Name: a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate
Uploaded: 2018-07-10T15:00:00.000+00:00
Duration: 4 min 48 s
Description: Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

著者は、郵便局バカロレア学者プログラムそしてノバルティス バイオメディカル研究本研究を資金調達のための全体として感謝したいと思います。

1. 細胞の調製
注: このプロトコルは、データの生成を含むエンコーディング NR1 と NR2A 細胞バキュロ ウイルスで導入した HEK293 細胞を使用します。
<ol><li>HEK293 細胞の適切な数をシードし、適切な最終濃度 (各 1.00 μ L) で NR1 および NR2A のウイルスを追加します。384 ウェル プレート 10,000 30 μ L の最終巻で細胞/ウェルを使用します。</li>
	<li>また、適切な細胞数と (384 ウェル プレート使用 10,000 細胞/ウェル 30 μ L の最終巻で) のためのボリュームで HEK293 NR1 NR2A 細胞をシードします。NR2A 式を誘導する化合物の保護を追加する 2 μ G/ml の濃度でテトラサイクリンを追加 (100 μ M MDL105、519 または 5 μ M CGP07066724)。</li>
	<li>注: エンコード NR1 と NR2 バキュロ ウイルス 5 x 109 - 10 x 109感染単位ミリリットル24間概算価が必要です。</li>
</ol>2. NMDA 受容体活性の測定
<ol><li>いずれかの保護化合物存在下での NR1/NR2 HEK293 NR1 NR2A セル増殖型 HEK293 細胞で 384 ウェル プレートを準備します。透明な底、黒フレーム、ポリ-D-リジン コーティング プレートを使用します。 注: 保護 100 μ m MDL105, 519 5 μ M 中のグリシンに感度を与えるため CGP070667 グルタミン酸感受性を付与するために使用します。</li>
	<li>16 時間 (夜間) 37 ° C および 5% の CO2でプレートを孵化させなさい。</li>
	<li>インキュベーターからプレートを削除し、優しくプレートをひっくり返す互換性存在コンテナーを遅くことによって携帯メディアを破棄します。プレートの端から媒体をきれいにし、残りのメディアをドレイン紙タオルの上にプレートを軽くたたきます。</li>
	<li>Calcium6 色素 (1 プレート サイズ) の 1 つのバイアルを可でインキュベーション バッファー (HBSS pH 7.5、20 mM HEPES、1 mM MgCl2、1 mM プロベネシド) 10 mL に calcium6 染料を準備します。プレートを 37 ° C、5% CO2で 2 時間インキュベートします。</li>
	<li>インキュベーターからプレートを削除し、10 分間部屋の温度に調整する細胞。</li>
	<li>優しく 2.3 の手順で説明されているように calcium6 染料を注ぐ、アッセイバッファー (HBSS pH 7.5、20 mM HEPES、CaCl2、1.8 mM 1 mM プロベネシド) の 30 μ L を追加します。</li>
	<li>3 洗浄の合計のために二度 2.6 手順を繰り返します。最後の洗浄後、セルにアッセイバッファーの 30 μ L を残します。</li>
	<li>室温で 5 分間、残りの細胞ができます。</li>
	<li>400 μ M のグリシンと 400 μ M グルタミン酸 (最終濃度 100 μ m; 下記のとおり決定する配位子の最適濃度) の 4 倍在庫することによって配位子板を準備します。V 底 384 ウェル プレートにリガンド株式の 25 μ L の最小値を転送します。</li>
