著者は、専門的な技術支援のためのC. Roderigoに感謝したいです。私たちは、ビデオ制作で助けをS.ケーラー（拡張イメージングのセンター、ハインリヒ・ハイネ大学デュッセルドルフ）をお願いいたします。著者の研究室での研究は、ドイツ研究協会（DFG：CRRにRoの2327 / 8-1）によって資金を供給されている（NH）は、ハインリッヒ・ハイネ大学デュッセルドルフと国立衛生研究所によって（MC）にR01NS032123を付与します。

<ol>
	<li>Somjen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+regulation+in+the+brain:+implications+for+pathophysiology.">Ion regulation in the brain: implications for pathophysiology.</a> Neuroscientist. 8, 254-267 (2002).</li><li>Dietzel, I., Heinemann, U., Hofmeier, G., Lux, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-induced+changes+in+extracellular+Na++and+Cl-+concentration+in+relation+to+changes+in+the+size+of+the+extracellular+space.">Stimulus-induced changes in extracellular Na+ and Cl- concentration in relation to changes in the size of the extracellular space.</a> Exp Brain Res. 46, 73-84 (1982).</li><li>Nicholson, C., ten Bruggencate, G., Stockle, H., Steinberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+potassium+changes+in+extracellular+microenvironment+of+cat+cerebellar+cortex.">Calcium and potassium changes in extracellular microenvironment of cat cerebellar cortex.</a> J Neurophysiol. 41, 1026-1039 (1978).</li><li>Rice, M. E., Nicholson, 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+aspartate-induced+extracellular+potassium+and+calcium+shifts+and+their+relation+to+those+of+kainate,+quisqualate+and+N-methyl-D-aspartate+in+the+isolated+turtle+cerebellum.">Glutamate- and aspartate-induced extracellular potassium and calcium shifts and their relation to those of kainate, quisqualate and N-methyl-D-aspartate in the isolated turtle cerebellum.</a> Neuroscience. 38, 295-310 (1990).</li><li>Prince, D. A., Lux, H. D., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+extracellular+potassium+activity+in+cat+cortex.">Measurement of extracellular potassium activity in cat cortex.</a> Brain Res. 50, 489-495 (1973).</li><li>Deitmer, J. W., Rose, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+regulation+and+proton+signalling+by+glial+cells.">pH regulation and proton signalling by glial cells.</a> Prog Neurobiol. 48, 73-103 (1996).</li><li>Chesler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+modulation+of+pH+in+the+brain.">Regulation and modulation of pH in the brain.</a> Physiol Rev. 83, 1183-1221 (2003).</li><li>Kofuji, P., Newman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Regulation of potassium by glial cells in the central nervous system. , (2009).</li><li>Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-selective+microelectrodes+and+diffusion+measurements+as+tools+to+explore+the+brain+cell+microenvironment.">Ion-selective microelectrodes and diffusion measurements as tools to explore the brain cell microenvironment.</a> J Neurosci Methods. 48, 199-213 (1993).</li><li>Fedirko, N., Svichar, N., Chesler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+use+of+high-speed,+concentric+h+-+and+Ca2+-selective+microelectrodes+suitable+for+in+vitro+extracellular+recording.">Fabrication and use of high-speed, concentric h+- and Ca2+-selective microelectrodes suitable for in vitro extracellular recording.</a> J Neurophysiol. 96, 919-924 (2006).</li><li>Stephan, 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=Kir4.1+channels+mediate+a+depolarization+of+hippocampal+astrocytes+under+hyperammonemic+conditions+in+situ.">Kir4.1 channels mediate a depolarization of hippocampal astrocytes under hyperammonemic conditions in situ.</a> Glia. 60, 965-978 (2012).</li><li>Haack, N., Rose, C. R., Lei, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+Calibration+and+Application+of+Potassium-Selective+Microelectrodes.">Preparation, Calibration and Application of Potassium-Selective Microelectrodes.</a> Microelectrodes. , 87-105 (2014).</li><li>Roos, A., Boron, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pH.">Intracellular pH.</a> Physiol Rev. 61, 296-434 (1981).</li><li>Kleinhans, C., Kafitz, K. W., Rose, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+Intracellular+Sodium+Imaging+Combined+with+UV-mediated+Focal+Uncaging+of+Glutamate+in+CA1+Pyramidal+Neurons.">Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons.</a> Journal of visualized experiments : JoVE. , (2014).</li><li>Hertz, L., 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=Roles+of+astrocytic+Na+,K+-ATPase+and+glycogenolysis+for+K+homeostasis+in+mammalian+brain.">Roles of astrocytic Na ,K -ATPase and glycogenolysis for K homeostasis in mammalian brain.</a> J Neurosci Res. , (2014).</li><li>Pumain, R., Heinemann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-+and+amino+acid-induced+calcium+and+potassium+changes+in+rat+neocortex.">Stimulus- and amino acid-induced calcium and potassium changes in rat neocortex.</a> J Neurophysiol. 53, 1-16 (1985).