The authors would like to acknowledge contributions from the entire Gereau Lab for manuscript editing. This work is supported by NINDS funds 1F31NS078852 to DSB and NINDS fund NS42595 to RWG.

すべてのマウスプロトコルは、健康のガイドラインの国立研究所に従ったとワシントン大学医学部の動物実験委員会（ミズーリ州セントルイス）によって承認された。 1.テストプレートとエンクロージャの準備<ol><li>ガラス表面をきれい。 </li><li>実験用テープでガラス板の中央に表面にT型フィラメント熱電対プローブを固定する。 </li><li>プレートの中央に沿って一列にガラス板上の動物のエンクロージャを置きます。 </li><li>中央動物囲いを通して熱電対プローブを通し、データロガーに接続します。自動シャットダウン機能を無効化しながら、上のデータロガーの電源を入れ、付属のケーブルでコンピュータにデータロガーを添付。 <ol><li>実験中のプレート温度を記録する場合は、プレート温度の記録を開始するために、データロガーソフトウェアを開きます。 </li><li>必要であれば、再ソフトウェアを調整コー​​ドプレート温度を一度毎秒。 </li><li>熱データロガーでパッケージソフトを使用して温度の記録を開始。 </li></ol></li><li>マウスとの間の視覚的相互作用を防止するために、黒挿入物を有する筐体を区切ります。 </li><li>位置は、エンクロージャの下面が快適着座位置から見えるようにガラスの下に反映しています。 </li></ol> 2.温暖化/ガラス板を冷却<ol><li>温め、水、湿潤氷またはドライアイスアルミニウムボックスを充填し、ガラス板上に適切に配置する（ドライアイスを充填したアルミ箔のパケットは、ガラスを冷却するために使用することができ、 図1）2。 <ol><li> 30℃でのテストでは、離れて、動物のエンクロージャ（ 図2B）2から「約0.25」アルミボックスを配置します。 <ol><li>ガラス板の両側に加熱された水循環器を設定する。 45にサーキュレータを設定 - 60°C、と私たちお湯（ 図1C）2の安定した流れでアルミボックスを埋めるために、それをメール。 </li><li>サーキュレータを配置し、アルミニウムボックスからお湯を直接戻ってそれぞれの側（ 図1C）上のサーキュレータの貯水池に排出すること2。 </li></ol></li><li> RTでのテストのために、（ 図2）2空のボックスを残す。 </li><li> 17℃でのテストでは、いずれかの側に離れて、動物のエンクロージャから&#39;&#39; 0.25約の箱を置き、湿った氷（ 図2）2で満たす。 </li><li> 12℃でのテストでは、離れて両側の筐体から&#39;&#39; 1.25約の箱を置き、ドライアイス（ 図2）2で満たす。 </li><li> （ 図2）5℃でのテストでは、約0.25 &#39;&#39;離れていずれかの側の筐体からのボックスを配置し、ドライアイスで埋める2。<ol><li>ドライアイスを用いてガラスを冷却すると、部屋中のCO 2の蓄積を防止するのに十分な換気があることを確認してください。 </li></ol></li></ol></li><li>所望の温度範囲に到達するためにガラスのを待ちます。 </li><li>プレート上のエンクロージャにマウスを追加します。 注記：白色雑音発生器は、ノイズ障害を減少させるために使用することができる。 </li><li>マウスが順応するのを待ちます。 注：私たちの施設では、これはおよそ2.5時間かかりますが、これは、動物の収容及び取扱い条件に基づいて大幅に異なる場合があります。 </li><li>ボックスは温め水、湿った氷、またはドライアイスの完全に保たれていることを確実にすることにより、所望の温度範囲でガラスを維持します。 注：私たちの装置では箱が氷おおよそすべての90分を再充填する必要がある。 注：17℃の条件では、それは氷でそれを再充填する前に排水孔を通じてアルミボックスからほとんどの水を空にしておくと便利です。これは、より良い温度を安定し、PRしますイベントのオーバーフロー 注：ドライアイスの正確な量は季節変化するが、一般に温度を一定に維持するボックスの全長に沿って完全以上のボックスを¼保ちます。 </li></ol> 3.固定温度でのマウスのテスト<ol><li>行動の部屋の外では、ドライアイスの完全な約半分のアイスバケツを埋める。 </li><li>ハンマーやマレットを使用して、微粉末にドライアイスを粉砕。 注：バケツを過剰充填することは、困難な完全に粉末にドライアイスを粉砕するようになります。 </li><li>ストレートカミソリの刃やはさみを使って、3ミリリットル注射器をトップを遮断。 </li><li> 21 G針を使用して、シリンジ（6穴の合計）の両側に3つの穴を突く。 注：これらの穴は、ドライアイスを圧縮しながら昇華により発生する圧力が減少します。カットオフ注射器は、複数の実験のために再利用することができる。 </li><li>注射器、ドライアイス粉末、および行動部屋に手持ち式ストップウォッチを取る。 </li><li>ドライアイス粉末の半分シリンジ室を埋める。フラットオブジェクトに対して注射器の切断端を持ち、しっかりとプランジャーを用いた粉末を圧縮する。注意してください;プラスチック製のプランジャーは、曲げや圧力から破損する可能性があります。このような場合は、新しい注射器からプランジャーを交換してください。 </li><li>注射器の端を超えて圧縮されたドライアイスペレットの先端を拡張します。 </li><li>安静時に完全にある試験マウス。 <ol><li> 30℃、23℃、17℃では、すべての4ガラス上の足としないを持って試験マウスは動くが、19完全に眠っていない。 </li><li> 12℃、5℃で、移動やジャンプ2足または4足としない上に試験マウス。 </li></ol></li><li>マウスの後肢（ 図1A）2の下にガラス面に対して平らペレットフラッシュを押して慎重にしっかり、ターゲティングのためにミラーを使用。手タイマーを起動します。 </li><li>タイマーを停止し、マウスが離れて冷却されたガラスから移動したときのペレットを削除します。 NOTE：撤退運動が垂直または水平であることができる。 <ol><li>マウスは非常に簡単に足を移動し、冷却面にそれを返す場合、マウスが離れて完全移籍するまで、タイミングと刺激を続ける。 注：私たちの研究室では、ほとんどの場合において、マウスのための20秒の最大刺激時間を使用しています。 </li></ol></li><li>各動物の各足に少なくとも3つの値が収集されるまで、このテスト手順を繰り返します。少なくとも15分までに、任意の単一の足に少なくとも7分であり、個別の連続した​​試験により、同じマウスの反対の足をテストする個別の試験。 </li><li>必要に応じて、（ 図3）1の冷却速度の違いを生成するために、ガラスの異なる厚さを使用する。 注：冷却速度が反比例ガラスの厚さと相関している。 </li></ol> 4.低温適応中にマウスのテスト注：これはガラスPLATとしてテストを可能にする別のプロトコルですeはむしろプレートが安定したとマウスが完全に寒冷環境に適応した後も、冷却される。 <ol><li>装置をセットアップするための第1節に記載されている指示に従ってください。 </li><li> RT（ 図7A）2でベースライン測定を行うためにセクション3に記載されている指示に従ってください。 </li><li>ドライアイス入りのアルミボックスを事前に冷却する。 </li><li>ベースライン引っ込め潜伏時間が測定された後、離れてエンクロージャから両側にプレート約1.25 &#39;&#39;に予め冷却ボックスを配置する（ 図7（a）は、矢印がラベル「ドライアイスを追加」）2。 </li><li>できるだけ頻繁に測定を行う、ガラス板を冷却するように測定を行う第3節に記載されている指示に従ってください。 </li></ol>

