우리는 또한 중요 한 독서에 대 한 가브리엘 손을 감사합니다.

모든 실험은 오사카 대학 및 일본 신경 과학 학회의 동물 복지 위원회에 의해 설립 하는 지침에 따라 수행 했다.
1. Organotypic cocultures의 시상과 대뇌 피 질
참고: 자세한 절차를 참조 원래 간행물7,8,13. 모든 절차는 무 균 조건 하에서 수행 되어야 합니다. Sprague-Dawley (SD) 쥐 신경 문화 사용 됩니다.
<ol><li>
시 약의 준비 참고: 다음 시 약의 한 쥐 두뇌에 대 한 있습니다.<ol>
<li>
수정 된 n 2 보충 x 10 준비 13: 인슐린 (50 μ g/mL), 황 체 호르몬 (200 nM), 하이드로 코 티 손 (200 nM), 나트륨 selenite (300 nM), 올려진다 (1 mg/mL), putrescine (1mm), 포도 (60 mg/mL)</li>
		<li>
준비 포함 하는 혈 청 문화 매체 (10 mL): 85 %DMEM / F12, 10% 수정 10 x n 2 보충, 5% 태아 둔감 한 혈 청 (FBS)</li>
		<li>
준비 혈 청 무료 문화 매체 (20 mL): 90 %DMEM / F12, 10% 수정 10 x n 2 보충</li>
		<li>쥐 꼬리 콜라겐 솔루션 (0.5 mL)을 준비 합니다. 간단히, 무 균 조건 하에서 쥐 꼬리에서 부드러운 섬유를 초 산 솔루션에서 밤새 품 어. 원심, 후 상쾌한 (몇 mg/mL)를 수집 하 고 냉장고13에 저장 합니다.</li>
	</ol>
</li>
	<li>
문화 요리 준비
	<ol>
<li>35 mm 페 트리 접시에 막 삽입을 놓습니다.</li>
		<li>막에 쥐 꼬리 콜라겐 솔루션의 40-50 μ를 추가 하 고 중심 콜라겐 솔루션을 확산.</li>
		<li>공기 깨끗 한 벤치에 완전히 막 건조.</li>
		<li>막이는 매체와 젖, 접시에 포함 하는 혈 청 문화 매체의 1.5-2 mL를 넣어.</li>
		<li>인큐베이터에서 (37 ° C, 습도 95%, 공기와 5% CO2) 도금 하기 전에 문화 접시를 놓습니다.</li>
	</ol>
</li>
	<li>
대뇌 피 질의 조각 준비
	<ol>
<li>모든 수술 기구 70% 에탄올과 10 분 이상 소독.</li>
		<li>출생 후 하루 (P) 2에서 신속 하 게 뇌를 해 부 (몇 분 동안 얼음 조각에 몰두) 하는 차가운 취 쥐.</li>
		<li>장소 100 mm 페 트리 접시 찬 행 크 스의 100 mL를 포함으로 뇌 균형 소금 솔루션.</li>
		<li>좋은 겸으로 두뇌에서 피아 문제를 제거 하 고 microsurgical가 위 시각과 somatosensory 피 질에서 대뇌 피 질의 조각 (300-400 μ m 두께)를 잘라 (세부 사항, 그림 1참조). 참고: 대뇌 피 질의 조각의 두께 쌍 안 현미경 격자의 규모에 의해 대략 추정 된다. 참고: 또는, 대뇌 피 질의 조각 무 균 조건 하에서 vibratome로 할 수 있다.</li>
		<li>일회용 플라스틱 피 펫으로 막 삽입에 몇 대뇌 피 질의 조각 전송 합니다.</li>
		<li>(자세한 내용은 참조8,13) 화재 광택 파스퇴르 피펫으로와 문화 삽입의 중앙 조각의 위치를 조정 하 고 삽입에서 초과 매체를 제거 합니다. 참고: 슬라이스 가스 매체의 충분 한 공급 받을 수 있도록 조각의 표면 보다 약간 중간의 레벨을 조정 합니다. 이것은 세포 생존 능력에 대 한 중요 합니다.</li>
		<li>혼자 하루 전에 thalamic 블록을 촬영 하 고 그것을 옆에 배치 되어 습도 95% 공기와 5% CO2, 그리고 문화의 환경에서 37 ° C에서 포함 하는 혈 청 문화 매체에서 분할 영역을 유지 합니다.</li>
	</ol>
</li>
	<li>
Thalamic 블록의 준비
	<ol>
<li>모든 수술 기구 70% 에탄올과 10 분 이상 소독.</li>
		<li>임신한 쥐 (배아 하루 15) 복 주사 pentobarbital (50 mg/kg) 마 취를 포함 하 여 anesthetize.</li>
		<li>자 궁 뿔에서 각 태아를 해 부 고 배아 100 mm 페 트리 접시 찬 행 크 스 균형된 소금 솔루션의 100 mL를 포함 합니다.</li>
		<li>쌍 안 현미경 각 배아에서 뇌를 제거 하 고 주로 microsurgical가 위 ( 그림 1참조)를 사용 하 여 ventrobasal 복잡 하 고 측면 geniculate 핵 (약 0.5 m m x 3)7 을 포함 하는 thalamic 블록을 잘라.</li>
	</ol>
</li>
	<li>
문화 및 organotypic cocultures를 유지
	<ol>
<li>파스퇴르 피 펫을 사용 하 여, 멤브레인 필터에 배치 되어 있는 대뇌 피 질의 조각의 심 실 표면 옆 thalamic 블록을 배치 합니다.</li>
