この作品は、国立精神衛生研究所、国立科学財団によってサポートされていました。専門家の魚の飼育のために多くの有益な議論や建設的な批判のためのSive研究室のメンバーに、オリヴィエPaugois氏に感謝します。

1。マイクロインジェクションのための準備<ol><li>サッター楽器ニードルプラーを使用してキャピラリーチューブを引いて、マイクロインジェクション針を準備します。 </li><li>蛍光色素（FITC-デキストラン）とマイクロインジェクションニードルをロードします。 </li><li>マイクロマニピュレータおよびマイクロインジェクション装​​置に針を取り付けます。 </li><li>慎重に幅約2μmに鉗子を用いてマイクロインジェクションニードルを破るが、これはあなたの微量注入器のセットアップに応じて異なります。私たちのマイクロインジェクション針の場合、これは曲がらない先端から針の第1の領域に対応しています。 </li><li>各注射は1 nlを提供できるように、噴射時間と圧力を調整、油の中にドロップサイズを測定します。ハーバード装置マイクロインジェクターの設定例は次のとおりです。Pバランス= 1.4 psiでは、P = 1.4 psiのうち、Pは0.4から0.7秒の噴射時間と= 22.9 PSIは、Pクリア= 67.8 psiを注入します。これらの設定を使用して私たちの針の直径はおよそ2μmである。ただし、設定はマイクロインジェクター具体的に、と変化します針径にccording。 </li></ol> 2。胚の準備<ol><li>各条件について、水に1％アガロースでコート2皿を、1から200μlのピペットチップでアガロースに穴を突く、アガロースプラグを取り外します。胚メディアで料理を記入してください。 </li><li>鉗子、18 HPFまたは実体顕微鏡下で古いdechorionate胚を用いた。胚は、キンメルらによるステージングされます11。 </li><li>第一アガロースコーティングされた皿にdechorionated胚を転送します。 </li><li>胚は（ウェスター12に従って製）移動を停止するまでの胚を麻酔し、皿にtricaine（0.1 mg / ml）を加える。 </li></ol> 3。脳室を注入<ol><li>オリエント胚従ってあなたは穴に胚の尾部を置くことによって、彼らの背側を見ている。あなたのマニピュレーターが右側にある場合は、前脳が右に左と後脳になるように、胚を移動します。 </li><li>無線で位置針後脳の脳室のdestポイント。 </li><li>卵黄（ 図1A）に脳の深さを通過しないようにしてある後脳室の慎重ピアス屋根板。 </li><li>色素が脳室の全長をいっぱいに確実に心室に蛍光色素1-2のNLを注入します。 </li><li>胚メディアに充填された第2のアガロースコーティングされた皿に胚を移し、再度麻酔として2.4で説明。 </li><li>セクション4で説明されるように直ちにゼロ時間の画像を得るために、イメージングを起動します。 </li></ol> 4。イメージング<ol><li> 3.1で説明したように穴の中の彼らの尾を持つオリエント胚。 </li><li>明視野像背を取るために、送信されたと蛍光の両方に光を解剖顕微鏡を使用しています。異なる胚のイメージングの間で倍率を一定に保つ。これは、イメージJを（5.2から6）を用いて実施し分析を直接比較することができます。 </li><li>胚、顕微鏡やシャーレを移動することなく、かかる対応する蛍光画像。 </li><li>希望する時点で各胚について、この手順を繰り返します。 </li></ol> 5。染料運動の定量化<ol><li>以前GutzmanとSive 10で説明したように、Photoshopで明視野と蛍光画像をマージします。 </li><li>距離をチェックの利用可能なイメージJソフトウェアの染料前面に移動測定<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/を</a> 。 </li><li>画像Jのマージされたファイルを開くと神経上皮（ 図1A）10から20°の角度で前方を染めるために前脳ヒンジ点から線を描画するためにラインツールを使用します。それは、野生型の神経上皮から漏れ出す色素の最初の、そして最も顕著な部位であるため、この地域が選択されまし​​た。 </li><li>ラインの長さを計算するために測定ツールを選択します。 </li><li>各時点について、この手順を繰り返します。 </li><li>染料前面が他の時点からt = 0での距離を減算することによって時間をかけて移動した正味の距離を計算します。 </li><li>上のプロットグラフ。 </li></ol> 6。代表的な結果野生型の胚を用いた神経上皮透過性アッセイで得られた結果の一例を図1B-Dに示されている。正確に透過性を区別するためには、野生型またはコントロール胚（ 図2）でわずかに漏れているサイズを識別異なる分子weightstoと染料をテストするのに便利です。これは、遺伝的変異または環境条件の識別を可能にし、そのいずれかの増加または減少は、透磁率（ 図1D、緑と赤の線）であった。 24 HPFゼブラフィッシュの神経上皮のために、2時間かけてゆっくりと70 kDaのFITCデキストランリークを、2000 kDaではないし、ほとんどすぐに10kDaの漏れる一方。したがって70 kDaの増加と両方神経上皮透過性を減少させるという条件を識別するための理想的な分子量である。 針は、心室腔、蛍光ワットを逃した場合病気にt = 0で（たとえばGutzmanとSive、2009 10を参照）で、脳の外側に表示される。注入された色素は最初に脳内に含まれていなかったとneuropeitheliumの染料と透磁率の移動に関する明確な結論が出せないので、これらの胚は破棄されるべきである。 胚は小さな心室または非膨張脳室を持っている場合、最終的には生理食塩水で、心室の注入前は、蛍光色素の注入前に行うことができます。これは、蛍光色素で注入した場合、心室の後続の可視化が容易に心室を膨らませます。適切なコントロールは、生理食塩水の注入が正常な神経管の発達を混乱させるかどうかを判断するために実行する必要があります。 <img alt="図1" src="/files/ftp_upload/4242/4242fig1.jpg" /> 図1。分子量の異なる染料の時間経過。（）実験図。最初の、蛍光色素が心室に注入される。 X注射用の針の位置=。次に背のイメージは時間をかけて取得されます。最後に、前脳ヒンジ点から染料フロントで移動距離が（赤線で表される）が測定されます。 （BC）22 HPF（t = 0分、B）と24 HPF（トン= 120分、C）で明視野と蛍光背画像を合併。白い線が前脳脳室から染料フロントの距離を示している。 （D）は架空のサンプル透過データ。青=野生型またはコントロール、コントロールと比較して増加透磁率の減少制御する相対浸透率、緑=サンプルで赤=サンプル。 <img alt="図2" src="/files/ftp_upload/4242/4242fig2.jpg" /> 図2。異なる分子量の染料に神経上皮透過性の測定。= 0分、FITC-dを使って注射後（AE）は背マージ明とトンで22 HPF野生型胚の蛍光画像10 kDaの（）、40 kDaの（B）は、70 kDaの（C）は、500kDaの（D）および2000 kDaの（E）は、次の分子量のエキストラン。トン= 24 HPFで120分でのように、（A&#39;-E &#39;）は同じ胚（AE）。左前方に。 F =前脳、中脳、M =、H =後脳。アスタリスク=耳。