	<li>フリーダイヤル (機能薬物スクリーニング システム) にリガンド板とセル板を読み込みます。</li>
	<li>30 秒間セル板のベースライン蛍光 (Fo) を測定します。5 分間 calcium6 色素蛍光を記録することによってフリーダイヤル調剤機能と測定カルシウム フラックスを使用して細胞板にリガンド株式の 10 μ L を転送します。</li>
	<li>最大蛍光比を計算する、ベースラインの蛍光 (F最大/Fo) によって得られる最大の蛍光を割る。</li>
</ol>3. NMDAR 発現レベルの最適化
<ol><li>使用するバキュロ ウイルスの量を調べるには、滴定、NR1 および NR2A のウイルスを実行します。HEK293 細胞 (10,000 細胞/ウェルの推薦、メディアの 30 μ L) で 384 ウェル プレートを準備し、手順 2.1 保護化合物が含まれます。</li>
	<li>プレートの異なる行の NR1 ウイルスの様々 な量を追加 (例: 行 A E 0 μ L、2 μ、1 μ L、0.5 μ L、0.25 μ L をそれぞれ追加)。データの正確性の重複を追加します。</li>
	<li>プレートの別の列に NR2A ウイルスの様々 な量を追加 (例: 行 A E 0 μ L、2 μ、1 μ L、0.5 μ L、0.25 μ L をそれぞれ追加)。データの正確性の重複を追加します。</li>
	<li>16 時間 (夜間) 37 ° C および 5% の CO2でプレートを孵化させなさい。</li>
	<li>フリーダイヤル (上記参照) にカルシウム フラックス測定を実行することによって、最適な比率と NR1 および NR2A のウイルスの量を決定します。</li>
</ol>4. 配位子の滴定
<ol><li>2.3 の手順で説明するように、セルを準備します。</li>
	<li>4 倍原液として他の配位子の飽和濃度 (100 μ M) の存在下でそれぞれの ligand の希釈液を準備します。たとえば: グリシン最終試験濃度 10 μ M、40 μ M の原液 400 μ M グルタミン酸を含むグリシンの準備します。</li>
	<li>フリーダイヤル測定 2.11 の手順で説明するように進みます。</li>
</ol>5. 複合試験
<ol><li>手順 1 で説明したように、セルを準備します。</li>
	<li>アッセイ バッファーに最終的なリガンド濃度の 5-fold 株式を持つリガンド プレートを準備します。</li>
	<li>アッセイ バッファーにおける化合物の所望の最終濃度の 4 倍の株式を持つ化合物のプレートを準備します。最終的な化合物の濃度、アッセイバッファーで 10 μ m、40 μ M の原液を準備します。<ol>
<li>ジメチルスルホキシド (DMSO) の濃度を一定に保つすべてのサンプル測定バッファーにさらに希薄化前に、の DMSO の化合物の前希釈を準備することによって。</li>
		<li>3 と 30 μ M の間で及ぶ最終試金の化合物の線量応答を 1 から 10 の mM まで及ぶ DMSO の化合物の前希釈を準備します。4 倍のストック濃度にアッセイバッファーで希釈します。DMSO の最終濃度 0.5% を超えてはなりません。 注: トリプリケートで各サンプルをテストすることをお勧めします。</li>
	</ol>
</li>
	<li>フリーダイヤルにリガンド、化合物、及び細胞板を読み込みます。</li>
	<li>30 秒のベースライン蛍光を測定します。</li>
	<li>化合物の在庫の 10 μ L を追加し、5 分の蛍光を測定します。</li>
	<li>リガンド株式の 10 μ L を追加し、5 分の蛍光を測定します。</li>
	<li>蛍光比の積分を測定の最後の 5 分間蛍光比の曲線下面積を計算することによって決定します。 注: また、蛍光リガンド添加後の最大比使用できます分析のため。</li>
</ol>

