This work was supported by the Australian Research Council Centre of Excellence for Integrative Brain Function (ARC Centre Grant CE140100007), an NHMRC project grant (#1086643). Prof. George Paxinos is supported by a Senior Principal Research Fellow NHMRC grant (#1043626).

Etik Beyanı: Hayvan denekleri tüm işlemler (onaylı ACEC numarası 14 / 94A ise) New South Wales Üniversitesi&#39;nde Hayvan Bakım ve Etik Komitesi (ACEC) yönergeleri izleyin. Şeffaf Fare Omurilik 1. Hazırlık <ol><li> Buz Hidrojel Çözüm hazırlanması <ol><li> % 16 paraformaldehit solüsyonunun hazırlanması (PFA) <ol><li> 70 ml önceden ısıtılmış damıtılmış su (50-55 ° C) içine 16 g paraformaldehit tozu eklenir ve paraformaldehit çözünene kadar ısıtılmış bir manyetik karıştırıcı üzerinde karıştırılmıştır. Not: çözüm üzerinde 55 ° C ısı ve paraformaldehit zehirli olduğunu unutmayın izin vermeyin. </li><li> bir silindir paraformaldehit çözeltisini ve 100 ml damıtılmış su ilave edilir. 4 ° C buzdolabı kullanarak paraformaldehit çözümü soğutun. </li></ol></li><li> su distile 26.25 ml 125 mg VA-044 başlatıcı eritin ve 4 çözümü serinC buzdolabı °. </li><li> Serin 5 mi akrilamid çözeltisi (% 40), 1.25 ml bis çözeltisi (% 2) (DİKKAT: Bis ve akrilamid çözeltiler genetik bozukluklar veya kanser neden olabilir, toksik), 5 mi 10x PBS, 12.5 mi% 16 PFA çözeltisi ve buz üzerinde VA-044 başlatıcı çözeltisi. </li><li> buz üzerinde yukarıdaki çözümler karıştırın. Not: toplam hacim 50 ml. </li></ol></li><li> Fare Omurilik Perfüzyon ve Koleksiyon <ol><li> % 0.9 normal serum fizyolojik içinde seyreltildi ketamin (80 mg / kg) ve xyzaline (5 mg / kg) intraperitonal enjeksiyonu ile fare anestezi uygulayın. Fare dozaj fareler için daha düşük olabilir. </li><li> Fare ayak derisine bir tutam yanıt vermiyor bir kez davlumbaz uygun bir plastik yüzeyde, (yapışkan bant etkilidir) vücuttan uzak fare uzuvlarını düzeltmek. </li><li> göğüs üzerine fare deri kesme ve daha sonra kemik göğüs makas ile kalp maruz. </li><li> 25 G iğne th bağlanmış olan, bir peristaltik pompa kullanılarakboru e ucu, kalbin sol ventrikül içine iğneyi yaklaşık olarak 10 ml / dk bir oranda kan için bir çıkış noktası ve daha sonra 40 ml% 0.9 normal serum fizyolojik ile yıkanarak başlatmak için sağ atrium makasla kesme. </li><li> Tamamlandığında, 35 ml buzlu soğuk hidrojel solüsyonu ile fare serpmek. Not: 10 ml&#39;lik bir hızda serpmek / dakika sinir dokusunun şişmesini önlemek için. </li><li> Bir bıçak ile arka üzerindeki deriyi insizyon ve daha sonra omurgalı yanındaki kasları kaldırın. Bir tarafta vertebra kemerler kesme ve diğer tarafa, onları saygısız sonra, ince bir makas ile vertebral kolonun dışına omurilik alır. </li></ol></li><li> Fare Omurilik Takas <ol><li> gece boyunca 4 ° C &#39;de hidrojel çözeltisi 15 ml omurilik yerleştirin. </li><li> Hidrojel çözeltisi 10 ml çıkarın ve 5 ml tüp omurilik segmentleri ile çözeltinin geri kalanı dökün. tamamen dolana kadar hidrojel çözeltisi ile 5 ml tüp doldurun. </li><li> 5 ml tüp üstünden parafilm küçük bir parça gerin ve sonra tüpün boynuna parafilm sarın. Not: parafilm kontaklar hidrojel çözümü sağlayın ve hidrojel çözüm ve parafilm arasında hiçbir kabarcıkları vardır. </li><li> 37 5 ml tüp koymak &#160; ° C fırın gece boyunca karıştırılmıştır. Bir jel olana dek hidrojel çözümü gözlemleyin. </li><li> fırın dışına 5 ml tüpü