이 연구는 입슨 재단에 의해 일부 자금했다. IMV는 분자 생물학의 미국 암 협회 교수이며, 모범적인 생명 과학의 어윈 제이콥스와 조안의 의자를 보유하고 있습니다. 이 작품은 NIH, Leducq 재단 Meriaux 재단 엘리슨 의료 재단 입슨 / Biomeasure, Sanofi Aventis, 전립선 암 재단과 HN과 프랜시스 C. 버거 재단의 보조금에 의해 일부 지원되었다. PAS은 신경 과학 및 약물 남용 (R01 DA019022)에있는 국립 연구소에 대한 맥나이트 엔다우먼트 기금 후원됩니다. 저자 마크 슈미트 감사하고 싶습니다. 그래픽 아트는 제이미 T. 사이먼에 의해 디자인되었다. 스폰서가 친절하게 Invitrogen에 의해 제공되었다.

1. Organotypic 소뇌 문화 : <ol><li> 성공적으로 생성하려면 organotypic 소뇌 문화 P0 - P3 또는 P8 - P12 사이에 마우스를 사용합니다. 이 발달 단계가 우리의 연구에 대한 더 적절한했기 때문에 우리가 실험을 위해 우리는 P8 - P12 범위에서 마우스를 사용합니다. </li><li> 해부 및 배지 준비 : <ol><li> 뇌 해부와 슬라이스 준비는 1mM의 염화칼슘을 포함 낮은 나트륨 인공 뇌척수 (ACSF), 10mM D - 포도당, 4mm짜리 칼륨 염화물, 5mM 마그네슘 염화물, 26mM 나트륨 중탄산염, 246mM 자당 및 페놀 레드 용액 (1:1000)에서 이루어졌습니다 , 산도 7.3. 솔루션을 소독하기 위해 4 0.22 μm의 필터와 저장소를 통해 필터 ° C.를 </li><li> 소뇌 조각은 75 % 최소 필수 중간 이글 (멤), 25 % 열 inactivated 말 혈청, 25mM HEPES, 1mM 글루타민, 5mg / ML 포도당, 페니실린 (100 U / ML)과 스트렙토 마이신 (100 U / ML)를 양식하고 있습니다. 2 주간 최대 4 0.22 μm의 필터와 매장 ° C를 통해 매체를 필터링합니다. </li></ol></li><li> 그것이 빨간색에서 주황색으로 이동 있도록 이전 Carbogen (95 % 산소와 5 % 이산화탄소) 참고로 부풀어 오른 얼음처럼 차가운 ACSF 매체에서 머리를 절개하다, Carbogen 가진 채도는 솔루션의 산도가 변경됩니다. 절개는 뇌의 구조를 손상하지 않도록주의하고 가능한 시간의 최소 금액 수행하여야한다. </li><li> vibratome를 사용하여 400 μm의의 화살 섹션에 머리를 슬라이스. <ol><li> 소뇌의 가능한 한 조금 잘라 하나씩 반구에서 면도날과 화살 절단을 확인,이 부러 뜨리 평평한 표면을 제공합니다. 대뇌 피질은 깔끔히 쉽게 지원에 대해서만 사용하지만, 시간이 중요한 문제이기 때문에 그것은 면도날과 피질의 주동이의 절반을 잘라 것이 더 편리입니다. </li><li> 금속 vibratome 판에있는 두뇌의 위치를​​ 수정하는 (Superglue) cyanoacrylate 사용합니다. 접착제를 추가하기 전에 얼음 접시를 진정하고 얇은 접착 층을 형성하기 위해 튜브의 끝부분을 사용하여 잘 확산하는 것이 중요합니다. Superglue의 초과가있다면 그것이 ACSF 및 샘플이 손실됩니다 접촉되면, 그것은 접시에서 분리합니다. </li><li> 일단 두뇌는 두뇌를 커버하고 차가운 유지하는 접시 아래에 얼음을 넣어 충분히 ACSF 미디어를 추가 vibratome 접시에 첨부되어 있습니다. </li><li> 400 μm의 슬라이스 컷 얼음에 ACSF를 포함하는 6 자 세포 배양 판에 플라스틱 파스퇴르 피펫 (그것을 넓혀과 조각에 손상을 피하기 위해 팁을 절단)으로 그들을 전송합니다. </li><li> 해부 현미경이 인슐린 주사기 (소형 직경 28 게이지 바늘과 주사기)의 도움으로 두뇌의 나머지 부분에서 소뇌를 구분한다. 