この仕事のための資金は、R.リムとAJキャンプにガーネットパッシーとロドニーウィリアムズ記念財団プロジェクトの助成金によって提供されていました。

1。動物<ol><li>マウスは、オーストラリアの齧歯類センター（ARC、パース、オーストラリア）から入手し、環境エンリッチメントとノーマルの12時間の明/暗サイクルでシドニーボッシュ動物施設の大学で開催された。記載されたすべての実験は、シドニーの動物倫理委員会の大学によって承認された。 </li><li>この菌株は一般に遺伝子操作の背景として使用され、 野生型 8-9と同等とみなすことができるので、雄と雌のマウス（C57/BL6）は、すべての実験に使用した。 </li></ol> 2。組織標本<ol><li> 26のNaHCO 3、11グルコース、250グリセロール、2.5のKCl、1.2のNaH 2 PO 4、1.2のMgCl 2、及び2.5のCaCl 2から成るグリセロールベースの人工脳脊髄液（ACSF）（単位：mm）300mlのを準備します。 CaCl 2を添加する前に、ガスカーボゲン（95％O 2および5％CO 2）を含む溶液は、APを確立する7.4のHとカルシウム沈殿（曇り）を回避。氷スラリーが形成されるように、45分間-80℃の冷凍庫内の溶液を凍結。 </li><li>深く腹腔内注射を介して、ケタミン（100 mg / kg）を（パーネル、アレクサンドリア、オーストラリア）をマウスにanaesthetize。 </li><li>一度後肢ピンチ反射が鋭いステンレス製のハサミを使用して不在首マウスであり、頭蓋骨を露出するためにカミソリの刃を（＃22を四捨五入）を使用して、矢状皮膚切開を行います。この時点で、2.3から2.8までと手順を通して頭蓋、脳、および基礎となる前庭装置は、組織上の氷冷ACSFの定期的なアプリケーションで、できるだけクールに保つ必要があります。 </li><li>標準パターンのはさみの尖った腕（FST、ノースバンクーバー、カナダ）を使用するとラムダで頭蓋骨に小さな切開をし、矢状縫合に沿ってカット。脳は、このステップの間にせん断刃で &quot;ドラッグ&quot;されていないことを確認してください。 </li><li>慎重に横方向に離れて頭頂骨と後頭骨posterioピールRLY浅い屈曲ピアソン骨鉗子（FST）を用いて。 </li><li>小さなステンレス鋼へらがゆっくり真ん中と後頭蓋窩の表面から脳を持ち上げるために使用され、公開されるvestibulocochlea神経（CNVIII）内耳と細かい虹彩はさみのペアで脳幹の中間をカット。この神経を切断すると、有毛細胞を使用して、プライマリ求心性とその接続の軸索上の過度の緊張を最小限に抑えることができます。 </li><li> CN VIIIの離断の後脳トトで除去される。 </li><li>前庭迷路蝸牛は前内側方向に指して、今では中頭蓋窩にはっきりと見える。そっと前方半規管を把持し、横方向に引っ張って切除する前に前庭迷路の両側に骨鉗子。 </li><li> 2.1節で説明した氷のように冷たい、連続毒ガスACSFを含む解剖皿に切除迷路（ 図1）を浸します。実体顕微鏡下に迷路を保持蝸牛を把持によって料理のベース。前半規管の膨大部を覆う骨で離れてスクラッチする細かい鉗子を使用してください。骨に小さな穴が達成されたら離れて膨大部から骨をフリックし始める。注意これは、基礎となる膜迷路と感覚上皮への損傷を引き起こす可能性がありますように骨を通して鉗子をプッシュしないように注意してください。前方および隣接水平半円膜ダクトと膨大部の両方が（ 図2）が露出されるまでこの手順を続けます。 </li></ol><img alt="図1" src="/files/ftp_upload/50471/50471fig1.jpg" /> 図1。隔離されたマウス前庭迷路。A.左パネル）孤立マウス前庭迷路の概略図。前庭感覚上皮にアクセスするための基準の重要なポイントは、蝸牛、前方、および水平半規管は、ラボであるELED。アスタリスクは、前庭感覚上皮を含む半規管の膨大部を示している。B.右パネル）1ヶ月齢のマウスから単離された前庭迷路の顕微鏡写真。 <img alt="図2" src="/files/ftp_upload/50471/50471fig2.jpg" /> 図2。膜性前庭迷路の露出。前方と水平半規管の膨大部を覆う骨はブラック/ブラウンの斑点の膜膨大部と関連する膨大部神経（CNVIII）を明らかにするために離れて傷れています。下のパネルの回路図は、顕微鏡写真のハイライト領域内の構造を表しており、膨大部とCNVIIIに半規管ダクトの関係を示しています。 <ol start="10"><li>細かい鉗子を使用して、静かに確実に、骨迷路から離れて膨大部および関連卵形嚢を持ち上げその膨大部の中心領域（コンタ）感覚上皮をiningが破損していない。いくつかのケースでは半円形膜管の近位部は、骨から膨大部を解放するためにアイリスハサミでカットする必要があるかもしれません。 </li><li>リーボビッツのL-15培地（Sigma-Aldrich社、セントルイス、MO）で満たされたペトリ皿に2膨大部と卵形嚢を含むトライアドを転送します。 &quot;バックライト&quot;組織への光ファイバを使用してください。これは、膨大部内感覚上皮を含む稜の明確な可視化を可能にします。 </li><li>注意深く細かいアイリスはさみで卵形嚢の斑点 &quot;屋根&quot;で切開を行います。