alın ve (örneğin bir shoveled 1 ml&#39;lik pipet gibi) bir anahtarla tüpten jel çıkarın. jele kaba doku yapışmasını tarafından omurilik dokusu jel çıkarın ve sonra (jel dokusuna sadık olacaktır ve bu nedenle doku tarafından silinecektir) uzakta kaba doku alır. </li><li> omurilik dokusu jelin çıkarılmasından sonra, bir çalkalayıcı üzerinde 24 saat boyunca 1 x PBS (pH 7.4) ile Omurilik 4 kez yıkayın. </li><li> 3.85 gr üre ve 3.85 g N, N, N &#39;, N&#39;-tetrakis- (2-hidroksipropil), etilendiamintetraasetat eritilmesi kübik temizleme çözeltisi hazırlayın5.38 ml ine damıtılmış su. Not: Bir sıcak karıştırıcı kullanılır. açık ve oda sıcaklığına kadar soğutulduktan sonra solüsyona 2.31 g polietilen glikol mono-p-isooctylphenyl eter / Triton X-100 ilave edin. Not: Bu, üç kimyasal 10 g bir fare için geçerlidir. </li><li> bir jilet ile 2-3 mm uzunluğunda bölümler halinde koronal fare omurilik kesin ve yukarıda KÜP temizleme çözeltisi, 5 ml içine koydu. 37 ° C&#39;de bir fırında, bir karıştırıcı üzerine yerleştirin &#160; 3 gün boyunca ° C. </li><li> Üç gün sonra, yeni bir birine takas çözümü değiştirin. Not: Yukarıdaki çözelti, kullanımdan önce hazırlanmıştır. </li><li> İki üç gün sonra KÜBİK takas çözümü ferahlatıcı sonra, yazı 8 harflerle bir kağıda karşı doku şeffaflık kontrol edin. şeffaf ise, harfler temizlenir doku yoluyla görülebilir. (% 0.1 Triton-X100) Doku günde 4 kez (her 6 saatte bir) yıkamak için temizleme çözeltisini çıkarın ve PBST 4 ml. </li></ol></li></ol><ol><li> 37 ° C fırın içinde bir karıştırıcı üzerinde 3 gün: Primer antikor solüsyonu omurilik segmentleri inkübe (PBST içinde 100, anti-serotonin, tavşan içinde yetiştirilmiş, 1 seyreltilmiş). </li><li> Antikor çözeltisi çıkarın ve PBST 4 ml (% 0.1 Triton-X100) 37 ° C bir fırında spinal kord günde 4 kez (her 6 saat) yıkayın. </li><li> 37 ° C fırın içinde bir karıştırıcı üzerinde, 3 gün boyunca doku inkübe etmek: PBST solüsyonu çıkarın ve ikincil antikor solüsyonu (PBST içinde 100 594 konjuge keçi anti-tavşan IgG, 1 seyreltilmiş). </li><li> ikincil antikor çözeltisi çıkarın ve 37 ° C fırın içinde bir karıştırıcı üzerinde günde spinal kord 4 kez yıkama (her 6 saatte bir) PBST 4 ml. Ertesi gün doku görüntüleme için hazırdır. </li></ol> 3. Görüntüleme <ol><li> PBST solüsyonu çıkartın ve doku kırılma indeksi yapmak için% 85 gliserol 4 ml. </li><li&gt; doku netlik kontrol edin. yaklaşık 1 saat açık olduğunda, çoklu fotoğraf floresan mikroskop tutma çerçevesini uyan bir büyük, ince cam slayt üzerine doku koydu. </li><li> Omurilik dokusu yanında% 85 gliserol birkaç damla koyun ve bir 22 x 50 mm, 2 lamel camının ile lamel. Not: omurilik oryantasyon görüntü benziyor nasıl karar verir. </li><li> çoklu foton floresan mikroskop tutma çerçevesi üzerine cam slayt koyun ve hafif yolun içine doku taşıyın. </li><li> Helyum Neon lazer hattı 594 nm seçin ve canlı görüntüde olumlu bir sinyal parlaklığını kontrol ederek optimum seviyeye lazer yoğunluğunu ayarlayın. </li><li> (Suda, NA 0.7) 20X objektif kullanılarak omurilik dokusu tarama alanı seçin ve z-yığını oluşturmak için hazırlamak, ve 3 mikron ayarlanmış her adımda derinliği. </li><li> 20X objec altında z yığının alt üst doku tarayıntif ve (yağda, NA 1.4) sonra 63X objektif sırasıyla (adım büyüklüğü 1 mikron idi). 3D yazılımını 28 kullanarak 3D videoları yeniden yapılandırma. </li></ol>

