本作品は、国立衛生研究所 HD043680、MH106392、DA013137、および NS100624 の健康 (NIH) の補助金によって支えられました。部分的な基金は、医用生体行動科学 NIH T32 のトレーニングの許可によって提供されました。

実験プロトコルは、動物のケアおよび使用委員会 (IACUC)、サウスカロライナ州の大学によって承認された (連邦保証番号: A3049-01)。
1. 新鮮な冷凍脳セクションの準備
<ol><li>N/F344 ラット系統を使用: 各性のラット 3 匹 13 ヵ月齢、本体重量約 320 g。</li>
	<li>5% (セボフルランの過剰摂取) のセボフルラン濃度を調整します。呼吸停止の追加分、後セボフルラン投与を続行します。</li>
	<li>ネズミの首をはねるし、脳を削除します。</li>
	<li>水没 15 液体窒素でラット脳組織収穫の 5 分以内で s。</li>
	<li>1 h 用クライオスタットの-20 ° C と脳を平衡します。</li>
	<li>30 μ m のセクションをカットし、スライド (材料の表を参照してください) に転送します。</li>
	<li>核側坐地域、約 2.76 mm ~ 前14前方 2.28 mm からセクションを選択します。</li>
	<li>継続的にラットの脳定位固定脳の構造によると核側坐地域の嗅球からラット脳をスライスします。</li>
	<li>スライドにサンプルをマウントします。セクションを保つため、-20 ° c で 10 分間乾燥します。</li>
	<li>4 ° C で 1 時間前に冷蔵 4% のパラホルムアルデヒドのスライドをすぐに没頭します。</li>
	<li>室温 (RT) で増加するエタノール勾配にスライドを配置: 50% エタノールに 5 分。70% エタノールに 5 分。100% エタノールに 5 分。</li>
	<li>新鮮な 100% リピート江藤。</li>
	<li>吸水紙の上のスライドを配置し、空気が乾燥。</li>
	<li>バリア ペンで各セクションに障壁を描画 (材料の表を参照してください)。1 分間完全に乾燥バリアができます。</li>
</ol>2. 脳の前処理
<ol><li>オーブンをオンにし、温度を 40 ° C に設定</li>
	<li>4 前処理試薬 3 滴 (90 μ L) を追加 (材料表参照) 各脳セクション (スライドごとの 1 つのセクション) に。</li>
	<li>セクション 30 分室温で孵化させなさい</li>
	<li>新鮮な 1 × PBS で RT。 繰り返し 1 分の 1x PBS でスライドが水没します。 注: スライドに 15 分よりも長く 1x PBS で滞在しないでください。</li>
</ol>3. RNA In Situハイブリダイゼーション蛍光多重アッセイ
<ol><li>暖かい水のお風呂で 40 ° C で 10 分間のターゲット プローブと右に冷却してから</li>
	<li>RNA 試薬の場所 (例えば、アンプ 1 4 FL) 室温</li>
	<li>スライド ラックの背面吸水紙と場所でスライドから余分な液体を削除する (テーブルの材料を参照してください)。</li>
	<li>サンプルのスライスに Drd1α プローブ (C1) の 3 滴 (90 μ L) を追加します。40 ° C で 2 時間インキュベートします。</li>
	<li>洗浄バッファー x 新鮮な 1 としたリピートで 2 分間洗浄バッファー x 1 のスライドが水没します。</li>
	<li>スライド ラックの背面吸水紙と場所でスライドから余分な液体を削除する (テーブルの材料を参照してください)。</li>
	<li>サンプルのスライス上アンプ 1 フロリダの 3 滴 (90 μ L) を追加します。40 ° C で 30 分間インキュベートします。</li>
	<li>新鮮な 1wash バッファーを使用したリピートで 2 分間 1 洗浄バッファーのスライドが水没します。</li>
	<li>スライド ラックの背面吸水紙と場所でスライドから余分な液体を削除する (テーブルの材料を参照してください)。</li>
	<li>サンプルのスライスにアンプ 2 fl (90 μ L) を 3 滴を追加します。40 ° C で 15 分間インキュベートします。</li>
	<li>洗浄バッファー x 新鮮な 1 としたリピートで 2 分間洗浄バッファー x 1 のスライドが水没します。</li>
	<li>スライド ラックの背面吸水紙と場所でスライドから余分な液体を削除する (テーブルの材料を参照してください)。</li>
	<li>サンプルのスライス上アンプ 3 FL の 3 滴 (90 μ L) を追加します。40 ° C で 30 分間インキュベートします。</li>
	<li>洗浄バッファー x 新鮮な 1 としたリピートで 2 分間洗浄バッファー x 1 のスライドが水没します。</li>
	<li>スライド ラックの背面吸水紙と場所でスライドから余分な液体を削除する (テーブルの材料を参照してください)。</li>
	<li>アンプ 4-フロリダ-オルト A サンプルのスライス上の 3 滴 (90 μ L) を追加します。40 ° C で 15 分間インキュベートします。</li>
	<li>洗浄バッファー x 新鮮な 1 としたリピートで 2 分間洗浄バッファー x 1 のスライドが水没します。</li>
	<li>スライドから余分な液体を削除する、すぐに取付試薬 2 滴 (材料表参照) 各セクションに。</li>
	<li>22 mm 22 mm coverslip の場所 (材料表参照) 各脳セクションの上。</li>
	<li>乾燥するまで、2-8 ° C で暗闇の中でスライドを保存します。</li>
	<li>共焦点顕微鏡をオンにし、60 X 目的に切り替えます。</li>
	<li>共焦点顕微鏡画像を Z スタックを取得します。共焦点レーザー顕微鏡の詳細手順の補足のビデオを参照してください。</li>
	<li>Drd1α 核側坐領域内のセルのラベルの画像を取得 (励起: 488 nm;放出: 515/30 nm)。 注: セクションが潜伏ステップ間乾く脳をてはいけないです。</li>
</ol>4. データ解析
