Biz onların yardım için Karine Kindbeiter, Muriel bozon ve Florie Reynaud teşekkür ederim. Iş CNRS, ANR Yoda program ve Labex DevWeCan tarafından desteklenmektedir.

1. GÜN 1.. E12.5 fare embriyoları Omurilik Kat Plate (FP) diseksiyonu Not: Tüm prosedür steril koşullar kullanımını gerektirir. Bu kirlenmeyi önlemek için bir diseksiyon kaputun altında diseksiyon tercih edilir. Kaput yüzeyi etanol ile temizlenmelidir. Tüm diseksiyon aletleri steril bir Petri kabındaki sterilize ve tutulmalıdır. Sıvılar (orta, diseksiyon orta) kapalı tutulur ve bir buz banyosu koymak gerekir. Her embriyo bireysel taze damla alınmalıdır. Bu doku korunması için önemlidir. Diseksiyon Dumont # 5 forseps ile yapılır. Yemekler, kavrama arka beyin herhangi bir zarar vermeden göbek kordonu veya başı arasında embriyolar transfer için. Bu başarıyla diseksiyonu tamamlamak için omurilik zarar kritik değildir. Hazırlama soğuk Fosfat Tamponlu Salin (PBS), soğuk Hank Dengeli Tuz Çözeltisi (HBSS) -% 6.5 glikoz ve oda sıcaklığı neurobasal ortamı. 1.1 Omurilik diseksiyonu Hayvanlar Avrupa direktiflerine aşağıdaki Centre National de la Recherche Scientifique bir hayvan bakım kurallarına göre tedavi edilir. Ötenazi yöntem servikal dislokasyon, bu boyun basınç uygulayarak ve beyin omurga dislocating oluşur. Bu yöntem, fare hayvan modelleri için tavsiye edilir, ve bir hızlı ve verimli bir şekilde gerçekleştirilebilir. <ol><li> Gebelik (E0.5 = ilk gün aşağıdaki paspas) ve E12.5 hamile bir fareyi Euthanize. Fare ventral yüzü yukarı yerleştirin. </li><li> % 70 etanol ile karın ıslatın. Adson forseps ile karın cildi sıkın ve cerrahi makas ile deri ve periton katmanları kesti. Bir kesi yapmak ve karın boşluğuna maruz fare her iki tarafında kesmek için kullanabilirsiniz. </li><li> Embriyoların bir dizi, fare her bir tarafında bulunur. Ute bir boynuz kavramakiki embriyo ve dış kısmından başlayarak onu teşrih arasındaki dokuda rus. Buz gibi soğuk PBS ile bir 100 mm Petri tabağına sağlam rahim aktarın. </li><li> Ekstra-embriyonik dokulardan embriyolar ayıklayın. Forseps iki çift ile plasenta dokusu (koyu kırmızı) kavrayın ve doku gözyaşı ve rahim kesesi embriyo çıkarmak için yavaşça çekin. </li></ol> Not: Daha iyi bir koruma için, onların rahim kese tek tek gelen embriyolar toplamak. <ol start="5"><li> Sonraki adımlar binoküler büyüteç ve diseksiyon başlık altında yapılır. Soğuk HBSS-% 6.5 glikoz bir damla tek embriyo yerleştirin. </li><li> Forseps kullanarak, embriyo başını kesmek. </li></ol> Not: Alt çene ve arka beyin arasında kesin, arka beyin ve arka kısmı bir sonraki adım için önemlidir. <ol start="7"><li> Deneyci bakan ön kısmı ile embriyo ventral yüzü aşağı bakacak şekilde yerleştirin. </li><li> Cilt çimdikforseps bir çift ve başka çift embriyo deri çekmeye. </li></ol> Not: Bu adım, cilt tamamen embriyo ön kısmı çıkarılır kadar tekrar edilmesi gerekir. <ol start="9"><li> Sola embriyo ön yan çevirin. Bir forseps ile ön kısmında embriyo &quot;aşağı pin&quot;. Diğer forseps cildi kapmak ve embriyonun rostral tarafına doğru çekin ile. Omurilik sonra erişilebilir. </li><li> Hala omurilik sarın meninks kesmek için forseps bir tarafını kullanın. Omurilik artık açıktır. </li><li> Sağa doğru embriyo ön yan çevirin. </li><li> Embriyonun servikal tarafındaki başlangıç ​​omurilikten doku çıkarın. Omurilik (forseps omurilik hasarları önlemek için kapalı tutulmalıdır) altında forseps kaydırın. Forseps omurilik altında görünür olmak zorunda. Omurilik altında küçük rotasyon hareketleri ayırmak için yeterli olandoku ve dorsal kök ganglionlar (DRG) çevreleyen. </li></ol> Not: Ayrılma bağlı çevreleyen doku, böylece bir sonraki adım sırasında kırılma olasılığını arttırarak, omurilik ağırlık ekleyin. <ol start="13"><li> Soğuk HBSS-% 6.5 glikoz taze bir damla izole spinal kord yerleştirin. </li><li> Düz bir pozisyonda, deplasmanda deneyci gelen üst ve ön tarafta meninkslerde omurilik yerleştirin. </li><li> Bir forseps ile kalan arka beyin kullanarak omurilik &quot;aşağı pin&quot; ve ikinci forseps meninks kapmak ile. Omurilik rostral yakasından başlayarak meninks sıyırın. </li></ol> Not: hareket omurilik herhangi kırılmasını engellemek için yavaş ve sabit olmalıdır. Titreşimli hareketleri menenjlerin kopmasını kolaylaştırabilir. 1.2 FP diseksiyonu <ol><li> Bir forseps ile arka beyin çimdik. </li><li> FP merkezi tradoku nsparent parçasıdır. Bir neşter kullanılarak tabanını kesip. </li></ol> Not: aynı kalitede iki tarafı kesmek için, rostral taraftan başlar ve sırayla sağ tarafı ve zemin plakasının sol tarafını kesti. <ol start="3"><li> Oda sıcaklığında, Neuro temelli ortam içinde parçalara FP koruyun. </li></ol> 2. FP Kültür Not: Tüm adımlar, doku kültürü başlığı içinde steril koşullar altında gerçekleştirilmelidir. Taze orta ve taze çözülmüş takviyeleri ve reaktifleri kullanın. Steril cam lamelleri, sıcak Neurobasal + B27, plazma ve HBSS-trombin hazırlayın. 2.1 FP kültür <ol><li> Bir 100 mm Petri çanağı ve her bir lamel merkezinde pipet 20 ul plazma içindeki steril lamelleri yerleştirin. </li><li> Her plazma içinde damla 2-4 FP aktarın ve plazma damla yanında 20 ul HBSS-trombin ekleyin ve iyice karıştırın. Lamelleri tutmakOda sıcaklığında on dakika minimum plazma pıhtılaşmasını sağlamak için. </li></ol> Not: Kısmi diseksiyon durumunda tam diseksiyon ve daha halinde Transferi iki FP. Bir pipet ile yavaş yavaş karıştırın ve FP eksplantlarından ile temasından kaçının. Plazma pıhtı koagülasyon bir işareti olarak birkaç mikron çeker. Koagülasyon plazma pıhtısı ayrılmasını önlemek için, zamanından önce durdurulması gerekir. <ol start="3"><li> 24 oyuklu bir plaka içerisinde her bir lamel aktarın ve Neurobasal + B27 500 ul ekle. 37 ° C&#39;de 48 saat inkübe </li></ol> 3. GÜN 3. FP koşullandırılmış ortam (FP cm) Hasat Not: Hasat bir doku kültürü başlığı içinde steril koşullar altında gerçekleştirilmelidir. Yüzer kültür peri sırasında kirlenmiş veya zemin plakaları (yani yüzen eğer onlar ölmüş olabilir varsaod), daha sonra bu kuyudan FP cm hasat yok. 3.1 FP cm hasat <ol><li> Her kuyudan dikkatli bir şekilde ortam hasat. </li></ol> Not: polimerize plazma trombin karışımı veya herhangi bir FP parçaları pipetle etmeyin. <ol start="2"><li> -80 ° C&#39;de 100 ul alikotları Mağaza </li></ol> 4. GÜN 4. E12.5 fare embriyoları Omurilik Commissural Nöronlar Diseksiyon Not: Bu preparat, steril koşullar altında gerçekleştirilebilir gerek yoktur. Embriyoların toplanması için yukarıda tarif edilen prosedür takip edin. Hazırlayın soğuk PBS, soğuk HBSS-% 6.5 glikoz ve soğuk neurobasal. 4.1 Omurilik diseksiyonu <ol><li> Forseps kullanarak, embriyo başını kesmek. </li></ol> Not: Alt çene ve arka beyin arasında kesin, arka beyin ve posterior bölümü için önemlidirsonraki adımlar. <ol start="2"><li> Deneyci bakan ön kısmı ile taze bir düşüş embriyo ventral yüzü aşağı bakacak şekilde yerleştirin. </li><li> Bir forseps ile embriyo cilt sıkın ve diğeri ile cilt uzak çekin. </li></ol> Not: Bu adım, cilt tamamen embriyo ön kısmı çıkarılır kadar tekrar edilmesi gerekir. <ol start="4"><li> Sola embriyo ön yan çevirin. Bir forseps ile ön kısmında embriyo &quot;aşağı pin&quot;. Diğer