MSB is supported by the Alcohol Beverage Medical Research Foundation, a Center for Translational Research Award (UL1 TR000058), the National Institutes for Alcohol Abuse and Alcoholism (P50 AA022537), and startup funds provided by the Virginia Higher Education Equipment Trust Fund and the VCU School of Medicine.

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None of the authors have competing or conflicting interests.

ここで紹介する手順は、マイクロインジェクションカニューレと動機付け行動の分子基質の解明に役立ちますmicroinjectorsを製造するための効率的な手段です。この方法は、いくつかの利点を提供しています。まず、自分のインプラントおよびmicroinjectorsを製造することにより、新たな実験パラメータは、急速に最適化することができ、 すなわち、1つは、コンポーネントが到着するカスタムメイドのを待つ必要はありません。第二に、カニューレの小径のため、多くのカニューレを同時に注入することができます。これは、生存率を向上させることができ、必要な外科手術時間が、短縮し、また、動物ごとに複数のインプラントを可能にします。固定比パラダイムを迅速に簡単に所望の強化スケジュールを含むイベント遷移パラメータリストを適用することにより、プログレッシブ比パラダイムに変換することができるので、第三に、オペラントチャンバーを制御するために使用されるソフトウェアは、容易に、プログレッシブ比スケジュールを収容します。 であるために広く便利な、汎用のマイクロインジェクションの手順は、現在利用可能なほぼすべての試薬のマイクロインジェクションのために広く適用されるべきであることを提示しました。そのため、私たちは、この技術はマイナーな修正を加えて、将来的に同様の高い有用性であり続けることを期待しています。唯一のいくつかの変数を変化させることにより、この方法は、試薬の広い数に適用することができます。最も一般的に操作することになるパラメータは、マイクロインジェクターがカニューレ、注射の量、注入速度から突出長さがあります。例えば、1は、インジェクタは、通常、慢性移植片の周りに形成グリア性瘢痕を避けるために、カニューレ先端からさらに突出することができます。さらに、一つはより大きな体積を注入することを望むかもしれません。線条体ウイルスマイクロインジェクションのために、1μLの量は、一般的に使用され、このボリュームは、一般的に、より長い時間かけて注入される使用と比較して（頻繁に7から10分の追加の拡散時間 - 10分プラス3）薬理学的試薬用のD（通常は0.3から3分プラス1 - - 2オーバー0.5μL3分の追加の拡散時間）。ユーザーは文献を参照してくださいおよび/または経験的に自分のニーズに最適なパラメータを決定する必要があります。 1）カニューレの長さ、2）マイクロインジェクターの長さ、3）マイクロインジェクターの噴霧パターンの品質、及び4）注射前に、システムの整合性：いずれにしても、この手順の成功は、4つの変数に決定的に依存しています。マイクロインジェクションの場所はマイクロインジェクタがカニューレから突出した深さに依存しているため、それは両方のカニューレ（ステップ1.2.8）と、マイクロインジェクターの長さ（ポスト曲げ、ステップ2.2.1）の両方を正確既知およびすべての被験者の間で均一であることが肝要です。これは、簡単に簡単に任意のはそれが最後の再測定に必要な長さではありません実現拒否することによって制御することができます。これはガイドカニューレの直下に発生した場合、また、注入位置は、予測することができます。 DOEこのように、任意のマイクロインジェクターテストの際に長い、細い流れ（ステップ2.4​​.6）が拒否されるべき噴霧しませんよ。質の注入はまた、注入前に、システムの完全性に関連しています。インジェクタからすべての水を分配した後（前の試薬で充填する）は、複数のスポットをラボワイプ上で観測されている場合、リークは（ステップ2.4​​.8に注意してください）​​是正する必要があります。さらに、（試薬とマイクロインジェクターを充填した後）PE20チューブ内の水からの薬物が一つではない分離バブル（ステップ2.4​​.9）、単一のバブル場合、インジェクタは、部分的に詰まっています。この詰まりは防止または注射をそらすことができどちらか。これも（ステップ2.4​​.8に注意）は、容易に改善することができます。 1は3つの選択肢がある動物が定位フレームにある間に顕微注入することを望むべきです。第1に、それは定位マニピュレータによってしっかりと保持し、また、PE20チューブとの接続を可能にするために十分遠くまで延長することができるように、マイクロインジェクターの襟の長さを増加させることができます。第二に、上eは、時間的にカニューレを移植し、ここで紹介する標準マイクロインジェクターを使用することができます。第三に、人は描かれ、研磨されたガラスピペットを使用することができます。16,17 ここで紹介する手順の重要な制限は、それが最善の手順を熟知しているだけでなく、扱いラットで実施されていることです。同じ研究者が2ヶ月以上毎日ラットを取り扱うため、結果のセクションで説明したデータに使用したラットは、特別な取り扱い手順を必要としません。これは、少なくとも2週間、外科用インプラントの毎日の観察および操作が含まれていました。しかし、ラットは急速に応力が作用することができるプレパルス阻害アッセイの前に使用されている多数の技術により馴化され得ます。これらの特別な馴化技術がうまく以前に詳述されている。43これらの手順に加えて、ラットが短くmicroinjectorsはデュ使用されているマイクロインジェクションの手順に慣らしすることをお勧めしますリング「偽」噴射。これらの偽注射中に、マイクロインジェクターは、組織損傷を制限するために組織内に突出しないことが重要です。つまり、マイクロインジェクターは、もはや14ミリメートル以上曲げてはなりません。したがって、この手法の最適な実施のために必要な完全な慣れは、限定と見なすことができます。 いくつかの行動パラダイムは動機を測定するために存在するが、進行性の比率は、一般的に、被験者が強化子を得るために発揮していく所存です努力を定量化するために使用されます。プログレッシブ比パラダイムは、多くの場合、最後に完了した比率のレバー押しの最大数として定義されたブレークポイントとして知られている指標を生成;。。 すなわち 、最大のものは強化子を生成応答21プログレッシブ比は大きさを補強するために敏感です。例えば、より高いコカイン（またはスクロース）の用量は、より高いブレークポイントと低いコカイン（またはスクロース）を生成低いbreakpをもたらす用量OINT。21,22により、ブレークポイントがモチベーションおよび/ ​​または有効性を補強するための日常的に使用するプロキシです。21,23-26ブレークポイントの意図は、動物が応答を停止したときを決定することであるため、プログレッシブ比パラダイムの重要なパラメータでありますセッションの長さ。有限セッションの長さは、ブレークポイントの値に偽のキャップを置くことができ、これは異常に自己投与またはその増加後の補強休止の速度を減少させる前処理によって悪化することができます。この交絡は、アプローチの任意の数によって克服することができる;例えば、動物は平均間の注入間隔の倍数によって応答する源泉徴収したときに終了するセッションは、このアプローチの44より一般的に適用される変異体は、セッションが一度した応答終了することです。被験者全体で一定に保持されているいくつかの実験的に決定された値によって源泉徴収されて。私たちは、ステップ4.4.9.11にこの手法を適用する方法を提供しています。

