The work was funded by the University of Zurich. The authors would like to thank Estrela Neto and Dr. Meriem Lamghari for helping in establishing the co-culture conditions.

Tüm fareler İsviçre Hayvan Refahı Kanunu ve Kanton Veteriner ofisi, Zürih düzenlemelere uygun olarak uygun muhafaza ve ele alınmıştır. Diseksiyon Malzeme, Kültür Medya, mikroakışkan Cihazlar 1. Hazırlık <ol><li> Otoklav mikro diseksiyon forseps ve makas (121 ° C, sterilizasyon süresi: 20 dk) ve steril bir kapta saklayın. </li><li> 37 ° C&#39;de bir orbital çalkalayıcı üzerinde 24 saat süre ile, 1 M HCI onları kuluçka cam lamelleri (24 mm x 24 mm) sterilize edin. Etanol% 99 ile steril distile H 2 O, onlara üç kez üç kez yıkayın. Kuru daha sonra 37 ° C&#39;de ya da steril bir akış hunisi altına lamelleri. Son olarak, otoklav veya sterilizasyon tamamlamak için UV ışığı (30 dakika) lamelleri maruz kalmaktadır. Lameller sonra etanol% 70 saklanabilir. </li><li> Steril forseps kullanarak dikkatlice paketinden AXIS Akson İzolasyon Aygıtlar çıkarın ve steril bir Petri kabı koyun. </li&gt; <li> Steril bir biyopsi yumruk kullanarak (ø: 1mm) kültür odaları yazışmalarda numune başına bir delik kültüre edilmesi (Şekil 1) oluşturun. NOT: uygulanan basınç tarafından zarar olabileceği gibi, mikro oluklarda çok yakın yumruk yapmayın. </li><li> Etanol% 70 bunları yükselmekte tarafından AXIS Akson İzolasyon Cihazları sterilize edin. Tamamen steril bir akış kaputu altında kuru ardından EKSEN Akson İzolasyon Cihazları ve lamelleri. Geçmeden önce 3 saat, en az bekleyin. NOT: Eksik kurutma mikroakışkan cihazların arızalı mecliste neden olacaktır. </li><li> 6-kuyu plaka içindeki bir 35 mm Petri kabı içine veya iyi olarak her bir lamel yerleştirin. </li><li> Izolasyon cihazı ve cam lamel arasında tam yapışma sağlamak için bükülmüş uçları ile forseps ile yavaşça ama sıkıca lamel ve basın üzerine AXIS Akson İzolasyon Cihazı yerleştirin. </li><li> Her bir kültür odası içinde, bir pipet, poli-D-lisin, 150 ul (steril 0.1 mg / ml, H 2 damıtılmışO). Kültür odaları tüm havayı çıkarmak için, 5 dakika vakum altında mikroakışkan cihazlar yerleştirin. </li><li> Hala hava odaları, yeniden pipet olan odalarına poli-D-lisin solüsyonu görülebilir gerekebilir. </li><li> 37 ° C &#39;de, poli-D-lizin O / N cihazları inkübe edin. </li><li> Steril distile H 2 O ile bölmeleri üç defa yıkamak </li><li> 150 (PBS içinde, Sigma-Aldrich, 5 ug / ml ya da serum barındırmayan ortam) il, laminin çözelti ile odaları doldurun ve 37 ° C &#39;de O / N inkübe edin. </li><li> Şöyle oluşan trigeminal ganglion kültürleri 37 için 50 ml hazırlayın: 48 mi, Neuro temelli vasat, 1 mi B-27 / ml penisilin / streptomisin, 2 mM L-glutamin, 5 ng / ml sinir büyüme faktörü 100 U (NGF) , 12:25 sitosin arabinosid. </li><li> 40 mi DMEM-F12, 10 ml cenin sığır serumu (FBS, nihai konsantrasyon:% 20), aşağıdaki gibi oluşan, diş mikrop kültürlerinden 37 için 50 ml hazırlayın, 100 U / ml penisilin / streptomisin, 2 mM L-glutamin, 150 ug / ml askorbik asit. </li></ol> 2. Fare Embriyo Üretimi ve Diseksiyon <ol><li> (Vajinal fişi: geliştirme 0.5, E0.5 embriyonik gün) vajinal fiş göre embriyonik yaşını belirlemek ve morfolojik kriterlere aracılığıyla onaylayın. Bu protokol için, genellikle E14.5-E17.5 fare embriyoları kullanmak. </li><li> Diseksiyon alanı ve etanol% 70 ile stereoskop temizleyin. </li><li> Servikal dislokasyon yoluyla hamile anne Kurban. Bir ızgara üzerine birinci ve ikinci parmak ile fare boyun engelleyin ve kararı kuyruğu ile