This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.

주 : 모든 동물의 절차는 지침을 따르고 코네티컷 대학의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었습니다. 참고 : YFP 기자 20 (JAX 번호 006148) 선이 각각 사용되었다 : NG2cre 18 구성 적 (JAX # 008533)와 NG2creER 16 유도 이러한 실험 (JAX 번호 008538)의 경우 형질 전환 마우스는 19 (JAX은 # 003920) 또는 gtRosa26 / EG Z에 교차 화상 NG2 세포 및 그 자손. 희소 돌기 아교 세포로 성숙 화상, PLPDsRed 트랜스 제닉 마우스 (21)를 사용 하였다. 일관성 생존을 위해, 조각까지 전뇌와 소뇌 모두에서 출생 일 10 생쥐에서 분리 할 수​​ 있습니다. 1 조각 준비 <ol><li> 문화 후드에서 여섯 잘 접시에 조직 배양 삽입을 배치하고 (시약 섹션에서 레시피) 1 ml의 슬라이스 문화 매체를 추가 할 수 있습니다. 5 %와 37 ° C에서 해부하기 전에 CO 2 적어도 2 시간을 세포 배양 인큐베이터에서 접시를 넣습니다. </li><li> 70 % 에탄올에서 모든 도구와 조직 초퍼 멸균. </li><li> 이전 해부의 시작 적어도 15 분 동안 얼음에 95 % O 2, 5 % CO 2와 버블 해부 버퍼 (시약 섹션에서 레시피). </li><li> 승인 된 동물 프로토콜에 따라 5 ~ 10 분 동안 얼음에 배치하여 마우스 새끼를 마취. 쥐 꼬리와 발가락 핀치에 응답하지 않는 것을 확인하여 마취의 적절한 깊이를 확인하라. </li><li> 동물 프로토콜을 다음 예리한 가위를 사용하여 동물을 참살. 빨리 첫번째 살균 도구를 사용하여 횡 절개 다음에 시상 컷을하여 전뇌와 소뇌를 통해 두개골을 제거합니다. 조심스럽게 옆으로 조직을 압연하여 뇌와 소뇌의 복부 표면에서 두개골 신경을 잘라. </li><li> 얼음처럼 차가운 산소 해부 버퍼를 포함하는 멸균 35mm 접시에 조직을 배치합니다. </li><li> 면도날을 사용하여, 소뇌 및 전뇌 단절. 그런 다음 중간 시상 컷 죽을반구를 분리 우 전뇌와 소뇌. </li><li> 수동 조직 헬기와 전뇌와 소뇌의 300 μm의 두께의 관상 및 시상 부분을 잘라. 참고 : 수동 조직 헬기 아가로 오스 삽입 할 필요없이 문화 부화 해부에서 신속하게 처리 할 수​​ 있습니다. 앞서 설명한 바와 같이 9 vibratome도 사용될 수있다. </li><li> 얼음에 갓 버블 차가운 해부 버퍼로 분리 된 조각을 전송합니다. </li><li> 작은 해부를 사용하거나 개별 섹션을 분리하고 배양 삽입과 여섯 잘 요리를 미리 배양로 전송 주걱 무게. </li><li> 일회용 전송 피펫 또는 P 200 피펫 팁을 사용하여 문화 삽입물의 표면에서 초과 해부 버퍼를 제거합니다. 세 전뇌 또는 세 소뇌 조각에 두 하나의 문화 삽입에 배치 할 수 있습니다. 참고 : 전뇌 문화와 일치하는 결과를 얻기 위해서 (때문에)는 전방에게 뇌량의 3 분의 1에 걸쳐 조각을 위해 이상적입니다동일한 슬라이스에 모두 회색과 흰색 문제 영역을 공부하기 위해 (그림 1A). 각 동물은 일반적으로 6 전뇌 슬라이스와 6 소뇌 조각을 얻을 수 있습니다. </li><li> 인큐베이터에있는 여섯 잘 접시를 놓고 다음 일 일일 슬라이스 준비 후 슬라이스 매체의 ML 슬라이스 고정 될 때까지 다른 모든 일에 슬라이스 매체를 교체합니다. </li><li> 실험에 따라, 문화 5-7 일 후에 조각을 사용합니다. 처음 몇 일 후에 투명하게 조각을 사용하여 요철이 불투명 한 영역을 가지고 그 사람들을 버린다. 참고 : 슬라이스 내에서 불투명 한 영역은 눈으로 볼 수 있으며, 위상차에서 어두운 세포의 덩어리로 나타납니다. 기술이 습득 한 후에, 죽은 불투명 조각 조각 미만 1-5%에서는 거의 발생하지 않는다. </li></ol> NG2의 세포 분열 및 희소 돌기 아교 세포 분화 2 타임 랩스 영상 참고 : 경제의 제로 성장 생쥐 16 : 우리가 NG2cre를 사용 NG2 세포의 증식과 분화의 시간 경과 이미징을 수행하려면NG2 세포 및 그 자손 (도 1C-E)에서 EGFP를 발현한다. 