</li><li>Vargova, L., Jendelova, P., Chvatal, A., Sykova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate,+AMPA,+NMDA+Induced+Changes+in+Extracellular+Space+Volume+and+Tortuosity+in+the+Rat+Spinal+Cord.">Glutamate, AMPA, NMDA Induced Changes in Extracellular Space Volume and Tortuosity in the Rat Spinal Cord.</a> J Cereb Blood Flow Metab. 21, 1077-1089 (2001).</li><li>Fellin, T., Gomez-Gonzalo, M., Gobbo, S., Carmignoto, G., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+glutamate+is+not+necessary+for+the+generation+of+epileptiform+neuronal+activity+in+hippocampal+slices.">Astrocytic glutamate is not necessary for the generation of epileptiform neuronal activity in hippocampal slices.</a> J Neurosci. 26, 9312-9322 (2006).</li><li>Rouach, N., Koulakoff, A., Abudara, V., Willecke, K., Giaume, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglial+metabolic+networks+sustain+hippocampal+synaptic+transmission.">Astroglial metabolic networks sustain hippocampal synaptic transmission.</a> Science. 322, 1551-1555 (2008).</li><li>Lux, H. D., Heinemann, U., Dietzel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+changes+and+alterations+in+the+size+of+the+extracellular+space+during+epileptic+activity.">Ionic changes and alterations in the size of the extracellular space during epileptic activity.</a> Adv Neurol. 44, 619-639 (1986).</li><li>Zanotto, L., Heinemann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartate+and+glutamate+induced+reductions+in+extracellular+free+calcium+and+sodium+concentration+in+area+CA1+of+'in+vitro'+hippocampal+slices+of+rats.">Aspartate and glutamate induced reductions in extracellular free calcium and sodium concentration in area CA1 of 'in vitro' hippocampal slices of rats.</a> Neurosci Lett. 35, 79-84 (1983).</li><li>Borrelli, M. J., Carlini, W. G., Dewey, W. C., Ransom, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+making+ion-selective+microelectrodes+suitable+for+intracellular+recording+in+vertebrate+cells.">A simple method for making ion-selective microelectrodes suitable for intracellular recording in vertebrate cells.</a> J Neurosci Methods. 15, 141-154 (1985).</li><li>Ammann, D., Anker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutral+carrier+sodium+ion-selective+microelectrode+for+extracellular+studies.">Neutral carrier sodium ion-selective microelectrode for extracellular studies.</a> Neurosci Lett. 57, 267-271 (1985).</li><li>Deitmer, J. W., Schlue, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Na++and+Ca2++in+leech+Retzius+neurones+during+inhibition+of+the+Na+-K++pump.">Intracellular Na+ and Ca2+ in leech Retzius neurones during inhibition of the Na+-K+ pump.</a> Pflugers Arch. 397, 195-201 (1983).</li><li>Schwiening, C. J., Thomas, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+intracellular+calcium+and+its+muffling+measured+by+calcium+iontophoresis+in+snail+neurones.">Relationship between intracellular calcium and its muffling measured by calcium iontophoresis in snail neurones.</a> J Physiol. 491 (Pt 3), 621-633 (1996).</li><li>Chesler, M., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pH+of+astrocytes+increases+rapidly+with+cortical+stimulation.">Intracellular pH of astrocytes increases rapidly with cortical stimulation.</a> Am J Physiol. 253, R666-R670 (1987).</li><li>Ammann, D., Chao, P. S., Simon, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valinomycin-based+K++selective+microelectrodes+with+low+electrical+membrane+resistance.">Valinomycin-based K+ selective microelectrodes with low electrical membrane resistance.</a> Neurosci Lett. 74, 221-226 (1987).</li><li>Deitmer, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicarbonate-dependent+changes+of+intracellular+sodium+and+pH+in+identified+leech+glial+cells.">Bicarbonate-dependent changes of intracellular sodium and pH in identified leech glial cells.</a> Pflugers Arch. 420, 584-589 (1992).</li><li>Lux, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+recording+ion+specific+microelectrodes:+their+use+in+pharmacological+studies+in+the+CNS.">Fast recording ion specific microelectrodes: their use in pharmacological studies in the CNS.</a> Neuropharmacology. 13, 509-517 (1974).</li><li>Nicholson, C., Phillips, 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=Ion+diffusion+modified+by+tortuosity+and+volume+fraction+in+the+extracellular+microenvironment+of+the+rat+cerebellum.">Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum.</a> J Physiol. 321, 225-257 (1981).</li><li>Chesler, M., Chan, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-induced+extracellular+pH+transients+in+the+in+vitro+turtle+cerebellum.">Stimulus-induced extracellular pH transients in the in vitro turtle cerebellum.</a> Neuroscience. 27, 941-948 (1988).</li><li>Thomas, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+sodium+activity+and+the+sodium+pump+in+snail+neurones.">Intracellular sodium activity and the sodium pump in snail neurones.</a> J Physiol. 220, 55-71 (1972).</li></ol>

