仕事の大半は、関係職員は、NIH助成金NS060677によって、一部はNIHがMH099559と（BSKへ）MH10406​​9の補助金によってサポートされていました。作品の一部は、CHDI財団によってサポートされていました。

全ての動物プロトコルは、実験動物の管理と使用に関する健康ガイドの米国国立研究所に従ったとUCLAの施設内動物管理使用委員会によって承認された。 1.1）マイクロピペットおよびAAV2 / 5ウイルスの読み込みを準備<ol><li>ウイルスの注射のために細かいホウケイ酸ガラスマイクロピペットを使用してください。垂直プラーで二段階引っ張っプログラムを使用してマイクロピペットを引き出します。ベベルピペットグラインダーを用いて40°の角度でピペット。が40μmおよび6のシャンクの長さ - - 7mmでの使用に最適ですピペット20の先端径を持つことになります。ピペットや手術器具をオートクレーブ。ドリルは、70％エタノールで拭くことによって滅菌される。 </li><li> 10μlアリコートで-80℃でウイルスAAV2 / 5-GfaABC 1 D.cyto-GCaMP3.SV40（1.5×10 13 GC / ml）を、保管してください。ちょうど手術の前に、冷凍庫から一定分量を取り出し、目に充填するために使用するまで氷上で保存する電子マイクロピペット。 </li><li>ウイルスは、シリンジポンプの使用にマイクロピペットを埋める。しっかりと補強されたプランジャーを有するガラスシリンジに、フィッティング、適切なサイズのチューブ圧縮により、チューブの一片の一端を接続します。フィッティング適切なサイズの取り外し可能なニードル圧縮によりチューブの一片の他端にオートクレーブ処理マイクロピペットを取り付けます。 </li><li>ウイルスベクターをロードする前に、針を用いて1mlシリンジでスダン赤IV（約1ミリグラム/ 50ml）で着色されたミネラルオイルシステム（27 Gを充填することにより、チューブ、注射器およびマイクロピペットを含むポンプシステムから気泡を除去する）。 </li><li>ガラスマイクロピペットの下にパラフィルムのきれいな作品を置き、5分注 - パラフィルム上にウイルスベクターを10μl。 </li><li>後方を0.5μl/分でシリンジのプランジャを移動させることにより、ウイルスベクターを吸引し、ガラスピペット上のベクトルと油との間の境界をマークする。 </li></ol> 1.2）麻酔、ヘアーカット、H開頭のためのEAD固定と準備<ol><li> N 2 OとO 2および5％イソフルランで満たされたチャンバー内にマウス（P49-63、C57BL / 6）を置く。意図的な運動の喪失と鈍化呼吸数（ - 90 /分〜60）によって麻酔の深さを評価する。マウスを麻酔されたときに頭の上に髪を切った。 </li><li>その頭には麻酔および換気システムに置か鈍い耳バーおよびその鼻で固定して、定位フレームにマウスを合わせます。 2から3パーセントで連続イソフルランを維持する。マウスは麻酔下で彼らのまばたき反射を失うので、両眼に人工涙液軟膏を適用します。 </li><li>疼痛緩和のための皮下注射によりブプレノルフィン（0.1 mg / mlで）を0.05mlを投与する。 </li><li>二つの溶液間で交互にポビドンヨードを皮切りに、10％ポビドンヨードおよび70％アルコールで手術領域を清掃して三回。正面から起動し、すでにカット毛を除去するために戻って向かって拭いてください。 </li><li>トップO上の皮膚切開を作る耳の間の目と端部の間に開始され頭F。 </li></ol> 1.3）開頭およびマイクロインジェクション<ol><li>綿棒でスワイプして骨膜を削除してから歯科綿球で頭蓋骨の表面を乾燥させます。注射部位の周りにマークを作成し、高速ドリルを搭載し、小さなスチールバリを使用して、マークのエッジの周りにドリル。 </li><li>骨を削除し、生理食塩水で表面を洗浄する。 </li><li>前後0.8、内側-外側：2.0、背腹軟膜表面から：-2.4 13以下の座標（mm）の左背側線条体に横方向のピペットを置きます。これらの座標は、マイクロインジェクション針の先端は、それが少しだけ、この核内で浸透していることを意味し、ちょうど背外側線条体（ 図1A、B）の上に座っていることを意味していることに注意してください。損傷血管を避けてください。ピペットが血管の上で右であることを起こる場合は、若干の調整0.2mmの - 〜0.1によって調整します。 </li><li> 0.2μL/分に設定注入速度で注入を開始し、一般的に、ウイルスの1〜1.5μlのを注入する。 </li><li> 10分間注入後に所定の位置にピペットを残し、その後、ゆっくりとピペットを撤回。 </li><li>連続縫合外部ナイロン縫合糸で手術創を閉じて終了します。 </li></ol> 1.4）回復<ol><li> 24時間加熱パッド上に清潔なケージにマウスを置く。完全に回復するまで、他の動物の会社に手術を受けたマウスを返さないでください。それは胸骨横臥位を維持するのに十分な意識を取り戻したまでマウス無人のまま放置しないでください。 </li><li>週間水（40mgのトリメトプリムと200mgのsulfamethoxiazoleを含む水500mlあたり5ミリリットル、）トリメトプリムSulfamethoxiazoleを追加します。手術後最大3日間ブプレノルフィンを1日2回投与する。 </li><li>供給され、毎日の水やり、12時間の明暗サイクルの上でマウスを維持、AND手術後3日目まで一日一回重み付けされる。これらの3日間で、10％以上の体重損失を持つマウスが侵害とみなされ、そして安楽死させる代わりに、実験に供される。 </li><li>これとは別に、1週間に2回、マウスのケージを変更し、1日あたり少なくとも1回、一般的な健康状態を監視する。定位微量注入後3週間 - 典型的には、マウスを2内で使用されている。 </li></ol>共焦点のCa 2+イメージング用2.急性脳スライスの準備 2.1）切削や録音溶液の調製を。 <ol><li> （ミリモル）を含む溶液を切断する準備：194ショ糖、30のNaCl、4.5のKCl、10 D-グルコース、1のMgCl 2、1.2のNaH 2 PO 4、および95％O 2および5％CO 2で飽和し26のNaHCO 3、。 </li><li> （mm）を含む記録液を調製：124のNaCl、4.5のKCl、1のMgCl 2、10 D-グルコース、2のCaCl 2、1.2のNaH 2 PO 4 </サブ&gt;、および26のNaHCO 3; pHは7.3から7.4、290 - 95％O 2および5％CO 2で飽和さ295ミリオスモル。記録液の入ったビーカーを保持する脳スライスを記入し、34℃で保管してください。 </li></ol> 2.2）スライス<ol><li>深く末端定位微量注入後2〜3週間、マウスを麻酔し、それを首を切る。 </li><li>脳を抽出して、非注射右半球を削除するには、ブレードを使用し、スーパー接着剤を使用してビブラトームトレイ左折1をマウントします。氷冷間切断緩衝液でビブラトームトレイを充填し、95％O 2および5％CO 2でそれを飽和保つ。 </li><li> 300μmの厚さで冠状または矢状線条体の脳切片をカット。通常、4から5の冠状、または6から7矢状の線条体切片を収集することができる。 </li><li>その後のrecordi室温でそれらを格納する前に34℃で温めスライス保持ビーカーにスライスを移し、30分間そこにそれらを保つNG。 </li><li>の[Ca 2+] i の画像化の前に少なくとも30分間室温で酸素記録バッファ内の脳切片をインキュベートする。 </li></ol> 3.免疫組織化学（IHC） <ol><li>二週間、ウイルス注射後、PBSをPBS中の10％ホルマリンで経続いマウスを灌流する。 </li><li> 、取り外し一晩後固定し、そして少なくとも2日間、バッファリングされた30％スクロース中の脳をcryoprotect。 </li><li>クリオミクロトームを用いて40μmのセクションを準備します。 </li><li> 10分間隔でセクションをPBSで3回すすいでください。 </li><li> 10％正常ヤギ血清を含むPBS中で1時間インキュベートし、0.5％トリトンX-100で切片をインキュベートする。 </li><li> 0.5％トリトンX-100を含むPBS中で調製した一次抗体で4℃で一晩切片をインキュベートする。以下のように用いた一次抗体であった：ニワトリ抗GFP 1：1000マウス抗S100β1：400、マウス抗NeuNの1：600、マウス抗グルタミンシンテ1：300。 </li><li> 10メートルでセクションをPBSで3回すすぎ間隔で。 </li><li>室温で2時間、10％正常ヤギ血清を含むPBS中で調製した二次抗体とともに切片をインキュベートする。次のように二次抗体は以下の通りであった：ヤギ抗マウスAlexa546、1：500、ヤギ抗ニワトリAlexa488で1：500。 </li><li> 、スライドガラス上のセクションをマウントし、乾燥し、退色防止封入剤を数滴を適用し、その後ゆっくりとガラスカバースリップでセクションをカバーしています。 4℃でスライドを保存してください。 </li><li> 1.3の開口数を40倍油浸レンズを共焦点顕微鏡で画像を撮影する。 </li></ol> 4.共焦点の[Ca 2+] iのイメージング NOTE：[Ca 2+] iの 0.8の開口数40X水浸対物レンズを備えた共焦点顕微鏡を用いて行った撮像する。 <ol><li>スライス後の1および5時間の間、所定の位置のスライス1の流速で室温で酸素記録バッファと記録チャンバーとsuperfuse中 - あたり2mlの分。その後、実験中に動きを最小限に抑えるために、スライスの上にナイロン弦の白金ハープを置く。 </li><li> 10X水浸対物レンズの下に明視野発光と背側線条体を視覚化し、検索します。 </li><li> 40X水浸対物レンズに切り替え、アルゴンレーザーの488nmのラインをオンにします。焦点面を変化させることで、細胞体、枝や小枝で基底蛍光を表示し、約20μmのスライス面の下に位置するセルを見つける。注目すべきは、損なわれた細胞は、通常は避けるべきで平均以上の蛍光レベルを表示する。 </li><li>スキャンを開始するには、 &#39;フレームスキャン」モードを使用してください。通常、0.5に調整し、レーザ強度-最大出力の5％の[Ca 2+] iの CYTO-GCaMP3の画像のに十分である。アストロサイトの全領土での[Ca 2+] iの動態を監視するために、フレームあたり1秒のスキャン速度を持つ「フレームスキャン」または高速でrecommeですndedが、これは、問題の実験に応じて決定する必要がある。 </li><li>視野全体で2アストロサイト- 1を監視するために3倍に-必要に応じて、画像には[Ca 2+]のプロセスでは、私は 、2のデジタル倍率を使用しています。 </li><li>録音中に飽和画素を回避するために、細胞を疑似カラーには、 &#39;ヒロ「ルックアップ·モードを使用します。常に赤い色は彩度の指標として表示されているかどうかをテストするために、約5分間、「パイロット録画」で始まります。その場合、レーザ出力パワーを下げるか、または下げるPMT /ゲイン/オフセット値を画素値をテストするために、別のパイロット記録を行う非飽和の範囲である。次のように一般的には、の[Ca 2+] iの線条体における撮像アストロサイトのために使用される組み合わせパラメータは次のとおりです。レーザ出力パワー0.5 - （10ミリワットの）5％、PMT 550から650ボルト、ゲイン2.0 - 3.5X、およびオフセット0 - 5％。 </li></ol>

