This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

여기에 설명 된 모든 실험은 영국의 동물 (과학적인 절차) 1986 년 법에 따라 수행 하였다. 악기, 문화 미디어, 그리고 요리 1. 준비 <ol><li> 두 스테인레스 스틸 실험실 해부 가위와 두 개의 스테인레스 스틸 실험실 집게를 준비합니다. 문화 후드 O의 모든 악기와 장소를 압​​력솥 / N 살균 자외선 (UV) 빛 아래. </li><li> 최소 필수 매체 (MEM) 배지 (500 mL로 10 % 태아 소 혈청 [FBS, 20 mM의 KCl을 25 mM의 NaHCO3를, 30 mM의 D- 글루코오스, 2 mM L- 글루타민, 100 U / ㎖ 페니실린, 100 μg의 / ㎖ 스트렙토 마이신, 6 μg의 / ㎖ 암피실린) 및 필터 소독. 달 동안 4 ℃에서 보관하십시오. </li><li> 14mm, 번호 1 두께 된 커버를 포함하는 24 잘 배양 플레이트를 준비합니다. 이전에 3 설명한 바와 같이 100 ㎎ / ℓ 폴리 D 라이신 (PDL)의 커버 슬립과 코트를 압력솥. 자외선은 O / N을 소독. </li></ol> CGC 문화 솔루션 2. 준비 <ol><li> 처리 된 오토 클레이브, UV 광, 멸균 DH에서 &#39;용액 A&#39;(100 ㎖)를 준비 포도당 250 ㎎, 소 혈청 알부민 [BSA] 300 mg을 인산염 완충 염수 [PBS (칼슘 및 955 ㎎을 함유하는 2 O 마그네슘) + 무료, 3.8 %의 1 ml의 황산 4 · 7H 2 O </li><li> A. 용액 9 ㎖에 희석 / ㎖ 트립신 5 mg을 1 ㎖를 함유하는 &#39;용액 B&#39;(10 ㎖)를 준비 </li><li> &#39;용액 C&#39;1000 유닛의 DNase, 대두 트립신 억제제의 0.5 ㎎, A. 용액 10 mL로 희석하여 3.8 % 황산 · 7H 2 O 100 ㎕를 함유하는 (10 ㎖)를 준비 </li><li> A. 용액으로 20 ㎖로 희석 용액 3.2 ml의 C를 함유 &#39;용액 D&#39;(20 ㎖)를 준비 </li><li> · 7H 2 O EBSS, 25 mM의 NaHCO3를, 3 mM의 황산을 포함 BSA / 얼의 균형 소금 솔루션 (EBSS) 솔루션을 준비하고,4 % BSA, pH를 7.4으로 이전에 3을 설명했다. </li><li> 150 mM의 LME의 최종 농도를 수득 MEM 배지 5 ㎖에 순수한 LME 추가하여 LME 준비 벤치 탑 pH 미터를 사용하여 pH를 7.4까지 용액을 복귀하고, 필터는 28mm의 폴리 에테르 설폰 (PES), 0.2 μm의 주사기를 사용하여 소독 필터. </li></ol> 3. CGCs을 배양 <ol><li> 이전에 3 설명한대로 4-7 일 된 쥐 새끼에서 소뇌를 수집합니다. 얼음에 5 ml의 솔루션을 포함하는 배양 접시에 바로 배치합니다. </li><li> 페트리 접시 뚜껑에 소뇌와 장소 조직에서 과잉 솔루션을 붓고. </li><li> 적어도 세 개의 서로 다른 방향에서 잘 골조 면도날 미세 조직을 잘라. </li><li> 5 분 동안 37 ℃에서 물을 욕조에 솔루션 (손실 제한 조직을 위해 솔루션 페트리 접시를 씻어) B와 장소에 다진 조직을 추가합니다. 