이 작품은 넉넉한 신경 질환 및 뇌졸중 (NIH / NINDS) 부여 1R01NS086981과 콘래드 N. 힐튼 재단의 보건 / 국립 연구소의 국립 연구소에 의해 지원되었다. 우리는 공 초점 현미경과의 귀중한 도움 박사 로랑 Bentolila 박사 마태 복음 Schibler 감사합니다. 우리는 CLARITY 포럼 (http://forum.claritytechniques.org)에 기여한 것들 감사합니다. 우리는 특히이 매혹적인 기술을 가르치는 그의 연구실을 개방 박사 칼 Deisseroth 감사합니다. 저자는 뇌 매핑 의료 연구 조직, 뇌 매핑 지원 재단, 피어슨 - 난봉꾼 재단, Ahmanson 재단, 캐피탈 그룹 회사 자선 재단, 윌리엄 M. 린다 R. Dietel 자선 기금, 그리고 노스 스타 펀드의 관대 한 지원에 감사 . 이 책에서보고 된 연구는 부분적으로 연구 자원을위한 국립 센터와 국가의 이사의 사무실에 의해 지원되었다보너스 번호 C06RR012169, C06RR015431 및 S10OD011939에서 건강의 알 연구소. 내용은 전적으로 저자의 책임이며 반드시 국립 보건원의 공식 견해를 대변하지 않습니다.