이 줄 리포터 발현 즉시 적어도 4주의 기간 동안 후 단면 슬라이스 라이브 GFP + 세포의 이미지를 획득 할 수있을만큼 충분히 튼튼하다. 깨끗한 이미지는 문화의 첫 번째 3-5일을 통해 발생하는 슬라이스의 초기 숱이 기간 이후에 얻을 수있다. <ol><li> 실험을하는 동안, 37 ° C에서 세포 배양 인큐베이터에서 조각을 유지하고 시간의 짧은 기간 만 제거 (슬라이스 미만 15 분), 육 웰 플레이트 동안 영상에 뚜껑을 유지. </li><li> 디지털 카메라를 구비 한 반전 된 형광 현미경을 사용하여, 10 배 대물 렌즈를 사용하여 첫 번째 시점에서 이미지를 캡처. 참고 : 처음 지점이 실험에 의해 달라지며 조각이 제조되었다 일 또는 후 어느 시점이 될 수 있습니다. </li><li> 슬라이스의 두 회색과 흰색 물질 분야에서 시점 일에 여러 지역의 이미지를 캡처합니다. 주 일슬라이스 내의 특정하여 도시 및 후속 이미징 세션에 사용할 수있는 이미지에 개략적 위치를 매핑하여 전자 묘화 영역. 유용한 랜드 마크 백질 책자의 조각 및 / 또는 방향의 모서리를 포함 할 수 있습니다. 콘텐츠 각 시점에서 각 조각에 위치 4-8. </li><li> 4-6 시간 간격으로 연속 된 이미지를 캡처 NG2의 세포 분열 및 희소 돌기 아교 세포의 분화, 이상적 통해 5~7일 검사하는. </li><li> 모든 지역의 최종 이미지를 캡처하여 즉시 조각을 수정. 다음 조각 위에 다른 하나를 가하여, 문화 삽입의 바닥 (0.1M L-라이신 0.01 M 나트륨 메타 요오드와 4 % PFA) 고정액의 한 ML을 추가합니다. 그것은 막에서 분리 할 수​​ 있습니다로 슬라이스에 직접 정착 물총 않도록주의하십시오. 30 분 동안 조직을 수정하고 4 절에 설명 된대로 발달 단계 특정 마커를 사용하여 면역 조직 화학 진행합니다. </li><li> 0.2 M 인산 나트륨 완충액 3 × 10m 씻을 조각인치 1-3일 0.2 M 인산 나트륨 완충액에서 4 ° C에서 필요한 저장하기 전에 조각은 면역 염색한다면 이상적으로 24 시간 내에 염색을 수행합니다. 희소 돌기 아교 세포 (그림 2D-F)로 분화 한 세포의 비율을 결정하기 위해 4 장에 아래와 같이 면역 조직 화학 염색을 진행합니다. </li></ol> 슬라이스 문화에 유도 성 기자 형질 전환 마우스를 사용 NG2 세포 자손의 3 운명 매핑 주 : 문화 특정 시점에서 NG2 세포의 운명을 추적하기 위해, 유도 NG2creER : YFP 트랜스 제닉 마우스를 사용할 수있다. <ol><li> 배지에 에탄올 100 나노 4OHT 추가하여 Cre 호텔 재조합 및 리포터 발현을 유도한다. NOTE : 리포터 양성 세포는 모든 NG2 세포 단계에서 세포 것이다 ~ 20-25% YFP + NG2 + / NG2 + 세포의 유도 효율이 시점에서 1~2일 내에 표시한다 (도 2A-C) </li><li> 수정하고 슬라이스를 씻어상이한 시점에서의 각종 조작 후 (섹션 2.5 및 2.6에 기재되어 있음). </li></ol> 4 슬라이스 면역 조직 화학 <ol><li> 메스를 사용하여 삽입에서 부착 된 조각 세포막을 잘라. </li><li> 멤브레인 픽업시 슬라이스의 조성물을 방해하지 않도록주의하면서, 핀셋의 세트를 이용하여 24 웰 플레이트에 함유하는 인산염 완충 식염수 (PBS) 중 각각의 웰에 조각을 전송. </li><li> 1 시간 동안 PBS에서 5 % 정상 염소 혈청 (NGS)와 0.1 % 트리톤 - X100을 포함 차단 솔루션과 24 웰 플레이트에 조각을 전송합니다. </li><li> 4 ° C에서 PBS에서 5 % NGS에 / 차 항체 O에서 N을 슬라이스를 품어. NG2 세포 단계에서 세포를 식별하기위한, 그리고 NG2 PDGFRα 대한 항체를 사용. 희소 돌기 아교 세포 분화를 확인하기위한, 선종 성 용종증 콜라이 항원에 대한 항체를 사용하여 (APC; 클론을 CC1). </li><li> PBS 3 X15의 분에서 두 번째 일 세척 슬라이스에서 다음 차 antibo에 품다RT에서 1 시간 동안 PBS 중 5 %에​​서 NGS 죽는다. PBS 3 X15 최소의 조각을 세척하고 슬라이드에 탑재합니다. 멤브레인 대물과 조직 사이되지 않도록 슬라이드 대향 조각 위로 막 마운트. </li></ol>

