이 연구 프로젝트는 ARRA 보충, 5 R01 NS056380에 의해 지원되었다. 추가 지원은 바이러스 성 벡터 코어와 모리 신경 NINDS 핵심 시설 보조금, P30NS055077의 현미경 코어를 통해 제공되었다. 안정 SSTR3-GFP IMCD3 세포 라인에 대한 그렉 Pazour; 우리는 GENSAT에서 마우스 선을 파생시키기위한 모리 형질 전환 마우스 및 유전자 타겟팅 코어를 감사하고 Sstr3-GFP 렌티 바이러스에 대한 브래들리 Yoder는 구성. 단일 클론 항체는 NICHD의 후원하에 개발 된 발달 연구 브리 도마 은행에서 얻은 아이오와 대학교 생물 과학부, 아이오와 시티, 아이오와 52242에 의해 유지되었다. 모든 동물 절차 IACUC에 모리 대학의 바이오 안전성위원회에 의해 승인되었습니다.

성인 마우스는 기계 자궁 전위에 의해 안락사 있습니다. 모든 동물 절차 IACUC에 모리 대학의 바이오 안전성위원회에 의해 승인되었습니다. 1. 배아 생성 <ol><li> 크로스 타목시펜 유도 크레 라인 CAGGCreER 및 dsRedCre 기자 선 (전이 (CAG-Bgeo, -는 DsRed * MST) 1Nagy) (그림 1) 5,6. 딸 세포, 간 CAGGCreER 및 Olig2-EGFP BAC 형질 전환 생쥐 (전이 (Olig2-EGFP) EK23Gsat) (그림 1) 7,8과 DsRed를 줄 쉬 응답 상대 타이밍을 모니터링 할 수 있습니다. </li><li> 100 % 에탄올에 타목시펜을 녹여 크레 발현과 세포의 작은 하위 집합에서 DsRed를 라벨을 유도하는 배아 6.5 (E6.5)에서 임신 여성에 체중 40g 당 2.5 밀리그램 타목시펜의 복강 내 주입을 수행합니다. </li><li> 48 시간 나중에, 배아를 해부 형광 현미경을 사용하여 DsRed를 긍정적 인 배아를 식별 문화 접시로 전송하고 observ생체 이미징 동안 전자 단일 세포 분열 (아래 참조). </li></ol> 2. 전체 마우스 배아 문화 <ol><li> 10 %의 신생아 송아지 혈청과 1 % 페니실린 / 스트렙토 마이신 (P / S) 9로 보충 DMEM/F12 (1:1)를 포함하는 미리 예열 세척 매체에 E8.5 배아를 해부. </li><li> 직접 형광 현미경 37 ° C 가열 무대에서 해부, 장소 배아 후와 GFP 및 / 또는 DsRed를 플러스 (아래 참조)로 식별합니다. </li><li> L-글루타민과 보충 페놀 레드없이 50 %의 Sprague-Dawley 계 수컷에서 혈청 쥐와 50 %의 DMEM/F12 (1:1)를 포함하는 사전 평형 배지 500 μL 강하의 1 %에 2 배아까지 이동 0.85 % 염화나트륨 및 P / S 9 1 M의 HEPES. </li><li> 증발을 방지하고 배양 접시는 37 ° C, 5 % CO 2 배양기에 배아를 포함하는 전송 매체를 통해 평형 가벼운 미네랄 오일의 얇은 층 (0.1 cm)를 적용합니다. </li></ol> 3.바이러스 감염 <ol><li> neuroepithelium의 레이블 섬모하기 위해서는, E8.5 배아를 포함하는 배양액 500 μL 평형 하락 Sstr3-GFP 렌티 5 ~ 10 μL, 약 2 백만 비리를 추가합니다. </li><li> 문화의 18 시간 후, 바이러스를 씻어 내고 신경관 슬라이스를 준비하는 세척 매체의 몇 방울에 감염된 배아를 전송합니다. </li></ol> 4. 라이브 영상에 대한 신경 튜브 슬라이스 준비 <ol><li> 1 %의 한천 코팅 접시에, 마이크로 칼 크기 0.025 mm를 사용하여 미리 예열 세척 매체 E8.5 태아의 신경관을 해부하다. </li><li> 35mm 폴리-L-라이신 코팅 유리 바닥 접시에 페놀 레드 않고 평형 배양액 150 μL 드롭 고립 된 신경관 복부 측면을 놓습니다. </li><li> 를 위해 유리 커버 슬립의 좁은 조각에 의해 100 % 순수 석유 젤리 및 장착 신경관 주위에 녹은 초 왁스 (IKEA에서 촛불) 부드럽게 눌러 만든 1:1 혼합물의 소량을 넣어샘플을 고정시킨다. </li><li> 평형 가벼운 미네랄 오일의 얇은 층 (~ 0.1 cm) (그림 2)와 접시를 커버. </li></ol> 5. 