이 연구는 밴더빌트 - 잉그램 암 센터 지원 그랜트 (P30 CA068485), 아동 뇌종양 재단과 국립 보건원 (NS042205)에서 보조금에 의해 지원되었다.

1. 종양 - 베어링 소뇌 및 종양 조직의 분리의 마이크로 해부 <ol><li> 종양 조직 검색 <ol><li> medulloblastoma 베어링 병든 생쥐는 종종 runted 및 hydrocephaly 및 사후 마비 및 전복시 자세를 회복하기 위해 실패를 포함하여 전형적인 신경 증상을 표시하고 있습니다. 종양 조직을 검색하려면, 이산화탄소 흡입하여 쥐를 안락사. 그것은 자궁 경부 전위, 뒷부분 두개골에 압력을 생성하고 종양 조직 무결성을 손상 수있는 절차를 수행하지 imporant이다. </li><li> 잘린은 두개골의 좋은 시각화를위한 머리 근육 조직 최대한 제거, 가위를 사용하여 죽은 후 즉시 수행됩니다. 95 % 에탄올로 적셔 Kimwipe와 두개골의 표면을 청소하십시오. </li><li> 두개골의 정중선을 따라 개방 잘라하고, 어느 시점에 종양 베어링 소뇌를 포함하여 전체 두뇌가 노출, 괜찮아요 핀셋을 사용하여 두개골 조직을 제거하는 미세 가위를 사용합니다. </li><li> 건강한 성인의 cerebella 잘 정의 반구와 vermis를 표시하지만, 종양이 베어링 생쥐의 cerebella은 종종 매끄러운 표면과 눈에 혈관과 비정질, 확대하고 있습니다. 무균 기술을 사용하여 얼음처럼 차가운 인산에 핀셋과 장소를 사용하여 소뇌 종양을 검색 호수 (PBS)가 MG이없이 버퍼 + 및 CA 2 +. </li></ol></li></ol> 참고 : 모든 악기가 사용하기 전에 autoclaved 95 %의 에탄올과 증기로 소독하고 있습니다. <ol start="2"><li> 종양 조직의 분리 <ol><li> 4 분간 37 인큐베이션 다음 실온에서 3 분 고급 가위, ° C로 조직을 말하다, PBS에서 종양 조직의 4 배 볼륨 약 50 % Accutase (PBS에 희석)에 종양 조직을 전송 , 그 이후 조직은 추가 3 분 1 - ML Pipetman와 반복 pipeting를 겪습. 이 방법은 단일 세포와 작은 세포 집합체의 혼합물을 얻을 것입니다. </li><li> 세포 현탁액 PBS 및 1000xg에서 펠릿 세포 5 분 동안 원심 분리기 3 배 희석. (위의 절차는 목성은 언론에서 &quot;medulloblastoma의 줄기 세포의 분리, 농축 및 유지 보수&quot;에 설명되어있다) </li></ol></li></ol> 글루타민 페니실린 - 스트렙토 마이신, N2, B27, 인간 EGF (25 NG / ML)과 기본 FGF (25 NG / ML)로 Neurobasal 매체의 구성 신선한 신경 줄기 세포 매체에있는 세포 펠렛을 Resuspend. 솔루션의 세포 밀도를 계산하고 하나는 멸균 베벨 - 스쳐 10μL 주사기로, dissociated 종양 세포의 적절한 숫자, 예를 들어 5x10 5 솔루션 4 μL를 로드할 수있는 등 추가로 신경 줄기 세포 매체와 적절한 추가 dilutions합니다. 주사하는 세포의 정확한 숫자는 특정 실험 조건에 따라 연구자에 의해 결정되어야합니다. 미니 200μL microcentrifuge 관의 세포 솔루션 (PCR 튜브) 얼음에 보관하십시오. 2. 받는 마우스에 대한 마취 절차 수술 들어, 설치류 수술 전용 소독 지역은 절차의 기간 동안 필요합니다. 이상적으로, 동물 준비, 운영 분야 및 동물 복구를 위해 별도하지만 인접한 영역 긴 벤치를 설정합니다. 수술 장갑이 아닌 시험 장갑은 수술이 필요합니다. 무균 기술은 허리 위에 장갑 낀 손을 잡고하여 유지하고 건조 멸균 표면 살균 개체를 처리하기 위해서만 사용됩니다. <ol><li> 마취제 (마우스 중량 1kg 당 100 MG의 마취제)와 xylazine (마우스 중량 1kg 당 10 MG xylazine)의 혼합물은 마취에 사용됩니다. anesthetics의 intraperitoneally을 관리할 수 있습니다. </li><li> 그것은 일반적으로 전체 적용하기 위해 anesthetics에 대한 3~5분 걸립니다. 마우스 발끝 / 꼬리 핀치에 의해 완전히 anesthetized 있는지 확인합니다. 마우스의 눈 안과 연고의 작은 금액을 넣으십시오. 끊임없이 마우스가 정상적으로 호흡 있는지 확인하기 위해 모니터링, 운영 영역에 완전히 anesthetized 마우스를 가져와. </li></ol> 3. 