forseps ile cilt kavramak ve omurilik ortaya çıkarmak için, embriyonun rostral tarafına doğru çekin. </li><li> Hala omurilik sarın meninks kesmek için forseps bir tarafını kullanın. Omurilik şimdi maruz ve açık olduğunu. </li><li> Sağa doğru embriyo ön tarafı yerleştirin. </li><li> Embriyonun servikal tarafındaki başlangıç ​​omurilikten doku çıkarın. Omurilik (kapalı forseps tutmak) altında forseps kaydırın. Içinceps omurilik altında görülebilir olmalıdır. Omurilik altında küçük döner hareketler, komşu doku ve dorsal kök gangliyon (DRG) ayırmak için yeterlidir. </li><li> Soğuk HBSS-% 6.5 glikoz, yeni bir damla olarak izole edilmiş omurilik aktarın. </li><li> Düz bir pozisyonda, deplasmanda deneyci gelen üst ve ön tarafta meninkslerde omurilik yerleştirin. </li><li> Ikinci forseps meninks kapmak ile, bir forseps ile kalan arka beyin kullanarak omurilik &quot;aşağı pin&quot;. Omurilik rostral yakasından başlayarak meninks sıyırın. </li></ol> 4.2 Commissural Nöron diseksiyonu <ol><li> Bir forseps ile arka beyin çimdik. </li><li> Commissural nöronlar omurilik (dorsal tarafı) en yan kısmında yer almaktadır. Ince bir neşter ile bu yanal bölümünü kesmek. </li></ol> Not: omurilik dorsal kısmı hücre d farkla ventral kısmından ayırt edilebilirdensite, ventral kısmı daha opak olmak. Not: aynı kalitede iki tarafı kesmek için, rostral taraftan başlar ve sırayla sağ tarafında ve sol tarafında küçük bir uzunluğa kesti. <ol start="3"><li> Acil kullanım için buz üzerinde Neurobasal ortam içinde parçalara doku koruyun. </li></ol> 5. FP cm Commissural Tissue tedavisi Not: Bu prosedür steril koşullar gerektirmez. Bu doku, bireysel 1.5 ml tüpler içinde toplanmalıdır. Dondurulmuş FP cm sadece bir kez kullanılmalıdır. Birden soğutulmasından kaçının. Sıcak FP cm hazırlayın (37 ° C). Commissural nöronların 5,1 Tedavisi <ol><li> Küçük bir makas ile commissural doku Dilacerate. </li></ol> Not: olabildiğince küçük doku kesmek, hücrelerin erişilebilirlik kuvvetlendirmektir. <ol start="2"><li> Oldukça farklı deneysel koşullar kontrol tedavi ve FP cm tedavi arasındaki doku dağıtın. </li><li> 4 ° C&#39;de 1 dakika boyunca 800 x g doku parçaları santrifüj </li><li> Borular arasındaki bölümlerini yeniden kontrol edin. </li></ol> Not: fragmanları kadar tekrar gerekli redistribute ve santrifüj eşit olarak dağıtılır iseniz. <ol start="5"><li> Süpernatant kaldırmak. </li><li> Dokulara sıcak FP cm ve kontrol tedavileri ekleyin. </li></ol> Not: hacmi topağın miktarına göre ayarlanması gerekir ve en az iki katı pelet hacmi olması gerekir. <ol start="7"><li> 30 dakika boyunca 37 ° C&#39;de inkübe edin. </li></ol> Not: Yavaşça her 10-15 dk sallamak <ol start="8"><li> 1 dakika boyunca 800 x g&#39;de santrifüj ve tedavi çıkarın. </li><li> Liziz tamponu ekleme </li></ol> Not: hacim a göre ayarlanması gerekirpelet montaj ve en az iki katı pelet hacmi olması gerekir. <ol start="10"><li> Pipet ve aşağı yukarı pelet tekrar süspansiyon. </li></ol> Not: A girdap da kullanılabilir. <ol start="11"><li> Buz üzerinde 15 dakika inkübe edin. </li><li> 4 ° C&#39;de 15 dakika boyunca 14,000 x g&#39;de santrifüje </li><li> Süpernatantı tutun ve Bradford tahlili 9 protein miktarını ölçer. </li><li> 6x Laemmli Buffer ekleyin. </li><li> Protein içeriği denatüre etmek için 5 dakika boyunca 95 ° C&#39;de inkübe edilir. </li></ol> Not: Örnekler hemen kullanılabilir ya da -80 ° C&#39;de saklanabilir <ol start="16"><li> Western blot 10 ile tedavisinin etkilerini değerlendirmek. </li></ol> Not: toplam proteinlerin en az 50 mg her kulvar için yüklü olmalıdır.