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The authors declare that they have no competing financial interests

중추 신경계의 수초화 효율적으로 신경 세포의 통신 및 축삭의 생존 22 필수적이다. 25 - 대부분의 뇌 영역 16,23의 거주 인구를 유지하면서 NG2 세포는 지속적으로 성인에 myelinating 희소 돌기 아교 세포를 생성합니다. 이러한 세포의 분화를 조절 일부 유전 및 분자 메커니즘을 설명했지만 많이 발견되고 남아있다. Organotypic 슬라이스 배양 인해 해부학 영역, 세포 외 환경의 쉬?...

4 - 중추 신경계 Organoytpic 슬라이스 배양 semiintact 시스템 (1)의 뉴런 및 신경교 세포 생물학을 연구하는데 매우 유용한 것으로 입증되었다. 9 - 이러한 문화를 채택하고 세포 외 환경의 조작을 반복 장기 라이브 세포 이미징에 대한 쉬운 접근과 전기 생리 레코딩 (5)이 기본으로 해리 된 세포 배양의 많은 혜택을 유지하기가 비교적 간단하다. 또한, 슬라이스 배양 3 차원 조직 cytoarchitecture 지역 신경 연결을 유지하고, 가장 중요한 세포 유형은 시스템에 존재한다. 이러한 특성은 세포와 환경의 상호 작용으로 단일 세포의 행동과 생리를 조사하기 위해 이들 문화에 고유 한 편리한 시스템을 확인합니다. NG2 세포 증식 및 m을 계속 생성 포유류의 중추 신경계에서 신경교 세​​포 집단이다배아 및 출생 후의 개발 10 동안 희소 돌기 아교 세포를 yelinating. 이들은 광범위 해리 일차 세포 배양에서 연구되었으며, NG2 형광 단백질의 세포 특이 식 트랜스 제닉 마우스 라인의 최근의 발전은 급성 슬라이스 제제의 생체 내 거동 매핑 및 전기 생리 레코딩에서 촉진했다. 비록 이러한 연구와 거의 NG2 세포 증식 및 희소 돌기 아교 세포의 분화의 시간적 역학에 대해 공지되어있다. 해리 된 세포 배양 물을 광범위하게 약리 유전 조작에 대하여 설비를 위해 사용되지만, 그것은 세포의 미세 환경의 컨텍스트를 유지하는 것이 바람직하다 특히 다른 뇌 영역에서 이들 세포의 기능 차이를 질의에 적합하지 않다. 슬라이스 배양 세포 약리 조작에 대한 의무이며, 희소 돌기 아교 세포의 수초화 (11, 12)을 조사하기 위해 사용 된 간단한 대안을 제공(LPC)을 lysolecithin 또는 항체 약물 치료 15를 통해 탈수 초화 13, 14, 및 재유 수화 유도를 유도 한 후 신경근 응답. 방법은 조사 및 라이브 영상 및 고정 조직 (또는 postfixation) 전뇌와 소뇌에서 모두 촬영 Organotypic 슬라이스 문화의 NG2 세포의 증식 및 희소 돌기 아교 세포의 분화의 분석 설명을 수행 할 수 있습니다. 이 16 분할 한 후 NG2 세포 세포의 운명을 연구하고 성장 인자 유도 NG2 세포 증식 17 인 지역, 연령 의존적 차이를 발견하는데 사용될 수있는 강력한 방법이다. 이 상대적으로 간단한 기술은 또한 고유 및 / 또는 환경이 아교 세포의 생리학 및 신경 세포의 활동이나 수초의 손상에 대한 반응을 조절하는 메커니즘을 세포를 조사하기 위해 광범위하게 액세스 할 수 있습니다.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Slice Culture Medium&lt;/strong&gt;</td></tr><tr><td>Minimum Essential Medium</td><td>Invitrogen</td><td>11090</td><td>50%</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Invitrogen</td><td>14175</td><td>25%</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H-4034</td><td>25mM</td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030</td><td>1mM</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td>1mg/L</td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A-0278</td><td>0.4mM</td></tr><tr><td>Horse Serum</td><td>Hyclone</td><td>SH30074.03</td><td>25%</td></tr><tr><td colspan="4">Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td></tr><tr><td colspan="4">&lt;strong&gt;Dissection Buffer&lt;/strong&gt;</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td>124mM</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P5405</td><td>3.004mM</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td>1.25mM</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (anhydrous)</td><td>Sigma-Aldrich</td><td>M7506</td><td>4.004mM</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C7902</td><td>2mM</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761</td><td>26mM</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td>10mM</td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A-0278</td><td>2.0mM</td></tr><tr><td>Adenosine</td><td>Sigma-Aldrich</td><td>A4036</td><td>0.075mM</td></tr><tr><td