<p class="jove_content"&gt;私たちは、エレクトロポレーション、イラストのために博士ユバールゴットリーブ·ドロールに感謝します。この作品は、イスラエルの精神生物学研究所からニーダーザクセン·イスラエル研究協力プログラムからDSDへの補助金によって、イスラエル科学財団、健康のイスラエル大臣、そして卓越-レガシー遺産センターからAKへの補助金によって支えられてバイオメディカルサイエンス·パートナーシップ。</p&gt;

1。後脳のエレクトロ 1.1卵の取り扱い<ol><li>加湿インキュベーター（37から38.5℃）に水平に卵を置きます。胚は、彼らは16-17（HH）ステージ（25〜30体節）に達したときに、インキュベーションの65-70時間後にエレクトロポレーションされています。 </li><li>インキュベーターから卵を外し、彼らは水平位置に残ります。 </li></ol> 1.2準備<ol><li>ガラスキャピラリー（直径0.5mm）を引き出します。 </li><li>パルス発生器にアダプタ保持して屈曲L字状の金Genetrodes電極（直径3mm）を接続する。エレクトロポレーション·パラメータは、25ボルト、5パルス数、45ミリ秒のパルス長、300ミリ秒パルス間隔が含まれています。 </li><li>口のピペット、10ミリリットルの注射針、パラフィルムストリップ、柔らかいブラシ、鋭い解剖はさみ、25 mlの滅菌PBS溶液を準備します。この量は、通常、卵トレイ（18卵）を処理するのに十分である。 </li><li> DNAプラスミドの適切な混合物（5μgの/μlのそれぞれ）を準備し、DNA注入を可視化するために約0.025％ファストグリーン染料を追加します。通常4μLの容積は、トレイの注射のために十分である。 </li></ol> 1.3ウィンドウイング、注射とエレクトロ<ol><li> 70％エタノールで卵の殻をきれいにします。 </li><li>空気に胚の露光時間を最小限にするために一つずつ卵を扱う。 </li><li>はさみ両端卵ポールに穴を作り、シリンジでアルブミン3-4ミリリットル取り除く。それは可能性を減少させるのでアルブミン多量のを削除しないでください。 </li><li>解剖ハサミを使って卵の殻の上部中央にある小さな楕円形の窓（2.5×2センチサイズ）を開きます。 </li><li>全体のプロセス中に乾燥を防ぐために、胚の上にPBSを数滴垂らし。 </li><li>ガラス中にDNA混合物の少量を読み込む口のピペットを用いてキャピラリ。 </li><li>あなたに向かって、その後端に胚を置きます。胚がはっきり見えるようにインク噴射がこの段階で必要とされない。インク注入が増大を回避すること胚の生存。 〜45°でキャピラリーを保持することによって尾後脳に浸透。胚の周りからの膜を除去しないでください。気泡を形成することなく、後脳内腔と呼気に慎重にDNA溶液を注入する。 DNA溶液は、前方に広がる。 </li><li>すぐにDNA注入後、ターゲットを目指して正確なAPとDV後脳の位置で平行電極を配置。それは可能性を減らす可能性があるので、心に触れることなく少し腹の電極を押してください。エレクトロをパルス。電極（ 図1A）電気伝導性を示すために、泡で覆われるべきである。 </li><li>慎重に電極を除去し、胚を冷却するために、泡を除去するためにPBSを数滴垂らし。インキュベーション中に乾燥を防ぐためにパラフィルムストリップで正しく開く卵を密封。柔らかいブラシで、PBS中の電極を洗浄します。 </li><li>時間の希望の長さのためにインキュベーターに卵を置きます。胚の生存率は約90％の2-3日後ですエレクトロポレーション、およびエレクトロポレーション後に50％の12日まで減少します。 </li></ol> 2。胚の分析 2.1フラットマウントの準備と免疫<ol><li>後脳のフラットマウントの準備が簡単に顕微鏡やステレオ顕微鏡下で軸索を可視化するためにE6.5まで行うことができます。 </li><li>膜及び血管周囲から分離によって注意深く胚を解剖。 PBSを含むコーティングされたペトリ皿-小さなシリコーンで胚を置きます。背側に面しタングステンピンと皿に胚を取り付けます。後脳鋭いガラスキャピラリーを用いた屋根板に背スリットを実行します。 </li><li>鋭いピンセットとマイクロはさみを使用すると、頭の上部を保持し、吻側から尾側に非常に慎重に後脳組織を引っ張る。後脳を分離した後、クリーン準備に達するために、残りの腹側組織を削除します。 </li><li> 4と後脳をインキュベート％パラホルムアルデヒド（PFA）室温（RT）で1〜2時間のためのソリューション。 PFAを削除するには、PBT（PBS/0.1％トリトンX-100）と後脳を洗ってください。 </li><li>フラットマウントhindbrainsは4でON 1 回目の抗体°PBTとで洗浄（15分×3）でRTインキュベートで2時間PBTとインキュベートでブロッキング溶液を500μl（ヤギ血清の5％を追加します。インキュベートで免疫用室温で2時間で2 回目の抗体。PBT（15分×3）で洗うこと。 </li><li>スライドガラス上に背側に面し後脳を置きます。後脳の上に蛍光灯取り付けメディアを追加します。スライド上に後脳を平らにガラスキャピラリーやタングステン針を使用してください。 </li><li>カバースリップによる組織の圧搾を防止するために、各角部にグリースを少量加える。後脳の上にカバースリップを慎重に配置し、気泡を避ける。遵守のためのガラスのエッジをカバーするためにマニキュアを追加します。 </li></ol> 2.2クライオセクションと免疫<ol><li>脳幹CAのクライオセクションnは軸索軌道を可視化する任意の段階で行われる。しかし、E6.5後これはあまりにも厚くなり、フラットマウント胚で軸索を可視化することが困難であるため好ましい方法でなければなりません。また、シナプスのデモは、組織と顕微鏡下で高倍率ビューの切片が必要です。 </li><li>胚を解剖し、すべての周囲の組織を削除します。 PBSですすいでください。マイクロはさみと鋭いピンセットを使用して延髄の尾部分に胚をカットします。小脳と中脳の間に広い切開を行い、全体の脳幹を削除します。組織が髄質、橋と小脳を含むべきである。 </li><li> PBS（10分×3）ですすぎ、4℃で一晩4％PFA（ON）°Cで脳幹を修正し、ON 4℃で沈没するまで、30％ショ糖でインキュベート。 </li><li> 10月（最適切削温度）化合物と低温型で後脳を埋め込 ​​むと、他の場所で11説明されているように、凍結まで-20°Cでそれを配置します。 </li><li&gt;希望の断面軸でクライオスタットでのOCTブロックを置きます。セクション幅は12μmである。 </li><li>スライドを乾燥させます。室温で2時間、各スライドに100μlのブロッキング溶液を追加。抗体100μlの（所望の濃度にブロッキング溶液で希釈した）を追加します。 1回目と2 回目の抗体についての潜伏期間は4でONになっているそれぞれRT℃でまたは2時間、。それぞれのインキュベーション時間は、穏やかに乾燥を防ぐために、スライドの上にパラフィルムストリップを追加します。抗体間のPBS（10分×3）で洗浄します。必要に応じて、RTで20分間PBSで希釈1:1000 DAPI（4&#39;-6 - ジアミジノ-2 - フェニルインドール）を加える。 PBSですすいでください。 </li><li>蛍光灯取り付けメディアとスライドをマウントします。分析の前に1時間乾燥させます。 </li></ol>

