Name: protocol for studying extinction of conditioned fear in naturally cycling female rats
Uploaded: 2015-02-23T14:00:00.000+00:00
Duration: 9 min 7 s
Description: Watch this Scientific Journal Video about Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats at JoVE.com

Mohammed R. Milad is supported by NIMH grant R01 MH097880 and institutional funds from the Department of Psychiatry at MGH.

注：このプロトコルですべての手順は、マサチューセッツ総合病院のための施設内動物管理使用委員会（IACUC）となって研究動物管理小委員会によって承認され、健康のガイドラインの国立研究所に準拠しているされています。 1.動物住宅及び取扱い手順<ol><li>木材チップの寝具3-4インチのプラスチックビンのグループで到着すると、家の大人のSprague Dawley雌ラット（200〜225グラムの重さ）、年齢の約8〜10週間。固形飼料および水に自由摂取へのアクセスの食事と12時間の明/暗サイクルでそれらを維持する。 1週間の最低住宅事情と動物施設に順応するために、ラットのままにしておきます。 注：この原稿のすべての手順は、光サイクル中に行われている。 </li><li>この順応期間の後、2〜3日間、5分ごとに、すべての動物を扱う。適切に（胴体によってませ尾でサポートされている）一度にラット1を拾う、確実に固定して保持し、この時間の間に、それらをペット。激しく揺れる、またはより長い期間のために心配して、ストレスをより多く表示された動物を扱う。このプロトコルでは、次の手順に影響を与える可能性があり、ストレスを扱う最小化するためにこれを行います。 注記：動物の取り扱い時の日数および量は、異なる研究室間で変化させることができる。しかし、これらは、同じラボ内での実験の間で一貫している必要があります。 </li></ol>膣スワブスミアと染色手順：発情周期を監視する2. <ol><li> 0.9％食塩水（塩化ナトリウムを蒸留水に溶解）を作る。この溶液中で綿棒を湿らせます。不十分なサンプル収集または細胞の喪失につながる可能性が過飽和を回避するためにペーパータオルを使用して先端ブロット。上向きに離れて膣口から尾を持ち、静かに緩い膣の細胞を捕捉するために、壁の周りの膣管とロール状に綿棒を挿入します。 </li><li>動物はベッドに排尿した場合尿汚染が相同定のために、それが困難になりますようにトンの先端は、新しいものと交換してください。ストレスへの暴露が発情周期を混乱されるため、この手順の間に動物に苦痛を与えないよう十分に注意してください。拭き取りすると、偽妊娠を誘導することができる子宮頸部への余分な刺激を最小にする深すぎないが、迅速挿抜を実現することを目指しています。 注：偽妊娠、 すなわち典型的には約12日間続く ​​非環発情周期、発情25の連続した日として同定することができる。 </li><li>慎重にアプリケータ先端部を除去し、予め標識顕微鏡スライド上にそれをロール。スライド上あまりにも多くの流体としてあまりにもハードダウン押すとドライ一度相同定を困難にすることができないようにしてください。 注：これは個人の好みに応じて行うことができるが、一つのスライドは、各動物の複数の日を含むことができる。 </li><li>スライド上のサンプルの転送を確認してください。断つためのサンプルを収集1スライド上のAl日間連続、より追跡できるようにし、毎日の発情期の変更を識別します。 注：可能な場合はサンプルを収集する人々は、すべての動物のために同じままなら、それはまた、理想的です。 </li><li>スライドが乾燥していると、位相識別のための染色プロトコルを開始する。染色キット（材料の表を参照）を使用し、固定液（水色）のスライドを10回浸す、（ピンク）染色液で10倍、と対比溶液中の5倍（紫）。流水で軽くスライドをすすぐ。顕微鏡下で表示する前にスライドを完全に空気乾燥してみましょう。 NOTE：発情（E）、発情（M）、発情間期（D）、および発情前期（P）26：一般的に4~5日長い発情周期を（この順序で）で構成されている。発情期は、青、角化細胞によって特徴付けられる白血球による発情及び角化および有核細胞の組み合わせ、それらは非常に疎である以外は、発情間期細胞型の存在下での発情と同様であり、紫色に染色された凝集によって発情さの有核細胞（ 図1）。膣スミア診のために等しく有効である上述のものに加えて、種々の染色手順は、がある。 </li><li>実験前に正確に位相を監視するために、少なくとも2つの完全なサイクル（〜8〜10日）のためにサンプルを収集する。サンプルが取られる時間帯と一致している。全体と実験の終了まで毎日の膣スメアサンプルを収集します。 </li></ol> 3.事前暴露<ol><li>実験の開始前に、チャンバーは動物がどんな行動試験の前にコンテキスト（空調室）に順応できるように調節する動物​​を事前に公開します。 <ol><li>すべての実験の前に3日間、一日あたり20〜30分に家のライトが付いている（音減衰ボックスにあります）空調室にメスを事前に公開します。彼らは研究のための彼らの室と事前にさらされているボックスを割り当てます。完全に洗浄トン彼らは行動と発情周期に影響を与えることができるので、臭いを除去するためのセッションと動物の間、彼は箱（壁やトレー）。 </li></ol></li></ol> 4. 1日目：馴化/恐怖条件付け<ol><li>動物は彼らの発情周期の発情期にあることが確認された後、3日間の実験の馴化/コンディショニングフェーズを開始。 <ol><li>実験前の各日に刺激（CS）のプレゼンテーションを条件付け最初のトーンに凍結の前トーンベースライン対策を行ってください。 CS発症前の最初のCS-単独の裁判の開始時に刺激不在の時間間隔の間に（100を掛けた裁判の期間によって不動費やし秒で除して算出、）％の凍結のスコアを取得することによってこれを行います。 