著者は、拡張された条件付け場所嗜好プロトコルを確立する彼らの助けのためにアンニリーとモーリーンバーンに感謝したいと思います。

すべての手順は、ワイルコーネル医科大学制度動物実験委員会の規則に従って行われている。 1。ウイルスベクターの定位配信のための準備とセットアップ<ol><li>楽器、滅菌綿棒、または滅菌手袋を着用して手が非滅菌表面に接触し、破棄するかホットビーズ滅菌器を使用して再滅菌する器具によって汚染されている場合は、新しい滅菌手袋に綿棒または変更を破棄します。 </li><li>術後の回復のために温めるヒートブロックで寝具とマウスのケージを置きます。 </li><li>頭皮を殺菌するために頭とエタノールとヨウ素を剃るための電気かみそりを設定します。 </li><li> 70％エタノールでstereotaxの耳のバーや口のホールドを拭う。 </li><li> 5μlのハミルトンシリンジを用いて、ウイルスベクター（1×10 6ウイルス粒子/μl）を5μlのを策定し、stereotaxにシリンジホルダーに設置した。 </li><li>マイクロ波加熱パッドまたは上の電気パッドを回すstereotax上番目の場所。火傷のリスクを軽減するために、クリーン吸収パッドでカバーしています。モニタ、マウス体温（97から99.5°Fの間に）頻繁に手順全体。 </li><li>ケタミン（100 mg / kg体重）およびキシラジン（20 mg / kg体重）の混合物でマウス（22〜35グラムの間で計量）を注入。マウスを麻酔したら、マウスの反射神経をチェックして、マウスが十分に麻酔であることを保証するために、任意の動きや反応を注意するの尾先端をつまむ。 </li><li>耳と目の間に頭を剃る。 </li><li>エタノールとヨウ素滅菌綿アプリケーターを交互に坊主頭を清掃します。 </li><li> stereotax上の加熱パッドにマウスを転送します。 </li></ol> 2。ウイルスベクターの定位マイクロインジェクション<ol><li>手術全体に滅菌手袋を着用してください。手袋非滅菌表面に触れることにより、任意の時点で汚染されている場合は、手袋を変更してください。 </li><li>ゼロの歯バーと耳のバースケール。 </li><li>歯バーに歯をセットします。 </li><li>銃口のネジ。揺らすまたはウィスカ移動した場合リーメントはいつでも注目される、ケタミン（100 mg / kg体重）およびキシラジン（20 mg / kg体重）の混合物の腹腔内ブースター用量（0.05〜cc）で管理する。 </li><li>耳バーのネジ。 </li><li> PuraLubeで目に注油。 </li><li>メスでただ後ろに耳に開いて頭皮をカットします。 </li><li>皮膚剥がれ頭蓋骨から頭皮の角の上に置いてブルドッグクリップ。ブレグマとラムダが簡単に表示されるはずです。 </li><li>ブレグマからラムダへの縫合糸がまっすぐに整列していることを確認してください。縫合糸は、頭部の曲がっ再調整ポジショニングである場合。 </li><li> stereotax上のすべての設定がゼロにされていることを確認してください。 </li><li>ブレグマで所定の位置に針先を入れてください。 </li><li>ブレグマとラムダの両方のすべての座標を測定します。これは、腹側/背内側/横と前方/後方の座標が含まれています。 </li><li>ブレグマとラムダのために腹座標が互いに0.02 mm以内になるまで頭を調整します。 </li><li>一度内側/横（M / L）座標に内側に出かけるブレグマから、整列。ことを確認してください左右両側が互いに0.02 mm以内に整列される。 </li><li>ブレグマ上で、そこから再センター針の先端は、目的の前方/後方の座標に（/ P）前方/後方方向に移動する。 </li><li>必要に応じて、角度一方向に針とstereotaxがロックされていることを確認してください。 （ - 1.52 + / - ）左M / Lの座標に出て行く</li><li>ボーリング孔を掘削した後にリファレンスとして使用するために頭蓋骨の上部の腹の位置を測定します。 </li><li>希望の座標に無菌のドリルビットを使用して掘削孔を開けます。針が頭蓋骨を通して中断なく入力することができますことを確認します。 </li><li>希望の腹側の座標に針を下ろし。 </li><li>一度座標はディップ0.015ミリメートルはウイルスベクターのための小さなポケットを作成するために〜10秒以下、到達する。 </li><li> 〜0.1μL/分の速度でウイルスベクターの所望の体積を注入する。完全に拡散するウイルスベクターのために3分待ちます。針先0.015ミリメートルを上げ、さらに2分待つ。 </li><li>反対方向に針を（斜め注射を行う場合）を浸します。繰り返しになりますが、頭蓋骨の上部の基準として腹位置を記録。 </li><li> （二国間の注射をしている場合）の反対側にウイルスベクターを注入する第一の側と同じ手順を繰り返します。 </li><li>滅菌綿棒先端の木製の終わりを使用してそれらを密封するために、両方の穴に骨ワックスを適用します。 </li><li>頭皮を縫合。 </li><li>負傷者の領域に神経ブロックを適用します。 </li><li> 45分間回復するためのヒートブロック上で温めケージにstereotaxから、マウスと場所を削除します。 </li><li>含む痛みの兆候を監視します、全体的な活動や落ち着きのなさを減少させた食品や水の消費または増大発声を減少させた。ブプレノルフィン（0.05から0.2 mg / kg体重）は、痛みのため、必要に応じて一週間、毎日二回投与することもできる。許容可能な選択肢が含まれます：メペリジン（20-60 mg / kg）を、ペンタゾシン（10 mg / kg）を、ナルブフィン（4-8 mg / kg）を。 </li></ol> 3。条件付け場所の人種：Acquisitionは、絶滅、そして復活<ol><li>ギロチンドアが60秒の馴化期間の3室の場所嗜好装置（メッドアソシエーツ社、セントオールバンズ、VT、USA）の中央の室へ場所マウスは閉じた。 </li><li>馴化後、オープンギロチンドアやMedPC IVソフトウェア（メッドアソシエイツ社）を用いて記録されている各チャンバーに費やされる時間と1200のためのすべての3室のマウス自由探査秒、許す。 </li><li>プレテストの間に彼らのパフォーマンスに基づいて空調室にマウスを割り当てます。ベースラインの事前テスト中に最も好まれたコンパートメントとのその後の3日間、バイアス設計、ペア薬物投与を使用。 </li><li>午前中のセッション中に、注射の直後1200秒、割り当てられた部屋でそれらを閉じ込める、次の3のコンディショニング日の間に、コカイン（IP 10 mg / kg体重）をマウスに注入する。セッションの後に自分のホームケージにマウスを返します。 4時間後、生理食塩水（0.01ミリリットル/ gのマウスに注入する体重）と1,200秒午後のセッションのために反対室にそれらを閉じ込める。 </li><li>取得のためにテストするには、ギロチンドアと中央のチャンバー内の場所のマウスは60秒の馴化期間のため閉鎖。オープンギロチンドアやMedPC IVソフトウェア（メッドアソシエイツ社）によって記録されたすべての室で過ごした時間を1,200秒のための無料の探査を可能にします。コカインペア室に費やされる時間の量から生理食塩水対のチャンバ内で費やされた時間を減算することにより優先順位を計算する。 </li><li>同一装置で毎日無料探査の2 20分のセッションにマウスを公開、場所嗜好を消​​すためには、買収のためのテスト後24時間を開始し、少なくとも2時間で区切られています。すべてのチャンバに費やされた時間は再度記録し、初期CPPの大きさがそれぞれ消光セッションのそれと比較されるべきである。 </li><li>マウスはPAR用かどうかの好みを評価することによって、薬剤誘発性の好みを消滅しているかどうかを判断する二つの連続消光日にわたるticular室は、取得嗜好に比べて著しく低い。 </li><li>コカインのプライミング用量（5または10 mg / kg体重、腹腔内）でマウスを元に戻すと無料探査を許可装置に再公開し、各区画にかかった時間を記録します。 </li><li> Euthasol安楽死溶液（150 mg / kg）を管理します。 Transcardially 4％パラホルムアルデヒド（PFA）をマウスに灌流し、正しいマイクロインジェクションの配置を検証するための組織学的検査を行う。 </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+activation+of+lateral+amygdala+pyramidal+cells+instructs+associative+fear+learning.">Optical activation of lateral amygdala pyramidal cells instructs associative fear learning.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (28), (2010).</li><li>Zhang, F., Gradinaru, V., Adamantidis, A. R., Durand, R., Airan, R. D., de Lecea, L., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogation+of+neural+circuits:+technology+for+probing+mammalian+brain+structures.">Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures.</a> Nature Protocols. 5 (3), (2010).</li></ol>