이미징 및 시간 경과 공 촛점 현미경을 살고 <ol><li> 온도 조절 환경 챔버를 갖추고 공 초점 거꾸로 현미경 스캐닝 니콘의 A1R 레이저 아래 장소 접시, 37으로 설정 ° C, 5 % CO 2. </li><li> 40X 기름 침지 목적 사용 Olig2-GFP와 DsRed를 양성 세포를 모니터링하는 동안 GFP 표시 섬모와 DsRed를 양성 세포를 기록 60X 기름 침지 목표를 사용합니다. </li><li> 시간 경과 이미징 조건을 설정하는 NIS 요소 소프트웨어를 엽니 다. 매 10 분 1.5 μm의 (40X 목적)와 0.4 μm의 (60X 목적)의 간격이 최대 8 ㎛의 간격으로 Z-스택에 25 μm의 최대를 획득. 필요한 경우 488와 561 nm의 여기 파장 및 전송 채널을 사용합니다. 512 X 512 사이즈로 이미지를 획득. INT의 여러 사용자 정의 영역을 설정erest 동시에 녹음을 수행 할 수 있습니다. 스캔 속도 1, 선 평균 2 및 핀 구멍의 크기 (61, 이미지의 레이저 전력 및 밝기 최적의 노출로 사용 낮은 레이저 강도 (4.5 %까지 EGFP 488분의 405 mCherry 561의 경우 최대 11 %)로 조정되었다 DsRed를위한 μM, Olig2 28.1 μM, SSTR3GFP 72 μm의, DsRed를하고 SSTR3GFP을위한 61.3 μm의, DsRed를하고 Olig2 위해 58 μm의). 유전자 인코딩 형광 기자의 사용은 우리가 낮은 레이저 파워의 밝기를 감지 할 수있었습니다. </li><li> Imaris 3D 재구성 소프트웨어를 사용하여 기록 된 데이터를 분석합니다. </li></ol> 6. 면역 <ol><li> 유리 접시에 1 시간 동안 얼음에 4 % 파라 포름 알데히드 / 0.1 M 인산 버퍼 (4 ° C)에서 배아 또는 격리 된 신경 튜브를 고정합니다. </li><li> 샘플에게 얼음에 PBS에서 2 시간 (4 ° C) (변경 PBS 몇 번)와 하룻밤 또는 배아 싱크까지 30 % 자당 / 0.1 M 인산 버퍼에 넣어 씻는다. </li><li> -80에서 드라이 아이스 및 저장 ° C.에, 10 월에 동결을 포함 피그라 이오 스탯 (10 ㎜)에 단면을 erform. </li><li> 1 % 열 - 불 활성화 된 양 혈청을 포함하는 세척 용액에 10 분간 0.1 % PBS에 트리톤 X-100에 대한 슬라이드를 씻으십시오. </li><li> 농도를 다음에서 세척 용액에 기본 항체를 희석 : 쥐 단일 클론 항-RFP (5F8), 1:200, 토끼 반대로 Arl13b 혈청 1:1500 및 마우스 단클론 항-Arl13b 1시 5분 (295B/54) 토끼 반대로 Olig2 1:300, 마우스 monoclonals PAX6, 쉬와 Nkx2.2 모두 1:10과 Ki67 1:500 클론 토끼. 평면 가습 챔버 슬라이드 당 150 ㎕의 주위에 추가 건조 방지와 4에서 밤에 남겨 ° C로 파라 필름 (parafilm)와 커버 </li><li> 세 번, 20 분마다 상온에서 세척 용액에 슬라이드를 씻으십시오. </li><li> 1:200 농도 세척 솔루션 차 항체 알렉사 플 루어 488, 568, 350 희석. 핵 얼룩 훽스트 1:3,000 또는 TO-PRO-3 1:500을 사용합니다. 슬라이드 당 150 μl를 추가, 빛으로부터 보호 가습 챔버 실온에서 1 시간을 둡니다. </li><li> 세척 용액 TW에서 슬라이드를 씻으십시오오 시간, 실온에서 30 분마다. </li><li> 마운트는 24 시간 내에 골드 안티 - 페이드 시약 및 뷰를 연장 80 %에 슬라이드. </li></ol>

