우리는 우수한 기술 지원을위한 콜라겐 프로토콜과 Jutta 페이와 우르술라 Hinz을 공유 Urmas의 Arumae 감사드립니다. 아울러 우리는 친절 비디오 shoting에 도움을 기독교 F. Ackermann, Ulrike 엥겔과 하이델베르크 (Heidelberg) 대학에서 니콘 이미징 센터와도 요아킴 Kirsch 감사드립니다. 작품은 부분적으로 독일 Forschungsgemeinschaft (SFB 592)를 통해 기금을 마련했다.

1. 콜라겐 젤의 작성 <ol><li> 455 μl 10X 가상, 112 μl 7.5 % NaHCO 3, 50 μl 글루타민과 NaOH를 포함 주식 매체를 준비합니다. 농도와 NaOH의 양이 쥐 꼬리 콜라겐 준비 (1.2 참조), 1,000 μl되는 주식 매체의 마지막 볼륨에 따라 달라집니다. </li><li> Ebendal (1)에 따라 쥐 - 꼬리 콜라겐을 준비합니다. 4 ° C.에있는 콜라겐 솔루션을 저장 콜라겐 솔루션의 저하가 관찰 할 수 없습니다. 새로운 배치의 준비 후에 (1.1 참조) 주식 매체에 필요한 NaOH 농도를 추정. NaOH의 양 및 농도의 적정을위한 매체의 pH-표시기를 사용합니다. 800 μl 산성 쥐 - 꼬리 콜라겐은 재고 매체의 210 μl를 혼합 할 때 붉은 색의 그늘을 돌려해야합니다. NaOH의 농도가 너무 낮 으면 콜라겐 솔루션은 매체의 산도 표시기로 인해 노란색으로 바뀝니다. 찌르다없이 매체와 혼합에 콜라겐을 추가거품 ucing. 얼음에서 다음 단계를 수행합니다. 중합은 콜라겐 솔루션과 재고 매체가 혼합 된 후 2 시간 이내에 발생하고 새로운 솔루션은 37 ° C.에 incubated 수 있습니다 실험을 시작하기 전에이 작업을 테스트합니다. 1.3) 재고 매체의 210 μl (1.2 참조)과 콜라겐 솔루션 800 μl를 섞는다. 8 잘 챔버 슬라이드의 우물이 솔루션의 장소 100 μl. 37 슬라이드를 길러 ° C, 5 % CO 2 및 세포 문화 인큐베이터에서 습기 조건. 세포 배양 벤치 및 무균 조건 하에서 콜라겐 젤을 준비합니다. 콜라겐은 2 시간 이내에 gelatinizes. </li></ol> 2. 배아 SCGs의 해부 <ol><li> 시간이 임신 자궁의 쥐 (E16-18) 및 추가 필요 할 때까지 PBS에 보관 수확의 배아. </li><li> 양막에서 하나 배아를 해부. 쇄골의 수준의 배아 꼬리 목을 벨. &quot;곤충 - 바늘 &#39;로 실리콘 바닥 요리에 머리를 수정합니다. 머리의 복부 측면 여러분에게 직면하는당신은 턱밑 지역을 볼 수 있습니다 싶네요. 고정 쉽게 준비 할 수 있습니다. </li><li> 왼쪽 및 오른쪽 턱밑 지역의 피부를 제거하고 하위 피부 결합 조직과 함께. </li><li> 목 뿔뼈, infrahyoidal 근육과 목 빗근에보기를 삭제하려면 자신의 위치에서 턱밑 침샘을 제거합니다. </li><li> 후두 및 기관에보기를 삭제하려면 신중하게 이러한 근육을 제거합니다. 기도와 후두를 제거합니다. 경동맥 동맥는 prevertebral 근육의 양쪽에 볼 수 있습니다. SCG는 경동맥 동맥의 분기에 위치하고 있으며, 타원형 모양을 갖추고 있습니다. 조심스럽게 신경절을 제거하고 PBS에 배치합니다. 그들이 지키는 경우 분해 도구 부드럽게 신경절을 제거합니다. 이 집게뿐만 아니라 SCG 제거 &quot;곤충 바늘 &#39;로 장착 바늘 홀더를 사용하여 유용합니다. </li><li> 당신이 enou를 해부 할 때까지 한 번에 PBS에 PBS에 실온에서 신경절을 저장GH 신경절. 그런 다음 혈관과 결합 조직에서 신경절을 청소하십시오. 층류 벤치에서 위치 해부 현미경으로 SCG 해부 및 청소를 수행합니다. </li><li> 마지막으로, 쉽게 처리 할 수​​있는 주사기에 장착 주사기 바늘의 도움으로 gelatinized 콜라겐 젤에 explanted 신경절를 놓습니다. </li><li> 5 % CO 2와 습기 조건, 37 ° C에서 세포 배양 인큐베이터에서 gelatinized 콜라겐의 SCG explants를 품다. </li></ol> 3. SCG Explants의 치료 <ol><li> 1-2 시간의 배양 후, B27 보충제, 글루타민과 항생제를 포함하는 100 μl Neurobasal 세포 배양 매체와 SCG explant가 포함 된 콜라겐을 다룹니다. 