<ol>
	<li>Agulhon, 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=What+is+the+role+of+astrocyte+calcium+in+neurophysiology.">What is the role of astrocyte calcium in neurophysiology.</a> Neuron. 59, 932-946 (2008).</li><li>Khakh, B. S., North, 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=Neuromodulation+by+extracellular+ATP+and+P2X+receptors+in+the+CNS.">Neuromodulation by extracellular ATP and P2X receptors in the CNS.</a> Neuron. 76, 51-69 (2012).</li><li>Gourine, A. V., 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=Astrocytes+control+breathing+through+pH-dependent+release+of+ATP.">Astrocytes control breathing through pH-dependent release of ATP.</a> Science. 329, 571-575 (2010).</li><li>Florian, C., Vecsey, C. G., Halassa, M. M., Haydon, P. G., Abel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-derived+adenosine+and+A1+receptor+activity+contribute+to+sleep+loss-induced+deficits+in+hippocampal+synaptic+plasticity+and+memory+in+mice.">Astrocyte-derived adenosine and A1 receptor activity contribute to sleep loss-induced deficits in hippocampal synaptic plasticity and memory in mice.</a> J Neurosci. 31, 6956-6962 (2011).</li><li>Shigetomi, E., Jackson-Weaver, O., Huckstepp, R. T., O'Dell, T. J., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPA1+channels+are+regulators+of+astrocyte+basal+calcium+levels+and+long-term+potentiation+via+constitutive+D-serine+release.">TRPA1 channels are regulators of astrocyte basal calcium levels and long-term potentiation via constitutive D-serine release.</a> J Neurosci. 33, 10143-10153 (2013).</li><li>Tong, X., Shigetomi, E., Looger, L. L., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+calcium+indicators+and+astrocyte+calcium+microdomains.">Genetically encoded calcium indicators and astrocyte calcium microdomains.</a> Neuroscientist. 19, 274-291 (2013).</li><li>Shigetomi, E., Kracun, S., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+targeted+optical+sensor+to+monitor+calcium+signals+in+astrocyte+processes.">A genetically targeted optical sensor to monitor calcium signals in astrocyte processes.</a> Nat Neurosci. 13, 759-766 (2010).</li><li>Shigetomi, 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=Imaging+calcium+microdomains+within+entire+astrocyte+territories+and+endfeet+with+GCaMPs+expressed+using+adeno-associated+viruses.">Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.</a> J Gen Physiol. 141, 633-647 (2013).</li><li>Shigetomi, E., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+near+plasma+membrane+and+global+intracellular+calcium+dynamics+in+astrocytes.">Measuring near plasma membrane and global intracellular calcium dynamics in astrocytes.</a> J Vis Exp. 26, (2009).</li><li>Reeves, A. M., Shigetomi, E., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+loading+of+calcium+indicator+dyes+to+study+astrocyte+physiology:+key+limitations+and+improvements+using+morphological+maps.">Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps.</a> J Neurosci. 31, 9353-9358 (2011).</li><li>Li, D. D., Agulhon, C., Schmidt, E., Oheim, M., Ropert, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+investigating+astrocyte-to-neuron+communication.">New tools for investigating astrocyte-to-neuron communication.</a> Frontiers in Cellular Neuroscience. 7, (2013).</li><li>Davila, D., Thibault, K., Fiacco, T. A., Agulhon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+molecular+approaches+to+understanding+astrocyte+function+in+vivo.">Recent molecular approaches to understanding astrocyte function in vivo.</a> Front Cell Neurosci. 7, 272 (2013).</li><li>Paxinos, G., Franklin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2012).</li><li>Perea, G., Yang, A., Boyden, E. S., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+astrocyte+activation+modulates+response+selectivity+of+visual+cortex+neurons+in+vivo.">Optogenetic astrocyte activation modulates response selectivity of visual cortex neurons in vivo.</a> Nat Commun. 5, 3262 (2014).</li><li>Sofroniew, M. V., Vinters, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes:+biology+and+pathology.">Astrocytes: biology and pathology.</a> Acta Neuropathol. 119, 7-35 (2010).</li><li>Eid, T., 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=Loss+of+glutamine+synthetase+in+the+human+epileptogenic+hippocampus:+possible+mechanism+for+raised+extracellular+glutamate+in+mesial+temporal+lobe+epilepsy.">Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.</a> Lancet. 363, 28-37 (2004).</li><li>Eid, T., Williamson, A., Lee, T. S., Petroff, O. A., de Lanerolle, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+astrocytes--key+players+in+human+mesial+temporal+lobe+epilepsy.">Glutamate and astrocytes--key players in human mesial temporal lobe epilepsy.</a> Epilepsia. 49 Suppl 2, 42-52 (2008).</li><li>Tong, X., 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=Astrocyte+Kir4.1+ion+channel+deficits+contribute+to+neuronal+dysfunction+in+Huntington's+disease+model+mice.">Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice.</a> Nat Neurosci. 17, 694-703 (2014).</li><li>Ortinski, P. 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=Selective+induction+of+astrocytic+gliosis+generates+deficits+in+neuronal+inhibition.">Selective induction of astrocytic gliosis generates deficits in neuronal inhibition.</a> Nat Neurosci. 13, 584-591 (2010).</li><li>Zhang, Y., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+heterogeneity:+an+underappreciated+topic+in+neurobiology.">Astrocyte heterogeneity: an underappreciated topic in neurobiology.</a> Curr Opin Neurobiol. 20, 588-594 (2010).</li><li>Reimsnider, S., Manfredsson, F. P., Muzyczka, N., Mandel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+transgene+expression+after+intrastriatal+pseudotyped+rAAV2/1,+rAAV2/2,+rAAV2/5,+and+rAAV2/8+transduction+in+the+rat.">Time course of transgene expression after intrastriatal pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat.</a> Mol Ther. 15, 1504-1511 (2007).</li></ol>

