本研究は、湖北省重点研究所開設プロジェクト(助成金番号2021KFY055)、湖北省自然科学財団(助成金番号2020CFB240)、および中央大学の基礎研究資金(助成金番号2042020kf0065)から資金提供を受けました。

全ての工程は、特に断らない限り室温で実施される。使用したすべてのC57BL / 6Jマウスは武漢大学の実験動物センターから入手し、関連するすべての実験は武漢大学の動物実験倫理委員会によって承認されました。マウスの苦痛を最小限に抑えるためにあらゆる努力がなされた。
1.目の核摘出と固定
<ol><li>二酸化炭素窒息でマウスを安楽死させ、歯のないピンセットを使用して眼球を穏やかに摘出します。</li>
	<li>眼球をリン酸緩衝生理食塩水(PBS)で一度すすぎます。</li>
	<li>顕微鏡下で角膜に注射針で眼球を穿刺し(接眼レンズWF:10倍/22、連続倍率対物レンズ:0.67x-4.5x)、眼球への固定を促進し、固定効率を向上させます。</li>
	<li>4%パラホルムアルデヒド(PFA)を含む24ウェルプレートに移します。</li>
	<li>眼球を4%PFAで45分間固定した後、1x PBSに移して、すぐに余分なPFAを除去します(3回洗浄、5分/回)。</li>
</ol>2.網膜の分離と平坦化
<ol><li>眼球をスライドガラスに移し、解剖顕微鏡(接眼レンズWF:10x/22、連続倍率対物レンズ:0.67x-4.5x)の下に置きます。</li>
	<li>鉗子を片手で持ち、角膜の縁を固定して眼球の動きを防ぎます。もう一方の手で解剖用ハサミを持ち、角膜強膜辺部を通して角膜の周りを約1mmの深さで切ります。レンズと硝子体を取り外します。</li>
	<li>視神経乳頭を中央に持ち、解剖ハサミを使用して、網膜の半径の約2/3に達する4つの象限に沿って網膜を放射状に切断します。</li>
	<li>歯付き鉗子を使用して強膜エッジの1つをクランプします。一方、歯のない鉗子で強膜の端を持ち、歯付き鉗子で強膜が固定されている場所から強膜の基部に向かって移動します。網膜を慎重に剥がします。</li>
	<li>ピペット(200 μL)を使用してPBSを吸引し、網膜を洗い流して濡らしてから、小さな吸収紙で余分なPBSを取り除きます。必要に応じて、小さな毛ブラシで網膜をそっと平らにします。</li>
	<li>冷たいメタノール(-20°C冷凍庫で予冷)をゆっくりとピペットで網膜の内面に滴下します(網膜がメタノールで完全に覆われている限り、メタノールの量は固定されていません)。網膜は白くなり、粘り強さが増します。</li>
	<li>網膜をやさしく平らにし、残った色素膜や硝子体などの不純物を小さな毛ブラシで取り除きます。小さな毛ブラシを使用して網膜を反転させ、上記のプロセスを繰り返します。</li>
	<li>メタノール処理した網膜を24ウェルプレートのウェルに移し、メタノールに浸したままにします。</li>
	<li>即時免疫染色のために-20°Cで1〜2時間保存するか、網膜をメタノール中に放置し、-20°Cで長期保存します。</li>
	<li>24ウェルプレートからメタノールを取り出し、1x PBSで網膜をすすぎます。</li>
</ol>3. RGCの免疫蛍光染色
<ol><li>プラスチックパスツールピペットで網膜を24ウェル赤血球凝集プレートに注意深く移し、5%PBS-TX-BSA(100 mLの1x PBST溶液に溶解した5 gのウシ血清アルブミン粉末;PBSTは、10 mLのTriton X-100を2,000 mLの1 x PBSに添加することによって形成されます)で室温で4時間または4°Cで一晩ブロッキングします。 神経節側を上に保ちます。</li>
	<li>PBS-TX-BSAを除去し、網膜を4°Cで48〜96時間インキュベートし、1:400希釈液を使用して選択した一次抗体(モルモット抗RBPMS抗体)100 μLを加えます。</li>
	<li>一次抗体を除去し、室温で振とうしながら、1x PBSで網膜を3回(各20分)すすぎます。</li>
	<li>1:400希釈液を使用して、対応する二次抗体100 μL( 材料表を参照)、シアニンCy3 affiniPureロバ抗モルモットIgG(H + L)を使用して、4°Cで一晩穏やかに振とうしながら網膜をインキュベートします。</li>
	<li>網膜を1x PBSで3回(各20分)すすぎます。</li>
	<li>神経節細胞層を上に向けてスライド上に網膜を平らに置き、適量の抗蛍光消光剤をピペットで網膜周囲に滴下し、スライドをカバーガラスで覆います(気泡が入らないように注意してください)。 注意: 抗蛍光消光剤の適切な量を決定するには、それが網膜全体を覆っていることを確認してください。その後、網膜が浮いて簡単に動かないようにスライドを覆います。</li>
	<li>スライドが動かないように、スライドをシール剤で全面的にシールします。サンプルは-20°Cで保存し、光から保護してください。</li>
	<li>蛍光/共焦点顕微鏡を使用して網膜を画像化します。</li>
</ol>