이제 챔버 슬라이드 우물 200 μl (100 μl 젤과 100 μl 매체)의 볼륨을 포함하고 있습니다. , 매체의 또 다른 200 μl를 추가하지만 지금은 axonal 성장을 촉진하기 NGF를 (60 NG / ML) 포함. 따라서 최종 NGF 농도는 30 NG / ML입니다. </li><li><ol class="lalpha"><li> SC 이전의 발병을 조사하기 위해 체외 0 (DIV0)에 일에 직접 추가 요소를 추가 할 수 있습니다. 필요한 더블 농도 (2) NGF가 포함 된 미디어의 요소를 분해. </li><li> 이미 axons 따라 이주 시작 SCS에 미치는 영향을 분석하기 위해, DIV4 (실험의 잠재적 정류장)까지 D​​IV3에서 추가 요소를 추가 할 수 있습니다. 이를 위해 신중하게 DIV3에서 솔루션 180 μl를 제거 200 μl 새로운 매체에 포함 된 NGF와 더블 농도 (2)에 대한 관심의 추가 요소를 추가 할 수 있습니다. </li></ol></li></ol> 4. 시간 경과 영상 분석을위한 역 현미경을 사용하십시오. 다양한 목표를보기 및 확대의 필드를 정의 할 수 있습니다. SCG explants의 이미지는 유리 슬라이드와 콜라겐을 통해 수행되기 때문에 하나의 중요한 측면은 그러나, 사용 목적의 작동 거리입니다젤. 1/10-30 분 녹화 프레임 속도는 좋은 결과 (2) 보여 주었다. 그러나이 점은 과학 질문에 대한 조정되어야한다. 일반 CCD 카메라는 이미지 수집에 사용할 수 있습니다. 시간 경과 영상에 대한 세포 배양 배양 챔버는 현미경의 무대에 첨부되어야한다. 5 % CO 2와 습기 조건, 37 ° C에서 영상 동안 조직을 품다. 영상이 시작되기 전에 부화 시스템하다 시간을 시작합니다. 이 챔버 슬라이드를 포함하여 현미경 부품 (예 : 목표)의 온도에 적응 할 수 있으며, 온도 유도 앉를 방지 할 수 있습니다. 현미경 소프트웨어의 도움과 SCG explants에 다른 적용 조건의 동시 분석을위한 소프트웨어 제어 동력 단계 (multiposition 설치)에 분석의 특정 영역 (explant에 내 서로 다른 explants 사이) 정의합니다. 사용할 수있는 시간 경과 영상 wildtype 조직뿐만 아니라 유전자 변형 동물의 조직에 대한(3) : SCS (GFP s100b)을 표시. 영상 fluorophores를 들어 형광등 광원 micropscope 설정에서 구현해야합니다. 표준 필터를 사용합니다. 5. SC 마이그레이션 거리의 정량화 <ol><li> 끝 지점 (예를 들어 DIV4)에, 3-4 시간의 챔버 슬라이드에 4% PFA과 콜라겐 젤을 해결할 수 있습니다. 연속 PBS로 10 분의 콜라겐 젤 다섯 번 씻는다. immunocytochemistry (2)에 대한 표준 프로토콜을 적용합니다. 당신은 SCGs의 준비에 새로하는 경우, 티로신 hydroxylase에 대한 immunohistochemistry에 의해 교감 신경절 (TH) catecholaminergic 세포를위한 공통의 마커로 신경절의 신원을 확인합니다. immunohistochemistry 동안 (주 항체 후), 젤을 세정에 도움이되는 더 큰 볼륨을 가지고 24 잘 접시에 explant 포함 콜라겐 젤을 전송할 수 있습니다. </li><li> immunohistochemistry 후, 유리 슬라이드에 콜라겐 젤을주의 깊게 배치합니다. 주의하여나머지 솔루션을 제거하고 콜라겐이 건조 / 2 크기로 축소 보자. 이 거리를 (2) 쉽게 측정 할 수 있습니다. 이 후, 샘플을 탑재합니다. </li><li> 마지막으로, 피지 (NIH) 소프트웨어의 도움으로 마이그레이션 거리를 측정합니다. 특별한 플러그인이 끝 부분에 필요하지 않습니다. μm의 정확한 측정을 허용하려면 올바른 이미지 메타 데이터 및 피지 아래의 설정, 이미지와 규모를 확인합니다. 세포의 정량화에도 피지를 사용하십시오. </li></ol>

