この作品は、チャン庚医学財団 (許可番号 CMRPG3C1771、CMRPG3C1772、CMRPG3E0631、CMRPG3E0632、および CMRPG3E0633) によって支えられました。

研究は、制度のレビュー委員会の長庚記念病院 Linkuo、桃園、台湾 (102-2577A3) で承認されました。
1. NR1 GFP 発現プラスミドの作製
<ol><li>遠心分離機管、ピペットで移しなさい混合物の優しくまで上下のエシェリヒア属大腸菌の有能なセルひずみ DH5α 滅菌 1.5 mL の 100 μ L の NR1 GFP プラスミドのミックス 10 ng 4-6 回、20 分間氷の上管を孵化させなさいと。</li>
	<li>水のお風呂で 1 分の 42 ° C でチューブをインキュベート水浴からチューブを削除、管に 1 mL ホストゲノム スープ (LB) 媒体を追加し、37 ° c 1 時間振とうしながらインキュベーターでチューブを孵化させなさい。</li>
	<li>アンピシリン (0.1 Mg/ml) を含む LB 寒天培地プレート上に混合物の 50 μ L を広がり、インキュベーターの 37 ° C で LB 寒天培地プレートを一晩インキュベートします。</li>
	<li>一晩 LB 寒天培地滅菌チップを使用して単一コロニーを拾うし、渦の先端を 5 mL の LB 培地とアンピシリン (0.1 mg/mL) の管内細菌培養の。一晩中揺れでインキュベーターで 37 ° C でチューブを孵化させなさい。</li>
	<li>250 mL 滅菌 LB 培地とアンピシリン (0.1 mg/mL) が含まれています 500 mL フラスコに一晩培養分注 3 mL。揺れで 37 ° C でフラスコを孵化させなさい。600 の波長で細菌培養の光学濃度 (OD) を定期的にチェック、OD600 0.6 1.0 に達するまでに分光器を用いた nm。 注: は、グリセロール ストックを準備し、グリセロール ストック将来使用するため-80 ° C 以下での保存を 200 μ l 添加グリセリンと一晩培養も 800 μ L を混ぜます。</li>
	<li>250 mL の細菌文化から NR1 GFP 発現プラスミドを準備します。<ol>
<li>4 ° C で 15 分間 6,000 × g で遠心分離によって細胞を収集します。</li>
		<li>RNase A を含む 10 mL 再懸濁バッファーで細菌のペレットを再懸濁します渦塊は見られないまでに徹底的に。</li>
		<li>細菌懸濁液を 10 mL の溶解バッファーを追加、穏やかに 4-6 回、混合物を反転し、室温 (RT) 5 分でライセートを孵化させなさい。</li>
		<li>やさしく転倒させる混合物 4-6 回、ライセートを 10 mL 中古冷蔵沈殿バッファーを追加します。</li>
		<li>キャップとフィルター カートリッジの樽の中に溶解液を注ぐ室温 10 分待つ</li>
		<li>カートリッジ出口ノズルのキャップを外し、ピストンをカートリッジに挿入し、ピストンを軽く押します。フィルタ リングされた収集 50 mL のチューブに溶解。</li>
		<li>2.5 mL 独自バッファーの製造元からフィルタ リングされたライセートを加えてチューブ約 10 倍の反転によって混合物を混ぜる混合物 30 分間氷の上を孵化させなさい。</li>
		<li>適用 10 mL 平衡バッファーで平衡は事前フィルター列をフィルター選択されたライセート混合物。重力によって排出する列を許可します。</li>
		<li>30 mL 洗浄バッファーの列を 2 回洗浄し、15 mL の溶出バッファーを使用して、列から DNA を溶出します。</li>
		<li>10.5 mL (0.7 ボリューム) イソプロパノールを加える溶離された DNA バッファー、よく混ぜ、4 ° C で 30 分間 20,000 × g で混合物を遠心分離機</li>
		<li>慎重に上清をデカントし、一度、5 mL エンドトキシン フリー 70% エタノールによる DNA ペレットを洗浄し、10 分 20,000 × g で遠心。</li>
		<li>上清をデカント、化学フードで 5-10 分のペレットを乾燥、エンドトキシン フリー バッファーの 300-500 μ L のペレットを溶解します。</li>
		<li>紫外線 260/280 nm の吸光度で溶液の吸収を測定することによってプラスミド濃度を決定します。 注: は、同じプロトコルを使用して GFP 発現プラスミドを準備します。</li>
