Name: organotypic slice cultures for studies of postnatal neurogenesis
Uploaded: 2015-03-04T13:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

참고 : 모든 동물의 절차는 토론토 대학에서 비교 의학과의 동물의 건강과 복지 가이드 라인을 따른다. 해마 조각 1. 준비 <ol><li> 125 ° C에서 건조 오토 클레이브를 사용하여 다음과 같은기구를 소독 : 메스 핸들 (# 3) (2), 표준 패턴 집게, 대형 (1), 작은 해부학자 시저 (좌우로 각도) (1), 마이크로 스푼 (숟가락과 평면 주걱 끝) (1), 둥근 마이크로 주걱 (및 테이퍼 끝을 둥글게) (2), 미세 붓 (1), 화재 광택 파스퇴르 피펫 (2), 거즈 사각형, 2 × 2 인치 (5). </li><li> 때 살균, 멸균 용기에 악기를 넣어 사용할 때까지 덮여 유지. 즉시 절개하기 전에, 70 % 에탄올 용액에 담궈 악기. </li><li> 35 ° C의 배양기에서 플레이트를 웰 / 배지 1 ㎖를 첨가하고 저장함으로써 절개 절차를 시작하기 전에 배양 인서트 6 웰 배양 플레이트를 준비차 5 % CO 2. </li></ol> 2. 멸균 층류 후드의 해부 도구를 정렬 <ol><li> 70 % 에탄올과 층류 후드 스프레이 및 알코올 멸균 해부 악기를 제거합니다. 멸균 페트리 접시에 휴식하는 알코올과 해부 뇌 조직 사이의 접촉을 피하도록하면서 악기가 건조하도록 허용합니다. </li><li> 2 개의 대형 멸균 페트리 접시에 살균 얼음처럼 차가운 해부 솔루션의 예금 5-7 ml의. 한 접시 차리고 머리 (더러운), 냉각 밖으로 훔쳐 뇌 (청소)을 세척을 위해 다른 하나를 정리합니다. 뇌를 해부에 대한 페트리 접시 뚜껑 중 하나에 살균 필터 종이를 놓습니다. </li><li> 해마를 해부에 대한 작은 페트리 접시 뚜껑 중 하나에 작은, 살균 필터 종이를 놓습니다. 이 작은, 멸균 페트리 접시에 살균 얼음처럼 차가운 해부 솔루션의 예금 3-5 ml의. 한 접시 하나는 해마 부분의 분리시 섹션을 개최, 밖으로 훔쳐 해마를 개최합니다해부 현미경. </li><li> 절삭 단계 멸균 여과지를 테이핑하고 멸균 면도날을 장착함으로써 조직 초퍼를 준비한다. 멸균 해부 솔루션 필터 종이를 젖은. </li><li> 알코올로 청소 바이오 가방 스프레이와 층류 클린 벤치에 넣습니다. </li></ol> 3. 해마 해부 <ol><li> 층류 클린 벤치의 외부 70 % 에탄올과 P7 스프 라그 돌리 래트 새끼 스프레이 신속 층류 벤치 내부에 대형 멸균 수술 가위를 사용하여 동물을 참살. 머리는 페트리 접시 중 하나에 얼음처럼 차가운 해부 솔루션으로 떨어 뜨려. </li><li> 페트리 접시에서 피를 씻어하고 빨리 살균 필터 종이에 머리, 복부 측면을 전송합니다. </li><li> 시상면에서 등쪽 표면을 따라 잘라 메스를 사용하면 기본 두개골을 노출. 부드럽고 t의 쥐에서 쉽게 꿰뚫어입니다 기본 뼈, 피부를 통해 잘라 내기,하지만그의 나이. 옆이 &quot;더러운&quot;메스를 설정하고 뇌 조직에 사용하지 마십시오. </li><li> 작은 해부학자의 (좌우로 각도) 가위와 집게를 사용하여, 브레 그마에 두개골의 시상 봉합을 따라 관상 봉합이 시상 봉합에 의해 수직으로 교차하는 두개골의 해부학 적 지점을 두개골을 오픈. 