網膜神経節細胞を研究するためのマウス網膜単離とメタノール処理

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brn3a+as+a+marker+of+retinal+ganglion+cells:+qualitative+and+quantitative+time+course+studies+in+naive+and+optic+nerve-injured+retinas.">Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.</a> Investigative Ophthalmology &amp; Visual Science. 50 (8), 3860-3868 (2009).</li><li>Kwong, J. M., Caprioli, J., Piri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+binding+protein+with+multiple+splicing:+A+new+marker+for+retinal+ganglion+cells.">RNA binding protein with multiple splicing: A new marker for retinal ganglion cells.</a> Investigative Ophthalmology &amp; Visual Science. 51 (2), 1052-1058 (2010).</li><li>Stradleigh, T. W., Ishida, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+strategies+for+retinal+immunohistochemistry.">Fixation strategies for retinal immunohistochemistry.</a> Progress in Retinal and Eye Research. 48, 181-202 (2015).</li><li>Stradleigh, T. W., Greenberg, K. P., Partida, G. J., Pham, A., Ishida, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moniliform+deformation+of+retinal+ganglion+cells+by+formaldehyde-based+fixatives.">Moniliform deformation of retinal ganglion cells by formaldehyde-based fixatives.</a> Journal of Comparative Neurology. 523 (4), 545-564 (2015).</li><li>Bucher, D., Scholz, M., Stetter, M., Obermayer, K., Pfl&#252;ger, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+methods+for+three-dimensional+reconstructions+from+confocal+images:+I.+Tissue+shrinking+and+axial+scaling.">Correction methods for three-dimensional reconstructions from confocal images: I. Tissue shrinking and axial scaling.</a> Journal of Neuroscience Methods. 100 (1-2), 135-143 (2000).</li><li>Chidlow, G., Daymon, M., Wood, J. P., Casson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+a+wide-ranging+panel+of+antigens+in+the+rat+retina+by+immunohistochemistry:+Comparison+of+Davidson's+solution+and+formalin+as+fixatives.">Localization of a wide-ranging panel of antigens in the rat retina by immunohistochemistry: Comparison of Davidson's solution and formalin as fixatives.</a> Journal of Histochemistry &amp; Cytochemistry. 59 (10), 884-898 (2011).</li><li>Tokuda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+fixative+solution+for+retinal+morphology:+A+comparison+with+Davidson's+fixative+and+other+fixation+solutions.">Optimization of fixative solution for retinal morphology: A comparison with Davidson's fixative and other fixation solutions.</a> Japanese Journal of Ophthalmology. 62 (4), 481-490 (2018).</li><li>Miki, M., Ohishi, N., Nakamura, E., Furumi, A., Mizuhashi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+fixation+of+the+whole+bodies+of+fish+by+a+double-fixation+method+with+formalin+solution+and+Bouin's+fluid+or+Davidson's+fluid.">Improved fixation of the whole bodies of fish by a double-fixation method with formalin solution and Bouin's fluid or Davidson's fluid.</a> Journal of Toxicologic Pathology. 31 (3), 201-206 (2018).</li><li>Zanini, C., Gerbaudo, E., Ercole, E., Vendramin, A., Forni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+two+commercial+and+three+home-made+fixatives+for+the+substitution+of+formalin:+A+formaldehyde-free+laboratory+is+possible.">Evaluation of two commercial and three home-made fixatives for the substitution of formalin: A formaldehyde-free laboratory is possible.</a> Environmental Health. 11, 59 (2012).</li><li>Tang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+to+visualize+the+goblet+cell-associated+antigen+passages+and+identify+goblet+cells+in+the+intestine,+conjunctiva,+and+airway.">An optimized method to visualize the goblet cell-associated antigen passages and identify goblet cells in the intestine, conjunctiva, and airway.</a> Immunobiology. 227 (6), 152260 (2022).</li><li>Brock, R., Hamelers, I. H., Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fixation+protocols+for+adherent+cultured+cells+applied+to+a+GFP+fusion+protein+of+the+epidermal+growth+factor+receptor.">Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor.</a> Cytometry. 35 (4), 353-362 (1999).</li><li>Baykal, B., Korkmaz, C., Kocabiyik, N., Ceylan, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+post-fixation+on+visualising+vimentin+in+the+retina+using+immunofluorescence+method.">The influence of post-fixation on visualising vimentin in the retina using immunofluorescence method.</a> Folia Morphologica. 77 (2), 246-252 (2018).</li><li>Powner, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+gene+expression+in+whole+mouse+retina+by+in+situ+hybridization.">Visualization of gene expression in whole mouse retina by in situ hybridization.</a> Nature Protocols. 7 (6), 1086-1096 (2012).</li><li>Zhang, N., Cao, W., He, X., Xing, Y., Yang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+methanol+to+preserve+retinas+for+immunostaining.">Using methanol to preserve retinas for immunostaining.</a> Clinical and Experimental Ophthalmology. 50 (3), 325-333 (2022).</li><li>Kalesnykas, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cell+morphology+after+optic+nerve+crush+and+experimental+glaucoma.">Retinal ganglion cell morphology after optic nerve crush and experimental glaucoma.</a> Investigative Ophthalmology &amp; Visual Science. 53 (7), 3847-3857 (2012).</li><li>Parrilla-Reverter, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-course+of+the+retinal+nerve+fibre+layer+degeneration+after+complete+intra-orbital+optic+nerve+transection+or+crush:+a+comparative+study.">Time-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: a comparative study.</a> Vision Research. 49 (23), 2808-2825 (2009).</li></ol>

