デモで助けるため支援・ カルメラ ・ ヴィターレ ・ f ・ Benfenati (Istituto イタリアーノ ・ ディ ・技術、情報技術学院) をありがちましょう。この作品は、IIT とアラコエリ サン ・ パオロ (LAC にグラント号 9734) によって賄われていた。

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-phosphate-mediated+Transfection+of+Eukaryotic+Cells+with+Plasmid+DNAs.">Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs.</a> CSH Protoc. 2006 (1), (2006).</li><li>Chung, K. H., 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=Polycistronic+RNA+polymerase+II+expression+vectors+for+RNA+interference+based+on+BIC/miR-155.">Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155.</a> Nucleic Acids Res. 34 (7), e53 (2006).</li><li>McClure, C., Cole, K. L., Wulff, P., Klugmann, M., Murray, A. 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(92), e51845 (2014).</li><li>Vandesompele, J., 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=Accurate+normalization+of+real-time+quantitative+RT-PCR+data+by+geometric+averaging+of+multiple+internal+control+genes.">Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.</a> Genome Biol. 3 (7), (2002).</li><li>Cetin, A., Komai, S., Eliava, M., Seeburg, P. H., Osten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nat Protoc. 1 (6), 3166-3173 (2006).</li><li>Paxinos, G., Franklin, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2012).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (2013).</li><li>Gunaydin, L. A., 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=Ultrafast+optogenetic+control.">Ultrafast optogenetic control.</a> Nat Neurosci. 13 (3), 387-392 (2010).</li><li>Bourinet, E., 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=Splicing+of+alpha+1A+subunit+gene+generates+phenotypic+variants+of+P-+and+Q-type+calcium+channels.">Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.</a> Nat Neurosci. 2 (5), 407-415 (1999).</li><li>Chaudhuri, D., 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=Alternative+splicing+as+a+molecular+switch+for+Ca2+/calmodulin-dependent+facilitation+of+P/Q-type+Ca2++channels.">Alternative splicing as a molecular switch for Ca2+/calmodulin-dependent facilitation of P/Q-type Ca2+ channels.</a> J Neurosci. 24 (28), 6334-6342 (2004).</li><li>Soong, T. W., 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=Systematic+identification+of+splice+variants+in+human+P/Q-type+channel+alpha1(2.1)+subunits:+implications+for+current+density+and+Ca2+-dependent+inactivation.">Systematic identification of splice variants in human P/Q-type channel alpha1(2.1) subunits: implications for current density and Ca2+-dependent inactivation.</a> J Neurosci. 22 (23), 10142-10152 (2002).</li><li>Berndt, A., 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=High-efficiency+channelrhodopsins+for+fast+neuronal+stimulation+at+low+light+levels.">High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels.</a> P Natl Acad Sci USA. 108 (18), 7595-7600 (2011).</li><li>Klapoetke, N. C., 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nat Methods. 11 (3), 338-346 (2014).</li><li>Lin, J. Y., Lin, M. Z., Steinbach, P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+engineered+channelrhodopsin+variants+with+improved+properties+and+kinetics.">Characterization of engineered channelrhodopsin variants with improved properties and kinetics.</a> Biophys J. 96 (5), 1803-1814 (2009).</li><li>Zhang, Y. P., Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+induction+of+synaptic+plasticity+using+a+light-sensitive+channel.">Optical induction of synaptic plasticity using a light-sensitive channel.</a> Nat Methods. 4 (2), 139-141 (2007).</li><li>Fowler, D. K., Williams, C., Gerritsen, A. T., Washbourne, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+knockdown+from+artificial+microRNAs+in+an+enhanced+miR-155+backbone:+a+designer's+guide+to+potent+multi-target+RNAi.">Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.</a> Nucleic Acids Res. 44 (5), e48 (2016).</li></ol>