著者らは、開示することは何もない。

本明細書に記載の方法は、私たちは、その場で 、後続の[Ca 2+] iのイメージングのためのin vivoでの線条体アストロサイトにおけるCYTO-GCaMP3を表現することができました。この方法は、遺伝子組換えを使用して、またはノックイン実験的な実装および解剖学的特異性の標的タンパク質、迅速性と柔軟性の強固な発現を含むマウスに比べて利点を有する。 AAV2 / 5を使?...

アストロサイトは、脳のユビキタスと豊富なグリア細胞である。これは、十分にアストロサイトが重要なサポートおよび細胞外空間におけるK +濃度のバッファリング、神経伝達物質の取り込みだけでなく、栄養素を提供することを含む恒常的な役割を果たしていることが確立されている。しかし、最近の研究では、それらはまた、自然に発生し、ニューロンの活動を1だけ増加された[Ca 2+] iの信号を表示することを示している。アストロサイトの存在は、の[Ca 2+] i のシグナリングはますますニューロンとのコミュニケーションを誘発すると考えられてきた、そのようなものとして、アストロサイト内の「Ca 2+の興奮性」の形式として解釈されてきた。最後の20年間の利用可能なデータは、アストロサイトとニューロンは、おそらく、双方向の方法で、通信することが可能な2つの設定が示唆された。まず、アストロサイトは、多くの場合は[Ca 2+] iは神経伝達物質によって活性化されたときの増加に対応し、ニューロン2から放出された神経修飾物質。第二に、アストロサイト内の[Ca 2+] i の増加は、順番に、ニューロンおよび血管に影響を与える可能性アストロサイトからのシグナル伝達分子の放出を引き起こす。証拠は、神経膠星状細胞から放出された分子は、アストロサイトへの神経シグナリングを介してシナプス回路、最終的に行動3-5の機能に変化をもたらすことを示唆している。しかし、これは急速に発展して研究領域のままであり、それがより良いとアストロサイトの詳細な理解がの[Ca 2+] iは 、現在の不確定要素6のいくつかを解決するために必要であると主張されています。 過去の研究では、アストロサイトへの有機物のCa 2+指示色素のバルク·ロードを確実に、培養中および in situ 7-10 全体の星状細胞内の[Ca 2+] i の信号の検出に失敗したことが示された。これらの知見は、米国および他の6,11,12によって議論されてきた。 emergingの画像は、ニューロンおよび血管との相互作用のための主要な部位であるアストロサイトのプロセス内の[Ca 2+] iの信号（ 例えば、枝や小枝）、ということで、めったに詳細に検討されていない。近年、細胞質ゾルGCaMP3などの遺伝的にコードされたカルシウム指示薬（GECIs）の使用は、GCaMP5GとGCaMP6とプラズマ膜テザーのバージョン（ 例えば 、Lckは-GCaMP3）は、そのようなアストロサイトの小区画内の[Ca 2+] iの信号の研究が可能になった薄いプロセスなどの、原形質膜に近いと7,8全体の領土内である。しかし、GECIs有機のCa 2+指示色素上の1つの欠点があり、GECIsが適切であることに発現する遺伝的方法は、数週間の期間にわたって、インビボでの星状細胞に選択的にコード化遺伝子を送達することが要件である。 in vivoでの発現は、典型的にノックインマウスまたはウイルスベースの送達アプリで、トランスジェニックマウスを使用して達成されるゴキブリ。現在のJoveの記事では、アデノ随伴ウイルスを使用して、線条体アスト​​ロサイトにGECIsを配信するために用いられる方法と手順を報告します。ここでは、例として、CYTO-GCaMP3に焦点を当てるが、同じ基本的な手順は、他のGECIまたは蛍光タンパク質ベースのレポーターのために働く。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>704506</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Capillaries</td>
			<td >World Precision Instruments</td>
			<td >1B100-4</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette puller</td>
			<td >Narishige</td>
			<td >PC-10</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette grinder</td>
			<td >Narishige</td>
			<td >EG-40</td>
			<td></td>
		</tr>
		<tr >
			<td >pZac2.1 GfaABC1D.cyto-GCaMP3</td>
			<td >Addgene</td>
			<td>44331</td>
			<td >a plasmid sent to UPenn Vector Core for virus packaging</td>
		</tr>
		<tr >
			<td >I mL syringe</td>
			<td >BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe needle</td>
			<td >BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr >
			<td >AAV2/5 virus</td>
			<td >UPenn vector core</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >Sudan red IV</td>
			<td >Sigma-Aldrich</td>
			<td >67386</td>
			<td></td>
		</tr>
		<tr >
			<td >Mineral oil</td>
			<td >CVS Pharmacy</td>
			<td>152355</td>
			<td></td>
		</tr>
		<tr >
			<td >Cryostat</td>
			<td >Leica</td>
			<td>CM3050 S</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
			<td >900LS</td>
			<td></td>
		</tr>
		<tr >
			<td >High Speed Rotary Micromotor Kit</td>
			<td >FOREDOM</td>
			<td >K.1070</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Santa cruz biotechnology</td>
			<td >sc-281692</td>
			<td></td>
		</tr>
		<tr >
			<td >Super Glue</td>
			<td>Krazy&#174;Glue</td>
			<td>KG925</td>
			<td></td>
		</tr>
		<tr >
			<td >Microslicer</td>
			<td >Ted Pella</td>
			<td >DTK-Zero 1</td>
			<td></td>
		</tr>
		<tr >
			<td >Confocal microscopes</td>
			<td >Olympus</td>
			<td >FV300 and FV1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Normal goat serum</td>
			<td >Vector</td>
			<td >S-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >chicken anti-GFP</td>
			<td >Abcam</td>
			<td >ab13970</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-s100&#946;</td>
			<td >Sigma-Aldrich</td>
			<td >S2532</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-NeuN</td>
			<td >Millipore</td>
			<td >MAB377</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-glutamine synthetase</td>
			<td >Millipore</td>
			<td >MAB302</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-mouse-Alexa546</td>
			<td >Invitrogen</td>
			<td >A11003</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-chicken-Alexa488</td>
			<td >Invitrogen</td>
			<td >A11039</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope Slides</td>
			<td >Fisher Scientific</td>
			<td >12-550-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover Glass</td>
			<td >Fisher Scientific</td>
			<td >12-548-5J</td>
			<td></td>
		</tr>
		<tr >
			<td >Mounting Medium</td>
			<td >Vector</td>
			<td >H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging intracellular ca2+ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

