Name: imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique
Uploaded: 2016-02-26T14:00:00.000+00:00
Duration: 9 min 54 s
Description: Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

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).

윤리 문 : 동물 과목을 포함하는 모든 절차 (승인 ACEC 번호 14 / 94A입니다) 뉴 사우스 웨일즈 대학의 동물 관리 및 윤리위원회 (ACEC)의 지침을 따르십시오. 투명 마우스 척수 1. 준비 <ol><li> 아이스 콜드 하이드로 겔 용액의 조제 <ol><li> 16 % 파라 포름 알데히드 용액의 조제 (PFA) <ol><li> 70 ml의 미리 예열 증류수 (50-55 °에 C)에 16g 파라 포름 알데히드 분말을 추가하고 파라 포름 알데히드가 용해 될 때까지 가열 자석 교반기에서 교반한다. 참고 :이 솔루션은 이상 55 ° C 열 및 파라 포름 알데히드는 독성이 알고 있어야하는 것을 허용하지 않습니다. </li><li> 실린더에 파라 포름 알데히드 용액을 전송하고 100 ㎖에 증류수를 추가합니다. 4 ℃의 냉장고를 사용하여 파라 포름 알데히드 용액을 냉각. </li></ol></li><li> 증류수 26.25 mL에 125 mg의 VA-044 개시제를 용해 4 용액을 냉각C의 냉장고 °. </li><li> 차가운 5 ㎖ 아크릴 아미드 수용액 (40 %) 1.25 mL의 비스 용액 (2 %) (주의 : 비스 아크릴 아미드 솔루션은 유전 적 결함이나 암을 유발할 수 있고, 독성), 5 ㎖의 10 배 PBS, 12.5 ml의 16 % PFA 용액 및 얼음 VA-044의 개시제 용액. </li><li> 얼음 위의 솔루션을 섞는다. 주 : 전량을 50 ㎖이다. </li></ol></li><li> 마우스 척수의 관류 및 컬렉션 <ol><li> 0.9 % 생리 식염수에 희석 케타민 (80 ㎎ / ㎏) 및 xyzaline (5 ㎎ / ㎏)의 복강 내 주사로 마우스를 마취. 마우스의 투여 량은 생쥐의 경우보다 더 낮을 수있다. </li><li> 마우스가 다리의 피부에 핀치에 응답하지 않습니다 일단 흄 후드에 적합한 플라스틱 표면에, (접착 테이프가 효과가있다) 떨어져 몸에서 마우스의 사지를 고정합니다. </li><li> 가슴 위에 마우스 피부를 잘라 후 뼈 가슴은 가위로 가슴을 노출. </li><li> 25 G 바늘 토륨에 부착하여, 연동 펌프를 사용하여튜브의 E 단부는 심장의 좌심실로 니들을 삽입 약 10 ㎖ / 분의 속도를 피 종료점을 만든 다음 40 ml의 0.9 % 생리 식염수로 세정을 시작 우심방 자르지. </li><li> 완료되면, 35 ml의 차가운 얼음 하이드로 겔 용액으로 마우스를 관류. 참고 : 10 ML의 속도로 관류 / 분 신경 조직의 부종을 방지 할 수 있습니다. </li><li> 블레이드와 뒷면에 피부를 절개하고 척추 옆 근육을 제거합니다. 일측에 척추 아치를 절단하고, 다른 쪽에게 튀기는 후 미세 시저와 척주에서 척수를 취할. </li></ol></li><li> 마우스 척수 지우기 <ol><li> 밤새 4 ℃에서 하이드로 겔 용액을 15 ml의에서 척수를 놓습니다. </li><li> 겔 용액 10 ㎖를 제거하고 5 ㎖ 튜브에 척수 세그먼트 용액의 나머지를 붓는다. 그것이 완전히 찰 때까지 겔 용액 5 ㎖ 튜브를 채우기. </li><li> 5 ㎖ 튜브의 맨 위에 파라 필름의 작은 조각을 스트레칭 후 튜브 목 파라 필름 랩. 참고 : 파라 필름 접촉을 하이드로 겔 솔루션을 확인하고 하이드로 겔 용액과 파라 필름 사이에 거품이 없습니다. </li><li> 37 년 5 ML 튜브를 넣어 &#160; ° C 오븐에서 밤새. 