Rodents and humans respond in remarkably similar ways to behaviorally relevant stimuli1-3. This suggests that rodents are appropriate subjects for elucidating the molecular substrates of behavior and complex psychiatric conditions4. Understanding the molecular substrates of complex behavioral processes, such as motivation, frequently requires brain microinjection. Both the brain microinjection technique and a primary motivation assay will be presented here. Rats will be used as subjects, but these procedures can readily be adapted to well-handled mice. Included herein are procedures for the manufacture of the required cannulae, obturators (dummy cannulae or stylets), and microinjectors. The method presented is significantly more flexible and more cost-efficient than prefabricated implements. This flexibility will prove valuable when optimizing conditions. Importantly, because the microinjection procedure can be used to test a myriad of hypotheses; the techniques presented here should be broadly applicable. For example, receptor ligands can be microinjected to understand neurochemistry3,5,6; cell-permeable peptides and small-molecules can be microinjected to understand intracellular signaling pathways7-10; toxins, ion channel blockers, or antagonist cocktails can be microinjected to understand circuitry1,11,12.
While the generic protocol presented here can be readily adapted by the user for their particular needs, the procedure is particularly well suited for behavioral assays since microinjection occurs in awake rodents that are only under mild hand restraint. No anesthesia or special restraints are required. This is possible because the brain itself lacks pain sensation. However, if anesthesia is not used, microinjection must occur through cannulae that were previously stereotaxically implanted. This is because nociceptors are present on the scalp, meninges,13 which are the membranes surrounding the brain, and the periosteum,14 which is the membrane covering the skull. It should be noted that microinjection under anesthesia is sometimes desirable. One example is when the virus is being injected, and one may wish to inject virus directly through either stainless steal needles15 or glass pipettes because this can reduce tissue damage and improve transduction efficiency.16,17 The microinjectors described below can be modified for this purpose and suggestions on how to do this can be found in the Discussion. Because other JoVE articles have demonstrated stereotaxic brain cannula implantation,18-20 these procedures will not be covered here.
We present these microinjection procedures together with an assay that quantifies motivation. Several rodent models of motivated behavior are currently in use, such as the runway box and barrier scaling. Here, we describe how to use an operant progressive ratio schedule of reinforcement to quantify motivation where operant responding is being maintained by a reinforcer. Responding on the progressive ratio is responsive to reinforcer magnitude.21,22 Accordingly, this assay is routinely used as a proxy for motivation and/or reinforcing efficacy. 21,23-30 Because several excellent reviews have covered this topic in detail,21,24 we will focus mainly on practical concerns.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cannula Tubing</td>
			<td>Amazon Supply/ Small Parts</td>
			<td>HTXX-26T-60</td>
			<td>26 gauge, Hypotube S/S 316-TW 26GA</td>
		</tr>
		<tr>
			<td>Obturator</td>
			<td>Amazon Supply/ Small Parts</td>
			<td>GWXX-0080-30-05</td>
			<td>33 gauge, Wire S/S 316LVM 0.008 IN</td>
		</tr>
		<tr>
			<td>Microinjector Wire</td>
			<td>MicroGroup</td>
			<td>33RW 304</td>
			<td>33 gauge</td>
		</tr>
		<tr>
			<td>Super Glue</td>
			<td>Loctite</td>
			<td>3924AC</td>
			<td>Liquid, Non-gel, can be autoclaved</td>
		</tr>
		<tr>
			<td>Microinjector Plastic Tubing</td>
			<td>Becton Dickson</td>
			<td>427406</td>
			<td>PE20</td>
		</tr>
		<tr>
			<td>Medium Weight Hemostats</td>
			<td>World Precision Instruments</td>
			<td>501241-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Fisher</td>
			<td>09-016</td>
			<td>150 mm</td>
		</tr>
		<tr>
			<td>#7 Forceps</td>
			<td>Stoelting</td>
			<td>52100-77</td>
			<td>Dumont, Dumostar</td>
		</tr>
		<tr>
			<td>Rotary Tool</td>
			<td>Dremmel</td>
			<td>285</td>
			<td>Two-speeds</td>
		</tr>
		<tr>
			<td>Cut-off Disc</td>
			<td>McMaster Carr</td>
			<td>3602</td>
			<td>15/16&#34; x 0.025&#34;</td>
		</tr>
		<tr>
			<td>Microinjection Pump</td>
			<td>Harvard Apparatus</td>
			<td>PhD 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ul Glass Syringe</td>
			<td>Hamilton</td>
			<td>7001KH</td>
			<td>Needle Style: 25s/2.75&#34;/3</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicator</td>
			<td>Fisher</td>
			<td>23-400-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Wipes</td>
			<td>Kimwipes</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr>
			<td>Operant Software</td>
			<td>Coulbourn</td>
			<td>Graphic State</td>
			<td></td>
		</tr>
		<tr>
			<td>Operant Chambers</td>
			<td>Coulborun</td>
			<td>Habitest</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

