Name: live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons
Uploaded: 2015-06-24T14:00:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-&#945;4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.

全ての動物実験は、実験動物の管理と使用に関する健康ガイドの国立研究所に従って行った（NIH出版番号80から23は、2012年改訂）し、研究は施設内動物実験によって承認されたとストーニーの研究委員会のために使用しますブルック大学（＃1618と＃1792）。 1. vHipp-NACCシナプス共培養<ol><li>犠牲マウス（生後0日-野生型（WT）またはα7nAChR類トランスジェニックマウスラインから3）CO 2を。各子犬の首をはねると事後遺伝子型決定のための1.5ミリリットル遠心管に尾を保存します。滅菌フードで次の手順を実行します。 </li><li>脳全体を露出させるためにハサミやピンセットで頭蓋骨の上部を取り外します。互いに尾側からの大脳皮質の部分の接合部から開始して、解剖顕微鏡下vHipp 5,21を含む後部皮質が含まれている冠状断面を得ます。 </li><li> Vを分析発展途上のマウス脳アトラス22（ 図1A）によればCA1-海馬台領域を含むヒップ組織片。 （100ミクロンmicroslices×100ミクロン程度）の小さな薄いスライスにカット冷培地（4℃）を含む35mmの培養皿に移します。 </li><li> 37℃のインキュベーター内に少なくとも30分間、培地（〜500μl）を、24​​ウェル組織培養プレートの内側12ミリメートルポリ-D-リジン/ラミニン被覆カバーガラスをプレインキュベートします。 microslicesをメッキする前にメディアを取り出します。 </li><li>外植片の付着を容易にするために（microslicesの〜20個を含む〜50μL、）メディアの最小量でカバースリップの中央に火造りパスツールピペットでプレート。 1匹の動物に由来するプレートvHippのmicroslices（どちらか+ / +または - / - トランスジェニックα7ラインの​​遺伝子型）各カバースリップ上。 </li><li>外植片がカバースリップ上に沈降した後、静かに目によって追加メディア（〜100μl）を追加電子壁メディアのレベルは完全に周囲の外植片ではなく、カバースリップの中央にそれらをカバーするのに十分に高いようにします。 </li><li> 37℃でO / Nインキュベートした後、5％CO 2インキュベーター、生後1日目WTマウスに胚日18からNACCニューロンを分散させ、外植片に加えます。 <ol><li>発展途上のマウス脳アトラス20（ 図1B）に応じてNACC組織を解剖。 </li><li> （500ミクロンmicroslices×500ミクロン程度）を小片に切断し、15 mlチューブに移します。 </li><li> 37℃で15分間、0.25％トリプシン（2ml）で処理します。冷培地で1回洗浄（5 mlで4°C）、続いて冷洗浄培地（5 mlで4°C）での洗浄組織塊三回。サスペンションは、各洗浄工程における5分間の休憩、その後、上清を慎重に取り除くことを可能にします。 </li><li>軽くファイアーポリッシュパスツールピペットで培養培地2mlに優しい粉砕して細胞を解離します。 </li><li>トランス5分間、2,000 X rpmでさらに15 mlチューブと遠心分離機に細胞懸濁液FER。上清を除去し、6子犬ごとに1 mlの培養培地で細胞を再懸濁します。穏やかファイアーポリッシュパスツールピペットで培地で数回ピペッティングして細胞を分散。 </li><li> vHipp移植片と各カバースリップに分散NACC細胞の0.25ミリリットルを追加します。 </li><li>加湿37℃、5％CO 2インキュベーター内で培養を維持します。機械的にシナプス形成を促進する、湿度を安定して維持するために、湿らせた滅菌ガーゼパッド上に培養プレートを置きます。 </li></ol></li></ol> 2.免疫細胞化学<ol><li> 4％パラホルムアルデヒド/ 4％スクロース/ PBS（カバースリップあたり〜500μL、20分、RT）における- （in vitroで 7日5）文化を修正しました。 </li><li> 0.25％トリトンX-100 / PBSで透過処理培養（〜カバースリップあたり500μL、5分、RT）。 </li><li>不格好なやつの前に、PBS中の10％正常ロバ血清（カバースリップあたり〜500μL、30分、RT）でブロック文化Y染色。 4℃でO / N一次抗体でインキュベートします。 