注：これらの対策は、馴化、コンディショニング、消滅、およびリコール時とベースライン凍結レベルを評価し、比較することができる。 </li><li>発情後期フェーズの間に絶滅の研修を実施する、発情期の彼らの最後の日に、ラットを条件付け。綿棒と識別する発情期（ステップ2）実験プロトコル（空調/絶滅/リコール）を開始する前に、毎日。発情期は1日より長く続くことのような動物がトレーニングを開始する準備ができたら、使用サイクル履歴が決定するために、位相の長さを予測する。 </li></ol></li><li>空調室では、電気刺激発生器に足ショックグリッドの床に接続します。無条件刺激（US）として0.5〜0.6ミリアンペアの刺激レベルを使用します。条件刺激（CS）として4 kHzの、80デシベルの音を使用してください。 注：これらのパラメータは、実験室間で異なることができ、最適なコンディショニングのために修飾することができる。 <ol><li>凍結解析プログラムは、視覚的な手がかりを（一時的ではなく）が必要な場合は、トーンにLED光を共活性化する。ビデオフレーム内の、オペラントチャンバーの外側に光を置きます。各試験の開始時に、LEDライトを活性化し、それが目の終わりまで照らされ続けるEトライアル。 注：この光は動物では表示されませんとだけ開始及び（凍結挙動を測定するために使用される）を記録したビデオファイル内の試験終了のためのマーカーとして役立つ。 </li></ol></li><li>慣れの5 CS-単独の試験のための空調室に動物を置きます。すぐに慣れセッション以下、恐怖条件付けの7ペアCS-USの試験を行う。 CS音が30秒間持続し、0.5秒、米国のショックにcoterminatesていることを確認してください。 3分を平均可変試行間間隔ですべてのセッション（慣れ、エアコン、絶滅、およびリコール）で試験を実行します。 </li><li>コンディショニングセッションの最後に、次の日まで、ホームケージに及び動物施設に動物を返す。 </li></ol> 5. 2日目：エストラジオール準備<ol><li>このプロトコルの1日目を受けたすべての動物のための発情期を綿棒識別する。エストラジオールを準備します（またE2、、17β-エストラジオール、またはとして知られているビヒクルとしてゴマ油または0.9％生理食塩水を用いて15μgの/ kgの用量の皮下注射のためのベータ - エストラジオール）。 </li><li>注射の準備をそれらがこのプロトコルの1日目に調整したときに測定し、記録した全ての動物の体重を使用するエストラジオールの量を算出する。 </li><li>ラットあたりエストラジオール（ すなわち 0.2ミリリットル）の投与量を標準化するために計算を行う。エストラジオールは、その水不溶性に溶解させることは困難であり、少量のために大量の溶媒を必要とするので、溶解するまで、弱火ゴマ油中のエストラジオールを混合することにより原液を調製。次いで、溶液を汚染物質を除去するために0.22μmのフィルターを通過させることができる。例えば、動物当たり0.2ミリリットル注入量では15μg/ kgの用量を達成するために、ごま油の合計に必要な体積に3000μgのエストラジオール/ 1ミリリットルゴマ油原液の計算されたボリュームを追加する。 注：あるいは、エストロゲンTHAの他の形態を使用する<html> tは、水溶性および溶解が容易である。 </li></ol> 6. 2日目：エストラジオール管理/絶滅トレーニング<ol><li>エストラジオールを皮下投与する。穏やかなピンチに首、肩甲骨の間でたるんだ皮膚を持ち上げ、皮膚折り目によって形成される三角形に注射針を挿入します。 </li><li>エストラジオールの皮下注射後30分、再びコンディショニング室で動物を配置し、絶滅のトレーニングを始める。この絶滅セッションでは、3分の平均を可変試行間間隔で発表され、30秒、4 kHzの、80デシベルの音、からなる各試行で、20 CS-単独の試験を行う。 注：エストラジオール前絶滅の訓練にか、すぐに絶滅後に投与されたときにリコールに対するエストラジオールの効果が発生します。効果は4時間後に絶滅を観察していないので、それはトレーニングセッション15の開始または終了に時間的に近い注入することをお勧めします。 </li></ol>ル &quot;&gt; 7 3日目：絶滅リコール<ol><li>絶滅のトレーニング後24時間、1日目とこのプロトコルの2を完了した動物が綿棒。 </li><li> （1日目と2と同じ）、割り当てられた空調室に動物を置きます。ステップ6.2で説明したように絶滅の訓練のために投与20試行に似て試行間間隔を持つ唯一の3.80デシベルCS-単独の臨床試験で構成されて3 CS-単独の臨床試験（絶滅のリコールの3件の試験）を、提示することによって、絶滅リコールセッションを開始します。 </li><li>リコールセッションが完了すると、ホームケージに動物を返す。 </li></ol> 8.データ解析<ol><li>映像を通じて行動データを取得し、コンピュータソフトウェアを使用して分析する。 注：凍結スコアは手動でも各試行のCSプレゼンテーション中に凍結発作のタイミングによる薬物治療に盲目である実験者によってカウントすることができ、音の間に凍結した時間の割合として表さ。凍結がある30秒CS-USの試験中の呼吸を除いて、運動の完全な欠如によって定義される。 100を掛けた30秒（トライアルの期間）によって（不動）を凍結費やし秒で割って％の凍結を計算します。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17&#946;+Estradiol+Increases+Resilience+and+Improves+Hippocampal+Synaptic+Function+in+Helpless+Ovariectomized+Rats.">17&#946; Estradiol Increases Resilience and Improves Hippocampal Synaptic Function in Helpless Ovariectomized Rats.