이 작품은 부분적으로 Université 라발의 교제에 의해 지원되었다 ASJK에 건강 연구 (CIHR) 교부금의 캐나다 연구소에 의해 지원되었다. AS 출생 후의 neurogenesis에 캐나다 연구 의자의 수신자입니다.

<ol>
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C., Heintz, T., Young, S., Gordon, V., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonic+activation+of+GLUK5+kainate+receptors+decreases+neuroblast+migration+in+whole-mounts+of+the+subventricular+zone.">Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone.</a> J. Physiol. 586, 3783-3793 (2008).</li><li>Nam, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+features+of+postnatal+subventricular+zone+cell+motility:+a+two-photon+time-lapse+study.">Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study.</a> J. Comp. Neurol. 505, 190-208 (2007).</li><li>Kim, Y., Comte, I., Szabo, G., Hockberger, P., Szele, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mouse+subventricular+zone+stem+and+progenitor+cells+are+sessile+and+epidermal+growth+factor+receptor+negatively+regulates+neuroblast+migration.">Adult mouse subventricular zone stem and progenitor cells are sessile and epidermal growth factor receptor negatively regulates neuroblast migration.</a> PLoS One. 4, e8122 (2009).</li><li>Bortone, D., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCC2+expression+promotes+the+termination+of+cortical+interneuron+migration+in+a+voltage-sensitive+calcium-dependent+manner.">KCC2 expression promotes the termination of cortical interneuron migration in a voltage-sensitive calcium-dependent manner.</a> Neuron. 62, 53-71 (2009).</li><li>Comte, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin-3+maintains+cell+motility+from+the+subventricular+zone+to+the+olfactory+bulb.">Galectin-3 maintains cell motility from the subventricular zone to the olfactory bulb.</a> J. Cell Sci. 124, 2438-2447 (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

적절한 뇌 지역에 neuronal 전구체의 올바른 타겟팅은 신경 회로의 적절한 조립과 기능의 근간이 기본 과정입니다. 세포의 대부분은 배아 발달 동안 마이그레이션 만 등의 OB, 이가있는 이랑과 소뇌 등의 몇 지역에서 출생 후의 두뇌에 neuronal 변위는 아직 이루어집니다. 출생 후의 뇌의 세포 마이그레이션을 꾸미고 메커니즘이 저조한 이​​해하지만, 남아 있습니다. 성숙한 신경 조직에 세포 마이그...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 자당 </td><td> 시그마 </td><td> S9378 </td></tr><tr><td> 포도당 (ACSF) </td><td> EMD </td><td> DX0145-3 </td></tr><tr><td> NaCI </td><td> 시그마 </td><td> S9625 </td></tr><tr><td> KCI </td><td> 시그마 </td><td> P9541 </td></tr><tr><td> MgCI이 x6H 2 O </td><td> 시그마 - 알드리치 </td><td> M2670 </td></tr><tr><td> NaHCO 3 </td><td> 시그마 </td><td> S5761 </td></tr><tr><td> 아뇨 2 PO 4 xH 2 O </td><td> EMD </td><td> SX0710-1 </td></tr><tr><td> CaCI이 x2H 2 O </td><td> 시그마 - 알드리치 </td><td> C3881 </td></tr><tr><td> Dextran은 TexasRed </td><td> Invitrogen </td><td> D1864 </td></tr><tr><td> Dextran CascadeBlue </td><td> Invitrogen </td><td> D1976 </td></tr><tr><td> 포도당 (40X 솔루션) </td><td> 시그마 </td><td> G8769 </td></tr><tr><td> 나트륨 pyruvat </td><td> Gibco </td><td> 11360-070 </td></tr><tr><td> HEPES </td><td> 시그마 </td><td> H3375 </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 14170-112 </td></tr><tr><td> DNase I </td><td> 시그마 </td><td> D-5025 </td></tr><tr><td> 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) </td><td> Gibco </td><td> 25300-054 </td></tr><tr><td> Neurobasal 매체 </td><td> Gibco </td><td> 21103-049 </td></tr><tr><td> BSA </td><td> EMD </td><td> 2930 </td></tr><tr><td> 펜 / 패 혈성 </td><td> 생명 기술 </td><td> 15140-122 </td></tr><tr><td> 케타민 / Xylazine </td><td> CDMV </td><td> 5230 </td></tr><tr><td> 파스퇴르 피펫 </td><td> VWR </td><td> 14672-380 </td></tr><tr><td> 15 ML 원뿔 관 </td><tD&gt; Sarstedt </td><td> 62.553.205 </td></tr><tr><td> 50 ML 원뿔 관 </td><td> Sarstedt </td><td> 62.547.205 </td></tr><tr><td> 유리 모세관 (stereotaxic 분사) </td><td> WPI </td><td> 4878 </td></tr><tr><td> 파라핀 유 </td><td> EMD </td><td> PX0045-3 </td></tr><tr><td> Proviodine </td><td> Rougier </td><td> 65655-1370 </td></tr><tr><td> 상처의 봉합 </td><td> Stoelting </td><td> 50,487 </td></tr><tr><td> Anafen </td><td> CDMV </td><td> 11,508 </td></tr><tr><td> 20 CC의 주사기 </td><td> VWR </td><td> SS-20L2 </td></tr><tr><td> 페트리 접시 </td><td> VWR </td><td> 25384-094 </td></tr><tr><td> 한천 </td><td> Laboratoire 매트 </td><td> AP-0108 </td></tr><tr><td> 아교 </td><td> Permabond </td><td> 910 </td></tr><tr><td> 95% O 5분의 2 %의 CO 2 </td><td> Linde </td><td> 24068835 </td></tr><tr><td> 블레이드 &lt;/ TD&gt; <td> WPI </td><td> 501901 </td></tr><tr><td> 나일론 메쉬 </td><td> 워너 악기 </td><td> 64-0198 </td></tr><tr><td> 원심 분리기 </td><td> Eppendorf </td><td> 5702 000.019 </td></tr><tr><td> 피펫 풀러 </td><td> 셔터 악기 </td><td> P-97 </td></tr><tr><td> Nanoliter 주입기 </td><td> WPI </td><td> B203MC4 </td></tr><tr><td> Stereotaxic 주입 장치 </td><td> WPI </td><td> 502900 </td></tr><tr><td> 마이크로 드릴 시스템 </td><td> WPI </td><td> 501819 </td></tr><tr><td> Vibratome </td><td> 열 과학 </td><td> 920110 </td></tr><tr><td> 폭 넓은 분야 형광 현미경 </td><td> 하늘 </td><td> BX61WIF </td></tr><tr><td> CCD 카메라 </td><td> Photometrics </td><td> CS-HQ2-D </td></tr><tr><td> 울트라 조용한 이미징 챔버 </td><td> 하버드 장치 </td><td> 64-1487 </td></tr><tr><td> PH-1시리즈 20 히터 플랫폼 </td><td> 하버드 장치 </td><td> 64-0284 </td></tr><tr><td> 난방 시스템 </td><td> 워너 악기 </td><td> TC-344B </td></tr><tr><td> 40X 물 침지 목표 </td><td> 하늘 </td><td> 1 UM587 </td></tr><tr><td> 10X 물 침지 목표 </td><td> 하늘 </td><td> 1 UM583 </td></tr><tr><td> 람다 DG-4 </td><td> 셔터 악기 </td><td> DG-4/OF </td></tr><tr><td> MetaMorph 소프트웨어 </td><td> 분자 장치 </td><td> 40,000 </td></tr><tr><td> Imaris 소프트웨어 </td><td> Bitplane </td><td> BPI-IM70-F1 </td></tr></tbody></table>