çekin. </li><li> Alt karın çevresindeki deri teşrih ve makas kullanılarak karın açın. Rahim bulun: Gebeliğin böyle geç evrelerinde, rahim bol karın boşluğuna doldurun. </li><li> Buz üzerinde PBS ile dolu bir tüp içinde rahim ve yeri parçalara ayır. Zaman buz üzerinde doku birçok saat boyunca bırakılabilir. Kurumsal kurallarına göre anne cesedi atın </li> &lt;li&gt; rahim embriyolar inceleyin ve onların extraembryonic dokulardan onları serbest. Buz üzerinde PBS embriyolar yerleştirin. </li><li> Makas kullanarak embriyolar decapitate ve mikro diseksiyon makas (Şekil 2A) kullanarak baş geri kalanından alt çene ayırın. Trigeminal ganglionlar zarar vermeden, tam alt çene çıkarın; İkinci alt çene yakın lokalize olarak, onların kaza sonucu hasar mümkündür. Buz üzerinde alt çene ve soğuk PBS başın kalan, koruyun. </li><li> TG incelemek için, başını alıp önce soğuk PBS ile dolu bir diseksiyon cam Petri kabı üzerine yerleştirin. Forseps kullanarak, deri ve kafatası çıkarın. Daha sonra telencephalon ve telencephalon ve asansör altındaki forseps koyarak beyincik çıkarın; telencephalon ve beyincik maruz kafatası alt bırakarak, birlikte çevirmek olacaktır. </li><li> (Şekil 2B&#39;de gösterildiği gibi) trigeminal ganglionları yerelleştirilmesine. Forseps kullanıntrigeminal sinirler TG ayırmak için. Bıçaklar olarak diseksiyonu iğneler kullanılarak trigeminal projeksiyonlar kalıntılarını ortadan kaldırın. Soğuk PBS ile dolu bir Petri kabındaki disseke TG koyun ve buz üzerinde saklayın. </li><li> Soğuk PBS ile dolu önceden diseksiyon cam Petri kabı üzerine, kafatasından ayrılan alt çene, koyun, embriyonik dişleri incelemek için. Bıçaklar olarak diseksiyonu iğneler kullanarak, dil ve çene etrafındaki cilt kaldırmak. Çene orta hat boyunca keserek sol ve sağ hemi-çeneleri ayırın. Şekil 1C gösterildiği gibi diş mikrop kolaylıkla görebilir. Diseksiyon iğneler kullanarak diş mikropları yalıtmak ve olmayan diş dokularının fazlalığı kaldırın. Soğuk PBS ile dolu bir Petri kabındaki disseke diş mikropları yerleştirin ve buz üzerinde saklayın. </li></ol> 3. mikroakışkan Co-kültürler <ol><li> Diseksiyon sonra, mikroakışkan cihazlardan laminin çıkarın. Saygımdan 200 ul odaları doldurunmedyayı ettik. </li><li> Forseps ile (Şekil 1D) delme tarafından oluşturulan deliklere hafifçe disseke TG ve diş mikropları aktarın. Diş mikrop yüzer yok emin olun ve onlar lamelleri temas edene kadar onlar lavabo söyledi. </li><li> Kültür, 37 ° C&#39;de inkübatör içinde örnekleri,% 5 CO2. </li><li> Kültür ortamı, her 48 saat değiştirin. Tamamen odaları boşaltmak etmeyin ve kültür odalarına doğrudan pipetle yok. Odaları içindeki hava kabarcıklarının oluşmasına neden olur bölmelerin tam boşalması; odasına doğrudan pipetleme aksonal hasarla sonuçlanır. Kuyuların dış tarafına doğru pipet işaret orta kaldırmak, bu sorunları önlemek için; Benzer şekilde, odalarına karşısında yer kuyuların yanında taze orta pipetle. </li><li> Kültür döneminde, eş-kültürleri kolayca time-lapse mikroskobu ile görüntülenebilir. Eş-kültür 10 gün boyunca muhafaza edilebilir. </li><li> Kültür perio sonrad odasının başına bir kuyuya PBS 150 ul pipetle ve PBS odaları aracılığıyla üç kez akış sağlayarak odaları yıkayın. </li><li> PBS çıkarın ve odanın başına bir kuyudaki paraformaldehit 150 ul (PBS)% 4 pipetleme örnekleri düzeltmek; 15 dakika boyunca oda sıcaklığında inkübe edilir. </li><li> 3.6 de tarif edildiği gibi PBS ile iki kez odaları yıkayın. </li><li> Daha fazla analiz ile devam edin. </li></ol>

