Bu çalışma cömertçe Nörolojik Bozukluklar ve İnme (NIH / NINDS) hibe 1R01NS086981 ve Conrad N. Hilton Vakfı Sağlık / Ulusal Enstitüsü Ulusal Sağlık Enstitüleri tarafından desteklenmiştir. Biz konfokal mikroskobu ile onların çok değerli yardım için Dr Laurent Bentolila ve Dr Matthew Schibler teşekkür ederim. Biz AÇIKLIK forumu (http://forum.claritytechniques.org) katkıda bulunmuş olanlar teşekkür ederim. Biz özellikle bu büyüleyici tekniğini öğretmek onun laboratuvarını açılması için Dr. Karl Deisseroth teşekkür ederim. Yazarlar Beyin Haritalama Tıbbi Araştırma Örgütü, Beyin Haritalama Destekleme Vakfı, Pierson-Lovelace Vakfı, Ahmanson Vakfı, Capital Group Şirketleri Hayırsever Vakfı, William M. ve Linda R. Dietel Hayırsever Fonu ve Northstar Fonundan cömert destek için minnettarız . Bu yayında bildirilen araştırma da kısmen Ulusal Araştırma Kaynakları Merkezi tarafından ve Ulusun Başkanlığınca desteklenenÖdül numaraları C06RR012169, C06RR015431 ve S10OD011939 altında Sağlık al Enstitüleri. Içeriği sadece yazarların sorumluluğundadır ve mutlaka Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmemektedir.

