エリザベスタウン・カレッジ(R.J.W,M.L.,L.M.),NSF大学院フェローシップ(R.J.W.),イェール大学医学部(N.A.)の支援を受けました。

すべての実験は、国立衛生研究所(NIH)実験動物のケアと使用ガイドに従って行われ、エリザベスタウン・カレッジとイェール大学の制度的動物ケアと使用委員会(IACUC)の両方によって承認されました。このプロトコルは、CIS-FSCVを利用する麻酔ラット調製に固有のものです。
1. 術前の準備
<ol><li>電極溶液調製<ol>
<li>電極バックフィル溶液を作るために、140 mM塩化カリウム16で4M酢酸カリウムの溶液を調製する。</li>
	</ol>
</li>
	<li>電極準備<ol>
<li>真空吸引を使用して、T-650炭素繊維(直径7μm)をホウケイ酸ガラスキャピラリー(長さ=100mm、直径=1.0mm、内径=0.5mm)に挿入します。</li>
		<li>カーボンファイバーをガラスキャピラリーの内部に配置したら、ガラスキャピラリーを垂直電極引き手に入れ、熱要素をキャピラリーの真ん中に入れます。磁石をオフにしてヒーターを55に設定します。</li>
		<li>毛細管を引っ張った後、電極の先端が発熱体で囲まれないように上の毛細管ホルダーを慎重に上げる。</li>
		<li>鋭利なはさみを使用して、キャピラリーの2つの部分をまだ接続している炭素繊維をカットします。これにより、2つの別々の炭素繊維マイクロ電極が生じる。</li>
		<li>光顕微鏡下で、露出した炭素繊維を鋭いメスで丁寧に切断し、ガラスの端を越えて約75〜100μmの炭素繊維が伸びるようにします。</li>
		<li>軽顕微鏡を使用して、電極に毛細血管に沿った亀裂がないことを確認してください。また、炭素繊維が毛細血管から出るシールが気づきにくく、亀裂がないようにしてください。 メモ:シールが良いと、録音中のノイズを低減できます。詳細なプロトコルについては、発表された研究17、18、19を参照してください。</li>
	</ol>
</li>
	<li>参照電極製作<ol>
<li>5 cmの銀線に金のピンをはんだ。</li>
		<li>アノードを金属製のクリップまたは他の導体に取り付け、カソードをピンに取り付け、0.1 M HClに水没させながら電圧(~2V)を印加します。</li>
		<li>銀線に白色のコーティング(AgCl)が現れたら電圧を止める。</li>
	</ol>
</li>
	<li>埋め込み用電極の準備<ol>
<li>薄い絶縁ワイヤー(長さ10cm、直径0.50mm<)に金ピンをはんだ付けする。</li>
		<li>金ピンとは反対側のワイヤーから絶縁体の〜5cmを取り除きます。</li>
		<li>電極溶液をほぼ半分に充填します。</li>
		<li>絶縁線を電極に挿入します。 メモ:ワイヤは電極内部の炭素繊維と接触する必要があります。</li>
	</ol>
</li>
</ol>2. 電極注入
<ol><li>無菌生理食糸に溶解した0.5g/mLウレタンの腹腔内注射(1.5g/kgまたは1mL/kg量)の成人、オス、スプレイグ・ドーレーラット(250-450g)を与える。1.0-1.2 g/kgの初期ウレタン線量から始めます。ウレタン投与後20分間、有害刺激試験(テールピンチ)に対して動物が反応する場合は、1.5 g/kgの総用量で0.3-0.5 g/kgウレタンを追加投与する。 注:0.5 g/mLウレタン溶液の調製のために、10gのウレタンを10g(〜10 mL)の生理食音に加えます。ウレタンは発がん性物質であり、注意して取り扱う必要があります。ウレタンは、ドーパミンのレベルを変化させないように重要な麻酔薬であり、ケタミン/キシラジンおよびクロラルハイドレート20,21などの他の麻酔薬と同様に。</li>
	<li>動物が深く麻酔され、有害な刺激(例えば、つま先ピンチ)に反応しない場合は、ステレオタキシックフレームに置きます。ラットの各眼に眼用潤滑剤を塗布する。 注:これは非生存手術ですが、良い無菌技術が奨励されています。</li>
	<li>2段階のスクラブを使用してラットの頭皮を洗浄します(すなわち、ヨドポビトンスクラブに続いて70%エタノールスクラブを行い、3サイクルの繰り返しで実行します)。</li>
	<li>滅菌針鼻ピンセットと手術用はさみを使用して頭皮組織を切り取ります。以下に概説する様々な移植のためのスペースを作るために、かなりの量の組織を取り除きます。</li>
	<li>滅菌綿の先端アプリケーターを使用して、頭蓋骨の表面を静かにきれいにします。次に、λとブレグマを識別するために、過酸化水素3%の2〜3滴を適用します。</li>
	<li>ステレオタキシックドリルまたはハンドドリル(1.0 mm、〜20,000 rpm)を使用して、直径1.5mmの穴をブレグマに2.5mm、横に3.5mmの横に掘削します。部分的に(中途半端に、しっかりと所定の位置に入るまで)、この穴にネジ(1.59 mm O.D.、長さ3.2mm)を埋め込みます。熱損傷を防ぐために掘削中に灌漑するために滅菌生理布を使用することをお勧めします。</li>
	<li>参照電極の場合、直径1.0mmの穴を左半球のブレグマに1.5mmの前部と3.5mmの横に開きます。</li>
	<li>この穴には、約2mmのリファレンスワイヤを挿入し、インプラントねじの頭の周りと下に参照ワイヤを巻き付けます。</li>