	</ol>
</li>
</ol>2. NR1 GFP 発現プラスミドを HEK293 細胞のトランスフェクション
<ol><li>それぞれに 2% のゼラチン溶液 200 μ L 分注 48 ウェル培養プレートのと少なくとも 30 分の 37 ° C でプレートを孵化させなさい。</li>
	<li>10% 牛胎児血清 (FBS) 各井戸を含む細胞培養液 200 μ L でよくとシード 5 × 104 HEK293 細胞からゼラチン溶液を吸引します。一晩 5% CO2付属の加湿インキュベーターで 37 ° C でプレートを孵化させなさい。 注: 診断、セルがカウントされます。</li>
	<li>10% を含む新鮮な培養液 200 μ L で過ごした培を交換して次の日にもあたり FBS。</li>
	<li>各シングルの 100 を混合することによって、溶液を準備 20 μ L の培養液と 20 μ L の培養液中で 0.8 μ L のトランスフェクション試薬を混合することによってソリューション B NR1 tGFP プラスミドの ng。各実験のための総容積の準備に必要な井戸の数を乗算します。</li>
	<li>ソリューション A と B のソリューションを混合することによって C のソリューションを準備し、RT で 20-30 分のための混合物を孵化させなさい。</li>
	<li>C の解決策も HEK293 細胞を含むそれぞれの 40 μ L 分注プレートを軽く、旋回し、一晩 5% CO2付属の加湿インキュベーターで 37 ° C でプレートを孵化させなさい。</li>
	<li>40 X-200 X 倍率と蛍光顕微鏡下で宿主細胞の NR1 GFP 遺伝子組換えタンパク質の発現を確認 (励起/蛍光: 482/502 nm)、細胞を用いた蛍光抗体法に進みます。 注: を使って同じプロトコルを使用して HEK293 細胞に発現プラスミド GFP。</li>
</ol>3. 細胞を用いた蛍光抗体法
<ol><li>井戸から使用済の培地を吸引、その後洗ってリン酸を 200 μ l 添加の各ウェル バッファー生理食塩水 (PBS) 3 回。</li>
	<li>4% パラホルムアルデヒド溶液 200 μ L を加えるごとによく15 分間常温プレートを孵化させなさい。</li>
	<li>、井戸からパラホルムアルデヒド ソリューションを吸引させ、各 200 μ L の PBS を 3 回も。</li>
	<li>10% スキムミルク 200 μ L を加えて PBST (0.1 %pbs でトゥイーン 20) それぞれがよく、穏やかな揺れで 1 h の RT でプレートをインキュベートするソリューション。</li>
	<li>PBST ソリューションだけでなくからの 10% のスキムミルクを吸引して 200 μ L PBST で井戸を一度洗ってください。</li>
	<li>1 h の RT で穏やかな揺れで一次抗体として希薄プラズマ (pbst; で 1: 100) と井戸を孵化させなさい。 注: プラズマが自己免疫性脳炎が疑われる患者の血液から得られます。</li>
	<li>、井戸から希薄プラズマを吸引し、それぞれを洗ってよく PBST の 200 μ L で 3 回。</li>
	<li>ヤギ抗ひと IgG が Alexa Fluor 594 (1:1, 000 PBST 希釈) を各ウェルに共役の 200 μ L を追加し、1 h の穏やかな揺れで常温培養プレートを孵化させなさい。</li>
	<li>ヤギ抗ひと IgG 共役 Alexa Fluor 594 ソリューションを吸引し、それぞれを洗ってよく PBST の 200 μ L で 3 回。</li>
	<li>各ウェルに 100 μ L のグリセロールの解決 (PBS と 4', 6-diamidino-2-phenylindole (DAPI) の 1: 100,000 の 50% のグリセロール) を追加します。</li>
	<li>観察し、接眼レンズと 4 X、10 X、20 X 目標 X 10 を使用して蛍光顕微鏡下で細胞をイメージします。 注: 正しいフィルターを使用して、各染料のため: NR1 GFP の (励起/蛍光 = 482/502 nm)、Alexa Fluor 594 の (励起/蛍光 = 561/594 nm)、DAPI のため (励起/蛍光 = 358/461 nm)。同じ方法で GFP を発現する HEK 細胞を使用して負の制御を行います。</li>
</ol>