著者は、この研究の客観性に影響を与える可能性のある所属、メンバーシップ、資金、または金銭的保有を認識していません。

固定は網膜を保存するための重要なステップであり、形態に基づくその後のRGC調査に影響を与える可能性があります。固定が成功すると、網膜を固定媒体にさらした瞬間の網膜の構造と状態が迅速にキャプチャされ、これはさらなる分析にとって重要です。ホルムアルデヒドは、組織および細胞の固定および保存のための最も一般的な固定剤の1つと見なされてきましたが、ホルムアルデヒド...

網膜神経節細胞(RGC)は網膜内の唯一の投射ニューロンであり、外部の視覚情報を統合して脳に伝達します1。緑内障や外傷性視神経障害などの多くの神経変性疾患は、不可逆的な損傷とRGCの喪失を特徴としています2,3。RGCの形態学的および量的変化を分析することは、神経変性疾患がどのように発症し進行するかを決定する上で重要なステップです4,5。
間接免疫蛍光アッセイは、タンパク質の分布と細胞カウントをモニターするために広く受け入れられている方法です。実験室では、網膜の生理学的および病理学的状態を評価するために、RGCの実験的研究で全マウント網膜免疫染色が一般的に使用されています6。網膜全体でRGC定量に使用される最も一般的なマーカーには、Brn3a、多重スプライシングを伴うRNA結合タンパク質(RBPMS)などがあります7,8。RGCの量と分布を特徴付けるには、高品質の全マウント網膜免疫染色が必要です。一般に、免疫染色プロトコルでは、網膜は抗体でインキュベートされる前に化学固定液に浸されます。理想的な固定剤は、細胞の形状、抗体に対するエピトープの接近性または親和性、または組織の直線寸法を変えるべきではない9、10。
網膜の構造が複雑なため、網膜膜の損傷や折り畳みなどの問題や、細胞の収縮や核の不明瞭さなどの一般的な問題が発生しやすく、実験研究に悪影響を及ぼします。さらに、すべての網膜がすぐに免疫染色されるわけではなく、特に貴重な起源の高価な価格のトランスジェニックマウスの網膜に関しては、さらなる使用のために余分な網膜サンプルを保存する必要があります。
適切な固定液は、組織を迅速に固定し、組織の自己消化を回避し、組織細胞の正常な形態と構造を維持し、タンパク質やその他の物質の抗原性を維持することができます10。現在、ホルムアルデヒドベースの固定は、分離された網膜、半切除されたアイカップ、および眼球全体を含む様々な組織において広く使用されている10。組織の収縮と細胞の形態学的変化は、ホルムアルデヒド11に浸した後に直面する2つの重要な課題です。加えて、網膜および標的細胞の本来の特性の保持を最大化するために、修正された固定製剤がますます出現している9、10。異なる網膜固定治療は、網膜構造、タンパク質免疫原性、蛍光励起、および減弱消光サイクルに異なる影響を与える可能性があります12,13。デビッドソンの溶液で固定された網膜は、ホルマリンで固定されたものと比較して形態学的に無傷ですが、デビッドソンの溶液は、ミクログリアマーカーイオン化カルシウム結合アダプター分子112などの一部の抗体との適合性が低くなります。網膜の脆弱な性質を考えると、研究者は当然、網膜の完全性、ならびに標的細胞の特性と形態が長期保存後に変化するかどうか疑問に思うでしょう。しかし、数ヶ月保存後の網膜およびRGC細胞の形態に対する固定液の影響の可能性はほとんど報告されていません。網膜固定の最適化は、RGCの評価と保存に不可欠です。
全マウントマウス網膜染色に使用する信頼性が高く技術的に簡単な方法の詳細な説明を提供します。私たちの方法は、網膜組織の長期保存の必要性と蛍光色素の形成または分解の特定の側面を考慮して、RGC調査のための網膜の適切な調製と保存を強調しています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well cell culture cluster</td>
			<td>Costar</td>
			<td></td>
			<td>Eyeball fixation</td>
		</tr>
		<tr>
			<td>24-well hemagglutination plate</td>
			<td>Labedit Company</td>
			<td></td>