<ol>
	<li>Yamada, T., Pfaff, S. L., Edlund, T., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+pattern+in+the+neural+tube:+motor+neuron+induction+by+diffusible+factors+from+notochord+and+floor+plate.">Control of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate.</a> Cell. 73, 673-686 (1993).</li><li>Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M., Edlund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+induces+the+differentiation+of+ventral+forebrain+neurons:+A+common+signal+for+ventral+patterning+within+the+neural+tube.">Sonic hedgehog induces the differentiation of ventral forebrain neurons: A common signal for ventral patterning within the neural tube.</a> Cell. 81, 747-756 (1995).</li><li>Briscoe, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeodomain+Protein+Code+Specifies+Progenitor+Cell+Identity+and+Neuronal+Fate+in+the+Ventral+Neural+Tube.">Homeodomain Protein Code Specifies Progenitor Cell Identity and Neuronal Fate in the Ventral Neural Tube.</a> Cell. 101, 435-445 (2000).</li><li>Berbari, N. F., Johnson, A. D., Lewis, J. S., Askwith, C. 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관심 없음 충돌 선언하지 않습니다.

우리 전직 생체 시스템은 우리가 직접 실시간으로 개발 neuroepithelium 내에서 단일 세포 분열을 관찰 할 수 있습니다. 예를 들어 우리는 마우스 배아 신경관에서 세포 분열을 검토하고 섬모 형성이나 쉬 응답 중 하나를 감시. 우리는 우리의 기술 생리 학적으로 관련 데이터를 제공 나타내는 고정 부분 (n은 = 178)의 결과와 일치 하였다 우리의 이미징 결과 (N = 24)을 확인했다. <p c...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Z/RED line (STOCK Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J</td>
			<td>Jackson Laboratory</td>
			<td>005438</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olig2-eGFP line (STOCK Tg(Olig2-EGFP)EK23Gsat/Mmcd</td>
			<td>MMRRC,</td>
			<td>010555-UCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CAGGCreER</td>
			<td>Jackson Laboratory</td>
			<td>003724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1)</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Lonza</td>
			<td>14-416F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat Serum SD male</td>
			<td>Harlan Bioproducts</td>
			<td>4520</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1M Hepes</td>
			<td>BioWhittaker</td>
			<td>17-737E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sstr3-GFP lentivirus</td>
			<td>Emory Viral Core</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro-knife, size 0.025 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>62091</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-L-lysine coated glass bottom dish</td>
			<td>MatTek</td>
			<td>P35GC-0-10-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100% petroleum jelly</td>
			<td>Kroger</td>
			<td>FL9958c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A1R Laser Scanning Confocal Inverted Microscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NIS Elements software</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Imaris 3D software</td>
			<td>Bitplane AG</td>
			<td>Imaris 7.2.3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat-inactivated sheep serum</td>
			<td>Invitrogen</td>
			<td>16210-072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Parafolmaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffer</td>
			<td>Lab made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-RFP (5F8)</td>
			<td>Chromotek</td>
			<td>110411</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Arl13b serum</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-Arl13b 1:5</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Olig2</td>
			<td>Chemicon</td>
			<td>AB9610</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Pax6</td>
			<td>Developmental Hybridoma Bank</td>
			<td>Pax6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonalsShh</td>
			<td>Developmental Hybridoma Bank</td>
			<td>5E1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Nkx2.2</td>
			<td>Developmental Hybridoma Bank</td>
			<td>74.5A5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal Ki67</td>
			<td>Abcam</td>
			<td>AB15580</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568</td>
			<td>Molecular Probes</td>
			<td>A11031</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 350</td>
			<td>Molecular Probes</td>
			<td>A11046</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Fisher</td>
			<td>AC22989</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TO-PRO-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ProLong Gold anti-fade reagent</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

