This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.

NOT: Tüm hayvan prosedürleri yönergeleri izleyerek ve Connecticut Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. NOT: YFP muhabiri 20 (JAX # 006148) çizgiler sırasıyla kullanıldı: NG2cre 18 bünye (JAX # 008533) ve NG2creER 16 uyarılabilir bu deneylerde (JAX # 008538) için transgenik fareler 19 (JAX # 003920) veya gtRosa26 / EG Z&#39;ye geçti Görüntü NG2 hücreleri ve soylarına. Görüntü olgun oligodendrositlere, PLPDsRed transgenik farenin 21 kullanılmıştır. Tutarlı hayatta kalmak için, dilimler ön beyin ve beyincik hem doğum sonrası gün 10 fareden izole edilebilir. 1. Dilim Hazırlık <ol><li> Bir kültürü kaputu, altı iyi plakaları doku kültürü yerlefltirmede ve (reaktif bölümünde tarifi) 1 ml dilim kültür ortamı ekleyin. % 5 ile 37 ° C&#39;de diseksiyon önce CO2 en az 2 saat, hücre kültürü inkübatöründe plakaları koyun. </li><li> % 70 etanol ile tüm araçları ve doku chopper sterilize edin. </li><li> Diseksiyon önce başlamasından en az 15 dakika boyunca buz üzerinde% 95 O2,% 5 CO2 ile kabarcık tampon diseksiyon (reaktif bölümünde tarif). </li><li> Onaylanmış hayvan protokollere göre 5-10 dakika süreyle buz üzerinde yerleştirerek fare yavrular anestezisi. Farenin kuyruk ve ayak sıkıştığı yanıt vermeyen onaylayarak anestezi uygun derinliğini teyit edin. </li><li> Hayvan protokolleri aşağıdaki keskin bir makas kullanarak hayvanları başını kesmek. Hızla ilk sterilize araçlarını kullanarak lateral insizyon ardından sagital kesim, yaparak ön beyin ve beyincik üzerinde kafatası çıkarın. Dikkatli bir tarafına doku yuvarlayarak beyin ve beyincik ventral yüzeyinden kafatası sinirlerinde kesin. </li><li> Buzla soğutulmuş, oksijenli diseksiyon tampon içeren steril bir 35 mm çanak doku yerleştirin. </li><li> Bir jilet kullanarak, beyincik ve ön beyin sever. Sonra mid-sagital kesim uçtan bir uca yapmakhemisferleri ayıran öf ön beyin ve beyincik. </li><li> Manuel doku kıyıcı ile ön beyin ve beyincik 300 mikron kalınlığında koronal ve sagital kesitler halinde kesilmiştir. NOT: Manuel doku kesici ve agaroz yerleştirilmesi için gerek kalmadan kültür kuluçkalamadan diseksiyon ikinci hızlı bir şekilde işlenebilirlik sağlar. Daha önce tarif edildiği gibi, 9 bir vibratome da kullanılabilir. </li><li> Buz üzerinde taze kabarmış soğuk diseksiyon tampon ayrılır dilim aktarın. </li><li> Küçük diseksiyon kullanın veya bireysel bölümleri ayırmak ve kültürü ekler ile altı iyi yemekler preinkübe aktarmak için spatulası ağırlığında. </li><li> Tek kullanımlık bir transfer pipeti veya P 200 pipet ucu kullanılarak kültür uç yüzeyinden herhangi bir aşırı diseksiyon tamponunu çıkarın. Üç önbeyinde üç serebellar dilim iki bir kültür insert yerleştirilebilir. NOT: ön beyin kültürleri ile tutarlı sonuçlar için, anterior korpus kallosum üçte birini kapsayan dilim almak idealdirAynı dilim içinde hem gri hem de beyaz cevher bölgelerini incelemek için (Şekil 1A). Her hayvan genellikle 6 ön beyin dilimleri ve 6 beyincik dilimleri verir. </li><li> Inkübatör altı havuzlu plakalar yerleştirin ve daha sonra 1 1 gün sonra dilim dilim hazırlanması ortamının mililitresi ve dilim sabitleme kadar her gün ile dilim orta yerine. </li><li> Deneye bağlı olarak, kültür 5-7 gün sonra dilimler kullanın. Sadece ilk birkaç gün sonra şeffaf hale dilimleri kullanın ve düzensiz opak bölgeler var olanlar atın. NOT: dilimler içinde Opak bölgeler gözle görünür ve faz kontrast altında koyu hücrelerin bir yığın olarak görünür. Teknik hakim olmuştur sonra ölü opak dilimler dilim az% 1-5, nadiren meydana gelir. </li></ol> NG2 hücre Bölümü ve Oligodendrosit Farklılaşma 2. Time-Lapse Görüntüleme NOT: ZEG farenin 16: biz NG2cre kullanmak NG2 hücre çoğalması ve farklılaşması zaman atlamalı görüntüleme gerçekleştirmek içinNG2 hücreleri ve soyu (Şekil 1C-E) EGFP ifade edilmektedir. Bu hat Raportör ekspresyonu, kısa bir sürede en az dört haftalık bir süre boyunca kesit sonra dilimler canlı GFP görüntüleri + hücreler elde etmek