<ol>
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著者が明らかに何もありません。

生きている細胞を用いた蛍光アッセイ11、固定細胞を用いた蛍光抗体法9 を含む文献で報告された NMDA 受容体に対する自己抗体の存在を画面にいくつか細胞を用いた免疫学的アッセイには、流れの cytometry ベース測定14。生きている細胞を用いた蛍光抗体法は、流れの cytometry ベース試金は、訓練された人員および特別な装置を必要とする間...

抗 NMDA 受容体自己免疫性脳炎は、すべての年齢の患者に発生することができます、主に女性患者1,2に影響を与える新たに認められた疾患エンティティです。脳炎3の初期原因不明患者の間で最も頻繁に診断された脳炎の一つです。通常抗 NMDA 受容体脳炎で被害を受けた患者は、頭痛や発熱、意識レベルの変化、様々 な撹拌、過敏症など急性の神経精神症状の迅速な開発が続くの前駆症状を持っています。、不安、不眠、幻覚、妄想、侵略、奇妙な行動、運動異常、自律神経の調節不全や発作の攻撃4,5。この条件とタイミング療法と免疫療法を早期より良い結果と影響を受ける患者6も完全復旧のため重要です。したがって、抗 NMDA 受容体自己免疫性脳炎は急性または新規発症の精神機能7、8を呈する患者の鑑別診断として考慮されるべきことをお勧めします。
臨床的特徴のほかに血液や脳脊髄液の NMDA 受容体に対する自己抗体の検出は抗 NMDA 受容体自己免疫性脳炎9の正確な診断に不可欠です。ほとんどの抗 NMDA 受容体抗体を検出する免疫学的テストがいくつか研究所10,11, で利用可能なスクリーニングのための 1 つだけの市販細胞を用いた蛍光抗体法、抗 NMDA 受容体抗体12。本研究の目的は抗 NMDA 受容体の臨床研究を促進する抗 NMDA 受容体抗体の存在感を画面に実験室で便利に使用できる単純な社内細胞を用いた蛍光抗体を開発するには自己免疫性脳炎。NMDA 受容体は heterotetramer イオン チャネル蛋白質の複合体、脳で特異的に発現します。それはの 2 つの強制 NR1 サブユニットと NR2A、NR2B、NR2C、NR2D の13の 1 つまたは 2 つのサブユニットの組み合わせでください。以前の研究は、その抗体の対象となる主なエピトープは NR1 サブユニット5の細胞外 N 末端ドメインを報告しました。したがって、このプロトコルでは我々 は人間の萌芽期の腎臓上皮細胞株 (HEK293) で緑色蛍光タンパク質 (GFP) が付いた NMDA 受容体の人間の組換えの NR1 サブユニット蛋白質を表現し、検出する細胞を用いた蛍光抗体法を開発血液中の抗 NMDA 受容体抗体の IgG クラス。

<table>
	<tbody>
		<tr>
			<td>GRIN1 (GFP-tagged) - Human glutamate receptor, ionotropic, N-methyl D-aspartate 1 (GRIN1), transcript variant NR1-1</td>
			<td>Origene (Rockville, MD, USA)</td>
			<td>RG219368</td>
			<td>NR1-cDNA clone tagged with C-terminal tGFP sequences</td>
		</tr>
		<tr>
			<td>pCMV6-AC-GFP</td>
			<td>Origene (Rockville, MD, USA)</td>
			<td>PS100010</td>
			<td>mammalian vector with C-terminal tGFP tag</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen (Hilden Germany)</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Invitrogen (Carlsbad, CA, USA)</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose, Pyruvate</td>
			<td>Thermo Fisher</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>Characterized Fetal Bovine Serum, US Origin</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Human IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td>
			<td>Thermo Fisher</td>
			<td>A-11014</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive control: anti-glutamate receptor (type NMDA)</td>
			<td>Euroimmun AG, L&#252;beck, Germany</td>
			<td>CA 112d-0101</td>
			<td></td>
		</tr>
		<tr>
			<td>Euroimmun Assay Kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple cell-based immunofluorescence assay to detect autoantibody against the n-methyl-d-aspartate (nmda) receptor in blood