time-lapse imaging of neuroblast migration in acute slices of the adult mouse forebrain

새로운 기능 뉴런이 constitutively 성인 포유류의 뇌의 제한된 영역에서 신경 줄기 세포의 내생 풀에서 생성을 나타내는 증거의 상당한 몸이있다. subventricular 영역 (SVZ)에서 신생아 neuroblasts는 후각 망울 (OB) 1에서의 최종 목적지까지의 뱃 부리 장식이있는 철새 스트림 (RMS)을 함께 마이그레이션. RMS에서 neuroblasts은 구조 지원 및 마이그레이션 4,5에 필요한 분자 요소의 소스로 혈관을 사용하여 astrocytic 프로세스 2,3에 의해 ensheathed 체인에 tangentially 마이그레이션. OB에서 neuroblasts는 체인에서 분리하고 interneurons로 차별화하고 기존의 네트워크 1, 6에 통합 다른 안구 레이어로 방사상으로 마이그레이션됩니다. 이 원고에서 우리는 쥐의 뇌의 급성 조각의 모니터링 셀 마이그레이션에 대한 절차를 설명합니다. 급성 조각의 사용은 assessm 수 있습니다밀접하게 생체 조건과 생체 이미징에 대해 액세스하기 어렵 뇌 영역에서에 유사한하는 microenvironment의 세포 마이그레이션 다닐. 또한, 결국 세포의 이주 속성을 변경할 수도 있습니다 organotypic과 셀 문화의 경우에서와 같이 긴 배양 조건을 방지합니다. 급성 조각의 Neuronal 전구체는 DIC 광학 또는 형광 단백질을 사용하여 시각화 할 수 있습니다. 야생 형 생쥐의 SVZ에 기자 마우스에서 neuroblasts을 접목하고, neuroblasts에 형광 단백질을 표현하는 유전자 변형 쥐를 사용하여 SVZ의 neuronal 전구체의 바이러스 성 라벨은 neuroblasts을 표시하고 이전에 따라에 대한 모든 적합한 방법입니다. 나중에 방법은 그러나, 개별 셀 때문에 분류 세포의 높은 밀도의 장기간 추적 할 수 없습니다. 우리는 비교적 빠른 수집 간격 (한 IMA 달성하기 위해 CCD 카메라가 장착 된 넓은 분야 형광 수직 현미경을 사용GE 매 15 또는 30 초)가 안정적으로 고정와 철새 단계를 식별합니다. 고정와 철새 단계 기간의 정확한 식별 결과의 모호 해석에 매우 중요합니다. 우리는 또한 3D로 neuroblasts 마이그레이션을 모니터링하기 위해 여러 Z 단계 인수를 수행. 폭 넓은 분야 형광 이미징은 neuronal 마이그레이션에게 7-10를 시각적으로 광범위하게 사용되어왔다. 여기, 우리는 neuroblasts 라벨을 성인 마우스 forebrain의 급성 조각에 neuroblast 이주의 실시간 비디오 영상을 수행하고, 세포 마이그레이션을 분석에 대한 자세한 절차를 설명합니다. 설명 프로토콜 성인 RMS의 neuroblasts의 마이그레이션을 예시 있지만, 또한 배아 및 조기 출생 후의 두뇌에 세포 이주를 수행하는 데 사용할 수 있습니다.