	<li>ネジを完全に埋め込み、参照電極を所定の位置に固定します。</li>
	<li>右半球では、直径1.5mmの穴を前部1.2mm、横1.4mmの横に掘削します。</li>
	<li>ピンセットを使用して硬膜をそっと取り除きます。</li>
	<li>刺激電極の場合は、5.2mm後方に中央に5.2mm、横1.0mmの方角をブレグマに中心とする正方形の穴(2mm前側後部、5mm内側内側)を掘削します。</li>
	<li>立体性アームバーを使用して、双極刺激電極/ガイドカニューレを硬膜下5mm下げます。電極の移植中に出血した場合は、滅菌綿棒とガーゼを使用して出血を最小限に抑えます。 注:この方法で使用されるバイポーラ刺激電極はガイドカニューレ(材料表)を備えています。このアイテムで使用される内部カニューレは、ガイドカニューレに完全に挿入された場合、バイポーラ刺激電極のプロングで洗い流す必要があります。これにより、内部カニューレは、約1mm離れた刺激器の2つのプロングの間に直接座ることができます。同様のプロトコルは、他の場所で説明されています 14.</li>
	<li>定位アームバーを使用して、炭素繊維マイクロ電極を硬膜下4mm下げる。この場所は、線条体の最も後ろの部分にあります。</li>
	<li>リファレンスワイヤと炭素繊維をポテンショスタットに接続します。</li>
	<li>60 Hzで15分間、10分間、10 Hzで10分間、三角波(-0.4−1.3 V、400 V/s)を適用します。 注:通常、脳内の炭素繊維微小電極に波形を適用する場合、酸化物基が炭素繊維の表面に添加されます。この反応の平衡は、記録する前に到達する必要があります。それ以外の場合は、有意なドリフトが発生します19.より高い周波数(60 Hz)で電極をサイクリングすることで、炭素繊維はより高速に平衡を達成することができます。</li>
</ol>3. 炭素繊維の最適化と電極/ガイドカニューレの位置の活性化
<ol><li>60 Hz、24パルス、300μA電流、パルス幅2ms/位相の周波数で、バイポーラ電気波形を生成するように刺激器を設定します。</li>
	<li>刺激装置を、5mmから7.8mmまで0.2mmずつ徐に下げます。各増分で、VTAを刺激します。 注:より多くの後ろの深さ(5-6 mm)では、脳の刺激は通常(時間の80%程度)ラットのウィスカーをぴくぴくさせます。さらに深さで、ウィスカーは硬膜の下7.5-8.2 mmの間で起こるぴくぴくを止めます。ウィスカーがぴくぴくしなくなると、刺激電極はVTAの近くまたは近くにあります。これはすべてのラットで発生するものではなく、ウィスカーのけいれんの欠如は、双極性刺激電極/注入カニューレが見当違いであることを示すものとして取られるべきではありません。すべての麻酔薬(例えば、イオブルラン)に対してウィスカーのけいれんが起こるわけではない。</li>
	<li>刺激が炭素繊維微小電極(現在は側側線条体)でフェジックDA放出を生じるまで、バイポーラ刺激電極/ガイドカニューレを下げ続ける。 注:双極性電極がVTAに埋め込まれた場合、後回線でのDA放出は必ずしも起こるわけではありませんが、VTA刺激時のDA放出の観察は、通常、NAcコアに良好な信号が観察されることを示す良好な兆候です。</li>
	<li>炭素繊維マイクロ電極を、硬膜の下に6.0mm以上になるまで下げてください。これはNAcコアの最も後ろ側の部分です。</li>
	<li>VTAを刺激し、DAピークのピーク振幅を記録します。</li>
	<li>DA放出が最も大きい部位で炭素繊維マイクロ電極を下げるか、または上げる。</li>
	<li>DA応答のピークが0.6Vでの明確な酸化ピーク、-0.2 Vでの還元ピークであることを確認してください。これらのピークはDAを示しています。</li>
</ol>4. 組み合わせ注入と刺激FSCV記録
注: 図 1 は、VTA マイクロインフュージョンの前後の記録のタイムラインを示しています。
<ol><li>炭素繊維と刺激電極/ガイドカニューレの位置が最適化されたら、〜20〜30分の刺激を与えます。 注:現在の刺激パラメータの下で、22の静脈リローディングを可能にするために、3分ごとに1回以上刺激しないでください。</li>
	<li>安定したベースライン(5回の刺激に対して<20%の変動)を達成した後、バイポーラ刺激器にプリフィットされたガイドカニューレに手で内側のカニューレを穏やかに下げる。</li>
	<li>カニューレ挿入自体が呼び起こされる信号に変化を起こさないように、さらに2-3のベースライン録音を取ります。場合によっては、内部カニューレの挿入と取り外しがVTAに損傷を与える可能性があります。このベースライン期間(>20%)で信号が大幅に変化した場合は、ベースラインが再び不安定になるまで3~4の記録を追加します。</li>
	<li>シリンジポンプとマイクロシリンジを使用して、0.5 μLの溶液(例えば、0.9%生理食い、N-メチル-D-アスパラギン酸[NMDA]、(2R)-アミノ-5-ホスホノ球酸[AP5])を2分にわたってVTAに注入する。</li>
	<li>注入後、除去する前に少なくとも1分間、内カニューレを残す。 注:一部の薬物は、薬物の動態に基づいてより長い時間、内部カニューレを残すことを必要とし、内部カニューレの除去は、薬物が内部カニューレを通って戻って移動する可能性があります。懸念がある場合は、記録全体の間にガイドカニューレに内部カニューレを残すことができます。それ以外の場合は、この 1 分間隔の後に録画を開始できます。</li>