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D., Bianco, F., Matteoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+on+microfluidics:+multimodal+analysis+for+studying+celll-cell+interaction.">Pharmacology on microfluidics: multimodal analysis for studying celll-cell interaction.</a> Current opinion in pharmacology. 13 (5), 821-828 (2013).</li><li>Neto, E., Alves, C. J., 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=Sensory+neurons+and+osteoblasts:+close+partners+in+a+microfluidic+environment.">Sensory neurons and osteoblasts: close partners in a microfluidic environment.</a> Integrative Biology. , (2014).</li><li>Pagella, P., Neto, E., Jim&#233;nez-Rojo, L., Lamghari, M., Mitsiadis, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+co-culture+systems+for+studying+tooth+innervation.">Microfluidics co-culture systems for studying tooth innervation.</a> Frontiers in physiology. 5 (August), (2014).</li><li>Connor, R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+maxillary+factor,+a+maxillary+process-derived+chemoattractant+for+developing+trigeminal+sensory+axons.">Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons.</a> Neuron. 24, 165-178 (1999).</li></ol>

The authors declare that they have no competing financial interests.

Diş innervasyon in vitro çalışmalar mi, trigeminal ganglionların diş dokuları veya hücreleri 26,28,29 geleneksel ko-kültürler dayandırılmıştır. Bu çalışmalar çoğunlukla duyusal aksonlar 38 bu hücrelerin veya dokuların çekici etkisini araştırmak amacıyla yapılmıştır. Alanda önemli ilerlemeler getirerek rağmen, çeşitli teknik sorunlar dile getirildi. Diş mikroplar kültür 37 birkaç gün sonra dejenere başlar. Bu gözlemlere dayanarak, aynı k?...

