We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission&#8217;s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the &#8220;Stem Cells Call for Proposals.&#8221; This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by &#8220;Ente Cassa di Risparmio di Firenze&#8221;.

1. 축삭 라벨링 <ol><li> 클라이밍 섬유는 고 분자량 덱스 트란 또는 형광 단백질 26-29의 발현을 유도하는 플라스미드 /의 바이러스에 접합 된 유기 염료 하나를 주사하여 표시 할 수있다. 이 프로토콜, 유기 염료 알렉사 플 루어 488 덱스 트란은 등반 섬유 레이블 (그림 1) 소뇌 피질을 시각화하기 위해 하부 올리브에 주입된다. 여기에 설명 된 모든 절차는 건강의 이탈리아 교육부에 의해 승인되었습니다. </li><li> 마이크로 피펫 풀러에 당겨 유리 모세관 튜브를 준비한다. 외부 직경이 대략 30 μM이다까지 가위 유리 모세관 튜브의 팁을 잘라. </li><li> 덱스 트란 - 복합 염료의 1-2 μL와 모세관을로드합니다. </li><li> 시작하기 전에, 오토 클레이브의 모든 수술기구를 소독. 절차를 수행하는 동안, 유리 구슬 살균기를 사용에 의한 오염의 위험을 줄일 수 있습니다. </li><li> 마우스 (9-12 마취케타민 (90 ㎎ / ㎏)과 자일 라진 (9 ㎎ / ㎏)의 복강 내 주사 주). 동물이 완전히 꼬리 및 / 또는 발가락 핀치를 사용하여 침착되어 있는지 확인합니다. 마취 동안 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. </li><li> 면도날이나 제모 크림 하나와 목 위의 털을 면도. 에탄올 70 % 번갈아 베타 딘, 회 수술 부위를 면봉. </li><li> 정위 홀더에 마우스를 놓습니다. 가열 패드로 동물을 따뜻하게 유지. </li><li> 머리가 단단히 고정되도록 조심스럽게 옆에 귀를 뼈에 earbars을 누릅니다. 몸을 140 ° 각도를 형성하기 위해 머리를 돌립니다. 원하는 방향으로 부드럽게 정위 홀더 상 코 홀더로드의 높이를 변경함으로써 얻을 수있다. </li><li> 귀 사이의 피부에 리도카인의 몇 방울을 적용합니다. 후두부 아래의 피부에 약 1 ㎝의 수직 절개를 수행하기 위해 집게 나 면도날을 사용합니다. </li><li> 조심스럽게 무딘 트위터에 피하 조직과 근육을 분리해부 현미경의. 수평 근육 번들을 아래로 누르고 난원 매그넘을 통해 경질을 노출하기 위해 두 개의 수직 근육 다발을 따로 보유. </li><li> 극도의주의를 사용하여 뇌간을 노출하는 주사기 바늘 또는 매우 날카로운 집게로 경질을 해부하다. 경질이 절단되었을 때 뇌 - 척수의 작은 양이 쏟아 질 수있다. </li><li> 정위 미세 조작기에서 수직에서 모세관 45 °의 홀더를 회전합니다. 소뇌 피질의 꼬리 가장자리와 첫 번째 경추 사이의 중간에, 중간 선에서 모세관을 놓습니다. 표면에서 2mm의 깊이에 피펫을 삽입합니다. </li><li> 10 ~ 15 분에 걸쳐 염료의 0.5 ~ 1.5 ㎕의 볼륨을 제공합니다. 미세 주입 분배 시스템은 모세관에 액체의 제어 압력 전달을 할 수 있습니다 (펄스 압력 = 20 PSI, 펄스 지속 시간 = 3 ~ 4 밀리 초, 펄스 전달 = 12 Hz에서의 주파수). </li><li> 다음, 15 분 동안 자리에 모세관을 남겨 slowlY는 (2 ~ 3 분에)를 제거합니다. 섬세하게 근육을 다시 정렬하고 위의 피부를 봉합. 물론 전체 절차를 통해 식염수를 적용하여 수화 근육을 유지합니다. </li><li> 탈수를 방지하기 위해 피하에 0.5 ㎖의 0.9 % NaCl을 주입. 마취에서 완전히 회복 될 때까지 가열 새장에 동물을 유지합니다. </li><li> 피하 수술 후 2 ~ 3 일 동안 매일 Carprofen (5 ㎎ / ㎏)을 관리 할 수​​ 있습니다. </li></ol> 소뇌 피질에 2. 광학 창 참고 : 염료 (그림 1 참조)의 CF 단자 소뇌 피질 하부 olivary 핵까지에서 2 주에 전송됩니다. 띄엄 띄엄 표시의 CF는 다음과 같이 수행 할 수있는 영구적 인 뇌 창에서 시각화 할 수 있습니다 : <ol><li> 모든 수술기구를 소독. </li><li> 상술 한 바와 같이 마우스를 마취. 동물을 확인하기 위해 발가락 핀치를 사용하여 완전히 진정된다. </li><li> SK 닦아 머리 위에 머리를 면도70 % 알코올, 베타 딘의 교류 강타로 눈 연고를 적용합니다. 고정대에 마우스를 놓고 단단히 머리를 유지하기 위해 귀 막대를 놓습니다. </li><li> 피하 두개골 창 사이트의 뇌 가능한 염증의 붓기를 방지하기 위해 덱사메타손 (0.2 ㎎ / ㎏)과 Carprofen (5 ㎎ / ㎏)을 관리 할 수​​ 있습니다. </li><li> 귀 사이에서 눈 위를, 두개골 위의 피부에 리도카인의 드롭을 적용하고 플랩을 잘라. 