、抗小胞性グルタミン酸トランスポーター1（1：250）、抗GAD65（1：100）：;：抗nAChR類（抗α5サブユニット、1 500 500α4 -ECD 1：）以下の一次抗体を使用してください。 </li><li> PBSで一次抗体を洗浄（カバースリップあたり3回/〜500μL、5分間）した後、アレクサ488（カバースリップあたり〜500μlを、1：500）に結合した二次抗体で培養インキュベートまたはアレクサ594（カバースリップあたり〜500μLを、1：37℃で1時間、500）。 </li><li>標識前表面α7*のnAChRのための固定を37℃で45分間：（培地1,000〜カバースリップあたり500μlの、1）のAlexa 594にコンジュゲートαBgTxで培養をインキュベートします。 </li><li>マウントとシールカバースリップ。 </li><li>プランアポクロマート対物レンズ（NA 0.8または1.4 NAの63X油で20X）を備えた蛍光顕微鏡とCCDカメラで画像をキャプチャします。 </li></ol> 3. FM1-43ベースの水疱性の融合<ol><li>ディスク共焦点顕微鏡を紡糸上室マウント、撮像チャンバ内に- （in vitroで 7日、5）の文化を維持します。連続RTでHBSカクテル（1ml /分）灌流。 </li><li> 90秒間の56 mMのK + ACSF（〜500μL）（染色）で10μMのFM1-43で灌流し、負荷の文化を停止、のCa 2+ ADVASEP-7（0.1 mm）を含む無HBSで15分間離れて外部染料を洗います膜結合FM1-43を除去します。 </li><li> 、120秒（脱色）のためにFM1-43ずに56 mMのK + ACSF（〜500μL）と文化を刺激するのと同じ条件を用いて、FM1-43と文化を再ロード、およびCaで再び洗浄2+のためADVASEP-7を含む無HBS 15分。 </li><li> （1.4 NA、励起488 nmの発光530 nmのと60X水）プランアポクロマート対物レンズを搭載した回転ディスク共焦点顕微鏡でFM1-43の蛍光画像を取得し、CCDカメラで画像をキャプチャします。 </li><li> 1分間のベースラインFM1-43蛍光画像（2秒ごとに集めます）プリニコチンコントロールとして; 1分間の急速な灌流（2 ml /分）によってニコチン（1μM）を適用し、アウトHBSカクテルでニコチンを洗います。 5分ごとに2秒の間、およびニコチン適用後に、前にキャプチャタイムラプス画像を保管してください。 </li><li> （F 染色 ）の前に、各シナプスブートンにおける（F 脱色 ）ニコチン適用後FM1-43の蛍光強度を定量化します。 </li><li>ニコチンアプリケーション（ΔF= F 染色 -Fの脱色 ）の前と後のFM1-43蛍光強度の差を定量化することによってvHipp軸索に沿って解放可能FM1-43蛍光の総量を計算します。次式を有するニコチン後の蛍光強度の減少の割合を分析：F 減少 ％=ΔF/ F 染色 。 </li></ol> 4.カルシウムイメージング<ol><li>次に、（5μMのFluo-4のCa 2+指示薬と文化を読み込む、通常のHBSで迅速- （in vitroで 7日5）文化をすすぎますAMエステル）、37℃で30分間、5％CO 2のためにHBSで0.02％プルロニックF-127（2ml）中。 </li><li> HBSとのFluo-4溶液（3回/ 5分）洗い流します。 （37℃/ 5％CO 2）は、少なくとも30分間インキュベーターに文化を返します。 </li><li>同じ撮像チャンバーとFM1-43ベース小胞の融合の項に記載と同様の条件で培養を維持する（ステップ3.1を参照してください）​​。 </li><li>プランアポクロマート対物レンズ（1.4 NAの60X水、励起488 nmの発光530 nm）をCCDカメラでvHippマイクロスライスから軸索突起のフルオ4蛍光画像を収集します。 </li><li> 1分間の急速な灌流（2 ml /分）によってニコチン（1μM）を適用、事前ニコチンコントロールとして、ベースラインのFluo-4の蛍光画像（2分10秒ごと）を採取し、アウトHBSカクテルでニコチンを洗います。 30分ごとに10秒の間、およびニコチン適用後に、前にキャプチャタイムラプス画像を保管してください。 </li><li>一連の生のfluo-4の蛍光画像、輸出のすべてのフレームを保存さらなる分析のためのTIFF形式の画像。 </li><li>ニコチン適用前と後のvHipp軸索に沿ってFLUO-4の蛍光の積分強度を収集します。 </li><li> ΔF/ F F 0は、バックグラウンド補正前ニコチン積分蛍光強度であり、Fは、各時点におけるvHipp軸索に沿って統合された蛍光強度が0 =（FF 0）/ F 0、以下の式を有する集積蛍光強度を正規化ニコチン適用後。正規化された統合された蛍光強度を分析し、プロットします。 </li></ol>

