NIH R01MH084894は、この研究の資金調達に参加しました。我々は、博士に感謝したいと思います。マウスの寄付と、この作品の撮影時に、その手術や行動施設の利用のためのマウントサイナイ医科大学でヤスミンHurd氏とスコット・ルッソ。

注：動物の飼育や心配事のためのすべての手順は、マウントサイナイ医科大学の施設内動物管理使用委員会（IACUC）規則に従って行いました。作業中は滅菌手袋を使用してください。 1.薬剤およびウイルスの準備<ol><li> 医薬品の準備 <ol><li> 0.9％の食塩水の12.9ミリリットルで100 mg / mlでケタミンおよび20mg / mlのキシラジンの0.75ミリリットルの1.35ミリリットルを溶解することにより15.0ミリリットルのケタミン/キシラジン麻酔薬を準備します。溶液を十分に混合します。 </li></ol></li><li> ウイルス調製物 <ol><li> mGlu2のクローンを作成し、mGlu2ΔTM4Nは、標準的なプロトコルは、以前に6を説明し、次のシストロン性単純ヘルペスウイルス（HSV）ベクター内に構築します。以前6,13,14に記載されているようにウイルス粒子をパッケージ化します。残基の置換アラ-677 4.40、mGlu2でのAla-681 4.44およびAla685 4.48 Ser686 4.40、pHについてE690 4.44とのGly-694 4.48 mGlu3（HA-mGlu2ΔTM4N）に以前6に記載されています。 注：これは、以前にキメラ構築HA-mGlu2ΔTM4Nが無傷のGタンパク質依存6シグナル伝達と細胞膜で発現することが示されました。 </li><li> C使用しないときには-80℃でウイルスベクターを保管してください。氷の上でウイルスベクターを解凍してから、10μlのアリコートに分注し。外科的手順については、氷上に保ちます。 </li></ol></li></ol> 2.手術<ol><li> 手術の準備 <ol><li>マウスを計量し、（詳細については、1.1.1を参照）をケタミン/キシラジンのカクテルの適切な用量でマウスを注入。 </li><li>適切に麻酔をかけた場合、疼痛応答のための足と尾を絞る参照するには、マウスをチェックし、応答を誘発することができない場合は、マウスが適切に麻酔されます。 </li><li>バリカンを使用して、鼻の先端に頭蓋底からマウスの頭を剃ります。マウスAFに眼科ゲルを適用しますマウスの失明を防ぐためにterward。 </li><li>定位フレーム上にそれぞれ注射器をロードします。彼らは10度法線から離れているように、そして、定位フレームの各アームの垂直部分を傾けます。針がお互いに直面しているように、アームが傾いていることを確認してください。 </li><li> 70％エタノールで針を充填することにより、各注射器を清掃してください。注射器がクリーンであることを確実にするために、針に少なくとも3回を埋めます。 </li><li>針が洗浄された後、二重蒸留H 2 Oで針を充填することによって針をフラッシュフラッシュたら、二重蒸留H 2 Oの1.3μlの各針を埋めます二重蒸留H 2 Oの0.3μLを解放するために、シリンジのプランジャーをねじります針の先端での水のビーズは、慎重に水を拭き取る場合。何も注射器から出てきていない場合は、完全にダウンしてプランジャーを押し、その後、注射器の洗浄を繰り返します。 </li><li>水を充填した後、次いで、シリンジを充填注射器を引き上げ空気の0.5μlの。 </li><li>空気や水が注射器に入ると、慎重にウイルス液の1.3μlのシリンジを埋めます。この時点で、シリンジ内の総容量は2.8μlであることを確認してください。再びウイルスの0.3μLを解放するために、注射器の先端をねじります。針の先端の液体ビーズは、慎重に液体を拭き取る場合。何も注射器から出てきていない場合は、完全にダウンしてプランジャーを押し、その後、注射器の洗浄を繰り返します。 </li></ol></li><li> 手術 <ol><li>頭蓋骨が水平かつ平坦になるように、定位フレームを調整することを確認して、定位フレームにマウスを接続します。露出した頭皮にpovodine - ヨウ素を適用します。メスを用いて、露出した剃ったエリア内の頭蓋骨の正中線に沿って矢状切開を行います。その後、頭蓋骨が露出したままであることを確認するために切開部位の皮膚にビュレットクランプを取り付けます。 </li><li>の縫合糸を露出させるために骨膜を溶解除去するH 2 O 2を使用して頭蓋骨。今ブレグマおよび縫合糸が見えること、頭蓋骨が水平であることを確認するために、定位フレームを調整するようにしてください。 </li><li>ブレグマと注射器の針の先端の位置を合わせ、ブレグマの座標を記録します。針が挿入されようとしているの座標を計算します。 <ol><li>吻側-Cauldal（RC）面については、記録されたRCのブレグマ座標（ブレグマから1.6）に1.6ミリメートルを追加します。背腹（DV）面については、記録されたDVのブレグマ座標（ブレグマから-2.4）から2.4ミリメートルを引きます。 </li><li>最後に、内側側横方向（ML）面のために、記録されたMLのブレグマ座標（ブレグマから2.6）に2.6ミリメートルを追加します。これは二国間の注射であるように、すべての座標は、左右両方の座標を記録するようにしてください。 </li></ol></li><li>希望の座標に針を持参してください。針を挿入し、ドリルで、マークされた領域をドリルダウンしようとしている場所の場所をマークします。 </li><li>綿の先端アプリケータのwiと余分な血液や骨片を離れPE。 </li><li>針の先端は、脳の表面に触れている頭蓋骨に針を持参してください。その後、ゆっくりとそれらを下げる目的の座標に針を下げます。 </li><li>針は、所望の座標になったら、ゆっくりと（合計0.5μLで）5分にわたって毎分針0.1μlとプランジャーをねじることによって、注射器の内容物を注入します。 </li><li>注入が行われた後、別の5分間の皮質に注射器を残します。 </li></ol></li><li> アップクローズ/ケア <ol><li>着実に、ゆっくりとマウスの皮質から針を外します。そして、定位フレームからマウスを削除します。 </li><li>切開部から皮膚のベースフラップにシアノアクリレート（皮膚接着剤）を塗布した後、ピンセットで皮膚のフラップをつかみ、それらを一緒に配置します。 </li><li>シアノアクリレートが乾燥することができます。加熱パッドの上にケージにマウスを置きます（加熱パッドはオプションです外科場合それ以外の場合は不要） - iCalのスイートは、37℃の室温下に保たれています。寝具は、手術部位に付着しないことを確認するために、ペーパータオルの上にマウスを置くようにしてください。 </li><li>処置後60分 - 外科処置の長さに応じて、マウスが30以内に麻酔から外れていることを確認してください。手術後、動物はそれ自身のケージに入れ、それを回復するためにその部屋に戻る前に、意識的になるまで監視されています。彼らは脳の生化学的経路の一部を変更することによって、私たちの実験の結果を変更することができるので、無鎮痛薬は、手術後に使用されていません。しかし、彼らは手術の日に、麻酔から回復するまで、動物が監視され、感染および痛み/苦痛の評価の徴候について毎日術後。 </li></ol></li></ol> 3.ヘッド単収縮応答実験<ol><li> セットアップ <ol><li> 3日stereota後 - 2、午前10:00と午後2:00の間のすべての行動試験を実施ウイルス粒子のCTIC注射。 </li><li>ディゾルブ（±）-1-（2,5-ジメトキシ-4-ヨードフェニル）2.0ミリグラム/ kgの0.9％生理食塩水に-2-アミノプロパン塩酸塩（DOI）。また、0.9％生理食塩水溶液を調製。 </li><li>任意の寝具なしホームケージ（28×18×15センチ）を準備し、三脚を使用して、ビデオカメラのビューが直接ホームケージの上にあるようにカメラを調整します。 </li><li>実験の開始に先立って、少なくとも4時間、部屋にマウスを慣らします。 </li><li>ヘッド単収縮を記録するビデオカメラを設定します。 </li></ol></li><li> 実験 <ol><li>それはホームケージの真上になるようにカメラを置きます。全体のホームケージが視野に入るようにカメラを調整します。 </li><li>マウスを計量し、0.9％生理食塩水またはDOI（0.01ミリリットル/ g）のいずれかの適切な用量で腹腔内にマウスを注入します。注：マウスは25グラムの重量を量る場合、0.25ミリリットルの全容積に用量を投与。 </li><li> foのホームケージに戻って各マウスを置きますR 10分間。 10分後、空のホームケージの中央にマウスを置いて、カメラの視野内に死角がないことを確認します。ビデオカメラを押してレコード。部屋のままにしておきます。 注：マウスの動きやその中の様々な行動反応（。。。頭がけいれん、耳の傷などは DOIによって誘発される行動応答の完全なリストについてのゴンザレス-Maeso ら 2007補足データテーブル1を参照してください）3、のために記録されます30分。したがって、記録された視野内に死角が存在しないことが重要です。 </li><li> 30分には元のホームケージにバックカメラと場所マウスの録音を停止した後。各マウスについて、このプロセスを繰り返します。 </li></ol></li><li> レビュー <ol><li>テープは、マウスの実験条件に盲目各審判のレビューを持っている）（ すなわち 、ヘッドの間に使用される薬剤は、実験や頭蓋内注射時に使用されるウイルスをけいれん）。手動ですべてのヘッドがthrougけいれん記録ビデオハウト。 注：ヘッド単収縮は、マウス（補足ビデオ）が実施し、迅速な振とうヘッドの動きとして定義されています。 </li><li>各マウスについて、ブラインド審判の合計3からの最終的なHTR応答を平均します。実験条件によってその後のグループ、これらの値を、統計分析を行う（ すなわち 、t検定またはANOVA）。 </li></ol></li></ol>