için yeterince sağlamdır. Açık görüntüler kültüründe ilk 3-5 gün içinde gerçekleşir dilim, ilk incelmesi döneminden sonra elde edilir. <ol><li> Deney boyunca, 37 ° C&#39;de hücre kültürü inkübatöründe dilimleri tutmak ve kısa süreler için, sadece kaldırma (dilim başına en az 15 dakika), altı yuvalı plaka sırasında görüntüleme üzerindeki kapağı tutmak. </li><li> Bir dijital kamera ile donatılmış bir ters floresan mikroskop kullanılarak, bir 10X objektif kullanılarak ilk kez noktasında görüntüleri yakalayabilir. NOT: İlk kez puan deney göre değişir ve dilimler hazırlanmıştır gün veya sonrasında herhangi bir zaman noktası olabilir. </li><li> Dilim hem gri hem de beyaz cevher alanlarında zaman noktasında bir çoklu bölgelerden görüntüler yakalayın. Not thdilim içinde belirli noktalara ile ve daha sonra bir görüntüleme oturumları için kullanılabilecek bir şema üzerinde görüntü konumu eşleyerek görüntülenmiş e bölgesi. Faydalı yerlerine beyaz cevher yollarının dilimleri ve / veya yönlendirme kenarlarını içerebilir. Görüntü her zaman noktasında her dilim 4-8 yerleri. </li><li> 4-6 saat arayla sonraki fotoğrafları çekmek NG2 hücre bölünmesini ve olgodendrosit farklılaşma, ideal üzerinde 5-7 gün inceleyerek eğer. </li><li> Tüm bölgelerin nihai bir görüntü yakalamak ve hemen dilimleri düzeltmek. Daha sonra dilimler üzerine başka bir 1 ml ilave edilir, kültür ucun tabanına (0.1 M L-lisin, 0.01 M sodyum meta-periodat ile% 4 PFA), sabitleyici 1 ml ilave edilir. Bu membran ayırmak gibi dilim doğrudan Fiksatif fışkırtma dikkat edin. 30 dakika boyunca doku saptamak ve bölüm 4&#39;te tarif edildiği gibi gelişimsel aşama spesifik markörleri kullanılarak immünohistokimya ile devam edin. </li><li> 0.2 M sodyum fosfat tamponu, 3 x 10 m ile yıkayın dilimleriolarak. 1-3 gün boyunca 0.2 M sodyum fosfat tampon maddesi içinde 4 ° C&#39;de, gerekli deposu dilimleri daha önce immüno-boyama ve ancak ideal olanı ise, 24 saat içinde ve boyama gibi. Oligodendrositler (Şekil 2D,-F) farklılaşmış olan hücrelerin oranını belirlemek için bölüm 4, aşağıda tarif edildiği gibi immünohistokimya ile devam edin. </li></ol> Dilim Kültürlerde indüklenebi- Muhabir transgenik fareler kullanılarak NG2 hücre Döl 3. Kader Haritalama Not: kültüründe belirli bir zaman noktasında NG2 hücrelerin kaderini izlemek için, uyarılabilir NG2creER: YFP transgenik fareler kullanılabilir. <ol><li> Kültür ortamına, etanol içerisinde çözündürüldü, 100 nM 4OHT ekleyerek, Cre rekombinasyonunu ve bildirici ifadesini teşvik eder. Not: Raportör pozitif hücreler, bütün NG2 hücre aşamasında hücreler olacaktır ~% 20-25, YFP + NG2 + / + hücrelerinin NG2 bir indüksiyon verimle ve bu zaman noktasında 1-2 gün içinde görünecektir (Şekil 2A-C) </li><li> Fix ve dilim yıkayınFarklı zaman noktalarında in çeşitli değişiklikler yapıldıktan sonra (Bölüm 2.5 ve 2.6 &#39;da tarif edilmiştir). </li></ol> 4. Dilim İmmünhistokimya <ol><li> Bir neşter kullanılarak ekler dışarı ekli dilimleri ile membranlar kesti. </li><li> Membran taşırken dilimin bileşimi bozmak için özen cımbız bir dizi kullanılarak 24 gözenekli bir plaka ihtiva eden fosfat tamponlu tuzlu su (PBS), tek tek oyuklara dilimleri aktarın. </li><li> 1 saat boyunca PBS içerisinde% 5 normal keçi serumu (NGS) ve% 0.1 Triton-X100 içeren bloke etme çözeltisi, 24 gözlü bir levhaya dilimleri aktarın. </li><li> 4 ° C&#39;de PBS içinde% 5 NGS / birincil antikor O&#39;da N dilimleri inkübe edin. NG2 hücre safhasında hücrelerin belirlenmesi için, ve NG2 PDGFRα antikorlar kullanır. Diferansiye oligodendrositlerin belirlenmesi için, adenomatus poliposis koli antijene karşı bir antikorun kullanılması (APC klon CC1). </li><li> PBS 3 x15 dakikada ikinci gün yıkama dilimleri, daha sonra ikincil Antibo kuluçkaya yatmaktadıroda sıcaklığında 1 saat boyunca PBS içerisinde% 5 NGS ölür. PBS 3 x15 dakika dilimleri yıkayın ve slaytlar monte. Membran objektif ve doku arasındaki olmayacak şekilde slayt karşı karşıya dilimleri yukarı ve membran ile monte edin. </li></ol>