<ol>
	<li>Changeux, J. 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=Brain+nicotinic+receptors:+structure+and+regulation,+role+in+learning+and+reinforcement.">Brain nicotinic receptors: structure and regulation, role in learning and reinforcement.</a> Brain Res Brain Res Rev. 26 (2-3), 198-216 (1998).</li><li>Levin, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+receptor+subtypes+and+cognitive+function.">Nicotinic receptor subtypes and cognitive function.</a> J Neurobiol. 53 (4), 633-640 (2002).</li><li>Dani, J. A., Bertrand, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+acetylcholine+receptors+and+nicotinic+cholinergic+mechanisms+of+the+central+nervous+system.">Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system.</a> Annu Rev Pharmacol Toxicol. 47, 699-729 (2007).</li><li>Mineur, Y. S., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+receptors+and+depression:+revisiting+and+revising+the+cholinergic+hypothesis.">Nicotine receptors and depression: revisiting and revising the cholinergic hypothesis.</a> Trends Pharmacol Sci. 31 (12), 580-586 (2010).</li><li>Zhong, C., Talmage, D. A., Role, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+elicits+prolonged+calcium+signaling+along+ventral+hippocampal+axons.">Nicotine elicits prolonged calcium signaling along ventral hippocampal axons.</a> PloS one. 8 (12), e82719 (2013).</li><li>McGehee, D. S., Heath, M. J., Gelber, S., Devay, P., Role, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+enhancement+of+fast+excitatory+synaptic+transmission+in+CNS+by+presynaptic+receptors.">Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors.</a> Science. 269 (5231), 1692-1696 (1995).</li><li>Gray, R., Rajan, A. S., Radcliffe, K. A., Yakehiro, M., Dani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+synaptic+transmission+enhanced+by+low+concentrations+of+nicotine.">Hippocampal synaptic transmission enhanced by low concentrations of nicotine.</a> Nature. 383 (6602), 713-716 (1996).</li><li>Dickinson, J. A., Kew, J. N., Wonnacott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+alpha+7-+and+beta+2-containing+nicotinic+acetylcholine+receptors+modulate+excitatory+amino+acid+release+from+rat+prefrontal+cortex+nerve+terminals+via+distinct+cellular+mechanisms.">Presynaptic alpha 7- and beta 2-containing nicotinic acetylcholine receptors modulate excitatory amino acid release from rat prefrontal cortex nerve terminals via distinct cellular mechanisms.</a> Mol Pharmacol. 74 (2), 348-359 (2008).</li><li>Zappettini, S., Grilli, M., Salamone, A., Fedele, E., Marchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-synaptic+nicotinic+receptors+evoke+endogenous+glutamate+and+aspartate+release+from+hippocampal+synaptosomes+by+way+of+distinct+coupling+mechanisms.">Pre-synaptic nicotinic receptors evoke endogenous glutamate and aspartate release from hippocampal synaptosomes by way of distinct coupling mechanisms.</a> Br J Pharmacol. 161 (5), 1161-1171 (2010).</li><li>Zappettini, 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=Presynaptic+nicotinic+alpha7+and+non-alpha7+receptors+stimulate+endogenous+GABA+release+from+rat+hippocampal+synaptosomes+through+two+mechanisms+of+action.">Presynaptic nicotinic alpha7 and non-alpha7 receptors stimulate endogenous GABA release from rat hippocampal synaptosomes through two mechanisms of action.</a> PloS one. 6 (2), e16911 (2011).</li><li>Fabian-Fine, R., 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=Ultrastructural+distribution+of+the+alpha7+nicotinic+acetylcholine+receptor+subunit+in+rat+hippocampus.">Ultrastructural distribution of the alpha7 nicotinic acetylcholine receptor subunit in rat hippocampus.</a> J Neurosci. 21 (20), 7993-8003 (2001).</li><li>Jones, I. W., Barik, J., O'Neill, M. J., Wonnacott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha+bungarotoxin-1.4+nm+gold:+a+novel+conjugate+for+visualising+the+precise+subcellular+distribution+of+alpha+7*+nicotinic+acetylcholine+receptors.">Alpha bungarotoxin-1.4 nm gold: a novel conjugate for visualising the precise subcellular distribution of alpha 7* nicotinic acetylcholine receptors.</a> J Neurosci Methods. 134 (1), 65-74 (2004).</li><li>Nashmi, R., 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=Assembly+of+alpha4beta2+nicotinic+acetylcholine+receptors+assessed+with+functional+fluorescently+labeled+subunits:+effects+of+localization,+trafficking,+and+nicotine-induced+upregulation+in+clonal+mammalian+cells+and+in+cultured+midbrain+neurons.">Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons.</a> J Neurosci. 23 (37), 11554-11567 (2003).