<ol>
	<li>Farrall, A. J., Wardlaw, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier:+ageing+and+microvascular+disease--systematic+review+and+meta-analysis.">Blood-brain barrier: ageing and microvascular disease--systematic review and meta-analysis.</a> Neurobiology of Aging. 30 (3), 337-352 (2009).</li><li>Walhovd, K. B., Fjell, A. M., Espeseth, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+decline+and+brain+pathology+in+aging--need+for+a+dimensional,+lifespan+and+systems+vulnerability+view.">Cognitive decline and brain pathology in aging--need for a dimensional, lifespan and systems vulnerability view.</a> Scandinavian Journal of Psychology. 55 (3), 244-254 (2014).</li><li>Wittchen, H. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+size+and+burden+of+mental+disorders+and+other+disorders+of+the+brain+in+Europe+2010.">The size and burden of mental disorders and other disorders of the brain in Europe 2010.</a> European Neuropsychopharmacology. 21 (9), 655-679 (2011).</li><li>Traynelis, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor+ion+channels:+structure,+regulation,+and+function.">Glutamate receptor ion channels: structure, regulation, and function.</a> Pharmacological Reviews. 62 (3), 405-496 (2010).</li><li>Mony, L., Kew, J. N., Gunthorpe, M. J., Paoletti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+modulators+of+NR2B-containing+NMDA+receptors:+molecular+mechanisms+and+therapeutic+potential.">Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential.</a> British Journal of Pharmacology. 157 (8), 1301-1317 (2009).</li><li>Lau, C. G., Zukin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor+trafficking+in+synaptic+plasticity+and+neuropsychiatric+disorders.">NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders.</a> Nature Reviews Neuroscience. 8 (6), 413-426 (2007).</li><li>Zhou, Q., Sheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptors+in+nervous+system+diseases.">NMDA receptors in nervous system diseases.</a> Neuropharmacology. 74, 69-75 (2013).</li><li>Paoletti, P., Bellone, C., Zhou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor+subunit+diversity:+impact+on+receptor+properties,+synaptic+plasticity+and+disease.">NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease.</a> Nature Reviews Neuroscience. 14, 383 (2013).</li><li>Sanz-Clemente, A., Nicoll, R. A., Roche, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+in+NMDA+receptor+composition:+many+regulators,+many+consequences.">Diversity in NMDA receptor composition: many regulators, many consequences.</a> Neuroscientist. 19 (1), 62-75 (2013).</li><li>Neyton, J., Paoletti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relating+NMDA+receptor+function+to+receptor+subunit+composition:+limitations+of+the+pharmacological+approach.">Relating NMDA receptor function to receptor subunit composition: limitations of the pharmacological approach.</a> Journal of Neuroscience. 26 (5), 1331-1333 (2006).</li><li>Hunt, D. L., Castillo, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+of+NMDA+receptors:+mechanisms+and+functional+implications.">Synaptic plasticity of NMDA receptors: mechanisms and functional implications.</a> Current Opinion in Neurobiology. 22 (3), 496-508 (2012).</li><li>Huganir, R. L., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPARs+and+synaptic+plasticity:+the+last+25+years.">AMPARs and synaptic plasticity: the last 25 years.</a> Neuron. 80 (3), 704-717 (2013).</li><li>Shimomura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycine+plays+a+crucial+role+as+a+co-agonist+of+NMDA+receptors+in+the+neuronal+circuit+generating+body+movements+in+rat+fetuses.">Glycine plays a crucial role as a co-agonist of NMDA receptors in the neuronal circuit generating body movements in rat fetuses.</a> Neuroscience Research. 97, 13-19 (2015).</li><li>Tajima, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+NMDA+receptors+and+the+mechanism+of+inhibition+by+ifenprodil.">Activation of NMDA receptors and the mechanism of inhibition by ifenprodil.</a> Nature. 534 (7605), 63-68 (2016).</li><li>Mayer, M. L., Westbrook, G. L., Guthrie, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+block+by+Mg2++of+NMDA+responses+in+spinal+cord+neurones.">Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones.</a> Nature. 309 (5965), 261-263 (1984).</li><li>Zhu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+NMDA+Receptor+Inhibition+and+Activation.">Mechanism of NMDA Receptor Inhibition and Activation.</a> Cell. 165 (3), 704-714 (2016).</li><li>Yildiz-Unal, A., Korulu, S., Karabay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+strategies+against+calpain-mediated+neurodegeneration.">Neuroprotective strategies against calpain-mediated neurodegeneration.