ここでは、上記の手順で得られる結果の例を示します。示す第一は、ウイルスベクター（ 図3）によってトランスフェクトされた典型的な領域です。一般的に、約1 から 3mmのトランスフェクトされた体積は、線条体組織で得られます。トランスフェクトされた領域の体積は、カバリエリのメソッドを使用して、連続切片全体で定量することができる32重要なのは、トランスフェクトされたボリュームは、このようなトランスフェクトされる組織の種類など、多くの要因に依存します。ウイルス血清型;プロモーター;速度と容量注入。注入された粒子の数;注入液のpHは、およびマンニトールなど高浸透圧性試薬、かどうか、使用された。33,34は一般的に、我々は10分かけて10 12粒子/ ml、1μL/側を顕微注入すると、閉塞具を交換する7追加分前に許可します。また、注入液は、pHが8次の周りに一般的ですが、私たちはモチベーションがいずれかの導入遺伝子発現によって操作することができることを示している（ 図4）または薬理学的試薬マイクロインジェクション（ 図5）。 図4では 、我々は排他的デザイナー薬（DREADDs）によって活性化デザイナー受容体を発現しました。 DREADD受容体コード領域は、内部リボソーム侵入部位とmCitrineカセットが続きました。 mCitrineは、トランスフェクトされた細胞の便利な可視化を可能にします。 DREADDは、ヘテロ三量体Gタンパク質Gαqに結合させました。アストロサイト、28,35とDREADD自体を刺激することができるGαq結合DREADDの活性化は、クロザピン-N-オキシド（CNO、3ミリグラム/ kg、腹腔内）の全身投与により活性化することができる。14,22,36ラットはに訓練されましたレバーは、3レバー押しが60個の連続日間、毎日1時間のセッション中に飲む1のチャンスを得たエタノールの強化のために応答します。次に、ラットを禁欲を余儀なくされ、Gαq結合DREADDsは、コアアストロサイトの側坐核で発現させました。 3週間の禁欲後、自己ADMIに意欲<html> nisterエタノールがブレークポイントで測定した。核の21,27-30活性化は、コアアストロサイトを側坐核、全身CNOの投与を介して、車両に比べて禁欲後エタノール自己投与を再開するために、ラットのモチベーションを低下させました。重要なことは、CNOは、代わりにDREADDの緑色蛍光タンパク質を発現していた同じように訓練されたコホートでは効果がなかったです。 <img alt="図3" src="/files/ftp_upload/53018/53018fig3.jpg" /> マイクロインジェクション、ウイルスによってトランスフェクト図3.代表腹側線条体の領域が。この画像は、核の領域が上記の方法に従うことにより、トランスフェクションしたアストロサイトを側坐核示しています。データは、著作権者の許可を得てブルら13から転載されています。詳細は、出版物および付随する補足資料に記載されています。「ブランク&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図4" src="/files/ftp_upload/53018/53018fig4.jpg" /> 自己投与エタノールが核内で過剰発現された導入遺伝子を活性化することにより、減少したのラットの図4.動機は、コアアストロサイトを側坐核。ウイルスを表現するために、ウイルスのために1週間後にマイクロインジェクション可能とモチベーションがブレークポイントを経由して測定しました。導入遺伝子は、排他的デザイナー薬（DREADDs）によってデザイナー受容体活性化された表現しました。一方向ANOVA（F（2,30）= 3.29、P = 0.04）シェッフェ事後によって明らかに続いているクロザピン-N-オキシドの全身投与によるDREADDの活性化（CNO、3ミリグラム/ kg、腹腔内）大幅CNOはなく、DRの緑色蛍光タンパク質（GFP）を発現しても同様に訓練を受けたコホートでは効果がなかった車両に比べて禁欲後に自己投与エタノールにラットのモチベーションを低下させましたEADD。データは、著作権者の許可を得てブルら13から転載されています。詳細は、出版物および付随する補足資料に記載されています。 <img alt="図5" src="/files/ftp_upload/53018/53018fig5.jpg" /> 図5.ギャップ結合ブロッカーのマイクロインジェクションは、禁欲後に自己投与エタノールにラットの意欲を増加させた二つのギャップ結合ブロッカーは、自己投与エタノールへのラットの（ブレークポイントを介して測定）モチベーションに及ぼす影響について評価した：メフロキンと18- α-グリチルレチン酸（18-α）。二元配置ANOVAは、核内へのギャップ接合ブロッカーのマイクロインジェクションは、コアが禁欲（F（3,40）= 5.56、P = 0.003）の後に自己投与エタノールにラットのモチベーションを増加側坐核ことを明らかにしました。データは、著作権者の許可を得てブルら13から転載されています。詳細は同公報に記載されていますし、添付補足資料。