The authors declare no competing interests.

液体キャリアベースは、イオン選択電極が正常に何十年もの間、多くのイオンのために用いられてきた、非常に特異的なセンサは、22〜26が用意されています 。 ECSの幅はわずか約20〜50nmで、イオン選択の直径である間：脊椎動物の脳標本の細胞外空間（ECS）で使用する場合、一つは、これは非常に侵襲的技術であること、しかし、心に留めておく必要があります微小電極は、約1μm（二重の電極）以上（同心円状の電極）です。イオン選択性微小電極の先端は、このようにだけでなく、組織の彼らの突き刺しの間に組織を損傷するだけでなく、イオンの過渡現象の過小評価に有利、ECSを拡大します。これらの落とし穴にもかかわらず、神経活動に応じて、細胞外イオンの過渡現象は、この方法の信頼性を証明する、異なる研究室7,8間で著しく一致しています。 イオン選択性電極の性能と適合性使用されるセンサカクテル（ &#39;液膜のイオノフォア&#39;）によって定義され、それらの感度と選択性に依存します。センサーのカクテルは、 例えば 、カリウム27に対して高い選択性を示すK +選択微小電極のためのバリノマイシン、特別な担体分子が含まれています。それにもかかわらず、他のイオンとの交差反応性が発生する可能性がありますし、テストする必要があります。バリノマイシンは、結果を解釈する際に考慮しなければならアンモニウムに対する有意な交差反応性（例えば、11,12）を示します 。イオノフォアの電圧応答は、ネルンスト動作（参照方程式1）に従うため、また、信号対ノイズ比、検出閾値は、測定されるイオンの濃度に依存します。このように、しばらく小[K +] oをトランジェントトランジェントがはるかに難しいこんにちはに対して検出するようにしているoを低ベースライン[K +] oを、小さな[ の Na +]に対して大きな電圧変化を呼び起こしますGHベースライン[ の Na +] O（参照： 図5、図6）。 イオン選択性電極の性能も大きく、その電気的時定数によって支配される時間分解能によって決定されます。後者は主に、センサの軸方向の抵抗によって決定され、その内部の溶液と外部流体との間のピペットの長さに沿って分布容量によるものです。二重の構成では、抵抗が充填イオンセンサの長い列のために、高いです。 （この場合はホウケイ酸ガラスで）指定された絶縁誘電体の場合、容量は誘電体の厚さによって支配されます。二重の電極では、誘電体の幅は、ピペットのガラス壁に達します。ガラスが先端に近い薄くなるように、誘電体幅が低下し、静電容量が増加します。これらの要因には、数百ミリ秒から数の範囲の応答時間を有する電極を生成するために結合秒は、これらの要因は様々です。 同心の設計の主な利点は、軸方向の抵抗とバスの静電容量の両方が大幅に減少していることです。先端の前の最後の数マイクロメートルで唯一の名残を残して埋め戻しイオン交換体の抵抗の最も同心ピペットシャント、。また、同心のピペット内の充填液が物理的に非常に容量の低減、二枚のガラスの壁の厚さによって分離され、浴から離れています。 10先に示したように、低抵抗およびキャパシタンスの複合効果は、二桁の時間分解能の向上があります。同心の Ca 2+およびpH微小電極の場合には、90％の応答時間は10~20秒10と同様に低かったです。同心の設計の関連する利点は、より低いノイズレベル（参照： 図8）です。任意のアンビエンから大幅に減少し、抵抗のため、電圧過渡Tノイズが最小化されます。また、このような過渡状態からの回復が速いため、時定数の、迅速です。このようなアーティファクトは、したがって、小さく高速であり、生理的な録音（参照： 図8）にはあまり破壊的な効果を持っています。 同心の技術の欠点もあります。まず、それらのアセンブリは、より複雑で、時間がかかります。第2の欠点は、独立したマイクロマニピュレーターや特殊な二重のマニピュレータのいずれかの使用を伴う、その先端に別個の基準微小電極を配置する必要があることです。