두개골의 정중선에서 위로 떨어져 두개골 플랩을 끌어 집게를 사용합니다. </li><li> 부드럽게 두개골의 두뇌를 리프트, 뇌간 뇌에 아래의 아래쪽에 마이크로 스푼을 넣습니다. 뇌의 기저 표면에 시신경과 후각 망울을 노출 뇌를 들어 올립니다. 완전히 두개골에서 뇌를 분리 작은 가위로 이러한 구조를 잘라. 제거하고 해부 빙냉 용액을 함유하는 다른 큰 페트리 접시에 뇌 손상을 전송. </li><li> 마이크로 숟가락을 사용하여, 멸균 여과지를 함유하는 작은 페트리 접시 뚜껑에 뇌 옮긴다. 멸균 파스퇴르 피펫으로, DIS 몇 방울 뇌를 씻어솔루션을 secting하는 조직 촉촉한 유지합니다. </li><li> &quot;깨끗한&quot;메스 블레이드를 사용하여 떨어져 두 개의 반구를 잘라. 마이크로 스푼으로 큰 페트리 접시로 다시 왼쪽 반구를 전송하고 이후 사용을 위해 얼음처럼 차가운 해부 솔루션 아래 반구 피아 측면을 배치합니다. </li><li> 오른쪽 반구의 내측 얼굴을보고 가장자리 원개, 해마의 안쪽 가장자리를 따라 백질의 눈에 잘 띄는 밴드를 식별합니다. 멸균 메스를 사용하면, 원개를 통해 시상 상처를 만들지 만, 메스 팁 0.5 cm가 원개를 절단하기에 충분하기 때문에주의하십시오. </li><li> 2 마이크로 주걱을 사용하여, 뇌 줄기에 오른쪽 손으로 주걱을 배치하고 왼쪽 주걱 위에 놓인 피질을 들어 올려 오른쪽 반구에서 첫 번째 해마를 제거합니다. 부드럽게 해마의 뇌실 및 내측 표면을 공개하는 피질를 들어 올립니다. 흰색 곡선, fimbria, 지금은 볼 수 있어야합니다. </li><li> curva와 주걱의 곡률을 맞 춥니 다fimbria의 헌과 부드럽게는 fimbria 아래 주걱을 누릅니다. 왼쪽 주걱을 밀어 주동이의 - 꼬리 축을 따라 타고 다음 해마를 제거하는 등의 방향으로 주걱을 들어 올립니다. </li><li> 얼음처럼 차가운 해부 솔루션으로 2 차 작은 페트리 접시에 해마를 전송합니다. 좌측 해마를 제거 좌반구에 동일한 절차를 반복한다. </li><li> 마이크로 주걱을 사용하여 조심스럽게 조직 헬기 단계로 해마를 전송합니다. 초퍼 블레이드의 축이 서로 직교하고 그들에 인접하고 평행 배열. 조직의 위치와 해마의 상단에 해부 솔루션의 몇 방울을 추가하는 붓을 사용합니다. </li><li> 개별 조각을 제거하기 위해 일시​​ 정지하지 않고 400 μm의 조각의 조직을 잘라 (보통 칼날을 준수하지 않을 것이다). 전체 해마가 절단 된 후, 부드럽게 해부 솔루션으로 2 차 작은 페트리 접시에 섹션을 전송하기 위해 붓을 사용합니다. </li><li> 광고에서현미경을 issecting, 조심스럽게 amicro-주걱 및 페인트 브러시를 사용하여 부동 조각을 분리합니다. </li><li> 층류 후드의 보육과 장소에서 문화 인서트 사전 준비 배양 플레이트를 제거합니다. </li><li> 화재 광택 파스퇴르 피펫을 사용하여, 문화 삽입 멤브레인의 혀끝의 표면에 피펫 및 전송 조각으로 4 ~ 5 조각을 그립니다. 다음으로, 페인트 브러시와 위치를 조정하고 개별 섹션과 문화 삽입의 경계 사이의 공간을 남겨. </li><li> 멸균 파스퇴르 피펫을 사용하여, 막 혀끝의 표면에서 과잉 해부 솔루션을 제거합니다. 