이 작품은 국립 정신 건강 연구소와 국립 과학 재단 (National Science Foundation)에서 지원되었습니다. 전문가 생선 축산에 대한 많은 유용한 토론과 건설적인 비판, 그리고 올리버 Paugois에 대한 Sive 연구소 회원에게 특별 감사드립니다.

1. Microinjection 준비 <ol><li> 셔터 계기 바늘 풀러를 사용하여 모세관 튜브를 당겨 microinjection의 바늘을 준비합니다. </li><li> 형광 염료와로드 microinjection의 바늘 (FITC-Dextran). </li><li> micromanipulator 및 microinjection 장치에 바늘을 탑재합니다. </li><li> 조심스럽게 폭 약 2 μm로 집게를 사용하여 microinjection의 바늘을 부러 그러나, 이렇게하면 microinjector 설정에 따라 달라집니다. 우리 microinjection의 바늘이 구부리하지 않는 끝에서 바늘의 첫 번째 지역에 해당합니다. </li><li> 각각의 주입은 1 NL을 제공합니다 있도록 분사 시간과 압력을 조정, 기름에 방울 크기를 측정합니다. 하버드 장치 microinjector 예를 들어 설정은 다음과 같습니다 P 잔액 = 1.4 PSI, P = 1.4 PSI 아웃, P는 0.4-0.7 초의 분사 시간 = 22.9 PSI, P 명확 = 67.8 PSI를 삽입. 이러한 설정을 사용하여 Google 바늘의 직경은 약 2 μm이다. 그러나, 설정 microinjector 구체적으로, 그리고이 달라집니다바늘 직경 쪽에서 그러는데 마이애미의 해변 에선. </li></ol> 2. 태아 준비 <ol><li> 코트는 2 각 조건 물에 1% 아가로 오스와 요리 1-200 μl 피펫 팁과 아가로 오스에 포크 구멍, 그리고 아가로 오스 플러그를 제거합니다. 배아 미디어와 요리를 채 웁니다. </li><li> 사용 포셉, stereomicroscope 18 명의 hpf 세 이상 dechorionate 배아. 태아도 Kimmel 외에 따라 개최됩니다. 11. </li><li> 첫째 아가로 오스 코팅 그릇에 dechorionated 배아를 전송합니다. </li><li> 배아는 (Westerfield 12에 따라 변경) 이동 중지 할 때까지 배아를 마취시키기 위해서, 음식에 tricaine을 (0.1 밀리그램 / ML)를 추가합니다. </li></ol> 3. 뇌 심실을 주입 <ol><li> 동양의 배아 그래서 당신은 구멍에 배의 꼬리를 넣어 자신의 등쪽 옆에 찾고 있습니다. 귀하의 micromanipulator가 오른쪽에 있으면 forebrain의 오른쪽에 왼쪽과 hindbrain에이되도록 한 다음 배아를 이동합니다. </li><li> 무선의 위치 바늘hindbrain의 심실의 목적지 가리 킵니다. </li><li> 난황 (그림 1A)로 뇌의 깊이 통과하지 않도록 것 hindbrain의 심실의주의 깊게 관통 지붕 판. </li><li> 염료는 뇌 심실의 전체 길이를 채우고 확인하는 심실에 형광 염료를 1-2 NL를 삽입. </li><li> 배아 미디어로 가득 번째 아가로 오스 코팅 접시에 배아를 전송하고 다시 마취로 2.4에 설명되어 있습니다. </li><li> 즉시로 시간 제로 이미지를 위해 제 4에 설명 된 이미지를 시작합니다. </li></ol> 4. 영상 <ol><li> 구멍에 자신의 꼬리 동양의 배아는 3.1에 ​​설명되어 있습니다. </li><li> brightfield의 ​​등쪽 이미지를 취할 전송 및 형광등을 모두 빛으로 해부 현미경을 사용합니다. 다른 배아의 이미지 사이의 배율이 일정하게 유지. 이 이미지 J (5.2-6)를 사용하여 수행 분석의 직접적인 비교를 할 수 있습니다. </li><li> 태아의 현미경이나 접시를 이동하지 않고,을해당 형광 이미지. </li><li> 원하는 시간 지점에서 각각의 배아에 대해 반복합니다. </li></ol> 5. 