</a> Neuropsychiatric Disease and Treatment. 11, 297-310 (2015).</li><li>Gascon, S., Sobrado, M., Roda, J. M., Rodriguez-Pena, A., Diaz-Guerra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxicity+and+focal+cerebral+ischemia+induce+truncation+of+the+NR2A+and+NR2B+subunits+of+the+NMDA+receptor+and+cleavage+of+the+scaffolding+protein+PSD-95.">Excitotoxicity and focal cerebral ischemia induce truncation of the NR2A and NR2B subunits of the NMDA receptor and cleavage of the scaffolding protein PSD-95.</a> Molecular Psychiatry. 13 (1), 99-114 (2008).</li><li>Uttara, B., Singh, A. V., Zamboni, P., Mahajan, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+neurodegenerative+diseases:+a+review+of+upstream+and+downstream+antioxidant+therapeutic+options.">Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options.</a> Current Neuropharmacology. 7 (1), 65-74 (2009).</li><li>Hansen, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+a+fluorescence-based+screening+assay+identifies+histamine+H3+receptor+antagonists+clobenpropit+and+iodophenpropit+as+subunit-selective+N-methyl-D-aspartate+receptor+antagonists.">Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.</a> Journal of Pharmacology and Experimental Therapeutics. 333 (3), 650-662 (2010).</li><li>Bettini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+novel+NMDA+receptor+antagonists+selective+for+NR2A-+over+NR2B-containing+receptors.">Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors.</a> Journal of Pharmacology and Experimental Therapeutics. 335 (3), 636-644 (2010).</li><li>Feuerbach, D., Loetscher, E., Neurdin, S., Koller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+pharmacology+of+the+human+NMDA-receptor+subtypes+R1-2A,+R1-2B,+R1-2C+and+R1-2D+using+an+inducible+expression+system.">Comparative pharmacology of the human NMDA-receptor subtypes R1-2A, R1-2B, R1-2C and R1-2D using an inducible expression system.</a> European Journal of Pharmacology. 637 (1-3), 46-54 (2010).</li><li>Hansen, K. B., Brauner-Osborne, H., Egebjerg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+characterization+of+ligands+at+recombinant+NMDA+receptor+subtypes+by+electrophysiological+recordings+and+intracellular+calcium+measurements.">Pharmacological characterization of ligands at recombinant NMDA receptor subtypes by electrophysiological recordings and intracellular calcium measurements.</a> Combinatorial Chemistry and High Throughput Screening. 11 (4), 304-315 (2008).</li><li>Guo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+NMDA-receptor+calcium+influx+assay+sensitive+to+stimulation+by+glutamate+and+glycine/D-serine.">A NMDA-receptor calcium influx assay sensitive to stimulation by glutamate and glycine/D-serine.</a> Scientific Reports. 7 (1), 11608 (2017).</li><li>Hackos, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+Allosteric+Modulators+of+GluN2A-Containing+NMDARs+with+Distinct+Modes+of+Action+and+Impacts+on+Circuit+Function.">Positive Allosteric Modulators of GluN2A-Containing NMDARs with Distinct Modes of Action and Impacts on Circuit Function.</a> Neuron. 89 (5), 983-999 (2016).</li><li>Romero-Hernandez, A., Furukawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Mode+of+Antagonist+Binding+in+NMDA+Receptors+Revealed+by+the+Crystal+Structure+of+the+GluN1-GluN2A+Ligand-Binding+Domain+Complexed+to+NVP-AAM077.">Novel Mode of Antagonist Binding in NMDA Receptors Revealed by the Crystal Structure of the GluN1-GluN2A Ligand-Binding Domain Complexed to NVP-AAM077.</a> Molecular Pharmacology. 92 (1), 22-29 (2017).</li><li>Auberson, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Phosphonomethylquinoxalinediones+as+competitive+NMDA+receptor+antagonists+with+a+preference+for+the+human+1A/2A,+rather+than+1A/2B+receptor+composition.">5-Phosphonomethylquinoxalinediones as competitive NMDA receptor antagonists with a preference for the human 1A/2A, rather than 1A/2B receptor composition.</a> Bioorganic and Medicinal Chemistry Letters. 12 (7), 1099-1102 (2002).</li><li>Danysz, W., Parsons, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycine+and+N-methyl-D-aspartate+receptors:+physiological+significance+and+possible+therapeutic+applications.">Glycine and N-methyl-D-aspartate receptors: physiological significance and possible therapeutic applications.</a> Pharmacological Reviews. 50 (4), 597-664 (1998).</li><li>Liu, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+II+Inhibitors+Can+Enhance+Baculovirus-Mediated+Gene+Expression+in+Mammalian+Cells+through+the+DNA+Damage+Response.">Topoisomerase II Inhibitors Can Enhance Baculovirus-Mediated Gene Expression in Mammalian Cells through the DNA Damage Response.</a> International Journal of Molecular Science. 17 (6), (2016).</li></ol>