	<li>3分ごとに記録を続けて、輸液後の効果を測定します。 注:制御溶液を注入し、効果が見られない場合は、2回目の10を注入することが可能です。内部カニューレまたは生理食糸の挿入によって起こされるDA放出が変化した場合、シグナルは通常30分以内にベースラインに回復する。</li>
</ol>5. 電極配置の組織学的検証
<ol><li>実験の最後に、炭素繊維マイクロ電極を用いて記録部位に小さな病変を作る。<ol>
<li>実験後のキャリブレーションのために電極を保存する必要がある場合は、キャピラリー先端を越えて約100μm突出したガラスキャピラリーに配置されたタングステン線を使用してください。この場合、脳から電極を上げ、記録電極をタングステン電極に置き換え、それを同じドーソベントラ座標に下げる。 注:炭素繊維は、同様に脳を病変するために使用することができ、記録部位のより正確な表現を提供します。しかし、実験者はこれらの電極を較正する能力を失うことになる。</li>
	</ol>
</li>
	<li>記録部位を病変するには、電源を使用して電圧を印加する。1Vから始めて、10 Vに達するまで10 sごとに1 Vずつ増加します。</li>
	<li>ペントバルビタールの致死的な腹腔内注射(150mg/kg)を使用して動物を安楽死させる。</li>
	<li>4%ホルマリン溶液を使用してラットを浸透させます。</li>
	<li>鋭利なギロチンを使用してラットから頭部を取り除きます。</li>
	<li>ロンガを使用して、脳を取り巻く結合組織と頭蓋骨を取り除き、残りの組織から脳を静かに取り除きます。</li>
	<li>脳を4%ホルマリンに1日間保存し、それを30%スクロースに移す。 注:4%ホルマリンを灌流することは病変部位を見る必要はないが、ベストプラクティスとしては病変部位の再建を改善する。</li>
	<li>クライオスタットを使用して脳の30 μmのスライスを作成します。</li>
	<li>スライドにスライスを取り付け、カバースリップで覆います。</li>
	<li>光顕微鏡を用いた炭素繊維微小電極病変及びバイポーラ刺激/注入カニューレの位置を示す。</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+L-type+calcium+channel+blocker,+isradipine,+attenuates+cue-induced+cocaine-seeking+by+enhancing+dopaminergic+activity+in+the+ventral+tegmental+area+to+nucleus+accumbens+pathway.">The L-type calcium channel blocker, isradipine, attenuates cue-induced cocaine-seeking by enhancing dopaminergic activity in the ventral tegmental area to nucleus accumbens pathway.</a> Neuropsychopharmacology. 43 (12), 2361-2372 (2018).</li><li>Hermans, A., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conical+tungsten+tips+as+substrates+for+the+preparation+of+ultramicroelectrodes.">Conical tungsten tips as substrates for the preparation of ultramicroelectrodes.</a> Langmuir. 22 (25), 10348-10353 (2006).</li><li>Borland, L. M., Michael, A. C., Borland, L. M., Michael, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+to+Electrochemical+Methods+in+Neuroscience.">An Introduction to Electrochemical Methods in Neuroscience.</a> Electrochemical Methods for Neuroscience. , (2007).</li><li>Mundroff, M. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amperometry+and+cyclic+voltammetry+with+carbon+fiber+microelectrodes+at+single+cells.">Amperometry and cyclic voltammetry with carbon fiber microelectrodes at single cells.</a> Current Protocols in Neuroscience. 6 (6), 14 (2002).</li><li>Rodeberg, N. 