염료 운동의 정량화 <ol><li> 이전 Gutzman와 Sive (10)에 의해 설명 된대로 포토샵에서 brightfield 및 형광 이미지를 병합합니다. </li><li> 거리에게에서 제공 이미지 J 소프트웨어의 염료 전면 이동 측정 <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/을</a> . </li><li> 이미지 J와 neuroepithelium에서 10-20 ° 각도 (그림 1A)에서 전면 색깔을 할 수있는 forebrain 힌지 점에서 라인을 그릴 줄 도구를 사용에서 열기 병합 파일을 만듭니다. 이 야생 유형 neuroepithelium 밖으로 누출되는 염료의 최초이자 가장 눈에 띄는 사이트입니다 때문에이 지역은 선정되었습니다. </li><li> 라인의 길이를 계산하기 위해 측정 도구를 선택합니다. </li><li> 할 때마다 포인트에 대해 반복합니다. </li><li> 염료 앞에 다른 시간 지점에서 t = 0에서 거리를 차감하여 시간이 지남에 따라 이동 순 거리를 계산합니다. </li><li> 에 플롯그래프. </li></ol> 6. 대표 결과 야생 유형 배아를 사용하여 neuroepithelial 투자율 분석에서 얻은 결과의 예는 그림 1B-D로 표시됩니다. 정확하게 투과성을 차별화하는 데, 그것은 야생 유형 또는 제어 배아 (그림 2)에서만 약간 새는있는 크기를 식별하는 다른 분자 weightsto와 염료를 테스트하는 데 유용합니다. 이 유전자 돌연변이 나 환경 조건의 식별을 허용 그 중 증가 또는 감소는 투자율 (그림 1D, 녹색과 붉은 라인을 각각). 24 hpf의 zebrafish의 neuroepithelium를 들어, 70 kDa FITC Dextran 누출 천천히 2 시간 이상, 2,000 kDa이되지 않고 즉시 10 kDa 누수가 아웃 반면. 따라서 70 kDa 모두 증가하고는 neuroepithelial 투자율이 감소하는 조건을 식별 할 수있는 이상적인 분자량입니다. 바늘은 심실 내강, 형광 w를 그리워하는 경우병 t에서 뇌 이외의 표시 = 0 (예를 들어 Gutzman과 Sive 2009 년 10 참조). 주입 된 색소는 처음 뇌에 포함되지 않은 및 neuropeithelium의 염료 및 투자율의 움직임에 대한 명확한 결론이 내려 수 없습니다 때문에 이러한 배아는 폐기해야합니다. 마지막으로, 배아 전에 형광 염료의 주입을 수행 할 수있는 작은 심실 또는 해제 - 팽창 뇌 심실, 생리 식염수로 심실의 사전 주입이있는 경우. 이 형광 염료를 주입하면 쉽게 심실의 후속 시각화하는 심실 웃기 구 있네. 적절한 컨트롤은 생리의 주입이 정상 신경 튜브의 개발을 방해 여부를 판단하기 위해 수행해야합니다. <img alt="그림 1" src="/files/ftp_upload/4242/4242fig1.jpg" /> 1 그림. 다른 분자량의 염료의 시간 코스입니다. (A) 실험 다이어그램. 처음으로, 형광 색소는 심실에 주입되어 있습니다. X 분사에 대한 바늘의 = 위치. 다음 등쪽의 이미지는 시간이 지남에 캡처됩니다. 마지막으로, 거리 경첩 점은 (빨간색 선으로 표시) 측정 forebrain에서 염료를 전면으로 옮겼습니다. (BC) 22 hpf (t = 0 분, B), 24 hpf (t = 120 분, C)에서 brightfield 및 형광 등의 이미지를 흡수 합병. 화이트 라인은 forebrain 심실에서 염료 앞의 거리를 나타냅니다. (D) 가정 해봐 샘플 투자율 데이터입니다. 블루 = 야생 종류 나 컨트롤 제어 상대적으로 증가 투자율과 감소 제어 할 상대 투자율, 그리고 녹색 = 샘플과 붉은 색 = 샘플. <img alt="그림 2" src="/files/ftp_upload/4242/4242fig2.jpg" /> 그림 2. 다른 분자량의 염료에 neuroepithelial 투자율의 측정. (AE) 등의 병합 brightfield과 t 22 hpf 야생 유형 배아의 형광 이미지 = 0 분 FITC-D로 주입 후10 kDa (A), 40 kDa (B), 70 kDa (C), 500 kDa (D)와 2,000 kDa (E) 다음 분자량의 extran. t = 24 hpf의 120 분의에서와 같이 (A&#39;-E &#39;)과 동일 배아 (AE). 왼쪽 앞. F = forebrain, M = midbrain, H = hindbrain. 별표 = 귀.

