Supported by grants from CNRS and INSERM. PM acknowledges support from the Fondation pour la Recherche M&#233;dicale en France (Equipe FRM 2009). SC acknowledges support from the Fondation de France.

이 작품에 사용 된 마우스는 보살핌과 유럽 연합위원회의 가이드 라인을 실험 동물의 사용에 대한 (609분의 86 / EU)에 따라 처리 하였다. 필요 솔루션의 1 준비 <ol><li> 주식 솔루션 <ol><li> 10 배 폴리-L 오르니 틴 (PLO) 해결책 : PLO 브롬화 수소 산염 10 ㎎을 무게 (분자량 = 30,000-70,000) 및 멸균 물 70 ㎖에 녹인다. -20 ° C에서 0.2 μm의 주사기 필터, 나누어지는 솔루션을 사용하고, 가게를 필터링합니다. </li><li> 30 % 포도당 해결책 : 30 D (+)의 g을 달아 - 포도당을 100 ml의 총 부피에 멸균 물에 용해. 4 ° C에서 필터, 나누어지는 및 저장. </li><li> 5X 둘 베코 변형 이글 배지에 (DMEM) / 영양 혼합물 햄 F-12 : DMEM / 햄 F-12 분말 6g를 달아, 100 ml의 총 부피를 멸균 수에 용해. 4 ° C에서 필터, 나누어지는 및 저장. </li><li> 7.5 % 중탄산 나트륨 (탄산 수소 나트륨) Soluti에 : 탄산 수소 나트륨 7.5 g을 계량하고 100 ㎖의 총 부피로 멸균 수에 용해. 4 ° C에서 필터, 나누어지는 및 저장. </li><li> 1 M HEPES 용액 : HEPES 23.8 g을 달아 100 ml의 총 부피를 멸균 수에 용해. 4 ° C에서 필터, 나누어지는 및 저장. </li><li> 70 % (v / v) 에탄올 : 멸균 수 30 ㎖의 무수 에탄올 70 ㎖에 희석. </li></ol></li><li> 포도당과 항생제 (PBS-GAB)와 인산염 완충 생리 식염수 : 10 30 % 포도당의 수용액 및 둘 베코 인산염 완충 생리 식염수 500 mL에 페니실린 - 스트렙토 마이신 용액 5 ㎖를 추가합니다. 4 ℃에서 보관하십시오. </li><li> 비활성화 된 소 태아 혈청 (IFBS가) : 4 ° C에서 하룻밤 소 혈청을 해동 30 분 동안 56 ° C에서 비활성화. -20 ° C에서 50 ML로 나누어지는 및 저장. </li><li> 문화 미디엄 : 멸균에서 5 배 DMEM / F12 햄의 연속 40 ML, 1 1 M HEPES ml의 2 ML 200 mm의 L-글루타민, 페니실린 - 스트렙토 마이신 (streptomycin) 2 ㎖, 4 ㎖의 30 %의 포도당을 혼합ND 180 ml의 최종 부피로 7.5 %의 NaHCO3 3 ㎖. 필터 및 당일 사용. </li><li> 호르몬 믹스 <ol><li> 문화 매체의 180 ML를 준비합니다. 이 매체에 아포 트랜스페린의 200 mg을 녹인다. </li><li> 인슐린을 50mg 칭량하고, 0.1 M 염산 2 ㎖에 녹이고. 부드럽게 혼합하면서 멸균 물을 천천히 8 ML을 추가합니다. 배지에서이 솔루션을 희석. </li><li> 퓨 트레 염산염 19.3 mg의 밖으로 달아 멸균 수 10ml에 용해. 호르몬 믹스로 10 ml의 솔루션을 추가합니다. </li><li> 멸균 수에 3 mM의 아 셀렌 산 나트륨 용액 및 무수 에탄올에 2 mM의 프로게스테론 솔루션을 준비합니다. 호르몬 믹스 각 솔루션의 20 μl를 추가합니다. </li><li> -20 ° C에서 호르몬 믹스, 나누어지는 10 ML에 저장을 필터링합니다. </li></ol></li></ol> 문화 접시와 인스트루먼트의 2 준비 <ol><li> 문화 판의 FBS 코팅 : 해부 전에 날, 배지 180 ㎖의 준비를합니다. 10 ML을 추가배지 90 mL에 IFBS. 