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>
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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.

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			<td height="46" rowspan="2" style="height:47px;width:265px;">Name of Reagent/ Equipment</td>
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			<td rowspan="2" style="width:135px;">Catalog Number</td>
			<td rowspan="2" style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">5-chloro-2&#39;-deoxyuridine (CldU)</td>
			<td style="width:131px;">MP Biomedicals</td>
			<td align="right" style="width:135px;">105478</td>
			<td style="width:239px;">Hazardous, Carcinogenic</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Cell culture inserts, 30mm diameter, 0.4&#181;m pore size</td>
			<td style="width:131px;">Thermo scientific&#160;</td>
			<td align="right" style="width:135px;">140660</td>
			<td style="width:239px;">Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Conical Centrifuge tubes (sterile)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">14-432-22</td>
			<td style="width:239px;"></td>
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			<td height="40" style="height:40px;width:265px;">Dissector scissors (angled to side)</td>
			<td style="width:131px;">Fine Science Tools&#160;</td>
			<td style="width:135px;">14082-09</td>
			<td style="width:239px;"></td>
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			<td height="20" style="height:20px;width:265px;">Minimum essential medium (MEM)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">11095; liquid</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Eclipse Ni-U fluorescent microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
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			<td height="80" style="height:80px;width:265px;">Glue for tissue</td>
			<td style="width:131px;">Krazy Glue</td>
			<td style="width:135px;">KG585</td>
			<td style="width:239px;">Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td>
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			<td height="40" style="height:40px;width:265px;">Hank&#8217;s Balanced Salt Solution (HBSS) (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">14025-092</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Horse Serum Heat Inactivated (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">16050-122</td>
			<td style="width:239px;">Make 50 mL aliquots and store at -20&#176;C</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Kimwipes</td>
			<td style="width:131px;">Kimberly-Clarke</td>
			<td style="width:135px;">TW 31KYPBX</td>
			<td style="width:239px;"></td>
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			<td height="60" style="height:60px;width:265px;">Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Petri Dish (100mm x 15mm) and (60mm x 15mm)</td>
			<td style="width:131px;">Fisher Brand</td>
			<td style="width:135px;">FB0875712 and FB0875713A</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel blades #11</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10011-00</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel handle #3</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10003-12</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Serological Pipettes</td>
			<td style="width:131px;">Sorfa Medical Plastic Co.</td>
			<td style="width:135px;">P8050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Standard Pattern forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Sterile vacuum filter</td>
			<td style="width:131px;">Thermo-Scientific</td>
			<td style="width:135px;">565-0020</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Surgical Scissors</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">14054-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Syringe driven filter unit</td>
			<td style="width:131px;">Millipore-Millex</td>
			<td style="width:135px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Tissue chopper with moveable stage</td>
			<td style="width:131px;">Stoelting&#160;</td>
			<td align="right" style="width:135px;">51425</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Fine tip paintbrush</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td></td>
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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

Research

JoVE Journal

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

마우스 해마 조직의 예 자궁 Electroporation에와 Organotypic 슬라이스 문화

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

연구

발생학

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

약화 된 인간의 진피를 사용하여 유전자 변형의 Organotypic 피부 문화의 생성

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

마우스 귀와 꼬리 조직에서 생성 차 섬유 아세포 배양

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

영상은 단일 셀 해상도에서 배아 및 출생 후의 마음을 해제

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

출생 후 심근 세포의 세포주기 변화의 시각화 및 핵의 결정

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

해 부 및 마우스 뇌 개발의 코로나 슬라이스 준비

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Vestibular 및 청각 감각 기관의 3 차원 Organotypic 문화

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

마우스 췌 장 내 분 비 세포의 단일 셀 Transcriptomic 분석

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Neurogenesis Using P19 Embryonal Carcinoma Cells

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of retinoic acid (RA), the suspension cultured P19 cell line is induced to differentiate into neurons. This phenomenon is extensively investigated as a neurogenesis model in vitro. Therefore, the P19 cell line is very useful for molecular and cellular studies associated with neurogenesis. However, protocols for neuronal differentiation of P19 cell line described in the literature are very complex. The method developed in this study are simple and will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

P19 배아 암종을 이용한 신경 발생

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

뮤린 신경 발생 시 노치 신호 역학의 실시간 생물 발광 이미징

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...