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=Hitchhiker's+Guide+to+Voltammetry:+Acute+and+Chronic+Electrodes+for+in+vivo+Fast-Scan+Cyclic+Voltammetry.">Hitchhiker's Guide to Voltammetry: Acute and Chronic Electrodes for in vivo Fast-Scan Cyclic Voltammetry.</a> ACS Chemical Neuroscience. 8 (2), 221-234 (2017).</li><li>Sabeti, J., Gerhardt, G. A., Zahniser, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloral+hydrate+and+ethanol,+but+not+urethane,+alter+the+clearance+of+exogenous+dopamine+recorded+by+chronoamperometry+in+striatum+of+unrestrained+rats.">Chloral hydrate and ethanol, but not urethane, alter the clearance of exogenous dopamine recorded by chronoamperometry in striatum of unrestrained rats.</a> Neuroscience Letters. 343 (1), 9-12 (2003).</li><li>Masuzawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pentobarbital+inhibits+ketamine-induced+dopamine+release+in+the+rat+nucleus+accumbens:+a+microdialysis+study.">Pentobarbital inhibits ketamine-induced dopamine release in the rat nucleus accumbens: a microdialysis study.</a> Anesthesia &amp; Analgesia. 96 (1), 148-152 (2003).</li><li>Montague, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+gain+control+of+dopamine+delivery+in+freely+moving+animals.">Dynamic gain control of dopamine delivery in freely moving animals.</a> Journal of Neuroscience. 24 (7), 1754-1759 (2004).</li><li>Keithley, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+sensitivity+dopamine+measurements+with+faster-scan+cyclic+voltammetry.">Higher sensitivity dopamine measurements with faster-scan cyclic voltammetry.</a> Analytical Chemistry. 83 (9), 3563-3571 (2011).</li><li>Jackson, B. P., Dietz, S. M., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast-scan+cyclic+voltammetry+of+5-hydroxytryptamine.">Fast-scan cyclic voltammetry of 5-hydroxytryptamine.</a> Analytical Chemistry. 67 (6), 1115-1120 (1995).</li><li>Park, J., Takmakov, P., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparison+of+norepinephrine+and+dopamine+release+in+rat+brain+by+simultaneous+measurements+with+fast-scan+cyclic+voltammetry.">In vivo comparison of norepinephrine and dopamine release in rat brain by simultaneous measurements with fast-scan cyclic voltammetry.</a> Journal of Neurochemistry. 119 (5), 932-944 (2011).