiyon büyük ve küçük makas, büyük dişli forseps, kavisli bir forseps ve Dumont No.5 dört setleri de dahil olmak üzere diseksiyon için gerekli cerrahi aletler yatıyordu ( detaylar) özel reaktifler ve ekipman tabloya bakın. 100 mm Sylgard kaplı Petri kabındaki filtre kağıdı bir sayfa yerleştirin. 12 plaka, 100 mm petri ve 12 ml tüp kuyulardan soğuk PBS dökün ve buz üzerinde bırakın. Buz üzerinde ikinci bir 12 plaka yeri ve her iyi fiksasyonu tampon Pipet 2ml (PBS, pH 7.4% 4 paraformaldehid). </li><li> Anesthetization sonra E12.5 (vajinal fişi tespit E0.5 olarak belirlenmiştir), zaman hamile baraj kurban, fare yerleştirinüç kağıt havlu, çarşaf ve% 70 etanol ile karın bölgesine emmek ventral yüzü yukarı. </li><li> Abdominopelvik boşluğuna açın, bilateral uterin boynuz izole etmek ve buz PBS ile 100 mm yemekler birine aktarabilirsiniz. </li><li> Mikroskopik kontrolü altında, küçük, düz makas ile rahim duvarına uzunlamasına bir kesi yapmak her amniyon kesesi ve plasenta kesip. </li><li> Uzakta Peel her embriyo amniyon kesesi ve göbek kordonu kesmek. </li><li> Küçük bir makas kullanarak, embriyoların başını kesmek ve genomik DNA izolasyonu için genotipleme gerekli olmalıdır başkanları veya bunların bir kısmını bir kenara koymak. Daha sonra, 12 plaka, soğuk PBS ile ilgili kuyu torsi aktarın. </li><li> PBS birkaç damla ile Sylgard çanak filtre kağıdı ıslak ve kağıt üzerinde, dorsal yüzü embriyonik gövde yukarı konumda. Daha iyi denge için, vücut uzak, kuyruk ve ekstremitelerde düzeltin. </li><li> Çalışma16x büyütme ile yaklaşık bir diseksiyon mikroskobu altında, iki çift ince uçlu forseps ile omurilik üzerinde embriyo cilt dikkatlice çimdik ve nazikçe onu parçalamaktır. Ortalarından başlayarak, ilk kuyruk doğru devam edin ve sonra ön tarafına doğru orta sürdürme. </li><li> Embriyo, kurumasını önlemek için iki veya üç damla PBS zaman zaman ıslatın. </li><li> Cilalı iç-ipucu ile ince bir forseps bıçak ile yatay kayma hareketleri çevreleyen kıkırdak vertebral kolon DRG ve omurilik ayırmak embriyo kaldırıldığında bu DRG önlemek için omurilik kapalı teared. Ön sonuna kadar embriyo sağ tarafının ortasında başlayarak, daha sonra kuyruk doğru şekilde çalışır ve. Daha sonra, diğer tarafta işlemi tekrarlayın. Ilişkili DRG rip off için dikkatli olun. </li><li> Embriyo gevşetti omurilik tamamen ayırmak için, onun cervic pick upal ince forseps ile kaudal sonuna doğru tek parça halinde dışarı doğru çekin. </li><li> Izole spinal fiksasyon tamponu ile dolu 12 plaka iyi kablosunu bağlı DRG olan ve geri kalan embriyolar omurilik hazırlık ile devam Batmak. </li><li> Omurilik Fix buz üzerinde en az iki saat. </li></ol> 2. DRG nöronlar DII etiketleme <ol><li> Etiketli her omurilik için bir mikropipet çektirmesi ile iki cam iğneler hazırlar. 2 ISI = 600, VEL = 25 = 175, 3, ZAMAN ISI = 630, VEL = 1 ISI = 625, VEL = 35 = 175, TIME: Sutter model P-97 elektrot çektirmesi (program ayarı 30, ZAMAN = 150). </li><li> Mikroskobik kontrolü altında, etanol içinde DII% 5 (w / v) çözeltisi 3-5 kez, her bir cam iğne ucu batırın. Buharlaşma etanol DII kristalleri iğne üzerinde ince bir tabaka oluşturur. </li><li> Yaklaşık 40x büyütme ile diseksiyon mikroskobu altında çalışan, v ile spinal kordentral yüzü yukarı bir slayt. Yaylı makas kullanılarak, daha sonra ters bir açık-book modunda (bkz. kısım C) hazırlık montaj izin Taban ile keserek bütün uzunluğu ventral tarafta kordon açmak. </li><li> Bir slayt dorsal yüzü omurilik yerleştirin. Sonra mikroskobik görselleştirme DII kaplı altında cam iğne ucu ve omurilik bir tarafında dikkatle delmek her saniye DRG elle kablosu yaklaşım. DII hemen hemen hiçbir iz sadece küçük bir nöron sayısı etiketleme sağlamak için deldi DRG görünür olmalıdır. Kablonun diğer tarafına ikinci bir iğne götürün. Sadece