最後に、二重の微小電極は、同心電極に不可能であると同時に28、2つの異なるイオン種の検出を可能にする、三連の設計に拡張することができます。 最も一般的な落とし穴非効率的なシラン化。 任意の液体-SENSの製造において最も重要なステップ、および主要な障害物またはベースのイオン選択微小電極は、シラン化手順です。電極は、特定のイオン濃度の変化に応答する、またはサブネルンスト応答（10倍の濃度差あたりすなわち 、よく未満58 mVの）に応答しない場合には、シラン化の乏しい有効性は、一般的な原因です。大気中の湿度がそれぞれ、夏の高さ、または冬の条件の典型的な、高すぎる、または低すぎると我々の経験では、これが発生する可能性があります。それは室内湿度をある程度制御を発揮することが可能である場合、これらの問題を克服することができます。 電極抵抗は高すぎます。 必要に応じて、イオン感受性バレルの抵抗が面取りによって低減することができます。この目的を達成するために、数秒間水に懸濁させた研磨の強いジェットへのヒントを公開します。これは、最上の先端が破損し、所望の値に抵抗を下げることになります。 塩橋。 ソルトイオンと参照バレル間のブリ​​ッジが不十分またはnone応答の電極をもたらし、ひいては大幅キャリブレーションでその性能を混乱することができます。 （ポイント1.6を参照してください）​​。上述したように、これは主に二重のシータガラスが選択されている問題ですが、ここで説明したオフセット、ツイストバレル技術を使用する場合にまれです。 念頭に置いて製造の容易さで、ラックス29の元の二重のデザインは、多くの場合、有利に使用することができます。この方法は、（30,31参照）、また先端を経由して、イオン交換を組み込むことにより、以下の、先端からの吸引や除名によりシラン溶液への高速露出を塩溶液とイオンと参照バレルの事前充填を利用。これらの電極は、およそ10分で製造することができるが、その先端の大きさは、典型的には4μm以上であり、彼らは、実験中に失敗する傾向です。対照的に、その方法は、シラン化シラン蒸気および熱への暴露を含みますingが小さい日最後のヒント、そして時には数週間で電極を製造することができます。 まとめると、イオン選択性微小電極を準備する方法についてのいくつかのプロトコルとアプローチがあります。ここでは、100％に近いの全体的な成功率で、私たちの研究室でよく、かつ確実に動作ツイスト銃身のダブルと同様に同心円状の微小電極の製造のための2つの主要な手順を記載しています。重要なことは、これらの技術は、pH又はカルシウムを含む他のイオン種の測定に転送されます、また、一般的に液体で満たされた空洞または流体などの脳以外の製剤に適用されます。少なくとも最後のではなく、イオン選択的微小電極は、細胞内イオン濃度の決意を可能にします。なぜなら、それらの比較的大きなチップサイズ（約1ミクロン）で、これは、しかし、唯一の大きな細胞体を有する細胞に無脊椎動物の調製物28,32に見られるように、例えば 、そのようなことが可能となります。

An author's middle initial was omitted from the publication, <a href="http://www.jove.com/video/53058/double-barreled-concentric-microelectrodes-for-measurement">Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue</a>. The author&#39;s name has been updated to:
Christine R. Rose
from:
Christine Rose

A correction was made to Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue.