ex vivo live imaging of single cell divisions in mouse neuroepithelium

우리는 형광 마커와 배아 마우스 neuroepithelium의 배양 조각의 라이브 영상을 통합하는 시스템을 개발했다. 우리는 추적 혈통 유전 셀에 대한 기존의 마우스 선을 이용했다 : 타목시펜 유도 크레 라인과 크레-매개 재조합에 DsRed를 표현하는 크레 기자 라인. 타목시펜의 상대적으로 낮은 수준을 사용함으로써, 우리는 우리가 각각의 세포 분열을 따르도록 허용하는 세포의 작은 수의 재조합을 유도 할 수 있었다. 또한, 우리는 Olig2-EGFP 유전자 변형 라인 1-3를 사용하여 소닉 고슴도치 (쉬) 신호에 대한 전사 반응을 관찰하고 우리는 섬모 마커, Sstr3-GFP 4를 표현 바이러스 배양 슬라이스를 감염시켜 섬모의 형성을 감시. 이미지 순서 neuroepithelium, 우리는 신경 튜브를 절연, E8.5에서 배아를 수확, 이미징 챔버에 적절한 배양 조건에서 신경 슬라이스를 장착 시간 경과 공 촛점 이미징을 수행 하였다. 우리의 예생체 라이브 영상 방식은 우리가 생리학 관련 방식으로 차 섬모 형성과 쉬 응답 상대 타이밍을 평가하기 위해 단일 세포 분열을 추적 할 수 있습니다. 이 방법은 쉽게 구별 형광 마커를 사용하여 적응하고 필드에게 현장에서 실시간으로 세포의 행동을 모니터링되는 도구를 제공 할 수 있습니다.

여기에서 우리는 E8.5 마우스 neuroepithelium 내에서 단일 세포 분열의 생체 라이브 영상을 시행 하였다. 개별 셀에 레이블을 지정하려면, 우리는 재조합 5,6 (그림 3A)에 DsRed를 표현 크레 기자 라인을 포함하는 셀의 하위 집합에 Cre 호텔 재조합을 유도. 따라서, 48 시간 후에 우리는 생체 이미징 (그림 4A-D) 중 하나의 세포 분열을 관찰 할 수 있었다. 1-3,?...

Watch this Scientific Journal Video about Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium at JoVE.com

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

여기에서 우리는 필요한 도구를 개발<em&gt; 생체</em&gt; 라이브 영상은 마우스 E8.5 neuroepithelium에서 단일 세포 분열을 추적

생체 마우스 Neuroepithelium에서 단일 세포 분열의 라이브 영상

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

ex-vivo-live-imaging-of-single-cell-divisions-in-mouse-neuroepithelium

Research

JoVE Journal

Neuroscience

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

10.3K 조회수.  Emory University School of Medicine. 여기에서 우리는 필요한 도구를 개발 생체 라이브 영상은 마우스 E8.5 neuroepithelium에서 단일 세포 분열을 추적

비디오: Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

여러 마우스 Neuroanatomical 자기 공명 영상

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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. ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

개발 Neuroepithelium의 세포 문제의 고해상도 라이브 영상

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

개발 마우스 배아의 피질 유사 분열의 라이브 영상

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

두 광자<em&gt; 생체</em&gt; 시닝 두개골 준비를 사용하여 마우스 피질 돌기 쪽의 영상

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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 ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

영상 모니터 세포 생물학과 여러 신경 인구의 계보 진행을 추적 하는 단일 셀 다음 라이브

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

단일 셀 해상도 형광 애벌레 뇌 문화에 살고 있는 초파리 Circadian Clock의 이미징

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

생체 마우스 Neuroepithelium에서 단일 세포 분열의 라이브 영상

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