前方の屋根、その後水平膨大部を通じて、この切開を続ける。それ（ 図3A）に接触することなく、できるだけ感覚上皮の端の近くに切開を加えます。感覚上皮（ 図3B）を覆う膜のない部分が存在しないことを確認してください</li><li>小さなGLASに孤立半無傷準備を転送L-15メディアでいっぱいの底記録室。平坦化されたU字状の白金線（ 図3B）に固定された微細なナイロン繊維のグリッドを使用して調製を圧迫。理想的には、グリッドの繊維は感覚上皮を覆うべきではありません、しかし、より多くの安定性が必要とされるいくつかのケースでは、多分好ま感覚上皮を横断する単繊維。 </li></ol><img alt="図3" src="/files/ftp_upload/50471/50471fig3.jpg" /> 図3。半無傷準備と電極構成の前庭感覚上皮。A.回路図表現の孤立半無傷準備 。稜を覆う膨大部は、感覚上皮（緑）の表面を露出するために&quot;デ屋根&quot;となっています。B.前方を示す半無傷&#39;トライアド&#39;準備の顕微鏡写真（AC）と水平（HC）稜（卵形嚢こと隠さ後肢AC）。記録室のベースに準備を保護するために使用されるナイロン繊維に注意してください。スケールバー：100μmのC.個々の前庭有毛細胞上に位置する記録電極。スケールバー：15μmで。 3。電気生理学<ol><li>継続的に酸素L-15培地で半無傷準備を灌流。メディアは録音を通してモニターされるべきである培地のpH指示薬（フェノールレッド）と色が含まれています。酸素とpHは7.3から7.4であると赤の色に対応する必要があります。 </li><li>達成するために、マイクロピペットプラー（ナリシゲ、日本、モデルPP-830）上：;：ツーステップ·プロトコル（50および熱ステップ2 72ヒートステップ1）を用いて1.5ミリメートル（1.19ミリメートルのID）ホウケイ酸ガラスからの記録ピペットを準備3-4MΩの最後のインピーダンス。 </li><li>含まれているカリウムフッ化物系内部溶液（単位：mm）110 KF、12のKCl、27 KOH、1のNaCl、10 HEPES、10 EGTA、1.8のMgCl 2、3-4 mmにピペットのシャンクを埋める3 D-グルコース、および2のNa-ATP; KOHでpH 7.4。 </li><li>ピペット2-3回のシャンクをラップし、限りダウン電極を絶縁し、ピペット容量を低減するためにパラフィルムの薄いストリップできるだけ先端まで。 </li><li>正立顕微鏡（オリンパスBX51）で低消費電力の倍率（5倍）の下で感覚上皮の上にピペットを置きます。ハイパワー（40X）に切り替えて、付属のCCDカメラで、個々の前庭有毛細胞を可視化する。 </li><li>マニピュレーター（サッター·インスツルメンツ、カリフォルニア、USA）を用いて可視化有毛細胞（ 図3C）の膜上の位置ピペット。ギガオームシールが達成されると、破断ピペットホルダに吸引口を介して印加される負圧少量の細胞膜。 </li><li>標準的な技術を8-9を用いて全細胞電圧クランプ記録を行います。 </li></ol> 4。二光子顕微鏡<ol><li> 5mMのオレゴングリーンを含む0.9％生理食塩水を準備488 BAPTA-1（OGB-1;六カリウム塩、Invitrogen社、ドイツ）。 </li><li>感覚上皮が上から障害物がないことを保証する;まだ水没しながら、ろ紙の小片（ミリポア、ドイツ4X4ミリ、厚0.8μm）の上に &quot;デ屋根の&quot;半無傷トライアドの準備を配置。 </li><li>パルス発生器と広帯域増幅器の組み合わせからなるエレクトロの生理食塩水覆われ、ベース白金電極（7μl）を上にろ紙および準備を転送し、合成カルシウムの5 mMの溶液をさらに5μlのカバーインジケータ染料オレゴングリーン488 BAPTA-1（ 図4）。 </li></ol><img alt="図4" src="/files/ftp_upload/50471/50471fig4.jpg" /> 図4。バルクエレクトロ。A.エレクトロポレーション構成の断面を模式的に示す。半無傷の調製は、（*）カルシウムdyの中に浸漬濾紙上に配置される電子（オレゴングリーン488 BAPTA-1）と電流が2白金電極間に印加する。 Briggmanとオイラー、2011年10から適応。 <ol start="4"><li>オレゴングリーン488 BAPTA-1溶液が行われるとの接触時の準備の向きを妨害しないように注意して、約2mmの距離でベース電極と上部白金電極を平行に持参。 </li><li> 10-11;準備（+13 V、10ミリ秒のパルス幅は、1 Hzの周波数、10方形波パルス電流のパラメータ）を越え短い電流パルスを渡します。 </li><li> L-15培地で満たしたガラス底の記録室の底に、再び感覚上皮は上から障害物がないことを確実に、その上にシリコンオイルと位置の小軽く半無傷準備を配置。 （ポイント2.13を参照）上記のように画像全体の安定性を確保するために、準備上のナイロングリッドを交換してください。 </li><li>自発の光学録音を行う二光子顕微鏡を用いて標準撮像プロトコール10-11を使って感覚上皮（ 図7）でジニアスカルシウム活動。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