Erratum: Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical signaling in the brain is based on the flux of ions across plasma membranes. Major ion movements into and from the extracellular space are not only mediated by passage through voltage-gated ion channels, but also by postsynaptic ionotropic receptors as well as ion transporters. Neuronal activity is thus accompanied by complex changes in the extracellular concentration of all major ions 1. For example, influx of sodium into neurons during excitatory activity has been shown to result in a decrease in the extracellular sodium concentration ([Na+]o) 2. The same holds true for the extracellular calcium concentration because calcium ions rapidly enter both pre- and postsynaptic structures 3. At the same time, potassium moves the opposite way and this mediates an increase in the extracellular potassium concentration ([K+]o) in the low mM range 4,5. Synaptic activity also causes changes in extracellular pH that are partly mitigated by concomitant glial membrane fluxes that change intraglial pH 6,7. These activity-related changes in extracellular ion concentrations have significant functional consequences. For example, even small increases in [K+]o depolarize neurons as well as glial cells thereby altering neuronal excitability, and several mechanisms exist to remove excess potassium 8. Failure of these may result in epileptiform activity of neurons or phenomena like spreading depression 1. 
Because of their critical importance, quantitative determination of extracellular ion concentrations is often necessary and required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. For decades, double-barreled ion-selective microelectrodes have proven to be excellent tools for such measurements in brain tissue 9. For many ions, highly specific sensors with low cross-reactivity for other ions are available. In addition to the classical double-barreled electrodes, so-called concentric electrodes were recently introduced. The latter provide a superior time resolution, but take a little more time and effort to construct 10. 
In the following, we will describe the preparation and calibration of these two types of ion-selective microelectrodes. We then show how these electrodes can be employed in brain slice preparations for measurement of changes in [K+]o or [Na+]o induced by excitatory activity following different stimulation paradigms including bath and pressure application of drugs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Abrasive&#160;&#160;</td>
			<td>MicroPolish&#160;</td>
			<td>Buehler GmbH</td>
			<td>Dissolved in A.dest</td>
		</tr>
		<tr>
			<td>Borosilicate-glass capillaries</td>
			<td>1405059</td>
			<td>Hilgenberg</td>
			<td>Application pipette; 75 mm x 2 mm, wall thickness 0.3 mm</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC 150 F-15&#160;</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the sensor of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC100-F-15</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the reference of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-200TF-15&#160;</td>
			<td>Science Products</td>
			<td>Concentric, outer channel. o.d. 2.0 mm&#160;&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-120TF-10</td>
			<td>Science Products</td>
			<td>Concentric, inner channel. o.d. 1.2 mm</td>
		</tr>
		<tr>
			<td>Digidata</td>
			<td>1322A</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrometer amplifier with headstage</td>
			<td></td>
			<td>Custom-made</td>
			<td>Rin = 10T&#937; and Ibias=50fA-1pA (commercially available&#160; alternatives: e. g. Dagan IX2-700, with headstage (10 Gig feedback resistor) or EPMS-07, NPI, Tamm, Germany)</td>
		</tr>
		<tr>
			<td>Experimental chamber</td>
			<td></td>
			<td>Custom-made</td>
			<td>Commercially available from e.g.&#160; Warner Instruments,USA;&#160; Scientifica, UK</td>