참고 : 피펫으로 조직 절편을 그리기 솔루션을 피를 제거하는 동안. 또한, 천천히 용액을 제거 멸균 피펫 팁을 정기적으로 피펫 (P200 또는 P1000)를 사용합니다. </li><li> 혈청 함유 배지와 35 ° C에서 인큐베이터에 해마 슬라이스와 5 % CO 2와 문화 판을 놓습니다. 참고 : 실험 호출하는 경우여러 동물 문화를 생성, 철저하게 해부 사이의 층류 클린 벤치를 청소합니다. 알코올 용액에 악기를 돌려 모든 페트리 접시, 여과지와 두 번째 해부하기 전에 멸균 면도날을 교체합니다. </li></ol> 4. 수유와의 Organotypic 조각을 유지 <ol><li> 멸균 층류 클린 벤치에서 피드 문화. </li><li> 배양 섹션 이일 후 해부의 첫 공급을 수행합니다. 멸균 유리 피펫을 사용하여 대기음 된 배지. </li><li> 우물에 신선한, 멸균, 혈청 함유 배지 1 ㎖를 추가하는 멸균을 5 ml에게 혈청 피펫을 사용합니다. </li><li> 부드럽게 문화 삽입물을 장착하고 막 표면 아래에 형성 할 수있는 공기 방울을 제거하기 위해주의를 기울입니다. </li><li> 처음 공급 한 후, 매일 매체를 변경합니다. </li></ol> 티미 딘 유사체 5. 배양 조직 조각 신생아 뉴런에 레이블을 지정하려면 <ol><li> 공부를하기 위해성숙과 해마에 이가있는 과립 세포의 통합은 이러한 브로 모데 옥시 우리 딘 (BrdU의) 또는 Chlorodeoxyuridine (CldU)로 티미 딘 아날로그로의 Organotypic 슬라이스를 품어. 여기, 우리 때문에 식염수에서의 높은 용해도의 CldU을 사용합니다. </li><li> 3DIV 후, 10 ㎍ / ml의 최종 농도를위한 배양 배지 1 ㎖의 생리 식염수에 10 ㎎ / ㎖ CldU 스톡 용액을 추가 1 μL. BrdU를 사용하는 경우, 분자량의 차이에 대한 농도를 수정한다. 문화 우물에 CldU와 매체를 추가하고 35 ° C에서 2 시간 동안 매체를 CldU이 함유와 조직을 배양한다. </li><li> 35 ° C에서 배양 2 시간에 따라, CldU 함유 배지를 제거하고 (위에서 설명한) 정상 공급 일정을 다시 시작하는 일반 공급 배지로 교체합니다. </li></ol> 6. 조직 고정 및 저장 <ol><li> 치료를 적용하고 배양 실험 (그림 2B)를 시작하기 전에 조직 샘플을 고정하기위한 타임 라인을 설정합니다. </li> &lt;소정 일 후 해부에서 리&gt;, 실험실 흄 후드에서 다음과 같은 준비 : 10 ~ 50 비이커 인산염 완충 식염수 (PBS)에 4 % 파라 포름 알데히드 (PFA)를 포함하는 단계; 폐기 배지 빈 비커; 작은 집게; 일회용 팁과 1,000 mL의 피펫. </li><li> 흄 후드 배양 챔버 및 전송에서 배양 플레이트를 분리합니다. 집게로 개별 웰 플레이트 삽입을 기울입니다. 다음으로, 처분 비커에 문화 매체와 전송을 제거하기 위해 피펫을 사용합니다. 모든 문화 매체가 제거되었는지 확인하기 위해 각도로 문화 판을 기울입니다. </li><li> 한번에 한 배양 접시에 대한 매체를 제거 완료. 