バランス感覚のメカニズムは、視覚と聴覚のシステムを、たとえば 、他の感覚系と比較して限定され注目を集めている。前庭やバランス機能の変化を調べた研究のうち、ほとんどはバランス前庭有毛細胞自体の基本的なビルディングブロックの不完全な知識で、前庭動眼反射を含む行動の対策に焦点を当てている。彼らのネイティブ環境から削除急性孤立有毛細胞を用いたので、有毛...

私たちの日常生活への前庭系の重要な貢献にもかかわらず、加齢に伴う前庭機能の観察された減少の責任のプロセスを明確に理解することは、とらえどころのないまま。知識の欠如のための一つの理由は、その前庭機能の低下がほぼ独占的に前庭動眼反射（VOR）、外因性前庭機能の正確な指標を含む行動のアッセイを使用して検討されているですが、本質的な構成要素の変更に限定された洞察力を提供しています。これは、健康、病気、あるいは老化に前庭有毛細胞機能の理解への大きな障害である。 個々の前庭有毛細胞の多くの研究が行われてきたが、主要な欠点​​は、有毛細胞、さらには萼求心端子は、機械的および/または酵素的処理により、通常の環境から除去される急性有毛細胞調製物、に依存している。このようなアプローチinevitaブライ有毛細胞や萼、及び有毛細胞と支持細胞との間の微妙なマイクロアーキテクチャを混乱させる。半無傷の準備1-5、及び孤立マウス迷路の準備6の発展に伴い、より密接に、生体内でそれらの似ている条件の下でシナプスのコミュニケーションの様々な形態を勉強する機会が用意されました。実際、林ら （2011）I前庭有毛細胞は神経上皮内に埋め込 ​​まれて残ったものに比べて急性孤立型から記録された全細胞電流の著しい差が認められた。具体的には、カリウム有毛細胞と求心萼間の間隙内に蓄積すると考えられ、著しく有毛細胞応答7を変化させる。この種の情報は、ここで説明した前庭感覚上皮の半無傷準備なし得ることは不可能であろう。我々はマウス稜3の半無傷準備を実証および全細胞パッチ電気生理学から得られた代表的な結果、2光子カルシウムイメージングを示す。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Leibovitz medium L-15</td>
 <td>Sigma Aldrich</td>
 <td>L4386-10X1L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAPTA-1-oregon green</td>
 <td>Invitrogen</td>
 <td>O6806 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT </td>
 </tr>
 <tr>
 <td>Stereo microscope</td>
 <td>Leica Microsystems</td>
 <td>A60S</td>
 <td></td>
 </tr>
 <tr>
 <td>Upright microscope</td>
 <td>Olympus</td>
 <td>BX51WI</td>
 <td></td>
 </tr>
 <tr>
 <td>Two-photon microscope</td>
 <td>Olympus/La Vision</td>
 <td>BX51WI/ TriMScope II</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont #5 SF Forceps</td>
 <td>FST</td>
 <td>11252-00</td>
 <td></td>
 </tr>
 <tr>
 <td>Friedman-Pearson Rongeurs</td>
 <td>FST</td>
 <td>16221-14</td>
 <td></td>
 </tr>
 <tr>
 <td>Standard Pattern Scissors</td>
 <td>FST</td>
 <td>14001-12</td>
 <td></td>
 </tr>
 <tr>
 <td>InstraTECH A-D converter</td>
 <td>HEKA</td>
 <td>ITC-18</td>
 <td></td>
 </tr>
 <tr>
 <td>Sutter Micromanipulator</td>
 <td>Sutter</td>
 <td>MP-225/M</td>
 <td></td>
 </tr>
 <tr>
 <td>multiclamp amplifier</td>
 <td>Axon Instruments</td>
 <td>700B</td>
 <td></td>
 </tr>
 <tr>
 <td>Data acquisition software (electrophysiology)</td>
 <td>Axograph</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Imspector Data acquisition software (two-photon)</td>
 <td>Max Planck innovation</td>
 <td>N/A</td>
 <td></td>
 </tr>
 </tbody>
</table>