추가 300 μL / 웰의 배지 / 폴리-D-라이신 - 예비 코팅 된 24 웰 플레이트에 10 % IFBS. 37 ° C에서 밤새 품어. 4 ° C에서 (로와 IFBS없이) 남은 용지를 보관하십시오. </li><li> 악기와 파스퇴르 피펫을 소독. 클래스 II 층류 후드 및 CO 2 배양기뿐만 아니라 재료 표에 나열된 장비를 수집합니다. </li></ol> 마우스 중뇌의 3 해부 <ol><li> (제도적 지침에 따라) E13.5 임신 마우스를 희생. 70 % 에탄올로 마우스의 복부를 청소하고 복부 벽을 엽니 다. 섬세 가위를 사용하여 열, 자궁 뿔을 수집 자궁 뿔에서 각 배아를 해부하고, 양막을 제거합니다. 멸균 PBS-GAB를 포함하는 100mm 배양 접시에있는 배아를 놓고 집게를 사용하여 3 연속 동일 화장실에서 송금을 씻는다. 해부를 들어, 60mm 멸균 페트리 접시에 세에 의해 배아를 놓습니다. </li><li> mov 인실체 현미경을 이용하여 최종 페트리 접시를 전자. 배아를 참수하지 마십시오. Vannas 가위를 사용하여 뇌를 절제. <ol><li> 조심스럽게 제거하고 fore-와 후뇌 지역을 폐기합니다. 주동이의 측면으로, 지협 지역에서 꼬리 쪽 인하, 시상 지역에 가까운 잘라 우수한 둔덕을 제거합니다. </li><li> 복부 중뇌가 분리되면, 신중 초 미세 집게를 사용하여 수막을 제거합니다. PBS-GAB 가득 멸균 13 ML 튜브에, 수막없이 해부 세그먼트를 수집합니다. </li></ol></li><li> 70 % 에탄올로 철저하게 mesencephala를 포함하는 튜브를 청소하고 후드를 가져옵니다. </li></ol> 4 셀 해리 <ol><li> 세척은 멸균 PBS-GAB로 3 배 mesencephala. 뇌 조각이 각 세척 사이에 정착 조직을 방해하지 않도록 할 수 있습니다. 마지막 세척 후, 37 ° C에서 15 분 동안 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)의 3 ㎖에 뇌 세그먼트를 가능한 한 신중하게 많은 솔루션을 제거하고 배양이 창업 보육 센터 CO. </li><li> 불 닦으 파스퇴르 피펫은 비 - 연마 된 유리 피펫의 끝과 같은 뉴런의 생존을 최대화하고 날카로운 해리 단계에서 세포에 손상을 줄 수있다. 그것을 불을 연마하는 동안 약간 선단 직경 파스퇴르 피펫 (0.5 mm)을 줄일 수 있습니다. </li><li> 조심스럽게 최대한 트립신 / EDTA 용액을 제거하고 배지 10 ml의 / 10 %를 추가 IFBS. 워시 뇌 세그먼트는 배양 배지 / 10 % IFBS와 배. </li><li> 불 연마 파스퇴르 피펫을 사용하여, 배양 배지 / 10 % IFBS의 6 ㎖에 mesencephala의 분해를 시작합니다. 씹다 배 후 새로운 무균 13 ㎖의 튜브에서 매체를 수집 청크 정착 할 수 있도록, (기포를 방지). </li><li> , 배지 6 ㎖에 추가하면 이전 단계를 반복하고 13 ㎖의 동일 관에 해리 된 세포를 포함하는 배지를 수집한다. </li><li> 5 분 동안 160g에서 원심 분리기 세포. 뜨는을 취소하고 보충 된 배지에서 세포를 재현 탁10 % 호르몬 믹스. </li></ol> 5 셀 도금 <ol><li> Malassez 세포에 세포 현탁액을 카운트하고 600,000 세포 / ml의 농도로 매체의 볼륨을 조정한다. 약 1,700,000 세포는 하나의 마우스 중뇌에서 얻을 수 있습니다. </li><li> 24 웰 플레이트에서 코팅 매체를 FBS 함유를 제거하고 각 웰에 세포 현탁액 1 ㎖에 추가 할 (600,000 세포 / 웰, TH + 세포 2-4 %). </li><li> 37 ° C에서 접시를 품어 5 % CO 95분의 2 % 대기. 어떤 매체 변화는 필요하지 않습니다. 도파민 신경 세포는 체외에서 약 5-7일 (도파민 수송의 일관성있는 표현) 후 성숙. 