</a> Psychoneuroendocrinology. 42, 77-88 (2014).</li><li>Grueso, 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=Early,+but+not+late+onset+estrogen+replacement+therapy+prevents+oxidative+stress+and+metabolic+alterations+caused+by+ovariectomy.">Early, but not late onset estrogen replacement therapy prevents oxidative stress and metabolic alterations caused by ovariectomy.</a> Antioxidants and Redox Signaling. 20 (2), 236-246 (2014).</li><li>Shughrue, P. J., Bushnell, C. D., Dorsa, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+messenger+ribonucleic+acid+in+female+rat+brain+during+the+estrous+cycle:+a+comparison+with+ovariectomized+females+and+intact+males.">Estrogen receptor messenger ribonucleic acid in female rat brain during the estrous cycle: a comparison with ovariectomized females and intact males.</a> Endocrinology. 131 (1), 381-388 (1992).</li><li>Frye, C. A., Erskine, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+time+of+mating+and+paced+copulation+on+induction+of+pseudopregnancy+in+cyclic+female+rats.">Influence of time of mating and paced copulation on induction of pseudopregnancy in cyclic female rats.</a> Journal of Reproduction and Fertility. 90 (2), 375-385 (1990).</li><li>Becker, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+and+methods+for+research+on+sex+differences+in+brain+and+behavior.">Strategies and methods for research on sex differences in brain and behavior.</a> Endocrinology. 146, 1650-1673 (2005).</li><li>Westwood, F. 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+female+rat+reproductive+cycle:+a+practical+histological+guide+to+staging.">The female rat reproductive cycle: a practical histological guide to staging.</a> Toxicologic Pathology. 36 (3), 375-384 (2008).</li><li>Hurn, P. D., Macrae, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+as+a+Neuroprotectant+in+Stroke.">Estrogen as a Neuroprotectant in Stroke.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 20, 631-652 (2000).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology and Pharmacotherapeutics. 1 (2), 87-93 (2010).</li><li>Gillies, G. E., McArthur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+actions+in+the+brain+and+the+basis+for+differential+action+in+men+and+women:+a+case+for+sex-specific+medicines.">Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines.</a> Pharmacological Reviews. 62 (2), 155-198 (2010).</li><li>Tolin, D. F., Foa, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+trauma+and+posttraumatic+stress+disorder:+a+quantitative+review+of+25+years+of+research.">Sex differences in trauma and posttraumatic stress disorder: a quantitative review of 25 years of research.</a> Psychological Bulletin. 132, 959-992 (2006).</li></ol>