분의 부드럽게 모든 커플을 흔들어. </li><li> 트립신, 흔들림 센트를 중화하기 위해 튜브에 용액 D (20 ml)에 추가5 분 동안 65 XG에 rifuge. </li><li> 상층 액을 붓는다. </li><li> 현탁액 가능한 균질해질 때까지, 직경을 감소시키는 세 골조 유리 피펫으로 잘 용액 C. 씹다 4 ㎖ (약 10 회)에 재현 탁. </li><li> 천천히 부드럽게 BSA / EBSS 위에이 균질 몇 방울을 추가합니다. 이 BSA / EBSS를 통해 싱크 시작하면, 더 씹다 및 / 또는 균질는 BSA / EBSS의 상단에 레이어에 앉아 있어야 좀 더 솔루션 C를 추가 할 수 있습니다. 흔들지 마십시오. 5 분 동안 100 XG 스핀. </li><li> 상층 액과 MEM 배지 1 ㎖에 재현 탁 펠릿 (소프트)를 제거합니다. </li><li> MEM 배지 500 μL에 80 만 셀 / 커버 슬립에서 혈구와 플레이트를 사용하여 세포를 계산합니다. 6 % CO 2와 37 ° C에서 인큐베이터에있는 장소. </li><li> 매체에게 다음 날 (준비 후 24 ~ 36 시간)을 변경합니다. 세포주기 억제제 아라 비노 푸라 노실 시티 딘 (ARAÇ) 10 μM을 함유하는 MEM 배지 용액을 제조. <ol><li> 각각커버 슬립은 다시 세포와 정상까지 오래된 매체의 250 μl를 추가, 신선한 24 웰 플레이트에서 기존 매체의 250 μl를 유지 나머지를 대기음, 정상 MEM 배지 250 μl를 추가 한 다음, 세포를 씻어이 대기음 ARAÇ 함유 매체의 250 μL와. 참고 : 세포는 체외 (DIV)에서 육일에서 사용할 수 있습니다. </li></ol></li></ol> 4. 선택적으로 미세 아교 세포 고갈 (6 DIV에서 수행) <ol><li> 150 mM의 LME의 최종 농도를 수득 MEM 배지 별개 분취 량 5 ml의 순수한 LME 추가. </li><li> pH 7.4의 용액으로 돌아가서 필터 28mm 폴리 에테르 술폰 (PES), 0.2 μm의 주사기 필터를 이용하여 멸균. </li><li> 제어 배양 정상적인 MEM 배지와 함께 10 분 동안 37 O C에서 수욕 MEM 배지 자리에 50 mM의 (2 × LME)의 농도로 용액을 희석. </li><li> 반 (250 μL) CGC의 문화 MEM 배지를 제거하고 37 ℃에서 유지한다. </li><li> 취급 세포또는 제어 문화에 대한 LME없이 미리 예열 MEM 배지 (250 μL) 25 mm의 잘 농도를주는 1 : 즉, 2 × LME (250 μL)를 포함하는 MEM 배지로 1 희석. </li><li> 1 시간 동안 6 % CO 2의 가습 분위기에서 37 ℃에서 세포를 배양한다. </li><li> 두번 신선한 미리 예열 MEM 배지에서 세척 세포 LME - 함유 배지를 제거하고 배양 배지를 유지 및 해당 웰 신선한 미리 예열 MEM 매체의 동일 부피를 대체한다. </li><li> (37 ° C에서 6 % CO 2 가습) 인큐베이터에 문화를 반환하고 추가 처리하기 전에 24 시간 동안 나머지 둡니다. </li></ol>