<ol>
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特別な利害関係は宣言されません。

私たちは、与えられた分子量が注入された色素に決定されるように生きている胚ゼブラフィッシュの脳の透過性を定量化する能力を示す。ゼブラフィッシュ胚性神経上皮は分子量の異なる染料の差動透過性であるという我々の観察は、色素が傍細胞透過性を介して移動していることを示唆している。しかし、我々は、観測された透過性に経貢献の可能性を排除することはできません。この手?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>デキストラン、フルオレセイン、アニオン、リジン修正可能</td><td>インビトロジェン</td><td> D7136、D7137、D1822、D1820、D1845 </td></tr><tr><td> Tricaine粉</td><td>シグマ</td><td> A5040 </td></tr><tr><td>毛細管</td><td> FHC社</td><td> 30-30-1 </td></tr></tbody></table>

an assay for permeability of the zebrafish embryonic neuroepithelium

脳室系は、脊椎動物間で保存されていると脳の発達の初期段階で形成し、その動物の生涯にわたって維持されている脳室と呼ばれる相互接続された空洞のシリーズで構成されています。脳室系は、脊椎動物で発見され、心室は中央の管腔は脳脊髄液（CSF）の1,2を満たすと、神経管形成後に開発されています。 CSFは、正常な脳の発達と機能3-6必須であるタンパク質に富む流体である。 神経管が閉じられた後、ゼブラフィッシュでは、脳室のインフレ率は、約18時間後に受精（HPF）で始まる。複数のプロセスを透磁率とCSF産生を調節する神経上皮、タイトジャンクショ​​ン形成の形成を含む脳室形成、関連付けられています。我々は、Na、K-ATPaseのこれらのすべてのプロセスに影響を与える、脳室のインフレのために必要であることを示したクロー5aはタイトジャンクションの形成9のために必要である間ES 7,8、。さらに、当社は、ミオシンの阻害を介して、胚性神経上皮のその &quot;緩和&quot;を示した脳室のインフレ率に関連付けられています。 ゼブラフィッシュの脳室の膨張時に透過性の調節を調べるために、我々は、心室色素保持アッセイを開発した。この方法では、蛍光脳脊髄液を標識するために、生きているゼブラフィッシュ胚、以前に我々の研究室で開発された10の技術で脳室注入を使用しています。胚は、その後、脳室と神経上皮を通して蛍光染料が移動すると時間をかけて結像される。染料前面が離れて時間をかけて神経上皮の基底（非管腔）側から移動する距離を定量化して、神経上皮透過性（ 図1）の尺度である。我々は染料70 kDaと小さいが、神経上皮を通って移動することを観察し、detecteすることができます24 HPF（ 図2）で、ゼブラフィッシュ胚の脳の外側D。 この色素保持アッセイを開発中に、異なる時間に、異なる遺伝的背景の様々な神経上皮透過性を分析するために使用され、環境変動の後にすることができます。また、CSFの病的集積を検討する上で役に立つかもしれません。全体的に、この手法は、研究者が開発と疾患の間に透磁率の役割と規制を分析することができます。

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

我々は、ゼブラフィッシュ胚の脳の透磁率のためのライブ全体の動物定量的な測定について説明します。技術は神経管管腔内に異なる分子量の脳脊髄液や分子を保持する能力を分析し、心室の彼らの動きを定量化します。この方法では、開発や病気時の上皮透過性と成熟の違いを判別するのに役立ちます。

ゼブラフィッシュ胚性神経上皮の透水性に関するアッセイ

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

an-assay-for-permeability-of-the-zebrafish-embryonic-neuroepithelium

Research

JoVE Journal

Neuroscience

9.4K ビュー.  Massachusetts Institute of Technology. 我々は、ゼブラフィッシュ胚の脳の透磁率のためのライブ全体の動物定量的な測定について説明します。技術は神経管管腔内に異なる分子量の脳脊髄液や分子を保持する能力を分析し、心室の彼らの動きを定量化します。この方法では、開発や病気時の上皮透過性と成熟の違いを判別するのに役立ちます。

ビデオ: 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.

ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立

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 ...

神経科学

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.

神経芽細胞アッセイ：フローサイトメトリーを用いて神経幹細胞の子孫を差別から神経前駆細胞の生成と濃縮のためのアッセイ

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.

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

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.

ゼブラフィッシュ胚の脳室の手動排水

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

ex vivoでマウス神経上皮における単一細胞分裂のライブイメージング

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.

胚ゼブラフィッシュマウスナー細胞からパッチクランプ記録

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.

発生中のマウス胚性皮質における有糸分裂のライブイメージング

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.

無傷の神経上皮におけるマウス嗅細胞の穿孔パッチクランプ記録：同定された嗅覚受容体を発現するニューロンの機能解析

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.

の歩行速度に対するエタノールの効果を測定するためのアッセイ<em&gt;線虫（Caenorhabditis elegans）</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.