이 작품은 웨인 S. Sossin으로 보건 연구 (CIHR) 부여 캐나다 연구소에 의해 지원되었다. 캐롤 A. 파라이 드 라 퐁 물리 엉 쌍떼 두 퀘벡 (FRSQ)와 콘라드 해링턴 교제에서 박사 후 교제의 수상자이다. 저자는 그래픽 개요 도움 비디오 촬영과 매들린 리치몬드 풀에 대한 도움 조애너 부기, 마가렛 헤이스팅스와 마가렛 Labban 감사하고 싶습니다.

1. 준비 및 Sf9 세포 Monolayer 문화의 유지 보수 <ol><li> Sf9 세포 배양 및 유지 관리는 조직 문화 후드에서 수행됩니다. </li><li> Sf9 세포 Spodoptera frugiperda의 난소 세포 (Sf21 세포)에서 파생되며 Invitrogen에서 그레이스의 미디어에 냉동 세포로 구입할 수 있습니다. </li><li> 보충 그레이스의 곤충 매체 (1X), 30 % 태아 소 혈청 (FBS) canted 목이 75cm 2 세포 배양 플라스크에 추가되었습니다. 장소 8 ML </li><li> (1-2 분) 앞뒤로 튜브를 이동하여 37 ° C의 물을 욕조에 빠르게 Sf9 세포의 냉동 튜브를 녹여. </li><li> 고르게 세포를 배포 손으로 부드럽게 75cm 2 세포 배양 플라스크와 암석 플레이트에 Sf9 세포를 전송합니다. </li><li> 세포가 첨부 ° C가까지 27 1-2 시간 품어. </li><li> 오래된 매체를 제거 30 % FBS와 freshGrace의 곤충 중간의 10 ML로 바꿉니다. </li><li> 세포가 합류 아르 ° C까지 27 알을 품다. </li><li> monolayer 문화를 유지하려면, Sf9 세포의 합류 술병에서 이전 미디어를 제거와 10 % FBS와 그레이스의 곤충 중간의 5 ML를 추가합니다. </li><li> 가볍게 술병의 측면에 Sf9 세포를 분리하기 위해 여러 번 누릅니다. </li><li> 피펫은 아래 pipetting 동안 플라스크 벽을 분사 한번 부드럽게, 위아래 10 ML 피펫과 pipettor을 미디어 사용합니다. 멸균 15 ML Polysterene 튜브에 세포를 전송합니다. </li><li> 손으로 부드럽게 새로운 75cm 2 세포 배양 플라스크와 바위 플레이트에서 10 % FBS (로그 위상 성장을 유지하기 위해 1시 5분 희석)와 그레이스의 곤충 중간 8 ML에 Sf9 세포 현탁액 2 ML을 추가합니다. </li><li> 세포가 합류 아르 ° C까지 27 알을 품다. </li></ol> 2. Sf9 세포에서 찬란 태그 PKCS 표현 <ol><li> 에 대한 라이브 영상 실험, 문화 35mm MatTek 유리 바닥 문화 disheswith 14mm와 0.16-0.19 mm의 coverslip 두께의 유리 표면에 Sf9 세포. </li><li> 형광 단백질 태그 PKCS는 아래에 설명된 수정 제조 업체의 Cellfectin II의 transfection 시약 다음과 같은 권장 사항을 사용 Sf9 세포에 표현됩니다. </li><li> 1 일 : 전에 세포를 도금하기 위해 최소 30 분 10 % FBS와 그레이스의 곤충 중간 각 MatTek 요리를 취급. </li><li> hemacytometer를 사용하여 11 단계 (위)에서 Sf9 세포를 계산합니다. </li><li> 플레이트 0.05 150 μL의 총 볼륨의 각 MatTek 유리 coverslip에 X 10 6 세포. 세포가 실온에서 30 분 첨부할 수 있습니다. </li><li> 그레이스는 세포를 분리하지 않도록 천천히 한 번에 하나씩 각 접시 하나 ML 10 % FBS와 보충의 2 ML을 추가합니다. </li><li> ° C 하루 27 알을 품다. </li><li> 이 시점부터, 플라스미드 DNA와 Sf9 세포의 transfection에 대한 제조 업체의 추천을 사용합니다. </li><li> 일 2 : Cellfectin II의 transfection 시약을 사용하여 찬란 태그 PKCS와 Transfect Sf9 세포. 