			<td>Incubation antibody</td>
		</tr>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>Citotest</td>
			<td></td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Anti-fluorescent quenching mountant</td>
			<td>Servicebio</td>
			<td>G1401</td>
			<td>Slow down fluorescence quenching</td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumin)</td>
			<td>Servicebio</td>
			<td>GC305010</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>OLYMPUS</td>
			<td></td>
			<td>Apply 40x objective lens</td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>RWD Life science Co.,LTD&#160;</td>
			<td>77001S</td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>20210624</td>
			<td>GC&#8805;99.5%</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>SecheVite</td>
			<td></td>
			<td>Sealing agent</td>
		</tr>
		<tr>
			<td>Needles&#160;</td>
			<td>Shanghai Kindly Enterprises Development Group Co., Ltd.</td>
			<td></td>
			<td>Accelerate the fixation</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution</td>
			<td>Servicebio</td>
			<td>G1101</td>
			<td>Eyeball fixation</td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline pH 7.4)</td>
			<td>Servicebio</td>
			<td>G0002</td>
			<td>Rinse the eyeball&#160;</td>
		</tr>
		<tr>
			<td>Primary antibody: guinea pig anti-RNA-binding protein with multiple splicing (RBPMS)</td>
			<td>PhosphoSolutions</td>
			<td>Cat. #1832-RBPMS</td>
			<td>For immunofluorescence. Used at 1:400</td>
		</tr>
		<tr>
			<td>Secondary antibody: Cy3 affiniPure donkey anti-guinea pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>706-165-148</td>
			<td>For immunofluorescence. Used at 1:400</td>
		</tr>
		<tr>
			<td>Straight scissors</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
	</tbody>
</table>

methanol-based whole-mount preparation for the investigation of retinal ganglion cells