Inervasyonu organ ve dokularda 1,2 geliştirilmesi, homeostasis ve rejenerasyonda önemli bir rol oynar. 5 - Ayrıca, innervasyon kök hücre proliferasyonu, mobilizasyon ve farklılaşma 3 düzenlenmesinde rol oynar. Gerçekten de, orofasyal kompleks dokularda gerçekleştirilen son çalışmalar parasempatik sinir tükürük geliştirilmesi ve rejenerasyonu 6,7 bezleri içinde epitel progenitör hücrelerin fonksiyonu için gerekli olduğunu göstermiştir. 11 - Aynı şekilde, innervasyon damak 8 gelişimi ve korunması için gerekli olduğu gösterilmiştir. Bu nedenle, bu gibi dişler gibi diğer önemli orofasyal organ ve dokuların gelişimi innervasyon yapılmamış ihmal rolünü analiz etmek önemlidir. Yetişkin dişi zengin innervasyon rağmen, ve buna karşılık vücut develo tüm diğer organ ve dokularaping dişler erken doğum aşamalarında innerve başlanacak. Dişler ağız ektoderm ve kranial nöral krest kökenli mezenşimin arasındaki sıralı ve karşılıklı etkileşimleri sonucunda gelişir. Bu etkileşimler, epitel türevli ameloblast ve mine ve dentine sırasıyla 12 oluşumundan sorumlu olan mezenşimin türetilmiş odontoblast yol açar. 15 - Üstün servikal ganglionlar gelen trigeminal ganglia ve sempatik sinirler duyu sinirleri yetişkin dişleri 13 innerve. Embriyogenez sırasında sinir lifleri gelişmekte diş mikrop karşı trigeminal ganglion projeden kaynaklanan ve giderek onları çevreleyen ama diş papilla mezenşimin 13 içine nüfuz etmez. Sinir lifleri odontoblast farklılaşma ve dentin matriks birikimi 16 ile ilişkili daha ileri gelişim aşamalarında diş pulpa mezenşimi girin. Diş hamuru innervasyon completed yakında ağız boşluğunda 13 diş patlaması sonucu. 19 - Çeşitli semaphorins ve nörotrofinler Odontogenez 16 sırasında innervasyon düzenlenmesinde rol oynadığını Önceki çalışmalar ortaya koymuştur. Daha önceki çalışmalarda açıkça innervasyon balıkların 20 diş oluşumu için bir ön koşul olduğunu göstermiştir. Daha yeni çalışmalar sonik kirpi (sus) 21 salgılanması yoluyla duyusal sinirler tarafından düzenlenir fare kesici diş mezenşimin kök hücrelerin bu homeostasisi göstermiştir. 24 - Bununla birlikte, diş başlatma, geliştirme ve yenilenme içinde innervasyon rolü hala memelilerde 22 derece tartışmalıdır. In vivo çalışmalarda bir bolluk, çeşitli hayvan modellerinde 13,25,26 geliştirme ve onarım işlemleri sırasında diş dokularının innervasyon desen hakkında önemli bilgiler vermiştir. Bununla birlikte, bu en APPROağrıları sinir lifleri ve hedef organ ve doku arasındaki etkileşimin moleküler temelini vurgulamak için optimal değildir. 29 - Co-kültürler araştırılması ve kontrollü ve yalıtılmış bir ortamda 26 sinir lifleri ve dişlerin arasındaki etkileşimleri işlemek için değerli bir yöntem teşkil etmektedir. Aynı zamanda, eş-kültürleme çeşitli teknik ayarlamalar tabidir. 32 - Örneğin, sinirler ve spesifik diş dokularının (örneğin, diş hamuru, diş folikül, diş epiteli) için çoğu zaman 30 uzun süreler için doku hayatta garanti etmek amacıyla farklı bir kültür ortamı gerektirir. 29 - Son yıllarda, aynı kültür ortamı kullanılarak geleneksel eş-kültürler duyusal sinir lifleri 27 ağız ve diş dokularının gelişiminin çekici veya itici etkilerini araştırmak için çok kısa bir süre (örneğin, iki gün) için yapılmıştır.Ancak, kültür süresinin uzatma diş morfolojilerinden ve hücrelerdeki farklılaşmanın üzerindeki innervasyonunun etkilerini araştırmak ve hedef organlarda içinde dallanma sinir liflerinin dinamiklerini incelemek için gereklidir. Bu nedenle, bitişik olmayan ko-kültürler nöronal-diş dokuları etkileşimler çalışmalar yapmak daha uygun olacaktır. Microfluidics sistemleri, uygun bir kültür ortamı nöronların ve farklı hücre tipleri arasında ko-kültürler izin verir. Hedef doku 33 içeren bölmeye doğru mikrokanallar boyunca nöral hücre kütlelerinden aksonlarının gelişimini izin verirken, bu cihazlarda, diş dokuları ve nöronlar farklı bölümlerinde ayrılır. Mikroakışkan cihaz zaten kanser ve neovaskülarizasyon 35 hücre etkileşimleri nöronlar ve mikroglia 34,35 arasındaki etkileşimleri hem de hücre incelemek için kullanılmıştır. Ayrıca, bu sistemler Dors&#39;a arasındaki etkileşimi incelemek için kullanılmıştıral kök gangliyon ve osteoblastlar 36. Biz son zamanlarda trigeminal gangliyon (TG) ve diş uzun süre hayatta kalmak için mümkün olduğunu göstermiştir zaman birlikte kültüre mikroakışkan cihazlar 37. Ayrıca, biz farklı gelişim aşamalarında dişler bu in vitro koşullarda bunlar, in vivo 37 göstermek trigeminal inervasyon aynı itici ya da çekici efektler korumak olduğunu göstermiştir. Bu protokol, basit, güçlü ve esnek bir şekilde birlikte kültür ganglionlar / sinirler ve hedef doku ve kontrollü ve yalıtılmış bir ortamda bu tür etkileşimler ile ilgili özel moleküllerin rollerini incelemek için hakkında bilgi sağlar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AXIS Axon Isolation Devices</td>
			<td>Millipore</td>
			<td>AX15010-TC</td>
			<td>Microchannels of different lenght are available</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse beta-NGF</td>
			<td>R&#38;D Systems</td>
			<td>1156-NG-100</td>
			<td>Human and Rat beta-NGF (R&#38;D Systems) are equivalent</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of developing tooth germ innervation using microfluidic co-culture devices