著者があるない利益相反とは何を開示します。

この試金の成功は、使用される HEK 細胞の状態によって大きく異なります。細胞の急激な増加を受けて、低通過数を使用する必要があります。このアッセイ多く転送と警告を使用して、ソリューションの追加はその結果で高い精度を確保します。化合物およびその他のすべての試薬の濃度は、エラーを最小限に抑えるためにクロス チェックもする必要があります。アッセイバッファー カルシ...

医学の現在の進歩、平均寿命が大幅に増加します。しかし、加齢に伴う疾患の有病率がのであります。中枢神経系 (CNS)、他の中で、パーキンソン病、アルツハイマー病、筋萎縮性側索硬化症 (ALS) 統合失調症などの疾患は例外ではありません、次の十年1,で増加する予測されています。2,3N メチル D アスパラギン酸塩の受容器 (NMDAR) として知られているイオン型グルタミン酸受容体の機能不全は、アルツハイマー病、統合失調症、外傷性脳損傷、脳卒中、糖尿病、緑内障等保証のにリンクされている、。効果的な疾患修飾療法4,5,6、7の開発のための生物を勉強する必要があります。
NMDARs は、4 つのモノマーまたはサブユニット4,8,9で構成されます。NMDAR の構造は、脳7,10内の発達と地域の変動を示しています。NMDARs は、シナプス可塑性と認知及び呼吸と歩行11,12,13のリズムの生成に関与しています。電位依存性チャネルとしてそれは、安静時の膜電位伝導非主 (-70 mV) し、さらにイオンの透過を防ぐためにマグネシウムによってブロックします。2 配位子、グルタミン酸およびグリシン/D-セリン、バインドによってチャネルがアクティブになるし、シナプス膜に同時脱分極は AMPA 受容体、イオン型グルタミン酸受容体の別のサブクラスによって仲介されます。分極防止作用は、特にカルシウム14,,1516陽イオンの流入を有効にする、NMDAR のマグネシウム閉塞を削除します。にもかかわらず NMDAR の活性化は細胞の生存に不可欠な過剰な活性化は細胞死17,18,19応需型につながります。これは、受容体の複雑な性質に加えて難しく研究を実施する効果的な治療法を開発するために必要です。
別の方法は、NMDAR の研究に開発されています。ただし、それぞれの注意事項が付属しています。たとえば、1 つの広く使われている技術は安定したセルライン テトラサイクリン誘導性プロモーター (テトの)20の制御の下での細胞内カルシウム NMDAR 依存性の変化を測定する蛍光ベースの試金であります。ただし、このシステムに必要な配位子の激濃度と NMDAR の阻害剤が測定中に存在している要件不可能になります競争力の拮抗筋の活動を検出します。他の同様のシステムは、機能的受容体の発現は、ケタミン21,22細胞培養を維持するなどの拮抗を必要とする毒性を引き起こします。これらのチャネルのブロッカーは、受容体のコアに座るし、受容体の機能解析と干渉すること特にプレート ベースの形式で、洗いにくい。最後に、パッチなどの電気生理学的計測、スループットの制限があるし、大規模な研究は、非常に高価な23。にもかかわらず、上記のシステムはグリシンの刺激に敏感したがって、課題となる、NMDAR のグリシン依存性活性を勉強します。
ここでは、議論の制限を克服 NMDAR の勉強への新しいアプローチについて述べる。バキュロ ウイルス発現系としてはほとんど 16 時間で受容体のサブユニットの最適比の機能レベルを表現する技術を使用します。さらに、バキュロ ウイルスの使用によりシンプルかつコンビナトリアル アプローチは、異なる組換え NMDAR のサブタイプの広範な評価を提供します。他のアッセイとは異なり、このプロトコルには弱いアンタゴニストの使用のためチャネル遮断薬は必要ありません。メソッドの最も強い利点は弱い拮抗薬の後のウォッシュ アウトはグリシン/D-セリン、グルタミン酸の両方のデュアルの変調に加えて個々 グリシンとグルタミン酸結合部位の変調に敏感で受容体リガンド結合サイト。試金は NMDAR 受容体の既知の薬理学とその既知の正と負の変調器の効果を繰り返します。最後に、この生体外細胞アッセイの生成過剰なカルシウムの流入によって引き起こされる細胞毒性を克服でき、NMDAR の変調器の発見を加速することができます高スループット方法で受容体の機能解析病にある州。