著者らは、開示することは何もありません。

絶滅と復職段階を含むCPPと組み合わせてウイルスベクターの定位マイクロインジェクション経由地域と時間的に特異的な遺伝子アブレーションは中毒性の​​ような行動の3つの異なる段階に遺伝子の特定の貢献の調査を可能にします。伝統的なCRE-のLoxPシステムを利用して達成され、条件付きノックアウトは空間、時間的制限遺伝子アブレーションに提供していますが、定位loxP導入マウスの...

コカインは非常に強化精神刺激薬である。反復暴露に続いて、いくつかの分子や細胞適応が強迫的になると考えられている報酬関連脳回路で発生する薬物探索重大な臨床的問題1ポーズ再発率の高さを促し、行動を。コカインは、遺伝子発現を調節することによって、これらの長期的な行動効果を発揮する。慢性コカインの使用から生じる適応を研究するために、前臨床げっ歯類モデルは、広く使用されている。そのようなモデルは、条件付け場所嗜好（CPP）パラダイムである。このモデルは、以前に中立的な環境とコカインのやりがいの特性と学び関連の開発が含まれます。特定の室とコカインのいくつかのペアリング後、動物を自由にコカインペアと非コカインペア環境を探索するために許可されていると、彼らは薬物ペアコンパートメントを好む場合は、それらは、コカイン誘発性のプラクを取得していると言われています電子好み。また、絶滅の訓練期間の後、このパラダイムはコカイン探索行動のコンテキスト固有の復職を研究するために使用することができます。 より正確に中毒性の模倣するように考えられているような長期的なコカイン誘発行動と分子可塑2,3と自己管理（SA）を研究するために使用されている精神感、などの中毒性のような行動、他のビヘイビア·モデルに比べのような人間に見られる行動、CPPパラダイムは、コカイン文脈学習4を勉強するための簡単な手順です。 CPPプロトコルは便利に薬物渇望のメカニズムと再発5-7との調査を可能にSA、同様に、絶滅と復職段階を含むように拡張することができ、人間の薬物探索行動と薬物で発生するものの側面を要約すると考えられている-とキュー誘発再発8-10。 その根底に一つのメカニズム買収、絶滅、そして復職など、CPPの異なる相のそれぞれの特徴であるコカイン誘発性行動の可塑性、異なる脳領域内の遺伝子発現のユニークな署名の活性化である。直接コカイン誘発性の行動変化を仲介報酬関連脳領域内のどの遺伝子テストするには、それは、地域固有の方法で選択的にそれらを操作できるようにすると便利です。これを達成する1つの方法は明白CreリコンビナーゼはloxP部位（loxPにはさまれたマウス）に挟まれた標的遺伝子を有するマウスの脳の異なる領域に、定位手術を使用している組換えアデノ随伴ウイルス（rAAVの）ベクターを顕微注入することである。この方法では、いつ、どこで遺伝子が11-13免疫応答を誘発することなく、両方の分割および非分割ニューロンにおいて、アブレーションさにわたって非常に正確な時間的·地域的に制御できます。このレベルの制御は、loxP導入マウスのw繁殖の伝統的なCRE-のLoxP技術上の重要な利点を表しという点で、内因性遺伝子プロモーターの制御下でCreリコンビナーゼを発現するマウスは、i番目のタイミングおよび遺伝子欠失の分布をより強固に規制することができる。また、ウイルスベクター媒介遺伝子ノックアウトは、伝統的なノックアウト戦略を使用して発生する可能性のある潜在的な発達代償効果を回避。 ここに記載されているコカインCPPに加えて、特定の遺伝子の役割を評価するためのloxPにはさまれたマウスの脳内のrAAV-Creのマイクロインジェクションは、自己投与及び精神運動感作を含む別々の相を含む行動のパラダイムに遍在的に適用することができる。例えば、我々の研究室では、コカイン精神運動感14中のCa V 1.2 L型Ca 2 +チャネルの役割を研究するためのrAAV-Creの技術を利用しています。具体的には、するrAAV-Creをを実証するためにコードする遺伝子のCa V 1.2 loxP導入によるマウスの側坐核（NAC）にマイクロインジェクションしたことのCa V </suロ&gt; 1.2この領域を仲介に精神感14,15の発現位相を演技。精神感中のCa V 1.3 L型Ca 2 +チャネルの役割を評価する際に、目的のloxP導入遺伝子を持つマウスが存在しない場合は、するrAAV-Creの戦略を使用することができない、などの私たちの経験であった。条件付きマウスは、関心のある特定の遺伝子のために存在しない場合、したがって、のrAAV-Creリコンビナーゼを用いた制限は、それ自身を示す。しかし、我々はカルシウムバージョン 1.3チャンネル14,15の役割を調べるために行ったように明白なsiRNAは、ノックダウンの標的遺伝子に使用することができることrAAVs。 loxP導入マウスの離散的な脳領域にするrAAV-Creををマイクロインジェクションした後、CPPパラダイムでそれらをテストする中毒性のような行動の様々な段階を媒介し、彼らが行動している特定の遺伝子の調査を可能にします。このパラダイムを使用するには、本質的に繰り返しコカイン投与がBrをハイジャックする方法についての我々の理解で支援しているAINSの報酬回路は中毒状態1,16,17につながることを分子シグナル伝達経路と遺伝子発現の不適応な変化を引き起こす。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Conditioned place preference activity chambers</td>
			<td>Med Associates, Inc., St. Albans, VT, USA</td>
			<td>MED-CPP-MS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereotaxic alignment system for mouse</td>
			<td>David Kopf Instruments, Tujunga, CA, USA</td>
			<td>model 900</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hamilton syringes</td>
			<td>Hamilton Company, Reno, Nevada, USA</td>
			<td>7634-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-Cre-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7004</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