매체가 제거 된 경우, 잘 각각의 문화에 4 % PFA 1 ㎖를 넣고 잘 파라 필름과 판 인감. 24 시간 동안 4 ° C에서 냉장고에 필요하고 전송 플레이트 많은 문화 판을 반복합니다. </li><li> 24 시간 후, 흄 후드에서 다음을 준비 : 10 ~ 50 PBS의 0.1 % 아 지드 화 나트륨을 함유하는 비이커; 빈폐기 PFA (독성 물질을 폐기 할 때는 안전주의 사항을 준수)에 대한 비커; 작은 집게; 일회용 팁을 1,000 ㎖를 피펫. </li><li> PFA를 추가하는 6.4에 설명 된 절차에 따라, 대신에 아 지드 화 나트륨과 1 ㎖의 PBS를 추가합니다. 일단 완전히 파라 필름의 가장자리를 포장하고 4 ° C에서 냉장고에 저장하여 나중에 사용하기 위해, 인감 웰 플레이트를 옮겼다. </li></ol> 7. 면역 조직에 대한 조직을 단면 <ol><li> 에 vibratome를 사용하여 단면 조직을 수행합니다. 다음과 같은 일련의 단계가의 Organotypic 문화에서 사용 가능한 조직 섹션의 수율을 극대화 할 수 있도록 도움을줍니다. 주 : 즉시 해마 절제술 및 슬라이스 도금 다음, 조직은 대략 400 ㎛의 두께를 갖는다. 그러나, 배양 챔버 2 ~ 3 주 후, 조직 슬라이스 150-300 ㎛의 마지막 부분 두께의 결과로, 평평하기 시작합니다. </li><li> 섹션 배양 조직에 다음 항목을 준비 : # 11 메스 B를짐을 싣다 및 처리, 조직을 탑재 얼음 차가운 PBS, 마이크로 해부 포셉, 깨끗한에 vibratome 절단 단계를 포함하는 유리 페트리 접시. </li><li> 이 플라스틱 인서트 하우징으로부터 분리 될 수 있도록 메스 조심스럽게 원형 인서트 막의 주변을 따라 절단한다. 배양 슬라이스와 메스 사이에 충분한 공간을 두십시오. </li><li> PBS를 함유하는 페트리 접시로 삽입 분리 멤브레인을 전송. PBS에 세척 한 후,에 vibratome 설치 단계로 막을 전송하는 집게를 사용합니다. </li><li> 다음, 배양 한 조각을 둘러싼 초과 멤브레인을 제거하고 깨끗한 가장자리를 만들기 위해 여분의 재료를 절단하기 위해 메스를 사용합니다. 멤브레인을 보장이 단계는 평평하고 쉽게 절삭면에 부착 할 수있다. </li><li> 에 vibratome 절단 단계에서 접착제의 1 ~ 2 방울을 넣고도 22 G 바늘을 사용하여 계층에 확산. 에 vibratome의 칼날 길이가 긴 변에 평행 한 직사각형 형상으로 접착제를 확산. 이 단계를 수행빨리 건조되는 접착제를 방지한다. </li><li> 절단 단계로 해마 슬라이스를 포함 트리밍 멤브레인을 전송하고 부드럽게 접착제에 막을 놓고 기포가 없는지 확인하기 위해 집게를 사용합니다. </li><li> superglue가 건조로, 다시 PBS 포함하는 페트리 접시에 붙어 막을에 vibratome 단계를 전송할 수 있습니다. 에 vibratome 블레이드와 조직 섹션을 저장하기 위해 48 웰 플레이트 함유 아 지드 화 나트륨을 준비한다. </li><li> 스토리지 및 이후의 면역 조직 화학 염색에 대한 48 웰 플레이트 포함, 아 지드 화 나트륨에의 Organotypic 슬라이스 조직 및 전송의 30 μm의 섹션을 생성하기에 vibratome를 사용합니다. </li><li> 면역 조직 화학 염색의 26,29위한 프로토콜 기존 사전을 참조하십시오. </li></ol>