종양 세포의 이식 Intracranial <ol><li> midbrain과 소뇌 위의 후부 두개골을 나타내기 위해 머리 깎기를 사용하여 마우스 등의 사후 헤드 영역을, 면도. </li><li> 알코올 패드로 닦아 다음 면화 팁 작은 주걱을 사용하여 노출된 두피에 Betadine 솔루션을 적용합니다. 이 두 살균 단계를 두 번 반복합니다. </li><li> 멸균 메스를 사용하여 사후 두피 안에 25 인치 절개를합니다. 오른쪽으로 0.5 mm 버 구멍 2mm 및 살균 치과 드릴을 사용하여 람다에 후부 2mm를 드릴. </li><li> 프레임 개최에 해당 incisors 넣었지하여 stereotactic 프레임에 마우스를 놓습니다. 조심스럽게 내려가면 및 버 구멍에 종양 세포 솔루션 4 μL와 함께 로드된 베벨 - 스쳐 10 μL 주사기 위치. syri의 일단 사각nge 니들은 두개골 표면 아래 다른 3mm에 대한 하강 후 0.5 mm를 위해 승천. 천천히받는 사람의 소뇌로, 30 번째 프레임에서 꾸준한 강제로, 종양 세포의 원하는 금액을 주입. 추가 2 분 동안 주입 완료 후에 그 자리에 바늘을 둡니다. 그런 다음 바늘과 주사기를 제거합니다. autoclip를 사용하여 상처를 클립. </li><li> 의 체온을 유지하기 위해 수술 후 온난 화 담요 위에 마우스를 유지. 이동성과 호흡 패턴이 지속적으로 관찰됩니다. 마우스가 마취에서 회복되면 살균 주택 케이지에 다시 그것을 놓으십시오. 음식과 물 섭취, 행동, 정리, 그리고 눈이 붉은 얼룩에 대해 매일 모니터링합니다. 절개 사이트에서 감염의 흔적을 살펴보세요. 일 6.25 게시 수술에 autoclips를 제거합니다. 2-6개월의 평균에 대한 쥐를을 유지하고 더욱 연구에 대한 전형적인 medulloblastoma 증상을 표시하는 사람들을 희생. </li></ol> 4. 대표 결과 받는 생쥐에서 개발된 보조 medulloblastomas는 매우 기본 종양의 문제들을 닮은. 전체 - 마운트로 볼 때 이차 종양 베어링 cerebella 종종 겉보기 이소성 혈관과 비정질, 확대되었다. 보조 종양 조직의 Immunohistochemical 분석은 종양 세포가 강한 표현 SmoM2 - YFP과 같은 Math1 및 Pax6 같은 소뇌 과립 신경 세포의 엽 성의 전구 물질의 표시 높은 proliferative 있다고 밝혔다. dissociated과 교양을 사용 신고 방법 ( &quot;분리, 농축 및 medulloblastoma의 줄기 세포의 유지 보수&quot;언론의 조브), 보조 medulloblastoma 종양 세포가 급속하게 확대 될 수있을 때, 표현 여러 줄기 세포 마커는 clonogenic하고 때 더욱 종양 형성을 시작할 수 있습니다 추가 immunocompromised받는 사람에 이식. <img src="/files/ftp_upload/2153/2153fig1.jpg" alt="그림 1" /> 그림 1. 일반 전체 장착 및 보조 medulloblastoma 조직의 단면보기 보조 medulloblastoma 조직의 전형적인 예를 들어 26 일 5x10 5 일차 종양 세포의 주사 후 개발했습니다. Hematoxylin의 얼룩이 높은 세포질 / 핵 비율과 핵 다형성 포함 medulloblastoma의 전형적인 세포 형태를 보여준다. Ki67 표현으로 표시 이러한 보조 종양 세포는 매우 proliferative하고 그들은 또한 견고 SmoM2 - YFP 막의 표현한다. <img src="/files/ftp_upload/2153/2153fig2.jpg" alt="그림 2" /> 그림 2. 보조 medulloblastoma 세포는 교양과 체외에서 확장 가능 기본 마우스 medulloblastoma 세포 ( &quot;분리, 농축 및 medulloblastoma의 줄기 세포의 유지 보수&quot;언론의 조브)에 설명된 프로토콜을 사용하여 이러한 보조 medulloblastoma 세포는 교양 여러 구절에 대한 확장하실 수 있습니다. 그림 4 일 14 일에서 교양 같은 대표적인 종양의 식민지를 보여줍니다. 교양 종양 세포는 세포 골재의 고밀도 코어에서 부족한 세포질과 자주 방출로 길쭉한 바이폴라입니다. 그들은 성장 접촉 억제를 전시하지 않습니다. 작은 원형 세포는 적혈구 수 있습니다.

