Name: purkinje cell survival in organotypic cerebellar slice cultures
Uploaded: 2019-12-18T12:00:00
Description: Watch this Scientific Journal Video about Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures at JoVE.com

화상 진찰 일은 로버트 H 루리 포괄적인 암 센터에 수여된 NCI CCSG P30 CA060553에 의해 관대하게 지원되는 고급 현미경 검사법에 대한 노스웨스턴 대학 센터에서 행해졌습니다. 숀 맥더모트의 기술 지원과 지원에 감사드리며, 그림 1에나타난 손으로 그린 그림에 대해 마야 엡스타인에게 감사드립니다.

동물과 관련된 모든 실험은 노스웨스턴 대학 동물 연구 위원회에 따라 수행되었다.
1. 오르가노티픽 소뇌 슬라이스 배양 전 준비
<ol><li>사전에 70% 에탄올로 분무된 세포 배양 후드에서 멸균 병 수신기에 부착된 250 mL 병 상판 진공 필터에 배양 배지 200 mL를 준비합니다. 기저 중간 이글 100 mL(BME), 행크스 균형 소금 용액 50 mL(HBSS), 50 mL의 열 불활성화 말 세럼, 200 mM L-글루타민 1mL(?...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mifepristone+(RU486)+protects+Purkinje+cells+from+cell+death+in+organotypic+slice+cultures+of+postnatal+rat+and+mouse+cerebellum.">Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (13), 7953-7958 (2003).</li><li>Labombarda, F., 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=Neuroprotection+by+steroids+after+neurotrauma+in+organotypic+spinal+cord+cultures:+a+key+role+for+progesterone+receptors+and+steroidal+modulators+of+GABA(A)+receptors.">Neuroprotection by steroids after neurotrauma in organotypic spinal cord cultures: a key role for progesterone receptors and steroidal modulators of GABA(A) receptors.</a> Neuropharmacology. 71, 46-55 (2013).</li><li>Gao, P., 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=P2X7+receptor-sensitivity+of+astrocytes+and+neurons+in+the+substantia+gelatinosa+of+organotypic+spinal+cord+slices+of+the+mouse+depends+on+the+length+of+the+culture+period.">P2X7 receptor-sensitivity of astrocytes and neurons in the substantia gelatinosa of organotypic spinal cord slices of the mouse depends on the length of the culture period.</a> Neuroscience. , 195-207 (2017).</li><li>Sundstrom, L., Morrison, B., Bradley, M., Pringle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+cultures+as+tools+for+functional+screening+in+the+CNS.">Organotypic cultures as tools for functional screening in the CNS.</a> Drug Discovery Today. 10 (14), 993-1000 (2005).</li><li>Jankowski, J., Miething, A., Schilling, K., Baader, S. 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I., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+protein+kinase+C+prevents+Purkinje+cell+death+but+does+not+affect+axonal+regeneration.">Inhibition of protein kinase C prevents Purkinje cell death but does not affect axonal regeneration.</a> Journal of Neuroscience. 22 (9), 3531-3542 (2002).</li><li>Ghoumari, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+mifepristone+involves+neuron+depolarization.">Neuroprotective effect of mifepristone involves neuron depolarization.</a> FASEB Journal. 20 (9), 1377-1386 (2006).</li><li>Repici, M., Wehrle, R., Antoniou, X., Borsello, T., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Jun+N-terminal+kinase+(JNK)+and+p38+play+different+roles+in+age-related+Purkinje+cell+death+in+murine+organotypic+culture.">c-Jun N-terminal kinase (JNK) and p38 play different roles in age-related Purkinje cell death in murine organotypic culture.</a> Cerebellum. 10 (2), 281-290 (2011).</li><li>Rakotomamonjy, J., Ghoumari, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-Derived+Neurotrophic+Factor+Is+Required+for+the+Neuroprotective+Effect+of+Mifepristone+on+Immature+Purkinje+Cells+in+Cerebellar+Slice+Culture.">Brain-Derived Neurotrophic Factor Is Required for the Neuroprotective Effect of Mifepristone on Immature Purkinje Cells in Cerebellar Slice Culture.</a> International Journal of Molecular Sciences. 20 (2), (2019).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Linkert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Uesaka, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+coculture+preparation+for+the+study+of+developmental+synapse+elimination+in+mammalian+brain.">Organotypic coculture preparation for the study of developmental synapse elimination in mammalian brain.</a> Journal of Neuroscience. 32 (34), 11657-11670 (2012).</li><li>Falsig, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+pathogenesis+is+faithfully+reproduced+in+cerebellar+organotypic+slice+cultures.">Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures.</a> PLoS Pathogens. 8 (11), 1002985 (2012).</li><li>Bouslama-Oueghlani, L., Wehrle, R., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+loss+of+the+ability+of+Purkinje+cells+to+regenerate+their+axons+occurs+in+the+absence+of+myelin:+an+in+vitro+model+to+prevent+myelination.">The developmental loss of the ability of Purkinje cells to regenerate their axons occurs in the absence of myelin: an in vitro model to prevent myelination.</a> Journal of Neuroscience. 23 (23), 8318-8329 (2003).</li><li>Apuschkin, M., Ougaard, M., Rekling, J. 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소뇌 슬라이스 배양은 산후 발달 중에 프로그램된 Purkinje 세포 사멸을 연구하는 강력한 도구입니다. 이 기술은 그들의 신경 보호 잠재력을 위한 후보 분자를 급속하게 스크리는 것을 이용될 수 있습니다. 주요 장점은 설정이 간단하고 매우 비용 효율적이며, 단지 장비에 적당한 투자가 필요하다는 것입니다 (진동은 조직 헬기보다 최대 3 배 더 많은 비용이 들 수 있습니다). 또한, 10 ~15개의 건강한 ?...