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The authors have nothing to disclose

The CPA can be used to assess cold sensitivity and cold adaptation in mice. It provides an affordable, efficient way to measure cold responses in unrestrained, acclimated animals at a wide variety of temperature ranges. It also provides an unambiguous behavioral response with an easily quantified and analyzed output variable. It has already been used to assess changes in cold sensitivity induced by inflammation1, neuropathic injury1, analgesics1, genetic knockouts20, and ge...

げっ歯類で冷たい反応性を測定することは、正常および病的両方の条件下で、ヒトにおける低温感受性の潜在的なメカニズムの理解を改善するのに重要である。コールド足底アッセイ（CPA）、もともと数年前に1を開発し、RTで配信寒冷刺激に再現可能な、明確なネズミ行動反応を生成するように設計されています。このアッセイのより最近の強化は、温度が2の広い範囲での低温感受性の再現性のある測定を可能にしてきた。どちらのバージョンも、比較的高いスループット、および使用する安価であるように設計されている。 大きな進歩は、他の行動の方法を使用して低温感受性のメカニズムを理解する上でなされた。一つの方法は、軽くたたくまたは噴霧アセトンをマウスの足に、マウスが足3,4フリック費やす時間の量を測定することを含むアセトンの蒸発試験である。残念ながら、アセトンの蒸発に対する応答は、湿式感覚とアセトンの匂いによって混乱している。また、アセトンの蒸発試験に適用される冷熱刺激を印加アセトンの量に基づいて変化し、定量化が困難であることができる。最後に、無傷のマウスは、ベースライン時にアセトンに最小限の応答を持っていることは不可能この方法で過敏症が存在しない場合に鎮痛を測定すること。 冷応答のための別の古典的なアッセイは、テール冷水5,6に浸漬された後に撤退する待ち時間が測定されるテイルフリックアッセイである。このアッセイにおける行動応答があいまいであり、アッセイは、特定の温度に対する応答を測定している間、動物は、十分に記載ストレス誘発鎮痛メカニズム7を通じて冷たい応答性を変えることができ、試験中に拘束されなければならない。 もう一つの一般的に使用されるツールは、行動を測定コールドプレート試験であり、彼らはペルチェ冷却板8-10上に置かれた後のマウスの応答。このツールは、特定の温度での動物の応答に関する情報を提供していますが、それはまた、一貫性なく使用されてきた。別のグループは、ジャンプ8,11数、最初の応答8,11- 13に待ち時間を含む応答の異なるタイプを測定して、足の数は非常に異なる結果を11,13,14を持ち上げる。コー​​ルドプレートアッセイはまた、一度に試験することができる唯一の​​動物のような比較的低いスループットであり、それは高価で脆弱なペルチェ素子を必要とする。 2プレート温度嗜好試験は、動物が、異なる温度9,15- 17の2接続プレートに費やす時間の相対量を測定するコールドプレート試験の一般的に使用される誘導体である。別の類似の一般的に使用されるアッセイは、熱勾配アッセイ、マウスは、異なる温度帯で過ごす時間の量である長い金属板上に5°Cと45°Cの範囲では16を測定する。これらのアッセイは、温度の比較を可能にしながら、それは行動が温度嫌悪や温度の好みにを表すかどうかは不明である。 最後に、動的コールドプレートアッセイは、マウスは、周囲温度18の変化に対応するかを測定するために使用されてきた。この方法は、RTペルチェ素子にマウスを置き、マウスがジャンプしたり、異なるプレート温度で彼らの足をなめるどの程度測定しながら、1℃にそれをランプダウンが含まれます。これはマウスが冷却環境に適応する方法をテストが、それはマウスがクーラー周囲温度の設定で冷たい刺激に反応するかテストするための方法を提供していません。さらに、それは実行するために高価な設備を必要とし、その低温感受性を測定する前に検査機器にマウスを順応する方法を提供していません。 これらのアッセイを補完するために、CPAはacclimaをテストテッド温度範囲において、種々の明確に定義された冷刺激に対する応答、または冷たい周囲温度に適応する過程。これは、安価に高スループットテストのためにスケールアップされる可能性を、私たちの現在の装置との時間で14匹のマウスまでテストすることができます。