her saniyede DRG hedefleyen bireysel akson farklılaşma zorlaştıran komşu DRG etiketli aksonal projeksiyonlar bir örtüşme önler. </li><li> Ortam sıcaklığı en az altı saat ya da 4 gece, karanlıkta fiksasyon tampon ve inkübe omurilik dön ° C di boya için zaman vermekplazma zarı içinde akson boyunca ffuse. Omuriliğe teminat, büyüme analizi isteniyorsa daha uzun bir kuluçka dönemi (iki gün kadar 4 ° C) için izin ver. </li></ol> 3. Montaj ve mikroskopik analizler <ol><li> Dorsal yüzü tek bir omurilik PBS açılan bir lamel aşağı boya difüzyon, pozisyon sonrasında. Ekli DRG laterale doğru yönde ve karışmaz özen gösterin. Aşırı sıvı aspire ve forseps kullanarak ters bir açık-book modunda kablosu düzleştirmek. </li><li> Hazırlık PBS kullanarak bir mikroskop lamı üzerine monte edin. </li><li> Etiketli aksonal projeksiyonlar istenmeyen arka plan boyama floresan mikroskopik analiz artışı önlemek için montaj gün yapılmalıdır. 4 slaytlar karanlıkta tutmak kadar ° C </li></ol> 4. Temsilcisi Sonuçlar: Fare omurilik alır afferent prservikal 8 çift ojections, 13 çift torakal, lomber ve sakral DRG 4 çift 60 spinal ganglionlar olmak üzere toplam 5 çift. Bir eğitimden sonra en DRG ile hala bağlı bir omurilik beş dakika altında bir embriyo izole edilebilir. Bu prosedür E11.5 E13.5 fare embriyoları bağlı DRG omurilik izolasyonu için uygundur. Ancak, en iyi sonuçları E12.5 elde edilir. Burada açıklanan etiketleme prosedürü Örnek sonuçları Şekil 3&#39;te gösterilmiştir. Spinal kord uzunluğu boyunca DRG etiketleme sonra farklı vertebra seviyelerinde (Şekil 4) aksonal dallanma davranışını ölçmek için kullanılıyor olabilir. <img alt="Şekil 1" src="/files/ftp_upload/3667/3667fig1.jpg" /> Şekil 1: Programı aksonal dallanma iki temel modları tasvir. Burada belirtildiği gibi - (A) bir çatallanma yol açabileceği büyüme konisi faaliyetleri ile Dallanma yanı sıra karmaşık terminali milleri ya da(B) kollateral oluşumu akson mil (interstisyel dallanma). Ikinci modu 6,7 kortikal ve talamokortikal aksonlar projelendirme hakim dallanma tipi. <img alt="Şekil 2" src="/files/ftp_upload/3667/3667fig2.jpg" /> Şekil 2 Afferent projeksiyonları embriyonik omuriliğe DRG nöronların aksonal dallanma her iki tip ekran: çatallanma tarafından DREZ (1) ve aksonlar ilk şubesini interstisyel dallanma (2) bir bekleme süresinden sonra kızı dalları teminatlar formu. <img alt="Şekil 3" src="/files/ftp_upload/3667/3667fig3.jpg" /> Şekil 3, embriyonik fare DRG nöronları tek aksonal yörüngeleri Görselleştirme . (A) bağlı DRG Omurilik bir E12.5 fare embriyo hazırlanmıştır. Artan büyütme wild-tip farelerden DII etiketli DRG (Ölçek çubuğu, 1 mm) (BD) Dorsal kez gösterilmiştir. B her saniye DRG labeled DII tarafından. Floresan görüntüleri ters, kaudal sol ve yanal C ve D alt kısmında. C küçük bir sayıda akson etiketli ve yüksek büyütme T gibi dalların omurilik DREZ varlığı tespit edilebilir. (Ölçek barlar, 250 mikron (B), 100 mikron (C) ve 50 mm (D).) <img alt="Şekil 4" src="/files/ftp_upload/3667/3667fig4.jpg" /> Şekil 4 T-biçimli dalları, yabani tip tek rostral veya kaudal döner ve C-tipi natriüretik peptid (CNP) eksik E13.5 fareler Kantitasyonu. Sayılır tek bir akson sayısı, her bir genotip için farklı gövde düzeyi için parantez içinde verilmiştir. C veya R-döner, ya da rostral kaudal yönde sadece büyüme, sırasıyla. <img alt="Şekil 5" src="/files/ftp_upload/3667/3667fig5.jpg" /> Şekil 5 cGMP bir sinyal yolağı omurilik DREZ duyusal akson çatallanma tetikler. (A) Programıligand CNP, alıcı guanil siklaz Npr2 ve serin / treonin kinaz cGKIα embriyonik DRG nöronlar oluşur cGMP sinyal yolağı. Npr2 GTP CNP tarafından uyarılması üzerine gelen cGMP üretir. (B), yabani tip ve CNP-eksik farelerde nöron DRG tek bir akson izleme DII. (Ölçek çubuğu, 25 mm.)