<table><tbody><tr><td>HEK-293</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td>Human NMDA (NR1/NR2A) Receptor Cell Line</td><td>ChanTest Corporation</td><td>CT6120</td><td></td></tr><tr><td>pFastBac Dual Expression Vector</td><td>ThermoFisher Scientific</td><td>10712-024</td><td></td></tr><tr><td>Corning 384-well Clear Flat Bottom Microplate</td><td>Corning Life Sciences</td><td>3844</td><td></td></tr><tr><td>FLIPR Calcium 6-QF Assay Kit</td><td>Molecular Devices</td><td>R8192</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Glutamate</td><td>Sigma-Aldrich</td><td>49621</td><td></td></tr><tr><td>D-serine</td><td>Sigma-Aldrich</td><td>S4250</td><td></td></tr><tr><td>L701,324</td><td>Tocris</td><td>907</td><td></td></tr><tr><td>HEPES Buffer</td><td>Boston Bio Product</td><td>BB-103</td><td></td></tr><tr><td>Magnesium Chloride Solution</td><td>Sigma-Aldrich</td><td>63069</td><td></td></tr><tr><td>Calcium Chloride</td><td>VWR</td><td>E506</td><td></td></tr><tr><td>HBSS</td><td>ThermoFisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Probenecid</td><td>ThermoFisher Scientific</td><td>P36400</td><td></td></tr><tr><td>DMEM/F-12, GlutaMAX media</td><td>ThermoFisher Scientific</td><td>10565018</td><td></td></tr><tr><td>MDL105,519</td><td>NIBR</td><td>Synthesized in house</td><td></td></tr><tr><td>NVP-AAM077</td><td>NIBR</td><td>Synthesized in house</td><td></td></tr><tr><td>CGP070667</td><td>NIBR</td><td>Synthesized in house</td><td></td></tr></tbody></table>

a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate

N メチル D アスパラギン酸 (NMDA) 受容体 (NMDAR) はグルタミン酸に分類され、学習と記憶に重要な役割を持っています。表現としてどちらかに- または下-activity の突然変異、発現、人身売買、またはローカリゼーションによる NMDAR の故障は、中枢神経系を中心に、多数の病気に貢献できます。したがって、化合物と低分子化合物の探索を促進することと同様、受容体の生物学を理解することは、神経疾患と闘うための継続的な努力で重要です。現在の受容体の勉強方法低スループット、高コスト NMDAR 仲介された毒性を防ぐためにチャネル遮断薬の必要な存在のためにその機能能力を研究することができないなどの制限があります。また、既存のアッセイ系のみグルタミン酸による刺激に敏感である、グリシン、NMDAR の他の共同のリガンドによる刺激に対する感受性を欠いています。両方共配位子、グルタミン酸や D セリン ・ グリシンへの感受性と NMDA 受容体を研究する高スループット力と最初のプレート ベースのアッセイを紹介します。このアプローチ様々 な NMDAR サブユニット組成の研究でき、受容体の機能解析グリシンやグルタミン酸に敏感なモードで。また、メソッドでは、測定中の阻害剤の存在は必要がないです。正と負のアロステリック変調器の効果は、このアッセイで検出できるし、NMDAR の既知の薬理学は、私たちのシステムにレプリケートされています。この手法は既存の方法の限界を克服、経済的であります。この手法は NMDAR を介した病態の治療の発見を加速すると考えています。

小分子の効果をテストする前に、1 つは NMDARs として最適なリガンド濃度の最適な発現レベルを定めなければなりません。前述、HEK293 細胞/ウェルの 384 ウェル プレートで 10,000 細胞播種 5 μ M の存在下で CGP060667、その後導入 NR1 と NR2A の変化量とウイルス。孵化翌日、リガンド誘導性カルシウム後フラックスを測定した (図 1)。これらの実験の結?...

Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

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

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

このプロトコルの目標より大きな規模での NMDA 受容器 (NMDAR) の研究と、低分子化合物の変調作用の検討と治療への応用です。

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

a-high-throughput-calcium-flux-assay-to-study-nmda-receptors-with

Research

JoVE Journal

Neuroscience

9.2K ビュー.  Novartis Institutes for BioMedical Research. このプロトコルの目標より大きな規模での NMDA 受容器 (NMDAR) の研究と、低分子化合物の変調作用の検討と治療への応用です。

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

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

遺伝的にコードされたセンサを用いたグルタミンフラックスイメージング

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

生物工学

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

CA1錐体ニューロンにおけるグルタミン酸のUV媒介焦点アンケージングと組み合わせることで、多光子細胞内ナトリウムイメージング

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

神経科学

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

組換えGタンパク質共役型受容体のハイスループットスクリーニングのための「二重付加」カルシウム蛍光アッセイ

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

生化学

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

アルツハイマー病のマウスモデルにおけるトニックと相性グルタミン酸を測定するための酵素ベースのバイオセンサーを使用して

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

AMPA の監視用高コンテンツ受容体トラフィッ キング

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

解離された原発性海馬培養におけるグルタミン酸受容体の密売を研究するための抗体摂食アプローチ

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常マウスおよびハンチントンマウスからの急性脳スライスにおけるグルタミン酸放出および取り込みの単一シナプス指標

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

主マウス中脳ニューロンにおける自発的Ca2+ フラックスとその下流効果の定量化

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

一チャネルセル付パッチクランプ法

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

生物学

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

筋萎縮性側索硬化症のモデルにおけるシナプス機能のリアルタイム蛍光測定

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

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

要約

さらに動画を探す

この動画の章

遺伝的にコードされたセンサを用いたグルタミンフラックスイメージング

CA1錐体ニューロンにおけるグルタミン酸のUV媒介焦点アンケージングと組み合わせることで、多光子細胞内ナトリウムイメージング

組換えGタンパク質共役型受容体のハイスループットスクリーニングのための「二重付加」カルシウム蛍光アッセイ

アルツハイマー病のマウスモデルにおけるトニックと相性グルタミン酸を測定するための酵素ベースのバイオセンサーを使用して

AMPA の監視用高コンテンツ受容体トラフィッキング

解離された原発性海馬培養におけるグルタミン酸受容体の密売を研究するための抗体摂食アプローチ

正常マウスおよびハンチントンマウスからの急性脳スライスにおけるグルタミン酸放出および取り込みの単一シナプス指標

主マウス中脳ニューロンにおける自発的^Ca2+ フラックスとその下流効果の定量化

一チャネルセル付パッチクランプ法

筋萎縮性側索硬化症のモデルにおけるシナプス機能のリアルタイム蛍光測定

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

要約

さらに動画を探す

この動画の章

遺伝的にコードされたセンサを用いたグルタミンフラックスイメージング

CA1錐体ニューロンにおけるグルタミン酸のUV媒介焦点アンケージングと組み合わせることで、多光子細胞内ナトリウムイメージング

組換えGタンパク質共役型受容体のハイスループットスクリーニングのための「二重付加」カルシウム蛍光アッセイ

アルツハイマー病のマウスモデルにおけるトニックと相性グルタミン酸を測定するための酵素ベースのバイオセンサーを使用して

AMPA の監視用高コンテンツ受容体トラフィッ キング

解離された原発性海馬培養におけるグルタミン酸受容体の密売を研究するための抗体摂食アプローチ

正常マウスおよびハンチントンマウスからの急性脳スライスにおけるグルタミン酸放出および取り込みの単一シナプス指標

主マウス中脳ニューロンにおける自発的Ca2+ フラックスとその下流効果の定量化

一チャネルセル付パッチクランプ法

筋萎縮性側索硬化症のモデルにおけるシナプス機能のリアルタイム蛍光測定

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

AMPA の監視用高コンテンツ受容体トラフィッキング

主マウス中脳ニューロンにおける自発的^Ca2+ フラックスとその下流効果の定量化