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Name</td>
			<td style="width:320px;">Company</td>
			<td style="width:353px;">Catalog Number</td>
			<td style="width:303px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-type thermocouple probe</td>
			<td>Physitemp</td>
			<td>IT-24p</td>
			<td>Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass plate</td>
			<td>Local glass company (in St. Louis, Stemmerich Inc)</td>
			<td>N/A</td>
			<td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#39;&#39;, but 3/16&#39;&#39; and 1/8&#39;&#39; are also useful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Data logger</td>
			<td>Extech</td>
			<td>EA15</td>
			<td>Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&#38;prodid=408)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3mL Syringe</td>
			<td>BD</td>
			<td align="right">309657</td>
			<td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td>If using Extech logger, any Pcwill work</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum boxes</td>
			<td>Washington University in St. Louis machine shop</td>
			<td>N/A</td>
			<td>boxes are 3&#39; long, 4.5&#39;&#39; wide, and 3&#39;&#39; tall with a sealed lid.&#160; There is a 1/2&#39;&#39; hole drilled into one short side of each box, near the bottom. These holes are filled with rubber stopcocks when the boxes are filled with wet ice or hot water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heated water circulator</td>
			<td>VWR</td>
			<td></td>
			<td>Any water circulator model with a pump will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 gauge needle</td>
			<td>BD</td>
			<td align="right">305165</td>
			<td>The exact needle size is not important</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hand timer</td>
			<td>N/A</td>
			<td></td>
			<td>Any hand timer will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mirror</td>
			<td>N/A</td>
			<td></td>
			<td>Any flat mirror will work</td>
		</tr>
	</tbody>
</table>

a simple and inexpensive method for determining cold sensitivity and adaptation in mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

30°C、23°C、17°C、12°Cで開始されたマウスから誘発される行動反応は、再現性の高い（ 図4A）20である。後足の下に生成された冷熱刺激を測定するために、マウスをケタミン/キシラジン/アセプロマジンのカクテルで麻酔し、それらの足をT型フィラメントの熱電対（ 図4B）20の上にガラス上に固定した。ガラスは、所望の検査範囲に冷却または加温し?...

Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

マウスにおける低温感受性と適応を決定するためのシンプルで安価な方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

a-simple-inexpensive-method-for-determining-cold-sensitivity

Research

JoVE Journal

Neuroscience

14.6K ビュー.  Washington University in St. Louis. The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

ビデオ: A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

春雨とカッペリーニの処理試験：ラットとマウスの器用な前足の機能の簡単な定量的指標

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

神経科学

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

ハエでエタノールの感度を測定するために簡単

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

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 ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

の長期嗅覚順応の分子読み出し<em&gt; Cエレガンス</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

exの子宮内エレクトロポレーションと全半球植：早期皮質の発達についての研究のための簡単な実験方法

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

エタノール鎮静感度との急速な寛容を測定するための安価な、スケーラブルな行動アッセイ<em&gt;ショウジョウバエ</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

方法は、標的とマウスにおけるアイソ気道-神経支配感覚ニューロンへ

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

大人のホールマウントの準備のためのシンプルなワンステップ解剖プロトコル<em&gt;ショウジョウバエ</em&gt;ブレインズ

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

冷痛覚のための新規アッセイ<em&gt;ショウジョウバエ</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

生体内培養-高速、シンプルで、安価な AWESAM プロトコルを使用してマウスのアストロ サイトのように

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...