이 겔하게까지 하이드로 겔 솔루션을 관찰한다. </li><li> 오븐에서 5 ML 튜브를 타고 (예 : 삽 1 ML의 피펫 팁 등) 스패너와 튜브에서 젤을 제거합니다. 겔에 거친 조직을 고집하여 척수 조직에서 젤을 제거하고 (겔 조직에 충실하고, 따라서 조직에 의해 제거됩니다) 떨어진 거친 조직을. </li><li> 척수 조직으로부터 겔을 제거 후, ​​쉐이커상에서 24 시간 동안 1X PBS (PH 7.4)와 척수 4 회 반복한다. </li><li> 3.85 g의 우레아 및 385 g의 N, N, N &#39;, N&#39;- 테트라 키스 (2- 히드 록시 프로필) ethylenediam 용해 CUBIC 클리어 용액을 준비5.38 용액에 오프라인은 증류수. 주 : 뜨거운 교반기가 사용됩니다. 분명히하고 실온까지 냉각 후, 용액에 2.31 g의 폴리에틸렌 글리콜 모노 메틸 -p- isooctylphenyl 에테르 / 트리톤 X-100을 추가한다. 참고 :이 세 가지 화학 물질 10 g을 하나의 마우스입니다. </li><li> 면도날 2-3 mm 길이 세그먼트로 coronally 마우스 척수를 잘라 위의 CUBIC 청소 용액 5 ㎖로했습니다. 37 ℃ 오븐에서 진탕 놓고 &#160; 3 일간 C를 °. </li><li> 3 일 후 새로운 하나에 청소 솔루션을 변경합니다. 주 : 상기 용액은 사용 전에 제조한다. </li><li> 2 ~ 3 일 후에 CUBIC 청소 솔루션을 새로 고침 후, 글꼴 8 문자 용지에 조직의 투명성을 확인합니다. 이 투명한 경우, 글자 삭제 조직을 통해 알 수있다. (0.1 % 트리톤-X100)는 조직 4 회 (6 시간마다)을 씻어 청소 용액을 제거하고 PBST의 4를 가하여. </li></ol></li></ol><ol><li> 37 ° C 오븐에서 진탕 기에서 3 일 : 차 항체 용액에서 척수 세그먼트를 품어 (PBST 100 항 세로토닌은 토끼에서 제기, 1 희석). </li><li> 항체 용액을 제거하고 4 PBST를 가하여 (0.1 % 트리톤 X100)는 37 ° C의 오븐에서 척수 세그먼트 4 회 (매 6 시간)을 씻어. </li><li> 37 ° C 오븐에서 진탕 기에서 3 일간 조직을 배양 : PBST 용액을 제거하고 이차 항체 솔루션을 추가 (PBST 100 594 복합 염소 항 - 토끼 IgG의를 1 희석). </li><li> 이차 항체 용액을 제거하고 37 ° C 오븐에서 통에 하루 척수 세그먼트 4 번 씻어 (6 시간마다) PBST 4를 가하여. 다음날 조직 이미징을위한 준비가되어 있습니다. </li></ol> 3. 이미지 <ol><li> PBST 용액을 제거하고, 심지어 조직의 굴절률을 85 % 글리세롤 (4)를 가하여. </li><l난&gt; 조직의 선명도를 확인합니다. 그것은 약 1 시간 분명되면 멀티 포토 형광 현미경 유지 프레임 맞는 크고 얇은 유리 슬라이드 상 조직을 넣어. </li><li> 척수 조직 옆에 85 % 글리세롤을 몇 방울을 넣어 22 X 50mm 유리 커버 슬립이 그것을 커버 슬립. 참고 : 척수의 방향은 이미지가 어떻게 보이는지를 결정한다. </li><li> 다중 광자 형광 현미경의 유지 프레임에 유리 슬라이드를 넣고 빛 경로로 조직을 이동합니다. </li><li> 헬륨 네온 레이저 라인 594 nm의 선택 및 라이브 화상에 양 신호의 밝기를 검사하여 최적의 레벨로 레이저의 세기를 조절한다. </li><li> (물, NA 0.7) 20X 대물 렌즈를 이용하여 척수 조직의 스캔 영역을 선택하고, Z 스택을 만들 준비하고,이 3 μm로 설정된 각 단계의 깊이. </li><li> 20X 미만의 objec Z 스택의 상단부터 하단까지의 조직을 검사적인하고 (오일 중, NA 1.4)를 63X 대물 각각 (스텝 크기는 1 ㎛였다). 3D 소프트웨어 (28)를 이용하여 3 차원 영상을 재구성한다. </li></ol>