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Molecular Cell Biology. 8 (10), 839-845 (2007).</li><li>Lubetzki, C., Demerens, 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=Even+in+culture,+oligodendrocytes+myelinate+solely+axons.">Even in culture, oligodendrocytes myelinate solely axons.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (14), 6820-6824 (1993).</li><li>Demerens, C., Stankoff, B., 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=Induction+of+myelination+in+the+central+nervous+system+by+electrical+activity.">Induction of myelination in the central nervous system by electrical activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (18), 9887-9892 (1996).</li><li>Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+sensory+axons+are+mitogenic+for+Schwann+cells.">Evidence that sensory axons are mitogenic for Schwann cells.</a> Nature. 256 (5519), 662-664 (1975).</li><li>Stevens, B., Tanner, S., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+myelination+by+specific+patterns+of+neural+impulses.">Control of myelination by specific patterns of neural impulses.</a> The Journal of Neuroscience. 18 (22), 9303-9311 (1998).</li><li>Stevens, B., Porta, S., Haak, L. L., Gallo, V., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine:+a+neuron-glial+transmitter+promoting+myelination+in+the+CNS+in+response+to+action+potentials.">Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action potentials.</a> Neuron. 36 (5), 855-868 (2002).</li><li>Wake, H., Lee, P. R., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+local+protein+synthesis+and+initial+events+in+myelination+by+action+potentials.">Control of local protein synthesis and initial events in myelination by action potentials.</a> Science. 333 (6049), 1647-1651 (2011).</li><li>Rosenberg, S. S., Kelland, E. E., Tokar, E., De la Torre, A. R., Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geometric+and+spatial+constraints+of+the+microenvironment+induce+oligodendrocyte+differentiation.">The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (38), 14662-14667 (2008).</li></ol>