<ol>
	<li>Ebendal, T., Rush, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Use of collagen gels to bioassay nerve growth factor activity. IBRO Handh, (1989).</li><li>Heermann, S., Schm&#195;cker, J., Hinz, U., Rickmann, M., Unterbarnscheidt, T., Schwab, M. H., Krieglstein, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuregulin+1+type+III/ErbB+signaling+is+crucial+for+Schwann+cell+colonization+of+sympathetic+axons.">Neuregulin 1 type III/ErbB signaling is crucial for Schwann cell colonization of sympathetic axons.</a> PloS One. 6 (12), e28692 (2011).</li><li>Zuo, Y., Lubischer, J. L., Kang, H., Tian, L., Mikesh, M., Marks, A., Scofield, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+expressed+in+mouse+transgenic+lines+mark+subsets+of+glia,+neurons,+macrophages,+and+dendritic+cells+for+vital+examination.">Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.</a> The Journal of Neuroscience. The Official Journal of the Society for Neuroscience. 24 (49), 10999-11009 (2004).</li><li>Levi-Montalcini, R., Booker, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EXCESSIVE+GROWTH+OF+THE+SYMPATHETIC+GANGLIA+EVOKED+BY+A+PROTEIN+ISOLATED+FROM+MOUSE+SALIVARY+GLANDS.">EXCESSIVE GROWTH OF THE SYMPATHETIC GANGLIA EVOKED BY A PROTEIN ISOLATED FROM MOUSE SALIVARY GLANDS.</a> Proceedings of the National Academy of Sciences of the United States of America. 46 (3), 373-384 (1960).</li><li>Meintanis, S., Thomaidou, D., Jessen, K. R., Mirsky, R., Matsas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuron-glia+signal+beta-neuregulin+promotes+Schwann+cell+motility+via+the+MAPK+pathway.">The neuron-glia signal beta-neuregulin promotes Schwann cell motility via the MAPK pathway.</a> Glia. 34 (1), 39-51 (2001).</li><li>Yamauchi, J., Miyamoto, Y., Chan, J. R., Tanoue, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB2+directly+activates+the+exchange+factor+Dock7+to+promote+Schwann+cell+migration.">ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration.</a> The Journal of cell biology. 181 (2), 351-365 (2008).</li><li>Anton, E. S., Weskamp, G., Reichardt, L. F., Matthew, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+and+its+low-affinity+receptor+promote+Schwann+cell+migration.">Nerve growth factor and its low-affinity receptor promote Schwann cell migration.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (7), 2795-2799 (1994).</li><li>Mahanthappa, N. K., Anton, E. S., Matthew, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+growth+factor+2,+a+soluble+neuregulin,+directly+increases+Schwann+cell+motility+and+indirectly+promotes+neurite+outgrowth.">Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 16 (15), 4673-4683 (1996).</li><li>Yamauchi, J., Chan, J. R., Shooter, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+regulate+Schwann+cell+migration+by+activating+divergent+signaling+pathways+dependent+on+Rho+GTPases.">Neurotrophins regulate Schwann cell migration by activating divergent signaling pathways dependent on Rho GTPases.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (23), 8774-8779 (2004).</li><li>Gumy, L. F., Bampton, E. T. W., Tolkovsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycaemia+inhibits+Schwann+cell+proliferation+and+migration+and+restricts+regeneration+of+axons+and+Schwann+cells+from+adult+murine+DRG.">Hyperglycaemia inhibits Schwann cell proliferation and migration and restricts regeneration of axons and Schwann cells from adult murine DRG.</a> Molecular and Cellular Neurosciences. 37 (2), 298-311 (2008).</li><li>Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., Kashiwagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+spatiotemporal+dynamics+of+multicellular+cell-cycle+progression.">Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.</a> Cell. 132 (3), 487-498 (2008).</li><li>Park, D., Don, A. S., Massamiri, T., Karwa, A., Warner, B., MacDonald, J., Hemenway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+imaging+of+cell+death+using+an+Hsp90+ligand.">Noninvasive imaging of cell death using an Hsp90 ligand.</a> Journal of the American Chemical Society. 133 (9), 2832-2835 (2011).</li><li>Shcherbo, D., Souslova, E. A., Goedhart, J., Chepurnykh, T. V., Gaintzeva, A., Shemiakina, I. I., Gadella, T. W. J., Lukyanov, S., Chudakov, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+and+reliable+FRET/FLIM+pair+of+fluorescent+proteins.">Practical and reliable FRET/FLIM pair of fluorescent proteins.</a> BMC Biotechnology. 9, 24 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