colspan="4">Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O&lt;sub&gt;2&lt;/sub&gt; 5%CO&lt;sub&gt;2&lt;/sub&gt; before use</td></tr><tr><td colspan="4">&lt;strong&gt;Other Reagents and Supplies&lt;/strong&gt;</td></tr><tr><td>4-hydroxy tamoxifen (4OHT)</td><td>Sigma-Aldrich</td><td>H7904</td><td>100nM</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>EM Sciences</td><td>19200</td><td>4%</td></tr><tr><td>L-Lysine</td><td>Sigma-Aldrich</td><td>L-6027</td><td>0.1M</td></tr><tr><td>Sodium Metaperiodate</td><td>Sigma-Aldrich</td><td>S-1878</td><td>0.01M</td></tr><tr><td>Rabbit anti NG2 antibody</td><td>Chemicon (Millipore)</td><td>AB5320</td><td>1:500</td></tr><tr><td>Mouse anti CC1 antibody</td><td>Calbiochem (Millipore)</td><td>OP80</td><td>1:200</td></tr><tr><td>Mouse anti Ki67 antibody</td><td>Vision Biosystems</td><td>NCL-Ki67-MM1</td><td>1:300</td></tr><tr><td>Mouse anti NeuN antibody</td><td>Chemicon (Millipore)</td><td>MAB377</td><td>1:500</td></tr><tr><td>Species specific secondary antibodies</td><td>Jackson Immunoresearch</td><td></td><td></td></tr><tr><td>Normal Goat Serum (NGS)</td><td></td><td></td><td>5%</td></tr><tr><td>Triton X-100</td><td></td><td></td><td>0.10%</td></tr><tr><td>Mounting medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Millicell culture membrane inserts 0.45&amp;mu;m pore size</td><td>Fisher Scientific</td><td>PICM03050</td><td></td></tr><tr><td>Bottle top filters</td><td>Fisher Scientific</td><td>09-740-25E</td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt; 5% CO&lt;sub&gt;2&lt;/sub&gt; gas mix</td><td></td><td></td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td></td><td></td><td></td></tr><tr><td>Sterile six well plates</td><td></td><td></td><td></td></tr><tr><td>Dissecting (hippocampal) or weighing spatulas</td><td></td><td></td><td></td></tr><tr><td>Manual or automatic tissue chopper</td><td></td><td></td><td></td></tr><tr><td>Razor blades</td><td></td><td></td><td></td></tr><tr><td>Disposable transfer pipettes</td><td></td><td></td><td></td></tr><tr><td>35mm and 60mm sterile Petri dishes</td><td></td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Inverted Phase Microscope</td><td></td><td></td><td></td></tr><tr><td>Laminar Flow Hood</td><td></td><td></td><td></td></tr><tr><td>Tissue Culture Incubator</td><td></td><td></td><td></td></tr><tr><td>Inverted Fluorescence Microcope</td><td></td><td></td><td></td></tr></tbody></table>