<ol>
	<li>Buchs, P. A., Stoppini, L., 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=Structural+modifications+associated+with+synaptic+development+in+area+CA1+of+rat+hippocampal+organotypic+cultures.">Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures.</a> Brain research. Developmental Brain Research. 71 (1), 81-91 (1993).</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> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Opitz-Araya, X., Barria, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures.">Organotypic hippocampal slice cultures.</a> Journal of Visualized Experiments. (48), (2011).</li><li>Muller, D., Buchs, P. A., Stoppini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+synaptic+development+in+hippocampal+organotypic+cultures.">Time course of synaptic development in hippocampal organotypic cultures.</a> Developmental Brain Research. 71 (1), 93-100 (1993).</li><li>Rio, J. A., Heimrich, B., Soriano, E., Schwegler, H., Frotscher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+differentiation+of+glial+fibrillary+acidic+protein-immunoreactive+glial+cells+in+organotypic+slice+cultures+of+rat+hippocampus.">Proliferation and differentiation of glial fibrillary acidic protein-immunoreactive glial cells in organotypic slice cultures of rat hippocampus.</a> Neuroscience. 43 (2-3), 335-347 (1991).</li><li>Ziemka-Nalecz, M., Stanaszek, L., Zalewska, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-glucose+deprivation+promotes+gliogenesis+and+microglia+activation+in+organotypic+hippocampal+slice+culture:+involvement+of+metalloproteinases.">Oxygen-glucose deprivation promotes gliogenesis and microglia activation in organotypic hippocampal slice culture: involvement of metalloproteinases.</a> Acta Neurobiologiae Experimentalis. 73 (1), 130-142 (2013).</li><li>Subramanian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+Lhx2+is+necessary+and+sufficient+to+suppress+astrogliogenesis+and+promote+neurogenesis+in+the+developing+hippocampus.">Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (27), E265-E274 (2011).</li><li>Strassburger, M., Braun, H., Reymann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+treatment+with+the+p38+mitogen-activated+protein+kinase+inhibitor+SB239063+is+neuroprotective,+decreases+the+number+of+activated+microglia+and+facilitates+neurogenesis+in+oxygen-glucose-deprived+hippocampal+slice+cultures.">Anti-inflammatory treatment with the p38 mitogen-activated protein kinase inhibitor SB239063 is neuroprotective, decreases the number of activated microglia and facilitates neurogenesis in oxygen-glucose-deprived hippocampal slice cultures.</a> European Journal Of Pharmacology. 592 (1-3), 55-61 (2008).</li><li>Sadgrove, M. P., Chad, J. E., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kainic+acid+induces+rapid+cell+death+followed+by+transiently+reduced+cell+proliferation+in+the+immature+granule+cell+layer+of+rat+organotypic+hippocampal+slice+cultures.">Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures.</a> Brain Research. 1035 (2), 111-119 (2005).</li><li>Wise-Faberowski, L., Robinson, P. N., Rich, S., Warner, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+and+glucose+deprivation+in+an+organotypic+hippocampal+slice+model+of+the+developing+rat+brain:+the+effects+on+N-methyl-D-aspartate+subunit+composition.">Oxygen and glucose deprivation in an organotypic hippocampal slice model of the developing rat brain: the effects on N-methyl-D-aspartate subunit composition.</a> Anesthesia and Analgesia. 109 (1), 205-210 (2009).</li><li>Cho, S., Wood, A., Brain Bowlby, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=slices+as+models+for+neurodegenerative+disease+and+screening+platforms+to+identify+novel+therapeutics.">slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics.</a> Current Neuropharmacology. 5 (1), 19-33 (2007).</li><li>Noraberg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures+for+studies+of+brain+damage,+neuroprotection+and+neurorepair.+Current+Drug+Targets.">Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Current Drug Targets.</a> CNS And Neurological Disorders. 4 (4), 435-452 (2005).</li><li>Berdichevsky, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K-Akt+signaling+activates+mTOR-mediated+epileptogenesis+in+organotypic+hippocampal+culture+model+of+post-traumatic+epilepsy.">PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 33 (21), 9056-9067 (2013).</li><li>Koyama, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+excitation+after+febrile+seizures+induces+ectopic+granule+cells+and+adult+epilepsy.">GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.</a> Nature Medicine. 18 (8), 1271 (2012).</li><li>Staley, K. J., White, A., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+spikes:+harbingers+or+causes+of+epilepsy.">Interictal spikes: harbingers or causes of epilepsy.</a> Neuroscience Letters. 497 (3), 247-250 (2011).</li><li>Lee, H., Lee, D., Park, C. H., Ho, W. K., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+mediates+the+network+activity-dependent+facilitation+of+axonal+outgrowth+from+the+newborn+granule+cells+in+the+early+postnatal+rat+hippocampus.">GABA mediates the network activity-dependent facilitation of axonal outgrowth from the newborn granule cells in the early postnatal rat hippocampus.</a> The European Journal Of Neuroscience. 36 (6), 2743-2752 (2012).</li><li>Raineteau, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+labeling+of+newborn+granule+cells+to+visualize+their+integration+into+established+circuits+in+hippocampal+slice+cultures.">Conditional labeling of newborn granule cells to visualize their integration into established circuits in hippocampal slice cultures.</a> Molecular and Cellular Neurosciences. 