주변 신경 시스템의 개발은 흥미로운 과정이다. 개발이 완료되면, axons는 인간에 자주 이상 100cm 할 수 있습니다 전체 길이를 따라 SCS에 의해 ensheathed 있습니다. 이를 위해 필요한 SCS의 정확한 숫자는 개발 중에 설립 할 수 있으며 SCS는 전체 axonal 범위를 확인하기 위해 주변에 확장 axons을 따라 이동해야합니다. 이 myelinated뿐만 아니라에 대한 unmeylinated axons에 대한 진정한 보유하고 있습니다. 두 경우...

<table border="1"><tbody><tr><td> 시약 / 재료의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 10X 가상 </td><td> Gibco </td><td> 21,430 </td><td></td></tr><tr><td> 나트륨 중탄산염 (7.5 %) </td><td> Gibco </td><td> 25,080 </td><td></td></tr><tr><td> 글루타민 </td><td> Gibco </td><td> 25,030 </td><td></td></tr><tr><td> NaOH </td><td> 머크 </td><td> 109137 </td><td></td></tr><tr><td> NGF </td><td> 로슈 </td><td> 1058231 </td><td> R &amp; D # 556-NG-100 </td></tr><tr><td> Neurobasal 중 </td><td> Gibco </td><td> 21,103 </td><td></td></tr><tr><td> B27 보충 </td><td> Gibco </td><td> 17,504 </td><td></td></tr><tr><td> 항생제 </td><td> Gibco </td><td> 15,640 </td><td></td></tr><tr><td> D-PBS </td><td> Gibco </td><td> 14,040 </td><td></td></tr><tr><td> 곤충 바늘 </td><td> FST </td><td> 26002-20 </td><td></td></tr><tr><td> 주사기 바늘 </td><td> 브라운 </td><td></td><td> BD # 300013 </td></tr><tr><td> 8 잘 챔버 슬라이드 </td><td> 랩 테크 </td><td> 177402 </td><td></td></tr></tbody></table>