우리는 종종 향상된 그린 형광 단백질 (eGFP) 및 monomeric 레드 형광 단백질 (mRFP) (플라스미드 건설에 대한 세부 이전 2,5 설명했습니다)로 태그가이 시스템 PKCS 공동 표현했습니다. 한 번에 하나의 형광 단백질의 표현이 70-80 % 정도 세포를 표현 72시간 게시물 - transfection을 산출. 이 형광 단백질의 공동 표현은 약 30 % 공동 표현 세포에 transfection 효율을 감소시킵니다. eGFP - 태그 PKC 및 mRFP - 태그 PKC를 공동 표현하면, 최적의 단백질 표현 mRFP - 태그 단백질에 대한 더 플라스미드 DNA를 2 배 사용합니다. </li><li> 수행 라이브 영상 48-72시간 포스트 transfection (최적 단백질 표현, 72 시간 기다). </li></ol> 3. Aplysia 감각 뉴런의 찬란 태그 PKCS 표현 <ol><li> Aplysia의 연결을 세포 배양의 준비는 조 및 동료 4의 비디오 문서에 설명되어있다. </li><li> 형광 단백질 태그 PKCS는 아래 자세한 microinjection 절차를 사용 Aplysia 감각 뉴런으로 표현됩니다. </li><li> 증류수에 1퍼센트 빠른 그린 (FCF)의 주식 솔루션을 준비, 주사기와 주사기 28mm 필터 (0.20 μm의)을 사용하여 필터링합니다. -20 ° C.에서 나누어지는 및 저장 </li><li> 감각 뉴런의 Microinjection은 뉴런를 분리 후 1 일 또는 2 일에서 수행하지만 우리는 이일 기다리는 것은 coverslip 더 나은 휴대 준수에 대한 허용 찾을 수 있습니다. </li><li> 일 2 감각 뉴런의 분리 후, 빠른 그린의 나누어지는를 해동하고 주사기와 주사기 28mm 필터 (0.20 μm의)를 사용하여 다시 그것을 필터링합니다. </li><li> 0.5 % 빠른 그린을 포함하는 증류수에 플라스미드 DNA의 솔루션을 준비합니다. μg / μL 0.4처럼 높은 플라스미드 DNA의 농도는 사용할 수 있지만 이러한 값을 위의 이동하지 않는 것이 좋습니다. </li><li> 1 ML의 주사기와 4mm 주사기 필터 (0.20 μm의)를 사용하여 플라스미드 DNA / 고속 그린 솔루션을 필터링합니다. </li><li> 마이크를 사용하여 15 분 동안 16,110 XG에서 플라스미드 DNA / 고속 그린 솔루션을 원심 분리기rocentrifuge. </li><li> microelectrode의 풀러를 사용하는 뉴런의 microinjection (서터 플라밍 / 브라운 Micropipette 풀러, 모델 P - 97)에 대한 날카로운 전극을 준비합니다. 상자 필라멘트를 사용하여 1mm 외경, 내부 직경 0.75 mm와 내부 필라멘트 (세계 정밀 계측기가 TW100F - 4)와 얇은 벽을 유리 모세관을 사용하여 100mm 길이로 유리 microelectrodes를 가져옵니다. microinjection 전극을 당기에 대한 자세한 내용은 서터 전극 요리책을 참조하십시오. </li><li> microloader 피펫 팁 (eppendorf CA32950 - 050)를 사용하여 플라스미드 DNA / 빠른 그린 솔루션 2 μL와 각각의 전극을 입력합니다. </li><li> microinjection 역에 Aplysia의 연결 문화를 포함하는 MatTek 유리 바닥 문화 요리를 전송합니다. </li><li> microinjection 역은 다음으로 구성되어 있습니다 : 집에서 만든 진동 절연 테이블 (운동 시스템 진동 절연 워크 스테이션), 외부 할로겐 조명과 스테레오 - 범위, micromanipulator (서터 헉슬리 월 종류 Micromanipulator의 문화 요리를 잡아 큰 유리 단계 하는)는 세 개의 축에 굵고 울트라 좋은 위치 모두를 허용, 공압 펌프 (세계 정밀 계측기 PV820 공압 피코 - 펌프)에 연결된 microelectrode 홀더. 