모든 실험은 기관 동물 관리 및 사용위원회 (IACUC) 지침에 따라 수행되었다. THY1-YFP, PLP-EGFP와 PV-TdTomato 마우스를 실험에 이용했지만, 형광 단백질을 발현하는 마우스를 성공적으로 삭제되고 묘화 될 수있다. 1. 조직 준비 주의 : 파라 포름 알데히드 (PFA) 및 아크릴 아미드는 독성 및 자극한다. 흄 후드와 적절한 개인 보호 장비 (실험실 코트, 장갑, 눈 보호) 이러한 시약을 이용하여 실험을 수행합니다. <ol><li> (- 3 분 ~ 2) 호흡이 중단 될 때까지 이소 플루 란의 ~ 1 ml의 안락사 챔버에서 동물을 희생. </li><li> (- 10 분 5) 혈액이 조직에서 제거 될 때까지 ~ ​​5ml를 설정 연동 펌프를 사용하여 / 분 intracardially 4 ° C에서 관류 액 (표 1)와 마우스를 관류한다. </li><li> 정착액 솔루션에 관류 변경 (표 1)(- 10 분 5) 4 ° C에서 목과 꼬리는 크게 보강 할 때까지. </li><li> 관류를 종료하고 동물 목을 벨. 조심스럽게 제 두개골 주변 결합 조직을 제거한 후, 두개골의 복부 표면으로부터 뼈를 분리 및 20에 기재된 바와 같이, (도 1a)에 뇌를 올려 두개골로부터 뇌를 절개. 참고 : 소뇌의 flocculonodular 로브와 후각 전구를 둘러싼 뼈의 제거에 특별한주의를주십시오. <ol><li> 조심스럽게 척추 및 척추 사이에 미세, 예리한 가위의 선단을 삽입 (21)에 기술 된 바와 같이, 거리 개별 척주 세그먼트를 절단함으로써 척추의 척추 해부. </li></ol></li><li> 플라스틱 마우스 뇌 매트릭스에 배치하고 매트릭스 블레이드와 중간 선을 따라 절단하여 뇌 Hemisect. </li><li> 원심 분리 욕조 50 ㎖를 분리하는 각각의 반구와 척수 추가함께 ES ~ 겔 용액 (표 1) 40ml를. 이 시점 앞으로 커버에서 알루미늄 호일로 튜브는 이후의 모든 단계 동안 형광을 보호 할 수 있습니다. </li><li> 부드러운 7 일 (~ 30 회전)을 진탕 4 ° C에서 하이드로 겔의 샘플이 들어있는 튜브를 품어. </li></ol> 2. 중합 <ol><li> 원심 분리 튜브 하이드로 ~ 25 mL의 시료를 보존하고, ~ 25 ㎖의 하이드로 겔을 버린다. </li><li> 뚜껑을 제거하고 건조기 항아리 원심 튜브를 배치하고, 진공 용기에 접속하여 샘플을 탈산 소화. 솔루션 및 양식 거품 나오는 용해 가스를 허용하기 위해 적어도 10 분 동안 진공을 적용합니다. 부드럽게 거품을 제거 흔들어. </li><li> 3 분 - 2 이상 100 % 질소 가스로 데시 케이 터 항아리에 진공을 교체합니다. 압력 (데시 케이 병의 상부가 개방 될 수있을 경우에) 등화 후 신속 산소의 재 도입을 방지하기위한 튜브 캡. </li><li> 중합체중합이 완료 될 때까지 3 시간 동안 37 ° C의 물 배스에서 샘플 튜브를 배치하여 하이드로 겔이지는. 중합 하이드로 겔은 겔 일관성 (그림 1b)에 걸립니다. 주 : 상단 미 중합 겔 소량 좋다. </li><li> 다음, 원심 분리기 튜브에서 중합 된 샘플을 제거 샘플에서 과량의 하이드로 겔을 절단하기 위해 주걱을 사용합니다. </li><li> 무료 닦아 보풀에 샘플을 배치하고 부드럽게 샘플에 중합 된 하이드로 겔의 얇은 층 뒤에 남겨두고, 마찰과의 조직을 압연하여 나머지 하이드로 겔을 제거합니다. </li></ol> 3. 지질 제거 참고 : 지질 삭제 조직이 취약, 취급에주의. 튜브를 배출 할 때 샘플 손실을 방지하기 위해 셀 스트레이너를 사용합니다. 또한, 샘플은 굴절률 매칭 프로세스 동안 반전 될 수 팽윤성 그러나, 소거 과정을 통해 팽창한다. 주의 : 소디음 도데 실 설페이트 (SDS)와 붕산을 자극한다. 흄 후드와 적절한 개인 보호 장비 (실험실 코트, 장갑과 눈 보호)로를 이용하여 실험을 수행합니다. <ol><li> 깨끗한 50 ㎖ 원심 분리 튜브에 중합 샘플을 전송하고 버퍼 (표 1) 삭제의 45 ml에 추가 할 수 있습니다. 부드러운 흔들림 45 ° C (~ 30 RPM)에 품어. 화학 폐기물에 사용 청산 버퍼를 붓는 신선한 청산 버퍼 ~ 45 ml에 추가하여 초기 7 일간 매일 버퍼를 변경합니다. 형광 물질을 보호하기 위해 알루미늄 호일로 덮여 샘플을 함유하는 원심 분리기 튜브를 유지한다. </li><li> 삼일 - 초기 7 일 후, 매 2 ~ 40 ° C 및 교환 청산 버퍼에서 샘플을 품어. 모든 지질을 용해하고 조직 투명성 (그림 1C)에 도달 할 때까지 청소를 계속합니다. 마우스 뇌 반구 6 주에서 2 - - 척수에 대한 사주이 4됩니다. </li><li> 조직이 삭제되면, 전송새로운 50 ML의 원심 분리 관에 시료 잔류 SDS를 제거하기 위해 24 시간 동안 부드러운 진동 (~ 30 RPM)와 40 ° C에서 PBST (표 1)에서 4 번 씻는다. </li></ol> 4. 분자 염색 주의 : 4 &#39;, 6-Diamidino -2- 페닐 인돌 다이 하이드로 클로라이드 (DAPI)의 자극과는 흄 후드와 적절한 개인 보호 장비 (실험실 코트, 장갑, 눈 보호)을 수행해야합니다 사용하는 실험이다. <ol><li> 깨끗한 15 ML의 원심 분리기 튜브에 샘플을 전송하고 1X PBST 1 μg의 / ㎖ DAPI, pH를 7.5로 입력합니다. 24 시간 동안 실온 (RT)에서 인큐베이션. 형광 물질을 보호하기 위해 알루미늄 호일로 덮여 샘플을 함유하는 원심 분리기 튜브를 유지한다. </li><li> 적어도 6 시간 / 세탁 동안 실온에서 PBST 3 회 반복한다. </li></ol> 5. 