<ol>
	<li>Nawabi, H., Castellani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+commissures+in+the+central+nervous+system:+how+to+cross+the+midline.">Axonal commissures in the central nervous system: how to cross the midline.</a> Cell. Mol. Life Sci. 68, 2539-2553 (2011).</li><li>Placzek, M., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+floor+plate:+multiple+cells,+multiple+signals.">The floor plate: multiple cells, multiple signals.</a> Nat. Rev. Neurosci. 6, 230-240 (2005).</li><li>Chedotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+tales+of+the+midline.">Further tales of the midline.</a> Curr. Opin. Neurobiol. 21, 68-75 (1016).</li><li>Nawabi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+midline+switch+of+receptor+processing+regulates+commissural+axon+guidance+in+vertebrates.">A midline switch of receptor processing regulates commissural axon guidance in vertebrates.</a> Genes Dev. 24, 396-410 (2010).</li><li>Ruiz de Almodovar, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+mediates+commissural+axon+chemoattraction+through+its+receptor+Flk1.">VEGF mediates commissural axon chemoattraction through its receptor Flk1.</a> Neuron. 70, 966-978 (2011).</li><li>Charoy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gdnf+activates+midline+repulsion+by+Semaphorin3B+via+NCAM+during+commissural+axon+guidance.">gdnf activates midline repulsion by Semaphorin3B via NCAM during commissural axon guidance.</a> Neuron. 75, 1051-1066 (2012).</li><li>Kiecker, C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+organizers+in+patterning+the+nervous+system.">The role of organizers in patterning the nervous system.</a> Annu. Rev. Neurosci. 35, 347-367 (2012).</li><li>Le Dreau, G., Marti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+of+the+neural+tube:+a+tale+of+three+signals.">Dorsal-ventral patterning of the neural tube: a tale of three signals.</a> Dev. Neurobiol. 72, 1471-1481 (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J. Vis. Exp. , (2010).</li><li>Salinas, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+collaborates+with+netrin-1+to+guide+axons+in+the+spinal+cord.">The morphogen sonic hedgehog collaborates with netrin-1 to guide axons in the spinal cord.</a> Trends Neurosci. 26, 641-643 (2003).</li><li>Bechara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAK-MAPK-dependent+adhesion+disassembly+downstream+of+L1+contributes+to+semaphorin3A-induced+collapse.">FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse.</a> EMBO J. 27, 1549-1562 (2008).</li></ol>