메스로 긁어 전에 골막에 다시 리도카인 솔루션을 적용합니다. </li><li> 소뇌 위의 두개골의 얇은 반원형의 영역으로 치과 드릴을 사용합니다. 두개골 깊이 반원의 둘레에 걸쳐 거의 일정하도록 윈도우는 일반적으로, 중앙과 뒤 봉합사를 향해 배치된다. 이 두꺼운 영역에 람다 봉합하고 반복 훈련에 너무 가까이 창을 배치하면 부종의 뇌 형성의 과열을 초래할 수 있습니다. </li><li> 지혈을 적용출혈의 경우 두개골에 스펀지. </li><li> 면도날과 족집게의 도움으로 두 개의 반쪽 커버 글라스를 잘라. 커버 글라스가 뼈의 섬보다 약간 큰이며, 핀셋 뼈의 플랩을 제거하기 전에 유사한 모양을 가지고 있는지 확인합니다; 하지 않을 경우, 커버 글라스 모양을 변경하거나 다른 하나를 선택합니다. 뼈를 그리는 동안 족집게로 경막을 만지지 마십시오. 조심스럽게 경질에 커버 글라스를 내려 놓으십시오. 참고 : 경질의 유리의 표면 전체의 준수가 오래 지속 두개골 창을 예고한다. </li><li> 반복 식염수로 세척하고면 스틱 건조가 유리 아래로 기포를 제거합니다. 이것은 또한 유리를 배치 한 후 작은 모세 혈관 밖으로 유출 될 수있는 몇 가지 잔여 혈액을 제거하는 데 도움이 될 것입니다. 아크릴 글루 치과 시멘트 (30)의 혼합물을 밀봉 창. </li><li> 완전 마취에서 회복 될 때까지 회수 챔버에서 가열 패드 위에 동물을 계속. 다시 사용하지 마십시오완전히 회복 될 때까지 홈 케이지에 마우스를 켠다; 그들은 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인를 두지 마십시오. </li><li> 피하 수술 후 2 ~ 3 일 동안 매일 Carprofen (5 ㎎ / ㎏)을 관리 할 수​​ 있습니다. </li></ol> 3. 생체 다 광자 레이저 Axotomy <ol><li> 첫 번째 영상 세션을 수행하기 전에 수술 2-7일 기다립니다. 사파이어 레이저 소스 (120 FS 폭 펄스, 90 MHz의 반복율) 우선 갈바 노 미러의 쌍에 의해 스캔 후 수침 (20X)에 의해 시료 상에 집중된다 : 여기에서 사용 이광자 현미경 내장 지정은 티타늄으로 제공된다 목적 (NA 0.95, WD 2mm). 폐쇄 루프 압전 스테이지는 대물 400 μM까지의 축 방향 변위를 수행한다. 광전자 배증 관은 형광 신호를 수집합니다. </li><li> 두개골 창 objecti에 평행하게 놓 이도록 동물을 마취 한 후 정위 홀더에 머리를 위치초점면을했습니다. </li><li> TPF는 두개골 창 아래 피질 이미징과 탐색. 밝은 사람 사이에 손상 될 수있는 축삭을 선택합니다. </li><li> Z-스택을 취득 대상 축삭의 3 차원 재구성을 얻기 위해 (일반적으로 시야는 512 X 512 픽셀, 2 μm의 Z-축 단계에서, 100 × 100 μm의 2). 이미지가 볼륨 내에서 초점면에 주로 병렬 자리 축삭 분기에 병변 사이트를 선택합니다. 이 방향은 신속하게 성공적으로 절개의 예측이다 형태 학적 변화를 득점 할 수 있습니다. </li><li> CF의 말단 부분에 선택한 점 복용량 높은 에너지를 조사. 이는 LabVIEW 소프트웨어를 통해 수행된다 : (1 ~ 4 라인 스캔을 수행하는 갈바 노 미러의 위치를​​ 설정, 영상 (대물 렌즈 후에 측정 ≈ 300 mW의)에 사용되는 전력보다 5 배 이상의 레이저 파워를 증가 - 10 병변 사이트에 μM) 및 (400)의 레이저 셔터를 엽니 다 - 600 밀리 초. 라인 스캔 배향CF의 평면에 수직 완전히 축삭을 절개합니다. 밝은 장소 (예를 들면., 정맥류)를 조사하는 것은 일반적으로 축삭을 해부하는 데 도움이됩니다. 레이저 axotomy에 대한 파장은 영상 (920 nm의)에 사용되는 동일합니다. </li><li> 일반적으로 조사 지역에있는 축삭의 형광 photobleaching에 일시적으로 인해 감소한다. 조사가 절제 제대로 수행되었는지를 보장하기 위해 후에 동일한 영역 몇 분의 스택을 획득. 지역 붓기 시작 축삭 구슬 같은 구조가 형성되면 신경 세포가 성공적으로 해부하고있다. 이 경우, 몇 시간 (분의 수십에서 24 시간에) 축삭의 말단 부분은 퇴화 완전히 사라집니다. </li><li> 불행히도 완전한 axotomy을 생성하는데 필요한 에너지 량은 항상 예측 가능하지 않다. 조사의 결과 만이 photobleaching에 경우, 형광이 빠르게 확산 염색에 의한 회복되어야하고, 방사선 조사 효과에 다시 반복해야LY는 축삭을 해부하다. 또한, 경우에 부종의 증상은 5에서 표시되지 않습니다 - 증가하는 새로운 검사와 함께 다시 시도 (. 유사 ​​레이저 dendrotomy에 기술 된 내용에, 알레그라 Mascaro 등 23 참조) 20 분, 또는 부종은 회복 다음에 시간 및 / 또는 레이저 파워 드웰. </li><li> 생체 내 이미징에서 두 광자와 레이저 axotomy을 따라 일 축삭의 최종 리모델링 모니터 (그림 2와 그림 3). 