著者が明らかに何もありません。

光プローブおよび興味のシナプス前遺伝子に対してミールの共発現遺伝子ノックダウンがそのまま神経回路内でのシナプス前機能に及ぼす影響を特徴付けるための強力なアプローチを提供しています。この実験の方法識別し非常に効率的なネイティブ システムの興味の遺伝子をノックダウンで選択 miRs を特徴付ける重要です。可能であれば、関心の同じ mRNA に対する 2 つ以上の独立した miRs は最終的なオフターゲット効果のコントロールに使用ください。レスキュー実験、ミール耐性遺伝子は、システムの再導入は、特異性のコントロールとして使用できます。
同じ構成要素を使用して、光を表現するプローブと、ミールは、光学的に操作されているシナプス前ニューロンのみを刺激するためできます。ために混合し、希釈の結果を起こす感染および非感染のニューロンを区別しないので、これは電気刺激で可能ではありません。光の刺激は、複数の活動電位15を引き起こすことができる、ので拡大パレット15,の19,20,21の中で、最速の光遺伝学的プローブのみを選択することが重要です。また、光刺激はシナプス前の研究の直接脱分極を誘導せず、シナプス伝達の手順の一部をバイパスするを避けるために 1 つは22を調査したいことを確保するため不可欠です。
ここで、述べたい実験的アプローチでは、並行限られた期間 (4 〜 6 ヶ月) 内の興味の複数のシナプス前遺伝子の生理学的な関連性を評価可能です。ただし、ノックダウンほとんど 100% に達することを留意することが重要です。さらに、rAAVs は初期の発達過程を調査するときに時間の制約を表す場合があります光プローブの最大のノックダウンと表現を可能にするのには、少なくとも 2 週間の表現する必要があります。シナプス前ニューロンに限るより多くの時間がかかり、条件付きのノックアウト マウスは通常結果が興味の遺伝子の削除を完了し、有効な相補的なアプローチを提供するため。
ミールの技術の特定の利点は、複数の miRs 同じプロモーターから発現できます。このプロパティは、同じミールまたは同じターゲット遺伝子に対する異なる miRs の複数のコピーを挿入することによってノックダウン効率化に主に使用されています。ただし、によってまた使用されるノックダウンする複数の遺伝子別のターゲット遺伝子7,23に対して miRs を表現します。このプロパティは、シナプス前のシグナル伝達経路を分析する複数のシナプス蛋白質の沈黙にされる可能性があります。
ここでは、我々 は急性海馬スライスにおけるシナプス前機能遺伝子の打撃の効果を特徴付ける人工 miRs で光遺伝学を組み合わせています。同様のアプローチは、操作し、プローブの体内の神経回路にも使用できます。さらに、人工 miRs と chemogenetic を組み合わせてアプローチにより、尋問の長い時間スケールの神経回路に 1 つ。

このプロトコルでは、遺伝子の打撃がそのまま神経回路内でのシナプス前機能に及ぼす影響を評価する新しいアプローチについて説明します。そのまま神経回路のシナプス前機能の調査多くのシナプス前のボタンが小さすぎるのでと遠く離れた複合分子と電気生理学的介入を許可する相馬からは困難です。RNA 干渉をノックダウン シナプス蛋白質1の強力で柔軟な手段が用意されています、このアプローチは、従来の打撃の効果を検出することは困難だからシナプス前機能を調査するため多用されていた区別しない電気刺激の操作と素朴なシナプス ボタン2。ここでは、人工マイクロ Rna (ミール) を結合する方法について説明-急性脳スライスの操作シナプス ボタンの選択的刺激を達成するために最近開発された光遺伝学的技術と RNA 干渉を介する。
電気生理学との組み合わせで条件付きノックアウト マウスは、シナプス蛋白質3,4の機能を調査するも使用できますが、我々 の戦略は不要開発と保守の遺伝子マウスの行を変更し、遺伝子の特定のアイソ フォームをノックダウンするも簡単に採用することができます。一般的に相対的な使用される短いヘアピン Rna (Shrna) ここを用いて、人工のミールはニューロンの打撃の主な利点を提供しています。Shrna とは異なり、彼らはポリメラーゼ II のプロモーター1の制御の下で表現できます。したがって、単一プロモーターは、ミールの蛍光レポーターと一緒にの光遺伝学的プローブ式をドライブに使用できます。このように、組換えアデノ随伴ウイルス (下さい rAAV、図 1 a) の包装範囲内で構造体のサイズを維持できます。また、単一の構造と単一のプロモーターを使用はミール、光遺伝学的プローブ、固定比率で蛍光レポーターの発現できるので実験的変動を低減します。
我々 は最近5海馬におけるシナプス前性カルシウム チャネルの交互スプライシング アイソ フォームの役割を調べるにこの技術を適用されます。このような戦略は、一般的に関心の任意の脳回路他ンエに表現された蛋白質の生理学的な関連性を勉強に適用されます。