</li><li>Drenan, R. M., 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=Subcellular+trafficking,+pentameric+assembly,+and+subunit+stoichiometry+of+neuronal+nicotinic+acetylcholine+receptors+containing+fluorescently+labeled+alpha6+and+beta3+subunits.">Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled alpha6 and beta3 subunits.</a> Mol Pharmacol. 73 (1), 27-41 (2008).</li><li>Wu, J., 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=Electrophysiological,+pharmacological,+and+molecular+evidence+for+alpha7-nicotinic+acetylcholine+receptors+in+rat+midbrain+dopamine+neurons.">Electrophysiological, pharmacological, and molecular evidence for alpha7-nicotinic acetylcholine receptors in rat midbrain dopamine neurons.</a> J Pharmacol Exp Ther. 311 (1), 80-91 (2004).</li><li>Parikh, V., Ji, J., Decker, M. W., Sarter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+beta2+subunit-containing+and+alpha7+nicotinic+acetylcholine+receptors+differentially+control+glutamatergic+and+cholinergic+signaling.">Prefrontal beta2 subunit-containing and alpha7 nicotinic acetylcholine receptors differentially control glutamatergic and cholinergic signaling.</a> J Neurosci. 30 (9), 3518-3530 (2010).</li><li>Nayak, S. V., Dougherty, J. J., McIntosh, J. M., Nichols, 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=Ca(2+)+changes+induced+by+different+presynaptic+nicotinic+receptors+in+separate+populations+of+individual+striatal+nerve+terminals.">Ca(2+) changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals.</a> J Neurochem. 76 (6), 1860-1870 (2001).</li><li>Richards, C. I., 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=Trafficking+of+alpha4*+nicotinic+receptors+revealed+by+superecliptic+phluorin:+effects+of+a+beta4+amyotrophic+lateral+sclerosis-associated+mutation+and+chronic+exposure+to+nicotine.">Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.</a> J Biol Chem. 286 (36), 31241-31249 (2011).</li><li>Colombo, S. F., Mazzo, F., Pistillo, F., Gotti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+trafficking+and+up-regulation+of+nicotinic+ACh+receptors.">Biogenesis, trafficking and up-regulation of nicotinic ACh receptors.</a> Biochem Pharmacol. 86 (8), 1063-1073 (2013).</li><li>St John, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+trafficking+of+nicotinic+acetylcholine+receptors.">Cellular trafficking of nicotinic acetylcholine receptors.</a> Acta Pharmacol Sin. 30 (6), 656-662 (2009).</li><li>Zhong, C., 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=Presynaptic+type+III+neuregulin+1+is+required+for+sustained+enhancement+of+hippocampal+transmission+by+nicotine+and+for+axonal+targeting+of+alpha7+nicotinic+acetylcholine+receptors.">Presynaptic type III neuregulin 1 is required for sustained enhancement of hippocampal transmission by nicotine and for axonal targeting of alpha7 nicotinic acetylcholine receptors.</a> J Neurosci. 28 (37), 9111-9116 (2008).</li><li>Jacobowitz, D. M., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemoarchitectonic Atlas of the Developing Mouse Brain. , (1998).</li><li>Betz, W. J., Mao, F., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+fluorescent+staining+and+destaining+of+living+vertebrate+motor+nerve+terminals.">Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals.</a> J Neurosci. 12 (2), 363-375 (1992).</li><li>Amaral, E., Guatimosim, S., Guatimosim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+fluorescent+styryl+dye+FM1-43+to+visualize+synaptic+vesicles+exocytosis+and+endocytosis+in+motor+nerve+terminals.">Using the fluorescent styryl dye FM1-43 to visualize synaptic vesicles exocytosis and endocytosis in motor nerve terminals.</a> Methods Mol Biol. 689, 137-148 (2011).</li><li>Garduno, J., 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=Presynaptic+alpha4beta2+nicotinic+acetylcholine+receptors+increase+glutamate+release+and+serotonin+neuron+excitability+in+the+dorsal+raphe+nucleus.">Presynaptic alpha4beta2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus.</a> J Neurosci. 32 (43), 15148-15157 (2012).</li><li>Guo, J. Z., Liu, Y., Sorenson, E. M., Chiappinelli, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+released+and+exogenous+ACh+activates+different+nicotinic+receptors+to+enhance+evoked+glutamatergic+transmission+in+the+lateral+geniculate+nucleus.">Synaptically released and exogenous ACh activates different nicotinic receptors to enhance evoked glutamatergic transmission in the lateral geniculate nucleus.</a> J Neurophysiol. 94 (4), 2549-2560 (2005).</li><li>Szabo, S. I., Zelles, T., Vizi, E. S., Lendvai, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+nicotine+on+spiking+activity+and+Ca2++dynamics+of+dendritic+spines+in+rat+CA1+pyramidal+neurons.">The effect of nicotine on spiking activity and Ca2+ dynamics of dendritic spines in rat CA1 pyramidal neurons.</a> Hippocampus. 18 (4), 376-385 (2008).</li></ol>