</li><li>Wenzel, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phasic+Dopamine+Signals+in+the+Nucleus+Accumbens+that+Cause+Active+Avoidance+Require+Endocannabinoid+Mobilization+in+the+Midbrain.">Phasic Dopamine Signals in the Nucleus Accumbens that Cause Active Avoidance Require Endocannabinoid Mobilization in the Midbrain.</a> Current Biology. 28 (9), 1392-1404 (2018).</li><li>Spanos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+Receptor-Dependent+Cholinergic+Modulation+of+Mesolimbic+Dopamine+Cell+Bodies:+Neurochemical+and+Behavioral+Studies.">NMDA Receptor-Dependent Cholinergic Modulation of Mesolimbic Dopamine Cell Bodies: Neurochemical and Behavioral Studies.</a> ACS Chemical Neuroscience. 10 (3), 1497-1505 (2019).</li><li>Cheer, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+enhance+subsecond+dopamine+release+in+the+nucleus+accumbens+of+awake+rats.">Cannabinoids enhance subsecond dopamine release in the nucleus accumbens of awake rats.</a> Journal of Neuroscience. 24 (18), 4393-4400 (2004).</li><li>Melchior, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+versus+electrical+stimulation+of+dopamine+terminals+in+the+nucleus+accumbens+reveals+local+modulation+of+presynaptic+release.">Optogenetic versus electrical stimulation of dopamine terminals in the nucleus accumbens reveals local modulation of presynaptic release.</a> Journal of Neurochemistry. 134 (5), 833-844 (2015).</li><li>Sun, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genetically+Encoded+Fluorescent+Sensor+Enables+Rapid+and+Specific+Detection+of+Dopamine+in+Flies,+Fish,+and+Mice.">A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice.</a> Cell. 174 (2), 481-496 (2018).</li><li>Robinson, D. 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=Monitoring+rapid+chemical+communication+in+the+brain.">Monitoring rapid chemical communication in the brain.</a> Chemical Reviews. 108 (7), 2554-2584 (2008).</li><li>Park, 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=Heterogeneous+extracellular+dopamine+regulation+in+the+subregions+of+the+olfactory+tubercle.">Heterogeneous extracellular dopamine regulation in the subregions of the olfactory tubercle.</a> Journal of Neurochemistry. 142 (3), 365-377 (2017).</li><li>Ganesana, M., Venton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+changes+in+transient+adenosine+during+cerebral+ischemia+and+reperfusion+injury.">Early changes in transient adenosine during cerebral ischemia and reperfusion injury.</a> PLoS One. 13 (5), e0196932 (2018).</li></ol>