抗 NMDA 受容体抗体の存在は、影響を受ける患者は、抗 NMDA 受容体自己免疫性脳炎と呼ばれるさまざまな神経精神症状を引き起こします。血液や脳脊髄液 (CSF) の NMDA 受容体に対する特定の自己抗体の検出は、この条件の正確な診断のために不可欠です。NMDA 受容体は、イオン チャネル蛋白質複合体 2 つ必須 NMDA 受容体サブユニット 1 (NR1) と 1 つまたは 2 つの NMDA 受容体を含む 4 つのサブユニットを含むサブユニット 2 a (NR2A) NMDA 受容体サブユニット 2B NR2B、NMDA 受容体サブユニット 2 C (NR2C) または NMDA 受容体サブユニット 2 D (NR2D)。抗 NMDA 受容体抗体のエピトープは、NMDA 受容体の NR1 サブユニットの細胞外 N 末端ドメインに存在すると報じられました。本研究の目的は臨床および基礎研究を容易にするために血液の NMDA 受容体の NR1 サブユニットに対する抗体の存在を検出するスクリーニング検査として使用できる単純な細胞を用いた蛍光抗体法を開発、します。抗 NMDA 受容体の自己免疫性脳炎。

平均では、プロトコルのセクション 1 で説明されている手順に従って 250 mL の細菌文化から 200 〜 300 μ エンドトキシン発現プラスミド NR1 GFP の GFP を取得私たちでした。100 ng/よく発現プラスミドの量は、プロトコルのセクション 2 の説明に従って 48 ウェル プレートで培養した HEK293 細胞のトランスフェクションに使用されました。トランスフェクション後 24-30 時間?...

Watch this Scientific Journal Video about A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood at JoVE.com

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

我々 は異所萌出へ自己免疫性脳炎が疑われる患者の血中 NMDA 受容体に対する自己抗体を検出する抗原としてヒト胚性細胞 (HEK293) で緑色蛍光タンパク質が付いた NMDA 受容体の NR1 サブユニットを表明しました。この単純な方法はスクリーニング目的のため臨床現場で適切な可能性があります。

血の N-メチル-D-アスパラギン酸 (NMDA) 受容体に対する自己抗体を検出する単純な細胞ベースの蛍光抗体法

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

a-simple-cell-based-immunofluorescence-assay-to-detect-autoantibody

Research

JoVE Journal

Neuroscience

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

9.8K ビュー.  Chang Gung Memorial Hospital-Linkou. 我々 は異所萌出へ自己免疫性脳炎が疑われる患者の血中 NMDA 受容体に対する自己抗体を検出する抗原としてヒト胚性細胞 (HEK293) で緑色蛍光タンパク質が付いた NMDA 受容体の NR1 サブユニットを表明しました。この単純な方法はスクリーニング目的のため臨床現場で適切な可能性があります。

ビデオ: A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

ダイナミッククランプ法を用いたラット中脳ドーパミン作動性ニューロンにおけるNMDA受容体のコンダクタンスのアプリケーション

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

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 ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

イメージング初代培養マウス神経細胞における形質膜に受容体の挿入をpHluorinタグ付き

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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 ...

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.

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 Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

使用して報酬を求める行動の変化を測定するための簡単な方法<em&gt;キイロショウジョウバエ</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

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 ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

機能と神経病理学的評価の c57bl/6 マウスの実験的自己免疫性視神経炎を誘導するための単純なアプローチ

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

検出技術その場で革新的な RNA 側坐齧歯動物の核内ドーパミン D1-アルファ受容体の同定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

感受性グリシン/D-セリン、グルタミン酸と NMDA 受容体を研究する高スループット カルシウム変化分析

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...