The authors declare that they have no competing financial interests

Merkezi sinir sisteminde etkin Myelinasyon nöronal iletişim ve aksonal hayatta 22 için gereklidir. 25 - En beyin bölgelerinde 16,23 içinde yerleşik nüfusu korurken NG2 hücreleri sürekli yetişkinlik içine Myelinating oligodendrositleri oluşturmak. Bu hücrelerin farklılaşmasını düzenleyen bazı genetik ve moleküler mekanizmalar tarif edilmiştir ama çok keşfedilmeyi beklemektedir. Organotipik dilim kültürleri nedeniyle anatomik bölge, hücre dışı ortama kolay man...

4 - merkezi sinir sistemi Organoytpic dilim kültürlerinin semiintact sistem 1 &#39;de nöron ve glial hücre biyolojisi çalışmak için çok yararlı olduğu kanıtlanmıştır. 9 - Bu kültürler, kabul ve bu hücre dışı ortamın bir manipülasyon ve tekrarlamalı uzun-süreli canlı hücre görüntüleme için kolay erişim ve elektrofizyolojik kayıtları 5, birincil hücre kültürleri ayrışmış birçok yararlarını korumak için nispeten basittir. Buna ek olarak, dilim kültürler, 3 boyutlu doku hücre mimarisini, bölgesel sinir bağlantısına sahip, ve en önemli hücre tiplerinin sistemde bulunmaktadır. Bu özellikler hücresel ve çevresel etkileşimleri ile tek hücre davranış ve fizyolojisini araştırmak için bu Kültürlerin benzersiz ve kullanışlı bir sistem yapmak. NG2 hücrelerinin çoğalmasını ve m oluşturmaya devam memelilerin merkezi sinir sistemindeki glial hücreler bir nüfusembriyonik ve postnatal gelişim 10 sırasında oligodendrositleri yelinating. Bunlar yaygın olarak ayrışmış primer hücre kültürlerinde incelenmiştir ve floresan proteinleri NG2, hücre spesifik ifadesi ile transgenik fare hatları son gelişmeler, akut dilim preparasyonlarında in vivo kaderi haritalama ve elektrofizyolojik kayıtları kolaylaştırmıştır. Hatta bu çalışmalar ile, küçük NG2 hücre çoğalması ve olgodendrosit farklılaşmasının zamansal dinamikleri hakkında bilinmektedir. Ayrışık hücre kültürü geniş farmakolojik ve genetik manipülasyon göreli tesis için kullanılır, ancak, bunun hücresel mikro-bağlamını sağlamak tercih edilir, özellikle, beynin farklı bölgelerinde bu hücrelerin fonksiyonel farklılıklar sorgulamak için uygun değildir. Dilim kültürleri, hücre farmakolojik manipülasyonlar mükellef ve olgodendrosit myelinleşmeyi 11,12 araştırmak için kullanılır olmuştur basit bir alternatif sağlamak(LPC) lisolesitin veya antikor farmakolojik tedavinin 15 üzeri demiyelinizasyonu 13,14 ve remiyelinizasyon indüklenmesini sonra ular yanıt. Bir yöntem araştırmak ve canlı görüntüleme ve sabit doku (veya Postfiksasyon) ön beyin ve beyincik hem de alınan organotipik dilim kültürlerde NG2 hücre çoğalması ve farklılaşması olgodendrosit analizini tarif gerçekleştirmek için. Bu bölünme sonrası 16 tek NG2 hücrelerinin hücre kaderine incelemek için ve büyüme faktörü ile uyarılan NG2 hücre çoğalması 17 bölge ve yaşa bağlı farka için kullanılabilir güçlü bir yöntemdir. Bu, nispeten basit teknik ayrıca doğuştan ve / veya çevresel, bu glial hücre fizyolojisi ve nöronal aktivite ya da mielin hasarına cevabı düzenleyen mekanizmaların hücre araştırılması yaygın erişilebilir.