<ol>
	<li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dil+and+diO:+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing.">Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends. Neurosci. 12 (9), 333-333 (1989).</li><li>Acebes, A., Ferrus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+features+of+axon+collaterals+and+dendrites.">Cellular and molecular features of axon collaterals and dendrites.</a> Trends. Neurosci. 23 (11), 557-557 (2000).</li><li>Ozaki, S., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+trajectories+of+sensory+axons+toward+laminar+targets+in+the+developing+mouse+spinal+cord.">Initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord.</a> J. Comp. Neurol. 380 (2), 215-215 (1997).</li><li>Schmidt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+receptor+guanylyl+cyclase+Npr2+is+essential+for+sensory+axon+bifurcation+within+the+spinal+cord.">The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord.</a> J. Cell Biol. 179 (2), 331-331 (2007).</li><li>Gibson, D. A., Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+axon+branching+in+the+vertebrate+nervous+system.">Developmental regulation of axon branching in the vertebrate nervous system.</a> Development. 138 (2), 183-183 (2011).</li><li>O'Leary, D. D., Terashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+axons+branch+to+multiple+subcortical+targets+by+interstitial+axon+budding:+implications+for+target+recognition+and+&amp;quot;waiting+periods&amp;quot;.">Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and &amp;quot;waiting periods&amp;quot;.</a> Neuron. 1 (10), 901-901 (1988).</li><li>Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+modes+of+axon+elaboration+in+the+developing+neocortex.">Diverse modes of axon elaboration in the developing neocortex.</a> PLoS. Biol. 3 (8), e272-e272 (2005).</li><li>Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+imaging+and+ultrastructural+analysis+of+individual+axon+terminals+labeled+by+iontophoretic+application+of+lipophilic+dye.">Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye.</a> J. Neurosci. Methods. 93 (1), 13-13 (1999).</li><li>Schmidt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-type+natriuretic+peptide+(CNP)+is+a+bifurcation+factor+for+sensory+neurons.">C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (39), 16847-16847 (2009).</li><li>Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulate+axon+branching+by+the+cyclic+GMP+pathway+via+inhibition+of+glycogen+synthase+kinase+3+in+dorsal+root+ganglion+sensory+neurons.">Regulate axon branching by the cyclic GMP pathway via inhibition of glycogen synthase kinase 3 in dorsal root ganglion sensory neurons.</a> Journal of Neuroscience. 29 (5), 1350-1350 (2009).</li><li>Zhao, Z., Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+axonal+development+by+natriuretic+peptide+hormones.">Regulation of axonal development by natriuretic peptide hormones.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (42), 18016-18016 (2009).</li><li>Schmidt, H., Rathjen, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+mechanisms+regulating+axonal+branching+in+vivo.">Signalling mechanisms regulating axonal branching in vivo.</a> Bioessays. , (2010).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28 (1), 41-41 (2000).</li><li>Livet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450 (7166), 56-56 (2007).</li></ol>