<ol><li>染色パターンを半定量的に解析:「不連続な点」。<ol>
<li>イメージから個々 のセルを識別するために DAPI 染色として核マーカーを実行します。しかし、まだそれは細胞の核や細胞の不規則な形状の種類を複数持っているので、脳の特定の部分を分析することは困難です。ここでは、2 つの経験豊富な研究者は個別にセル領域を定義する画像から各セルを割り当てます。</li>
		<li>視覚的に特定の基準 (図 1 b) を使用して、セルごとのドット数に基づく代表的な共焦点画像内の各セルをスコアします。</li>
		<li>各カテゴリーの総細胞数を決定し、すべてのティッシュ セクション (図 2 a) の男女別各部門内のセルのパーセントの周波数を表示する相対頻度グラフを作成する細胞数 x100 で割ります別に (図 2 e)。</li>
		<li>個別にすべてティッシュ セクション (図 2 b) と男性と女性のためのセルの累積度数を確認する細胞数 x100 で事前のすべてのカテゴリ内のセルの合計を除算 (図 2 f)。</li>
	</ol>
</li>
	<li>染色パターン (省略可能)「離散ドット」を統計的に分析します。<ol>
<li>自然 (すなわち、最高の電池式 (9) に最下位セル式 (1) から各ランクのカテゴリに該当するセルの数) の序数は、カテゴリカル変数を提供するセルあたりのドットの数を調べてさらに統計解析。</li>
		<li>記述統計量の検査の後、3 つの主なアプローチを使用: マントルピース Haenszel 統計, マン-ホイットニーの U 検定やクラスカル-ウォリス検定。正確な統計的手法に依存なりますが興味、統計的決定ツリー (図 4) の実験の設計と研究の質問は 1 つの分析的なアプローチに関する意思決定に役立つ可能性があります。</li>
	</ol>
</li>
	<li>染色パターンの利益 (率 ROI) の領域の信号強度の定量化:「クラスター」。<ol>
<li>次の式を使用して各画像の信号強度を計算します。 信号強度 = ROI 画像 - (平均背景強度 × 全体のイメージ領域) の合計強度</li>
		<li>セルあたりおおよそ信号式を計算する投資収益率イメージ内のセルの合計数をカウントします。セル/画像の数と同様、信号強度/画像のグループの違いは、信号式/携帯で見られるグループの相違に貢献するかもしれないメカニズムを考慮する興味のある (図 3B-3 D)。</li>
	</ol>
</li>
	<li>染色パターン (省略可能)「クラスター」統計的に分析。<ol>
<li>さらに統計分析の連続的な変数を提供する信号式/セルを調べます。デザインに含まれる独立したサンプルの t 検定、分散分析 (ANOVA)、被験者間要因の数に基づいてなど、2 つのアプローチを使用します。統計的決定ツリー (図 4) は、最も適切な統計方法についての意思決定に役立つ可能性があります。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Goldman-Rakic, P. S., Castner, S. A., Svensson, T. H., Siever, L. J., Williams, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+dopamine+D1+receptor+in+schizophrenia:+insights+for+cognitive+dysfunction.">Targeting the dopamine D1 receptor in schizophrenia: insights for cognitive dysfunction.</a> Psychopharmacology (Berl). 174 (1), 3-16 (2004).</li><li>Beaulieu, J. M., Gainetdinov, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology,+signaling,+and+pharmacology+of+dopamine+receptors.">The physiology, signaling, and pharmacology of dopamine receptors.</a> Pharmacol Rev. 63, 182-217 (2011).</li><li>Zahrt, J., Taylor, J. R., Mathew, R. G., Arnsten, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supranormal+stimulation+of+D1+dopamine+receptors+in+the+rodent+prefrontal+cortex+impairs+spatial+working+memory+performance.">Supranormal stimulation of D1 dopamine receptors in the rodent prefrontal cortex impairs spatial working memory performance.</a> J Neurosci. 17, 8528-8535 (1997).</li><li>Floresco, S. B., Magyar, O., Ghods-Sharifi, S., Vexelman, C., Tse, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+dopamine+receptor+subtypes+in+the+medial+prefrontal+cortex+of+the+rat+regulate+set-shifting.">Multiple dopamine receptor subtypes in the medial prefrontal cortex of the rat regulate set-shifting.</a> Neuropsychopharmacology. 31, 297-309 (2006).