<ol>
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<p class="jove_content"関心の&gt;いいえ衝突は宣言されていない。</p

OVOエレクトロポレーションでひよこ神経系の発達の間に20セルの仕様と軸索ガイダンスを検査するために、実現可能な信頼性の高い、効果的なツールです。このプロトコルでは、特定のニューロンの条件付きラベルを有効にエンハンサーエレメントを使用してE2.75でニワトリ後脳におけるエレクトロポレーションのモードを説明します。この戦略は、胚発生の高度な段階...

後脳は、神経回路網を昇順と降順を介して、中枢および末梢神経系の間で通信することにより、神経系の主要な中継ハブを表しています。それは呼吸、意識、聴覚、およびモータ協調1-3を含む基本的な機能を調節する。初期胚発生時には、脊椎動物の後脳を一過異なる神経細胞型が形成され、複数の脳幹核センター4が生成されている反復的な菱へとその前後（AP）軸に沿って分割されます。後脳はまた、個別の神経前駆細胞は、指定されたとなり、明確なDVの場所3,5,6で分化れる基底とエイラープレートにその背腹（DV）軸に沿って分割されています。どのように早期のAPとDV固有ニューロンのパターンが機能脳幹回路網の確立を支配していることはあまり知られていない。 この基本的な知識を得るために質問、ツールは早い後脳の神経細胞の特定のサブセットにラベルを付けると、より高度な段階でそれらの軸索の軌道と接続性をトレースするために必要です。我々は以前、特定のエンハンサーエレメント、および早期のニワトリ胚7-9に背側脊髄介在ニューロンの軸索軌道を追跡するためのCre /のLoxPベースの条件式のシステムを利用してきた。現在の原稿では、後脳を対象とし、修正されたエレクトロポレーション戦略とpiggyBacを使用して、後期胚後脳ニューロン、軸索とそのシナプスの標的を標識するための実験的なパラダイムをアップグレードした - 媒介DNAの転置を。私たちの新しい戦略は、エレクトロポレーション後2までの12日から、後脳の片側に異なる神経細胞サブタイプのタグ付け、様々な胚の段階でそれらの軸索投射やシナプスのサイトのトラッキングを可能にします。この方法に基づき、我々は後脳ニューロン（dA1/Atoh1 +の背ほとんどサブグループのラベルが付いた</sアップ&gt;細胞）および2対昇順軸索投射パターンを明らかにし、それぞれが異なる索内の別のAPの位置と伸長に由来しています。 DA1軸索は、プロジェクトと聴覚核、中脳および小脳10の複数の層のフォームシナプスに発見された。 ひよこのエレクトロポレーションの組み合わせは、遺伝的にはニューロンと開発の多くの先進的な段階で投影サイトの分析のトレース、脳内の神経ネットワークの形成を研究するために、回路形成を支配する分子メカニズムを解明するユニークなプラットフォームを提供します。

