This work was supported by a Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (T.I.) and Grants-in-Aid for Scientific Research (C) from MEXT (S.T. and K.N).

プロトコルおよび実験動物の使用は、山梨と早稲田大学大学の制度的動物のケアと使用委員会によって承認されました。動物のケアは、機関のガイドラインに従って行いました。 1. CPEC準備<ol><li>次の装置と材料を準備します。実体顕微鏡下から照明を送信できることが好ましいです。時計屋のピンセット（デュモン＃3または＃4）、火炎滅菌し、ストレート操作はさみ、火炎滅菌。 20ミリリットルの氷冷リーボビッツL-15培地を含む2の滅菌10cmのプラスチック皿; 2ミリリットルRTリーボビッツL-15培地を含む35mmのガラスボトムディッシュ。 70％エタノールを含有する100mLのビーカー。新生児仔マウス。 </li><li>簡単に言えば、70％エタノールで新生児マウスを浸すとオペレーティングはさみを使用して断頭により迅速に安楽死させます。 </li><li>滅菌10cmのディで氷冷リーボビッツL-15培地中ですぐに頭を置きSH。 </li><li>脳を露出するために頭蓋骨を開いてカットし、時計屋のピンセットを使用して、頭蓋冠から肌を削除し、全脳を分離するために脳神経をカット。 </li><li>氷冷リーボビッツL-15培地を含む新しい皿に脳を転送し、脳が完全に媒体に浸漬されることを確認して、実体顕微鏡下で観察します。 </li><li>上向きに脳の背側面を設定し、嗅球（右利きの人のために）3時の位置に存在するように脳を向けます。左手にピンセットで軽く脳を持ちます。 </li><li>細かい解剖鉗子（デュモン＃3または＃4）右手にを使用して、大脳の縦裂に沿って、大脳半球をつなぐ脳梁と実質の下をカット。 </li><li>静かに側面への大脳半球を押して横断脳亀裂を公開します。 </li><li>目をつまんで半球を分離半球と視床間の電子の実質。 </li><li>ゆっくりラミナaffixaによって海馬の側面に取り付けられている横心室脈絡叢を引き出します。 </li><li>新鮮リーボビッツL-15培地を含む35mmのガラスボトムディッシュに分離された脈絡叢を移し、重量（材料リスト）静かな場所に組織を保持するためにオーバーレイ。 </li></ol> CPEC繊毛の2ライブイメージング<ol><li>適切な紫外線（UV）と推論（IR）を確認して400nmから700nmのより長いより短い光を遮断するフィルタを切断し、その中性濃度（ND）フィルタ（25％および6％）を光路に挿入されています倒立顕微鏡の。 </li><li>おおよその目で、対物レンズの焦点を調整し、中心となるよう冷却器を調整し、ケーラー照明に適合するように焦点を当てています。李に適切な微分干渉コントラスト（DIC）プリズム、DICの要素だけでなく、分析器と偏光子の要素を挿入しますDIC光学系に適合するGHTパス。 </li><li>組織表面の構造が最も認識可能であるように、DICプリズム位置によってビューのコントラストを調整します。標的細胞の全ての繊毛運動がある場合は、運動性繊毛のクリアな視界は、その動きを目では得られません。 </li><li>ビデオカメラに光路を変更し、光パワーを増大させるためにNDフィルタを削除します。 </li><li>ビューとフォーカスのフィールドを調整するフォーカスモードでカメラを使用してください。ビデオ画像をモニタにリアルタイムで表示される、フォーカスの際、運動性繊毛のクリアな視界は使用できません。 </li><li>所望の期間（数秒から数分）、0.1ミリ秒の露光時間で200 Hzでカメラを使用してください。画像スタックを取得した後、単一のフレームが明確な繊毛構造が表示されます。繊毛エッジがぼやけている場合は、フレームレートを増加させるか、より短い露光時間を使用します。 </li><li>リーボビッツに安楽死の後、25〜60分以内にCPECの繊毛の動きを記録L-15培地。 </li></ol>毛様体運動の3分析<ol><li>手動でコンピュータのモニタ上の各繊毛のパターンを破って追跡します。各繊毛の軌跡については、組み立てられて、マウスポインタ、各フレーム内のマーク繊毛先端位置。いずれかのさらなる分析のために他のより一般的なアプリケーションに同じソフトウェアまたはエクスポートを使用して軌跡情報を分析。この分析工程の効率を議論に記載されています。 </li><li>目で、前後または回転、運動の二つのモードに軌道を分類します。 </li><li>以下の式を使用して繊毛拍動周波数（CBF）を計算する：[CBF =（秒あたりのフレーム数）/（シングルビート用フレームの平均数）]繊毛先端動作図から得ることができる14（ 図3 ）。同じセル上の他の繊毛は、各ciliuの動きを妨げる可能性があるため、複数の繊毛運動サイクルのため、この計算を繰り返しますM、凹凸が生じます。 </li><li>単一セル内の軸を破って、毛様体の角度均一性を分析するために、各軌道のための鼓動角度θ（ 図4）を定義します。前後の軌道は、直線に繊毛の先端の位置に合わせて、ラインがx軸となす角度としてθを定義します。回転軌跡は、楕円に位置に合わせて、楕円の長軸がx軸となす角度としてθを定義します。フィッティングの詳細は代表的な結果のセクションに記載されています。 </li><li>各軌道の定量的な説明については、一般的なアスペクト比ARを計算します。簡単に言うと、によって軌道を回転させる- θとXに沿っ分布の幅の比率としてARを定義-とy -axes（ 図4B）。詳細は代表的な結果のセクションに示した、と解釈されています、重要性、およびパラメータの制限が議論に記載されています。 </li></ol> SEM 4.サンプル調製注：SEMは総合的にCPECsに繊毛の状態を評価するための重要な方法です。 SEM用の試料を調製するために、標準的な手順は、以前に15がわずかな修正で採用されていると報告しました。 <ol><li>脳から組織を切開する前に、ポリエチレンキャップを5mLのガラスバイアル中で固定液を準備します。固定液を2％パラホルムアルデヒド、0.1Mリン酸緩衝液、pH 7.4中の2.5％グルタルアルデヒド（半Karnovskyの溶液16）からなります。 </li><li>ステップ1で説明したように、脳からの組織を解剖。 </li><li>簡単に言えば、新しい皿中ハンクス平衡塩溶液（HBSS）で単離された組織をリンスした後、室温で1時間ガラスバイアルに固定液で組織を固定します。使い捨て転送ピペットを使用し、試験片GEを扱いますntly。 HBSSで洗浄した後、組織がべたつきます。 <ol><li>固定液に検体を転送するには、ゆっくりと滴としてトランスファーピペットからの組織を含む少量の溶液を排出し、固定液に加えます。 </li></ol></li><li>固定後、固定液を廃棄し、リン酸緩衝液で3回組織をすすぎます。 </li><li>残りのアルデヒド類を洗浄するために、10％ショ糖溶液の組織を浸します。アルデヒドの完全な除去を確実にするために、新鮮な10％​​のスクロースを二回繰り返した後、10分間、溶液中に試料を浸漬し、。このステップは、後の工程で、適切後固定を達成することが重要です。 </li><li> 30分間、リン酸緩衝液中の1％四酸化オスミウムの溶液に組織を浸漬した後、ポスト固定氷上に置きます。サンプル色によってオスミウム酸染色法の程度を判断する：アルデヒドが完全に除去されている場合、サンプルは黒です。 </li><li>ダブルdistilleで広範囲に後固定した組織試料を洗浄Dの水で数回。 </li><li>エタノールの濃度を段階的に浸漬することによって試料を脱水、通常、65％、75％、85％、95％、99％、100％、10分それぞれについて。新しく購入したボトルから99.5％エタノールにモレキュラーシーブを配置することによって無水エタノールを得ます。無水エタノールで3回脱水を繰り返します。 </li><li> 10分間、酢酸イソアミル、臨界点乾燥の置換試薬に脱水サンプルを置きます。二回、この手順を繰り返します。この試薬は迅速に蒸発し、試料が表面張力によって破壊をもたらす、ドライになることができます。したがって、試料を完全に乾燥させません。 </li><li>酢酸イソアミルの最終交換の後、直ちにアルミホイルで開放ガラスバイアルをラップし、ドライアイス上にバイアルを置き、大部分の溶媒を除去します。液体二酸化炭素は、臨界点乾燥機にバイアル内に容易に流れるように、針や細かい鉗子を使用して、バイアルの口を覆うホイルにいくつかの穴を作ります。次のステップに進んでください可能な限り迅速に。 </li><li>この工程では、乾燥機のチャンバー内に酢酸イソアミルのキャリーオーバーを最小限に抑えるが、サンプルが完全に臨界点乾燥の前に乾燥させてください。また、バイアルに霜の形成を回避するために、不必要に長い時間のために、ドライアイス上のサンプルを放置しないでください。 </li><li>組織に含まれる水分を除去しながら、組織の表面構造は無傷のまま確実に臨界点乾燥機に組織サンプルを含むアルミホイルにくるまれたガラスバイアルを転送します。臨界点乾燥機を動作に関する詳細情報は、製造業者の説明書から得ることができます。 </li><li>慎重に機械的損傷を最小限にするためにつまようじを使用してサンプルを処理します。得られた乾燥組織サンプルが壊れやすいです。イオンスパッタを用いて、金 - パラジウムの金属スタブとコート上のサンプルをマウントします。 </li></ol> SEMによる観察5. <ol><li> SEMで観察し、デジタルカメラで画像を記録走査型電子顕微鏡に搭載しました。 </li><li>分析のためにPCにデジタル画像データを転送します。 </li></ol>

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著者は、彼らが競合する金融利害関係がないことを宣言します。本論文では、単離された脈絡叢の組織で繊毛の運動性を観察するための詳細な方法論を報告することを目的とします。科学的なノベルティは、以前の研究1,8で報告されています。

この方法の展望ここに記載された技術は、以前に公開された方法よりも繊毛のより詳細な分析を提供していませんが、この技術の重要性は、簡単に繊毛運動ex vivoでのあらゆる種類のスクリーニングに適用することができるシステムと費用対効果のシンプルさにあります。具体的には、TI Workbenchは、より簡単に繊毛運動を観察し、分析する研究者を可能にシンプル?...

Cilia are hair-like projections on the surface of most vertebrate cells, which have attracted attention by medical researchers because of a class of diseases termed ciliopathies2&#8211;4. Despite the ubiquitous expression of the organelle, a wide variety of ciliary functions have been reported, including motility and biosensing. For example, motile cilia on the mucoepithelial surface transport mucus5 and epithelial debris to the outlet of tracts, thereby preventing disease by clearing the surface of epithelia. Moreover, during early developmental periods and embryonic stages, cilia regulate the proliferation of stem cells6, and are involved in the determination of left&#8211;right asymmetry of the vertebrate body7. 
Choroid plexus epithelial cells (CPECs) are derivatives of neuroepithelial cells that cover the surface of the choroid plexus tissue in the brain, which play important roles in maintaining homeostasis of the intracranial environment by production of cerebrospinal fluid (CSF). It has been previously demonstrated that CPECs have multiple non-motile cilia that regulate the production of CSF through G-protein-coupled receptors that are specifically concentrated on the cilia8. Although these cilia had been regarded as quiescent non-motile cilia, it was discovered that some CPEC cilia exhibit transient motility during the neonatal period1. This finding was quite important because it revealed that so-called non-motile cilia are not necessarily immotile from the beginning of development and might display transient motility during specific time windows, possibly in response to specific physiological demands and functions9. To precisely describe the motile nature of CPEC cilia, it is necessary to develop an ex vivo observation system that encompasses analysis of the kinetic profiles unique to CPEC cilia. 
With respect to motility, although several technical reports have described observations of the motile cilia of the tracheal epithelium5,10, motile single-cell flagella11, so-called conventional motile cilia12, and nodal cilia13, detailed analytical methods applicable to relatively undulated structures such as the choroid plexus have not been well documented so far. Moreover, a high time resolution is required to analyze the ciliary movement of CPECs, in which expensive high-speed cameras are indispensable. To circumvent this necessity and simplify monitoring the ciliary motility of various cell types, a low cost, high-speed camera has been introduced, and an easily accessible and convenient method to record the motility of motile cilia, especially to describe the speed and pattern of motion of each cilium, has been developed1. Moreover, original image analysis software &#8220;TI Workbench&#8221; has been used here to facilitate detailed analysis of motility. Collectively, this method provides a new concise strategy to analyze ciliary motion together with correlative scanning electron microscopy (SEM), which can be adopted in a wide range of cilium research.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>for both live imaging and SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereo microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr>
			<td>flat paper towel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#934;10-cm plastic dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100-mL beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>straight operating scissors</td>
			<td>Sansyo</td>
			<td>S-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>watchmaker forceps</td>
			<td>Dumont</td>
			<td>No.DU-3 or -4, INOX</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass bottom dish</td>
			<td>Matsunami Glass</td>
			<td>D110300</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alminum foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-mL glass vial with a polyethylene cap</td>
			<td>Nichiden Rika-Glass</td>
			<td>PS-5A</td>
			<td></td>
		</tr>
		<tr>
			<td>transfer pipette</td>
			<td>Samco Scientific</td>
			<td>SM251-1S</td>
			<td>for specimen tranfer</td>
		</tr>
		<tr>
			<td>toothpick</td>
			<td></td>
			<td></td>
			<td>for specimen transfer</td>
		</tr>
		<tr>
			<td>ion sputter with gold-palladium</td>
			<td>Hitachi</td>
			<td>E-1030</td>
			<td></td>
		</tr>
		<tr>
			<td>critical point dryer</td>
			<td>Hitachi</td>
			<td>HCP-2</td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>100 W mercury lump housing and power supply</td>
			<td>Olympus</td>
			<td>U-ULH and BH2-RFL-T3</td>
			<td></td>
		</tr>
		<tr>
			<td>100 W mercury lamp</td>
			<td>Ushio</td>
			<td>USH103D</td>
			<td></td>
		</tr>
		<tr>
			<td>DIC condenser, n.a. 0.55</td>
			<td>Olympus</td>
			<td>IX-LWUCD</td>
			<td></td>
		</tr>
		<tr>
			<td>electrrical shutter</td>
			<td>Vincent Associates</td>
			<td>VS35S22M1R3-24 and VMM-D1</td>
			<td>manual shutter can be used.</td>
		</tr>
		<tr>
			<td>band-pass filter (400-700 nm, &#934;45 mm)</td>
			<td>Koshin Kagaku</td>
			<td>C10-110621-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ND filter (&#934;45 mm)</td>
			<td>Olympus</td>
			<td>45ND6, 45ND25</td>
			<td>combination of 25% and 6% ND filters are used</td>
		</tr>
		<tr>
			<td>objective lens (water immersion) with DIC element</td>
			<td>Olympus</td>
			<td>UApo 40XW/340, n.a., 1.15 with IX-DPAO40</td>
			<td></td>
		</tr>
		<tr>
			<td>high-speed video camera</td>
			<td>Allied Vision Technologies</td>
			<td>GE680</td>
			<td>&#62;= 200 Hz frame rate and 1 msec expose time</td>
		</tr>
		<tr>
			<td>image acquisition / analysis software</td>
			<td>in-hous software</td>
			<td>TI Workbench</td>
			<td>capable of acquisition at high frame rates.</td>
		</tr>
		<tr>
			<td>PC for camera control / analysis</td>
			<td>Apple</td>
			<td>Mac Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>vibration isolation table</td>
			<td>Meiritsu Seiki</td>
			<td>AD0806</td>
			<td></td>
		</tr>
		<tr>
			<td>weight for tissue</td>
			<td>Warner Instruments</td>
			<td>slice anchor kits</td>
			<td>It can be made with nylon mesh glued to a U-shape squashed &#934;0.5mm platinum wire.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>scanning electron microscope</td>
			<td>JEOL</td>
			<td>JSM-6510</td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol&#160;</td>
			<td>Wako Chemicals</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz L-15 medium</td>
			<td>Life Technologies</td>
			<td>11415-064</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol&#160;</td>
			<td>Wako Chemicals</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Life Technologies</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde&#160;</td>
			<td>Merck</td>
			<td>1040051000</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaraldehyde&#160;</td>
			<td>Nacalai tesque</td>
			<td>17003-05</td>
			<td></td>
		</tr>
		<tr>
			<td>isoamyl acetate</td>
			<td>Nacalai tesque</td>
			<td>02710-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Sieves 4A 1/8&#160;</td>
			<td>Wako Chemicals</td>
			<td>130-08655</td>
			<td>for preparation of anhydrous ethanol</td>
		</tr>
		<tr>
			<td>phosphate buffer saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer, 0.1 M</td>
			<td></td>
			<td></td>
			<td>To make 100 ml, mix 19.0 ml of 0.1 M NaH2PO4 and 81.0 ml of 0.1 M Na2HPO4&#160;</td>
		</tr>
		<tr>
			<td>monosodium phosphate (dihydrate)</td>
			<td>Nacalai tesque</td>
			<td>31718-15</td>
			<td></td>
		</tr>
		<tr>
			<td>disodium phosphate (anhydrous)</td>
			<td>Nacalai tesque</td>
			<td>31801-05</td>
			<td></td>
		</tr>
		<tr>
			<td>suclose</td>
			<td>Nacalai tesque</td>
			<td>30406-25</td>
			<td></td>
		</tr>
		<tr>
			<td>osmium tetroxide</td>
			<td>Nisshin EM</td>
			<td align="right">300</td>
			<td></td>
		</tr>
		<tr>
			<td>dry ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

observation of the ciliary movement of choroid plexus epithelial cells ex vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

ワークフローの概要は、装置の画像を含む、 図1に示されています。 CPECsのライブの動きの観察 映画1は、周産期のマウスから単離したCPECsの映画を示しており、 ムービー2ムービー1の画像の拡大図を示している。それは、個々の繊毛の先端は映画のものと比較して、静止画ではあまり明確であることに留意すべ?...

Watch this Scientific Journal Video about Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo at JoVE.com

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

脈絡叢上皮細胞の繊毛運動の観測<em&gt;エクスビボ</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

observation-ciliary-movement-choroid-plexus-epithelial-cells-ex

Research

JoVE Journal

Neuroscience

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

12.1K ビュー.  Waseda University. In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

ビデオ: Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

急性上皮単純ヘルペスウイルスI型感染の ex vivo器官角膜モデル

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

神経科学

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病ラットの熱侵害受容閾値の迅速定量

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

全体の開発、 ショウジョウバエの脳のex vivoで培養

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

ラットの脳から脳表面血管系および関連髄膜の解剖と脈絡叢のビジュアル解説

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

無傷の鋤鼻器官と副嗅球のex vivoでの準備

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;インビボ</em&gt;麻酔ラットにおけるHyperdirect経路からの局所場ポテンシャルとマルチユニット活性の電気生理学的記録

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

Ex Vivoセル固有カルシウム マウス ダイヤフラムの三者のシナプスでシグナル伝達のイメージング

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

インビボ マウス劣オリーブにおけるカルシウムイメージング

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents.

エクスビボ げっ歯類の三叉神経血管系からのカルシトニン遺伝子関連ペプチドの放出

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in ...

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

Ex Vivo神経生理学のためのラット坐骨神経の調製

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...

脈絡叢上皮細胞の繊毛運動の観測

要約

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この動画の章

急性上皮単純ヘルペスウイルスI型感染の ex vivo器官角膜モデル

糖尿病ラットの熱侵害受容閾値の迅速定量

全体の開発、ショウジョウバエの脳のex vivoで培養

ラットの脳から脳表面血管系および関連髄膜の解剖と脈絡叢のビジュアル解説

無傷の鋤鼻器官と副嗅球のex vivoでの準備

急性

Ex Vivoセル固有カルシウムマウスダイヤフラムの三者のシナプスでシグナル伝達のイメージング

インビボマウス劣オリーブにおけるカルシウムイメージング

エクスビボげっ歯類の三叉神経血管系からのカルシトニン遺伝子関連ペプチドの放出

Ex Vivo神経生理学のためのラット坐骨神経の調製

脈絡叢上皮細胞の繊毛運動の観測

要約

さらに動画を探す

この動画の章

急性上皮単純ヘルペスウイルスI型感染の ex vivo器官角膜モデル

糖尿病ラットの熱侵害受容閾値の迅速定量

全体の開発、 ショウジョウバエの脳のex vivoで培養

ラットの脳から脳表面血管系および関連髄膜の解剖と脈絡叢のビジュアル解説

無傷の鋤鼻器官と副嗅球のex vivoでの準備

急性

Ex Vivoセル固有カルシウム マウス ダイヤフラムの三者のシナプスでシグナル伝達のイメージング

インビボ マウス劣オリーブにおけるカルシウムイメージング

エクスビボ げっ歯類の三叉神経血管系からのカルシトニン遺伝子関連ペプチドの放出

Ex Vivo神経生理学のためのラット坐骨神経の調製

全体の開発、ショウジョウバエの脳のex vivoで培養

Ex Vivoセル固有カルシウムマウスダイヤフラムの三者のシナプスでシグナル伝達のイメージング

インビボマウス劣オリーブにおけるカルシウムイメージング

エクスビボげっ歯類の三叉神経血管系からのカルシトニン遺伝子関連ペプチドの放出