어떤 조각은 Superglue의 가장자리에 첨부해야합니다 : 이것은 슬라이스의 생존에 영향을 미칠 수로를 제거하려고하는 순간이다. 소뇌 구조를 손상시키지 않고 신중하게 그것을하는 것이 중요합니다. </li></ol></li><li> 0.4 μm의의 모공과 30mm 문화 플레이트 삽입을 포함하는 6 자 접시에 소뇌 조각 전송합니다. 한 문화 미디어의 ML 당 자 37 적어도 2 시간 배양하여 사전에 시설되었다 ° C와 5% CO 2 (세포 배양 인큐베이터에서). 추가 여기 아래에 갇혀있는 공기의 거품이되지 않습니다 그리고 그들이 완전히 확장되었는지 확인하고 삽입 위에 어떠한 액체가 그들을 다루는 없다는 것을 소뇌 조각 (삽입 당 1-3) 장소 확인하는 미디어의 상단에 삽입 놓으십시오. 필요한 경우 미디어의 초과를 대기음. 37 품어 ° C와 5% CO 2 문화 매체마다 2 ~ 3 일 변화. 사용 매체 세포 생존을위한 중요한 슬라이스 - 파생 영양 요소를 포함 이래, 한번에 매체의 일부만 (예 : ½ 볼륨) 교체하는 것이 좋습니다. </li></ol> 2. 파 처리 및 소뇌 조각의 스테 이닝 : <ol><li> apoptotic 도전 실험을 위해, 우리는 첫째 문화 체외 3 (DIV3)에서 하루에 미디어를 변경 오일에 대한 슬라이스. DIV5 문화 미디어의 변화 때문에 세포 죽음의 깨끗한 배경을하기 위해 미디어에 직접 추가하여 파의 작용제의 항체 Jo2 0.5 μg / ML로 처리하는이 문화의 일부 사망률을 일으킬 수 있습니다. 우물의 절반은 컨트롤로 치료 남아 있습니다. </li><li> 24 시간 흡입하여 삽입 아래 미디어를 제거한 후. 조각 해결하려면 아래 얼음 차가운 4% PFA 1 ML 및 삽입 위의 1 ML를 추가합니다. 실온에서 10 분 알을 품다. </li><li> 10 분 PFA를 제거하고 차가운 PBS로 세척 후 잠시. </li><li> 다음 PBS를 제거하고 실온에서 5 분간 PBS과 부화에 최고 20 % 차가운 메탄올의 삽입 위의 1 ML의 아래 1 ML를 추가합니다. </li><li> PBS로 다시 세척하고 아래 한 ML 및 permeabilization 위해 PBS에 트리톤 - X - 100 0.5 %의 삽입 위의 1 ML를 추가합니다. 4 12 시간 최소 품어 ° C. </li><li> permeabilization 후 최소 PBS에서 20 % BSA와 차단4 상온에서 시간 또는 4 박 ° C. 이 단계에서는 조각 4 적어도 삼일 ° C.에 대한 차단 용액에 보관 가능 </li><li> 슬라이스가 조심스럽게 소뇌 슬라이스에 연결된 삽입 막의 조각을 잘라 PBS를 포함하는 24 잘 접시로 전송 얼룩하기​​ 위해서. </li><li> immunostaining은 다음 1 시간 Calbidin 항체와 하룻밤을 위해 TUNEL 시약 처음에 순차적으로 이루어집니다. <ol><li> PBS를 대기음하고 각 잘는 슬라이스의 전체 표면을 커버해야하는 시약으로 TUNEL에서 250 μL를 추가합니다. 