stereotaxic microinjection of viral vectors expressing cre recombinase to study the role of target genes in cocaine conditioned place preference

興味のある選択的にノックアウト遺伝子に異なるマウス脳領域にCreリコンビナーゼを発現する組換えアデノ随伴ウイルス（rAAVの）ベクトルをマイクロインジェクションすると、既存の方法に比べ、遺伝子欠失の強化された時間的及び地域固有の制御を可能にします。条件付き欠失はまた、loxPにはさまれた遺伝子を有するマウスと特異的遺伝子プロモーターの制御下でマウスを発現するCreリコンビナーゼを交配することによって達成することができるが、定位マイクロインジェクションは、関心のある実験的に決定時点で離散的な脳の領域の標的を可能にする。コカイン条件付け場所嗜好のコンテキスト、および撤退、消滅、および/または復職段階を含むことができるような自己管理や精神運動感など他のコカイン行動のパラダイムでは、この手法では、これらの個別に標的遺伝子のユニークな貢献を探索に特に有用であるコカイン誘発性可塑性のビヘイビア·モデルの位相。具体的には、この手法は、時間を越え行動への貢献をテストするための行動の離散段階で標的遺伝子の選択的切除が可能になります。最終的には、このような理解は、中毒性の行動の各段階の間に自分自身を提示する最も強力なリスク要因に対処する最善のことができる、よりター​​ゲットを絞った治療を可能にします。

CPP マイクロインジェクションマウスでCPPを実行した後、一方は制御注入のコホート（のrAAV-GFP）マウスは、通常の薬物ペアリング室（ 図1A、1B）の優先順位を取得していることを確認する必要があります。マウスは、コカインの好み（生理食塩水ペアリング室で過ごしたコカインペア室マイナス時間で費やされた時間）がベースライン嗜好（ 図1B、対B）<...

Watch this Scientific Journal Video about Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference at JoVE.com

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

この記事では、マウスの脳にウイルスベクターを顕微注入すると、その後買収、絶滅と回復期を含む条件付け場所嗜好パラダイムでテストする方法を説明します。

コカイン条件付け場所嗜好における標的遺伝子の役割を研究するためにCreリコンビナーゼを発現するウイルスベクターの定位マイクロインジェクション

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

stereotaxic-microinjection-viral-vectors-expressing-cre-recombinase

Research

JoVE Journal

Behavior

18.5K ビュー.  Weill Cornell Graduate School of Biomedical Sciences. この記事では、マウスの脳にウイルスベクターを顕微注入すると、その後買収、絶滅と回復期を含む条件付け場所嗜好パラダイムでテストする方法を説明します。

ビデオ: Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

の定位注入マイクロRNA発現マウス海馬CA1領域にレンチウイルスと行動アウトカムの評価

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

行動学

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

禁欲ラットで探しているヘロインに長期の食事制限の影響を研究するための手順

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

脂肪嗜好：摂食行動の新規モデルラットにおける

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

ラットにおけるパブロフ付きアルコール探索行動の状況誘発性のリニューアルを観察する手順

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

動機付け行動の分子基質を研究するために齧歯類の脳マイクロインジェクション

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

コカイン誘発性行動感作の評価とマウスにおける条件付け場所嗜好

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

オープンフィールドタワー迷路に置き、レスポンス学習

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

エアコンの場所の好みのパラダイムを使用してマウスの薬物探索の復権

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

ラットにおける渇望のインキュベーションを測定するためエアコンの場所設定プロトコル

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

Assessing the Coherence of Parents&#039; Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

5分間の音声サンプル手順を用いた子どもに関する親の短い物語の一貫性の評価

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...