혈관 패턴은 다음 일 해부 축삭을 검색하기 위해 참조 패턴으로서 사용된다. </li><li> 피하 Carprofen (5 ㎎ / kg) 실험의 전체 지속 기간에 대한 모든 촬상 세션을 관리. </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+retraction+and+degeneration+in+development+and+disease.">Axon retraction and degeneration in development and disease.</a> Annu Rev Neurosci. 28, 127-156 (2005).</li><li>Chauvet, N., Prieto, M., Alonso, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tanycytes+present+in+the+adult+rat+mediobasal+hypothalamus+support+the+regeneration+of+monoaminergic+axons.">Tanycytes present in the adult rat mediobasal hypothalamus support the regeneration of monoaminergic axons.</a> Exp Neurol. 151, 1-13 (1998).</li><li>Morrison, E. E., Costanzo, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+olfactory+sensory+neurons+and+reconnection+in+the+aging+hamster+central+nervous+system.">Regeneration of olfactory sensory neurons and reconnection in the aging hamster central nervous system.</a> Neurosci Lett. 198, 213-217 (1995).</li><li>Cafferty, W. B., McGee, A. W., Strittmatter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+therapeutics+regeneration+or+sprouting+or+plasticity.">Axonal growth therapeutics regeneration or sprouting or plasticity.</a> Trends Neurosci. 31, 215-220 (2008).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Two-Photon+Excitation+Microscopy+and+Its+Applications+to+Neuroscience.">Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Feng, 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=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Tomomura, M., Rice, D. S., Morgan, J. I., Yuzaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Purkinje+cells+by+fluorescence-activated+cell+sorting+from+transgenic+mice+that+express+green+fluorescent+protein.">Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein.</a> Eur J Neurosci. 14, 57-63 (2001).</li><li>De Paola, V., Arber, S., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptors+regulate+dynamic+equilibrium+of+presynaptic+terminals+in+mature+hippocampal+networks.">AMPA receptors regulate dynamic equilibrium of presynaptic terminals in mature hippocampal networks.</a> Nat Neurosci. 6, 491-500 (2003).</li><li>Holtmaat, A., 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=Experience-dependent+structural+synaptic+plasticity+in+the+mammalian+brain.">Experience-dependent structural synaptic plasticity in the mammalian brain.</a> Nature reviews. Neuroscience. 10, 647-658 (2009).</li><li>De Paola, V., 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=Cell+type-specific+structural+plasticity+of+axonal+branches+and+boutons+in+the+adult+neocortex.">Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex.</a> Neuron. 49, 861-875 (2006).</li><li>Allegra Mascaro, A., Sacconi, L., Pavone, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+nanosurgery+in+live+brain.">Multi-photon nanosurgery in live brain.</a> Frontiers in neuroenergetics. 2, 21 (2010).</li><li>Rossi, F., van der Want, J., Wiklund, L., Strata, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinnervation+of+cerebellar+Purkinje+cells+by+climbing+fibres+surviving+a+subtotal+lesion+of+the+inferior+olive+in+the+adult+rat.+II.+Synaptic+organization+on+reinnervated+Purkinje+cells.">Reinnervation of cerebellar Purkinje cells by climbing fibres surviving a subtotal lesion of the inferior olive in the adult rat. II. Synaptic organization on reinnervated Purkinje cells.</a> The Journal of comparative neurology. 308, 536-554 (1991).</li><li>Carulli, D., Buffo, A., Strata, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reparative+mechanisms+in+the+cerebellar+cortex.">Reparative mechanisms in the cerebellar cortex.</a> Progress in neurobiology. 72, 373-398 (2004).</li><li>Grasselli, G., Mandolesi, G., Strata, P., Cesare, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+sprouting+and+axonal+atrophy+in+cerebellar+climbing+fibres+following+in+vivo+silencing+of+the+growth-associated+protein+GAP-43.">Impaired sprouting and axonal atrophy in cerebellar climbing fibres following in vivo silencing of the growth-associated protein GAP-43.</a> PLoS One. 6, e20791 (2011).</li><li>Nishiyama, H., Fukaya, M., Watanabe, M., Linden, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+motility+and+its+modulation+by+activity+are+branch-type+specific+in+the+intact+adult+cerebellum.">Axonal motility and its modulation by activity are branch-type specific in the intact adult cerebellum.</a> Neuron. 56, 472-487 (2007).</li><li>Shinoda, Y., Sugihara, I., Wu, H. S., Sugiuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+entire+trajectory+of+single+climbing+and+mossy+fibers+in+the+cerebellar+nuclei+and+cortex.">The entire trajectory of single climbing and mossy fibers in the cerebellar nuclei and cortex.</a> Prog Brain Res. 124, 173-186 (2000).</li><li>Strata, P., Tempia, F., Zagrebelsky, M., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+trophic+interactions+between+climbing+fibres+and+Purkinje+cells+in+the+rat+cerebellum.">Reciprocal trophic interactions between climbing fibres and Purkinje cells in the rat cerebellum.</a> Prog Brain Res. 114, 263-282 (1997).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. (12), (2008).</li></ol>