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		<tbody>
		 <tr>
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electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo

ニワトリ胚の神経管のエレクトロポレーションは、例えば、神経細胞への外来遺伝子の発現のための迅速かつ効率的なものとして多くの利点を有する。本稿で我々は、特に神経前駆細胞のサブセットにラベルを付けるために、E2.75で鳥類の後脳にDNAをエレクトロする方法を一意に示し方法、どのように開発のより高度な段階で、その軸索投射やシナプスの目標に従うことを提供しますまでE14.5まで。ベースのプラスミドおよび後脳細胞（ニューロン、DA1の背最もサブグループ）のサブタイプでGFPの発現を駆動するpiggyBacを媒介DNA転位システム - 私たちは、特定のエンハンサーエレメント、Creを/ロックス含む小説遺伝のツールを利用してきた。 DA1軸索の軸索軌道と目標は、さまざまな脳幹領域に初期および後期胚の段階で続いている。この戦略は、胚後脳における目的の細胞を標的とするためとTRAのための高度な技術に貢献開発の複数の段階での回路形成をcing。

このプロトコルは、最近ニワトリ後脳10におけるニューロンのDA1サブグループの軸索パターンと投影サイトを発見するために使用された。具体的には、これらの軸索を標識するために、予め脊髄ニューロン8,12,13 DI1に特異として特徴付けられているエンハンサーエレメント（ATOH1）は、後脳DA1セル10で発現することが確認された。要素は、Creリコンビナーゼとプラスミ?...

Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

発達神経生物学の根本的な問題はどのように神経細胞のネットワークが胚の脳の中で確立されています。ここでは、そのようなのCre / LOX-プラスミドおよび背側介在ニューロンにラベルを付け、様々な発達段階でそれらの軸索投射やシナプスの標的を追跡する鳥類の後脳におけるpiggyBacを媒介DNA転位システムなどの小説の遺伝的なツール、とエレクトロポレーション技術を組み合わせた。

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

electroporation-hindbrain-to-trace-axonal-trajectories-synaptic

Research

JoVE Journal

Neuroscience

10.8K ビュー.  The Hebrew University of Jerusalem. 発達神経生物学の根本的な問題はどのように神経細胞のネットワークが胚の脳の中で確立されています。ここでは、そのようなのCre / LOX-プラスミドおよび背側介在ニューロンにラベルを付け、様々な発達段階でそれらの軸索投射やシナプスの標的を追跡する鳥類の後脳におけるpiggyBacを媒介DNA転位システムなどの小説の遺伝的なツール、とエレクトロポ?...

ビデオ: Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

ゼブラフィッシュ後脳における標識およびイメージング細胞

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

神経科学

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

マウス神経堤幹細胞の培養のためのニワトリ胚抽出物の製造

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

細胞培養およびひよこ胚における軸索誘導受容体のダイナミクス評価におけるpHluorinの使用

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

モデルとしての嗅覚系は、軸索の成長パターンと形態を研究する<em&gt;インビボ</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同時2光子<em&gt;インビボ</emマウス脳梁膨大後部皮質におけるシナプス入力とシナプス後ターゲットの&gt;イメージング

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

胎児アルコール スペクトルの無秩序のモデルラットにおける海馬機能障害を評価するためにトレース瞬目反射条件づけの使用

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

少量循環、血流 - と水没型チャンバー システムで管理急性海馬スライスにおけるシナプス可塑性を記録

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

さまざまな地域と発展途上と大人の脳のラミナを明らかにするシナプス亜鉛化学の使用

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

オプトジェネティクスを組み合わせてそのまま神経回路内でのシナプス前機能に関する遺伝子ノックダウンの効果を特徴付ける人工マイクロ Rna

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...