<ol>
	<li>Herculano-Houzel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+remarkable,+yet+not+extraordinary,+human+brain+as+a+scaled-up+primate+brain+and+its+associated+cost.">The remarkable, yet not extraordinary, human brain as a scaled-up primate brain and its associated cost.</a> PNAS. 26 (109), 10661-10668 (2012).</li><li>Evans, G. J., &amp;Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+neurotransmitter+release+by+dihydropyridine-sensitive+calcium+channels+involves+tyrosine+phosphorylation.">Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation.</a> Eur J Neuro. 11 (1), 279-292 (1999).</li><li>Kingham, P. J., Cuzner, M. L., Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+pathways+mobilized+in+microglia+and+neurones+as+a+consequence+of+chromogranin+A-induced+microglial+activation.">Apoptotic pathways mobilized in microglia and neurones as a consequence of chromogranin A-induced microglial activation.</a> J Neurochem. 73 (2), 538-547 (1999).</li><li>Contestabile, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+granule+cells+as+a+model+to+study+mechanisms+of+neuronal+apoptosis+or+survival+in+vivo+and+in+vitro.">Cerebellar granule cells as a model to study mechanisms of neuronal apoptosis or survival in vivo and in vitro.</a> Cerebellum. 1 (1), 41-55 (2002).</li><li>Kramer, D., Minichiello, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+of+primary+cerebellar+granule+cells.">Cell culture of primary cerebellar granule cells.</a> Methods Mol Biol. 633, 233-239 (2010).</li><li>Facci, L., Skaper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+rat+cerebellar+granule+neurons+and+application+to+identify+neuroprotective+agents.">Culture of rat cerebellar granule neurons and application to identify neuroprotective agents.</a> Methods Mol Biol. 846, 23-37 (2012).</li><li>Thiele, D. L., Kurosaka, M., Lipsky, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+of+the+accessory+cell+necessary+for+mitogen-stimulated+T+and+B+cell+responses+in+human+peripheral+blood:+delineation+by+its+sensitivity+to+the+lysosomotropic+agent,+L-leucine+methyl+ester.">Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood: delineation by its sensitivity to the lysosomotropic agent, L-leucine methyl ester.</a> J Immunology. 131 (5), 2282-2290 (1983).</li><li>Giulian, D., Vaca, K., Corpuz, 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+glia+release+factors+with+opposing+actions+upon+neuronal+survival.">Brain glia release factors with opposing actions upon neuronal survival.</a> J Neurosci. 13 (1), 29-37 (1993).</li><li>Guillemin, G., 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=Obtention+and+characterization+of+primary+astrocyte+and+microglial+cultures+from+adult+monkey+brains.">Obtention and characterization of primary astrocyte and microglial cultures from adult monkey brains.</a> J Neurosci Res. 49 (5), 576-591 (1997).</li><li>Hewett, J. A., Hewett SJ, ., Winkler, S., Pfeiffer SE, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+nitric+oxide+synthase+expression+in+cultures+enriched+for+mature+oligodendrocytes+is+due+to+microglia.">Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia.</a> J Neurosci Res. 56 (2), 189-198 (1999).</li><li>Jebelli, J., Hooper, C., &amp;Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+p53+activation+is+detrimental+to+neuronal+sysnapses+during+activaton-induced+inflammation:+implications+for+neurodegeneration.">Microglial p53 activation is detrimental to neuronal sysnapses during activaton-induced inflammation: implications for neurodegeneration.</a> Neurosci Lett. 7 (583), 92-97 (2014).</li><li>Frade, 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=Glutamate+induces+release+of+glutathione+from+cultured+rats+astrocytes+&#8211;+a+possible+neuroprotective+mechanism.">Glutamate induces release of glutathione from cultured rats astrocytes &#8211; a possible neuroprotective mechanism.</a> J Neurochem. 105 (4), 1144-1152 (2008).</li><li>Hamby, M. E., Uliasz, T. F., Hewett, S. J., Hewett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+improved+procedure+for+the+removal+of+microglia+from+confluent+monolayers+of+primary+astrocytes.">Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes.</a> J Neurosci Methods. 150 (1), 128-137 (2006).</li><li>Morgan, S. C., Taylor, D. L., Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+release+activators+of+neuronal+proliferation+mediated+by+activation+of+mitogen-activated+protein+kinase,+phosphatidylinositol-3-kinase/Akt+and+delta-Notch+signalling+cascades.">Microglia release activators of neuronal proliferation mediated by activation of mitogen-activated protein kinase, phosphatidylinositol-3-kinase/Akt and delta-Notch signalling cascades.</a> J Neurochem. 90 (1), 89-101 (2004).</li><li>Crocker, S. J., Frausto, R. F., Whitton, J. L., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+establish+microglia-free+astrocyte+cultures:+comparison+of+matrix+metalloproteinase+expression+profiles+in+pure+cultures+of+astrocytes+and+microglia.">A novel method to establish microglia-free astrocyte cultures: comparison of matrix metalloproteinase expression profiles in pure cultures of astrocytes and microglia.</a> Glia. 56 (11), 1187-1198 (2008).</li><li>Kumamaru, H., 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=Liposomal+clodronate+selectively+eliminates+microglia+from+primary+astrocyte+cultures.">Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures.</a> J Neuroinflamm. 9, 116 (2012).</li></ol>