著者らは開示するものは何もない。

CIS-FSCVは、Phasic DA放出の基礎となるVTA受容体機構を調査するユニークな機会を提供します。適切な記録を確実にするためには、2つの重要なステップがあります。まず、安定したベースライン記録を達成する必要があります。安定した記録を確立する可能性を高める重要な方法は、電極が60 Hzと10 Hzの両方でサイクルするのに十分な時間を持っていることを確認することです(通常、60 Hzでは15分?...

腹側テグメンタル領域(VTA)から側坐核(NAc)への放出は報酬関連の行動において重要な役割を果たす。VTA DAニューロンは、強壮性の発火(3~8Hz)からバースト状の発火(>14Hz)1に切り替え、NAcでフェージDA放出を生み出します。VTAは、強壮剤からバースト焼成2、3、4、5への切り替えを制御するのに適した位置にある様々なソマト樹状受容体を発現する。これらの受容体のどれとそれぞれの入力を特定して、制御フェシカルDA放出は報酬関連回路がどのように組織されているかについての理解を深める。ここで説明する方法論の目的は、高速スキャン環状ボルタンメトリー(CIS-FSCV)との注入と刺激を組み合わせて、迅速かつ堅牢に駆動フェージックDA放出におけるVTA受容体の機能性を評価することである。
融合と刺激(CIS)を組み合わせた用語は、細胞のグループ(ここではVTA)上の受容体を薬理学的に操作し、それらのニューロンを刺激して受容体の機能を研究することを指します。麻酔ラットでは、高速スキャン環式ボルタンメトリー(FSCV)で測定したNAcコアに大きなフェージDA信号(1-2 μM)を呼び起こすためにVTAを電気的に刺激します。刺激部位における薬理学的薬剤(すなわち受容体アゴニスト/アンタゴニスト)の注入は、誘発されたフェースDA放出の後の変化を観察することによってVTA受容体の機能を測定するために使用することができる。FSCVは、高空間(50-100 μm)と時間(10Hz)の両方の解像度を楽しむ電気化学的アプローチであり、報酬関連のフェージックDAイベント6、7を測定するのに適しています。この解像度は、マイクロ透析などの他の生体内神経化学的測定よりも細かいものです。したがって、CIS-FSCVは、一緒に、フェースドーパミン放出のVTA受容体調節を評価するのに適している。
VTA受容体機能を調べる一般的な方法の1つは、それらの受容体がニューロン1,8の発火速度をどのように変化させるかを扱う電気生理学的アプローチの組み合わせを用いることである。これらの研究は、活性化時にDA発火を促進する際にどのような受容体が関与しているか理解する上で非常に貴重です。しかし,これらの研究は,軸索末端で下流に起こり得るものを示唆することしかできません(すなわち、神経伝達物質の放出)。CIS-FSCVは、VTAバースト焼成、フェーシックDA放出の出力がVTAデンドライトおよび細胞体上にある受容体によってどのように調節されているかに答えることによって、これらの電気生理学的研究に基づいています。したがって、CIS FSCVはこれらの電気生理学研究に基づいて構築するのに適しています。一例として、ニコチン受容体活性化はVTA9においてバースト発成を誘導することができ、そして麻酔ラットにおけるCIS-FSCVは、VTAにおけるニコチン性アセチルコリン受容体(nAChR)活性化もNAc10,11におけるフェージドDA放出を制御することを示すために使用された。
フェシカルDA規制の機械検査はまた、薬物の浴用途と一緒にスライス製剤を使用して一般的に研究されています.これらの研究は、多くの場合、細胞体がスライス12から除去されるようにドーパミン末端からのフェジーカルDA放出のシナプス前調節に焦点を当てる。これらの調製物はドーパミン末端に対するシナプス前受容体の効果を研究するのに価値があるのに対し、CIS-FSCVは、ドーパミンニューロンに対するソマトデン化受容体の効果、ならびにVTAへのシナプス前の入力を研究するのに適している。この区別は重要であり、VTAにおけるソマト樹状受容体活性化は、NAcシナプス前受容体活性化とは異なる効果を有し得るからである。実際、NAcにおけるドーパミン作動性シナプス前nAChRsを遮断すると、バースト焼成13中にパシズムドーパミン放出を上昇させることができるが、その反対はVTAソマトデンドリックnAChDrcnAChRs10,11に当てはまる。
CIS FSCVは、VTA受容体がフェースDA放出を調節する能力を研究するための理想的なアプローチです。重要なことに、このアプローチは、麻酔または自由移動のいずれか、無傷のラットで行うことができます。このアプローチは急性期研究に適し、そのベースライン状態10、14の受容体機能を研究するとともに、薬物暴露後または行動操作11、15の後の受容体の機能的変化を評価できる長期研究を行う。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electrode Filling Solution/Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>World Precision Instruments</td>
			<td>MF286-5 (28 gauge)</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Sigma</td>
			<td>236497-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P3911-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon fiber</td>
			<td>Thornel</td>
			<td>T650</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode puller</td>
			<td>Narishige International</td>
			<td>PE-22</td>
			<td>Note: horizontal pullers can be used as well</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>A-M systems</td>
			<td>626000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulated wires for electrodes</td>
			<td>Weico Wire and Cable Incorporated</td>
			<td>UL 1423</td>
			<td>Length; 10 cm; diameter,0.4mm; must get custom made; insulated material should cover 5 cm of the wire</td>
		</tr>
		<tr>
			<td>Light Microscope (for viewing and cutting electrode)</td>
			<td>Fischer Scientific</td>
			<td>M3700</td>
			<td></td>
		</tr>
		<tr>
			<td>Pin</td>
			<td>Phoenix Enterprises</td>
			<td>HWS1646</td>
			<td>To be soldered onto the insuled electrode wire and reference electrode; connects to headstage</td>
		</tr>
		<tr>
			<td>Putty</td>
			<td>Alcolin</td>
			<td><a target="_blank" href="https://www.dickblick.com/items/23922-1003/">23922-1003</a></td>
			<td>Used to place electrode on while cutting the carbon fiber</td>
		</tr>
		<tr>
			<td>Scalpal Blade</td>
			<td>World Precision Instruments</td>
			<td>500239</td>
			<td>For cutting carbon fiber to the apprpriate length</td>
		</tr>
		<tr>
			<td>Silver Wire</td>
			<td>Sigma</td>
			<td>327026-4G</td>
			<td></td>
		</tr>
		<tr>
			<td>FSCV Hardware/Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>U-Line</td>