굴절률 매칭 주의 : 쉬 2,2&#39;- 티 오디 에탄올 (TDE)는 자극이고 어떤 실험을 이용하여흄 후드와 적절한 개인 보호 장비 (실험실 코트, 장갑, 눈 보호)을 수행 할 수 울드. <ol><li> 깨끗한 15 ML의 원심 분리기 튜브에 샘플을 전송하고 30 % 1X PBS에서 (v / v)의 TDE, pH를 7.5로 입력합니다. 24 시간 동안 부드러운 흔들림 40 ° C (~ 30 RPM)에 품어. </li><li> 1X PBS, pH가 7.5에서 63 % (v / v)의 TDE와 교환 30 % TDE. 부드러운 24 시간 동안 진탕 40 ° C에서 품어. 참고 :이 샘플은 약 원래 크기로 다시 축소됩니다. </li><li> 편평하고 청결한 표면에 시료의 두께보다 단지 약간 큰 균일 한 직경을 갖는 실린더에 재사용 가능한 접착제의 일부를 롤. </li><li> 개방형 원으로 재사용 접착제 실린더를 마련 (~ 상단에 작은 구멍에 25mm 내경)는 40mm의 유리 바닥 접시에. 교정이 이루어질 필요하면 유리 실린더를 제거하고 면도날로 절단. </li><li> 하여 재사용 가능한 접착제와 유리 사이의 밀봉을 생성하기 위해 P1000 피펫 팁을 사용하여실린더의 외주 주위 압연. </li><li> 장소 ~ 100 ㎕의 63 % 유리 접시 상 TDE 재사용 가능한 접착제의 원 내에있는 유리 표면을 덮도록 주위에 전파. </li><li> 주걱을 사용하여 조심스럽게 기포를 형성하지 않도록주의하면서, 원의 중간 샘플을 놓는다. </li><li> 즉시 재사용 접착제를 통해 40mm 원형 커버 유리를 배치, 직접 샘플에 63 % TDE의 또 다른 100 μl를 피펫. </li><li> 커버 유리 샘플과 연락을 할 때까지 인덱스, 중간, 양손의 약지를 사용하여 조심스럽게 원의 둘레를 누릅니다. </li><li> 천천히, 표면에 휴식 수직 위치로 유리 바닥 접시를 가져 오는 솔루션은 상단에있는 작은 구멍에 도달 할 때까지 다음 피펫 63 % TDE를 추가합니다. 무료 솔루션이 유리에 떨어지지 않도록 피펫 팁을 건조 닦아 보풀을 사용합니다. 챔버에서 거품을 방지하기 위해, 피펫을 완전히 비울 수 없습니다. </li><li> 에작은 구멍은 원형을 넣고 밀폐 된 챔버를 만들기 위해 실리콘 엘라스토머를 추가합니다. 이 영상 전에 건조를 위해 5 분을 기다립니다. </li></ol> 6. 현미경 주의 : 공 초점, 단일 평면 조명과 빛 시트 현미경에 사용되는 레이저는 매우 강력하고 실명의 원인이 될 수 있습니다. 레이저를 이용한 모든 실험은 광범위한 훈련 후 큰주의와 적절한 눈 보호를 수행해야합니다. <ol><li> 레이저 스캐닝 공 초점 현미경의 무대에 내부의 샘플 이미징 챔버를 놓습니다. </li><li> 이미징 소프트웨어를 엽니 다 (재료의 표 참조). 아르곤 여기 레이저를 켜려면, 다음 프로그램 레이저 아이콘의 상단에있는 설정 탭을 클릭합니다. 레이저에 전력을 30 %로 슬라이드 규모를 이동 아르곤 옆의 확인란을 선택합니다. 포인터가 천천히 선택한 위치로 워밍업 관찰한다. <ol><li> 상단의 획득 탭​​으로 돌아갑니다. 큰 상자에서 &quot;빔 경로 설정 및# 34; / 부하로 이동 상자를 설정 저장 및 이미지 YFP (527 ㎚)에 대한 적절한 파장 채널을 선택 드롭 다운 메뉴를 선택합니다. </li></ol></li><li> 채널 설정 상자에서 &quot;현재 객체 목표&quot;라고 표시된 빨간색 하이퍼 링크를 배치함으로써 이미징을위한 적절한 목표를 선택합니다. 탭을 클릭하면 사용 가능한 모든 목표를 열고 선택은 터렛을 자동으로 회전합니다. <ol><li> 사용 목적 (예., 10X)이 큰 작동 거리 (조직의 두께보다 더는 묘화 될 예를 들면, 11mm)과 큰 개구 수 (예. 0.3)을 평면 이미지 해상도를 극대화하고 최소화 할 광학 부 두께. </li></ol></li><li> 1,024 X 1,024에 &quot;형식&quot;이라고 인수 행렬을 설정하는 창 또는 목표의 측면​​ 분해능 한계에 적절한 값 : &quot;해결 XY&quot;를 열고 메뉴를 드롭 다운의 왼쪽 열에서. (가능한 한 낮게 줌 배율을 예. 10.7). </li><li> 같은 창에서 따라 개별적으로 표시된 메뉴를 드롭하여 8 라인 평균 1 프레임 평균 매개 변수를 설정합니다. 신속한 취득이 높은 신호 대 잡음비와 작은 값의 큰 값을 사용한다. 주 : 8 라인 평균 1 프레임의 평균은 약 4 시간에서 마우스 뇌 반구의 고품질 이미지를 획득한다. </li><li> 무대 컨트롤을 사용하여 샘​​플을 찾습니다. 대표적인 필드가 표시되면, 이득 및 오프셋을 설정합니다. 이득 및 조직 유형, 형광에 따라 크게 변화 오프셋 (offset), 및 / 또는 해부학 적 기능을 이미지화되는 참고. YFP를 들어, 이득 780-760로부터 -1.0에서 오프셋을위한 -1.5 값을 설정합니다. </li><li> &quot;타일 스캔&quot;을 선택하고 관심 영역의 경계를 선택합니다. 는 z 스택의 설정 상한과 하한 취득합니다. 목적의 광학 부재의 분해능 한계에 기초하여 상기 광학 부재의 두께를 설정한다. 참고 : 목적의광학 부재의 분해능 한계는 주로 그 개구에 의해 규정된다. 자동 목표 값을 적절한 계산할 대부분 현미경 제어 소프트웨어가 사용된다. 0.3의 개구 수와 10X 대물 렌즈의 경우 즉, 약 11 μm의 것이다. </li><li> 인수를 시작합니다. 참고 :이 여러 시간이 걸릴 수 있습니다. </li><li> 관심의 특정 지역에서 높은 배율 목표를 사용하여 높은 해상도의 이미지를 수집합니다. </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.