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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>Ando, 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=Inside+Alzheimer+brain+with+CLARITY:+senile+plaques,+neurofibrillary+tangles+and+axons+in+3-D.">Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.</a> Acta Neuropathol. 128 (3), 457-459 (2014).</li><li>Zhang, 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>Lee, H., Park, J. 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H., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+application+of+the+electrophoretic+tissue+clearing+technology,+CLARITY,+to+intact+solid+organs+including+brain,+pancreas,+liver,+kidney,+lung,+and+intestine.">Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.</a> BMC Dev Biol. 14, 781 (2015).</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 (4), 945-958 (2014).</li><li>Rosset, A., Spadola, L., Ratib, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OsiriX:+an+open-source+software+for+navigating+in+multidimensional+DICOM+images.">OsiriX: an open-source software for navigating in multidimensional DICOM images.</a> J Digit Imaging. 17, 205-216 (2004).</li></ol>

The authors have nothing to disclose.

이 프로토콜은 결합 된 선명도와 CUBIC 기술로 마우스 척수의 이미지 세로토닌 섬유하는 방법을 보여줍니다 설명했다. 이 청 등. 14 토 메르 외. (15)에 의해 개발 된 수동적 지우기 프로토콜에 비해 빨리 소거 과정을 소개하고 척수 조직이 아니라 클리어시 하이드로 겔에 의해 지원 될 수있다. 청 등. (14)와 토 메르 등. (15)에 의해보고 된 마우스 척수의 고정시 중요한 단계는 모든 솔루션은 화학 물질의 중합을 방지 얼음에 냉각 유지하는 것입니다. 다른 중요한 단계는 신경 조직의 팽창을 방지하기 위해 하이드로 겔 용액으로 천천히 마우스 관류한다. 겔 용액을 겔화하기 위해서는 탈이 요구된다. 이에 대한 대안으로, 우리는 HYD 사이에 공기 방울이 튜브의 상단을 덮도록 파라 필름을 사용하지 않고 남겨rogel 솔루션 및 파라 필름. 이것은 매우 잘 작동하고 겔 용액을 몇 시간 후의 겔이되었다. 일부 기포 겔에서 발견되었지만, 그들은 척수 조직 중의 기포의 부재에 의해 입증되었다 다음 실험을 방해하지 않았다. 용지가 가장 중요한 단계이며, 시약의 조합을 필요로한다. 아미노 알코올을 함유하는 클리어 용액을 원래 CLARITY 프로토콜 (16)에 사용되는 SDS / 붕산 계 클리어 솔루션보다 훨씬 빠른 척수 조직의 지질을 제거한다. 기존의 면역 조직 화학적 기법을 통해 명확하고 CUBIC의 장점은 전체 조직 블록이 결과, 세로토닌 섬유의 연속성이 유지되어 조직 절편없이 묘화 될 수 있으며, 이는 먼 거리에 동일한 섬유를 수행 할 수있다 Z 스택 내의. 이러한 장점과 함께, 담보는 자신의 3D 이미지를 식별 할 수 있습니다,ND 신규 결론 따라서, 세로토닌 성 경로의 특성에 대해 이루어질 수있다. 기술 된 프로토콜은 단순히 특정 항체와 조직을 레이블링 섬유 시스템뿐만 아니라 핵 조사 편리한 도구이다. 이 기술은 이중 또는 삼중 표지를 사용하여 동일한 마우스 뇌의 다른 시스템을 질의에 이상적이다. 실험실 세팅에서, 다중 광자 형광 현미경은 약 300 ㎛의 작동 거리를 사용할 수 있었다. 조직의 양면을 스캔하는 경우이 600 μm의 최대 우리의 이미지를 제한합니다. 그러나, 가벼운 시트 형광 현미경, 자신의 전체 폭, 길이, 깊이 통해 이미지 전체 마우스 뇌와 척수를 할 수 있습니다. 이 기술의 다른 장점은 항체가 용출 가능하고 또 다른 항체 또는 항체가 동일한 조직에 다시 적용한다는 것이다. 이것은 동물 사용량 상당히뿐만 아니라 새로운 뇌 조직의 제조와 관련된 비용을 줄일각 실험. 같은 이유로,이 기술은 다른 조직에 적용될 수있다. 이미 폐, 신장, 심장, 소장, 및 종양 조직 (26, 27)에서 수행 연구가 있었다. 또한,이 기술에는 한계가 있으며, 예를 들어, 마우스 후뇌에서 솔기 핵 GABA 성 뉴런을 포함한 여러 유형의 신경 세포를 포함한다. CLARITY 및 CUBIC는 하나의 마우스 뇌와 척수에 raphespinal 섬유의 다양한 유형의 분포의 연구를 위해 수 있지만. 자신의 신경 전달 물질 또는 단백질 마커에 의해 구체적으로 확인되지 않은 그 뉴런은이 설정에서 확인 할 수 없습니다. 대안은 동일계 하이브리드 화에서와 같이 핵산 프로브 특정한 뉴런 또는 광섬유 시스템 라벨이다. 이 라벨링 시스템 및 형광 이미징 가능한 필터에 의해 제한 한편,이다. 모든 raphespinal 섬유 이미징하기 때문에 도전 남아있다.이러한 기술은 선행 성 트레이서의 두개 내 주사와 결합 된 경우 (예 : 강낭콩의 leucoagglutinin로 - PHA-L)은 세로토닌 섬유, GABA 성 섬유 등을 볼 수있을 수 있습니다.