		<li>습도 95% 공기와 5% CO2의 환경에서 37 ° C에서 포함 하는 혈 청 문화 매체에서 cocultures을 유지 관리 합니다.</li>
		<li>문화에 있는 2 일 후 혈 청 무료 문화 매체와 매체의 절반을 교환 합니다. 그 후, 매체 2-3 일 마다 교환 합니다. 참고: 매체의 수준으로 세포 생존에 대 한 중요 한 중간 교환 후 확인 되어야 한다.</li>
	</ol>
</li>
</ol>2입니다. Electroporation
모든 절차는 무 균 조건 하에서 수행 되어야 합니다.
참고: Electroporation 블록의 분리를 방지 하기 위해 thalamic 블록의 준비 후에 1 일을 수행 합니다.
<ol><li>
플라스 미드
	<ol>
<li>플라스 미드의 혼합물을 다음과 같이 확인: pCAGGS-DsRed 2 μ g/μ; pCAG SYP EGFP 3.5 μ g/μ. 참고: 내 무료 Maxiprep 키트를 사용 하 여 두 플라스 미드 정화 하 고 행 크 스 균형된 소금물에 용 해.</li>
	</ol>
</li>
	<li>
Electroporation 장비 설치 자극과는 아이 솔 레이 터 등 전기 악기는 그림 2와 같이 연결 되어야 합니다.<ol>
<li>복 형 아이 솔 레이 터에는 자극을 연결 합니다.</li>
		<li>전극 (실버 와이어 0.2 m m 직경)는 아이 솔 레이 터의 부정적인 터미널에 연결 합니다.</li>
		<li>전극 (실버 와이어 1 m m 직경) 시리즈는 100 Ω 저항 하는 아이 솔 레이 터의 긍정 맨끝에 연결 합니다.</li>
		<li>전기 전류 저항는 증폭기와 전압을 측정 하 여 전달 여부를 모니터링 하는 오실로스코프에 앰프를 연결 합니다.</li>
	</ol>
</li>
	<li>
유리 펫의 준비 참고: 두 가지 유형의 유리 펫 플라스 미드 솔루션의 방출 및 전기 펄스의 응용 프로그램에 대 한 사용 됩니다.<ol>
<li>전극 끌어당기는 사람와 유리 모 세관에서 펫을 확인 합니다.</li>
		<li>휴식 방출 피 펫에 대 한 팁 (팁 직경 20-50 μ m 이어야 한다).</li>
		<li>갈기 고 화재 연마 (내부 직경 50-200 μ m 이어야 한다) 다음 샌드 페이퍼와 전극 피 펫의 끝을 폴란드어. 참고: 전극 팁은 직접 조직 접촉으로 평평 하 고 부드러운, 이어야 한다.</li>
	</ol>
</li>
	<li>
Electroporation 절차
	<ol>
<li>배출 및 전극 펫은 컨트롤러에 연결 합니다.</li>
		<li>전극 피 펫에 실버 와이어 (0.2 m m 직경)를 삽입 합니다.</li>
		<li>플라스틱 튜브 1 mL 주사기를 방출 피 펫을 연결 합니다.</li>
		<li>차지 > 주사기 흡입에 의해 방출 피 펫에 플라스 미드 솔루션의 5 μ.</li>
		<li>Electroporation 단계에 coculture를 넣어 하 고 문화 매체에 1mm 직경 전극 삽입.</li>
		<li>Thalamic explant에 방출 피 펫을 놓습니다.</li>
		<li>팁 thalamic 블록의 표면 접촉 후 주사기를 수동으로 밀어. 참고: 플라스 미드의 0.5 μ에 대 한 솔루션으로 하나의 thalamic 블록 당 사용 됩니다.</li>
		<li>즉시 방출 피 펫의 철회 후 전극 피 펫을 thalamic 블록에 놓습니다.</li>
		<li>전기 펄스 (50-400 µ A, 200 Hz에서 1 ms 기간 200 평방 펄스의 3 ~ 5 열차)를 제공 합니다. 진폭 및 오실로스코프에 열차의 수를 모니터링 합니다. 참고: 전기 전류 진폭 전극 피 펫13의 내부 직경에 따라 다릅니다.</li>
		<li>전극 피 펫을 철회 하 고 인큐베이터에 접시를 반환 합니다.</li>
	</ol>
</li>
	<li>
현미경 관찰
	<ol>
<li>문화 요리 EGFP에 대 한 두 개의 필터 집합을 갖춘 올바른 confocal 현미경의 현미경 단계에 넣어 (여기, 488 nm; 방출, 515 nm) 및 DsRed (여기, 514 nm, 방출, 565 nm).</li>
		<li>1-7 µ m 단계 크기는 20 X 긴 작동 거리 대물 렌즈에서 10-25 광 섹션의 이미지를 가져가 라. DsRed와 SYP EGFP 표현 하는 개별적으로 구별할 수 레이블이 축 삭을 선택 합니다. 참고: 관찰된 지역 (1024 x 1024 픽셀, 즉 해당, 0.68 μ m/픽셀) 약 0.7 x 0.7 m m 이다.</li>
		<li>캡처 이미지 매일 이미징에 대 한 모든 24 h. 실 온에서 10 분 이내 완료 하 고 문화 인큐베이터에 반환. 짧은 간격 시간 경과 영상에 대 한의 환경 습도 95% 공기와 5% CO2 37 ° c.에 유지 문화 실에서 문화 유지</li>
	</ol>
</li>
</ol>