생체 외 모델링은 생물 의학 연구에 필수적인 도구입니다. 그것은 조사자가 공부하고 제한한 세포 모형에 있는 특정 기계장치를 단단히 통제할 수 있습니다, 또는 고립된 시스템/기관에서. 오르가노티픽 슬라이스 배양은 특히 신경과학 분야에서 시험관내 기술로 널리 사용되고있다 1. 이 방법은 먼저 롤러 튜브 기술2를사용하여 뇌 조각을 배양한 Gähwiler에 의해 처음 확립되었으며, 나중에 야마모토 등에서 수정되었으며, 이들은 피질 슬라이스 배양을 수행하기 위해 미세 다공성 멤브레인의 사용을도입했습니다 3. 1 차적인 세포 배양에 비해, organotypic 슬라이스 배양은 조직의 세포 아키텍처를 보존하는 이점을 제시, 조직 섹션의 평면에서 뿐만 아니라 네이티브 세포 세포 연결.
Organotypic 조각은 해마4,피질5,줄무늬6,소뇌4,7,척수8,9,다른 사람의 사이에서 와 같은 중추 신경계의 많은 부분에서 배양되었습니다. 그들은 약물 발견 연구에서 강력한 도구로 입증되었습니다10. 신경 활성 분자의 효과 여러 가지 방법으로 평가 될 수 있다: 면역 염색 및 생화학 분석, 신경 회로 형성, 또는 전기 생리학 및 라이브 이미징을 사용 하 여 중단을 사용 하 여 생존 및 신경 변성.
이 작업의 목적은 시험관에서 소뇌 발달을 모방하는 관련 모델로 알려져 있는 organotypic 소뇌 슬라이스 문화를 수행하는 간단한 방법을 설명하는 것입니다. 특히, 우리는 Purkinje 세포 발달 죽음의 연구 결과에 집중했습니다. 생체 내에서, Purkinje 세포는 출생 후 첫 번째 주 동안 세포 자멸을 겪고, 출생 후 일 3 (P3)11에피크. 소뇌 슬라이스 배양에서 동일한 패턴이 관찰되며, 세레베야가 P1과 P8 사이의 동물로부터 채취될 때, P34,12에서피크를 가진 후발성 세포사멸에 의해 죽어가는 Purkinje 뉴런과 함께 관찰된다. organotypic 소뇌 슬라이스 배양의 사용은 여러 신경 보호분자를식별 할 수 있다7,13,뿐만 아니라 이 프로그램 된 세포 사멸에 관련된 메커니즘의 일부를 이해14,15,16. 여기서, 우리는 해마에서 Stoppini et al.17의 연구에 기초한 프로토콜을 설명하고, Dusart et al.4에 의해 소뇌에 적응하여 산후 뇌의 급속한 해부 및 절단을 포함한다; 미세다공막을 함유하는 세포 배양 삽입체상에 배양물을 슬라이스하는 경우, 신경보호 치료 유무에 관계없이; 및 면역 형광 염색신경 생존을 평가하기 위해.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody</td>
			<td>ThermoFisher scientific</td>
			<td>A21203</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal Medium Eagle (BME)</td>
			<td>ThermoFisher scientific</td>
			<td>21010046</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet Class II, Type A2</td>
			<td>NuAire</td>
			<td>NU-540-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Calbindin D-28K antibody (CB-955)</td>
			<td>Abcam</td>
			<td>ab82812</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NuAire</td>
			<td>NU-5700</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom 6-well Cell Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 50 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing forceps, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8949</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge blades</td>
			<td>Fisher Scientific</td>
			<td>50949411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs, Inc</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-127</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 100X</td>
			<td>ThermoFisher scientific</td>
			<td>25030149</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose solution</td>
			<td>ThermoFisher scientific</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks' Balanced Salt Solution</td>
			<td>ThermoFisher scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>Fisher Scientific</td>
			<td>H21492</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum, heat inactivated, New Zealand origin</td>
			<td>ThermoFisher scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>Fisher Scientific</td>
			<td>NC9914528</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprobes</td>
			<td>Fisher Scientific</td>
			<td>08-850</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Inserts</td>
			<td>Millipore Sigma</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL</td>
			<td>ThermoFisher scientific</td>
			<td>168-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal laser microscope system</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Imaging Software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics</td>
			<td>41678-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher scientific</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8403</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker Belly Dancer</td>
			<td>IBI Scientific</td>
			<td>BDRLS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish, 60 mm w/ grip ring</td>
			<td>Fisher Scientific</td>
			<td>FB012921</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24 well</td>
			<td>Falcon/Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes, sterile</td>
			<td>ThermoFisher scientific</td>
			<td>13-711-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