매체 교체없이 십오일에 문화까지 세포를 보관하십시오. </li></ol> (6) 면역 형광 프로토콜 <ol><li> Coverslips는 청소 <ol><li> 해부 전날, 페트리 접시에 1 M 염산 10 ㎖를 추가합니다. 액체 12-15 유리 커버 슬립의 표면에 올려 놓고 15 분을 기다립니다. </li><li> 커버를 싱크액체로 미끄러 15 분 동안 기다립니다. </li><li> 염산을 제거하고 물로 배를 씻는다. </li><li> 한 번 순수 에탄올로 신속하게 된 커버를 세척 후 접시에 순수 에탄올을 추가하고 30 분 동안 기다립니다. </li><li> 후드, 에탄올 세정 된 커버를 제거하고 멸균 집게를 사용하여 12 - 웰 세포 배양 접시에 한 웰당 커버 슬립을 추가한다. </li><li> 세척 멸균 물 (1 ㎖ / 웰)로 두 번 된 커버. 그런 다음, 멸균 PBS로 한번 씻어. </li></ol></li><li> Coverslips는의 코팅 <ol><li> PBS를 제거하고 500 μL / 2 배 PLO의도 (PBS에 희석)를 추가합니다. CO 2 배양기에서 37 ° C에서 4 시간 동안 품어. </li><li> PLO의 솔루션을 제거하고 멸균 PBS로 3 회 씻는다. </li><li> PBS를 제거하고 500 μL / 웰의 20 % IFBS / 1g / ㎖ 라미닌을 추가합니다. CO 2 배양기에서 37 ° C에서 밤새 품어. </li></ol></li><li> 면역 형광의 셀 도금 <ol><li> 12 - 웰 플레이트에서 코팅 매체를 제거합니다. </li><li> 900,0 추가00 세포 / 웰이 ml의 부피와 배양기에서 37 ° C에서 세포를 성장. 세포는 중간 교체없이 십오일에 문화까지 유지 될 수있다. </li></ol></li><li> 면역 염색 <ol><li> 12 - 웰 플레이트에서 매체를 제거하고 잘 DMEM이 37 ° C로 예열 / 500 ㎕를 씻어. </li><li> / 잘 DMEM 500 ㎕를 37 ° C로 예열 추가, 부드럽게 160 μL / 웰 8 % 파라 포름 알데히드 (PFA, 2 % 최종 농도)를 추가하고 37 ° C에서 10 분 동안 배양한다. </li><li> PFA를 제거하고 PBS로 3 배 세척 / 0.1 M 글리신을 10 분 동안. </li><li> , PBS를 제거 300 μL / 웰의 PBS / 0.05 % 트리톤 X-100 / 20 % 염소 혈청을 추가하고, 실온에서 30 분 동안 배양한다. </li><li> 매체를 제거 300 μL / 웰의 PBS에 희석 차 항체 / 0.05 % 트리톤 X-100 / 1 % 염소 혈청을 추가하고 실온에서 2 시간 동안 배양한다. 도파민 성 신경 세포 배양의 특성의 검출에 사용될 수있는 일차 항체 재료 표에 나타내었다. 유지일 잘 차 항체없이 다른 차 항체의 배경에 대해 평가합니다. </li><li> 배지를 제거하고 PBS로 3 배 세척 / 0.2 % 젤라틴을 10 분 동안. </li><li> 매체 분리 300 μL / 웰의 PBS에 희석 차 항체 / 0.05 % 트리톤 X-100 / 1 % 염소 혈청을 추가하고 어두운 곳에서 실온에서 1 시간 동안 배양한다. 사용 된 차 항체는 재료 표에 표시됩니다. </li><li> 배지를 제거하고 PBS로 3 배 세척 / 0.2 % 젤라틴을 10 분 동안. </li><li> 매체 제거하고 PBS로 3 회 씻는다. </li><li> 다운 VECTASHIELD의 드롭 유리 슬라이드 셀 측 커버 슬립을 장착. 다음 4 ℃에서 보관, 설치 매체 건조 수 있도록 실온에서 밤새 슬라이드를 유지합니다. </li><li> 공 초점 현미경 표면 형광에 장착 된 위상차 현미경을 사용하여 이미지를 획득. 대표 이미지는 라이카 SP2 UV 공 초점 현미경으로 획득 하였다. </li></ol></li></ol>