網膜の投射ニューロンである網膜神経節細胞(RGC)は、外部の視覚情報を脳に伝播します。RGCの病理学的変化は、多くの網膜変性疾患と密接な関係があります。全マウント網膜免疫染色は、網膜の発生および病理学的状態を評価するためのRGCの実験的研究で頻繁に使用されます。状況によっては、トランスジェニックマウスからのものなど、いくつかの貴重な網膜サンプルを、RGCの形態や数に影響を与えずに長期間保持する必要がある場合があります。信頼できる再現性のある実験結果を得るには、効果的な保存培地の使用が不可欠です。ここでは、網膜ホールマウント製剤および長期保存のための補助固定培地としてのメタノールの効果について述べる。簡単に言うと、単離プロセス中に、冷たいメタノール(-20°C)を網膜の表面にピペットで移して組織を固定し、その透過性を促進し、免疫染色する前に網膜を冷メタノール(-20°C)に保存することができます。このプロトコルは、網膜分離ワークフローと組織サンプル保存プロトコルを記述しており、RGCの調査に有用で実用的です。

解剖後、網膜は平らな四つ葉のクローバーのように見えるはずです。この研究では、上記のプロトコルを使用して、メタノールを添加した後に網膜が白くなりました(図1)。その間、網膜は柔らかいものから柔軟で平らに変わりました。次に、RGCを抗RBPMS8で標識した。共焦点顕微鏡(接眼レンズ:10倍、対物レンズ:40倍)を用いて、全マウント網膜(n=3)の4つ?...

Watch this Scientific Journal Video about Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells at JoVE.com

著者スポットライト:網膜神経節細胞の研究のためのメタノールベースのホールマウント調製  

メタノールは、網膜ホールマウント製剤および長期保存のための補助固定培地として使用することができ、網膜神経節細胞の調査に有用である。

網膜神経節細胞の研究のためのメタノールベースのホールマウント調製

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

この研究は、RGCの調査に有用で実用的な、修正された網膜分離ワークフローと組織サンプル保存プロトコルを提供することを目的としています。 網膜は壊れやすく折りたたまれているため、全マウント網膜パッチを作成する際の実験研究が制限されます。さらに、すべての網膜は即時の免疫染色を必要としません。場合によっては、追加の網膜サンプルをさらに使用するために保存する必要があります。RGC調査のための網膜の適切な調製と長期保存の必要性を強調する、信頼性が高く技術的に簡単な全マウント網膜染色方法の詳細な説明を提供します。今後も変性視神経障害の基礎研究に取り組んでいきます。さらに、視神経損傷と事故再生の分子メカニズムと、神経変性疾患の診断と治療のための潜在的な臨床標的を探ります。

methanol-based-whole-mount-preparation-for-investigation-retinal

Research

JoVE Journal

Neuroscience

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

2.9K ビュー.  Renmin Hospital of Wuhan University. この研究は、RGCの調査に有用で実用的な、修正された網膜分離ワークフローと組織サンプル保存プロトコルを提供することを目的としています。 網膜は壊れやすく折りたたまれているため、全マウント網膜パッチを作成する際の実験研究が制限されます。さらに、すべての網膜は即時の免疫染色を必要としません。場合によっては、追加の網膜サンプルをさらに使...

ビデオ: 著者スポットライト:網膜神経節細胞の研究のためのメタノールベースのホールマウント調製  

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

神経科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

網膜色素上皮の全マウントするためのマウスの眼の解剖

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

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 ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人の全体のマウントの準備ショウジョウバエ</emジャイアント繊維染料注入のための>腹神経索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

マウスの網膜神経節細胞におけるシナプスと人工コンダクタンスによるダイナミッククランプの実装

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全マウント神経筋接合解析のための トランス対アブドミン筋 の解剖

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

における嗅覚受容ニューロンのホールマウント免疫標識<em&gt;ショウジョウバエ</em&gt;アンテナ

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

大人のホールマウントの準備のためのシンプルなワンステップ解剖プロトコル<em&gt;ショウジョウバエ</em&gt;ブレインズ

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

網膜神経節細胞の定義されたサブセットの単一細胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光干渉断層計: マウス網膜神経節細胞In Vivoイメージング

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...