この原稿の著者は、開示することはなく、競合する経済的利益やその他の利害関係はありません。

恐怖の絶滅は、様々な操作の下で特定され、評価され、馴化恐怖消去を仲介する神経機構と雄ラットで広く研究されてきた。比較的少数の研究は、雌ラットや恐怖消去における性腺ホルモンの役割を検討した。特に恐怖消去に対するエストロゲンの効果を研究するために、自然に循環する雌ラットは、3日間の行動パラダイムに供される。この手順では、馴化/エアコン、絶滅、絶滅リコール?...

固有性差は、ヒトとげっ歯類の両方で様々な認知行動や学習パラダイム間で観察される。例えば、男性は、良好な空間能力1-3を有し、一方、女性は、一般的に、より強力な口頭および詳細指向能力を有することが報告されている。これらの性差は、性腺ホルモンの影響に一部起因することができる。高いエストラジオールレベルは、女性がより良いである、タスクのパフォーマンスを向上させることが、男性は一般的にうまく4-6を行うタスクのパフォーマンスを悪化させる。この証拠は説得力があるが、完全にヒトでの研究で、実験環境を制御するために、限られた能力は、行動上のこれらの効果はホルモンに特異的に起因することができた場合、それが困難に決定することができる。動物実験は、対照的に、完全に制御された状況を可能にする。 恐怖消去の基本的な神経機構が同定されており、よく男性で研究されているが、かどうかは不明であるこれらのシステムは、女性またはそれらがどのように発情周期にわたって変更は同じである。恐怖条件付けと絶滅広く不安障害に関連する研究を行うために、げっ歯類およびヒトにおける行動パラダイムを使用している。女性は、不安障害のリスクが高いだけでなく、より高い症状の持続期間および重症度7~13を有することを考えれば、これらの研究において、女性を含めることが重要である。本研究では、女性の過少は、性ホルモンの行動への影響のための発情期の監視と会計の課題に起因すると考えられる。しばしば報告されていない行くか、または彼らの研究のための方法に記載されていないこの点に関して遭遇問題にメスを調べてやる研究所。 げっ歯類で証拠を新興恐怖消去における性差は生殖腺ホルモン14-18で変調されていることを示唆している。恐怖条件付けでは、動物は特定の刺激を恐れるように訓練されている。の未強化の発表数の後刺激は、動物はキュー、絶滅と呼ばれるプロセスを恐れることはない学ぶ。どれだけ動物を学習し、絶滅のトレーニングの後にいくつかの時間を与えられた吸光リコールテスト中に観察することができ、このタスクを学習するのメモリを統合します。絶滅のリコール時の少ない恐怖式が良い絶滅メモリの統合を示しています。私たちの研究室からの最近の知見は、エストロゲンは恐怖消去メモリの統合を調節し、絶滅のリコールを改善できることを示唆している。具体的には、発情前期に消滅している雌ラットは、発情周期の位相はその間エストロゲンレベルのピーク、絶滅メモリの展示強化保持を循環させる。対照的に、前または直後の吸光トレーニング15,16の後、外因性エストラジオールの投与によって改善することができ、低エストロゲン発情後期相表示比較的乏しい吸光リコールに消滅する雌。絶滅メモリの統合の神経機構（税込雌の生殖腺ホルモンの役割を）udingすることは明らかではない。 実験動物では、ホルモンの役割は、去勢および卵巣摘出などの侵襲的な外科的除去の手順を使用して調べることができる。手術からの回復に続いて、性腺ホルモンは、多くの場合、外因的行動のタスク19の実行中に操作されます。このアプローチは、性ホルモンについての重要な情報を提供し、それが20〜23（タイミングと投与量の面で）性腺ホルモンのよく制御された操作を可能にしているため便利ですしている。しかし、このアプローチは、発情周期にわたって生じる、天然に存在する変動の影響を評価せず、また、彼らは、それによってヒトの研究に並進可能性を制限する、「正常な」動物を表しています。