<ol>
	<li>Schuller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+granule+neuron+precursor+identity+is+a+critical+determinant+of+progenitor+cell+competence+to+form+Shh-induced+medulloblastoma.">Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma.</a> Cancer Cell. 14, 123-134 (2008).</li><li>Yang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+alters+the+fate+of+preneoplastic+cells+in+a+mouse+model+of+medulloblastoma.">N-myc alters the fate of preneoplastic cells in a mouse model of medulloblastoma.</a> Genes Dev. 23, 157-170 (2009).</li><li>Flora, A., Klisch, T. J., Schuster, G., Zoghbi, H. Y., Y, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Atoh1+disrupts+Sonic+Hedgehog+signaling+in+the+developing+cerebellum+and+prevents+medulloblastoma.">Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma.</a> Science. 326, 1424-1427 (2009).</li><li>Salsano, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+neurogenic+basic+helix-loop-helix+transcription+factor+NEUROG1+identifies+a+subgroup+of+medulloblastomas+not+expressing+ATOH1.">Expression of the neurogenic basic helix-loop-helix transcription factor NEUROG1 identifies a subgroup of medulloblastomas not expressing ATOH1.</a> Neuro Oncol. 9, 298-307 (2007).</li></ol>

관심 없음 충돌 선언하지 않습니다.