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O., Persani, L., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+by+internalized+G-protein-coupled+receptors.">Signaling by internalized G-protein-coupled receptors.</a> Trends Pharmacol Sci. 31 (5), 221-228 (2010).</li><li>Celada, P., Puig, M. V., Diaz-Mataix, L., Artigas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallucinogen+DOI+reduces+low-frequency+oscillations+in+rat+prefrontal+cortex:+reversal+by+antipsychotic+drugs.">The hallucinogen DOI reduces low-frequency oscillations in rat prefrontal cortex: reversal by antipsychotic drugs.</a> Biol Psychiatry. 64 (5), 392-400 (2008).</li><li>B&#233;&#239;que, J. -. C., Imad, M., Mladenovic, L., Gingrich, J. A., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+5-hydroxytryptamine+2A+receptor-mediated+facilitation+of+synaptic+activity+in+prefrontal+cortex.">Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (23), 9870-9875 (2007).</li></ol>

The authors have nothing to disclose.

一緒にmGlu2-KOマウス5の前の所見と、培養細胞において5-HT 2A -mGlu2受容体複合体を形成しないmGlu2およびmGlu2 / mGlu3キメラ構築物を用いた結果は、5-HT 2A -mGlu2ヘテロマー受容体複合体ことを示唆していますマウスの前頭皮質は、LSDのような幻覚5-HT 2A受容体アゴニストによって頭部攣縮挙動を誘発するために必要とされます。この方法の制限は、それが天然の組織内...

そのようなLSD、サイロシビンやメスカリンなどの幻覚剤は、人間の意識、認知と感情7-9に大きな変化を引き起こします。セロトニンの不活性化は、遺伝的または薬理学的のいずれかの方法により、5-HT 2A受容体のシグナル伝達は、両方の齧歯類モデル3,10、およびヒト11で幻覚剤に著しく減衰行動反応を引き起こします。幻覚剤は、他の受容体サブタイプ8に結合するが、5-HT 2A受容体は、これらの化学物質の固有の行動活性に必要と考えられています。 グループII代謝型グルタミン酸受容体（ すなわち 、mGlu2およびmGlu3）は幻覚剤の分子機構に関するかなりの注目の対象と精神病12の基礎となる彼らの重要な役割となっています。これまでは、mGlu2タンパク質の発現なしでのマウス（mGlu2-KOマウス）はHALの細胞や行動への影響に鈍感であることが実証されていますlucinogens 5。また、5-HT 2AおよびmGlu2受容体は、セロトニンおよびグルタミン酸リガンドが生細胞1,2のGタンパク質結合のパターンを調節し、それを通して、特定のヘテロマー複合体を形成することが示唆されています。 構造的には、膜貫通（TM）ドメイン4およびmGlu2の5は、5-HT 2A受容体5とヘテロマー形成に重要な役割を果たしています。さらに、さらなる調査がmGlu2のTM4の細胞内の端部に位置する3つの残基が、細胞を6生体内5-HT 2A -mGlu2レセプターヘテロを形成するのに必要であることを実証しました。 異種発現系で観察されたこれらの知見に基づいて、ここでは、5-HT 2Aとの間のヘテロマー形成するかどうかをテストするために、野生型mGlu2およびmGlu2-KOマウスの前頭皮質におけるmGlu2 / mGlu3キメラ構築物のHSV媒介表現の使用を記載していますそして、mGlu2はのために必要です幻覚5-HT 2A受容体アゴニストによって誘導される頭部攣縮挙動。