これは、特定の発情周期の位相、及びサイクル内のエストロゲン受容体発現の変化で女性ホルモンのレベルがピークと衰退することを十分に文書化された、と卵巣摘出24の後。したがって、確実に高および低エストロゲン状態はそれらの寿命を通して、女性に与えることができるという効果を研究するために無傷の生殖腺とメスの研究を実施し、実験計画を改善する必要がある。 このプロトコルは、恐怖消去に関与神経生物学的システムでのエストロゲンの影響に焦点を当てています。 1）慎重に、発情周期を監視2）全身投与のためのエストラジオールの効果的な用量を準備し、3）恐怖条件、絶滅が含ま行動パラダイムに従っており、自然にサイクリング雌ラットをでリコール：それはどのように説明しています。このプロトコルは、より良好な消光挙動恐怖条件付けで観察された性差を理解するために研究を支援するために他の薬理学的操作および細胞および分子ツールを使用して修飾することができる。以下の手順は、私たちの研究室内で利用するものである、と私はこれらの手順の多くの変形が存在することに注意してくださいnの文学。

<table><tbody><tr><td>Fear conditioning chamber</td><td>Coulbourn Instruments</td><td></td><td></td></tr><tr><td>Graphic State</td><td>Coulbourn Instruments</td><td></td><td></td></tr><tr><td>Sound-attenuating box</td><td>Med Associates, Inc.</td><td>NIR-022MD</td><td></td></tr><tr><td>Estradiol</td><td>Sigma-Aldrich</td><td>E1024</td><td>In sesame oil for subcutaneous injection</td></tr><tr><td>Sesame oil</td><td>Sigma-Aldrich</td><td>S3547-250ML</td><td></td></tr><tr><td>Freezescan</td><td>Cleversys, Inc.</td><td></td><td></td></tr><tr><td>Dip quick stain</td><td>Jorgensen Laboratories, Inc.</td><td>J0322A1, J0322A2, J0322A3</td><td></td></tr><tr><td>Cotton-tipped applicators</td><td>Fisher Scientific</td><td>23-400-114</td><td>6-inch, sterile</td></tr><tr><td>0.9% saline</td><td>LabChem, Inc.</td><td>LC23460-2</td><td>Sodium chloride w/v</td></tr><tr><td>Selectfrost microscope slides</td><td>Fisher Scientific</td><td>12-550-003</td><td></td></tr><tr><td>Virex II 256</td><td>Diversey, Inc.</td><td>5019317</td><td>Dilute 1:256 with water</td></tr><tr><td>Luer-Lok Tip 1ml Syringes</td><td>Becton Dickinson</td><td>309628</td><td></td></tr><tr><td>Hypodermic disposable needles</td><td>Exelint International, Co.</td><td>26402</td><td>26-gauge</td></tr></tbody></table>