<table>
	<tbody>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma</td>
			<td>Acrylamide/Bis-acrylamide, 30% solution</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Animal Temperature Controller with heat pad</td>
			<td>WPI</td>
			<td>ATC2000</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>BLOCK-iT Pol II miR RNAi Expression Vector kit</td>
			<td>ThermoFisher Scientific</td>
			<td>K4936-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain slices Prechamber</td>
			<td>Harvard Apparatus</td>
			<td>BSC-PC</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera-based imager</td>
			<td>Bio-Rad</td>
			<td>ChemiDoc&#8482; MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture reagents</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescent substrate</td>
			<td>GE Helthcare</td>
			<td>ECL Western Blotting Reagents, RPN2106</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabinofuranoside</td>
			<td>Sigma</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Foredom</td>
			<td>K.1030</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin ointment</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>54459</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection micropipettes</td>
			<td>Narishige</td>
			<td>GD1</td>
			<td></td>
		</tr>
		<tr>
			<td>Inorganic salts &#38; detergents</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K2-creatine phosphate</td>
			<td>Calbiochem</td>
			<td>237911</td>
			<td></td>
		</tr>
		<tr>
			<td>KGluconate</td>
			<td>Fluka</td>
			<td>60245</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Laserglow Technologies</td>
			<td>LRS-0473-GFM-00100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane</td>
			<td>Amersham</td>
			<td>Protran&#8482; 0.2 &#181;m NC</td>
			<td></td>
		</tr>
		<tr>
			<td>MgATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Narishige</td>
			<td>IM-H1</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3GTP</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>NaPyruvate</td>
			<td>Sigma</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular lubricant</td>
			<td>Local pharmacy</td>
			<td>Lacrigel</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate inhibitors</td>
			<td>Sigma</td>
			<td>P0044, P5726</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Sigma</td>
			<td>cOmplete&#8482;, EDTA-free Protease Inhibitor Cocktail</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein gel electrophoresis and blotting devices</td>
			<td>Bio-Rad</td>
			<td>Mini-Protean III Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Providone iodine</td>
			<td>Local pharmacy</td>
			<td>Betadine</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiazol Lysis Reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>QuantiTect Reverse 
			Transcription Kit</td>
			<td>Qiagen</td>
			<td>205311</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic apparatus</td>
			<td>WPI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>1069</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
	</tbody>
</table>

combining optogenetics with artificial micrornas to characterize the effects of gene knockdown on presynaptic function within intact neuronal circuits

このプロトコルの目的は、そのまま神経回路内でのシナプス前機能に関する遺伝子の打撃の効果を特徴付けるためです。我々 は人工マイクロ Rna (ミール) を結合する方法のワークフローを記述する-急性脳スライスの操作シナプス ボタンの選択的刺激を達成するために研究と RNA 干渉を介する。実験的アプローチは、単一のウイルス構造と光プローブとシナプス前遺伝子に対して人工 miR(s) の両方の式を運転する単一ニューロン固有プロモーターの使用を含みます。興味の脳の領域に注入改良ときに、表現の構成要素、捜査遺伝子の発現低下と神経細胞のみを光で刺激することが可能になります。この戦略は、開発と遺伝子組み換えマウスのメンテナンスが必要ないと原則的に適用できる他の生物と選択のすべての神経の遺伝子。最近シナプス前 P/Q 型電位依存性カルシウム チャネル (VGCCs) の代替スプライス アイソ フォームのノックダウンが CA1 急性海馬スライスにおける興奮性シナプスに CA3 で短期的なシナプス可塑性を調節する方法を調査することを行った。同様のアプローチは、操作し、プローブのin vivo神経回路にも使用できます。