		</tr>
		<tr>
			<td>Furnace</td>
			<td></td>
			<td>Heraeus</td>
			<td>Must stay constant at 200&#176;C</td>
		</tr>
		<tr>
			<td>Hard sticky wax / dental wax&#160;</td>
			<td>Deiberit 502&#160;</td>
			<td>Siladent Dr. Boehme &#38; Schoeps GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td></td>
			<td>Custom-made</td>
			<td>Must stay constant at 40&#176;C</td>
		</tr>
		<tr>
			<td>Microelectrodes holder made of plexiglas</td>
			<td></td>
			<td>Custom-made</td>
			<td>Double-barreled: o.d.&#160; capillaries 1.5 mm, concentric: o.d.&#160; capillaries 2 mm</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td></td>
			<td>Leitz</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>MD4R</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>M205C</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Plan 0.8xLWD</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Model PP-830</td>
			<td>Narishige</td>
			<td>Concentric microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Model P-97</td>
			<td>Sutter Instruments&#160;</td>
			<td>Sensor of concentric microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Pneumatic drug ejection system</td>
			<td>Picospritzter Type II</td>
			<td>General Valve TM Corporation</td>
			<td></td>
		</tr>
		<tr>
			<td>Travel dovetail stage</td>
			<td>DT 25/M</td>
			<td>Thorlabs</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-component glue</td>
			<td>&#160;Araldite</td>
			<td>Huntsman advanced materials GmbH</td>
			<td>One may also use a small stripe of aluminum foil to stick the capillaries together&#160;</td>
		</tr>
		<tr>
			<td>Silverwire</td>
			<td>99.9%&#160;</td>
			<td>Wieland Edelmetalle</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicer / Vibratome</td>
			<td>Microm HM 650 V</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>AxoScope 8.1&#160;</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical puller&#160;</td>
			<td>Type PE-2</td>
			<td>Narishige Scientific Instruments</td>
			<td>With a revolvable chuck for&#160; double-barreled microelectrodes</td>
		</tr>
		<tr>
			<td>x/y translational stage</td>
			<td></td>
			<td>Custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Compound</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>1(S),9(R)-(&#8722;)-Bicuculline methiodide</td>
			<td>Sigma aldrich</td>
			<td>14343</td>
			<td>Competitive antagonist of GABAA receptors (light-sensitive); CAUTION toxic</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma aldrich</td>
			<td>C-127</td>
			<td>AMPA/kainate receptor antagonist; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-AP5</td>
			<td>Alfa Aesar</td>
			<td>J64210</td>
			<td>NMDA receptor antagonist; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Sigma aldrich</td>
			<td>440191</td>
			<td>CAUTION: Flammable, acute toxicity (oral, dermal, inhalation) and corrosive to metals and skin</td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma aldrich</td>
			<td>A9256</td>
			<td>Activates NMDA and non-NMDA and EAATs</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma aldrich</td>
			<td>G1626</td>
			<td>Activates NMDA-R, AMPA-R, QA-R and KA-R),&#160; mGluRs and EAATs</td>
		</tr>
		<tr>
			<td>Potassium ionophore I - cocktail B</td>
			<td>Fluka&#160;</td>
			<td>60403</td>
			<td>Based on valinomycin; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Sodium ionophore II - cocktail A</td>
			<td>Fluka&#160;&#160;</td>
			<td>71178</td>
			<td>Based on ETH 157</td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>Ascent Scientific</td>
			<td>Asc-055</td>
			<td>Inhibitor of voltage-dependent Na+ channels; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Water, ultra pure</td>
			<td>Sigma aldrich</td>
			<td>W3500&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