어두운 한 시간 동안 37 ° C에서 품어. </li><li> 한 시간 TUNEL 믹스를 제거하고 10 분 동안 PBS로 세 번 씻어 후. </li><li> 마지막 세척 후 PBS를 제거하고 PBS에서 Calbindin 항체의 1천분의 1 희석 250 μL를 추가하고 4 밤새 품어 ° 어둠 속에 C. </li><li> 기본 항체 솔루션을 제거하고 10 분 동안 PBS로 세 번 씻어. </li><li> 이차 항체의 추가 250 μL은 어둠 속에서 실온에서 최소한 세 시간은 PBS와 부화의 1 / 500 희석. 우리가 사용하는 TUNEL 키트는 TMR 빨간색으로 표시되므로 Calbindin위한 보조 항체는 알렉사 - 488에 복합이다. </li><li> PBS로 10 분 동안 세 번 씻고 DAPI를 포함하는 설치 미디어를 사용하여 유리 현미경 슬라이드에 슬라이스를 탑재합니다. </li><li> 각각 488 nm의과 Calbindin 및 TUNEL 위해 561 나노미터 여기 wavelenght를 사용하여 공촛점 현미경으로 이미지를 조각. </li></ol></li></ol> 3. 대표 결과 : 이 프로토콜의 주요 문제는 우리의 마지막 판독은 세포 죽음을 것입니다 특히 때문에, 건강한 소뇌 슬라이스 문화를 생성할 수 있도록합니다. 건강한 문화는 세포 몸 레이어는 투명 하나로 표시하고 현미경 (그림 1)에서 소뇌의 foliated 구조를 볼 수 있습니다. 문화의 몇 일 후에 조각이 얇고 비의 연결을 세포 시작은 조각의 가장자리로부터 멀리 마이 그 레이션 볼 수 있습니다. 문화의 몇 일 후에 조각 커버 어두운 원형 전지는 (대부분 macrophages), 결국 체외 (그림 2) 5 10-14일 후 번호 줄어 듭니다. 조각의 생존에 대한 궁극적인 증명은 Purkinje 세포와 같은 Calbindin 등 다양한 세포 마커에 대한 착색하는 것입니다. 그림 3은 몇 가지 TUNEL 긍정적인 핵으로 Purkinje 세포 층 대표 공촛점 현미경 이미지를 보여줍니다. 어떤 조각이 apoptotic 자극을받은 경우에도, 그들은 단 24 시간 동안 노출 있다고 생각하는 것이 중요합니다. 이것은 TUNEL의 얼룩과 죽어가는 세포의 DNA 조각을 관찰하기 위해 충분한 시간을 허용하지만 정의 Purkinje 세포 계층은 여전히​​ 존재했다. <img src="/files/ftp_upload/2564/2564fig1.jpg" alt="그림 1" /> 그림 1. DIV2에서 건강한 소뇌 슬라이스.이 이미지는 투명한 세포 몸 레이어와 건강 통에 foliated 소뇌 구조를 보여줍니다. <img src="/files/ftp_upload/2564/2564fig2.jpg" alt="그림 2" /> 그림 2. DIV 7 건강 소뇌 슬라이스.이 그림에서 우리는 여전히 소뇌와 어두운 세포 기관의 모양의 foliated 구조를 볼 수 있습니다. <img src="/files/ftp_upload/2564/2564fig3.jpg" alt="그림 3" /> 그림 3. 파 - 대우 소뇌 슬라이스의 대표 이미지는.이 공촛점 이미지 Calbindin (녹색) 및 TUNEL의 얼룩 (적색)에 대한 긍정적인 apoptotic 세포 물들일 Purkinje 세포를 보여줍니다. Purkinje 세포는 하나의 연속으로 표시되지 않지만 folium 같은 구조를 형성 클러스터 있습니다.