protocol for studying extinction of conditioned fear in naturally cycling female rats

条件付け恐怖の絶滅は広範囲に男性のげっ歯類で研究されてきた。最近では、特定の行動のタスクとレスポンス行動のための神経機構は、女性と男性では異なっていることを示す研究が増えてきた。調査研究にメスを使用すると、それらの発情周期中の性腺ホルモンの変化の挑戦を表すことができます。このプロトコルは、雌ラットの恐怖絶滅メモリの統合におけるエストロゲンの役割を調査するのに有用である十分に確立された手順を説明します。発情周期と外因性エストロゲンの投与の前に消光訓練の位相が2​​4時間後に吸光リコールに影響を与えることができる。ここで説明発情相同定のための膣拭き取り技術は自然に性腺ホルモンをサイクリングの検査と操作を補助する。この基本的な齧歯動物モデルの使用は、中恐怖消去メモリを調節することができるエストロゲンメカニズムを描写することができるメス。

この恐怖消リコールプロトコルでは、パーセント凍結は恐怖の指標として測定した。よく消滅と絶滅の訓練の記憶を保持した動物は、絶滅のリコール時の行動試験の最終日に、低恐怖を示した。オスとメスのラットは大幅にエアコン、絶滅の間、条件付け恐怖の表情が異なり、相（ 図2）覚えていません。動物は高および低エストロゲン発情位相群として別々に分析されている場...

Watch this Scientific Journal Video about Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats at JoVE.com

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

当然の雌ラットをサイクリングで馴化恐怖の消滅を研究するためのプロトコル

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

protocol-for-studying-extinction-conditioned-fear-naturally-cycling

Universidad San Sebastian

Research

JoVE Journal

Behavior

13.3K ビュー.  Massachusetts General Hospital. Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

ビデオ: Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

マウスにおける恐怖条件付けをトレース

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

行動学

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats 

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

実験用ラットに求めてコカインのフットショック誘発性回復を勉強するための手順

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

神経科学

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

頭蓋放射線療法によって引き起こされた定量認知デクリメント

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

医学

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

ツールとしての迷走神経刺激は、絶滅の学習のための関連経路の可塑性を誘導するために

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

再固定ウィンドウの間に絶滅の訓練は恐怖の回復を防ぎ

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

恐怖のストレス強化学習、心的外傷後ストレス障害の堅牢な齧歯動物モデル

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation&#8217;s behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

ラットの拡張恐怖調節プロトコルを用いた恐怖インキュベーション

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the ...

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.

Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders.

凍結と飛行行動を研究する新しいパブロビアン恐怖コンディショニングパラダイム

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat ...

An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. 
The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.

Cat skin/fur odor exposure provides a reliable method to investigate neural circuits and mechanisms of defensive responses in rodents and may offer insight into mechanisms that mediate fear in humans. Here, we describe a protocol for investigating the role of the interoceptive cortex in threat responses in rats.

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal ...

恐怖条件付けを修正したマウスの能動的防御行動の研究

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

著者スポットライト:恐怖学習と消滅における防御行動の神経相関の探索

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

マウスの飛翔行動を誘導するための修正された恐怖条件付け

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active ...