We have nothing to disclose.

이 프로토콜은 형광 염료와 하부 올리브의 뉴런 레이블을하는 방법을 보여줍니다. 이어서, 소뇌 피질에 두개 창을 수행하는 방법을 설명한다. 이 기술은 olivocerebellar 신경, 등산 섬유의 단자 부분에 광 액세스를 제공합니다. 불행하게도, 라벨 및 개두 수술 모두의 결과는 (일반적으로 3 마우스 중 1 표시, 1 3 중 두개골 창 1 ~ 2 주 후에 명확 유지됩니다)도 숙련 된 운영자의 손에 아주 낮다. <p class=...

기계적 손상, 독성 모욕 또는 신경 변성 질환으로 인한 절개 축삭은 보통 10-13 전지 본체에서 분리 축삭의 말단 부분의 변성에 의해 이어진다. 몇 가지 예외를 제외하고 2,7,14,15로, 성인 동물의 CNS에서 절단 축삭은 일반적으로 재성장 프로그램 (16)을 활성화 할 수 없습니다. 약간은 세포와 세포 이하의 수준에서 퇴행성 이벤트의 실시간 역학에 대한 알려져있다. 신경 세포의 손상을 제한하고 신경 세포의 재성장을 촉진하기위한 새로운 전략의 개발은 단독으로 부상당한 신경 세포가 퇴화하고 재생하는 메커니즘을 명확히, 첫 번째 단계로,이 필요합니다. 이 연구는 가장 직접적으로 생체 내에서 단일 신경 세포의 역학을 모니터링하여 해결됩니다. 일 광자 형광 이미징 기술은 가시광의 산란 강도에 의해 제한되는 반면, 두 개의 광자 여기가 리 깊은 피질 층에 도달세포 내 해상도 3,4,17와 마우스를했습니다. 형광 단백질을 선택적으로 신경 18-20의 모집단에서 발현되는 형질 전환 마우스의 활용, TPF 현미경은 생체 내 21, 22의 개발 과정에서 시냅스 가소성과 축삭 신장의 탐사에 적용되었습니다. 유일하게 손상된 신경 세포의 T 그 기능에 부상이 특히 관심의 축삭을 대상으로 상해의 모델 두 광자 이미징에 의해 생체 모니터링에 커플 링에 의해 조사 할 수 있습니다 후 다시 자라게. 펨토초 펄스의 다 광자 흡수는 하나의 수상 돌기 또는 한 쪽이 5,23을 방해하는 데 사용되었습니다. 또한,이 부상 패러다임은 연락 덴 드라이트 (dendrite) 6을 방해하지 않고 하나의 축삭 가지를 절단 할 수 있습니다. 한 번 손상, 소뇌 등산 섬유 (CFS) 자신의 축삭을 재생하는 특정 신경 세포의 인구 수 있도록 기능을 해부의 컨텍스트 내에서 유용한 모델 SI는후부 그들은 심지어 성인 동물 (24, 25)의 부상 후 뛰어난 플라스틱의 특성을 유지합니다. 최근 CF 및 장기 이미징은 이러한 축삭 레이저 axotomy 6에 따라 일 재성장 할 수 있다는 것을 보여 주었다. 이 프로토콜은 선행 성 추적을 통해 olivocerebellar 뉴런과 자신의 축삭 신장 레이블을하는 방법에 대해 설명합니다. 관심의 신경 세포가 형광 표지되면, 그들은 두개골 창 아래에 몇 주 또는 몇 달 임의의 시점에서 반복적으로 모니터링 할 수 있습니다. 생체 내에서 레이저 axotomy에 의해 하나의 축삭 가지를 해부하는 절차는 설명한다. 여기에 제시된 기술은 생체 내에서 축삭 리모델링의 메커니즘에 새로운 통찰력을 제공하고 신경 세포의 변성을 제한하고 축삭 재성장을 촉진하는 치료 전략의 개발을하는 데 도움이 될 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting&#160;</td>
			<td>51670</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament</td>
			<td>Sutter Instrument Inc</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (Glass bead sterilizer)</td>
			<td>Roboz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection dispense system</td>
			<td>Picospritzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R)</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0720</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser, 120 fs width pulses,&#160; 90 MHz repetition rate</td>
			<td>Coherent</td>
			<td>Chameleon&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spongostan, haemostatic sponge&#160;</td>
			<td>Ferrosan</td>
			<td>MS0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvanometric mirrors&#160;</td>
			<td>GSI Lumonics</td>
			<td>VM500+</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective&#160;</td>
			<td>&#160;Olympus</td>
			<td>XLUMPLFLN 20XW</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric stage&#160;</td>
			<td>Physik Instrumente</td>
			<td>P-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier modules&#160;</td>
			<td>Hamamatsu Photonics</td>
			<td>H7710-13</td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW System Design Software</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Voren, 1 mg/ml (dexamethasone-21- isonicotinate)</td>
			<td>Boheringer Ingelheim</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rymadil (carprofen)&#160;</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine clorohydrate 2%</td>
			<td>ATI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 dextran</td>
			<td>Life Technologies</td>
			<td>D22910</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser nanosurgery of cerebellar axons in vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