上記の手順は、どのようにシナプス伝達を評価する手法は、シナプス前ニューロンにおけるシナプス蛋白質の打撃によって影響を受けるを提供します。代替のノックダウン スプライスのシナプス前 Cav2.1 のアイソ フォームの代表的な結果 (P/q) VGCCs 例として下記の通り興奮性シナプス CA1 に CA3 で短期的なシナプス可塑性を調節します。
Cav2.1 (P/q) チャネルは、中枢神経系で最も速いシナプスで優勢なシナプス前 VGCCs。相互に排他的エクソンのスプライシング代替 37a と Cav2.1 (α1 a) の細孔形成 α1サブユニットの 37b 生成 2 つの主要な変形、Cav2.1 [EFa] と Cav2.1 [EFb]16,17 、18。かどうか Cav2.1 [EFa] と Cav2.1 [EFb] 差動を調節するシナプス伝達とラット海馬錐体ニューロンのシナプス可塑性を決定する開発したノックダウンするアイソ フォーム特異 miRs 選択的に Cav2.1 [EFa] または Cav2.1 [EFb]5。ショート サイズにもかかわらず (97 bp) とエクソン間類似性が高い (塩基配列レベルで id 61.86%) に対して 3 つのミール シーケンスを設計できるか 37b 37a、ラットの Cav2.1 [EFa] (ミール EFa1: TCCTTATAGTGAATGCGGCCG; ミール EFa2: ATGTCCTTATAGTGAATGCGG;ミール EFa3: TTGCAAGCAACCCTATGAGGA) および 2 Cav2.1 [EFb] ラットに対して (ミール EFb1: ATACATGTCCGGGTAAGGCAT; ミール EFb2: ATCTGATACATGTCCGGGTAA) 予測高ノックダウン効率。否定的な制御 (ミール コントロール) として、知られている脊椎動物の遺伝子をターゲットにしないシーケンスを含む pcDNA6.2 GW/EmGFP ミール neg プラスミドを使用しました。HEK 293 細胞異種チャンネルの最初の画面を基に、ミール EFa1、ミール EFa3 と EFb2 を選択しに 3' UTR の rAAVs の生産のために設計されている pAAV-Syn-ChETA-TdT-miR-X のベクトルの式カセットを複製と場所、synapsin プロモーター ドライブ式超高速チャネルロドプシン ケータ、赤の蛍光タンパク質 TdTomato と挿入されたミール (図 1)。我々 はまた、ミール EFb2 このミールのノックダウン効率を高めるための発現カセットを複製しました。
次に、上記の 4 つの構造の rAAV1/2 を作製し、ノックダウン効率化及びアイソ フォーム特異 qRT PCR を用いたラット初代神経文化における選択性を定量化しました。ミール EFa1 とミール EFa3 ネイティブ カリフォルニア州v2.1 [EFa] 〜 70% の mRNA はなく、Cav2.1 [EFb] ながら減少ミール EFb 減少ネイティブ カリフォルニア州v2.1 [EFb] ~ 60% の mRNA はなく、Cav2.1 [EFa] (図 2)。
我々、改良 4 rAAV1/2 のそれぞれに注入 (図 3 a-c)、P18 ラットの海馬の CA3 領域 (A ・ P/M L/D-V 前から) −2.6 の座標を持つ/± 2.9/−2.9。15 から 24 日後注入 rAAV1/2 注入ラット急性海馬スライスを作製し、TdTomato 蛍光式 (図 3 b) rAAVs の局在を確認するために使用します。[EFb] Cav2.1 [EFa] または Cav2.1 のシナプス前の打撃に CA1 シナプスに CA3 における短期的なシナプスの可塑性が影響を受けるかどうか調べるために、我々 は簡単な 473 nm レーザー光パルス (2 ms 間で選択的に感染の ca3 錐体細胞を刺激;図 3) と CA1 領域 (図 3) の内側の管に近位の錐体細胞のパッチによって結果の工を記録。Cav2.1 スプライス アイソ フォームのノックダウンに反対の方向で対パルス刺激に対する応答が影響を受けることが分かった: Cav2.1 のノックダウン [EFa] (ミール EFa1 またはミール EFa3) に対しペアパルス円滑化 (PPF) を後押しした Ca のノックダウンv2.1 [EFb] (ミール EFb2) はそれ (図 3 DE) を廃止しました。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57223/57223fig1.jpg"> 図 1: 超高速光プローブ ケータとアイソ フォーム特異 miRs Cav2.1 [EFa] と Cav2.1 [EFb] に対して組み合わせ式の構造の方式です。(A) 、下さい rAAV の地図作成 pAAV-Syn-ChETA-TdT-miR-X、synapsin プロモーター (Syn) を含む、超高速チャネルロドプシン ケータ融合 TdTomato し、近位 3' UTR、Cav2.1 スプライス ・ アイソ フォーム特異ミールで。表示の制限の酵素は、シングル カッターです。(B)式カセットのトップ、方式です。Synapsin のプロモーターは、ケータ TdTomato のアイソ フォーム特異 miR 式をドライブします。下、ミールのカセットの部分を形成する 21 ヌクレオチド ターゲット シーケンスの逆の補数。ミール EFb2 2 つの同一のミール カセットだった他の後タンデム 1 つで表されます。<a href="//cloudflare.jove.com/files/ftp_upload/57223/57223fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57223/57223fig2.jpg"> 図 2。ノックダウン効率と Cav2.1 [EFa] と Cav2.1 [EFb] のアイソ フォーム特異 miRs の選択性の評価します。MiRs Cav2.1 [EFa] (ミール EFa1 とミール EFa3) をターゲットを表現する rAAVs 6 DIV で感染 17 18 DIV 初代培養から分離された RNA のアイソ フォーム特異 qRT PCR 解析または Cav2.1 [EFb] (ミール EFb2)。データは、ネガティブ コントロール (ミール コントロール) に正規化されます。ミール EFa1 とミール EFa3 大幅にし、選択的に減らす [EFa] Cav2.1 の mRNA (n 8 と 7 の文化をそれぞれ =)、ミール EFb 大幅にし、選択的に減少 [EFb] Cav2.1 の mRNA (n = 4 文化; * * * p < 0.001; 一方向の解析分散テスト テューキー Kramer の事後テストが続く)。± SEM. を意味するようにデータが表示されます。この図は、Thalhammer、A.らから適応されています。5.<a href="//cloudflare.jove.com/files/ftp_upload/57223/57223fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57223/57223fig3.jpg"> 図 3。オプトジェネティクスをノックダウン ニューロンの刺激をターゲットの役割評価 Cav2.1 [EFa] と Cav2.1 [EFb] ネイティブ海馬。(A) 生体内で感染に使用下さい rAAV 構造の発現カセット法(B)その TdTomato 蛍光性を示す海馬のセクションは、CA3 領域とその予測に限定されます。(C)実験的構成: レーザービームが CA3 突起に指示された, CA1 錐体細胞からパッチ クランプ録音を行った。(D) 2 ms ロング ブルー (473 nm) レーザー光パルス 20 Hz で輝いていた呼び起こす工が PPF は miRs Cav2.1 [EFa] をターゲットにより増加し、ミール Cav2.1 [EFb] によって廃止されます。ミール コントロールする相対的なミール、EFb2 の減少とミール EFa3 ミール EFa1 PPF の増加を示すペアパルス比率 (D) のように実験のための(E)概要 (n = 9-11 録音; * p = 0.02; * * p = 0.01; * * * p < 0.0004; 共分散分析)。± SEM. を意味するようにデータが表示されます。この図は、Thalhammer、A.らから適応されています。5.<a href="//cloudflare.jove.com/files/ftp_upload/57223/57223fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits at JoVE