하이드로 겔 포함 된 조직 (수동 CLARITY)의 수동 청소 조직의 큰 조각을 삭제하기위한 간단하고 저렴한 방법이다. 이러한 접근 방식은 전용 설비를 필요로하지 않고, 용이하게 온도 제어 진탕 기에서 수행 될 수있다. 몇 주의 기간 동안, 같은 전체 뇌 또는 척수로도 큰, 매우 유수 조직, 투명하고 현미경에 적합하게 될 것이다. 이 보고서는 CNS 조직의 청산에 초점을 맞추고있다하더라도, 수동 CLARITY ?...

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.

뇌의 구조적 조직을 시각화하는 형태와 연결이 건강과 질병에서 뇌 기능에 미치는 영향을 이해하는 데 중요합니다. 광학 청소 기술은 우리가 응집력 형태와 연결을 연구 할 수 있도록, 손상 조직의 3 차원 이미지 세포 인구에 가능합니다. 세포가 뇌의 신경 연결의 복잡한 패턴을 떨어져 애타게 하나를 허용의 하위 집단에 대?...

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.

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 ...

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

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JoVE Journal

Neuroscience

13.4K 조회수.  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.

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

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

시신경의 절개 : 중추 신경계에서 신경 세포의 Apoptosis 성인의 모델

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

연구

신경과학

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.

마우스의 중추 신경계에서 줄기 세포 주입의 Multimodal 이미징

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

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.

중추 신경계에서 Transducing 전지 Lentiviral 벡터 제작

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.

의 중추 신경계에 단일 세포의 라벨<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.

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.

설치류 중추 신경계의 생체 Optogenetic 자극

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.

소설 수동 전체 CNS 조직에 광학 투명도의 급속 한 생산을 위한 방법 삭제

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.

제 3-탈피 초파리 Melanogaster 의 중앙 신 경계 활동의 electrophysiological 녹음

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.

화학유전학적으로 조작된 피질 인터뉴런 선조를 출생 후 마우스 뇌로 이식

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.

쥐 중추 신경계에서 안티 센스 올리고 뉴클레오티드의 내 전달

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