32 (4), 344-355 (2006).</li><li>Raineteau, O., Rietschin, L., Gradwohl, G., Guillemot, F., Gahwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+hippocampal+slice+cultures.">Neurogenesis in hippocampal slice cultures.</a> Molecular And Cellular Neurosciences. 26 (2), 241-250 (2004).</li><li>Kamada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+spontaneous+neurogenesis+in+the+postnatal+slice+culture+of+rat+hippocampus.">Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus.</a> The European Journal Of Neuroscience. 20 (10), 2499-2508 (2004).</li><li>Kim, H., Kim, E., Park, M., Lee, E., Namkoong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+culture+from+the+adult+mouse+brain:+a+versatile+tool+for+translational+neuropsychopharmacology.">Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.</a> Progress in Neuro-Psychopharmacology &amp; Biological Psychiatry. 41, 36-43 (2013).</li><li>Legradi, A., Varszegi, S., Szigeti, C., Gulya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+hippocampal+slices+as+in+vitro+models+for+neurodegeneration:+Studies+on+cell+viability+and+apoptotic+processes.">Adult rat hippocampal slices as in vitro models for neurodegeneration: Studies on cell viability and apoptotic processes.</a> Brain Research Bulletin. 84 (1), 39-44 (2011).</li><li>Sadgrove, M. P., Laskowski, A., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+granule+layer+cell+count,+cell+density,+and+single-pulse+BrdU+incorporation+in+rat+organotypic+hippocampal+slice+cultures+with+respect+to+culture+medium,+septotemporal+position,+and+time+in+vitro.">Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro.</a> The Journal of Comparative Neurology. 497 (3), 397-415 (2006).</li><li>Mielke, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal,+synaptic,+and+nuclear+protein+changes+associated+with+rat+interface+organotypic+hippocampal+slice+culture+development.">Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.</a> Developmental Brain Research. 160 (2), 275-286 (2005).</li><li>Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+pre-+and+postsynaptic+distribution+of+AMPA+receptors+at+synapses+in+CA3+stratum+radiatum+of+hippocampal+slice+cultures+compared+with+intact+brain.">Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.</a> European Journal of Neuroscience. 12 (10), 3687-3700 (2000).</li><li>Laplagne, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+neurons+generated+in+the+developing+and+adult+hippocampus.">Functional convergence of neurons generated in the developing and adult hippocampus.</a> PLoS Biology. 4 (12), e409 (2006).</li><li>McDonald, H. Y., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+neurogenesis+in+the+dentate+gyrus+of+adult+rats.">Dynamics of neurogenesis in the dentate gyrus of adult rats.</a> Neuroscience Letters. 385 (1), 70-75 (2005).</li><li>Stone, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+developmentally+and+adult-generated+granule+cells+in+dentate+gyrus+circuits+supporting+hippocampus-dependent+memory.">Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.</a> Hippocampus. 21 (12), 1348-1362 (2011).</li><li>Wang, S., Scott, B. W., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogenous+properties+of+dentate+granule+neurons+in+the+adult+rat.">Heterogenous properties of dentate granule neurons in the adult rat.</a> Journal of Neurobiology. 42 (2), 248-257 (2000).</li><li>Wojtowicz, J. M., Kee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+assay+for+neurogenesis+in+rodents.">BrdU assay for neurogenesis in rodents.</a> Nature Protocols. 1 (3), 1399-1405 (2006).</li><li>Fritsch, R. S. E. R., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+Methods,+Vol.+1:+Practical+Methods+for+Biological+Morphometry.">Stereological Methods, Vol. 1: Practical Methods for Biological Morphometry.</a> Zeitschrift f&#252;r Allgemeine Mikrobiologie. 21 (8), 630-630 (1981).</li><li>Bunk, E. C., Konig, H. G., Bonner, H. P., Kirby, B. P., Prehn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-induced+injury+of+mouse+organotypic+hippocampal+slice+cultures+triggers+delayed+neuroblast+proliferation+in+the+dentate+gyrus:+an+in+vitro+model+for+the+study+of+neural+precursor+cell+proliferation.">NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.</a> Brain Research. 1359, 22-32 (2010).</li><li>Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+postnatal+injections+of+bromodeoxyuridine+produce+age-dependent+morphological+and+behavioral+abnormalities.">Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 19 (6), 2337-2346 (1999).</li><li>Morris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+toxicology+of+5-bromodeoxyuridine+in+mammalian+cells.">The genetic toxicology of 5-bromodeoxyuridine in mammalian cells.</a> Mutation Research. 258 (2), 161-188 (1991).</li><li>Bannigan, J., Langman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+effect+of+5-bromodeoxyuridine+on+the+mammalian+embryo.">The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.</a> Journal Of Embryology And Experimental Morphology. 50, 123-135 (1979).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Duque, A., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+effects+of+bromodeoxyuridine+and+[3H]thymidine+incorporation+into+DNA+on+cell+proliferation,+position,+and+fate.">Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 31 (42), 15205-15217 (2011).</li><li>Namba, T., Mochizuki, H., Onodera, M., Namiki, H., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+in+hippocampal+slice+cultures:+early+in+vitro+labeling+of+neural+precursor+cells+leads+to+efficient+neuronal+production.">Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production.</a> Journal of Neuroscience Research. 85 (8), 1704-1712 (2007).</li></ol>