analyzing murine schwann cell development along growing axons

주변 신경의 발달은 흥미로운 과정이다. 뉴런은 인간의 이상 100cm 거리에 신경의 소마에서 자주 구체적인 목표를 신경을 분포시키다하기 위해 axons을 보낼 수 있습니다. 개발하는 동안 Neuronal 생존이 목표 - 유래 성장 요인에뿐만 아니라 Schwann 세포 (SCS)의 지원에 따라 달라집니다. 시냅스 또는 신경 근육 접합부에 neuronal 소마 (또는 중앙에서 주변 신경계로의 전환)의 지역에서이를 SC의 ensheath의 axons 있습니다. Schwann 세포는 각각의 문장의 파생이며, 전체 축삭은 SCS으로 덮여 때까지 새로운 axons 함께 전구체로 마이그레이션됩니다. 이 주변 신경 시스템의 개발을위한 SC 마이그레이션의 중요성을 보여줍니다이 과정을 조사 할 필요성을 강조. SC 발전을 분석하기 위해, 설치는 이전에 자신의 생리 기판, 축삭을 포함하는 SCS 옆에있는 필요합니다. 자궁 개발로 인해 (뮤스 musculus) 마우스와 같은 태반 vertebrates에 적합하지 않을 수 있습니다. 이 우회하기 위해, 우리는 우수한 자궁 경부 신경절 (SCG) explant 기술을 적응. 신경 성장 인자 (NGF)와 치료 후 SCG의 explants은 신경절에서 주변에 axons 따라 이전 SC의 전구체 다음 axons을 확장합니다. 이 시스템의 장점은 SC는 SC 내생의 풀에서하고 동시에 성장하고 자신의 생리 axons 따라 마이그레이션이 파생된다는 것입니다. axons 따라 SC 개발이 SC 마이그레이션에 통찰력을 얻기 위해 더 가능성을 열어, 시간 경과 영상으로 분석 할 수 있기 때문에이 시스템은 특히 흥미로운입니다.

Axonal 성장은 NGF (4) (영화 체계 S1 그림 1 방식)과 치료에 explants를 SCG에서 용이하게되어 있습니다. 이 과정은 어떤 역 현미경을 이용하여 쉽게 볼 수 있으며 시간 경과 영상 (영화 S2)에 의해 다음 수 있습니다. 과학자 마우스 배아에서 SCG을 해부에 새로운이라면 강력 간단한 안티 - 티로신 hydroxylase (TH) immunohistochemistry에 의해 기술의 유효성 검사를하는 것이 좋습니다. TH는 라...

Watch this Scientific Journal Video about Analyzing Murine Schwann Cell Development Along Growing Axons at JoVE.com

Analyzing Murine Schwann Cell Development Along Growing Axons

여기 SCS는 확장 axons을 함께 개발 할 수 있습니다하는 Schwann 세포 (SC) 마이그레이션 분석을 설명합니다.

성장 Axons 함께 손쥐 Schwann 세포 개발 분석

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

analyzing-murine-schwann-cell-development-along-growing-axons

Research

JoVE Journal

Neuroscience

11.6K 조회수.  University of Freiburg. 여기 SCS는 확장 axons을 함께 개발 할 수 있습니다하는 Schwann 세포 (SC) 마이그레이션 분석을 설명합니다.

비디오: Analyzing Murine Schwann Cell Development Along Growing Axons

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

의 형태학의 분석 Drosophila</em유전 모자이크 사용하기> 애벌레 주변 감각 신경 세포의 Dendrites 및 Axons

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

연구

신경과학

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Rhizotomy 후 도설 루트 Axons의 라이브 영상

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

개발하고 성숙한 망막 신경 세포의 단일 세포 프로 파일링

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

배아 Murine 신경 튜브에서 신경 크레스트 세포의 분리 및 문화

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

쥐과 장내 신경 전구 세포에 대한 양이 세포 이동의 분석

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

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.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

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 ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

니코틴 유도 칼슘 신호 전달 및 복부 해마 축삭을 따라 신경 전달 물질 방출의 라이브 영상

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Electroporation에 의해 쥐 좌골 신경의 슈반 세포로 생체 내 유전자 전달에

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

선형 아가로 오스 채널을 사용하여 공부하기<em&gt; 초파리</em&gt; 애벌레 크롤링 동작

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

열 및 레이어에 수지상 형태학 분석

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...