著者らは、開示することは何もありません。

共培養の準備は、in vitroで前シナプスのnAChRの活性化が強化されたグルタミン酸作動性伝達5、21を誘発することにより、空間的および時間的プロファイルのこの準備を許可比較的簡単で信頼性の高い検査を再降伏し、腹側海馬側坐核回路を説明しました。 共培養は、インビボ様の機能を実証するために、インビトロでの生理学的条件を提供す?...

回路の興奮性のコリン作動性調節は、認知機能の基本的な側面に寄与し、変更されたコリン作動性の調節は、アルツハイマー病、パーキンソン病、統合失調症及び中毒1-4を含む神経変性及び精神神経疾患の特徴です。 CNSにおけるシナプス伝達のコリン作動性促進の確立メカニズムは、シナプス前部位に局在するのnAChRの直接の活性化を介してです。 、間接的に特定のnAChRサブタイプ、および比較的高いカルシウムコンダクタンスの両方に直接起因を介した細胞内シグナル伝達カスケード-これらのシナプス前受容体の活性化は細胞内Ca 2+（[Ca 2+] i） のシナプス前末端での増加につながります図5は 、それによって神経伝達物質の放出を増強します。実際には、シナプス前のnAChRの活性化は、グルタミン酸、GABA、アセチルコリンなどの神経伝達物質の多種多様の放出の変化と関連している、ANDドーパミン6-10。このプロセスは、様々なシナプスにおける電気生理学的方法を用いて、間接的に研究されてきたが、の光学記者の[Ca 2+] iとシナプス小胞のリサイクルは、前シナプス現象のより直接的かつ時間的に正確な測定を可能にします。 nAChR類の前シナプス局在は、電子顕微鏡（EM）レベル11,12でのnAChRの直接の免疫金標識で説得力が実証されています。いくつかの他の技術もまた、培養ニューロン13,14でのnAChR subunit-蛍光タンパク質キメラの位置を検出するなど、間接的のnAChR局在に対処するために使用されている、シナプス端子15,16でのnAChR電流の電気生理学的記録、[内のCaニコチン誘発変化を監視しますによるシナプスターミナルでのライブセルイメージング17、及び神経伝達物質の放出を間接的に監視することにより、シナプス神経終末における2+] i のスチリル両親媒性FM色素（FM1-43とFM4-64）お​​よび/またはsynapto-pHluorinによって、そのようなグルタミン酸のためのAChとiGluSnFrためCNiFERsなどの特定の神経伝達物質の蛍光の記者、によって見シナプス小胞のエキソサイトーシスを含む蛍光指示薬と生細胞イメージング技術、 18-20。全体的に、nAChR類の前シナプス局在を同定するため、これらの現在の手法は複雑であり、信頼性の高い同定とプレシナプス活性の生理学的モニタリングを可能にするために、特別なシステム及び技術を必要とします。 識別し、シナプス伝達のシナプス前および後の両方の成分を分析するための直接アクセスを提供し、側坐核（NACC）回路-ここでは、腹側海馬（vHipp） の in vitro共培養システムのためのプロトコルおよび装置が記載されています。我々は、シナプス前のnAChRの局在およびCa 2+シグナル伝達と神経伝達物質の放出ALO媒介のnAChRのライブセルイメージングの例を示しますNG vHipp軸索。ここで紹介するプロトコルの自然（と簡単な）拡張は、異なる遺伝子型からの神経細胞からなる前と後シナプスの接点の製造です。このように、変調の前および/または後シナプスメカニズムの特定の遺伝子産物の寄与を直接評価することができます。