Yazarlar ifşa etmek başka bir şey var.

Kalıplaşmış projeksiyon modelleri DII etiketlenmesine yönelik sabit doku kullanımı ile birlikte hazırlık kolaylığı ile birlikte iki tip aksonal şube oluşumu oluşan aksonal dallanma çalışma için uygun bir model DRG bağlı embriyonik omurilik yapar. Kaplı cam iğneler kullanarak DII dakika miktarda uygulama sağlar DRG nöronlar küçük gruplar görselleştirme ve böylece bireysel akson ve dallanma desen analizi - DRG toplu etiketleme aksine. Etiketli nöronlar 8 sayı daha da azalır lip...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Stereomikroskopta STEMI DRC </td><td> Zeiss </td><td></td><td></td></tr><tr><td> Fosfat tampon çözeltisi (PBS) </td><td> Biochrom AG </td><td> L182-50 </td><td></td></tr><tr><td> Paraformaldehit </td><td> Merck </td><td> 8.18715.1000 </td><td></td></tr><tr><td> Standart cerrahi makas </td><td> Güzel Bilim Araçları </td><td> 14001-13 </td><td></td></tr><tr><td> Dişli standart forseps </td><td> Güzel Bilim Araçları </td><td> 11021-14 </td><td></td></tr><tr><td> Ekstra ince iris makası </td><td> Güzel Bilim Araçları </td><td> 14088-10 </td><td></td></tr><tr><td> Eğimli forseps </td><td> Güzel Bilim Araçları </td><td> 11003-13 </td><td></td></tr><tr><td> Dumont No.5 ince ipuçları forseps </td><td> Güzel Bilim Araçları </td><td> 11254-20 </td><td></td></tr><tr><td> Dumont No.5 ayna bitirmek forseps </td><td> Güzel Bilim Araçları </td><td> 11252-23 </td><td></td></tr><tr><td> Vannas-Tübingen yaylı makas </td><td> Güzel Bilim Araçları </td><td> 15008-08 </td><td></td></tr><tr><td> Filtre kağıdı </td><td> Fisher Scientific </td><td> FB59041 </td><td></td></tr><tr><td> Sylgard 184 </td><td> Dünya Hassas Aletler </td><td> SYLG184 </td><td></td></tr><tr><td> 100-mm Petri kapları </td><td> Greiner </td><td> 663102 </td><td></td></tr><tr><td> 12 ml&#39;lik polipropilen tüp </td><td> Carl Roth GmbH </td><td> ECO3.1 &lt;/ Td&gt; <td></td></tr><tr><td> 12 kuyu kültür plaka </td><td> Becton Dickinson </td><td> 35-3043 </td><td></td></tr><tr><td> Etanol </td><td> Merck </td><td> 1.00983.2500 </td><td></td></tr><tr><td> / Kahverengi mikropipet çektirmesi P-97 Flaming </td><td> Sutter Instrument Co. </td><td></td><td></td></tr><tr><td> Borosilikat cam kapilerleri </td><td> Harvard Apparatus </td><td> 30-0066 </td><td></td></tr><tr><td> DII (1,1 &#39;-Dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethyl - perklorat indocarbocyanine) </td><td> Sigma-Aldrich </td><td> 468495 </td><td></td></tr><tr><td> Mikroskop SuperFrost Plus, slaytlar </td><td> Carl Roth GmbH </td><td> H867.1 </td><td></td></tr><tr><td> Cam kapak fişleri </td><td> Carl Roth GmbH </td><td> 1870,2 </td><td></td&gt; </tr></tbody></table>