Watch this Scientific Journal Video about Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior at JoVE.com

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

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 ...

rodent-brain-microinjection-to-study-molecular-substrates-motivated

Research

JoVE Journal

Behavior

14.2K ビュー.  Virginia Commonwealth University School of Medicine. 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. 

ビデオ: Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

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

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

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 ...

行動学

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

ビート難聴を暴く：同期指タッピングと知覚タイミングタスクを検出リズム障害

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Dual Task Procedure Combined with Rapid Serial Visual Presentation to Test Attentional Blink for Nontargets

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the first target may be easy to spot but the second is likely to be missed. This phenomenon of attentional blink (AB) has been widely studied to probe the temporal capacity of attention for detecting visual targets. However, with the typical procedure of AB experiments, it is not possible to examine how the processing of non-target items in RSVP may be affected by attention. This paper describes a novel dual task procedure combined with RSVP to test effects of AB for nontargets at varied stimulus onset asynchronies (SOAs). In an exemplar experiment, a target category was first displayed, followed by a sequence of 8 nouns. If one of the nouns belonged to the target category, participants would respond &#8216;yes&#8217; at the end of the sequence, otherwise participants would respond &#8216;no&#8217;. Two 2-alternative forced choice memory tasks followed the response to determine if participants remembered the words immediately before or after the target, as well as a random word from another part of the sequence. In a second exemplar experiment, the same design was used, except that 1) the memory task was counterbalanced into two groups with SOAs of either 120 or 240 msec and 2) three memory tasks followed the sequence and tested remembrance for nontarget nouns in the sequence that could be anywhere from 3 items prior the target noun position to 3 items following the target noun position. Representative results from a previously published study demonstrate that our procedure can be used to examine divergent effects of attention that not only enhance targets but also suppress nontargets. Here we show results from a representative participant that replicated the previous finding.&#160;