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The authors have nothing to disclose.

protokol kombine CLARITY ve KÜP teknikleri ile fare omurilik görüntü serotonerjik lifleri nasıl gösterir nitelendirdi. Bu Cheung vd. 14 ve TÖMER&#39;ler ve ark., 15 tarafından geliştirilen pasif temizleme protokolüne göre daha hızlı bir temizleme işlemi sunar ve omurilik dokusu de temizleme esnasında hidrojel tarafından desteklenmesi sağlar. Cheung ve ark., 14 ve Tomer ark. 15 tarafından bildirilen fare omurilik sabitlenmesi sırasında önemli bir adım, tüm çözümler kimyasalların polimerizasyonu engelleyen buz üzerinde serin tutmaktır. Diğer önemli bir adım sinir dokusunun şişmesini önlemek için hidrojel çözeltisi yavaşça fare serpmek için. hidrojel çözüm jel olabilmesi için, gaz alma gereklidir. Buna alternatif olarak, hyd arasında hava kabarcıkları tüpünün üst kapak parafilm kullanılır ve solRogel çözeltisi ve Parafilm. Bu çok iyi çalıştı ve hidrojel çözüm birkaç saat sonra bir jel haline geldi. Bazı kabarcıklar jel bulundu rağmen, omurilik dokusu içinde kabarcıkların olmaması ile ortaya konmuştur, aşağıdaki deneylerde, müdahale etmedi. Takas en kritik adımdır ve reaktif bir arada gerektirir. Amino-alkol içeren temizleme çözümü, orijinal CLARITY protokolü 16 kullanılan SDS / borik asit bazlı temizleme solüsyonu çok daha hızlı omurilik dokusu lipid kaldırır. Geleneksel immünohistokimyasal tekniklerle üzerinde CLARITY ve CUBIC avantajı tüm doku bloğu bir sonucu olarak, serotonerjik liflerin süreklilik korunur, doku kesit olmadan görüntülü olabilir ki, ve uzun bir mesafeye aynı fiber takip etmek mümkündür z-yığınının içinde. Bu avantajla, teminat güvenle 3D görüntülerle tespit edilebilir, birND, yeni sonuçlar dolayısıyla serotonin yolaklarının doğası hakkında yapılabilir. açıklanan protokol sadece spesifik antikorlar ile doku etiketleme yoluyla lif sistemleri yanı sıra, çekirdeklerin araştırılması için uygun bir araçtır. Bu teknik aynı zamanda ikili ya da üçlü etiketleme kullanarak aynı fare beyninde farklı sistemleri sorgulamak için ideal bir seçimdir. Bizim laboratuvar ortamında, bir çoklu foton floresan mikroskop yaklaşık 300 mikron bir çalışma mesafesi ile kullanılabilir. doku her iki tarafı da taranır, bu 600 um&#39;lik bir en üst düzeye görseli sınırlar. Ancak, hafif bir tabaka floresan mikroskop, tam genişlik, uzunluk ve derinliği ile görüntü tüm fare beyni ve omuriliği yapabilirsiniz. Bu tekniğin diğer bir avantajı antikorlar elüte edilebilir ve daha sonra başka bir farklı antikor veya antikorlar, aynı doku tekrar uygulanmıştır olmasıdır. Bu hayvan kullanımını önemli ölçüde yanı sıra yeni beyin dokusunu hazırlanması ile ilgili maliyetlerini azaltırHer bir deney için. Aynı nedenle, bu teknik, diğer dokulara tatbik edilebilir. Zaten akciğerler, böbrekler, kalp, bağırsak, ve tümör dokusu 26,27 üzerinde yapılan çalışmalar olmuştur. Ayrıca bu tekniğin sınırlamalar vardır, örneğin, fare Beyin içinde rafe çekirdekleri GABAerjik nöronlar da dahil olmak üzere nöronların çok çeşitli içerir. AÇIKLIK ve CUBIC tek bir fare beyin ve omurilikte raphespinal liflerin farklı tiplerine göre dağılım çalışma için izin verecektir rağmen. onların nörotransmitter veya protein belirteçler tarafından özel olarak tespit edilmemiş olanlar nöronlar, bu ortamda tespit edilemez. Alternatif bir tür, in situ melezleme gibi nükleik asit probları ile belirli bir nöron veya lif sistemleri nitelemektedir. Bu etiketleme sistemi ve flüoresan görüntüleme için uygun filtreler ile sınırlı Öte yandan, bir. tüm raphespinal lifleri Görüntüleme nedenle bir sorun olmaya devam etmektedir.Bu teknikler, bir anterograde izleyici intrakranial enjeksiyonu ile kombine olsaydı (örneğin Phaseolus vulgaris leucoagglutinin olarak - PHA-L), serotonerjik lifler, GABAerjik lifleri ve diğerleri görmek mümkün olabilir.