Yazarlar ifşa hiçbir şey yok

Taban-plakası şartlandırılmış ortamın üretimi zemin plakası yayınlanan sinyallerin biyolojik özellikleri değerlendirmek için verimli ve kolay bir yol sağlar. Plazma trombin matris doku yaşam için mükemmel bir koşul sağlar. Bununla birlikte, bu zenginleştirilmiş bir ortam deneylerinin bazı türleri için bir sınırlama olabilir. Bu durumda, zemin levhası doku da agaroz matrisi içinde yetiştirilebilir. Taban plakası şartlandırılmış ortamın kalitesi, GDNF <stron...

Zemin plakası atalarıdır ve postmitotic hücre soylarının şartnamede kilit rol oynayan ve akson navigasyon 7,8 kontrol, gelişen omurilik tanınmış desenleme merkezidir. FP cm üretmek için burada açıklanan deney prosedürü araştırmacılar hücre desenlendirme ve hayatta kalma hücre ve akson göç, gelişimsel süreçleri çeşitli bağlamlarda zemin plaka kaynaklı sinyallerin fonksiyonel özelliklerini değerlendirmek için izin verir. Bu tür FP cm kullanımını göstermek için, commissural nöron içeren dorsal omurilik dokuları kesilmiş dilacerated ve FP cm ile uyarılır. Dokular daha sonra Western blot analizi için işlenebilir. Bu zemin plakası yayımlanan sinyalleri tarafından akson rehberlik makine düzenlemeleri araştıran sağlar. Dilacerated taze doku tedavisi için bir yöntem th içinde hücrelerin mikro korunması büyük bir avantaj sahiptire doku. Böylece FP cm tedavisi veya tedavinin herhangi bir türde sonuçları hücre ve doku kültürü koşullarında daha fizyolojik bir şekilde değerlendirilir.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS REQUIRED</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, Ca2+/Mg2+- free</td>
			<td>Invitrogen</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G-7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td>Sigma</td>
			<td>P-3266</td>
			<td>1mg/ml (Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T-4648</td>
			<td>10mg/ml(Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50X</td>
		</tr>
		<tr>
			<td>Hepes (260g/mol)</td>
			<td>Sigma</td>
			<td>H-7006</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E-5513</td>
			<td>0,5M pH8</td>
		</tr>
		<tr>
			<td>MgCL2</td>
			<td>Euromedex</td>
			<td>2189</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL</td>
			<td>Sigma</td>
			<td>I-3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Protease inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>04 693 116 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate (Na3VO4)</td>
			<td>Sigma</td>
			<td>S-6508</td>
			<td></td>
		</tr>
		<tr>
			<td>distilled H2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Reagents required</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>TOOLS AND MATERIAL</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Fine Tips forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro knives</td>
			<td>Fine Science Tools</td>
			<td>10136-14</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Tools and material</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			<table border="1">
				<tbody>
					<tr>
						<td colspan="2">Dissection and FP culture</td>
					</tr>
					<tr>
						<td>Ethanol 70%</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection hood</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection microscope</td>
						<td></td>
					</tr>
					<tr>
						<td>Petri dishes</td>
						<td></td>
					</tr>
					<tr>
						<td>glass coverslips</td>
						<td>18mm x 18mm</td>
					</tr>
					<tr>
						<td>Bunsen gas burner</td>
						<td></td>
					</tr>
					<tr>
						<td>24 well plate</td>
						<td></td>
					</tr>
					<tr>
						<td>tissue culture incubator (37&#176;C, 5% CO2, humidity controlled)</td>
						<td></td>
					</tr>
					<tr>
						<td>Centrifuge</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">RECIPES</td>
					</tr>
					<tr>
						<td colspan="2">FP culture media</td>
					</tr>
					<tr>
						<td>Neurobasal</td>
						<td></td>
					</tr>
					<tr>
						<td>B27</td>
						<td>1/50e dilution from stock</td>
					</tr>
					<tr>
						<td colspan="2">HBSS - Thrombin</td>
					</tr>
					<tr>
						<td>HBSS</td>
						<td>1 ml</td>
					</tr>
					<tr>
						<td>Thrombin 10mg/ml</td>
						<td>30 &#956;l</td>
					</tr>
					<tr>
						<td colspan="2">Lysis Buffer pH7,4</td>
					</tr>
					<tr>
						<td>Hepes (260 g/mol)</td>
						<td>25mM</td>
					</tr>
					<tr>
						<td>EDTA (pH8)</td>
						<td>5mM</td>
					</tr>
					<tr>
						<td>MgCL2</td>
						<td>1mM</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>10%</td>
					</tr>
					<tr>
						<td>IGEPAL (NP40)</td>
						<td>0.10%</td>
					</tr>
					<tr>
						<td>Protease Inhibitor Cocktail</td>
						<td>1X</td>
					</tr>
					<tr>
						<td>Sodium Orthovanadate</td>
						<td>0,2M</td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">Laemmli Buffer</td>
					</tr>
					<tr>
						<td>Tris 0,5M, pH 6,8</td>
						<td>375 mM</td>
					</tr>
					<tr>
						<td>SDS</td>
						<td>6%</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>60%</td>
					</tr>
					<tr>
						<td>DTT</td>
						<td>0,6M</td>
					</tr>
					<tr>
						<td>Bromophenol blue</td>
						<td></td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

culture of isolated floor plate tissue and production of conditioned medium to assess functional properties of floor plate-released signals