The authors declare that they have no competing financial interests.

CldU (또는 BrdU의) 투여 후, 약리 에이전트의 응용 프로그램의 타임 라인이 특히 발달이 진행되는 동안 신생아 DGCs을 대상으로 선택 될 수있다. 예를 들어, 에이전트는 가상의 GABA가 탈분극 곳 발달 단계에있는 미성숙 뉴런의 나이와 일치하는 것이 제안되어 두번째 주 사후 CldU 주입 동안 적용될 수있다. 이 프로토콜을 이용하여 향후 연구 약리 에이전트 및 &quot;맞춤형&quot;관심있는 특정 실험 질문 ?...

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain&#8217;s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in conditions, such as epilepsy, schizophrenia, and Alzheimer&#8217;s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.
Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.
Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

<table><tbody><tr><td>5-chloro-2&#039;-deoxyuridine (CldU)</td><td>MP Biomedicals</td><td>105478</td><td>Hazardous, Carcinogenic</td></tr><tr><td>Cell culture inserts, 30 mm diameter, 0.4 &amp;micro;m pore size</td><td>Thermo scientific&amp;nbsp;</td><td>140660</td><td>Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td></tr><tr><td>Conical Centrifuge tubes (sterile)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Dissector scissors (angled to side)</td><td>Fine Science Tools&amp;nbsp;</td><td>14082-09</td><td></td></tr><tr><td>Minimum essential medium (MEM)</td><td>Gibco</td><td>11095; liquid</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Eclipse Ni-U fluorescent microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Glue for tissue</td><td>Krazy Glue</td><td>KG585</td><td>Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS) (500 ml)</td><td>Gibco</td><td>14025-092</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Horse Serum Heat Inactivated (500 ml)</td><td>Gibco</td><td>16050-122</td><td>Make 50 ml aliquots and store at -20 &amp;deg;C</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clarke</td><td>TW 31KYPBX</td><td></td></tr><tr><td>Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td><td></td><td></td><td></td></tr><tr><td>Petri Dish (100 mm x 15 mm) and (60 mm x 15 mm)</td><td>Fisher Brand</td><td>FB0875712 and FB0875713A</td><td></td></tr><tr><td>Scalpel blades #11</td><td>Fine Science Tools</td><td>10011-00</td><td></td></tr><tr><td>Scalpel handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Serological Pipettes</td><td>Sorfa Medical Plastic Co.</td><td>P8050</td><td></td></tr><tr><td>Standard Pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Sterile vacuum filter</td><td>Thermo-Scientific</td><td>565-0020</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14054-13</td><td></td></tr><tr><td>Syringe driven filter unit</td><td>Millipore-Millex</td><td>SLGP033RS</td><td></td></tr><tr><td>Tissue chopper with moveable stage</td><td>Stoelting&amp;nbsp;</td><td>51425</td><td></td></tr><tr><td>Fine tip paintbrush</td><td></td><td></td><td></td></tr></tbody></table>