			<td>H-3618 (36&#34; x 24&#34; x 42&#34;)</td>
			<td></td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>Univ. of N. Carolina, Electronics Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulating electrode</td>
			<td>PlasticsOne</td>
			<td>MS303/2-A/SPC</td>
			<td>when ordering, request a 22 mm cut below pedestal</td>
		</tr>
		<tr>
			<td>TarHeel HDCV Software</td>
			<td>University of North Carolina-Chapel Hill</td>
			<td>-</td>
			<td><a href="https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/" target="_blank">https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/</a> for ordering information</td>
		</tr>
		<tr>
			<td>UEI breakout box</td>
			<td>Univ. of N. Carolina, Electronics Facility</td>
			<td></td>
			<td><a href="https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/" target="_blank">https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/</a> for ordering information</td>
		</tr>
		<tr>
			<td>UEI power supply</td>
			<td>Univ. of N. Carolina, Electronics Facility</td>
			<td></td>
			<td><a href="https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/" target="_blank">https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/</a> for ordering information</td>
		</tr>
		<tr>
			<td>Stimulator Hardware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurolog stimulus isolator</td>
			<td>Digitimer Ltd.</td>
			<td>DS4</td>
			<td>Neurolog 800A</td>
		</tr>
		<tr>
			<td>Infusion/Stimulation Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion Pump</td>
			<td>New Era Syringe Pump</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Internal Cannula</td>
			<td>PlasticsOne</td>
			<td>C315I/SPC INTERNAL 33GA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter Syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>PlasticsOne</td>
			<td>C313CT/ PKG TUBING 023 X 050 PE50</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula Holder</td>
			<td>Kopf Instruments</td>
			<td>1776 P-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tip Applicators</td>
			<td>Vitality Medical</td>
			<td>806</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Holder</td>
			<td>Kopf Instruments</td>
			<td>1770</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Kent Scientific</td>
			<td>RT-0501</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone Iodine</td>
			<td>Vitality Medical</td>
			<td>29906-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Screws</td>
			<td>Stoelting</td>
			<td>Bone Anchor Screws/Pkg.of 100</td>
			<td>1.59 mm O.D., 3.2 mm long</td>
		</tr>
		<tr>
			<td>Silver wire reference with AgCl</td>
			<td>InVivo Metric</td>
			<td>E255A</td>
			<td></td>
		</tr>
		<tr>
			<td>Square Gauze</td>
			<td>Vitality Medical</td>
			<td>441408</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotax</td>
			<td>Kopf Instruments</td>
			<td>Model 902 (Dual Arm Bar)</td>
			<td></td>
		</tr>
		<tr>
			<td>Histological Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formulin</td>
			<td>Sigma</td>
			<td>1004960700</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>BK Precision</td>
			<td>9110</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>80497</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten microelectrode</td>
			<td>MicroProbes</td>
			<td>WE30030.5A3</td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs for infusions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>((2R)-amino-5-phosphonovaleric acid</td>
			<td>Sigma Aldrich</td>
			<td>A5282</td>
			<td></td>
		</tr>
		<tr>
			<td>N-methyl-D-aspartate</td>
			<td>Sigma Aldrich</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Mecamylamine hydrochloride (M9020-5mg)</td>
			<td>Sigma Aldrich</td>
			<td>M9020</td>
			<td></td>
		</tr>
		<tr>
			<td>Scopolamine hydrobromide (S0929-1g)</td>
			<td>Sigma Aldrich</td>
			<td>S0929</td>
			<td></td>
		</tr>
	</tbody>
</table>