<table><tbody><tr><td colspan="4">1. Culture Media (50 ml)</td></tr><tr><td>Neurobasal&amp;nbsp;</td><td>GIBCO</td><td>10888022</td><td>48 ml</td></tr><tr><td>B-27 Supplements</td><td>GIBCO</td><td>0080085-SA</td><td>1 ml</td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO</td><td>10908-010</td><td>0.5 ml</td></tr><tr><td>GlutaMAX Supplement</td><td>GIBCO</td><td>35050-061</td><td>0.5 ml</td></tr><tr><td>Brain-derived neurotrophic factor (BDNF)</td><td>GIBCO</td><td>15140-122</td><td>20 ng/ml</td></tr><tr><td colspan="4">2. washing media (HBSS, 100 ml)</td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red&amp;nbsp;</td><td>GIBCO</td><td>14175-095</td><td>99 ml</td></tr><tr><td>HEPES ( 1M)</td><td>GIBCO</td><td>15630-130</td><td>1 ml</td></tr><tr><td colspan="4">3. HEPES buffered saline&amp;nbsp; (HBS)&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">4. HBS Cocktail for live imaging pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td>tetrodotoxin&amp;nbsp;</td><td>Tocris</td><td>1078</td><td>2 &amp;micro;M</td></tr><tr><td>bicuculline</td><td>Tocris</td><td>131</td><td>10 &amp;micro;M</td></tr><tr><td>D-AP-5</td><td>Tocris</td><td>105</td><td>50 &amp;micro;M</td></tr><tr><td>CNQX</td><td>Tocris</td><td>1045</td><td>20 &amp;micro;M</td></tr><tr><td>LY341495</td><td>Tocris</td><td>1209</td><td>10 &amp;micro;M</td></tr><tr><td colspan="4">5. Calcium-free&amp;nbsp; HBS&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">6. 56 mM Potassium ACSF pH=7.4</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td>119 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td>56 mM</td></tr><tr><td>MgSO4.7H</td><td>Sigma</td><td>M1880</td><td>1.3 mM</td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C1016</td><td>2.5 mM</td></tr><tr><td>NaH2PO4</td><td>Sigma</td><td>S8282</td><td>1 mM</td></tr><tr><td>NaHCO3</td><td>Sigma</td><td>S5761</td><td>26.2 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr></tbody></table>

live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

採用製剤は、 インビトロで vHipp-NACC回路の遺伝子キメラ共培養物から構成されています。 vHippのmicroslicesから発せられる投影は、前シナプスの軸索の入力として、シナプス後の目標、NACCから分散ニューロンとシナプス接触を行うことができます。ニコチンはvHipp軸索21とシナプス前α7を介しvHipp軸索5に沿ってシグナリング長期化カルシウム*のnAChRにより神経支配NACCニュ?...

Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

ニコチン誘導性カルシウムシグナリングおよび腹側海馬軸索に沿って神経伝達物質放出のライブイメージング

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

live-imaging-nicotine-induced-calcium-signaling-neurotransmitter

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Neuroscience

Unit 1

Drugs Acting on Autonomic Nervous System: Adrenergic Agonists and Antagonists Agents

SCIENTISTS IN ACTION

9.0K ビュー.  Stony Brook University. We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

ビデオ: Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

神経科学

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

慢性ニコチン投与によるマウスノックインで蛍光標識ニコチン性受容体のスペクトル共焦点イメージング

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

生きたマウスにおける後部海馬CA1の縦方向2光子イメージング

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

行動学

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

活性型のニコチンのレーザー フラッシュ ・ フォトリシス法によるマウス脳切片でのニコチン性アセチルコリン受容体の機能を探る

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

マウスの振る舞いの海馬における神経活動を可視化するための頭蓋骨管制法

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

インビボ 無傷の背根神経節における一次感覚ニューロンのネットワークにおけるニューロンアンサンブルのカルシウムイメージング

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Co-culturing of Ventral Hippocampal and Nucleus Accumbens

Source: Zhong, C., et al. <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates the process of preparing ventral hippocampal and nucleus accumbens tissues from mouse brains for co-culture. Neurons extend projections to form synaptic connections between the ventral hippocampal and nucleus accumbens tissues. This method allows studying neuronal interactions between these two brain regions, offering insights into cognition, behavior, and neurological disorders.

腹側海馬と側坐核の共培養

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

神経細胞培養技術

共培養と相互作用の研究

Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures

Source: Zhong, C., et al., <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates how nicotine-mediated activation of nicotinic acetylcholine receptors in the mouse ventral hippocampus results in sustained calcium signaling along the axons.

シナプス共培養における腹側海馬軸索に沿ったニコチン誘発性カルシウムシグナル伝達のイメージング

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Imaging

	Rinse ...

ニコチン誘導性カルシウムシグナリングおよび腹側海馬軸索に沿って神経伝達物質放出のライブイメージング

要約

さらに動画を探す

この動画の章

シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

慢性ニコチン投与によるマウスノックインで蛍光標識ニコチン性受容体のスペクトル共焦点イメージング

生きたマウスにおける後部海馬CA1の縦方向2光子イメージング

活性型のニコチンのレーザーフラッシュ・フォトリシス法によるマウス脳切片でのニコチン性アセチルコリン受容体の機能を探る

マウスの振る舞いの海馬における神経活動を可視化するための頭蓋骨管制法

インビボ無傷の背根神経節における一次感覚ニューロンのネットワークにおけるニューロンアンサンブルのカルシウムイメージング

腹側海馬と側坐核の共培養

シナプス共培養における腹側海馬軸索に沿ったニコチン誘発性カルシウムシグナル伝達のイメージング

ニコチン誘導性カルシウムシグナリングおよび腹側海馬軸索に沿って神経伝達物質放出のライブイメージング

要約

さらに動画を探す

この動画の章

シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

慢性ニコチン投与によるマウスノックインで蛍光標識ニコチン性受容体のスペクトル共焦点イメージング

生きたマウスにおける後部海馬CA1の縦方向2光子イメージング

活性型のニコチンのレーザー フラッシュ ・ フォトリシス法によるマウス脳切片でのニコチン性アセチルコリン受容体の機能を探る

マウスの振る舞いの海馬における神経活動を可視化するための頭蓋骨管制法

インビボ 無傷の背根神経節における一次感覚ニューロンのネットワークにおけるニューロンアンサンブルのカルシウムイメージング

腹側海馬と側坐核の共培養

シナプス共培養における腹側海馬軸索に沿ったニコチン誘発性カルシウムシグナル伝達のイメージング

活性型のニコチンのレーザーフラッシュ・フォトリシス法によるマウス脳切片でのニコチン性アセチルコリン受容体の機能を探る

インビボ無傷の背根神経節における一次感覚ニューロンのネットワークにおけるニューロンアンサンブルのカルシウムイメージング