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The authors have nothing to disclose.

이 프로토콜은 마우스 배아 도파민 성 신경 세포를 검출하는 면역 형광 절차에서 중간 뇌 신경 세포의 일차 배양을 준비하기 위해 필요한 절차 및 시약을 제공한다. 절차의 중요한 단계는 배아의 박리 및 회수 뇌 절편 기계적 해리이다. 고품질 해부 악기 해부 기술을 마스터하는 데 도움이됩니다. DA 뉴런 중뇌의 작은 부분을 구성한다. 따라서, 복부 중뇌의 오른쪽 부분을 수집하는 DA 뉴런 2-4 % 함?...

도파민, 필수적인 뇌의 신경 전달 물질 1,2 중 하나는, 주로 중뇌 도파민 (DA) 신경 세포에 의해 방출된다. DA 신경 세포의 대부분은 중뇌 2-6의 복부 부분에 상주. mesostriatal, mesolimbic 및 mesocortical 경로 2,5 : 도식적으로, 중뇌 DA 뉴런은 해부학 적 및 기능적으로 세에서 별개의 프로젝션 시스템을 나눌 수 있습니다. 전두엽 피질에 투사 도파민 경로는 인식이 연루되는 반면, 흑질 선조체 경로는 mesolimbic 경로 보강, 동기 부여, 학습에 중요한 역할을, 모터 동작에 참여하고있다. DA 뉴런은 정신 분열증, 주의력 결핍, 하이퍼 활동 장애, 파킨슨 병 (PD) 2,4 등 여러 가지 인간의 신경 질환에 참여하고 있습니다. PD는 흑질 접속 DA 신경 세포의 진행성 및 퇴행성 선택적 특징선조체에 갈 거예요의 compacta (SNC). PD의 운동 증상의 책임이있는 선조체 (striatum)에서 심각한 도파민 고갈에 nigro - 선조체 DA 뉴런 결과의 손실 (운동 완만, 휴식 진동, 강성) 7. 특발성 PD의 초기 원인은 확립되지 않았으며, 현재 치료 선조체 도파민 수준을 복원하는 목표 만 증상이다. 대부분의 처방 약은 L-도파 (레보도파), 도파민의 자연 전구 물질이다. 레보도파의 투여가 일정 시간 동안 도파민 손실을 보상하지만, 모터 후 합병증 (운동 이상증의 온 / 오프 상태) 장기간 치료 8,9 일어난다. 도파민 뉴런과 PD에 대한 연구가 진행되어 일정 강렬한 노력은 세포 이식, 유전자 치료, 또는 신경 보호제 10,11에 기반 치료법을 개발하기 위해 이루어지고있다. 단, 중요한 문제가 아닌 해명 남아 극단적 vulnerab의 원인이 무엇DA 뉴런의 ility? 답변의 일부는 DA 신경의 활성에서 찾을 수있다. 전기적 활동 및 DA 신경 세포의 흥분성의 감소는 12 퇴화하는 그들의 성향을 확대 할 것으로 보인다. 그럼에도 불구하고, PD의 병인의 복잡성 DA에 관여하는 메커니즘 신경 세포가 13 ~ 15 변성을 식별하기 위해 추가 연구가 필요합니다. 차 문화는 DA 신경 세포의 특성 16 ~ 19을 공부하고 신경 보호제 20-24의 평가를 위해 다양한 스트레스에 이러한 뉴런에 도전 특히 적합하다. 쥐 배양 모델은 대부분 쥐 배아 중뇌의 박리가 용이​​ 한, 마우스와 비교하고, 뉴런의 더 많은 양이 쥐에서 얻을 수있다 사용된다. 그러나, 병 (25)의 형질 전환 마우스 모델의 생성은 마우스에서 26-29 차 배양 용 신경학 커뮤니티의 관심을 상당히 증가하고있다. 홍보 문화 있지만신생아 동물로부터 epared은 뉴런 분화하는 능력을 유지 한 경우, 포스트 - 유사 분열 단계 (중뇌 신경 용 E13.5)에서 배아을 준비하는 것이 좋다, 사용될 수있다. 다음 프로토콜은 준비하기가 가장 어려운 마우스 배아 (E13.5)에서 차 문화에 고립 된 중뇌의 신경 세포를 제공합니다. 특히, 우리는 더 나은 재현성 위해 무 혈청 배지를 사용하는 프로토콜을 제공한다. 배양 제제 (해부 및 기계적 해리)에 두 개의 가장 중요한 단계는 신중 연관된 비디오에 설명 될 것이다.