산출 보조 종양 형성은 다음과 같은 것을 allografting 성공 medulloblastoma 세포 수 있도록 여러 가지 중요한 요소 : 첫 번째는 조직 분리에 대해서만 50 % Accutase를 사용하고 연장 효소 치료는 세포 생존에 상당한 감소에 이르게으로 조직을 덮어 소화하지 않습니다. 작은 팁 또한 dissociated 세포에 물리적 손상을 생성할 수 있습니다로도 오직 하나 ML 크기 Pipetman를 사용합니다. 그것은 allografting 하나의 세포...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Neurobasal medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103049</td>
<td>&nbsp;</td>
<tr>
<td>hEGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0311</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0023</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>N2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502048</td>
<td>1X</td>
</tr>
<tr>
<td>B27(-RA)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12587010</td>
<td>1X</td>
</tr>
<tr>
<td>Accutase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1110501</td>
<td>50% </td>
</tr>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>2mM</td>
</tr>
<tr>
<td>Stereotaxic instrument</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51730</td>
<td>&nbsp;</td>
<tr>
<td>Foredom flexible shaft drill, hang-up style</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>58650</td>
<td>&nbsp;</td>
<tr>
<td>Large probe holder</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51633</td>
<td>&nbsp;</td>
<tr>
<td>10 &mu;L syringe, 26 ga., bevel tip</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51105</td>
<td>&nbsp;</td>
<tr>
<td>Drill bit .75mm</td>
<td>&nbsp;</td>
<td>Stoelting</td>
<td>51455-3</td>
<td>&nbsp;</td>		</tbody>
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intracranial orthotopic allografting of medulloblastoma cells in immunocompromised mice

Medulloblastoma은 신경계의 가장 일반적인 소아 종​​양이다. 동물 연구의 큰 시체가 medulloblastoma 1-4에 대한 세포 수준의 기원으로 소뇌 과립 신경 세포의 엽 성의 전구 물질 (CGNPs)에 초점을 맞추고 있습니다. 그러나, 인간의 환자 (결절 desmoplastic, 고전 및 셀 / anaplastic 대형) 및 medulloblastoma가 CGNP 마커 5 노 이소성 표정으로 인간 환자의 일부에서 발견되는 사실에 medulloblastoma의 subtypes의 다양한 임상 프레 젠 테이션 것이 좋습니다 medulloblastoma의 세포 및 분자 기원은 지금까지 완전히 해독되지 않도록 더 복잡하고 있습니다. 따라서, 어떤 세포 유형 특정 치료 양상을 개발할 수에 따라 대체 medulloblastoma 종양 세포 수준의 원산지가 있는지 확인하는 것이 필수적입니다. 이를 위해 수신자의 이차 종양 개발의 후속 분석 다음에 표시된 유전자 종양 세포 유형의 allografting intracranial orthotopic는 종양 - 시작 세포의 세포 기원의 결정을하실 수 있습니다. 우리가 일차 종양 조직에서 파생된 medulloblastoma 세포의 allografting intracranial orthotopic에 대한 실험 프로토콜을 설명하고,이 절차는 또한 기존 휴대폰 라인에서 세포를 이식에 사용될 수 있습니다.

Watch this Scientific Journal Video about Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice at JoVE.com

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

이 프로토콜은 마우스 medulloblastoma 조직의 격리와 분리를 설명하고, 보조 medulloblastoma를 시작하기 위해 immunocompromised받는 생쥐에 종양 세포의 후속 allografting.

Immunocompromised 마우스의 Medulloblastoma 세포 Intracranial Orthotopic Allografting

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

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JoVE Journal

Neuroscience

12.1K 조회수.  Vanderbilt University. 이 프로토콜은 마우스 medulloblastoma 조직의 격리와 분리를 설명하고, 보조 medulloblastoma를 시작하기 위해 immunocompromised받는 생쥐에 종양 세포의 후속 allografting.

비디오: Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

절연, 강화하고, Medulloblastoma의 줄기 세포의 유지 보수

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

연구

신경과학

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Adeno - 관련 바이러스성 벡터의 Intracranial 분사

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

마우스에 콜드 민감도 및 적응을 결정하기위한 간단하고 저렴한 방법

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

신경 줄기 세포와 쥐의 뇌에서 5-하이드록시메틸시토신의 검출

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

쥐의 경내 합성 단백질의 정량적 측정

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

쥐에서 코필린을 표적으로 하는 아데노 관련 바이러스를 사용하여 조작된 대뇌 피질 지역의 전기 코르티코그래피 기록

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.