organotypic slice cultures to study oligodendrocyte dynamics and myelination

NG2 발현 세포 (polydendrocytes, 희소 돌기 아교 세포 전구 세포)는 중추 신경계에서 주요한 넷째 신경교 세​​포 집단이다. 배아 및 출생 후의 개발하는 동안 그들은 적극적으로 증식 myelinating 희소 돌기 아교 세포를 생성합니다. 이 세포는 일반적으로 기본 해리 문화, 신경 세포 공 배양 연구, 고정 조직되었다. 새로 사용 가능한 트랜스 제닉 마우스 선이 배양 시스템은 전뇌와 소뇌 모두 회색과 백질 영역에 희소 돌기 아교 세포 계통 세포의 증식과 분화를 조사하는데 사용될 수있는 슬라이스. 슬라이스 문화는 출생 초기 마우스에서 준비가되어 1 개월까지에 대한 문화에 보관됩니다. 이 조각은 세포의 행동과 상호 작용을 조사하기 위해 배양 기간 동안 여러 번 이미지를 만들 수 있습니다. 이 방법은 NG2의 세포 분열의 시각화와의 상세한 분석을 가능하게하고있는 동안 희소 돌기 아교 세포의 분화로 이어지는 단계 지역별 의존 할 수 있습니다천만에 NG2 세포 및 희소 돌기 아교 세포 기능 이질성. 이는 생체 내에서 밀접 발견 유사한 셀룰러 환경에서 시간 경과에 영향을 미치는 이러한 세포 및 극한 외부 신호를 조사하는데 사용될 수있는 강력한 기술이다.

대표 데이터의 예는 다음과 같습니다 P8 NG2cre의 전뇌와 소뇌에서 모두 슬라이스 문화를 사용하여 획득 한 것을 : 경제의 제로 성장, NG2creER : YFP를 형질 전환 마우스 라인을 PLPDsRed. NG2 세포 전뇌와 소뇌 조각 (그림 1B-D, 비디오 1) 이러한 세포의 표현형 일 동안 이미지를 만들 수는 고정 후 결정 NG2와 CC1 항체 (그림 1E)과 면역 염색 할 수 있습니다. 이미징 사는 또한 세포 증식?...

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Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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-slice-cultures-to-study-oligodendrocyte-dynamics

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Neuroscience

18.2K 조회수.  University of Connecticut. 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.

비디오: Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

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 ...

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Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

네크로 텍스의 Organotypic 슬라이스 준비에서 전체 셀 녹음

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

신경과학

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

<em&gt; 생체 내</em&gt; 급성 뇌 조각의 희소 돌기 아교 세포 지시 T 세포 공격의 모델

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

면역학 및 감염병학

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. ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

시각화 및 Adeno 관련 바이러스 및 Confocal 현미경 검사 법을 사용 하 여 Organotypic 뇌 조각에서 Oligodendrocyte 세포의 라이브 영상

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

준비 및 Immunostaining Myelinating Organotypic 소 뇌 조각 문화

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

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 ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

공동 문화 실험을 위한 올리고엔드로시테 및 올리고엔드로시테-컨디셔닝 배지 생성

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...