Watch this Scientific Journal Video about Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain at JoVE.com

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

우리는 마우스 forebrain의 neuronal 이주의 실시간 videoimaging을위한 프로토콜을 설명합니다. 급속도로 이루어질 - 표시 또는 이식 neuronal 전구체의 마이그레이션은 고정 및 마이그레이션 단계의 기간 및 속도 등의 세포 이주의 다른 단계를 공부하고 비교적 빠른 수집 간격과 폭 넓은 분야 형광 이미징을 사용하여 급성 라이브 슬라이스에 기록 된 이주.

성인 마우스 Forebrain의 급성 슬라이스에 Neuroblast 마이그레이션의 시간 경과 영상

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

time-lapse-imaging-neuroblast-migration-acute-slices-adult-mouse

Research

JoVE Journal

Neuroscience

14.8K 조회수.  Centre de Recherche Université Laval Robert-Giffard. 우리는 마우스 forebrain의 neuronal 이주의 실시간 videoimaging을위한 프로토콜을 설명합니다. 급속도로 이루어질 - 표시 또는 이식 neuronal 전구체의 마이그레이션은 고정 및 마이그레이션 단계의 기간 및 속도 등의 세포 이주의 다른 단계를 공부하고 비교적 빠른 수집 간격과 폭 넓은 분야 형광 이미징을 사용하여 급성 라이브 슬라이스에 기록 된 이주.

비디오: Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

출생 후의 두뇌에의 연결을 마이 그 레이션의 고해상도 시간 경과 이미징을위한 Organotypic 슬라이스 분석

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

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신경과학

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

칼슘 이미징에 대한 급성 Subventricular 지대 슬라이스의 작성

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

급성 Hippocampal 슬라이스에 Synaptically-evoked Astroglial와 Neuronal 응답의 듀얼 Electrophysiological 레코딩

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

마우스 급성 뇌 조각의 Neuroblast 마이그레이션의 생체 내 출생 일렉트로 및 시간 경과 영상

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

설치류 Neuroblasts의 Nucleofection는 Neuroblast 마이그레이션을 연구하는<em&gt; 체외</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

마우스 인공 와우의 Explants의 장기 시간 경과 영상

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

마우스 보습 코 기관의 급성 조직 조각에서 정의 된 세포 집단의 심층 생리 학적 분석

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

배아 마우스 두뇌의 organotypic 슬라이스 문화에서 마이 그 레이션 뉴런의 저속 공 촛점 이미징<em&gt; Utero에서</em&gt; 일렉트로 포 레이션

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial dysfunction has been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson&#39;s disease (PD), Huntington&#39;s disease, and Alzheimer&#39;s disease. To study the role of mitochondria in models of these diseases, we can measure mitochondrial respiration via oxygen consumption rate (OCR) as a proxy for mitochondrial function. OCR has already been successfully measured in cell cultures, as well as isolated mitochondria. However, these techniques are less physiologically relevant than measuring OCR in acute brain slices. To overcome this limitation, the authors developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice. The technique is optimized with a focus on the striatum, a brain area involved in PD and Huntington&#39;s disease. The analyzer performs a live cell assay using a 24-well plate, which allows the simultaneous kinetic measurement of 24 samples. The method uses circular-punched pieces of striatal brain slices as samples. We demonstrate the effectiveness of this technique by identifying a lower basal OCR in striatal slices of a mouse model of PD. This method will be of broad interest to researchers working in the field of PD and Huntington&#39;s disease.

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods.