彼女の優秀な技術的な支援のモデルとデリカ Bisignani の確立で彼女の科学的な入力をマーティン ツィンカーナーゲル、MD、PhD、ミリアム Reisenhofer、PhD を感謝いたします。

すべての実験眼科・視覚・眼科 (ARVO) 研究会のビジョン研究の動物の使用のための声明を遵守し、政府当局の関係法規を尊重します。
 1 です動物 <ol><li>維持 TgBAC (gfap:gfap-GFP) ゼブラフィッシュ 167 (AB) ひずみが 26.5 ° C の温度の水の標準的な条件下での 6-9 ヶ月と 14/10 h/明暗サイクル 18 歳です。</li>。
	<li>政府当局による承認後動物実験関係機関の動物のケアのガイドラインに従ってください</li>。
</ol> 2。リバーシブルの全身麻酔 <ol><li>97.9 mL タンクの水と 1 M トリスの 2.1 mL tricaine 粉の 400 mg を溶解することによりエチル 3-アミノ安息香酸メタンスルホン酸塩 (tricaine) の貯蔵液の準備緩衝生理食塩水 (TBS)。暗い 1 ヶ月までに 4 ° C で 1 M トリス (pH 9) とストア Ph7.0 に調整 。
	注: Tricaine は、動物の自然な生活条件のような水で準備されるべき、できれば元の水槽の水を使用必要があります</li>。
	<li>、原液 1:25 を水槽の水で希釈してすぐにします</li>。
	<li>10 cm シャーレまで不動なり外部刺激 (約 2-5 分、体重、年齢によって) に応答しない麻酔溶液 50 mL を含むにゼブラフィッシュを配置します</li>。
	<li>はレーザー治療 ( 図 1 a) のためのカスタムメイドのシリコン pin ホルダーにそれぞれの魚を手で転送します 。
	注意!魚は麻酔まで 10 分だけタンクの外側に残ることができる</li>。
	<li>逆に麻酔は治療および/またはイメージング後、タンクの水を含むコンテナーにゼブラフィッシュを配置します</li>。
	<li>回復をサポートする顔の上の新鮮なタンクの水の流れを水にゼブラフィッシュを移動することによって作成します</li>。
</ol> 3。網膜に焦点の傷害をレーザー 注: ゼブラフィッシュの網膜に焦点の光損傷を作成する A 532 nm ダイオード レーザーを使用します。レーザーの実験の設定により大人のゼブラフィッシュで再現可能な焦点網膜傷害を確立。
レーザーの出力を設定する <ol><li>: 70 mW; 空中直径: 50 μ m; パルス持続時間: 100 さん 注意!エリアの分類および適切な個人用保護具、レーザー光の使用が必要です</li>。
	<li>局所的に治療前に目で 2% ヒドロキシプロピルメチル セルロースの 1 〜 2 滴を適用し、2.0 mm 眼底レーザー レンズを使用して焦点を網膜にレーザー照準ビーム 。
	注意!ヒドロキシプロピルメチル セルロース滴は粘性、鰓に行く場合を呼吸に問題を引き起こす可能性があります</li>。
	<li>左の視神経乳頭周囲の場所 4 レーザー スポット目し、内部統制として、未処理の目を使用します</li>。
</ol> 4。生体内で網膜の形態のイメージング <ol><li>0 日に、ゼブラフィッシュの網膜レーザー誘導後に直接麻酔からそれらを復活することがなく視覚化します。他のすべての時間ポイントを用いる全身麻酔 (セクション 2 を参照してください: リバーシブルの全身麻酔)。( 図 1 B、B.1) カスタムメイドのシリコン pin ホルダーに固定されたゼブラフィッシュを配置します</li>。
	最適な画像を得るための <li>カット ゼブラフィッシュ眼に合わせて市販のハイドロゲル コンタクト レンズ (Ø 5.2 mm、r = = 2.70 mm、中央の厚さ = 0.4 mm) による穴パンチ。セルロースとレンズの凹面を埋めるし、角膜の上に置きます</li>。
	<li>78 D の非接触スリット ランプ レンズと OCT システムを装備します</li>。
	<li>フォーカス ir 赤外線 (IR) イメージ + 10 月モード ( 図 2 a) 眼の眼底を視覚化し、IR の写真をクリックして、&#34; 取得 &#34; ボタン ( 図 2 b) をローカライズするのにはレーザー システムを使用して網膜上のスポット &#39; s ソフトウェア</li>。
	<li>IR + OCT 網膜層部の三次元視覚化モードをクリックして写真を取るし、&#34; 取得 &#34; ボタン ( 図 2 b)。外の核層 (ONL) で損傷の重傷度を観察 (セクション 3 を参照してください: レーザー網膜に焦点の傷害) これらのイメージに</li>。
	<li>治療および/または画像はタンクの水を含むコンテナーに、ゼブラフィッシュを配置後、麻酔を逆にします</li>。
	<li>回復をサポートする顔の上の新鮮なタンクの水の流れを水にゼブラフィッシュを移動することによって作成します</li>。
	<li>網膜形態の似たような 生体内 イメージングを実行日 1、3、7、14、6 週目</li>。
</ol> 5。ヘマトキシリン &#38; エオシン (H &#38; E) 染色 <ol><li>冷 (4 ° C) に水没によるゼブラフィッシュを安楽死させる氷少なくとも 10 分のため、摘出に麻酔ソリューション目すぐににより小さいカーブタイプ鉗子</li>。
	<li>リン酸緩衝生理食塩水 (PBS) 20 h 4 ° C で 4% パラホルムアルデヒド (PFA) の全体の目の修正し、傾斜アルコール シリーズのサンプルを脱水 (キシレン 5 分二度 100%、エタノール 100 %5 分間 2 回、3 分 2 回エタノール 96%、エタノール 70 %3 min 回). 
	注意!PFA は、目、鼻、および上部の呼吸の経路を刺激することができます。PFA は、既知のひと発癌物質と疑われる生殖危険</li>。
	<li>パラフィン サンプルを埋め込む、視神経乳頭のレベルで 5 μ m のセクションをカット、スライド ガラスにそれらをマウントします</li>。
	<li>5 分 0.1% 酸ヘマトキシリン液と deparaffinized のセクションを染色し、塩酸ミックスでスライドを浸漬した後、蒸留水で 2 回スライドをディップ (2 mL 25 %250 mL に塩酸蒸留水) とミックス (250 mL の 2 mL 25% アンモニア アンモニア蒸留水)。ヘマトキシリンで少なくとも 10 分間水道水で染色の開発後エオシン G 水溶液 0.5 %3 分のセクションを染色</li>
	<li>アクリル樹脂取付中の脱水のスライドをマウントし、光学顕微鏡下でスライドを観察</li>。
</ol> 6。MC の活性化のための免疫組織化学 <ol><li>抗原検索バッファー (トリス EDTA + 0.05% 非イオン性洗剤、pH 9.0) 適切な蒸し器または電子レンジ 10-15 分で 3 分間の deparaffinized セクションを熱し、洗って 3 回 TBS 5各分</li>。
	<li>サークルであるシリコーン セクション ペンし、100 μ L を追加ブロック 1 における室温でのソリューション (TBS + 10% ヤギ血清 + 1% ウシ血清アルブミン、pH 7.6)</li>
	<li>染色抗グリア線維性酸性蛋白 (GFAP) ウサギ ポリクローナル抗体とモノクローナル抗体抗グルタミン合成酵素 (GS)、1: 200 希釈 (1 サンプルあたり 40 μ L) の両方。4 ° C で加湿チャンバー内でスライドを一晩インキュベートします。TBS + 0.1% で 3 回洗って 5 分トゥイーン 20</li>。
	<li>適切な仕上げ可視化二次抗体。このプロトコルは使用ヤギ抗うさぎ IgG H &#38; GFAP とヤギ抗マウス IgG H L 緑二次抗体 &#38; L 両方 1 時間室温で 1: 500 希釈で GS の明るい赤</li>
	<li>取付培 DAPI でスライドをマウントし、蛍光顕微鏡下でスライドを観察</li>。
</ol>

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ゼブラフィッシュの網膜再生/変性は、毒素による細胞死22、機械的損傷23、熱傷24などさまざまなアプローチが検討されています。ゼブラフィッシュの網膜を損傷する 532 nm ダイオード レーザーを採用しました。これにより、我々 のモデルは、いくつかの利点を提供しています。例えば、我々 は急速に PRs 層の具体的には、網膜の外側に?...

ビジョンは、おそらく人間の最も本質的な意味とその損傷が高い社会経済的影響。工業化された世界で網膜変性疾患は視力低下や失明成人人口の1間の大半を占めています。網膜色素変性症 (RP) は、約 150 万人世界2,3に影響を与える 60、および 20 の年齢間の人々 の失明の最も一般的な継承された原因です。遺伝性網膜疾患網膜色素上皮細胞と、その後神経膠症の変性が続くし、内部のニューロン4の改造 (PRs) 光受容細胞の進歩的な損失によって特徴付けられる異種族です。病気のコースは、通常、薄暗い光と色検査および視力5に欠かせないコーンで明暗視に責任がある棒で開始 2 つの PR セル型の増分の損失によって説明できます。単一の遺伝子異常は、RP を引き起こすのに十分です。これまでのところ以上 130 以上 45 遺伝子変異は6疾患に関連付けられています。これは、さまざまな疾患の表現型につながる、遺伝子療法は非汎化可能な理由の 1 つ、従って複雑な治療アプローチ。したがって、まばゆいばかりの疾患で網膜変性を治療する新しい一般的な治療方法を開発する急務があります。
しばしば網膜変性を含む PR 損失;そのため、PR 細胞死、7網膜の変性プロセスの特徴です。すでに、広報細胞死がミュラー グリア細胞 (MC) 活性化と増殖8を刺激することが実証されています。MCs、脊椎動物の網膜では、主要なグリア細胞型が網膜ニューロン間「接着剤」以外の何物であると考えた。近年、多くの研究は、MCs 行動以上の単なる構造は9をサポートすることを示しています。別の関数に MCs は神経新生にも関与し、10を修復します。確かに、変性網膜から拡散性因子に応答して、MCs は大幅、グリア線維性酸性蛋白 (GFAP) 表現を高めます。したがって、GFAP のラベリングは、網膜損傷し変性11二次応答として MC 活性化のマーカーとして使用できます。
最近では、(動脈分布) ゼブラフィッシュの網膜変性を誘発するレーザーを用いた焦点損傷のグラフィックノベルの適応を開発しました。焦点の損傷は、損傷部位に細胞の移動と網膜再生12中に行われるイベントの正確なタイミングなど特定の生物学的過程を研究するため有利です。さらに、ゼブラフィッシュは、その視覚システムと他の脊椎動物との間の類似性のため視覚研究で重要になりました。人間と魚類の網膜の総の形態学的および組織学的特徴は、いくつかの違いを表示します。人間とゼブラフィッシュの網膜が網膜投射神経細胞、神経節細胞は内側に存在しながら、光を感じる視細胞が最も外側の層を占める同じ層状パターンで構成されて同じの主要な細胞クラスを含むしたがって、レンズに近位の神経層。網膜の介在神経細胞、アマクリン細胞、バイポーラ、水平細胞の光受容細胞と神経節細胞層13の間にローカライズします。さらに、ゼブラフィッシュの網膜はコーンを支配し、したがってより、集中的に調査の齧歯動物の網膜など、人間の網膜に近い。硬骨魚と哺乳類の魅惑的な違いは、魚網膜と損傷後の網膜の再生に永続的な神経です。ゼブラフィッシュ、MCs が脱分化し、負傷した網膜14,15再生を仲介できます。鶏肉, MCs 脱分化して細胞周期を再入力にもいくつかの容量があります。成魚で網膜外傷後に MCs は前駆細胞および幹細胞の特定の特性を採用する、損傷した網膜組織に移行し、新しいニューロン16を生成します。網膜前駆細胞に予期しない類似点を明らかに哺乳類の MCs の遺伝子発現プロファイリングとチキン、齧歯動物、人間の網膜で MCs の組み込み神経因性潜在性のための証拠は17に成長しています。それにもかかわらず、鳥類や哺乳類の再生応答は魚で頑健な応答とに比べて低い理由はまだ分かっていません。したがって、ゼブラフィッシュの内因性の修復メカニズムを理解することは、哺乳類と人間の刺激的な網膜再生のための戦略を提案するかもしれない。網膜変性症患者の治療のため治療のツールとしては、MCs の内因性修復機構を採用私たちの社会の顕著な影響を与えるでしょう。
本明細書では、眼科研究の変性・再生モデルを採用するための手順を提供します。我々 はまず損傷部位と隣接する MCs の最後に可視化の関与を開発イベントのイメージングで、麻痺の網膜で焦点の損傷を誘発するのに焦点を当てた。一般的なプロトコルは、比較的簡単に行うさまざまな網膜をその後評価のための可能性を開きます。

<table>
	<tbody>
		<tr>
			<td>Acid hematoxylin solution</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2852</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A07030</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Pen</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI mounting medium</td>
			<td>Vector Labs, Burlingame, CA, USA</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin G aqueous solution 0.5%</td>
			<td>Carl Roth, Arlesheim, Switzerland</td>
			<td>X883.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene diamine tetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG H&#38;L Alexa Fluor&#174; 488</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG H&#38;L Alexa Fluor&#174; 594</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat normal serum</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>X0907</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogel contact lens</td>
			<td>Johnson &#38; Johnson AG, Zug, Switzerland</td>
			<td>n.a.</td>
			<td>1-Day Acuvue Moist</td>
		</tr>
		<tr>
			<td>Hydroxypropylmethylcellulose 2%</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>n.a.</td>
			<td>Methocel 2%</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A5040</td>
			<td>Tricaine, MS-222</td>
		</tr>
		<tr>
			<td>Visulas 532s</td>
			<td>Carl Zeiss Meditec AG, Oberkochen, Germany</td>
			<td>n.a.</td>
			<td>532 nm laser</td>
		</tr>
		<tr>
			<td>Mouse anti-GS monoclonal antibody</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>MAB302</td>
			<td></td>
		</tr>
		<tr>
			<td>HRA + OCT Imaging System</td>
			<td>Heidelberg Engineering, Heidelberg, Germany</td>
			<td>n.a.</td>
			<td>Spectralis</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Heidelberg, Germany</td>
			<td>n.a.</td>
			<td>Version 1.9.10.0</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFAP polyclonal antibody</td>
			<td>Invitrogen, Waltham, MA, USA</td>
			<td>180063</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone pin holder</td>
			<td>Huco Vision AG Switzerland</td>
			<td>n.a.</td>
			<td>Cut by hand from silicone pin mat of the sterilization tray accordingly.</td>
		</tr>
		<tr>
			<td>Slit lamp BM900</td>
			<td>Haag-Streit AG, Koeniz, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Slit lamp adapter</td>
			<td>Iridex Corp., Mountain View, CA, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus glass slides</td>
			<td>Gehard Menzel GmbH, Braunschweig, Germany</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>TgBAC (gfap:gfap-GFP) zf167 (AB) strain</td>
			<td>KIT, Karlsruhe, Germany</td>
			<td>15204</td>
			<td>http://zfin.org/ZDB-ALT-100308-3</td>
		</tr>
		<tr>
			<td>Tris buffered saline (TBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5912</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>78D non-contact slit lamp lens</td>
			<td>Volk Optical, Mentor, OH, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>534056</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular fundus laser lens</td>
			<td>Ocular Instruments, Bellevue, WA, USA</td>
			<td>OFA2-0</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Retriever</td>
			<td>Aptum Biologics Ltd., Southampton, United Kingdom</td>
			<td>R2100-EU</td>
			<td>Steamer</td>
		</tr>
	</tbody>
</table>

m&#252;ller glia cell activation in a laser-induced retinal degeneration and regeneration model in zebrafish

硬骨魚と哺乳類の魅惑的な違いは、魚類の網膜網膜神経新生と重度の損傷後の再生のための生涯の可能性です。ゼブラフィッシュにおける再生経路の調査は哺乳類の網膜変性疾患の治療のための革新的な戦略を開発する新しい洞察力をもたらす可能性があります。ここで、532 nm ダイオード レーザーによる大人のゼブラフィッシュの網膜の外側に病巣の誘導に注力しました。ローカライズされた損傷は、網膜変性・損傷の領域で直接再生時起こる生物学的プロセスを調査できます。我々 は、破損した領域とモニター以降再生の場所を定義するができた非侵襲的光コヒーレンストモグラフィ (OCT) を使用して、体内。確かに、OCT イメージングは、組織学的分析でだけ以前利用可能だった情報を提供するゼブラフィッシュ網膜の断面、高解像度の画像を生成します。組織切片を行ったリアルタイム 10 月からデータを確認するために、免疫組織化学による網膜損傷の誘導後の再生の応答を調べた。

リアルタイム 10 月: ゼブラフィッシュの網膜の損傷のよく線引きゾーンを誘導レーザー損傷モデルを用いて網膜の修復における MCs の役割を分析するために。(0 日) 損傷 (図 3) 60 分以内の初めて生体内でOCT による損傷のサイトをイメージしました。魚の眼の光学系を補うためにカスタムメイドのコンタクト レンズは、角膜に?...

Watch this Scientific Journal Video about MÃƒÂ¼ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish at JoVE.com

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

ゼブラフィッシュは、脊椎動物の網膜変性・再生のメカニズムを勉強する人気動物モデルです。このプロトコルでは、ローカライズされた外傷の内側の網膜に最小の被害で網膜の外側を中断することを誘導する方法について説明します。その後、我々 は生体内で網膜の形態と網膜再生を通してミュラー グリア細胞応答監視します。

ゼブラフィッシュの再生モデルと網膜変性のレーザー誘起のミュラー グリア細胞の活性化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

mller-glia-cell-activation-laser-induced-retinal-degeneration

Research

JoVE Journal

Neuroscience

9.7K ビュー.  University of Bern. ゼブラフィッシュは、脊椎動物の網膜変性・再生のメカニズムを勉強する人気動物モデルです。このプロトコルでは、ローカライズされた外傷の内側の網膜に最小の被害で網膜の外側を中断することを誘導する方法について説明します。その後、我々 は生体内で網膜の形態と網膜再生を通してミュラー グリア細胞応答監視します。

ビデオ: Müller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

神経科学

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

CHEF-tdTomatoを使用してゼブラフィッシュ体性感覚ニューロンのOptogeneticアクティベーション

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

大人のゼブラフィッシュにおける網膜再生研究で使用するための新規な光ダメージパラダイム

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

アン<em&gt;エクスビボ</em&gt;レーザー誘起脊髄損傷モデルがリアルタイムで軸索変性のメカニズムを評価するために

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

神経細胞死とマウス プライマリ小脳顆粒ニューロンの変性をモデリング

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

生体内でマルチ モーダル イメージングとマウス レーザー誘発脈絡膜新生血管モデルの解析

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

ラットの視神経の部分断裂: 網膜神経節細胞の二次変性を評価する新しい手術アプローチとの確立モデル

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脳スライスと一次細胞培養における錐体ニューロンの弾道標識

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

虚血におけるニューロングリア相互作用の役割を研究するための細胞培養モデル

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

ゼブラフィッシュの再生モデルと網膜変性のレーザー誘起のミュラー グリア細胞の活性化

ゼブラフィッシュの再生モデルと網膜変性のレーザー誘起のミュラーグリア細胞の活性化