<ol>
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관심 없음 충돌이 선언 없습니다.

우리는 주어진 분자 무게의 주입 염료에 대한 결정으로 생활 배아 zebrafish의 뇌의 투자율을 수량화 할 수있는 능력을 보여줍니다. 배아 zebrafish의 neuroepithelium는 분자 가중치를 서로 다른의 염료에 differentially 투과성있는 것으로 관찰 염료가 paracellular 투자율을 통해 이동하는 것이 좋습니다. 그러나, 우리는 관찰 투자율에 transcellular 공헌의 가능성을 배제 할 수 없습니다. 이 기술은 튜브의 내부와 ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Dextran, Fluorescein, 음이온, 리신 고칠 수 </td><td> Invitrogen </td><td> D7136, D7137, D1822, D1820, D1845 </td></tr><tr><td> Tricaine 분말 </td><td> 시그마 </td><td> A5040 </td></tr><tr><td> 모세관 튜브 </td><td> FHC 주식회사 </td><td> 30-30-1 </td></tr></tbody></table>

an assay for permeability of the zebrafish embryonic neuroepithelium

뇌 심실 시스템은 vertebrates 사이에 보존되어 있으며 뇌 개발의 초기 단계에서 형성과 동물의 삶을 통해 유지 뇌 심실라는 상호 충치의 일련의로 구성되어 있습니다. 뇌 심실 시스템은 vertebrates에서 발견하고, 심실은 중앙 루멘은 뇌척수 (CSF) 1,2로 채워 때, 신경 튜브 형성 한 후 개발을하고 있습니다. CSF는 정상적인 두뇌 개발과 기능 3-6을 위해 필수적이며 단백질 풍부한 액체이다. 신경 튜브가 폐쇄 된 후 zebrafish에서 뇌 심실 인플레이션은 약 18 시간 게시물 수정을 (hpf)에서 시작됩니다. 여러 프로세스가 neuroepithelium 형성, 투자율 및 CSF의 생산을 조절 긴밀한 접합 형성 등의 뇌 뇌실 형성와 연결되어 있습니다. 우리는이 모든 과정에 영향을 미치는, 오세영가 K-ATPase는 뇌 심실 인플레이션 필요하다고 보여claudin의 5A 꼭 접합부 형성 9 필요한 반면 에스 7,8. 또한, 우리는 마이 오신의 억제를 통해 배아 neuroepithelium의 &quot;휴식&quot;을 보여 뇌 심실 인플레이션과 연결되어 있습니다. zebrafish의 뇌 심실 인플레이션 동안 투자율의 규정을 조사하기 위해, 우리는 심실 염료 유지 분석을 개발했습니다. 이 방법은 휘황 중추 신경계에 라벨을, 거실 zebrafish의 배아, 이전에 우리 연구소 10 개발 기술에 뇌 뇌실 주입을 사용합니다. 태아는 다음 뇌 심실과 neuroepithelium을 통해 형광 염료 이동으로 시간이 지남에 따라 이미징 있습니다. 거리 염료 전면 정량 멀리 시간이 지남에 따라 neuroepithelium의 기초 (비 luminal) 측에서 이동하고 neuroepithelial 투자율의 측정 (그림 1)입니다. 우리는 염료 70 kDa과 작은이 neuroepithelium을 통해 이동한다는 관찰하고 detecte 할 수 있습니다24 hpf (그림 2)에서 배아 zebrafish의 뇌 이외의 D. 이 염료 유지 분석은 개발 기간 동안 다른 시간에 서로 다른 유전 배경을 다양한 neuroepithelial 투자율을 분석하는 데 사용, 환경 섭동 후 할 수 있습니다. 또한 CSF의 병적 인 축적을 조사에 유용 할 수 있습니다. 전반적으로,이 기법은 수사관 개발과 질병 중 투자율의 역할과 규정을 분석 할 수 있습니다.

Watch this Scientific Journal Video about An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium at JoVE.com

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

우리는 배아 zebrafish의 뇌의 투자율의 라이브 전체 동물 양적 측정을 설명합니다. 기술은 신경 튜브 내강 내에서 서로 다른 분자 무게의 중추 신경계와 분자를 보유 할 수있는 능력을 분석하고 심실의 움직임을 단정 지을. 이 방법은 개발과 질병 중 상피 투자율과 성숙의 차이를 결정하는 데 유용합니다.

Zebrafish 배아 Neuroepithelium의 투과율에 대한 분석

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

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Research

JoVE Journal

Neuroscience

9.4K 조회수.  Massachusetts Institute of Technology. 우리는 배아 zebrafish의 뇌의 투자율의 라이브 전체 동물 양적 측정을 설명합니다. 기술은 신경 튜브 내강 내에서 서로 다른 분자 무게의 중추 신경계와 분자를 보유 할 수있는 능력을 분석하고 심실의 움직임을 단정 지을. 이 방법은 개발과 질병 중 상피 투자율과 성숙의 차이를 결정하는 데 유용합니다.

비디오: An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neurosphere 분석을 사용하여 마우스 배아 신경 줄기 세포 문화 구축

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

연구

신경과학

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neuroblast 분석 : 유동세포계측법을 사용하여 차별화 신경 줄기 세포에서 자손의 연결 전구 세포의 생성과 강화에 대한 분석

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

개발 Neuroepithelium의 세포 문제의 고해상도 라이브 영상

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

Zebrafish 배아 뇌 심실의 수동 배수

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

생체 마우스 Neuroepithelium에서 단일 세포 분열의 라이브 영상

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

배아 Zebrafish의 MAUTHNER 세포에서 패치 클램프 녹음

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

개발 마우스 배아의 피질 유사 분열의 라이브 영상

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

본래 Neuroepithelium에서 마우스 강한 감각 뉴런의 천공 패치 클램프 녹음 : 확인 된 부 취제 수용체를 발현 뉴런의 기능 분석

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

의 로코 속도에 에탄올의 효과를 측정하는 분석<em&gt; 예쁜 꼬마 선충</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.