</li><li>Arnsten, A. F., Girgis, R. R., Gray, D. L., Mailman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+dopamine+therapeutics+for+cognitive+deficits+in+schizophrenia.">Novel dopamine therapeutics for cognitive deficits in schizophrenia.</a> Biol Psychiatry. 81, 67-77 (2017).</li><li>Ellenbroek, B. A., Budde, S., Cools, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prepulse+inhibition+and+latent+inhibition:+the+role+of+dopamine+in+the+medial+prefrontal+cortex.">Prepulse inhibition and latent inhibition: the role of dopamine in the medial prefrontal cortex.</a> Neuroscience. 75 (2), 535-542 (1996).</li><li>Parker, K. L., Alberico, S. L., Miller, A. D., Narayanan, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+D1+dopamine+signaling+is+necessary+for+temporal+expectation+during+reaction+time+performance.">Prefrontal D1 dopamine signaling is necessary for temporal expectation during reaction time performance.</a> Neuroscience. 255, 246-254 (2013).</li><li>Manduca, A., Servadio, M., Damsteegt, R., Campolongo, P., Vanderschuren, L. J., Trezza, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+Neurotransmission+in+the+Nucleus+Accumbens+Modulates+Social+Play+Behavior+in+Rats.">Dopaminergic Neurotransmission in the Nucleus Accumbens Modulates Social Play Behavior in Rats.</a> Neuropsychopharmacology. 41 (9), 2215-2223 (2016).</li><li>Okubo, Y., 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=Decreased+prefrontal+dopamine+D1+receptors+in+schizophrenia+revealed+by+PET.">Decreased prefrontal dopamine D1 receptors in schizophrenia revealed by PET.</a> Nature. 385 (6617), 634-636 (1997).</li><li>Abi-Dargham, 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=Prefrontal+dopamine+D1+receptors+and+working+memory+in+schizophrenia.">Prefrontal dopamine D1 receptors and working memory in schizophrenia.</a> J Neurosci. 22 (9), 3708-3719 (2002).</li><li>Shinohara, R., 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=Dopamine+D1+receptor+subtype+mediates+acute+stress-induced+dendritic+growth+in+excitatory+neurons+of+the+medial+prefrontal+cortex+and+contributes+to+suppression+of+stress+susceptibility+in+mice.">Dopamine D1 receptor subtype mediates acute stress-induced dendritic growth in excitatory neurons of the medial prefrontal cortex and contributes to suppression of stress susceptibility in mice.</a> Mol Psychiatry. 19, (2017).</li><li>Okubo, Y., 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=Decreased+prefrontal+dopamine+D1+receptors+in+schizophrenia+revealed+by+PET.">Decreased prefrontal dopamine D1 receptors in schizophrenia revealed by PET.</a> Nature. 385, 634-636 (1997).</li><li>Wang, F., 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J Mol Diagn. 14 (1), 22-29 (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. , (2014).</li></ol>