Gelişimi sırasında, atalarıdır ve post-mitotik nöronlar kendi kaderini düzenleyen komşu topraklardan sinyallerini alırsınız. Taban-plakası ventral pozisyonda ependimal kanal astar glial hücre grubudur. Zemin plakalı omurilik hücre soylarının dokusuna katkıda anahtar morphogens ifade eder. Sonraki gelişim aşamalarında, zemin plakası commissural akson 1 ipsilateral akson ve kontrol orta hat geçişi geçişi önlemek için bir bariyer olarak hareket, omurilikteki akson navigasyon düzenler. Bu işlevler, çeşitli rehberlik ipuçlarının salgılanması ile elde edilir. Diğerleri yerel rehberlik ipuçlarının 2,3 için akson duyarlılığını modüle rehberlik reseptörleri ve mansap sinyallerini regüle ise bu ipuçlarının bazıları büyüyen aksonlar için cezbedici ve kovucular gibi hareket ederler. Burada deve çeşitli taban plakalı türetilmiş sinyallerin özelliklerini araştıran izin veren bir yöntemi tarifZemin-Plaka klimalı ortamda (FP cm) 4-6 üretime dayalı lopmental bağlamları. Daha sonra akson rehberlik bağlamında bu FP cm kullanımını örnekler. İlk olarak, omurilik E12.5 fare embriyo izole edilir ve zemin plakası disseke ve plazma trombin matriks (Şekil 1) yetiştirilmektedir. İkinci olarak, iki gün sonra, doku commissural, E12.5 embriyolardan ayrılarak toz haline getirildi ve FP cm maruz kalmaktadır. Üçüncü olarak, doku commissural belirteçlerin Western blot analizi için işlenir.

Commissural dokular FP cm ile tedavi edildi ve numuneler Western blot reseptör seviyeleri analizi için işlenmiştir. FP cm uygulanması (Şekil 2) geçtikten sonra orta hat itici Semaphorin3B için commissural akson duyarlılığını aracılık eden bir yönlendirme reseptörü, PlexinA1, düzeylerini artırmak için gösterilmiştir. Bu FP tarafından yayınlanan sinyaller PlexinA1 seviyeleri 4,6 düzenleyen olduğunu ortaya koydu. <p class="jove_content" fo:kee...

Watch this Scientific Journal Video about Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals at JoVE.com

Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

Zemin plakası, omurilik gelişmekte atalarıdır yayılabilir sinyalleri veren ve ayırt nöronların önemli bir yapıdır. Biz, zemin plakası klimalı ortam üretmek için taze omurilik dokusu uygulamak için, ve biyokimya ile ilgi proteinler üzerinde sonuçlarını değerlendirmek için bir yöntem açıklanmaktadır.

Zemin Plaka yayımlanan Sinyalleri fonksiyonel özelliklerini değerlendirmek için klimalı ortamda İzole Zemin Plaka Doku ve Üretim Kültürü

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of ...

culture-isolated-floor-plate-tissue-production-conditioned-medium-to

Research

JoVE Journal

Neuroscience

7.9K Görüntüleme.  University of Lyon 1.  Zemin plakası, omurilik gelişmekte atalarıdır yayılabilir sinyalleri veren ve ayırt nöronların önemli bir yapıdır. Biz, zemin plakası klimalı ortam üretmek için taze omurilik dokusu uygulamak için, ve biyokimya ile ilgi proteinler üzerinde sonuçlarını değerlendirmek için bir yöntem açıklanmaktadır. 

Video: Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

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 ...