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Slice Culture Medium&lt;/strong&gt;</td></tr><tr><td>Minimum Essential Medium</td><td>Invitrogen</td><td>11090</td><td>50%</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Invitrogen</td><td>14175</td><td>25%</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H-4034</td><td>25mM</td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030</td><td>1mM</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td>1mg/L</td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A-0278</td><td>0.4mM</td></tr><tr><td>Horse Serum</td><td>Hyclone</td><td>SH30074.03</td><td>25%</td></tr><tr><td colspan="4">Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td></tr><tr><td colspan="4">&lt;strong&gt;Dissection Buffer&lt;/strong&gt;</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td>124mM</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P5405</td><td>3.004mM</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td>1.25mM</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (anhydrous)</td><td>Sigma-Aldrich</td><td>M7506</td><td>4.004mM</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C7902</td><td>2mM</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761</td><td>26mM</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td>10mM</td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A-0278</td><td>2.0mM</td></tr><tr><td>Adenosine</td><td>Sigma-Aldrich</td><td>A4036</td><td>0.075mM</td></tr><tr><td colspan="4">Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O&lt;sub&gt;2&lt;/sub&gt; 5%CO&lt;sub&gt;2&lt;/sub&gt; before use</td></tr><tr><td colspan="4">&lt;strong&gt;Other Reagents and Supplies&lt;/strong&gt;</td></tr><tr><td>4-hydroxy tamoxifen (4OHT)</td><td>Sigma-Aldrich</td><td>H7904</td><td>100nM</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>EM Sciences</td><td>19200</td><td>4%</td></tr><tr><td>L-Lysine</td><td>Sigma-Aldrich</td><td>L-6027</td><td>0.1M</td></tr><tr><td>Sodium Metaperiodate</td><td>Sigma-Aldrich</td><td>S-1878</td><td>0.01M</td></tr><tr><td>Rabbit anti NG2 antibody</td><td>Chemicon (Millipore)</td><td>AB5320</td><td>1:500</td></tr><tr><td>Mouse anti CC1 antibody</td><td>Calbiochem (Millipore)</td><td>OP80</td><td>1:200</td></tr><tr><td>Mouse anti Ki67 antibody</td><td>Vision Biosystems</td><td>NCL-Ki67-MM1</td><td>1:300</td></tr><tr><td>Mouse anti NeuN antibody</td><td>Chemicon (Millipore)</td><td>MAB377</td><td>1:500</td></tr><tr><td>Species specific secondary antibodies</td><td>Jackson Immunoresearch</td><td></td><td></td></tr><tr><td>Normal Goat Serum (NGS)</td><td></td><td></td><td>5%</td></tr><tr><td>Triton X-100</td><td></td><td></td><td>0.10%</td></tr><tr><td>Mounting medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Millicell culture membrane inserts 0.45&amp;mu;m pore size</td><td>Fisher Scientific</td><td>PICM03050</td><td></td></tr><tr><td>Bottle top filters</td><td>Fisher Scientific</td><td>09-740-25E</td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt; 5% CO&lt;sub&gt;2&lt;/sub&gt; gas mix</td><td></td><td></td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td></td><td></td><td></td></tr><tr><td>Sterile six well plates</td><td></td><td></td><td></td></tr><tr><td>Dissecting (hippocampal) or weighing spatulas</td><td></td><td></td><td></td></tr><tr><td>Manual or automatic tissue chopper</td><td></td><td></td><td></td></tr><tr><td>Razor blades</td><td></td><td></td><td></td></tr><tr><td>Disposable transfer pipettes</td><td></td><td></td><td></td></tr><tr><td>35mm and 60mm sterile Petri dishes</td><td></td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Inverted Phase Microscope</td><td></td><td></td><td></td></tr><tr><td>Laminar Flow Hood</td><td></td><td></td><td></td></tr><tr><td>Tissue Culture Incubator</td><td></td><td></td><td></td></tr><tr><td>Inverted Fluorescence Microcope</td><td></td><td></td><td></td></tr></tbody></table>