double-barreled and concentric microelectrodes for measurement of extracellular ion signals in brain tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

<html> [K +] o及び興奮活動に応答して、その中の変化を監視するには、二重のカリウム選択的微小電極は、CA1領域の放線層に挿入しました。数分突き刺した後、電極のイオン選択バレルは約2.8 mMで、使用ACSFの[K +]に近い値（のO [K +]に対応し、安定したベースライン（ 図7A）に達しました 2.5 mm）です。 10秒間0.5 mMのグルタミン酸のバスアプリケーションは、〜0.7ミリ（ 図7A）に相当するベースライン下のアンダーシュートに続いて、約4mmによってO [K +]で可逆的増加を誘導しました。 0.4、0.3にグルタミン酸濃度を下げる、および0.2 mMのは、一過性の上昇と[K +] O（ 図7A）でアンダーシュートの両方の振幅の対応する減少を引き起こしました。 OAD / 53058 / 53058fig7.jpg &quot;/&gt; 興奮活動に応じて、図7.外カリウムトランジェント。（A）[K +] oをトランジェント、示されるように、10秒間のグルタミン酸の異なる濃度の浴アプリケーションによって誘発されるベースライン以下のアンダーシュートに続いて上昇、からなります。 （B）グルタミン酸受容体遮断薬と灌流の影響（CNQX、100μM; APV、100μM）およびテトロドトキシン（TTX; 0.5μM）[K +] oをトランジェントに10秒間0.5 mMのグルタミン酸の浴アプリケーションによって誘発されます。 0Mg 2+ / BICの存在下で、（C）自発[K +] oをトランジェント。 ACに示される実験は、二重バレルの電極を用いて行きました。説明のため、トレースはSawitzky・ゴーレイフィルタ（幅20）で平滑化した。 <a href="https://www.jove.com/files/ftp_upload/53058/53058fig7large.jpg" target="_blank">をクリアしてください</a>この図の拡大版を表示するには、こちらICK。 以前の実験では、このようなグルタミン酸誘発[K +] O信号が大幅にTTXの適用中に変更されていないことを示し、したがって、電位依存性ナトリウムチャネルおよび神経活動電位発生15,16の開口部に概ね独立しています。グルタミン酸に応答して観察された[K +] Oの変化の根底にあるメカニズムを研究するために、我々は、グルタミン酸受容体遮断薬、すなわちCNQX（100μM; AMPA受容体遮断薬）を適用し、APVを（100μM; NMDA受容体遮断薬）の存在下でTTX（0.5μM）。 （; 図7B 例えば 17）、これらの遮断薬と一緒にお風呂灌流時には、グルタミン酸誘発[K +] oが変化する前に報告されているようにイオンチャネル型グルタミン酸受容体チャンネルの開口部への依存度を確認し、事実上廃止されました。 さらにトンを実証するために、彼外[K +]信号を生成するためのグルタミン酸作動性興奮の関連性、スライスは10μMのビククリンのメチオダイド（0Mg 2+ / BIC）を含む名目上のMg 2+を含まない生理食塩水で灌流しました。これは、NMDA受容体の電位依存性Mg 2+を-blockを緩和し、ネットワーク（ 例えば、18,19）に自発的再発性てんかん様活動を引き起こし、GABA A受容体を遮断することによって阻害を弱めます。予想されたように20、自発的、再発[K +] oをトランジェント、約1.5mmに相当するが0Mg 2+ / BIC生理食塩水（ 図7C）で検出されました。これらの応答の間には、0.2mm程度が発生した（ 図7C）の平均振幅と[K +] oを過渡に小さいです。 第二の実験セットにおいて、我々は、の適用により誘発【のNa +] Oの変化を決定するために【のNa +]感受性微小電極を使用しグルタミン酸アゴニスト。ここで、我々はまた、2つの異なるアプリケーションのパラダイムを採用し、二連と同心の微小電極の応答特性を比較しました。 [ の Na +]感受性微小電極は、約50μmの深さで放線層に配置しました。安定なベースラインが達成された後、グルタミン酸アゴニスト、L-アスパラギン酸（2.5ml /分の10mM、120秒間、浴灌流）浴潅流ごとに適用しました。以前の21観察されたように、アスパラギン酸の適用は、約5分続いたし、その後ベースラインに戻った （図8A）を回収し始めた約15 mMのでO [ の Na +]でゆっくりと減少を引き起こしました。両電極により決定【のNa +] O信号のピーク振幅と速度は、これらの条件下で実質的に同一であった注目すべきことに、同心の電極は、より安定したベースラインと低ノイズレベル（ 図8A）を示しました 。 Rをテストするには<html>より迅速なアプリケーションパラダイムの下で異なる電極の特性をesponse、我々はイオン選択性微小電極の先端から20〜40ミクロンの距離で放線層に位置し、微細ガラスピペットを介してグルタミン酸を、適 ​​用される圧力。 21予想されたように、200ミリ秒のためのグルタミン酸の適用（10 mM）を[ の Na +] O（ 図8B）の低下を引き起こしました。減少O [ の Na +]のピーク振幅は、（二連：4.5から13.5 mMであり、n = 14;同心：2.0から19.1 mmであり、N = 15）は、電極の両方のタイプの同じ範囲にありました。しかし、遅い浴灌流（上記参照）、信号対雑音比だけでなく、電極の2つのタイプによって検出【のNa +] O信号の経時変化のみならずで得られた結果とは対照的に、有意に異なりました。ピークまでの平均時間は、同心円状の微小電極のための銃身、ダブル、わずか1.3秒3.5秒でした。 ve_content &quot;&gt;このように、これらの結果は、またのCa 2+及びpH選択的対応10のために注目されたように、二重のの Na +対同心選択性微小電極、（参照： 図6Cおよび図8B）の速い応答速度を確認しますショートバーストシナプス刺激が速いシナプスによって誘発されるイオンの過渡現象を喚起するために実施した。