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Neurosci. 28 (37), 9117-9121 (2008).</li><li>Yamada, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+pre-+and+postsynaptic+activity+in+thalamocortical+axon+branching.">Role of pre- and postsynaptic activity in thalamocortical axon branching.</a> Proc Natl Acad Sci U S A. 107 (16), 7562-7567 (2010).</li></ol>

저자는 공개 없다.

현재 프로토콜은 또한 TC 프로젝션11의 다른 축 삭 성장 발달 측면을 연구 하는 강력한 도구입니다. 예를 들어, 대뇌 피 질의 조각 문화와 electroporation 기술은의 조합을 대뇌 피 질의 신경 세포와 장기 관찰9,18의 개별 axonal 형태를 시각화 수 있습니다.
현재 프로토콜을 사용 하 여 축 삭 분기 및 시 냅 스 형성에 흥미 ?...

포유류 두뇌에 있는 thalamocortical (TC) 투영 축 삭 지도 메커니즘을 대상으로 조사에 적합 한 시스템입니다. 개발 하는 동안 감각 TC axons 외피 격판덮개 및 양식 지점과 시 냅 스에 우선적으로 레이어 IV의 대뇌 피 질1,2차 감각 영역에서 성장 한다. 근본적인 연결의 설립 후에 axonal 아 버와 시 냅 스 터미널은 환경 변화3,4에 따라 리 모델링. 그러나, 어떻게 TC 축 삭 형태학 동적으로 변경 되는 제대로 이해. 주된 이유 중 하나는 단일 세포 수준에서 구조적인 변화를 관찰 하는 적절 한 기법의 부족 이다. 전반적인 TC 궤적5, 을 캡처에 대 한 여전히 기술적인 제한이 있다 현미경 검사 법, 2 광자 현미경 등 최근 개발 허용 생활 대뇌 피 질의 뉴런에 vivo에서의 직접 관찰, 비록 6. 따라서, TC 축 삭의 라이브 이미징 방법 체 외에서 축 삭 분기 및 시 냅 스 대형의 구조 분석을 위한 강력한 도구를 제공 것.
처음으로 우리 그룹 설립 투과성 막7정적 슬라이스 문화 방법. 이 방법을 사용 하 여, 쥐 대뇌 피 질의 조각 했다 감각 thalamic 블록, cocultured 그리고 lamina 특정 TC 연결 했다이 organotypic cocultures7,8지. 형광 단백질으로 스파스 라벨 추가 수 있었습니다 TC 축 삭 성장 및 지점 형성9,,1011관찰. 최근에, 분기의 동시 이미징에 대 한 새로운 방법을 개발 했습니다 그리고는 organotypic에서 개별 TC 축 삭 시 냅 스 형성 cocultures12. TC 축 삭과 연 접 사이트를 동시에 시각화할 DsRed EGFP 태그 synaptophysin (SYP EGFP) 했다 organotypic coculture의 electroporation에 의해 thalamic 뉴런의 작은 수로 공동 transfected. 현재 메서드는 TC axons의 형태소 분석을 용이 하 게 하 고 장기 관찰, 축 삭 분기 및 시 냅 스 형성 사이의 인과 관계를 표시 하는 데 사용할 수 있습니다 허용 합니다.