Tüm deneyler Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) kurallarına uygun olarak yapıldı. Thy1 YFP, PLP-EGFP PV-tdTomato farenin Bu deneyler için kullanılmıştır, ama floresan proteinleri eksprese bir farenin başarılı bir şekilde temizlenir ve görüntülenebilir. 1. Doku Hazırlanması Dikkat: Paraformaldehyde (PFA) ve akrilamid toksik ve tahriş edicidir. Davlumbaz ve uygun kişisel koruyucu ekipman (laboratuvar önlüğü, eldiven ve göz koruması) bu reaktifleri kullanılarak deneyler. <ol><li> (- 3 dakika ~ 2) solunum sona erene kadar izofluran ~ 1 ml ötenazi odasında hayvan kurban. </li><li> (- 10 dakika 5), kan dokulardan temizlenir kadar ~ 5 ml ayarlanmış bir peristaltik pompa kullanımı / dak intrakardiyak, 4 ° C de perfüzyon çözeltisi (Tablo 1) ile fare perfüze edilmesi için. </li><li> Fiksatif çözüm için perfüzat değiştirin (Tablo 1)(- 10 dakika 5), ​​4 ° C&#39;de, boyun ve kuyruk anlamlı katılaşana dek. </li><li> perfüzyon End ve hayvan başını kesmek. Dikkatle ilk kafatası çevreleyen bağ dokusu çıkarılması ve sonra da kafatasının ventral yüzeyinden kemik kaldırılması ve 20 de açıklandığı gibi, (Şekil 1a) beynini kaldırarak kafatası beyin teşrih. Not: beyincik flocculonodular lobları ve koku ampuller çevreleyen kemiğin çıkarılması için özel bakım verir. <ol><li> Nazikçe omurilik ve vertebral kolon arasında ince bir keskin makas ucu sokarak ve 21 de açıklandığı gibi, uzak bireysel omurga segmentleri keserek vertebra sütundan omurilik parçalara ayır. </li></ol></li><li> plastik bir fare beyin matrisi içine yerleştirerek ve bir matris bıçak ile orta hat boyunca keserek beyin Hemisect. </li><li> santrifüj küvet 50 ml ayırmak için her yarımkürede ve omurilik ekleES ~ hidrojel çözeltisi (Tablo 1) 40 ml. Bu noktada ileri kapaktan alüminyum folyo ile tüpler sonraki tüm adımları sırasında fluorophores korumak için. </li><li> yumuşak 7 gün boyunca (~ 30 rpm) çalkalayarak 4 ° C&#39;de hidrojel örneğini içeren tüp inkübe edin. </li></ol> 2. Polimerizasyon <ol><li> Santrifüj tüpü içinde ~ hidrojelin 25 ml numune ve koruyucu, ~ hidrojelin 25 mi atın. </li><li> kapağı çıkarmadan ve kurutucu kavanoza santrifüj tüpü yerleştirilmesi ve bir vakum kavanozu bağlayarak örnek oksilenlendirilmesi. çözüm ve form kabarcıklar çıkmaya çözünmüş gazlar izin için en az 10 dakika süreyle vakum uygulamak. Yavaşça kabarcıklarını çıkarmak için sallayın. </li><li> 3 dakika - 2 üzerinde% 100 azot gazı ile desiccator kavanoza vakum değiştirin. basıncı (kurutucu kavanozun üst açılabilir kez) eşitleyen sonra, hızlı bir şekilde oksijen reintroduction önlemek için tüp kap. </li><li> PolimerPolimerizasyon işlemi tamamlanana kadar, 3 saat için 37 ° C su banyosu içinde örnek tüpü yerleştirerek hidrojel ize. Polimerize hidrojel jel benzeri bir kıvam (Şekil 1b) alacak. Not: üst polimerleşmemiş hidrojel az miktarda kabul edilebilir. </li><li> Daha sonra, santrifüj tüpüne gelen polimerize örneği kaldırmak örnekten fazla hidrojel kesip bir spatula kullanın. </li><li> silip bir tüy bırakmayan üzerinde örnek yerleştirerek ve yavaşça örnek üzerinde polimerize hidrojel, sadece ince bir tabaka geride bırakarak, sürtünme ve üzerinde doku yuvarlayarak kalan hidrojel çıkarın. </li></ol> 3. Lipit Kaldırma Not: Lipid temizlenir doku kırılgan, dikkatli olun. tüp boşaltırken örnek kaybetmemek için bir hücre süzgeç kullanın. Ayrıca, örnek kırılma indisini uyumlu hale işlemi sırasında ters olabilir şişen, ancak, temizleme işlemi boyunca şişer. Dikkat: Sodium dodesil sülfat (SDS) ve borik asit tahriş edicidir. Davlumbaz ve uygun kişisel koruyucu ekipman (laboratuvar önlüğü, eldiven ve göz koruması) ile onları kullanan deneyler. <ol><li> Temiz bir 50 ml santrifüj tüpüne polimerize örnek aktarın ve tampon (Tablo 1) temizleme 45 ilave edin. hafifçe çalkalanarak 45 ° C (~ 30 rpm) inkübe edilir. kimyasal atık içine kullanılan takas tampon dökülen ve taze takas tampon ~ 45 ml ekleyerek ilk 7 gün boyunca günde tampon değiştirin. florofor korumak için alüminyum folyo ile kaplanmış numunelerin içeren santrifüj tüplerini muhafaza edin. </li><li> 3 gün - ilk 7 gün sonra, her 2 40 ° C ve döviz takas tamponu de örnek kuluçkaya yatmaktadır. Tüm lipidler çözünmüş ve doku şeffaflık (Şekil 1c) ulaşıncaya kadar takas devam edin. Bir fare beyin yarımkürede 6 hafta ve 2 - - Bir omurilik için 4 hafta Bu 4 olacak. </li><li> Doku temizlendikten sonra, aktarmakYeni bir 50 ml santrifüj tüpüne numune ve kalıntı SDS kaldırmak için 24 saat boyunca hafif bir çalkalama (~ 30 rpm) 40 ° C&#39;de PBST (Tablo 1) 4 kez yıkayın. </li></ol> 4. Moleküler Boyama Dikkat: 4 &#39;, 6-Diamidino-2-phenylindole dihidroklorür (DAPI) tahriş edici ve davlumbaz ve uygun kişisel koruyucu ekipman (laboratuvar önlüğü, eldiven ve göz koruması) ile yapılmalıdır kullanan herhangi deneyler olduğunu. <ol><li> Temiz bir 15 ml santrifüj tüpüne örnek transferi ve 1 x PBST içinde 1 ug / ml DAPI, pH 7.5 ile doldurun. 24 saat oda sıcaklığında (RT) enkübe edilir. florofor korumak için alüminyum folyo ile kaplanmış numunelerin içeren santrifüj tüplerini muhafaza edin. </li><li> en az 6 saat / yıkama için oda sıcaklığında PBST içinde 3 kez yıkanır. </li></ol> 5. Refraktif İndeks Eşleştirme Dikkat: sh 2,2&#39;-Thiodiethanol (TDE) tahriş edicidir ve herhangi deneyler onu kullananDavlumbaz ve uygun kişisel koruyucu ekipman (laboratuvar önlüğü, eldiven ve göz koruması) ile yapılabilir ould. <ol><li> Temiz bir 15 ml santrifüj tüpüne örnek aktarın ve% 30 1 x PBS içinde (hac / hac) TDE, pH 7.5 ile doldurun. 24 saat boyunca hafifçe çalkalanarak 40 ° C (~ 30 rpm) inkübe edilir. </li><li> 1 x PBS, pH 7.5 içinde% 63 (h / h) TDE Exchange% 30 TDE. yumuşak, 24 saat boyunca çalkalanarak 40 ° C&#39;de inkübe edin. Not: Örnek, yaklaşık orijinal boyutuna geri çekilmek olacaktır. </li><li> düz ve temiz bir yüzey üzerine numunenin kalınlığından sadece biraz daha düzgün bir çapa sahip bir silindirin içine yeniden bir yapışkan parça döndürün. </li><li> açık uçlu daire yeniden kullanılabilir yapışkan silindiri Lay (~ üst kısmında küçük bir açıklığı olan 25 mm iç çap) 40 mm cam alt çanak. düzeltmeler yapılması gerekiyorsa, cam silindir kaldırmak ve bir jilet ile kesti. </li><li> ile yeniden yapıştırıcı ile cam arasında bir sızdırmazlık oluşturmak için bir P1000 pipet kullanınsilindirin dış kenarı etrafında yuvarlayarak. </li><li> Yeri ~ 100 ul% 63 cam tabak üzerine TDE ve yeniden yapıştırıcı daire içinde cam yüzeyini kapsayacak şekilde yayıldı. </li><li> Bir spatula ile dikkatlice kabarcıkları oluşturmak için özen çemberin ortasında numuneyi. </li><li> hemen ardından tekrar kullanılabilir yapıştırıcı üzerinde 40 mm yuvarlak cam kapak yerleştirin, doğrudan numune üzerine% 63 TDE başka 100 ul pipetle. </li><li> cam kapak örneği ile temas yapmış kadar indeksi, orta ve iki elin yüzük parmağı kullanarak, dikkatli dairenin çevresinde basın. </li><li> Yavaş yavaş bir yüzeye karşı duran dik konuma cam alt yemek getirmek Çözelti en küçük