線条体におけるCYTO-GCaMP3の星状細胞特異的発現のために、我々は以前に、堅牢GCaMP3およびレポーター遺伝子を駆動することが示されているアデノ随伴ウイルス（AAV）5血清型、およびGFAP GfaABC 1 Dプロモーター（ 図1A）が 、使用海馬と皮質アストロサイト8,14で表現。二週間のマウスの線条体へのウイルスのマイクロインジェクション後、マウス（約10週齢）を灌流し...

Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

イメージング細胞内Ca<sup&gt; 2+</sup遺伝的にエンコードされたカルシウム指標を用いて成体マウスからの線条体アスト​​ロサイト&gt;シグナル

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

imaging-intracellular-ca2-signals-striatal-astrocytes-from-adult-mice

Research

JoVE Journal

Neuroscience

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

14.7K ビュー.  University of California Los Angeles. The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ. 

ビデオ: Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

遺伝的にコードされたカルシウムセンサーを発現する鋤鼻器のスライス標本のイメージング神経細胞応答

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

神経科学

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

同定されたニューロンと摂食行動からカルシウム信号の同時記録キイロショウジョウバエ</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

モデル星細胞腫発症機序<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;皮質アストロサイトまたは条件付き、遺伝子改変マウスから神経幹細胞を使用した

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

遺伝的にコードされた蛍光電圧センサーの2種類のイメージング膜電位

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

ニューロンに遺伝的にコードされた非天然アミノ酸を用いた神経タンパク質の光制御

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

膜をターゲットとした Ca2 +インジケーターによる培養細胞のローカル Ca2 +シグナルの解剖

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

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 ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

アストロサイトにおけるオルガネラおよび他の貨物の細胞内輸送の可視化と解析

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

成虫マウスおよび老化マウスからの海馬スライスにおける低酸素症の最小化

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.