<table><tbody><tr><td>DMEM/F12</td><td>GIBCO</td><td>11320-033</td><td></td></tr><tr><td>Hanks&amp;rsquo; balanced salt solution (HBSS)</td><td>Nissui</td><td>5905</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Scientific</td><td>SH30396-03</td><td>Hyclone</td></tr><tr><td>Insulin</td><td>Sigma</td><td>I6634</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma&amp;nbsp;</td><td>H0888</td><td></td></tr><tr><td>Sodium selenite</td><td>Wako Pure&lt;br/&gt; Chemical Industries</td><td>192-10843</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma</td><td>T1147</td><td></td></tr><tr><td>Putrescine&amp;nbsp;</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Glucose</td><td>Wako&lt;br/&gt; Pure Chemical Industries</td><td>16806-25</td><td></td></tr><tr><td>35 mm petri dishes</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>Millicell-CM insert</td><td>Millipore</td><td>PICMORG50</td><td></td></tr><tr><td>100 mm petri dishes</td><td>BIO-BIK</td><td>I-90-20</td><td>petri dish sterrile</td></tr><tr><td>HiPure Plasmid Maxiprep Kit</td><td>Invitrogen</td><td>K210006</td><td></td></tr><tr><td>Disposable sterile plastic pipettes</td><td></td><td>202-IS</td><td>transfer pipets sterile</td></tr><tr><td>Glass capillary: OD 1.2 mm</td><td>Narishige</td><td>&amp;nbsp;G-1.2</td><td>inner diameter, 1.2 mm</td></tr><tr><td>Silver wire: 0.2 and 1 mm&amp;nbsp;</td><td>Nilaco</td><td>AG-401265 (diameter, 0.2 mm), AG-401485 (diameter, 1.0 mm)</td><td></td></tr><tr><td>1 mL syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Stimulator&amp;nbsp;</td><td>A.M.P.I</td><td>Master 8</td><td></td></tr><tr><td>Biphasic isolator&amp;nbsp;</td><td>BAK ELECTRONICS</td><td>BSI-2</td><td></td></tr><tr><td>Amplifier&amp;nbsp;</td><td>A-M Systems</td><td>Model 1800</td><td></td></tr><tr><td>Oscilloscope</td><td>Hitachi</td><td>VC-6723</td><td></td></tr><tr><td>Manipulator</td><td>Narishige</td><td>SM-15</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MO-10</td><td></td></tr><tr><td>Stereomicroscope&amp;nbsp;</td><td>Olympus</td><td>SZ40</td><td></td></tr><tr><td>Universal stand&amp;nbsp;</td><td>Olympus</td><td>SZ-STU2</td><td></td></tr><tr><td>Light illumination system&amp;nbsp;</td><td>Olympus</td><td>LG-PS2, LG-DI, HLL301</td><td></td></tr><tr><td>Electrode puller&amp;nbsp;</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>Digital eclipse C1 laser</td><td></td></tr><tr><td>x20 objective</td><td>Nikon</td><td>ELWD 20x/0.45</td><td></td></tr><tr><td>Culture chamber</td><td>Tokai Hit</td><td>UK A16-U</td><td></td></tr><tr><td>Sprague-Dawley (SD) rat</td><td>Japan SLC and Nihon-Dobutsu</td><td></td><td></td></tr><tr><td>Microsurgery scissors</td><td>Natsume&amp;nbsp;</td><td>MB-54-1</td><td></td></tr></tbody></table>

visualization of thalamocortical axon branching and synapse formation in organotypic cocultures