açıklığı ulaşana kadar daha sonra bir pipet yardımıyla% 63 TDE ekleyin. ücretsiz bir çözüm camına damla kalmaması pipet kapalı kuru silin bir tüy bırakmayan kullanın. odasında kabarcıkları önlemek için, pipet tüm yol boş asla. </li><li> içinküçük açma, daire içine ve kapalı bir odasını oluşturmak için silikon elastomer ekleyin. o görüntüleme önce kurumaları için 5 dakika bekleyin. </li></ol> 6. Mikroskopi Dikkat: konfokal, tek düzlem aydınlatma ve ışık levha mikroskopi kullanılan lazerler çok güçlü ve körlüğe neden olabilir. lazerler kullanan herhangi deneyler geniş eğitimden sonra ve büyük bir dikkatle ve uygun göz koruması ile yapılmalıdır. <ol><li> Bir lazer tarama konfokal mikroskop sahnede iç numune ile görüntüleme odasına yerleştirin. </li><li> görüntüleme yazılımı açın (Malzeme Tablo). Argon uyarma lazer açmak için, daha sonra programın lazer simgesinin üstündeki yapılandırma sekmesini tıklatın. lazer güç ve% 30 slayt ölçekli taşımak için argon yanındaki kutuyu işaretleyin. işaretçi yavaşça seçilen pozisyona ısınmak gözlemleyin. <ol><li> üstündeki acquire sekmesine dönün. Büyük kutusuna &quot;Işın Yolu Ayarları &amp;# 34; / Yük gitmek kutusu ayarı kaydetmek ve görüntü YFP (527 nm) için uygun bir dalga boyu kanalı seçmek için açılır menüyü seçin. </li></ol></li><li> kanal ayarları kutusunun altında: &quot;Mevcut nesne objektif&quot; etiketli kırmızı köprü bularak görüntüleme için uygun hedefleri seçin. sekmesini tıklayarak mevcut tüm hedefleri açılır ve bir seçim taret otomatik olarak dönecektir. <ol><li> Kullanım amaçları (örn., 10X), büyük bir çalışma mesafesi ile (doku kalınlığından daha büyük, yansıması için, örneğin 11 mm) ve geniş bir sayısal açıklık (örn., 0.3) düzlem resim çözünürlüğü en üst düzeye çıkarmak ve en aza indirmek için optik kesit kalınlığı. </li></ol></li><li> 1024 x 1,024 &quot;biçim&quot; denilen toplama matriks ayarlamak için pencereyi, ya da objektif lateral çözünürlük sınırı için uygun bir değer: &quot;çözünürlük XY&quot; açık, açılır menüler sol sütun üzerinde. (Mümkün olduğunca düşük yakınlaştırma faktörünü ayarlamak gibi., 10,7). </li><li> Aynı pencerede, buna göre bireysel olarak etiketli menüleri aşağı bırakarak 8 hat ortalama ve 1 çerçeve ortalama parametreleri ayarlayın. hızlı elde edilmesi için yüksek bir sinyal-gürültü oranı ve küçük değerler için büyük değerler kullanın. Not: 8 hat ortalamaları ve 1 kare ortalama yaklaşık 4 saat içinde bir fare beyin yarımkürede yüksek kaliteli görüntüler elde edecektir. </li><li> sahne kontrollerini kullanarak örnek bulun. temsili bir alan görünür sonra, kazanç ve ofset ayarlayın. kazanç ve doku tipi, fluorophores dayalı ölçüde değişecektir ofset ve / veya anatomik özellik görüntülü Not:. YFP için kazanç için 780 760 den ve -1.0 ofset için -1.5 değerleri ayarlayın. </li><li> &quot;Karo tarama&quot; seçin ve ilgi bölgenin sınırlarını belirleyin. z-yığının Set alt ve üst sınırları elde edilecek. Amaç optik bölüm çözünürlük sınırına dayalı optik bölümün kalınlığı ayarlayın. Not: Bir amaç enoptik bölüm çözünürlük sınırı öncelikle sayısal açıklık ile tanımlanır. otomatik olarak objektif bir değer uygun olan hesaplar en mikroskop kontrol yazılımı kullanılmaktadır. 0.3 sayısal diyafram ile 10X objektif için bu yaklaşık 11 mikron olacaktır. </li><li> edinimi başlatın. Not: Bu, birden çok saat sürebilir. </li><li> özel ilgi bölgelerinden daha yüksek büyütme hedefleri kullanarak yüksek çözünürlüklü görüntüler toplayın. </li></ol>