combined infusion and stimulation with fast-scan cyclic voltammetry (cis-fscv) to assess ventral tegmental area receptor regulation of phasic dopamine

腹側テグメンタル領域(VTA)から側坐核へのPhasicドーパミン(DA)放出は、報酬処理および強化学習において極めて重要な役割を果たす。VTA制御フェーシックDAリリースへの多様な神経入力を理解することは、報酬処理と強化学習を制御する回路のより良い画像を提供することができます。ここでは、インビボ高速スキャン環状ボルタンメトリー(FSCV)で測定した刺激誘発フェシクスDA放出(融合注入および刺激、またはCIS)を有する薬理学的アゴニストおよびアンタゴニストのVTAカニューレ内注入を組み合わせた方法を説明する。麻酔下ラットにCIS-FSCVを用い、カナンベン核の中で記録しながらカニューレを取り付けた双極電極でVTAを電気的に刺激することによって、フェースDA応答を誘発することができる。薬理学的アゴニストまたはアンタゴニストは、刺激部位に直接注入して、Phasic DA放出を駆動する上での特定のVTA受容体の役割を調査することができる。CIS-FSCVの大きな利点は、VTA受容体機能がインビボで研究できることです, インビトロ研究に基づいて構築.

CIS-FSCVは、NAcコアにおけるフェスジカルDA放出を駆動するVTA N-メチル-D-アスパラギン酸受容体(NMDAR)、ニコチン性アセチルコリン受容体(nACHRs)、ムスカリン性アセチルコリン受容体(mACHRs)の機能を研究するために使用されました。 図2 は、陰性対照の代表的なデータを示し、生理食糸0.9%の注入、前(ベースライン)および9分のポストインフュージョン...

Watch this Scientific Journal Video about Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine at JoVE.c

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

このプロトコルの目標は、直接腹側テグメンタル領域受容体を操作して、第二次ドーパミン放出への寄与を研究することです。

高速スキャン環状ボルタンメトリー(CIS-FSCV)による注入と刺激を組み合わせて、パジカルドーパミンの腹側テグメンタル領域受容体調節を評価する

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...

combined-infusion-stimulation-with-fast-scan-cyclic-voltammetry-cis

Research

JoVE Journal

Neuroscience

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

3.0K ビュー.  Elizabethtown College. このプロトコルの目標は、直接腹側テグメンタル領域受容体を操作して、第二次ドーパミン放出への寄与を研究することです。

ビデオ: Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

デフォルトモードのネットワークを検査する経頭蓋磁気刺激とfMRIを組み合わせ

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

神経科学

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS：脆弱X症候群と自閉症の可塑性のMechasnismを探検するシータバーストプロトコルを使用する

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

高速スキャンサイクリックボルタンメトリーによる線条体の脳スライスにおけるシナプス前ドーパミンのダイナミクス

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

Voltage Biasing, Cyclic Voltammetry, &amp; Electrical Impedance Spectroscopy for Neural Interfaces

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive procedures, and can be used to monitor electrode performance over the long term. EIS measures electrical impedance at multiple frequencies, and increases in impedance indicate increased glial scar formation around the device, while cyclic voltammetry measures the charge carrying capacity of the electrode, and indicates how charge is transferred at different voltage levels. As implanted electrodes age, EIS and CV data change, and electrode sites that previously recorded spiking neurons often exhibit significantly lower efficacy for neural recording. The application of a brief voltage pulse to implanted electrode arrays, known as rejuvenation, can bring back spiking activity on otherwise silent electrode sites for a period of time. Rejuvenation alters EIS and CV, and can be monitored by these complementary methods. Typically, EIS is measured daily as an indication of the tissue response at the electrode site. If spikes are absent in a channel that previously had spikes, then CV is used to determine the charge carrying capacity of the electrode site, and rejuvenation can be applied to improve the interface efficacy. CV and EIS are then repeated to check the changes at the electrode-tissue interface, and neural recordings are collected. The overall goal of rejuvenation is to extend the functional lifetime of implanted arrays.

The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary.

ニューラルインタフェースの電圧バイアス、サイクリックボルタンメトリー、＆電気インピーダンス分光法

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

同時に電圧クランプとアンペロとドーパミン放出の単一細胞計測

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

黒質や腹側被蓋野のドーパミン規制の包括的なプロファイリング

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

レーザーキャプチャーマイクロダイセクション - 個々のドーパミンニューロンの単離と全体の腹側被蓋領域の実証

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

前根刺激のための斜め脊髄スライスの調製

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

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

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

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

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

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

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

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is implicated in diseases and disorders including attention deficit hyperactivity disorder, Parkinson's disease, and traumatic brain injury. Thus, DA neurotransmission and the methods to study it are of intense scientific interest. In vivo fast-scan cyclic voltammetry (FSCV) is a method that allows for selectively monitoring DA concentration changes with fine temporal and spatial resolution. This technique is commonly used in conjunction with electrical stimulations of ascending DAergic pathways to control the impulse flow of dopamine neurotransmission. Although the stimulated DA neurotransmission paradigm can produce robust DA responses with clear morphologies, making them amenable for kinetic analysis, there is still much debate on how to interpret the responses in terms of their DA release and clearance components. To address this concern, a quantitative neurobiological (QN) framework of stimulated DA neurotransmission was recently developed to realistically model the dynamics of DA release and reuptake over the course of a stimulated DA response. The foundations of this model are based on experimental data from stimulated DA neurotransmission and on principles of neurotransmission adopted from various lines of research. The QN model implements 12 parameters related to stimulated DA release and reuptake dynamics to model DA responses. This work describes how to simulate DA responses using QNsim1.0 and also details principles that have been implemented to systematically discern alterations in the stimulated dopamine release and reuptake dynamics.

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

QNsim1.0を用いた電気刺激されたドーパミン神経伝達データからの高速スキャンサイクリックボルタンメトリーデータのモデリング

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is ...