축 삭 분기 및 시 냅 스 형성 하는 정확한 신경 회로 설정 하기 위한 중요 한 프로세스입니다. 개발 하는 동안 감각 thalamocortical (TC) 축 삭 대뇌 피 질의 특정 레이어에 지점과 시 냅 스 형성합니다. 축 삭 분기 및 시 냅 스 형성 사이 명백한 공간 상관 관계에도 불구 하 고 그들 사이의 인과 관계가 제대로 이해 된다. 이 문제를 해결 하려면 우리는 최근 organotypic cocultures에서 개별 TC 축 삭의 분기 및 시 냅 스 대형의 동시 이미징 방법을 개발.
이 프로토콜 organotypic coculture 및 electroporation의 조합으로 구성 되는 방법을 설명 합니다. 시상과 대뇌 피 질의 Organotypic cocultures 유전자 조작 및 axonal 프로세스, 박판 모양 구성 등 독특한 구조를 보존의 관측을 촉진 한다. 두 가지 플라스 미드 DsRed EGFP 태그 synaptophysin (SYP EGFP) 인코딩 했다 electroporation 기술에 의해 thalamic 뉴런의 작은 수로 공동 transfected. 이 메서드를 사용 하 여 TC 뉴런의 개별 axonal 형태학 및 연 접 사이트를 동시에 시각화 수 있었습니다. 메서드는 또한 축 삭 분기 및 시 냅 스 형성 사이의 인과 관계를 밝혀 장기 관찰을 사용할 수 있습니다.

실험 여기 TC 축 삭 분기 및 시 냅 스 형성 사이의 관계를 공개 하는 것을 목표로 설명 합니다. 동시에 시각화 axonal 궤도 및 연 접 사이트, 단일의 위치 또는 몇 thalamic 셀 organotypic cocultures 인코딩 SYP EGFP 및 DsRed electroporation를 사용 하 여 두 개의 플라스 미드와 페 했다. 문화에서 두 번째 주 동안 개별적으로 구별할 수 TC axons 했다 DsRed (그림 3)에 의해 명?...

Watch this Scientific Journal Video about Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures at JoVE.com

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

이 프로토콜 thalamocortical 축 삭 분기 및 시 냅 스 대형의 organotypic cocultures에서 시상과 대뇌 피 질의 동시 이미징 하는 방법을 설명합니다. 개별 thalamocortical 축 삭 및 그들의 연 접 맨끝 DsRed와 GFP 태그 synaptophysin 단일 셀 electroporation 기법으로 시각화 됩니다.

분기 Thalamocortical 축 삭과 시 냅 스의 시각화 Organotypic Cocultures에서 형성

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

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Neuroscience

6.4K 조회수.  Kanazawa University. 이 프로토콜 thalamocortical 축 삭 분기 및 시 냅 스 대형의 organotypic cocultures에서 시상과 대뇌 피 질의 동시 이미징 하는 방법을 설명합니다. 개별 thalamocortical 축 삭 및 그들의 연 접 맨끝 DsRed와 GFP 태그 synaptophysin 단일 셀 electroporation 기법으로 시각화 됩니다.

비디오: Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

뇌의 Organotypic 슬라이스와 암 세포의 3 차원 회전 타원체와 공 배양 시스템

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

연구

의학

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

다중 구획 CNS 뉴런 - glia 공동 문화 Microfluidic 플랫폼

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

생물학

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

3D Cocultures 이다 인간 만능 줄기 세포에서 신경 세포의 시 냅 스 마이크로 회로 모델링

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

발생학

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

3 차원 조직 설계 정렬 사이토 네트워크 발달 메커니즘을 정리 하 고 신경 재생을 촉진 하

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

생체공학

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

2D 및 3D 인간 유도 만능 줄기 세포 기반 모델은 신코르티컬 개발 중 1 차용성 개입을 해부합니다.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

Colliculo-thalamocortical 마우스 뇌 조각의 변경, 3 차원 챔버 구성 요소의 인쇄 및 멀티 스케일 광학 이미징을 통합

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

신경과학

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

병합 된 포유류 외피가 대뇌 피 질의 모듈의 시각화

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

라인 피질-해마 오르가노티픽 슬라이스 배양에서 간질 발생의 모델

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

시투에서 Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures 시각화

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

<meta charset="utf8"></head><body>Neuronal Activity Mapping in Developing Mouse Sensory Cortex(생쥐 감각 피질 발달에서의 신경 활동 매핑)

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

<meta charset="utf8"></head><body>저자 스포트라이트: Deciphering Neural Circuit Formation from Two-Photon Microscopy and Single Neuron Imaging(이광자 현미경 및 단일 뉴런 이미징에서 신경 회로 형성 해독)

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

단일 뉴런 해상도에서 신생아 마우스 감각 피질의 자발적 활동에 대한 In vivo 시각화

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...