Supraspinal projections are responsible for the modulation of diverse behaviors such as pain perception. One of the projections carrying nociceptive information contains serotoninergic fibers, which originate from the hindbrain raphe and adjacent reticular nuclei1,2. Physiological and pharmacological studies have demonstrated an increased release of serotonin in the dorsal horn of the spinal cord after electrical stimulation of the raphe nuclei in the hindbrain3-5. In the rat and opossum, serotonergic raphespinal fibers have dense terminals, not only in the dorsal horn6-8, but also in the intermediate zone7,9,10, the ventral horn7,11, and even lamina 1012,13. There are no similar studies in the mouse. The present study aimed to map the termination pattern of serotonergic fibers arising from the hindbrain raphe nuclei and their adjacent reticular nuclei in the mouse spinal cord using the recently published CLARITY14 method and its modification - CUBIC15. 
Conventional fluorescence or peroxidase immunohistochemistry of the spinal cord clearly shows the distribution of serotonergic fibers in the gray matter of the spinal cord in 30-40 &#181;m thick cross-sections. However, this approach does not show the continuity of the serotonergic fiber tracts in the white matter and their collaterals in the gray matter. Although the 3D reconstruction of histological sections has advanced our knowledge of fiber tracts, it remains a challenge for histologists and anatomists to follow a single tract due to small distortions in the tissue caused by cutting. To circumvent this obstacle a number of researchers have developed various protocols for making the whole tissue structure transparent, and collecting an image of unaltered tissue in a single video file17-21. So far, the clear, lipid-exchanged, acrylamide-hybridized rigid, imaging/ immunostaining compatible, tissue hydrogel (CLARITY) technique, developed by Deisseroth's group14,15, as well as CUBIC, developed by Susaki et al16 are the most successful. Since the publication of the protocols, many researchers have started using these techniques to investigate various aspects of biological tissues, including, not only the brain22-25, but also the heart, kidneys, intestine, and the lungs26,27. 
By fixing the mouse spinal cord with the hydrogel solution (CLARITY) and clearing with the CUBIC reagents (which is a much faster method than that described by the original CLARITY protocol14,15), a spinal cord tissue block of 2-3 mm long was cleared within two weeks and immunofluorescence staining for serotonin completed in eight days. With just a combination of chemical agents, conventional immunohistochemistry can be used to create an image of individual fiber tracts in a 3D video file in approximately one month.

<table><tbody><tr><td>Photoinitiator VA044</td><td>Wako</td><td>va-044/225-02111</td><td>http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td></tr><tr><td>40% acrylamide solution</td><td>Bio Rad</td><td>161-0140</td><td>http://www.bio-rad.com/en-au/sku/161-0140-40-acrylamide-solution</td></tr><tr><td>2% Bis Solution</td><td>Bio Rad</td><td>161-0142</td><td>http://www.bio-rad.com/en-au/sku/161-0142-2-bis-solution?parentCategoryGUID=5e7a4f31-&lt;br/&gt;879c-4d63-ba0b-82556a0ccf1d</td></tr><tr><td>paraformaldehyde</td><td>Sigma</td><td>158127</td><td>http://www.sigmaaldrich.com/catalog/product/sial/158127?lang=en&amp;amp;region=AU</td></tr><tr><td>urea</td><td>Merck Millipore</td><td>66612</td><td>http://www.merckmillipore.com/AU/en/product/Urea---CAS-57-13-6---Calbiochem,EMD_BIO-66612</td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetrakis (2-hydroxypropyl) ethylenediamine</td><td>Merck Millipore</td><td>821940</td><td>http://www.merckmillipore.com/AU/en/product/Ethylenediamine-N,N,N&#039;,N&#039;-tetra-2-propanol,MDA_CHEM-821940</td></tr><tr><td>Triton-X 100</td><td>Merck Millipore</td><td>648462</td><td>http://www.merckmillipore.com/AU/en/product/TRITON&amp;reg;-X-100-Detergent---CAS-9002-93-1---Calbiochem,EMD_BIO-648462</td></tr><tr><td>sucrose</td><td>Sigma</td><td>S0389</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=en&amp;amp;region=AU</td></tr><tr><td>serotonin antibody</td><td>Merck Millipore</td><td>AB938</td><td>http://www.merckmillipore.com/AU/en/product/Anti-Serotonin-Antibody,MM_NF-AB938</td></tr><tr><td>goat anti rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&amp;reg; 594 conjugate</td><td>Life Technologies</td><td>&amp;nbsp;A-11012</td><td>https://www.lifetechnologies.com/order/genome-database/antibody/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/A-11012</td></tr><tr><td>multi-photon microscope</td><td>Leica</td><td>Leica TCS SP5 MP STED</td><td>http://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp5-mp/</td></tr></tbody></table>

imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique

omuriliğe uzun inen lifler lokomosyon, ağrının algılanması ve diğer davranışları için çok önemlidir. Bu lif sistemlerinin çoğunluğunun omurilik fiber sonlandırma desen iyice herhangi türde araştırılmamıştır. omurilik proje serotonerjik lifler, histolojik bölümlerde sıçanlar ve opossum incelenmiştir ve fonksiyonel önemi omurilikte kendi fiber sonlandırma desen çıkarılabilir dayanmaktadır. CLARITY ve KÜBİK tekniklerinin gelişmesiyle birlikte, lif sistemi ve serotonerjik supraspinal yolların önceden bilinmeyen özelliklerini ortaya çıkarmak için büyük olasılıkla omurilik dağıtımını, araştırmak mümkündür. Burada, kombine açıklık ve KÜP teknikleri kullanarak fare omurilik serotonerjik lifler görüntüleme için ayrıntılı bir protokol sağlar. yöntem combin ile doku hidrojel solüsyonu ve aydınlatılması ile bir fare perfüzyon gerektirirreaktifler temizleme tirme. Omurilik dokusu hemen altında iki hafta içinde temizlendi ve serotonin karşı sonraki immunofluorescent boyama az on gün içinde tamamlanmıştır. Bir çoklu foton floresan mikroskop ile, doku taranmış ve bir 3D görüntü OsiriX yazılımı kullanılarak yeniden inşa edildi.