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

척수 긴 하강 섬유 운동 통증 지각, 및 다른 동작을 위해 필수적이다. 이러한 섬유 시스템의 대부분의 척수 광섬유 종단 패턴 철저 종 조사되지 않았다. 척수 돌출 세로토닌 섬유, 조직 학적 섹션에 쥐 주머니쥐 연구되었으며 기능적 의미는 척수에서의 광섬유 종단 패턴에 기초하여 도출되었다. 명확성과 CUBIC 기술 개발과 함께,이 광 시스템 및 세로토닌 척 주상 경로의 알려지지 않은 특징을 밝힐 가능성 척수에서의 분포를 조사하는 것이 가능하다. 여기서는 결합 선명도 CUBIC 기술을 사용하여 마우스 척수 세로토닌 섬유 영상화 상세한 프로토콜을 제공한다. 방법은 COMBIN와 조직 겔 용액 설명과 마우스의 관류를 포함시약을 삭제의 ATION. 척수 조직 바로 아래 2 주 클리어하고, 세로토닌에 대한 후속 면역 염색 미만 십일 년에 완성되었다. 다중 광자 형광 현미경으로 조직을 검사하고, 3D 이미지 Osirix 소프트웨어를 사용하여 재구성 하였다.

이 섹션은 명확성과 CUBIC 프로토콜의 조합을 사용하여 투명 마우스 척수 세로토닌 항체 염색의 결과를 나타낸다. 우리는 (또한 비디오 (1) 그림 1 참조) 세로토닌 섬유는 복부 뿔의 복부 부분에서 우위와 척수의 모든 라미에 존재하는 것으로 나타났다. 제어 조직은 긍정적 인 섬유 (결과가 표시되지 않은)하지 않았다. 복부 호른에서 조밀 세로토닌 섬유 복부 혼 ventromedial 부분에 존재하고 이들이 복부 혼 측방으로 연장 (도 2,도 비디오 2 참조). 일부 면역 양성 섬유는 또한 후각 또는 중앙 운하쪽으로 복부 혼에서 확장 할 수 있습니다. 면역 양성 섬유 후각 모든 엽층에 존재하지만, 양의 섬유 사이의 작은 갭은, 특히 횡 방향으로 라미 나 (2, 4)에 존재후각의 일부. 후각에서 세로토닌 섬유의 대부분은 작은 직경이고 그 중 소수는 큰 직경 (비디오 1)의이다. 수평 섹션 복부 경적 조밀 세로토닌 섬유는 척수의 길이 방향 축을 따라 이동 큰 섬유 다발을 형성하는 것으로 관찰되었다. 가장 흥미로운 발견은이 큰 섬유 다발 섬유 다발에 수직 정기적 종축을 따라 분기를 발행한다는 것이다. 이러한 분기는 상기 나팔 복부 측 부분에 자신의 경로를 따라 할 담보. 이러한 분기에 비해 중간 선으로 연장하는 사람은 불규칙하고 작은 (그림 2)입니다. 63X 대물 렌즈에서 후각에서 세로토닌 섬유는 척수의 축을 따라 이동 및 관의 경로를 따라 다양한 지점에서 종료하는 것으로 관찰되었다. 두꺼운 섬유는 산발적으로 얇은 F 분산되어<html> ibers (그림 3, 또한 비디오 3 참조). <img alt="그림 1" src="/files/ftp_upload/53673/53673fig1.jpg" /> 그림 1 :. 200X 배율 왼쪽에서 요추 코드의 코로나 섹션에서 세로토닌 섬유는 바로 후각이, 지느러미는 척수의 중간 부분이며, 복부는 척수의 측면 부분, 복부 혼입니다. 스케일 바는 100 μm의입니다. 세로토닌 성 섬유는 특히 섬유 밀도가 높은 복부 혼 ventromedial 부분에서, 모든 엽층 존재한다. 이 섬유는 복부 뿔의 측면 부분과 중앙 운하 또는 등쪽 뿔 두쪽으로 확장 할 수 있습니다. 다른 라미의 섬유의 밀도는 낮다. 세로토닌 섬유의 대부분은 얇다. 오직 섬유의 소수 두꺼운 이들은 후각 및 복부 호른 (모두 보이는&gt; 또한 비디오 1 참조) (오른쪽 다운로드 클릭). <img alt="그림 2" src="/files/ftp_upload/53673/53673fig2.jpg" /> 그림 2 :. 복부 200X 배율 왼쪽에서 복부 뿔 부분과 오른쪽에서 세로토닌 섬유가 척수의 측면이며, 등 지느러미는 척수의 주동이의 일부이며, 복부는 척수의 꼬리 부분입니다. 스케일 바는 100 μm의입니다. 세로토닌 섬유는 척수의 종축을 따라 이동하는 도시가 두꺼운 섬유 다발을 형성 복부 혼의 중앙부에 포장된다. 중간 선을 향해 연장 다른 사람들이 불규칙하고 짧은 반면이 번들 가운데 일부 지점은 복부 뿔의 측면 부분으로 일정한 간격으로 연장된다. 