공압 펌프의 압력 입력은 압축된 질소 탱크에 연결됩니다. </li><li> microelectrode 홀더에 microelectrode를 넣습니다. </li><li> 스테레오 현미경으로 보는 세포의 핵에 micropipette의 끝부분을 삽입합니다. </li><li> 질소 부족 압력 펄스 제공 (기간이 10-100 MS를, 20파운드 / 2) 핵은 녹색 uniformely 될 때까지. </li><li> 실온에서 4 시간 동안 세포를 부화하고 ° C까지 사용 4 계속. 24시간 후 사출 - 최적 PKC translocation 들어, 라이브 영상 실험 20 수행합니다. </li></ol> 4. Sf9 세포를 생활과 Aplysia 감각 뉴런에서 PKC Translocation의 시각화 <ol><li> 이미징을 위해, 우리는 Axiovert 200 자이스 혈구 LSM510 거꾸로 공촛점 현미경을 사용합니다. </li><li> Aplysia 감각 뉴런은 x40 오일 침지 목적으로 몇 군데 반면 Sf9 세포 x63 기름 침지 목적으로 몇 군데 위치 : 영상 프로토콜은 다음을 제외하고 Sf9 세포와 Aplysia 감각 뉴런에 동일합니다. Aplysia 감각 뉴런이 18-20 O C. 주위 유지 실온에서 몇 군데 반면 Sf9 세포는 26-27 O C에서 유지 온도 제어 챔버에 몇 군데 아르 </li><li> 우리는 eGFP 단백질과 mRFP 단백질 태그 이미지 PKC에 HeNe 레이저 (543nm에서 여기)와 태그 이미지 PKC 50 %의 레이저 출력 30 MW 아르곤 레이저 (488nm에서 여진)을 사용합니다. 아르곤과 HeNe 레이저 라인 4 %로 감쇠하고 사전 이미징 및 핀홀을 각각 50 %의 전송 출력은 하나 조정됩니다. </li><li> 이미징 무대에서 안정화 문화 요리를하고 운동이나 진동을 방지하는 것이 중요합니다. </li><li> 부드럽게 접시 1 ML 전체 볼륨을 떠나는 10 ML의 피펫을 사용하여 요리의 문화 미디어 1 ML를 제거합니다. </li><li> 핵을 볼 수있는 세포의 중간 부분에 사진을 가져가라. </li><li> 사진 매 30 초 복용 10-20 공촛점 이미지의 시계열을 설정합니다. </li><li> 시간 시리즈를 시작합니다. </li><li> 이 그림은 (30 초 시점 이후) 촬영한 후, 부드럽게 약 1 ML (2X 농도) P1000 피펫을 사용하여 세포의 상단에 드롭하여 놓기를 추가합니다. 약물은 약 ​​30 초 동안 지속적으로 추가됩니다. </li><li> 일부 약물은 다른만이 최적의 5-10분 후 치료 (영화 1 영화 2)는 느린 translocation을 유도하는 동안 (60 초 시점에서) 바로 다음과 같은 약물 치료를 볼 수있는 빠른 translocation을 유도. </li><li> 약이 씻겨해야하는 경우, 접시 한쪽에 하나 피펫과 함께 부드럽게 아래로 세척 버퍼를 pipetting과 반대편에있는 미디어를 pipeting, 세탁을 2 X 10 ML의 pipets를 사용합니다. </li><li> 세척 효과되었으며 약물의 영향이 가능해 경우, PKC는 30-60초 이내 cytosol로 돌아갑니다.한다면 </li><li> LSM 파일로 동영상을 저장합니다. </li><li> LSM 파일을 열고 이미지 전후 약물 치료 수치 NIH 이미지 J 소프트웨어를 사용합니다. 부량는 다른 하나 설명되었습니다. </li></ol>