purkinje cell survival in organotypic cerebellar slice cultures

Organotypic 슬라이스 배양은 분리된 1차 세포 배양보다 생체 내 상태를 더 가깝게 모방하는 강력한 시험관 내 모델입니다. 초기 출생 후 발달에서, 소뇌 Purkinje 세포는 그(것)들이 프로그램한 세포 죽음을 겪는 동안 취약한 기간을 통과하는 것을 알려지고 있습니다. 여기서, 우리는 이 중요한 시간 동안 마우스 organotnotypic 소뇌 슬라이스 배양을 수행하기 위한 상세한 프로토콜을 제공한다. 슬라이스는 Purkinje 세포 생존및 신경 보호 처리의 효험을 평가하기 위하여 추가 표지됩니다. 이 방법은 새로운 신경 활성 분자를 선별하는 데 매우 유용 할 수 있습니다.

도 4에도시된 바와 같이, 이 프로토콜은 Purkinje 세포 생존이 면역 형광 및 이미지 획득 단계에 따라 평가될 수 있는 orgnotnotypic 소뇌 슬라이스 배양을 생성한다. 퍼킨제 세포는 항칼빈딘 D-28K(희석 1/200) 및 Alexa594 항마우스(희석 1/300) 항체의 조합으로 표지되었다. 이미지 스티칭은 전체 소뇌 슬라이스의 그림을 얻기 위해 현미경 수집 소프트웨어 (NIS-Elements)에 의해 자동으로 수?...