이 섬유는 기관의 경로를 따라 여러 지점에서 종료 ( <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53673/Video2.mp4">또한 비디오 2 참조</a>) (오른쪽 다운로드 클릭). <img alt="그림 3" src="/files/ftp_upload/53673/53673fig3.jpg" /> 그림 3 : 630X 배율에서 아래의 후각에 세로토닌 섬유 왼쪽 오른쪽 척수, 지느러미와 복부 각각 지느러미와 후각의 복부 부분을 참조의 중간 부분이고, 척수의 측면 부분입니다. . 스케일 바는 7 μm의입니다. 세로토닌 섬유는 척수의 길이 방향 축을 따라 이동하고 관의 경로를 따라 종료된다. 이들 섬유가 혼합 된 섬유의 두께 소수 주로 작은 직경이다. 이 섬유는 거의 서로 중첩되지 ( <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53673/Video3.mp4">도 비디오 3 참조 (오른쪽) 다운로드 클릭</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.

CLARITY를 사용하여 마우스 척수에서 세로토닌 섬유 이미징 / CUBIC 기술

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

10.4K 조회수.  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.

비디오: 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.

두 광자 현미경을 사용하여 마우스 척수의 생체내 이미징에

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 ...

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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.

종의 척추 상공 회의소를 주입하는 절차<em&gt; 생체</em&gt; 마우스 척수의 영상

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.

마우스 척수의 세포 역학의 두 광자 이미징

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.

수동 CLARITY를 사용하여 마우스 중추 신경계의 광학 지우기

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.

선명도 조직 클리어링 방법으로 전산 망막의 면역 염색

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.

선명도 조직 클리어링을 통한 손상되지 않은 뉴런 덴라이트의 이미징 및 정량화

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.

중시경에서 현미경 비늘에 이르기까지 신경 세포 이미징을위한 조직 제거 방법

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.

최소 침습 기술을 기반으로 한 마우스 타박상 척수 손상 모델 설정

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

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

C57BL/6J 마우스의 중심삭증후군 모델 확립

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

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.

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.

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