organotypic slice cultures for studies of postnatal neurogenesis

여기서 우리는 Organotypic 슬라이스 배양 기술을 이용하여 쥐의 뇌에서 해마 신경 산후을 연구하는 기술을 설명한다. 개발 해마 치아 이랑에되는 약물의 직접 적용을 허용하면서이 방법은 해마의 특징적인 지형 형태를 유지한다. 또한, 슬라이스 배양 물은 4 주까지 유지 될 수 있으며, 따라서, 일 그래뉼은 신생아 신경 세포의 성숙 과정을 연구 할 수있다. 복소 변수를 제외하면서 슬라이스 문화는 해마의 해부학 깊은 위치뿐만 아니라 혈액 뇌 장벽과 관련된 불확실성으로 해마 슬라이스 약리학 효율적인 조작이 가능. 이러한 이유로, 우리는 출생 후 신경 연구를 위해 특별히 Organotypic 슬라이스 문화를 최적화하기 위해 노력했다.

1) 조각은 체외에서 10~21일 (DIV) 후 해마 조각의 특성 형태 학적 특징을 유지하는 것이, 2) 신생아 DGCs 정량화 할 수있다 :의 Organotypic 문화가 성인 신경 연구에 적합 할 것인지 결정 그들은 두 가지 기준을 ​​만족하는 것이 필요 일반적으로 성인의 신경 연구에 이용 표준 면역 조직 화학적 기법을 사용. 첫 번째 기준과 관련하여,도 1a와 1b는 보존 해마 형태를 강조 표시합니다. 이...

Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

여기에서 우리는 Organotypic 슬라이스 문화 기술을 사용하여 해마 출생 후 신경 세포를 연구하는 기술에 대해 설명합니다. 이 방법은 성인 신경의 체외에서 조작이 가능하며 배양 된 해마되는 약물의 직접 적용이 가능합니다.

출생 후 신경 발생의 연구 Organotypic 슬라이스 문화

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

organotypic-slice-cultures-for-studies-of-postnatal-neurogenesis

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Developmental Biology

12.2K 조회수.  University of Toronto.  여기에서 우리는 Organotypic 슬라이스 문화 기술을 사용하여 해마 출생 후 신경 세포를 연구하는 기술에 대해 설명합니다. 이 방법은 성인 신경의 체외에서 조작이 가능하며 배양 된 해마되는 약물의 직접 적용이 가능합니다. 

비디오: Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18 랫 두뇌의 Organotypic 슬라이스 문화

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

연구

생물학

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

의 Organotypic 슬라이스 문화 GFP - 표현 주변 신경 가지의 실시간 이미징을위한 마우스 배아를

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

신경과학

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 ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

출생 후의 두뇌에의 연결을 마이 그 레이션의 고해상도 시간 경과 이미징을위한 Organotypic 슬라이스 분석

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

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 ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

의 연결을 Morphogenesis의 연구를위한 방법 :<em&gt; 예 생체내</em배아 Murine 대뇌 피질에서&gt; RNAi의 Electroporation

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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 ...

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 Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

배아 GFP 발현 마우스에서 전 Vivo Oculomotor 슬라이스 배양 Oculomotor 신경 파생의 시간 경과 화상 진찰을 위한

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

오르가노티픽 소뇌 슬라이스 배양에서 푸르킨제 세포 생존

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

출생 후 신경 발생의 연구 Organotypic 슬라이스 문화

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