Watch this Scientific Journal Video about Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures at JoVE.com

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic 슬라이스 문화는 신경 발달 또는 퇴행성 / 재생 과정을 연구할 수있는 강력한 도구입니다. 여기서, 우리는 마우스 소뇌 슬라이스 배양에서 Purkinje 세포의 신경 발달 죽음을 모델로 하는 프로토콜을 기술한다. 이 방법은 신경 보호 약물 발견에 대 한 연구 혜택을 받을 수 있습니다.

오르가노티픽 소뇌 슬라이스 배양에서 푸르킨제 세포 생존

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

Organotypic 슬라이스 문화는 신경 발달 및 퇴행성 또는 재생 과정을 연구하기위한 강력한 도구입니다. 이 기술은 그들의 신경 보호 잠재력에 대 한 후보 분자를 신속 하 게 검사 하는 데 사용할 수 있습니다. 이 방법은 조직 세트 아키텍처 및 네이티브 세포 세포 연결이 섹션의 평면 내에서 보존되기 때문에 해리된 1 차 세포 배양과 비교하여 생체 내 조건에서 밀접하게 모방됩니다.여기에서 우리는 발달소뇌에 있는 Purkinje 세포 죽음의 연구 결과 보여줍니다. 그러나 organotypic 슬라이스 배양은 거의 모든 중추 신경계 지역에서 신경 퇴행성 질환을 모델링하는 데 똑같이 적합합니다. 제니퍼 Rakotomamonjy와 절차를 시연 숀 맥더모트, 내 실험실에서 기술자가 될 것입니다.소뇌 조각을 수확하기 전에, 멸균 된 생물 안전 캐비닛에서, 진공 여과 배양 배지의 1 밀리리터로 6 웰 배양 판의 각 우물을 채우고, 치료 우물에 관심의 약리학제를 추가하고, 제어 우물에 차량의 동등한 볼륨을 추가합니다. 멸균 집게를 사용하여 각 우물에 0.4 마이크로미터 모공 크기의 PTFE 멤브레인 필터 를 배치하여 각 멤브레인과 배지의 인터페이스에서 거품을 피하고 배지를 섭씨 37도 및 5 %의 이산화탄소 인큐베이터로 2 시간 동안 상화합니다. 소뇌를 수확하려면 직선 드레싱 포셉을 사용하여 코로 새끼 머리를 잡고 직선 눈 가위를 사용하여 뒤쪽 끝에서 중간선으로 두피를 잘라냅니다.같은 방식으로 두개골을 열고, 소뇌를 손상시키지 않도록 가위 팁을 바깥쪽으로 가리키고, 주걱을 사용하여 감기 HBSS와 포도당 밀리리터 당 5 밀리그램을 포함하는 60mm 접시로 뇌를 옮기십시오. 직선 드레싱 집게를 사용하여 소뇌를 조심스럽게 해부하고 멸균 곡선미세 포셉을 사용하여 티슈 헬기의 절단 테이블에 플라스틱 디스크에 조직을 배치합니다. 절단 테이블을 회전하여 기생절 단면도의 획득을 허용하도록 조직을 방향을 지정하고, 블레이드가 조직의 가장자리에 위치 할 때까지 절단 테이블을 이동하는 테이블 방출 노브를 오른쪽으로 당깁니다.그런 다음 슬라이스 두께를 350 마이크로미터로 조정하고 블레이드 속도를 중간 크기로 조정하고 헬기를 시작합니다. 전체 소뇌가 슬라이스되었을 때 헬기를 끕니다. 멸균 집게를 사용하여 디스크를 새로운 60mm 접시 위에 고정하십시오.전달 파이펫을 사용하여 디스크 위에 HBSS 플러스 포도당을 플러시하여 슬라이스가 접시에 빠지게됩니다. 그런 다음 샘플을 가능한 한 최소화하고 마이크로 프로브를 사용하여 슬라이스를 분리합니다. 6웰 플레이트의 개별 세포 배양 삽입에 대한 소뇌의 부분을 선택하고 마이크로 프로브를 사용하여 각 삽입물의 중앙에 슬라이스를 배치합니다.모든 슬라이스가 배치되면, 신중하게 여분의 HBSS 플러스 포도당을 흡인하고, 세포 배양 인큐베이터에 접시를 반환합니다. 면역 형광 염색의 경우 각 우물에서 상체를 제거하고 PBS로 인서트를 세척하십시오. 각 인서치의 우물에 차가운 4% 파라포름알데히드 1밀리리터와 각 인서서 위에 500마이크로리터를 1시간 동안 고정합니다.고정이 끝나면 각 인서트 아래에 1 밀리리터의 신선한 PBS와 궤도 셰이커에 세척당 각 인서트 위에 500 마이크로리터의 신선한 PBS를 10분 동안 4회 세척합니다. PBS-TB의 500 마이크로리터로 24웰 플레이트의 각 웰을 미리 채우고, 페인트 브러시를 사용하여 각 세포 배양에서 소뇌 조각을 24웰 플레이트의 개별 우물로 옮기합니다. 