宣言する利害の対立がないです。

このプロトコルに交配その場で新鮮凍結脳スライス (NAc) 側坐核における Drd1α 受容体の発現と中脳 (SNR) 地域におけるチロシン水酸化酵素 (番目の) 式を評価するための手法について述べる。我々 はまたさまざまな染色パターンのためのデータ分析方法を提供:「離散ドット」または「クラスター」。
交配分析その場で成功した多重蛍光 RNA のための重要なス?...

線条体ドーパミン系機能障害は複数の神経認知症にみられる臨床症状の進行に関与しています。ドーパミン D1 受容体が前頭前皮質 (PFC) と脳の線条体領域に存在し、認知1メモリ、一時的な処理、および機関車の動作2,3を含むに大きく影響,4,5,6,7. 以前の研究は、ドパミン D1 受容体の変化は、注意欠陥多動性障害 (ADHD)8、統合失調症9,の神経認知症状の進行と関連付けられたことを解明10ストレス感受性11。具体的には、統合失調症、陽電子放射断層撮影 (PET) 研究に認知障害や陰性症状11の存在する前頭皮質におけるドパミン D1 受容体の結合能が関連性が高いことが示されました。前頭前皮質のドーパミン D1 受容体による規制で興奮性ニューロンの樹枝状結晶の成長には、ストレス感受性が軽減されます。さらに、内側前頭前皮質 (mPFC) ニューロンの D1 受容体のノックダウンは、社会的敗北ストレス誘発性の社会的回避12を高めることができます。
RNAの in situハイブリダイゼーション新鮮凍結組織サンプルでのセルに単一の RNA 分子を可視化する手法を紹介します。現在の技術では、現在の文献内に存在する方法上の複数の利点を持っています。まず、現在のプロシージャは組織の空間的および形態学的コンテキストを保存し、他のプロシージャは、新鮮なを必要とする、非埋め込まれた組織は、現在の方法と組み合わせることができるように新鮮凍結組織サンプルで行った。ホルマリン固定パラフィン包埋組織における同様の手順は、単一の転写の解像度は、RNAの in situハイブリダイゼーション テクニック13を使用して達成することができます説明しています。単一の転写レベルで RNA の検出は、低コピー数の表現だけでなく、核酸検出の他の方法では達成できない細胞レベルでの遺伝子発現を比較する機会に優れた感度を提供しますポリメラーゼの連鎖反応 (PCR) のテクニック」などさらに、現在のメソッドは、単一のターゲット RNA 転写産物にハイブリダイズと検出信号増幅の分子カスケードを順番にバインドされている、非常に特定の RNA プローブを介して高い信号対雑音比の画像を維持します。システム。最後に、現在の技術によるタンパク質検出などのシステム関連マーカーの 1 つだけのクラスに私たちの調査を制限するのではなく、そのターゲット固有の専用プローブでは、複数の生物学的システムを評価する機会を提供します免疫組織化学的方法。
私たちの研究では、側坐核 (NAc) Drd1α 受容体の発現と中脳 (SNR) でチロシン水酸化酵素 (番目の) 式を評価する男性と女性/N の F344 ラットの交配その場で新規 RNA を使いました。交配その場で革新的な RNA では、DA 取り込みと DA 放出の両方に同時に影響を及ぼす、線条体ドパミン DA システムの複雑さの理解を向上させるメカニズムを調査することができました。新鮮凍結脳をスライスし、さまざまな染色パターンのためのデータ分析方法を提供するため、我々 はプロシージャを記述するここでは、:「離散ドット」または「クラスター」。

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">HybEZ Oven system</td>
			<td style="width:205px;">Advanced Cell Diagnostics&#160;</td>
			<td style="width:135px;">310010</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">RNAscope Probe - Rn-Drd1a</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>317031</td>
			<td>Color channel 1, Green</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">RNAscope Probe - Rn-Th-C2</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>314651-C2</td>
			<td>Color channel 2, Orange</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">RNAscope Fluorescent Multiplex Reagent Kit</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">ImmEdge Hydrophobic Barrier Pen</td>
			<td>Vector Laboratory</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">4% paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Tissue-Tek vertical 24 slide rack</td>
			<td>Fisher Scientific</td>
			<td>NC9837976</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Tissue-Tek staining dish</td>
			<td>Fisher Scientific</td>
			<td>NC0731403</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Precision General Purpose Baths</td>
			<td>ThermoFisher Scientific</td>
			<td>TSGP28</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Pretreatment 4</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">ProLong Gold anti-fade reagent</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:135px;">P36930</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Amp 1-FL</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Amp 2-FL</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Amp 3-FL</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Amp 4-FL-Alt A</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">EZ-C1 software package</td>
			<td style="width:205px;">Nikon Instruments</td>
			<td style="width:135px;"></td>
			<td>version 3.81b</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">SAS/STAT Software</td>
			<td style="width:205px;">SAS Institute, Inc.,</td>
			<td style="width:135px;"></td>
			<td>version 9.4</td>
		</tr>
	</tbody>
</table>