<ol>
	<li>Farah, C. A., Nagakura, I., Weatherill, D., Fan, X. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+role+for+phosphatidic+acid+in+the+translocation+of+the+novel+protein+kinase+C+Apl+II+in+Aplysia+neurons.">Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons.</a> Mol Cell Biol. 28, 4719-4733 (2008).</li><li>Zhao, Y., Leal, K., Abi-Farah, C., Martin, K. C., Sossin, W. S. &. a. m. p. ;. a. m. p., Klein, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.Isoform+specificity+of+PKC+translocation+in+living+Aplysia+sensory+neurons+and+a+role+for+Ca2+-dependent+PKC+APL+I+in+the+induction+of+intermediate-term+facilitation.">M.Isoform specificity of PKC translocation in living Aplysia sensory neurons and a role for Ca2+-dependent PKC APL I in the induction of intermediate-term facilitation.</a> J Neurosci. 26, 8847-8856 (2006).</li><li>Farah, C. A., Weatherill, D., Dunn, T. W. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC+differentially+translocates+during+spaced+and+massed+training+in+Aplysia.">PKC differentially translocates during spaced and massed training in Aplysia.</a> J Neurosci. 29, 10281-10286 (2009).</li><li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures.">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J Vis Exp. , (2009).</li><li>Manseau, F., Fan, X., Hueftlein, T., Sossin, W. S., Castellucci, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-independent+PKC+Apl+II+mediates+the+serotonin+induced+facilitation+at+depressed+synapses+in+Aplysia.">Ca2+-independent PKC Apl II mediates the serotonin induced facilitation at depressed synapses in Aplysia.</a> J. Neuroscience. 21, 1247-1256 (2001).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리는 Sf9 세포와 감각 뉴런 Aplysia에서 실시간으로 찬란 태그 PKCS의 이미지 translocation에 기술을 설명합니다. culturing 그들을 transfecting 아주 스트레이트 포워드이기 때문에 Sf9 세포 이미지 PKC translocation하는 간단한 시스템을 제공합니다. 대조적으로, Aplysia 감각 뉴런의 microinjection은 주인에게 몇 시간이 걸립니다, 최대 몇 개월까지. 이 기법에 관한 어려움 전극 막히는가 포함되어 있습...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sf9 frozen cells</td>
<td>Spodoptera frugiperda ovarian cells</td>
<td>Invitrogen</td>
<td>B825-01</td>
<td>&nbsp;</td>
<tr>
<td>Grace&#x2019;s Insect Medium, Supplemented (1X), liquid</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11605-094</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Corning 75cm2 canted neck cell culture flask</td>
<td>Tool</td>
<td>Corning</td>
<td>430720</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>CanSera</td>
<td>CS-C08-500</td>
<td>Supplement Grace's media with FBS prior to use</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309604</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309602</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431229</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431212</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Glass Bottom Dish</td>
<td>Tool</td>
<td>Mattek</td>
<td>P35G-1.5-14-C</td>
<td>Use to culture Sf9 cells prior to transfection and imaging</td>
</tr>
<tr>
<td>Cellfectin II Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10362-100</td>
<td>Used to express fluorescently tagged PKCs in Sf9 cells</td>
</tr>
<tr>
<td>Fast Green FCF</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F-7252</td>
<td>Use to visualize microinjection of plasmid DNA solution into Aplysia sensory neurons</td>
</tr>
<tr>
<td>Thin wall glass capillaries</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>Use to make microelectrodes for microinjection</td>
</tr>
<tr>
<td>Microloader pipette tip</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>CA32950-050</td>
<td>Autoclave before using to fill up the microelectrodes for microinjection</td>
</tr>
<tr>
<td>PV820 Pneumatic PicoPump </td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
<td>Part of microinjection station</td>
</tr>
<tr>
<td>Microelectrode holder</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>MPH6S</td>
<td>Use to hold microelectrode </td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tool</td>
<td>Sutter</td>
<td>MP-85</td>
<td>Use to position microelectrode in the three-axis</td>
</tr>		</tbody>
		</table>
		</div>