organotypic slice cultures to study oligodendrocyte dynamics and myelination

NG2 ifade eden hücreler (polydendrocytes oligodendrosit öncü hücreleri), merkezi sinir sisteminde, dördüncü bir glial hücre içerisinde yer alırlar. Embriyonik ve postnatal gelişim sırasında aktif çoğalırlar ve Myelinating oligodendrositleri oluşturmak. Bu hücreler sık ​​primer ayrışmış kültürler, nöron cocultures okudu ve sabit dokuda edilmiştir. Kullanımı uygun yeni transgenik fare soyları kültür sistemleri ön beyin ve beyincik hem gri ve beyaz madde bölgelerde oligodendrosit soy hücrelerinin çoğalması ve farklılaşması araştırmak için kullanılabilir dilim. Dilim kültürleri erken doğum sonrası farelerde hazırlanan ve 1 aya kadar kültür içinde tutulur. Bu dilimler hücresel davranış ve etkileşimleri araştırmak için kültür dönemi boyunca birden fazla kez görüntülenmiş olabilir. Bu yöntem NG2 hücre bölünmesi görselleştirme ve ayrıntılı analizini sağlarken olgodendrosit farklılaşma giden adımlar bölgede-bağlıdır veriyorent NG2 hücre ve olgodendrosit fonksiyonel heterojenite. Bu ise, in vivo olarak bulunan andıran bir hücresel ortamda zaman bu hücreleri etkileyen iç ve dış sinyalleri araştırmak için kullanılan bir güçlü bir tekniktir.

Örnek veri örnekleri aşağıda verilmektedir P8 NG2cre bir ön beyin ve beyincik hem de dilim kültürleri kullanılarak elde edilmiştir ki: zeg, NG2creER: YFP ve transgenik fare hatları PLPDsRed. NG2 hücreleri ön beyin ve beyincik dilimleri (Şekil 1B-D, video 1) ve bu hücrelerin fenotip gün boyunca görüntülü olabilir tespit edilerek ve NG2 ve CC1 antikorları (Şekil 1E) ile immünboyama edilebilir. Canlı görüntülemeye ek olarak, hücre çoğalması aynı zamanda Ki67...

Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotipik Dilim Kültürler Oligodendrosit Dinamikler ve miyelinasyonun incelemek için

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

organotypic-slice-cultures-to-study-oligodendrocyte-dynamics

Research

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Neuroscience

18.3K Görüntüleme.  University of Connecticut. A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Video: Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

POSTNATAL nöron Çalışmaları için organotipik Dilim Kültürler

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Gelişim Biyolojisi

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

Neokorteks bir Organotipik Dilim Hazırlık Tüm Hücre Kaydı

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Nörobilim

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Embriyonik ventral orta beyin Organotipik Dilim Kültürler: dopaminerjik Nöronal Gelişim Eğitim Bir Sistem In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Bir<em&gt; Ex vivo</em&gt; Akut Beyin Dilimleri bir Oligodendrosit yönelik T-Hücre Saldırı Modeli

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

İmmünoloji ve Enfeksiyon

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hipokampal dilim kültürler çalışma Neuroprotection ve tümör hücrelerinin Invasiveness Model olarak

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Görselleştirme ve Oligodendrocyte organelleri Adeno ilişkili virüs ve Confocal mikroskobu kullanılarak Organotypic beyin dilimleri içinde canlı görüntüleme

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

Hazırlık ve Immunostaining Myelinating Organotypic serebellar dilim kültürlerin

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Olomotor sinir gelişimi için zaman aşımı görüntüleme için embriyonik GFP-Ifade fareler dan ex vivo okülomotor dilim kültürü

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

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 ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

<meta charset="utf8"></head><body>Gelişimsel ve Hastalıkla İlgili Manipülasyonlar için Kantitatif Bir Model Olarak Serebellar Dilim Kültürlerinde Ex Vivo Miyelinasyon ve Remiyelinasyon

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Gelişimsel ve Hastalıkla İlgili Manipülasyonlar için Kantitatif Bir Model Olarak Serebellar Dilim Kültürlerinde Ex Vivo Miyelinasyon ve Remiyelinasyon

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...