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

このプロトコルは、そのまま神経回路内で選択的な遺伝子の発現低下と特にシナプス研究を刺激するために光遺伝学と人工のマイクロ Rna を介した RNA 干渉を結合する方法のワークフローを提供します。

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

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

combining-optogenetics-with-artificial-micrornas-to-characterize

Research

JoVE Journal

Neuroscience

10.1K ビュー.  Istituto Italiano di Tecnologia. このプロトコルは、そのまま神経回路内で選択的な遺伝子の発現低下と特にシナプス研究を刺激するために光遺伝学と人工のマイクロ Rna を介した RNA 干渉を結合する方法のワークフローを提供します。

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

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

神経科学

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

レーザースキャニングの光刺激によって抑制神経回路のマッピング

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（<em&gt; Schistocercaアメリカーナ</em&gt;）嗅覚回路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

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

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

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

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

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

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

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

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

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

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

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

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

動物を振る舞う、覚醒中のシングルユニット記録のためにマイクロワイヤの組織化された配列を移植するための手順

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

齧歯類網膜とリモート四肢虚血プレコンディショニングの保護効果に機能を評価するために電図を用いて、

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

行動するマウスにおける海馬シータ振動の光同調

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

覚醒マウス ガラス Optrode を持つ神経型のオプトジェネティクス同定

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

ゼブラフィッシュ稚魚における側線有毛細胞の生体内カルシウム イメージング

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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

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シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

レーザースキャニングの光刺激によって抑制神経回路のマッピング

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（

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

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

動物を振る舞う、覚醒中のシングルユニット記録のためにマイクロワイヤの組織化された配列を移植するための手順

齧歯類網膜とリモート四肢虚血プレコンディショニングの保護効果に機能を評価するために電図を用いて、

行動するマウスにおける海馬シータ振動の光同調

覚醒マウスガラス Optrode を持つ神経型のオプトジェネティクス同定

ゼブラフィッシュ稚魚における側線有毛細胞の生体内カルシウムイメージング

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

要約

さらに動画を探す

この動画の章

シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

レーザースキャニングの光刺激によって抑制神経回路のマッピング

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（

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

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

動物を振る舞う、覚醒中のシングルユニット記録のためにマイクロワイヤの組織化された配列を移植するための手順

齧歯類網膜とリモート四肢虚血プレコンディショニングの保護効果に機能を評価するために電図を用いて、

行動するマウスにおける海馬シータ振動の光同調

覚醒マウス ガラス Optrode を持つ神経型のオプトジェネティクス同定

ゼブラフィッシュ稚魚における側線有毛細胞の生体内カルシウム イメージング

覚醒マウスガラス Optrode を持つ神経型のオプトジェネティクス同定

ゼブラフィッシュ稚魚における側線有毛細胞の生体内カルシウムイメージング