<ol>
	<li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+tomography:+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits.">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li><li>Kleinfeld, D., 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=Large-scale+automated+histology+in+the+pursuit+of+connectomes.">Large-scale automated histology in the pursuit of connectomes.</a> J Neurosci. 31, 16125-16138 (2011).</li><li>MacKenzie-Graham, 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=A+multimodal,+multidimensional+atlas+of+the+C57BL/6J+mouse+brain.">A multimodal, multidimensional atlas of the C57BL/6J mouse brain.</a> J Anat. 204, 93-102 (2004).</li><li>Denk, W., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+upmanship:+why+multiphoton+imaging+is+more+than+a+gimmick.">Photon upmanship: why multiphoton imaging is more than a gimmick.</a> Neuron. 18, 351-357 (1997).</li><li>Denk, W., 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=Anatomical+and+functional+imaging+of+neurons+using+2-photon+laser+scanning+microscopy.">Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.</a> J Neurosci Methods. 54, 151-162 (1994).</li><li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+magic:+multiphoton+microscopy+in+the+biosciences.">Nonlinear magic: multiphoton microscopy in the biosciences.</a> Nat Biotechnol. 21, 1369-1377 (2003).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Methods. 2, 932-940 (2005).</li><li>Theer, P., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+fundamental+imaging-depth+limit+in+two-photon+microscopy.">On the fundamental imaging-depth limit in two-photon microscopy.</a> J Opt Soc Am A Opt Image Sci. Vis. 23, 3139-3149 (2006).</li><li>Dodt, H. U., 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=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat Methods. 4, 331-336 (2007).</li><li>Erturk, 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=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat Protoc. 7, 1983-1995 (2012).</li><li>Hama, 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=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16, 1154-1161 (2013).</li><li>Susaki, E. 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=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</li><li>Chung, K., 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497, 332-337 (2013).</li><li>Spence, R. D., 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=Bringing+CLARITY+to+gray+matter+atrophy.">Bringing CLARITY to gray matter atrophy.</a> Neuroimage. 101, 625-632 (2014).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9, 1682-1697 (2014).</li><li>Yang, 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158, 945-958 (2014).</li><li>Costantini, I., 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+versatile+clearing+agent+for+multi-modal+brain+imaging.">A versatile clearing agent for multi-modal brain imaging.</a> Sci Rep. 5, 9808 (2015).</li><li>Zheng, H., Rinaman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+CLARITY+for+visualizing+immunofluorescence+labeling+in+the+developing+rat+brain.">Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain.</a> Brain Struct Funct. , (2015).</li><li>Parra, S. G., 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=Multiphoton+microscopy+of+cleared+mouse+brain+expressing+YFP.">Multiphoton microscopy of cleared mouse brain expressing YFP.</a> J Vis Exp. (67), e3848 (2012).</li><li>Weinger, J. G., 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=Two-photon+imaging+of+cellular+dynamics+in+the+mouse+spinal+cord.">Two-photon imaging of cellular dynamics in the mouse spinal cord.</a> J Viz Exp. (96), e52580 (2015).</li><li>Hama, 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=ScaleS:+an+optical+clearing+palette+for+biological+imaging.">ScaleS: an optical clearing palette for biological imaging.</a> Nat Neurosci. 18, 1518-1529 (2015).</li><li>Treweek, J. 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=Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping.">Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.</a> Nat Protoc. 10, 1860-1896 (2015).</li></ol>