前者の研究とは対照的に、唯一の傾向が、私たちのアプリケーションと同心と二重の電極間の平均ピーク振幅に有意差は認められていますパラダイム。これはおそらく、事実による2（20-40ミクロ​​ン）の要因によって変えるイオン選択微小電極の先端からアプリケーションピペットの距離ということであり、その結果、標的領域における最大のグルタミン酸濃度そのピーク振幅の既存の違いを妨害、別の実験では同じではありませんでした。 （A）トップ痕跡：ボルタの変更二重の電極（黒の点線のトレース）とバスの Na + 濃度の変化に応じて、同心円状の電極（グレーのトレース）のリファレンス・バレル（のV REF）のGE（[ の Na +]は b）に示すように。両電極の電圧応答が実質的に同一であることに注意してください。ボトムトレース：[ の Na +] Bの変化に応じて、二重の電極（黒のトレース）と同心円状の電極（グレーのトレース）のの Na + -電位（V のNa +）の電圧の変化。 （B）両電極のV のNa +対B [ の Na +]の半対数プロット。データの線形プロットは、両方の電極のための約48 mVでの傾きを明らかにしました。 70 mmの152 mMのから[ の Na +] Bの急速な変化に二重の（黒のトレース）のV のNa +（C）の対応と同心電極（グレーのトレース）。同心円状の電極の応答時間は著しくFASであることに注意してくださいター。例示の目的のために、トレースはSawitzky-ゴレイフィルタで平滑化した（幅20）。 <img alt="図8" src="/files/ftp_upload/53058/53058fig8.jpg" /> 図8：細胞外ナトリウムトランジェント・二連と同心の電極によって検出されました。 （A）過渡のV REFの変化や[ の Na +]バーで示されるように、O 120秒間の10mMアスパラギン酸と一緒にお風呂灌流により誘発されます。上部トレースは、二重の電極を用いて行った記録が、下側のトレースは、同心円状の電極を用いて記録した表示します。 （B）過渡のV REFの変化や[ の Na +]矢印で示すように、O 0.2秒間の10mMグルタミン酸と局所的な圧力の適用によって誘発されます。上部トレースは、二重の電極を用いて行った記録が、下側のトレースは、同心円状の電極を用いて記録した表示します。赤い点線の線は、その最大の信号の開始までの期間の線形フィットを表します。同心円状電極の応答時間は、この条件の下で大幅に高速であることに注意してください。説明のために、トレースがSawitzky・ゴーレイフィルタ（幅20）で平滑化した。 <a href="https://www.jove.com/files/ftp_upload/53058/53058fig8large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue at JoVE.com

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

脳組織における測定細胞外イオンの信号のための、二連と同心微小電極

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

double-barreled-concentric-microelectrodes-for-measurement

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JoVE Journal

Neuroscience

13.4K ビュー.  Heinrich Heine University Düsseldorf. We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations. 

ビデオ: Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

免疫電子顕微鏡のためのマウスの脳組織の準備

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

神経科学

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

集束イオンビームは、粉砕し、脳組織の走査型電子顕微鏡

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

小動物の慢性神経録音用超軽量電動マイクロドライブの設計およびアセンブリ

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

作成、テスト、および成人の脳の切片でカリウム イオン選択的な電極を使用して

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

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 ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶媒クリア脳組織における狂犬病ウイルス感染の高解像度3Dイメージング

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

天然ポリフェノールクルクミンを用いた脳組織におけるアミロイドプラークの標識とイメージング

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

マウス脳の水平海馬スライス

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

ラット脳組織の疎水性組織除去法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

急性脳スライスにおける内因性モノアミン放出測定用プレートベースアッセイ

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...