<table border="0" cellpadding="0" cellspacing="0" width="2329">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">mGlu2 bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td style="width:1716px;">mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mGlu2&#916;TM4N bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:423px;">xylazine&#160;</td>
			<td style="width:71px;">Lloyd</td>
			<td style="width:119px;">List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">ketamine&#160;</td>
			<td style="width:71px;">Vedco</td>
			<td style="width:119px;">KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ophthalmic gel</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC0550805</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">burret clips</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9268369</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Feather surgical blade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9032736</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Hydrogen Peroxide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>19-898-919&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton syringe</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14815203</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton&#8482; Small Hub Removable Needles (33 Ga)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14816206</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cordless Micro Drill</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9089241</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dermabond Dermal Adhesive</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC0690470</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">(&#177;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>42203-78-1</td>
			<td>Dissolved in .9% saline solution to the concentration of 2.0 mg/kg</td>
		</tr>
	</tbody>
</table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

以前の知見は、ヘッド単収縮マウスの行動応答を確実かつロバスト幻覚剤によって誘発され、それは、5-HT 2A -KOマウス3には存在しないことを示しています。また、DOIとLSD幻覚の5-HT 2Aアゴニストによって誘発される頭部攣縮応答が有意mGlu2-KOマウス5に減少したことが示されました。しかし、以前の知見は、説得力の5-HT 2Aおよびm...

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

この記事では、GPCRヘテロマーの形成を必要とする行動アッセイをテストするために、マウスの前頭皮質にウイルスベクターを注入する方法について説明します。

マウスにおけるGPCRヘテロマーの行動機能を研究するためのキメラ構築物のHSV媒介導入遺伝子発現

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Research

JoVE Journal

Neuroscience

7.2K ビュー.  Icahn School of Medicine at Mount Sinai. この記事では、GPCRヘテロマーの形成を必要とする行動アッセイをテストするために、マウスの前頭皮質にウイルスベクターを注入する方法について説明します。 

ビデオ: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

ラットのショ糖の渇望のインキュベーションを評価するための一般的な方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

神経科学

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

マウスの脳スライスにおけるニコチン性アセチルコリン受容体機能を研究するための薬物のローカルアプリケーション

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

げっ歯類におけるランニングホイール活動の概日リズムの記録および分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

テスト視機能のための簡単​​な行動アッセイ<em&gt;アフリカツメガエル</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

マウスの海馬領域の歯状回における樹状樹枝状分岐の評価

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

ラット皮質ニューロンにおける繊維状アクチン（F-アクチン）涙点の定量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

ヒトの嗅覚機能を研究する自由呼吸fMRI法

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

アデノ随伴ウイルスを介する遺伝子発現により脊髄のニューロンを遺伝的に定義されています。

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

刺激間隔の齧歯動物の一時的な処理の評価のための力

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脳スライスと一次細胞培養における錐体ニューロンの弾道標識

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

マウスにおけるGPCRヘテロマーの行動機能を研究するためのキメラ構築物のHSV媒介導入遺伝子発現

要約

さらに動画を探す

この動画の章

ラットのショ糖の渇望のインキュベーションを評価するための一般的な方法

マウスの脳スライスにおけるニコチン性アセチルコリン受容体機能を研究するための薬物のローカルアプリケーション

げっ歯類におけるランニングホイール活動の概日リズムの記録および分析

テスト視機能のための簡単な行動アッセイ

マウスの海馬領域の歯状回における樹状樹枝状分岐の評価

ラット皮質ニューロンにおける繊維状アクチン（F-アクチン）涙点の定量化

ヒトの嗅覚機能を研究する自由呼吸fMRI法

アデノ随伴ウイルスを介する遺伝子発現により脊髄のニューロンを遺伝的に定義されています。

刺激間隔の齧歯動物の一時的な処理の評価のための力

脳スライスと一次細胞培養における錐体ニューロンの弾道標識

マウスにおけるGPCRヘテロマーの行動機能を研究するためのキメラ構築物のHSV媒介導入遺伝子発現

要約

さらに動画を探す

この動画の章

ラットのショ糖の渇望のインキュベーションを評価するための一般的な方法

マウスの脳スライスにおけるニコチン性アセチルコリン受容体機能を研究するための薬物のローカルアプリケーション

げっ歯類におけるランニングホイール活動の概日リズムの記録および分析

テスト視機能のための簡単​​な行動アッセイ

マウスの海馬領域の歯状回における樹状樹枝状分岐の評価

ラット皮質ニューロンにおける繊維状アクチン（F-アクチン）涙点の定量化

ヒトの嗅覚機能を研究する自由呼吸fMRI法

アデノ随伴ウイルスを介する遺伝子発現により脊髄のニューロンを遺伝的に定義されています。

刺激間隔の齧歯動物の一時的な処理の評価のための力

脳スライスと一次細胞培養における錐体ニューロンの弾道標識

テスト視機能のための簡単な行動アッセイ