<table border="0" cellpadding="0" cellspacing="0" width="1291">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">DMEM 4.5g/L Glucose with L-Glutamine</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">0.05% Trypsin-EDTA (1X), Phenol Red&#160;</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td style="width:139px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine, 200 mM Solution</td>
			<td>Life Technologies</td>
			<td>25030123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Nutrient Mixture F-12 Ham</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D0547</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Laminin - 1 mg/mL in Tris buffered NaCl</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-Ornithine hydrobromide</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin from porcine pancreas</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">I5523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">apo-Transferrin human</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">T1147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Putrescine dihydrochloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">P5780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sodium selenite</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">S5261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">H4034&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">G7126</td>
			<td>Stock solution 1M in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">G9391</td>
			<td>Stock solution 2% (w/v) in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">T8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% in water</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:139px;">RT 15710-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrogen carbonate (NaHCO3)</td>
			<td>Merck Millipore</td>
			<td style="width:139px;">106329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D(+)-Glucose, Monohydrate</td>
			<td>Merck Millipore</td>
			<td>4074-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Hydrochloric acid - c(HCl) = 1 mol/l (1 N) Titripur</td>
			<td>Merck Millipore</td>
			<td style="width:139px;">109057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sterile water - Aqua B. Braun</td>
			<td style="width:176px;">Braun</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Ethanol absolute NORMAPUR analytical reagent</td>
			<td>VWR</td>
			<td style="width:139px;">20821.321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sterile Petri Dishes</td>
			<td>VWR</td>
			<td>82050-566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Pasteur pipettes plain glass - Wilhem Ulbrich GdbR.</td>
			<td>VWR</td>
			<td>612-2297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Counting chamber Malassez</td>
			<td>VWR</td>
			<td style="width:139px;">631-0975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum Acrodisc Syringe Filter with Supor Membrane, Sterile, GF/0.2 &#181;m, 37 mm</td>
			<td>PALL Life science</td>
			<td style="width:139px;">4525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors - Straight, sharp-sharp, 14.5 cm long</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td>14002-14</td>
			<td>To open the abdominal wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors - Straight, pointed, delicate, 10 cm long</td>
			<td style="width:176px;">MORIA</td>
			<td>4877A</td>
			<td>To open the uterine wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps - Curved, usual, serrated jaws 1 mm</td>
			<td style="width:176px;">MORIA</td>
			<td>2183</td>
			<td>To manipulate embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Vannas Scissors - Curved, pointed, 7 mm blades</td>
			<td style="width:176px;">MORIA</td>
			<td>MC50</td>
			<td>To take out the mesencephalon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Fine Forceps - Curved, delicate, 13 cm long</td>
			<td style="width:176px;">MORIA</td>
			<td>9987</td>
			<td>To remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD BioCoat Poly-D-Lysine 24-well Multiwell Plates</td>
			<td>BD Bioscience</td>
			<td style="width:139px;">356414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Falcon 12-well Cell Culture Plate, flat-bottom with lid</td>
			<td>BD Bioscience</td>
			<td style="width:139px;">353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperFrost Microscope Slides, Ground edges 90&#186;</td>
			<td>MENZEL-GL&#196;SER</td>
			<td style="width:139px;">AG00008032E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision cover glasses thickness No. 1.5H circular 18 mm &#216;</td>
			<td>MARIENFELD</td>
			<td>117580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyclonal Rabbit Anti-Microtubule-Associated Protein 2 (MAP2) Antibody</td>
			<td>Chemicon Millipore</td>
			<td>AB5622</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Mouse Anti-Glutamate Decarboxylase (GAD67) Antibody, clone 1G10.2</td>
			<td>Chemicon Millipore</td>
			<td>MAB5406</td>
			<td>1/400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Rat Anti-Dopamine Transporter (DAT) Antibody, clone DAT-Nt&#160;</td>
			<td>Chemicon Millipore</td>
			<td>MAB369</td>
			<td>1/500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Mouse Anti-5-HT Antibody</td>
			<td style="width:176px;"></td>
			<td style="width:139px;"></td>
			<td>1/8,000 - Generous gift from Yves Charnay (Swizerland, Yves.Charnay@hcuge.ch)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum, New Zealand Origin</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:139px;">16210-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 405 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:139px;">A-31556</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td>A-11001</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 Goat Anti-Rat IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td>A-11007</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD HardSet Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Stereomicroscope</td>
			<td>Carl Zeiss microscopy</td>
			<td>Stemi-2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Bunsen Burner FIREBOY</td>
			<td style="width:176px;">VWR</td>
			<td style="width:139px;">451-0136</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary culture of mouse dopaminergic neurons