Nörobilim

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), magnetoencephalography or functional magnetic-resonance imaging. These techniques have either inherently low temporal or low spatial resolution, and suffer from low signal-to-noise ratio and/or poor high-frequency sensitivity. Thus, they are suboptimal for exploring the short-lived spatio-temporal dynamics of many of the underlying brain processes. In contrast, the invasive technique of electrocorticography (ECoG) provides brain signals that have an exceptionally high signal-to-noise ratio, less susceptibility to artifacts than EEG, and a high spatial and temporal resolution (i.e., &lt;1 cm/&lt;1 millisecond, respectively). ECoG involves measurement of electrical brain signals using electrodes that are implanted subdurally on the surface of the brain. Recent studies have shown that ECoG amplitudes in certain frequency bands carry substantial information about task-related activity, such as motor execution and planning1, auditory processing2 and visual-spatial attention3. Most of this information is captured in the high gamma range (around 70-110 Hz). Thus, gamma activity has been proposed as a robust and general indicator of local cortical function1-5. ECoG can also reveal functional connectivity and resolve finer task-related spatial-temporal dynamics, thereby advancing our understanding of large-scale cortical processes. It has especially proven useful for advancing brain-computer interfacing (BCI) technology for decoding a user's intentions to enhance or improve communication6 and control7. Nevertheless, human ECoG data are often hard to obtain because of the risks and limitations of the invasive procedures involved, and the need to record within the constraints of clinical settings. Still, clinical monitoring to localize epileptic foci offers a unique and valuable opportunity to collect human ECoG data. We describe our methods for collecting recording ECoG, and demonstrate how to use these signals for important real-time applications such as clinical mapping and brain-computer interfacing. Our example uses the BCI2000 software platform8,9 and the SIGFRIED10 method, an application for real-time mapping of brain functions. This procedure yields information that clinicians can subsequently use to guide the complex and laborious process of functional mapping by electrical stimulation.
Prerequisites and Planning:
Patients with drug-resistant partial epilepsy may be candidates for resective surgery of an epileptic focus to minimize the frequency of seizures. Prior to resection, the patients undergo monitoring using subdural electrodes for two purposes: first, to localize the epileptic focus, and second, to identify nearby critical brain areas (i.e., eloquent cortex) where resection could result in long-term functional deficits. To implant electrodes, a craniotomy is performed to open the skull. Then, electrode grids and/or strips are placed on the cortex, usually beneath the dura. A typical grid has a set of 8 x 8 platinum-iridium electrodes of 4 mm diameter (2.3 mm exposed surface) embedded in silicon with an inter-electrode distance of 1cm. A strip typically contains 4 or 6 such electrodes in a single line. The locations for these grids/strips are planned by a team of neurologists and neurosurgeons, and are based on previous EEG monitoring, on a structural MRI of the patient's brain, and on relevant factors of the patient's history. Continuous recording over a period of 5-12 days serves to localize epileptic foci, and electrical stimulation via the implanted electrodes allows clinicians to map eloquent cortex. At the end of the monitoring period, explantation of the electrodes and therapeutic resection are performed together in one procedure.
In addition to its primary clinical purpose, invasive monitoring also provides a unique opportunity to acquire human ECoG data for neuroscientific research. The decision to include a prospective patient in the research is based on the planned location of their electrodes, on the patient's performance scores on neuropsychological assessments, and on their informed consent, which is predicated on their understanding that participation in research is optional and is not related to their treatment. As with all research involving human subjects, the research protocol must be approved by the hospital's institutional review board. The decision to perform individual experimental tasks is made day-by-day, and is contingent on the patient's endurance and willingness to participate. Some or all of the experiments may be prevented by problems with the clinical state of the patient, such as post-operative facial swelling, temporary aphasia, frequent seizures, post-ictal fatigue and confusion, and more general pain or discomfort.
At the Epilepsy Monitoring Unit at Albany Medical Center in Albany, New York, clinical monitoring is implemented around the clock using a 192-channel Nihon-Kohden Neurofax monitoring system. Research recordings are made in collaboration with the Wadsworth Center of the New York State Department of Health in Albany. Signals from the ECoG electrodes are fed simultaneously to the research and the clinical systems via splitter connectors. To ensure that the clinical and research systems do not interfere with each other, the two systems typically use separate grounds. In fact, an epidural strip of electrodes is sometimes implanted to provide a ground for the clinical system. Whether research or clinical recording system, the grounding electrode is chosen to be distant from the predicted epileptic focus and from cortical areas of interest for the research. Our research system consists of eight synchronized 16-channel g.USBamp amplifier/digitizer units (g.tec, Graz, Austria). These were chosen because they are safety-rated and FDA-approved for invasive recordings, they have a very low noise-floor in the high-frequency range in which the signals of interest are found, and they come with an SDK that allows them to be integrated with custom-written research software. In order to capture the high-gamma signal accurately, we acquire signals at 1200Hz sampling rate-considerably higher than that of the typical EEG experiment or that of many clinical monitoring systems. A built-in low-pass filter automatically prevents aliasing of signals higher than the digitizer can capture. The patient's eye gaze is tracked using a monitor with a built-in Tobii T-60 eye-tracking system (Tobii Tech., Stockholm, Sweden). Additional accessories such as joystick, bluetooth Wiimote (Nintendo Co.), data-glove (5th Dimension Technologies), keyboard, microphone, headphones, or video camera are connected depending on the requirements of the particular experiment.
Data collection, stimulus presentation, synchronization with the different input/output accessories, and real-time analysis and visualization are accomplished using our BCI2000 software8,9. BCI2000 is a freely available general-purpose software system for real-time biosignal data acquisition, processing and feedback. It includes an array of pre-built modules that can be flexibly configured for many different purposes, and that can be extended by researchers' own code in C++, MATLAB or Python. BCI2000 consists of four modules that communicate with each other via a network-capable protocol: a Source module that handles the acquisition of brain signals from one of 19 different hardware systems from different manufacturers; a Signal Processing module that extracts relevant ECoG features and translates them into output signals; an Application module that delivers stimuli and feedback to the subject; and the Operator module that provides a graphical interface to the investigator.
A number of different experiments may be conducted with any given patient. The priority of experiments will be determined by the location of the particular patient's electrodes. However, we usually begin our experimentation using the SIGFRIED (SIGnal modeling For Realtime Identification and Event Detection) mapping method, which detects and displays significant task-related activity in real time. The resulting functional map allows us to further tailor subsequent experimental protocols and may also prove as a useful starting point for traditional mapping by electrocortical stimulation (ECS).
Although ECS mapping remains the gold standard for predicting the clinical outcome of resection, the process of ECS mapping is time consuming and also has other problems, such as after-discharges or seizures. Thus, a passive functional mapping technique may prove valuable in providing an initial estimate of the locus of eloquent cortex, which may then be confirmed and refined by ECS. The results from our passive SIGFRIED mapping technique have been shown to exhibit substantial concurrence with the results derived using ECS mapping10.
The protocol described in this paper establishes a general methodology for gathering human ECoG data, before proceeding to illustrate how experiments can be initiated using the BCI2000 software platform. Finally, as a specific example, we describe how to perform passive functional mapping using the BCI2000-based SIGFRIED system.