live-imaging of pkc translocation in sf9 cells and in aplysia sensory neurons

단백질 키나제 고사 (PKCS)는 같은 세포 성장과 학습과 메모리로 휴대 프로세스의 다양한 규제에 중심 역할을 트레오닌 kinases 세린입니다. 척추 동물에서 PKC isoforms 네 가지 알려진 가족이 있습니다 : 고전 PKCS (α, βI, βII 및 γ), 소설 형식 내가 PKCS (ε와 η), 소설 유형 II PKCS (δ와 θ) 및 비정형 PKCS (ζ 및 ι ). 고전 PKCS는 칼슘이 활성화하는 소설 PKCS는 가리비 활성화하지만, 칼슘 2 + - 독립하는 동안 +와 diacylclycerol (가리비). 비정형 PKCS는 +이나 가리비 칼슘이도에 의해 활성화됩니다. Aplysia californica에서는, 메모리 형성을 공부하기 위해 모델 시스템, 즉 세 신경계 특정 PKC isoforms 각 주요 클래스에서 하나 기존의 PKC APL I, 소설 종류 I PKC APL II 및 비정형 PKC APL III있다. PKCS는 지질 활성화 kinases되며 따라서 세포 신호에 대한 응답으로 고전과 소설 PKCS의 활성화가 자주 세포질에서 플라즈마 막에 PKC translocation와 상관되었습니다. 따라서, 라이브 세포에서 실시간으로 PKC translocation을 시각화하는 것은 PKC 활성화로 이어질 신호 전달 경로를 elucidating위한 소중한 도구가되고있다. 예를 들어,이 기술은 PKC의 다른 isoforms은 시냅스 소성의 독특한 유형을 중재하기 위해 서로 다른 조건에서 이동시키다 및 PKC APL II의 세로토닌 (5HT) 활성화가 가리비와 phosphatidic 산성 (PA) 모두의 생산을 필요로 설립 우리에게 허용되었습니다 translocation 1-2. 중요한 것은, 같은 신경을 시각화하는 능력은 반복해서 우리가 예를 들어, 정교한 세부 3 PKC 응답의 desensitization를 측정할 수있다. 이 비디오에서는, 우리는 Sf9 세포 문화를 준비하는 각 단계를 보여, Aplysia 감각 뉴런의 문화가에 대한 응답으로 Sf9 세포에서 Aplysia 감각 뉴런과 PKC translocation 라이브 영상에 찬란 태그 PKCS을 표현, 다른 영상 제 4에 설명되어있다 레이저 스캐닝 현미경을 사용하여 다른 activators.

Watch this Scientific Journal Video about Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons at JoVE.com

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

이 비디오에서는, 우리는 찬란 태그 PKCS을 사용하여 살아있는 세포에서 PKC translocation의 시각화를 보여줍니다.

Sf9 세포에서 Aplysia 감각 뉴런에서 PKC Translocation의 라이브 영상

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

live-imaging-pkc-translocation-sf9-cells-aplysia-sensory

Research

JoVE Journal

Neuroscience

11.9K 조회수.  McGill University. 이 비디오에서는, 우리는 찬란 태그 PKCS을 사용하여 살아있는 세포에서 PKC translocation의 시각화를 보여줍니다.

비디오: Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

배아 척수에서 뉴런의 Commissural 해부와 문화

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

연구

신경과학

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation 및 Mitochondrial 교통 : 긴 기간이 이상 Hippocampal 뉴런의 라이브 영상

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

강한 감각 뉴런의 Lentivirus - 중재 유전자 조작 및 시각화 생체내에</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

강한 감각 뉴런의 속눈썹에 갇힌 화합물의 플래시 광분해

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Extracellularly의 근육 모터 풀에 모터 뉴런을 확인<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

의 감각 뉴런의 분리<em&gt; 군소 californica</em&gt; 글루타메이트 전류의 패치 클램프 녹음을위한

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

에있는 후각 수용체 뉴런의 전체 마운트 Immunolabeling<em&gt; 초파리</em&gt; 안테나

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Method는 목표와 마우스의 격리기도 - 근에 분포하는 감각 뉴런하기

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...