identification of dopamine d1-alpha receptor within rodent nucleus accumbens by an innovative rna in situ detection technology

中枢神経系 D1 α サブタイプ受容体 (Drd1α) は最も豊富なドーパミン (DA) の受容体で、神経の成長・発達の調節に重要な役割を果たしています。しかし、Drd1α 受容体異常行動応答を仲介してワーキング メモリ機能の変調メカニズムはまだ明らかではありません。交配アッセイの in situ新規 RNA を使用すると、現在の研究特定 RNA 発現核側坐 (NAc) エリアに位置し中脳領域 (SNR)、DA 関連回路から受容体とチロシン水酸化酵素 (TH) ドーパミン Drd1αそれぞれ。NAc の Drd1α 式は、「離散ドット」染色パターンを示しています。Drd1α 式で明確な性差が認められました。対照的に、第 5 回は、「クラスター化された」染色パターンを示します。番目の式について雌ラットはオスの動物と相対的にセルごとのより高い信号式を表示されます。紹介した方法は、中枢神経系疾患の進行中にドーパミン システムの機能不全の変化を調査するため小説の in situハイブリダイゼーション手法を提供します。

現在の研究は観察/N の F344 ラット (図 1 a) で NAc のドーパミン D1-アルファ受容体 (Drd1α) における RNA 発現のパターンを汚す「離散ドット」です。個々 の蛍光信号は容易に識別された、セル内の 1 つの RNA 転写産物を表す単一の「ドット」として見ることができます。「離散ドット」染色パターンを表示する、NAc からイメージの半定量分析 (<strong ...

Watch this Scientific Journal Video about Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology at JoVE.com

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

検出技術その場で革新的な RNA 側坐齧歯動物の核内ドーパミン D1-アルファ受容体の同定

側坐核ドーパミン D1-アルファ受容体の同定は、中枢神経系疾患の中に D1 受容体の機能不全を明らかにするため重要です。特定の脳領域の RNA 分子を可視化する交配アッセイの in situ新規 RNA を行った。

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

identification-dopamine-d1-alpha-receptor-within-rodent-nucleus

Research

JoVE Journal

Neuroscience

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

8.5K ビュー.  University of South Carolina. 側坐核ドーパミン D1-アルファ受容体の同定は、中枢神経系疾患の中に D1 受容体の機能不全を明らかにするため重要です。特定の脳領域の RNA 分子を可視化する交配アッセイの in situ新規 RNA を行った。

ビデオ: Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

齧歯類の脳における同時fMRIと電気生理学

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

神経科学

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker、眼球運動解析への革新的な自動化されたアプローチ

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

齧歯中枢神経系への薬物の直接脳室内デリバリー

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

蛍光による神経組織の持続的なDNAウイルスのゲノム転写物の検出その場でハイブリダイゼーションは、免疫染色と組み合わせる

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

高解像度を使用した脳切片における軸索のローカライズされたmRNAの検出<em&gt;その場での</em&gt;ハイブリダイゼーション

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

微小電極アレイへの3Dエンジニア神経文化のインタフェース：革新的な<em&gt;インビトロ</em&gt;実験モデル

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

RNA配列決定およびTH-プロモーター駆動EGFP発現を使用して中脳培養における生活ドーパミン作動性ニューロンの信頼性の高い同定

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Locust AntennaeにおけるRNAによる嗅覚受容体遺伝子の局在<em&gt;現場で</em&gt;ハイブリダイゼーション

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高速液体クロマトグラフィー分析とシナプトソーム ドーパミンによるマウスにおけるドーパミン作動性恒常性の評価

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

並列の単核 RNA 配列のため大人の脊髄核の分離

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...