<ol>
	<li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+ependymal+cells+in+tissue+cultures.">Human fetal ependymal cells in tissue cultures.</a> Anat Rec. 99, 523-529 (1947).</li><li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+studies+of+human+fetal+brain+cells+in+tissue+cultures.">Review of studies of human fetal brain cells in tissue cultures.</a> Etudes Neonatales. 1, 1-13 (1952).</li><li>Gogolla, N., Galimberti, I., DePaola, V., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+protocol+for+organotypic+hippocampal+slice+cultures.">Staining protocol for organotypic hippocampal slice cultures.</a> Nat Protoc. 1, 2452-2456 (2006).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Beat, H., G&#228;hwiler, S. M. T., McKinney, R. A., Debanne, D., Robertson, R. T., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic Slice Cultures of Neural Tissue in Culturing Nerve Cells. , 461-498 (1998).</li></ol>

진행 모든 동물의 작업은 IACUC에 의해 설립 정책에 따라 이루어졌다. 솔크 연구소는 AAALAC 인증 시설입니다.

이 방법의 연결 organotypic 문화의 여러 가지 응용 프로그램 중 하나를 설명하고 그것은 우리가 야생 유형 및 돌연변이 소뇌 조각에서 apoptotic 자극의 효과를 연구하기 위해 수 있습니다. 본 실험의 가장 중요한 부분은 지속적으로 건강 소뇌 슬라이스 문화를 생성할 수 있도록합니다. 핵심 요소는 해부하고 깔끔히 기술과 가능한 최소 시간에 프로토콜을 수행할 수있는 능력이 있지만, 동시에 신중하면 조각을 손상하지 않습니다. 또 다른 중요한 요인은 문화 미디어의 준비 및 구성이며, 이러한 문화는 매우 민감합니다, 그래서 우리는 미디어의 여러 조리법과 브랜드를 테스트했습니다. 혈청 또는 미디어의 다른 구성 요소도 다른 배치는 조각의 생존에 영향을 미칠 수 있습니다. 우리는 Invitrogen 롯 # 480116에서 호스의 세럼과 함께 최상의 결과가 나왔어요. 소뇌 슬라이스가 생성되면 하나는 소뇌 개발, 전기 생리학, 세포 생존 혼자 또는 apoptotic 자극, excitotoxicity 또는 산소가 결핍되어서에 대한 응답으로 공부를하실 수 있습니다. 야생 타입과 돌연변이 소뇌 조각을 사용하여 비교 연구는 소뇌 기능과 개발의 여러 측면에서 매우 통찰력이있다.

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		<div>
		<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>MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11090</td>
<td>&nbsp;</td>
<tr>
<td>Horse Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/ Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Superglue</td>
<td>&nbsp;</td>
<td>Krazy Glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well / 24 well cell culture plates</td>
<td>&nbsp;</td>
<td>Corning Incorporated</td>
<td>3506/ 3527</td>
<td>&nbsp;</td>
<tr>
<td>30mm culture plate inserts (0.4-&mu;m pore)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM03050</td>
<td>&nbsp;</td>
<tr>
<td>Purified NA/LE Hamster anti-mouse CD95 (Jo2)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>554254</td>
<td>&nbsp;</td>
<tr>
<td>In situ cell death detection kit, TMR red</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>12 156 792 910</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-Calbindin D-28k antibody</td>
<td>&nbsp;</td>
<td>Swant</td>
<td>CB-38a</td>
<td>&nbsp;</td>
<tr>
<td>Alexa 488-goat anti-rabbit IgG secondary antibody</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular probes)</td>
<td>A-11008</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield Mounting Medium with DAPI</td>
<td>&nbsp;</td>
<td>Vector labs</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Zeiss Stemi SV 11</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica TCS SP2 AOBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