an isolated semi-intact preparation of the mouse vestibular sensory epithelium for electrophysiology and high-resolution two-photon microscopy

理解前庭有毛細胞は、正常な条件の下で機能する、またはどのように外傷、疾患、およびこの機能を混乱させる老化、予防的アプローチ、および/または新たな治療戦略の開発に重要なステップです。しかし、異常な前庭機能を見て研究の大半は、細胞レベルではなかったが、このような歩行分析と前庭動眼反射性能として前庭機能障害の行動アッセイに主に焦点を当てた。この作品は物事がうまくいかないときに何が起こるかについての貴重なデータが得られているが、少し情報が障害の根本的な原因に関して収集されます。前庭機能の基礎となる細胞および細胞内のプロセスに焦点を当てている研究のうち、ほとんどは彼らのシナプス接続と支持細胞環境を欠い急性単離し有毛細胞、に頼ってきた。したがって、主要な技術的課題は、準備中で絶妙に敏感前庭有毛細胞にアクセスしている少なくとも生理的に、中断されaration。ここでは、有毛細胞/一次求心錯体を含む地元の微小環境を保持マウス前庭感覚上皮の半無傷の準備を示しています。

前庭有毛細胞の電気生理学的特性は、それらが07埋め込 ​​まれている内に複雑なマイクロアーキテクチャに依存している。 図半無傷の前庭上皮製剤は、タイプI有毛細胞（ 図5A）、タイプIIの有毛細胞を区別するために使用することができることを図5 （ 図5B）、そして特徴的な全細胞コンダクタンスに基づい萼一次求心（ 図...

Watch this Scientific Journal Video about An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy at JoVE.com

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

前庭有毛細胞機能の解析は頭蓋骨の最も困難な部分の奥深くの位置、側頭骨錐体によって複雑になる。最も機能有毛細胞研究は、急性単離された有毛細胞を使用している。ここでは、電気生理学的および二光子顕微鏡研究のためのマウス前庭上皮の半無傷の準備について説明します。

電気生理学および高分解能二光子顕微鏡用マウス前庭感覚上皮の切り抜き半無傷準備

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

an-isolated-semi-intact-preparation-mouse-vestibular-sensory

Research

JoVE Journal

Neuroscience

11.8K ビュー.  University of Sydney. 前庭有毛細胞機能の解析は頭蓋骨の最も困難な部分の奥深くの位置、側頭骨錐体によって複雑になる。最も機能有毛細胞研究は、急性単離された有毛細胞を使用している。ここでは、電気生理学的および二光子顕微鏡研究のためのマウス前庭上皮の半無傷の準備について説明します。

ビデオ: An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

免疫電子顕微鏡のためのマウスの脳組織の準備

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

神経科学

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

二光子顕微鏡を用いたマウス脊髄の in vivoイメージングで

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

スキンと四肢骨の神経支配マウス感覚神経線維の組織の調製と免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

不定冠詞<em&gt;インビトロ</em成体マウスの筋層間神経叢から分離腸ニューロンとグリアの&gt;準備

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

皮膚における単一神経終末のin vivo二光子顕微鏡で

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

二光子<em&gt; in vivoで</em&gt;間引き頭蓋骨の準備を使用して、マウス皮質における樹状突起棘のイメージング

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

マウス脊髄における細胞ダイナミクスの二光子イメージング

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

HIV誘導神経炎症のマウスモデルでは二光子顕微鏡を用いた脳血管のアーキテクチャの定量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.