dii-labeling of drg neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord

Burada lipofilik tracer 1,1 &#39;-dioctadecyl-3, 3,3&#39;, perklorat (DII) 1 3&#39;-tetramethylindocarbocyanine kullanarak uzadıya boyama embriyonik omurilik içine DRG nöronlar küçük gruplar yörüngeleri etiketlemek için bir teknik . Vahşi tip genleri mutasyona uğramış olan fare hatları ile aksonal yollarının karşılaştırma sinir sistemi kablolama önemli bir mekanizmadır aksonal dallanma kontrolü aday proteinlerin fonksiyonel bir rol için test sağlar. Aksonal dallanma ve böylece paralel bilgi işleme için fiziksel bir temel sağlayarak, çoklu hedefleri ile bağlanmak için tek bir nöron sağlar. Aksonal büyüme ara hedef bölgelerdeki etkileri terminali arborization ayırt edilebilir. Ayrıca, farklı mod aksonal şube oluşumu büyüme konisi (bölme ya da geç sutyen olmadığını dallanma sonuçları bağlı olarak sınıflandırılır olabilirnching) ya da bir süreç akson mili teminatların tomurcuklanan interstisyel dallanma 2 (Şekil 1) denir . Merkezi projeksiyonları DRG nöronların aksonal dallanma iki türlü incelemek için yararlı bir deneysel sistem sunuyoruz: afferent aksonlar 10 13 (E10 - E13) embriyonik gün arasında omurilik dorsal kök giriş bölgesi (DREZ) ulaştığınızda onlar basmakalıp desen T veya Y-şekilli çatallanma görüntüler. Daha sonra çıkan iki kızı aksonlar kablosunun dorsolateral marjı sırasıyla, rostral veya kaudal yönde ilerler ve bir bekleme süresi teminatlar gri madde (interstisyel dallanma) ve proje özel röle nöronlar nüfuz sonra bu kök aksonlar filiz spinal kord lamina daha da (terminal dallanma) 3 arborize. DII iz pr olduğu ortaya çıktı omurilik dorsal kök giriş bölgesi büyüme konileri ortaya koymuşturbu çatallanma düşündüren bölme ocess büyüme konisi kendisini 4 (Şekil 2) bölünmesi sebep olur, ancak, diğer seçenekler de 5 olarak tartışılmıştır var. Bu video DRG takılı bırakarak E12.5 farelerin omurilik nasıl incelemek için ilk olarak göstermektedir. Kılcal tüpler çıkarılmış cam iğneler kullanılarak DRG DII numune küçük miktarlarda sonra fiksasyon uygulanır. Bir inkübasyon adımdan sonra, etiketli omurilik floresan mikroskobu kullanarak bireysel aksonlar analiz etmek için ters bir açık-kitap hazırlık olarak monte edilir.

Watch this Scientific Journal Video about DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord at JoVE.com

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Kemirgen omuriliğe duyusal aferentlerde kalıplaşmış projeksiyonları tek akson izleme yoluyla aksonal dallanma incelemek için kolay ulaşılabilir bir deneysel sistem sunuyoruz.

Fare Embriyonik Omurilik Tüm Dağı Hazırlık aksonal dallanma Eğitim DRG Nöronlar DII-Etiketleme

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

dii-labeling-drg-neurons-to-study-axonal-branching-whole-mount

Research

JoVE Journal

Neuroscience

24.8K Görüntüleme.  Max Delbrück Center for Molecular Medicine. Kemirgen omuriliğe duyusal aferentlerde kalıplaşmış projeksiyonları tek akson izleme yoluyla aksonal dallanma incelemek için kolay ulaşılabilir bir deneysel sistem sunuyoruz.

Video: DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

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 ...

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Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Tüm montaj Embriyonik Sinir Sistemi Görselleştirme Drosophila Embriyolar

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

Yetişkin Tüm Dağı Hazırlık Drosophila Dev Fiber Boya Enjeksiyon Ventral Sinir Kablosu

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Bir<em&gt; Ex vivo</em&gt; Lazer kaynaklı Omurilik Felçlileri Modeli Gerçek zamanlı aksonal Dejenerasyon mekanizmaları değerlendirmek için

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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 ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Embriyonik Motor Nöronlarının Aksonal Projeksiyon Paterninin Görselleştirilmesi<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Farede Laminektomi ve Omurilik Penceresi İmplantasyonu

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Akut omurilik dilimleri hazırlanması gözlemliyorum Gelatinosa nöronlar bütün hücreli yama-kelepçe kayıt için

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

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 ...

Omurilik Stimülasyonuna Motor Nöron Yanıtlarını Anlama

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

Yazar Spotlight: Omurilik Stimülasyonunu İlerletmek - Yama-Kelepçe Elektrofizyolojisi Yoluyla Motor Nöronların Hücresel Tepkilerini Keşfetmek 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

Spinal Kord Stimülasyonu Sırasında Motor Nöronlarda Tüm Hücre Yama-Klemp Kaydı için Omurilik Diliminin Ex vivo Hazırlanması

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...