도파민 뉴런은 뇌 신경 세포의 총 수의 1 % 미만을 나타낸다. 신경이 적은 양의은 모터 제어, 동기 부여, 및 작업 메모리 중요 뇌 기능을 조절한다. 흑질 선조체 도파민 뉴런 선택적 파킨슨 병 (PD)에 퇴화. 이 진보적 인 신경 세포의 손실은 명백하게 병리의 모터 증상 (운동 완만, 휴식 진전, 근육 강도)와 연결되어 있습니다. 도파민 신경 세포의 변성을 담당하는 주요 에이전트는 아직 알 수 없습니다. 그러나 이러한 신경 세포는 다양한 조건에서 매우 취약한 것으로 나타납니다. 차 문화는 속성과 도파민 신경 세포의 특성을 조사하기 위해 가장 중요한 모델 중 하나를 구성한다. 이러한 문화는 중지하거나 신경 세포의 변성을 느리게하기 위해 PD 병리를 모방 다양한 스트레스 에이전트 및 신경 보호 화합물을 제공 할 수 있습니다. GENER왔다 PD의 수많은 형질 전환 마우스 모델지난 10 년간 나타나는데 더 도파민 신경 세포 배양에 대한 연구자들의 관심이 증가했다. 여기서, 비디오 프로토콜은 배아 마우스 뇌의 섬세한 절개에 초점을 맞추고있다. 복부 중뇌의 정확한 절제 후속 연구를 할 수 있도록 도파민 세포에서 충분히 풍부한 신경의 문화를 얻기 위해 매우 중요합니다. 이 프로토콜은 배아 형질 전환 마우스 실현 면역 형광 염색, 정량 PCR, 둘째 메신저 정량화, 또는 신경 세포의 죽음 / 생존 평가에 적합하다 할 수 있습니다.

중뇌 문화 단계의 그림 흐름도 그림 1에 나타나있다. 간단히, 임신 스위스 마우스에서 E13.5 배아를 수집 한 후, 복부 중뇌가 전체 배아 해부한다. 고립 된 뇌 조각이 연속적으로 소화 효소 및 기계적 분리에 제출됩니다. 해리 된 세포를 미리 코팅 된 12 - 또는 24 - 웰 플레이트에서 배양 배지에 재현 탁시키고, 도금, 원심 분리에 의해 펠렛 화한다. 세포는 중간 교체없이 십오일까지 유지?...

Watch this Scientific Journal Video about Primary Culture of Mouse Dopaminergic Neurons at JoVE.com

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

마우스 도파민 뉴런의 차 문화

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

primary-culture-of-mouse-dopaminergic-neurons

Research

JoVE Journal

Neuroscience

37.5K 조회수.  Institut de Génomique Fonctionnelle, Montpellier. Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

비디오: Primary Culture of Mouse Dopaminergic Neurons

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection 및 배아 마우스 Hippocampal과 두피 뉴런의 주요 문화

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

연구

신경과학

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

차 쥐 두피 뉴런과 Glia의 Bilaminar의 공동 문화

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

마우스 Dopaminergic 및 3 차원 콜라겐 매트릭스 Assays의 Striatal Explants의 해부와 문화

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

차 해마 신경 세포의 칼슘 인산염 형질

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

배아 쥐의 뇌에서 차 중뇌 도파민 신경 세포의 분리, 문화 및 장기 유지 보수

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

신경 죽음 및 마우스 기본 소 뇌과 립 신경에 변성을 모델링

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

마우스 기본 Hypothalamic 뉴런 Deconvolution 현미경을 사용 하 여 매매 GLUT4 단백질의 라이브 이미지

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

형광 활성 세포 선별을 통해 확립 된 정제 GABAergic 또는 Glutamatergic 뉴런의 일차 세포 배양

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

뇌 슬라이스와 기본 세포 배양에서 피라미드 뉴런의 탄도 라벨링

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...