This paper describes a novel dual task procedure combined with Rapid Serial Visual Presentation to look at attentional blink at varied stimulus onset asynchronies. This experimental procedure differed from others in that it tested attentional blink for nontargets.

非ターゲットのための注意の瞬きをテストするために迅速なシリアルビジュアルプレゼンテーションと組み合わせることで、デュアルタスク手順

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the ...

Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A challenging illness to treat, due to the lack of understanding of the underlying neurobiology, AN has the highest mortality rate among psychiatric illnesses. To address this need, neuroscientists are using an animal model to study how neural circuits may contribute toward vulnerability to AN and may be affected by AN. Activity-based anorexia (ABA) is a bio-behavioral phenomenon described in rodents that models the key symptoms of anorexia nervosa. When rodents with free access to voluntary exercise on a running wheel experience food restriction, they become hyperactive &#8211; running more than animals with free access to food. Here, we describe the procedures by which ABA is induced in adolescent female C57BL/6 mice. On postnatal day 36 (P36), the animal is housed with access to voluntary exercise on a running wheel. After 4 days of acclimation to the running wheel, on P40, all food is removed from the cage. For the next 3 days, food is returned to the cage (allowing animals free food access) for 2 hr daily. After the fourth day of food restriction, free access to food is returned and the running wheel is removed from the cage to allow the animals to recover. Continuous multi-day analysis of running wheel activity shows that mice become hyperactive within 24 hr following the onset of food restriction. The mice run even during the limited time during which they have access to food. Additionally, the circadian pattern of wheel running becomes disrupted by the experience of food restriction. We have been able to correlate neurobiological changes with various aspects of the animals&#8217; wheel running behavior to implicate particular brain regions and neurochemical changes with resilience and vulnerability to food-restriction induced hyperactivity.

Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa.

神経性食欲不振症の神経生物学的基礎を研究する活動ベースの食欲不振齧歯類モデルを用いました

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

知覚と学習を研究するためのツールと​​しての電気生理学的皮質の記録と刺激と組み合わせたシャトル・ボックストレーニング

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

げっ歯類精神運動警戒試験（rPVT）：ラットとマウスの神経行動のパフォーマンスを評価するための方法

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

リバーシブルの冷却誘起 Deactivations 歩行猫障害メモリへの皮質の貢献を研究するには

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

Central and Divided Visual Field Presentation of Emotional Images to Measure Hemispheric Differences in Motivated Attention

Two dominant theories on lateralized processing of emotional information exist in the literature. One theory posits that unpleasant emotions are processed by right frontal regions, while pleasant emotions are processed by left frontal regions. The other theory posits that the right hemisphere is more specialized for the processing of emotional information overall, particularly in posterior regions.
Assessing the different roles of the cerebral hemispheres in processing emotional information can be difficult without the use of neuroimaging methodologies, which are not accessible or affordable to all scientists. Divided visual field presentation of stimuli can allow for the investigation of lateralized processing of information without the use of neuroimaging technology. 
This study compared central versus divided visual field presentations of emotional images to assess differences in motivated attention between the two hemispheres. The late positive potential (LPP) was recorded using electroencephalography (EEG) and event-related potentials (ERPs) methodologies to assess motivated attention. Future work will pair this paradigm with a more active behavioral task to explore the behavioral impacts on the attentional differences found.

This study compared central versus divided visual field presentations of emotional images to assess differences in motivated attention between the two hemispheres. The late positive potential (LPP) was recorded using electroencephalography (EEG) and event-related potentials (ERPs) methodologies to assess motivated attention.

やる気のある注目の大脳半球機能差を測定する感情的な画像の中央と分かれて視野プレゼンテーション

Two dominant theories on lateralized processing of emotional information exist in the literature. One theory posits that unpleasant emotions are processed ...

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

定量的食物摂取の試金を組み合わせると強制的にショウジョウバエの食欲を勉強するニューロンを活性化

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

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 ...