이 프로토콜은 하나의 뉴런의 축삭 라벨, 생체 내 이미징 및 레이저 axotomy을 수행하는 방법을 설명했다. 실험의 타임 라인은도 1에 도시된다. 알렉사 형석 488 덱스 트란으로 표시하고 생체 이광자 현미경으로 두개 창 아래 시각의 CF의 예는도 2에보고된다. 이전 6,27보고 된 바와 같이, 상승하는 분기 수일의 관찰 기간에 걸쳐 높은...

Watch this Scientific Journal Video about Laser Nanosurgery of Cerebellar Axons In Vivo at JoVE.com

Laser Nanosurgery of Cerebellar Axons In Vivo

레이저 nanodissection 결합 이광자 이미징은, 세포 내 해상도 중추 신경계 퇴행성 및 재생 공정을 연구하는 유용한 도구이다. 이 프로토콜은, 라벨 이미지 및 생체 내에서 소뇌 피질에서 하나의 등반 섬유를 해부하는 방법을 보여줍니다.

소뇌 축삭의 레이저 Nanosurgery<em&gt; 생체</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

laser-nanosurgery-of-cerebellar-axons-in-vivo

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Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

8.1K 조회수.  University of Florence.  레이저 nanodissection 결합 이광자 이미징은, 세포 내 해상도 중추 신경계 퇴행성 및 재생 공정을 연구하는 유용한 도구이다. 이 프로토콜은, 라벨 이미지 및 생체 내에서 소뇌 피질에서 하나의 등반 섬유를 해부하는 방법을 보여줍니다. 

비디오: Laser Nanosurgery of Cerebellar Axons In Vivo

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Drosophila 애벌레 Segmental Axons 이내 시냅틱 Vesicles의 생체내 시각화에

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

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신경과학

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

C로 생체내 레이저 Axotomy에서 엘레간스

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

초음파 혈액 - 뇌 장벽 파괴 및 망간 향상된 MRI를 사용하여 기능성 Neuroimaging

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

소뇌 과립 신경 세포의 유전자 조작<em&gt; 체외</em&gt;와<em&gt; 생체</em&gt; 신경 세포의 형태와 마이그레이션을 연구하는

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

공 초점 Macroscopy을 사용하여 출생 후의 소뇌 과립 세포 이동의 생체 이미징

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

동시<em&gt; 생체</em두 망막의&gt; 기능 테스트로<em&gt; 생체</em&gt; 전위도 시스템

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

신경 죽음 및 마우스 기본 소 뇌과 립 신경에 변성을 모델링

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Vivo에서 복합 이미징 및 마우스 레이저 유도 안 Neovascularization 모델의 분석

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.