organotypic cerebellar cultures: apoptotic challenges and detection

의 연결 조직 Organotypic 문화가 처음 1947 년 1,2 호그에 의해 소개되었으며 신경 과학 분야의 주요 돌파구를 형성했습니다. 그 이후,이 기술은 더욱 발전하여 현재 organotypic 문화를 준비하는 방법에는 여러 가지가 있습니다했습니다. 방법은 Stoppini 외에 기술된 하나에서 적응되었다 여기에 제시했습니다. 슬라이스의 준비 및 Gogolla 외에서. 얼룩 절차 3,4하십시오. 이 기법의 독특한 기능은 모든 세포 조직과의 연결 네트워크가 중단되는 dissociated 문화 비해 큰 장점을 제공합니다, 당신이 원래 구조로 해마 또는 소뇌 등 뇌의 다른 부분을 공부 수 있도록하는 것입니다. 그것이 dissociated 기본 문화로 얻을 수 Purkinje 세포의 연구가 매우 어렵습 수 있기 때문에 소뇌의 경우는 훨씬 더 유리한 것입니다. 이 방법은 체외에서 소뇌의 특정 발달 기능을 연구뿐만 아니라, 야생의 종류와 돌연변이 생쥐 모두에서 electrophysiological 및 약리 실험하는 데 사용할 수 있습니다. 방법은 apoptotic 세포 죽음을 측정하는 TUNEL 염색법을 사용하여 이러한 개발 소뇌의 파의 리간드로 apoptotic 자극의 효과를 연구하기 위해 설계되었습니다 여기에 설명되어 있습니다. TUNEL의 얼룩이 Purkinje 세포에 대한 이러한 Calbindin 같은 세포 유형 특정 마커와 결합하는 경우, 그것은 세포 인구 구체적인 방식으로 세포 죽음을 평가하는 것이 가능합니다. Calbindin의 얼룩은 또한 소뇌 문화의 품질을 평가의 목적을 제공합니다.

Watch this Scientific Journal Video about Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection at JoVE.com

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

이 방법은 organotypic 소뇌 문화의 생성 및 다른 소뇌 세포 유형의 생존 능력에 특정 apoptotic 자극의 효과를 설명합니다.

Organotypic 소뇌 문화 : Apoptotic 도전과 감지

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

organotypic-cerebellar-cultures-apoptotic-challenges-and-detection

Research

JoVE Journal

Neuroscience

18.1K 조회수.  The Salk Institute for Biological Studies. 이 방법은 organotypic 소뇌 문화의 생성 및 다른 소뇌 세포 유형의 생존 능력에 특정 apoptotic 자극의 효과를 설명합니다.

비디오: Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotypic Hippocampal 슬라이스 문화

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

연구

신경과학

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Organotypic 문화에의 연결을 눈사태의 다중 전극 어레이 레코딩

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

배아 복부 Midbrain의 Organotypic 슬라이스 문화 : Dopaminergic의 연결을 개발 연구하는 시스템 체외에서</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Organotypic 슬라이스 문화의 Purkinje 세포 수지상의 형태학의 분석

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotypic 슬라이스는 문화 희소 돌기 아교 세포 역학 및 수초화을 공부하기

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

에서 신경 혈관 리모델링의 분석 Entorhino - 해마 Organotypic 슬라이스 문화

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

소뇌 과립 세포 배양에서 미세 아교 세포의 선택적 고갈 L - 류신 메틸 에스테르를 사용하여

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

다중 전극 배열에 성장의 Organotypic 척수 공동 문화 기능 재생 조사

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hippocampal 슬라이스 문화 연구 Neuroprotection 및 종양 세포의 침입을 모델로

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

전 Utero Electroporation 및 마이그레이션 GABAergic 수의 라이브 이미징 배아 마우스 두뇌의 Organotypic 조각 문화

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...