Bu bölüm, açıklık ve KÜP protokollerin bir kombinasyonu kullanılarak şeffaf bir fare omurilik serotonin antikor boyama sonuçlarını gösterir. Biz (aynı zamanda Video 1 bakınız Şekil 1) serotonerjik lifler ventral boynuz ventral kısmında üstünlüğü ile omuriliğin tüm lamina mevcut olduğunu göstermektedir. kontrol doku pozitif lifler (sonuç gösterilen değil) yoktu. Ventral boynuz olarak, yoğun dolu serotonerjik lifler ventral boynuz ventromedial kısmında bulunur ve bunlar ventral boynuz lateral kısmına doğru uzanan (Şekil 2, aynı zamanda video 2&#39;ye bakınız). Bazı immünopozitif lifler de dorsal boynuz veya merkezi kanalına doğru ventral boynuz uzanır. Immünopozitif lifler dorsal boynuzu tüm tabakalar içinde mevcut olan fakat pozitif lifler arasında küçük bir boşluk, özellikle yanal olarak, tabakanın 2 ve 4&#39;de olduğudorsal boynuz parçası. Dorsal boynuz serotonerjik liflerin çoğunluğu, küçük çaplı ve bunların sadece küçük bir büyük çaplı (Video 1) vardır. yatay bölümünde, ventral boynuzda yoğun şekilde paketlenmiş serotonerjik lifler, omurilikte uzunlamasına ekseni boyunca ve geniş bir elyaf demeti oluşturmak için gözlenmiştir. En ilginç bulgu bu büyük elyaf demeti elyaf demeti dik düzenli uzunlamasına eksen boyunca dalları, sorunları olmasıdır. Bu dallar daha ventral boynuz lateral kısmına kendi yolları boyunca collateralize. Bu dalları ile karşılaştırıldığında, orta hatta doğru uzanan olanlar düzensiz ve daha küçük (Şekil 2). 63x objektif altında, arka boynuzda serotonerjik lifler omurilik ekseni boyunca ve sistemin yol boyunca çeşitli noktalarda sonlandırmak için gözlenmiştir. Kalın lifler düzensiz ince f arasında dağıtılır<html> Ibers (Şekil 3, aynı zamanda video 3). <img alt="Şekil 1" src="/files/ftp_upload/53673/53673fig1.jpg" /> Şekil 1:. 200X büyütme Sol Lomber kablosunun bir koronal bölümünde serotonin lifler sağ dorsal boynuz, dorsal spinal kord medial parçası olduğunu ve ventral omurilik lateral parçasıdır ventral boynuz olduğunu. Ölçek çubuğu 100 mm. Serotonerjik lifler özellikle lif yoğunluğu yüksek ventral boynuz ventromedial bölümünde, tüm lamina bulunur. Bu lifler ventral boynuz lateral ve merkezi kanal veya dorsal boynuz hem doğru uzanır. Diğer tabakanın lif yoğunluğu düşüktür. Serotonerjik liflerin çoğunluğu incedir. Sadece bir lif az sayıda kalın ve dorsal boynuzu ve ventral boynuz (her ikisi de görülür&gt; Ayrıca video 1&#39;e bakınız) (Sağ indirmek için tıklayın). <img alt="şekil 2" src="/files/ftp_upload/53673/53673fig2.jpg" /> Şekil 2:. Ventral 200X büyütme Sol ventral boynuz kısmı ve sağ serotonin lifler, omurilikte lateral tarafı, sırt omuriliğin rostral parçası olduğunu ve ventral omurilik kuyruk kısmıdır. Ölçek çubuğu 100 mm. Serotonerjik lifler, omurilikte uzunlamasına ekseni boyunca hareket eden gösterilen ve kalın bir elyaf demeti oluşturur Karın boynuzuna mediyal kısmından paketlenir. orta hatta doğru uzanan, diğerleri düzensiz ve kısa iken bu paket dışında, bazı dalları, ventral boynuz lateral kısmına doğru düzenli aralıklarla genişletilmiştir. Bu lifler yolu yol boyunca birçok noktada sonlandırmak ( <a href="https://www.jove.com/files/ftp_upload/53673/Video2.mp4">ayrıca video 2 bakınız</a>) (Sağ indirmek için tıklayın). <img alt="Şekil 3," src="/files/ftp_upload/53673/53673fig3.jpg" /> Şekil 3: 630X büyütmede altında dorsal boynuzunda serotonin lifler Sol sağ omurilik, dorsal ve ventral sırasıyla dorsal ve dorsal boynuz ventral kısmına bakınız medial parçası, omurilik lateral parçasıdır. . Ölçek çubuğu 7 mm. Serotonerjik lifler, omurilikte uzunlamasına ekseni boyunca hareket eden ve sistem yolu boyunca uzanarak. Bu lifler içe kalın liflerin küçük bir sayı ile özellikle küçük çapa sahiptirler. Bu lifler nadiren birbiriyle örtüşen ( <a href="https://www.jove.com/files/ftp_upload/53673/Video3.mp4">aynı zamanda video 3&#39;e bakınız (Sağ) indirmek için tıklayınız</a> ).

Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Kullanımı Netlik Fare Omurilik içinde Serotonerjik Elyaf Görüntüleme / KÜBİK Tekniği

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

imaging-serotonergic-fibers-mouse-spinal-cord-using-claritycubic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.5K Görüntüleme.  Neuroscience Research Australia. Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Video: Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Fare Omurilik İki foton Mikroskop kullanarak in vivo Görüntüleme

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Nörobilim

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

Görüntüleme 3DISCO ile Hücresel Düzeyde Sağlam Biyolojik Sistemleri Temizlendi

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Boyuna için bir Spinal Odası implantasyon için bir Prosedürü<em&gt; İn Vivo</em&gt; Fare Omurilik Görüntüleme

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Fare Omurilik Hücresel Dynamics İki foton Görüntüleme

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

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.

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 ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

CLARITY Doku Temizleme Yöntemi ile Tüm Mount Retinaların İmmünasyonunun İmmünasyon

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

CLARITY Doku Temizleme Yoluyla Bozulmamış Nöronal Dendritlerin Görüntülenmesi ve Nicelleştirilmesi

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

Mezoskopikten Mikroskobik Ölçeklere Nöronal Görüntüleme için Bir Doku Temizleme Yöntemi

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Minimal İnvaziv Bir Tekniğe Dayalı Bir Fare Kontüzyonu Omurilik Yaralanması Modelinin Oluşturulması

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

<meta charset="utf8"></head><body>Bağırsakları "NETLİK" ile Görüntülemek

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

Bağırsakları "NETLİK" ile Görüntülemek

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...