The authors have nothing to disclose.

hidrojel gömülü dokularda (pasif CLARITY) pasif takas dokusunun büyük parçalar temizlenmesi için basit ve ucuz bir yöntemdir. Bu yaklaşım, özel ekipman gerektirmez ve kolay bir şekilde, sıcaklık kontrollü sallayıcı içinde gerçekleştirilebilir. Birkaç haftalık bir sürede içinde, örneğin bütün bir beyin ya da spinal kord bile büyük, çok miyelinli dokular, şeffaf ve mikroskopi için uygun olacaktır. Bu rapor, merkezi sinir sistemi doku temizleme odaklanmış olmasına rağmen, pasif AÇIKLI...

The ability to image complete neuroanatomical structures is immensely valuable for understanding the brain in health and disease. Traditionally, 3D imaging has required tissue sectioning to provide the axial resolution and to visualize deep anatomical structures. This approach can produce high-resolution data sets, but requires sophisticated image reconstruction techniques and is very labor intensive. As a result, it has been limited to the imaging of small volumes of tissue1-3. Optical sectioning, on the other hand, is well suited for the creation of high-resolution 3D images of fluorescently labeled tissues. Since optical sectioning is inherently three dimensional, it does not require extensive computation to produce a 3D image volume. However, light scattering and tissue opacity limit the depth at which tissues can be optically sectioned. The depth of imaging is limited to about 150 &#181;m in laser scanning confocal microscopy and to less than 800 &#181;m using two-photon excitation microscopy4-8.
In order to overcome these limitations, several optical clearing techniques have been recently developed and then further refined to permit the deep microscopic imaging of intact tissues. The use of benzyl alcohol and benzyl benzoate (BABB) to render fixed tissues transparent was among one of the earliest approaches9. However, this approach was limited by the quenching of fluorescence in the samples and incomplete clearing of highly myelinated structures10. Refinements of this technique, such as 3D Imaging of Solvent Cleared Organs (3DISCO), have led to very rapid and complete tissue clearing, but still suffer from rapid loss of fluorescent signals, especially yellow fluorescent protein (YFP)10. Water-based clearing solutions, such as Sca/e11 and SeeDB12, preserve fluorescent signals, but do not completely clear highly myelinated tissues. Clear, Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) is a promising new optical clearing technology that appears to overcome the limitations of previously developed water-based clearing solutions13. In contrast with other optical clearing techniques, Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY)14 embeds the brain in a porous matrix that provides structural integrity to proteins, nucleic acids and small molecules, while leaving lipids unbound. The lipids are then removed electrophoretically, culminating in an optically cleared brain that can be easily visualized and equally easily probed using commonly available techniques.
Electrophoretic tissue clearing (ETC) is used to remove lipids from hydrogel embedded tissues in CLARITY, however, ETC can be difficult to implement consistently and tissues subject to electrophoresis can exhibit tissue distortion, browning, loss of fluorescence and antibody reactivity. Passive lipid clearing avoids these limitations, but requires increased clearing times15-17. Passive clearing also permits the clearing of large numbers of samples in parallel, since it is not limited by the number of ETC apparatuses available. Decreasing the concentration of paraformaldehyde and excluding bis-acrylamide from the hydrogel have led to great increases in the speed of clearing at the expense of greater tissue expansion16. Less expensive alternatives to the refractive index matching solution used in the original CLARITY technique have been developed17-19. 2,2'-thiodiethanol (TDE) is an inexpensive and rapid brain clearing agent that reverses some of the tissue expansion that occurs during lipid removal18. This report incorporates passive clearing of hydrogel embedded tissue and the use of TDE as a refractive index matching solution to the original CLARITY technique to produce a highly reproducible and inexpensive protocol for the optical clearing of the mouse central nervous system.