The authors declare that they have no competing financial interests.

가장 중요한 단계는 CGC에서 미세 아교 세포의 성공적인 선택적 제거를 보장하기 위해 및 / 또는 혼합 된 문화가있다 : 1) 멸균 및 건강한 CGC 문화를 유지하는 단계; 2) 필터 LME 함유 배지를 살균하여 pH 7.4 용액을 복귀; 3) 유지 CGC 미디어를 유지하고 37 미디어 LME이 함유 열 충격을 방지하기 위해 C를 °; 4) 세포를 배양기 밖에 유지되는 시간을 줄이기 위해 빨리 작동. 이?...

인간의 두뇌는 추정 850 억 뉴런 및 아교 세포를 포함 하나 85000000000 다른 비 신경 세포를 포함한다. 영어로 번역 &#39;아교 세포&#39;의 따라서 그리스 어원 - 신경 과학자가 신경 세포 인구에 주로 초점을 맞추고있다 지난 100 년간의 큰 부분을 위해, 아교 세포를 믿는 것은 뉴런 구조 지원을 제공하는 수동적 인 지원 세포보다 조금 더 할 수 &#39;접착제&#39;. 최근에, 그러나, 신경 - 아교 상호 작용이 신경 생물학, 신경 생리학, 많은 신경 퇴행성 질환의 기원과 진행의 기본 측면에 훨씬 더 근본적인 될 수 있다는 것을 점점 더 분명되고있다. 소뇌 과립 세포 (CGCs), 인간의 두뇌에서 가장 풍부한 균일 신경 인구는 소뇌를 지배하고 세포 구성 성분의 90 % 이상을 차지한다. 따라서, 이러한 세포는 t위한 모델 시스템으로 in vitro에서 광범위하게 사용되어왔다그는 신경 발달, 기능, 병리 2-6의 연구. 그러나, CGC의 문화는 여전히 논란의 여지 상당한 비율의 미세 아교 세포 및 기타 아교 세포가 포함되어 있습니다. 그 결과, 추정되는 다른 세포 치료법에 직접 신경 반응을 표시 CGC 데이터가 실제로 발생할 수 - 부분적 또는 전체로 - 배양에서 신경교 이웃 간접 보조 응답으로부터. 이를 평가하기 위해, 우리는 선택적으로 L-류신 메틸 에스테르 (LME)의 도움으로 CGC의 연결을 문화에서 미세 아교를 제거. LME 원래 선택적 식세포 7을 파괴하는데 사용 lysomotropic 에이전트이며, 이후 선택적 신경, 성상 세포, 및 신경 교세포의 혼합 배양에서 8,9,10 미세 아교 세포를 고갈하는 데 사용되어왔다. LME는 리소좀 파쇄 및 후속 13,14 아폽토시스를 유발하며, 대 식세포와 미세 아교 세포에 의해 내재화된다. 대 식세포와 미세 아교 세포는 그들이 particula로 발생 리소좀에서 특징적으로 풍부LME 처리에 노출시 RLY 취약합니다. 이 프로토콜은 CGC 및 다른 신경 / 아교 세포 배양 시스템을 이용한 실험에서 미세 아교 세포의 기여를 확인 할 수있는 강력하면서도 간단하고 쉬운 방법을 제공합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>The curved end facilitates removal of the cerebellum&#160;</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146</td>
			<td>Straight, sharp point facilitates rodent P4-7 dissection&#160;</td>
		</tr>
		<tr>
			<td>L-leucine methyl ester hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>7517-19-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E7510-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>27964-99-4</td>
			<td>Coat coverslips 1 day before use&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ED1 antibody&#160;</td>
			<td>Abcam</td>
			<td>ab31630</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

선택적으로 CGC 및 / 또는 혼합 된 문화에서 미세 아교 세포를 제거하기 위해이 기술의 능력을 정확하게 파악하고 자신의 주변 세포에서 미세 아교 세포를 분화하는 연구자의 후속 능력에 의존한다. 도 1에 도시 된 바와 같이, 이것은, 예컨대 동종 렉틴-B4 같이, 소교 특정 셀 머신을 사용하여 달성 될 수있다.도 2에서 설명한 바와 같이, 성상 세포 및 신경 세포의 밀도 및 형?...

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

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

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 ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

Research

JoVE Journal

Neuroscience

8.6K 조회수.  University of Washington. 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. 

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

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. ...

연구

신경과학

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM 염료를 사용하여 양식 소뇌 과립 뉴런에서 시냅틱 소포 풀 보충의 정량 분석

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

에서 신경 Explant의 문화<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

소뇌 과립 신경 세포의 유전자 조작<em&gt; 체외</em&gt;와<em&gt; 생체</em&gt; 신경 세포의 형태와 마이그레이션을 연구하는

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

공 초점 Macroscopy을 사용하여 출생 후의 소뇌 과립 세포 이동의 생체 이미징

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

최적화 된 N를 사용 하 여 급성 뇌 조각의 준비-메 틸-D-glucamine 보호 복구 방법

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

소뇌 과립 신경 세포 배양에서 Fluorescein Diacetate-Propidium Iodide 이중 염색을 이용한 신경 생존력의 평가

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

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 ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

Microglia 형태학 Immunohistochemistry 조직 ImageJ를 사용 하 여 준비의 Photomicrographs에서 측정

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

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

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

준비 및 Immunostaining Myelinating Organotypic 소 뇌 조각 문화

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