1시간 동안 실온에서 슬라이스를 퍼메아빌리즈하고 차단합니다.인큐베이션의 끝에서, 궤도 셰이커에 섭씨 4도에서 하룻밤 동안 PBS-TB에서 희석 된 관심의 1 차 적인 항체의 200 마이크로 리터로 세포를 레이블. 다음 날에는 셰이커에서 세척당 10분 동안 4회, 시료 500마이크로리터를 세척한 후, 셰이커의 실온에서 2시간 동안 적절한 불소엽소 공조 보조 항체로 샘플을 표시하여 빛으로부터 보호합니다. 인큐베이션이 끝나면 실온에서 10분 동안 적절한 핵 얼룩 500마이크로리터를 장착하여 빛으로부터 보호합니다.그리고 유리 슬라이드에 조각을 장착하기 위해 전송 파이펫을 사용합니다. 그런 다음 PBS로 재수화하고 약 80 마이크로리터의 장착 매체로 코팅 된 커버립으로 조직을 장착하기 전에 섹션을 완전히 건조하게하십시오. 마운팅 매체가 경화되면 소뇌 섹션을 이미지화할 준비가 되었습니다.6일째산후, Purkinje 세포 생존은 그들의 알려진 취약성 창과 일치하는 통제 견본에서 낮습니다. 기증자 동물이 나이가 들고 이 중요한 시기를 종료함에 따라 생존율이 증가합니다. 염화칼륨의 고농도와 소뇌 슬라이스의 치료는 탈극화와 생존의 성공적인 유도결과.일관되고 재현 가능한 결과를 얻으려면 건강한 소뇌 섹션을 선택하고 가능한 한 효율적으로 문화 설정을 수행하는 것이 중요합니다. 배양 후 응용 분야는 면역 형광을 넘어섭을 넘어섭다. organotypic 슬라이스로 게놈 단백질 발현 연구에 사용할 수 있습니다., 그리고 전기 생리학 및 칼슘 라이브 이미징을 사용 하 여 신경 회로 활동을 모니터링 하기 위해.

purkinje-cell-survival-in-organotypic-cerebellar-slice-cultures

Research

JoVE Journal

Neuroscience

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

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

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

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

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

연구

신경과학

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotypic Hippocampal 슬라이스 문화

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic 소뇌 문화 : Apoptotic 도전과 감지

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

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 ...

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 ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Organotypic 슬라이스 문화의 Purkinje 세포 수지상의 형태학의 분석

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Hippocampal 슬라이스 문화의 시냅틱 단백질의 포스트 퍼가기 Immunogold 라벨링

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotypic 슬라이스는 문화 희소 돌기 아교 세포 역학 및 수초화을 공부하기

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

에서 신경 혈관 리모델링의 분석 Entorhino - 해마 Organotypic 슬라이스 문화

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

소뇌 과립 세포 배양에서 미세 아교 세포의 선택적 고갈 L - 류신 메틸 에스테르를 사용하여

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...