Recording Human Electrocorticographic ECoG Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

Nörobilimsel Araştırma ve Gerçek-zamanlı Fonksiyonel Kortikal Haritalama İnsan elektrokortikografik (ECoG) Sinyaller Kayıt

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), ...

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

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

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

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

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

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

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

Hücre Kültüründe ve Civciv Embriyosunda Akson Kılavuz Reseptörlerinin Dinamiklerini Değerlendirmek için pHluorin Kullanımı

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

Kapalı döngü Nöro-robotik Deneyler Nöronal Ağları Hesaplamalı Properties Test

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Beyin Dokusu Ölçme Ekstraselüler Ion Sinyalleri için Çift namlulu ve Konsantrik Mikroelektronlar

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

Atomik Kuvvet Mikroskobu, Darbe girinti ve reometre kullanarak Beyin Doku Ölçekli Mekanik Özellikleri karakterize

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Yalıtım ve dinamik bir teşvik etmek kemirgen Microglia kültürünü Morfoloji Serum-Alerjik orta Ramified

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Presynapse Formation Assay Using Presynapse Organizer Beads and &ldquo;Neuron Ball&rdquo; Culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

Presynapse formation is a dynamic process including accumulation of synaptic proteins in proper order. In this method, presynapse formation is triggered by beads conjugated with a presynapse organizer protein on axonal sheets of &#8220;neuron ball&#8221; culture, so that accumulation of synaptic proteins is easy to be analyzed during presynapse formation.

Presynapse oluşum assay kullanarak Presynapse Organizatör boncuk ve "neuron Ball" kültür

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Embriyonik Fare Beyincisinden İzole Edilen Nöronların Birincil Kültürü

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...