<table border="0" cellpadding="0" cellspacing="0" width="750">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10X phosphate buffered saline (PBS)</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">BP399-1</td>
			<td style="width:203px;">buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32% paraformaldehyde (PFA)</td>
			<td style="width:212px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714-5</td>
			<td>perfusion and hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,2&#39;-thiodiethanol (TDE)</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">166782-500G</td>
			<td>refractive index matching solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40% acrylamide</td>
			<td style="width:212px;">Bio-Rad</td>
			<td style="width:119px;">161-0140</td>
			<td>hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2% bis-acrylamide</td>
			<td style="width:212px;">Bio-Rad</td>
			<td style="width:119px;">161-0142</td>
			<td>hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VA-044 initiator</td>
			<td>Wako Pure Chemical Industries, Ltd.</td>
			<td>VA044</td>
			<td>hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">boric acid</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">B7901</td>
			<td>clearing buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium dodecyl sulfate (SDS)</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">BP166-5</td>
			<td>clearing buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium hydroxide</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">55255-1</td>
			<td>buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium azide</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">52002-100G</td>
			<td>preservative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">triton x-100</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">X100-500G</td>
			<td>buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">heparin</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3393-50KU</td>
			<td>perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium nitrate</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">BP360-500</td>
			<td>perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:212px;">Molecular Probes</td>
			<td style="width:119px;">D1306</td>
			<td>nuclear stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">99.9% isoflurane</td>
			<td style="width:212px;">Phoenix</td>
			<td style="width:119px;">57319-559-06</td>
			<td>anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">hydrocholoric acid</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">A1445-500</td>
			<td>buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glass bottom dish</td>
			<td style="width:212px;">Willco</td>
			<td style="width:119px;">HBSB-5040</td>
			<td>Willco dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">reusable adhesive</td>
			<td style="width:212px;">Bostick</td>
			<td style="width:119px;">371351</td>
			<td>Blu-Tack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">silicon elastomer</td>
			<td style="width:212px;">World Precision Instruments</td>
			<td style="width:119px;">KWIK-CAST</td>
			<td>Kwik-Cast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lint-free wipe</td>
			<td style="width:212px;">Kimberly-Clark</td>
			<td style="width:119px;">34120</td>
			<td>KimWipe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse brain matrix</td>
			<td style="width:212px;">Roboz</td>
			<td style="width:119px;">SA-2175</td>
			<td>sectioning tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">matrix blades</td>
			<td style="width:212px;">Roboz</td>
			<td style="width:119px;">RS-9887</td>
			<td>sectioning tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">peristaltic pump</td>
			<td style="width:212px;">Cole-Parmer</td>
			<td style="width:119px;">77122-24</td>
			<td>pefusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser scanning confocal microscope</td>
			<td>Leica</td>
			<td>SP5</td>
			<td>microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imaging software</td>
			<td style="width:212px;">Leica</td>
			<td style="width:119px;">LAS AF</td>
			<td>microscopy</td>
		</tr>
	</tbody>
</table>

optical clearing of the mouse central nervous system using passive clarity

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Beynin yapısal organizasyonu görselleştirme morfolojisi ve bağlantı sağlık ve hastalık beyin fonksiyonlarını nasıl etkilediğini anlamak için kritik öneme sahiptir. Optik takas teknikleri bize cohesively morfolojisi ve bağlantı incelemek için izin sağlam dokularda 3D görüntü hücre popülasyonu için mümkün kılar. Hücreler beyindeki sinir bağlantı karmaşık bir desen ayrı kızdırmak için bir izin ...

Watch this Scientific Journal Video about Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY at JoVE.com

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Pasif kullanımı Netlik Fare Merkezi Sinir Sistemi Optik Takas

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

optical-clearing-mouse-central-nervous-system-using-passive

Research

JoVE Journal

Neuroscience

13.4K Görüntüleme.  University of California, Los Angeles. Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Video: Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

Fareler ve Merkezi Sinir Sisteminde Kök Hücre İmplantasyonu Multimodal Görüntüleme

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Nörobilim

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Merkezi sinir sisteminin transducing Hücreleri için lentiviral Vektörler Üretimi

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Merkez Sinir Sistemi Tek Hücreler Etiketleme<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Kemirgen Merkezi Sinir Sistemi İlaçların doğrudan Intraventriküler teslim

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

Kemirgen Santral Sinir Sistemi vivo optogenetic Stimülasyon içinde

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

Roman pasif optik şeffaflık bütün sinir dokusunda hızlı üretim için yöntemleri Temizleme

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Üçüncü-biçim Drosophila Melanogaster merkezi sinir sisteminin etkinliğinin elektrofizyolojik kayıt

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Erken Doğum Sonrası Fare Beyinlerine Kemogenetik Mühendislik Kortikal İnkornöron Progenitorlarının Nakli

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

Sıçan Merkezi Sinir Sisteminde Antisense Oligonükleotidlerin İntinal Teslimi

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

Beş Omurgalı Grubu Arasında Merkezi Sinir Sistemi Mikrodamarlarının Güvenilir İzolasyon

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...