Inervasyonu organ ve dokuların gelişimi, homeostasis ve rejenerasyonda önemli bir rol oynar. Ancak bu olayları altta yatan mekanizmalar henüz tam olarak anlaşılamamıştır. Özellikle, diş gelişimi ve rejenerasyon innervasyonunun rolü ihmal edilmektedir. In vivo çalışmalar birçok değişik hayvan modellerinin geliştirilmesi ve onarım işlemleri sırasında diş dokularının innervasyon desen hakkında önemli bilgiler vermiştir. Ancak, bu yaklaşımların pek çok sinir lifleri ve hedef organ ve doku arasındaki etkileşimin moleküler temelini vurgulamak için optimal değildir. Eş-kültür araştırılması ve kontrollü ve yalıtılmış bir ortamda sinir lifleri ve dişlerin arasındaki etkileşimleri işlemek için değerli bir yöntem teşkil etmektedir. Son yıllarda, aynı kültür ortamı kullanılarak geleneksel eş-kültürleri çok kısa süreler için yapılmıştır (örneğin, iki gün)duyusal sinir lifleri üzerinde ağız ve diş dokularının gelişiminin çekici veya itici etkisini araştırmaktır. Ancak, kültür süresinin uzatma diş morfolojilerinden ve hücrelerdeki farklılaşmanın üzerindeki innervasyonunun etkilerini araştırmak için gereklidir. Microfluidics sistemleri, uygun bir kültür ortamı nöronların ve farklı hücre tipleri arasında ko-kültürler izin verir. Son zamanlarda, trigeminal gangliyon (TG) ve diş uzun bir süre hayatta mümkün olduğunu göstermiştir birlikte kültüre edildiğinde, mikroakışkan cihazlarda, ve bu koşullar altında da in vivo olarak gösterir, aynı innervasyon deseni korumak. Bu temelde, biz izole etmek ve bir mikroakışkan ko-kültürü sistemi kullanmaktadır.Bu protokolde trigeminal ganglion ve diş mikropları gelişmekte ko-kültür basit ve esnek bir yol için ortak kültür ganglionlar / sinirler ve hedef doku ve açıklar anlatan rolleri incelemek için Bir Contr tür etkileşimler üzerinde özel moleküllerolled ve izole bir ortam.

Bu sonuçlar, izole trigeminal ganglion izole diş mikrop gelişimi mikroakışkan cihazın diğer bölmeye uzun bir süre boyunca devam olduğu, ek olarak, mikro-akışkan cihazın bir bölmeye büyür ve olduğunu göstermektedir. Farklı kültür ortamı iki bölme kullanılır ve iki bölme arasında microgrooves geliştirilmesi diş mikrop doğru trigeminal ganglion gelen akson uzantısı sağlar. Şekil 3, bir fare, bir ko-kültür, immünofloresan 37 üzerinden nörofilament bir görse...

Watch this Scientific Journal Video about Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices at JoVE.com

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Mikroakışkan Co-kültür Aygıtları Kullanma Diş Germ innervasyon Geliştirme Analizi

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

analysis-developing-tooth-germ-innervation-using-microfluidic-co

Research

JoVE Journal

Neuroscience

8.2K Görüntüleme.  University of Zurich. Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Video: Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

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

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

İlköğretim Rat Kortikal Nöronlar ve Glia bilaminar Co-kültür

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

Nörobilim

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

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

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

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

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

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Mikroakışkan Kültür Platformu Glia Etkileşimi (MCP) tabanlı Nöronal Akson ve Glia Co-kültür Sistemi Nöron görüntülenmesi Analizi

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Frenik Motor Nöronlar tarafından Diyafram İNNERVASYONU Fonksiyonel ve Morfolojik Değerlendirmesi

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

Yüksek çözünürlüklü ses görüntüleme, nöronlar Floresans kullanım dışlama yöntemi tarafından ve adanmış mikrosıvısal aygıtları

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Farelerde beyin sapı uyarılmış yanıt odyometrisi veri edinme ve Analizi

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Göç, Kemo-Attraction, ve Co-Kültür Tahliller İnsan Kök Hücre Kaynaklı Endotel Hücreleri ve GABAerjik Nöronlar için

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

Oligodendrocytes ve Oligodendrocyte-Ko-Kültür Deneyleri için Şartlı Orta Üretimi

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Mikroakışkan Odacıksistemi Kullanılarak Motor Nöron Kültürlerinde Organellerin Aksonal Taşınması

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...