Name: combined infusion and stimulation with fast-scan cyclic voltammetry (cis-fscv) to assess ventral tegmental area receptor regulation of phasic dopamine
Uploaded: 2020-04-23T14:00:00
Description: 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

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<...

<ol>
	<li>Grace, A. A., Bunney, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+firing+pattern+in+nigral+dopamine+neurons:+burst+firing.">The control of firing pattern in nigral dopamine neurons: burst firing.</a> Journal of Neuroscience. 4 (11), 2877-2890 (1984).</li><li>Lester, D. 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=Midbrain+acetylcholine+and+glutamate+receptors+modulate+accumbal+dopamine+release.">Midbrain acetylcholine and glutamate receptors modulate accumbal dopamine release.</a> Neuroreport. 19 (9), 991-995 (2008).</li><li>Lodge, D. J., Grace, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laterodorsal+tegmentum+is+essential+for+burst+firing+of+ventral+tegmental+area+dopamine+neurons.">The laterodorsal tegmentum is essential for burst firing of ventral tegmental area dopamine neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (13), 5167-5172 (2006).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mu+Opioid+Receptor+Modulation+of+Dopamine+Neurons+in+the+Periaqueductal+Gray/Dorsal+Raphe:+A+Role+in+Regulation+of+Pain.">Mu Opioid Receptor Modulation of Dopamine Neurons in the Periaqueductal Gray/Dorsal Raphe: A Role in Regulation of Pain.</a> Neuropsychopharmacology. 41 (8), 2122-2132 (2016).</li><li>Zhang, H. Y., 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=Expression+of+functional+cannabinoid+CB2+receptor+in+VTA+dopamine+neurons+in+rats.">Expression of functional cannabinoid CB2 receptor in VTA dopamine neurons in rats.</a> Addiction Biology. 22 (3), 752-765 (2017).</li><li>Wickham, R. 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=Advances+in+studying+phasic+dopamine+signaling+in+brain+reward+mechanisms.">Advances in studying phasic dopamine signaling in brain reward mechanisms.</a> Frontiers in Bioscience. 5, 982-999 (2013).</li><li>Wightman, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+transmitter+metabolites+by+voltammetry+in+cerebrospinal+fluid+following+neural+pathway+stimulation.">Monitoring of transmitter metabolites by voltammetry in cerebrospinal fluid following neural pathway stimulation.</a> Nature. 262 (5564), 145-146 (1976).</li><li>Grace, A. A., Bunney, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+firing+pattern+in+nigral+dopamine+neurons:+single+spike+firing.">The control of firing pattern in nigral dopamine neurons: single spike firing.</a> Journal of Neuroscience. 4 (11), 2866-2876 (1984).</li><li>Mameli-Engvall, 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=Hierarchical+control+of+dopamine+neuron-firing+patterns+by+nicotinic+receptors.">Hierarchical control of dopamine neuron-firing patterns by nicotinic receptors.</a> Neuron. 50 (6), 911-921 (2006).</li><li>Wickham, 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=Ventral+tegmental+area+alpha6beta2+nicotinic+acetylcholine+receptors+modulate+phasic+dopamine+release+in+the+nucleus+accumbens+core.">Ventral tegmental area alpha6beta2 nicotinic acetylcholine receptors modulate phasic dopamine release in the nucleus accumbens core.</a> Psychopharmacology. 229 (1), 73-82 (2013).</li><li>Solecki, W., 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=Differential+role+of+ventral+tegmental+area+acetylcholine+and+N-methyl-D-aspartate+receptors+in+cocaine-seeking.">Differential role of ventral tegmental area acetylcholine and N-methyl-D-aspartate receptors in cocaine-seeking.</a> Neuropharmacology. 75, 9-18 (2013).</li><li>John, C. E., Jones, S. R., Michael, A. C., Borland, L. 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+Dopamine+and+Serotonin+in+Mouse+Brain+Slices.">Fast Scan Cyclic Voltammetry of Dopamine and Serotonin in Mouse Brain Slices.</a> Electrochemical Methods for Neuroscience. , (2007).</li><li>Rice, M. E., Cragg, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+amplifies+reward-related+dopamine+signals+in+striatum.">Nicotine amplifies reward-related dopamine signals in striatum.</a> Nature Neuroscience. 7 (6), 583-584 (2004).</li><li>Espana, R. 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=Hypocretin+1/orexin+A+in+the+ventral+tegmental+area+enhances+dopamine+responses+to+cocaine+and+promotes+cocaine+self-administration.">Hypocretin 1/orexin A in the ventral tegmental area enhances dopamine responses to cocaine and promotes cocaine self-administration.</a> Psychopharmacology. 214 (2), 415-426 (2011).</li><li>Addy, N. 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>

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 ...

This approach can help elucidate the role of specific ventral tegmental area, somatodendritic receptors and phasic dopamine release in the nucleus accumbens. The main advantage of this approach is that questions about receptor function control over phasic dopamine release can be addressed in the intact brain. Optimizing the carbon fiber microelectrode and bipolar stimulator location can be challenging, and some advice for beginners is starting recording in the dorsal striatum.Due to the use and manipulation of multiple types of electrodes, including recording, reference, and stimulating electrodes, a visual guide of when and where these electrodes are implanted will be useful. After confirming a lack of response to pedal reflex, clean the scalp of an anesthetized rat with three sequential iodopovidone and 70%ethanol scrubs. After the last scrub, use sterilized needle nose tweezers and surgical scissors to cut away enough scalp tissue to make room for the implanted electrodes.Gently clean the exposed skull surface with sterilized cotton tip applicators before applying two to three drops of 3%hydrogen peroxide to help identify lambda and bregma. Next, use a one millimeter drill bit set to approximately 20, 000 revolutions per minute to make a 1.5 millimeter diameter hole, 2.5 millimeters anterior and 3.5 millimeters lateral to the bregma. Implant a 1.59 millimeter outer diameter, 3.2 millimeter long screw about halfway into the hole until the screw is firmly in place.Next, drill a one millimeter diameter reference electrode hole in the left hemisphere, 1.5 millimeters anterior, and 3.5 millimeters lateral to the bregma. Insert approximately two millimeters of reference wire into the hole while wrapping the wire around and under the head of the implanted screw. Then fully implant the screw, pinning the reference electrode in place.In the right hemisphere, drill a 1.5 millimeter diameter hole, 1.2 millimeters anterior, and 1.4 millimeters lateral to the bregma, and use tweezers to carefully remove the dura. For the stimulating electrode, drill a square hole two millimeters anterior-posterior and five millimeters medial-lateral centered at 5.2 millimeters posterior and one millimeter lateral to the bregma. Using stereotactic arm bars, lower the bipolar stimulating electrode guide cannula five millimeters below the dura.Using the stereotactic arm bars, lower the carbon fiber microelectrode four millimeters below the dura at the most dorsal portion of the striatum and connect the reference wire and carbon fiber to a potentiostat. Then apply a minus 0.4 to 1.4 volt, 400 volt per second triangular waveform for 15 minutes at 60 Hertz, followed by a 10-minute stimulus at 10 Hertz. To optimize the stimulating electrode location, set the stimulator to produce a bipolar electrical waveform with a 60 Hertz frequency, 24 pulses, 300 microamp current, and two millisecond per phase pulse width.Gently lower the stimulator in 0.2 millimeter increments from 5 to 7.8 millimeters below the dura, stimulating the VTA at each increment. Continue to lower the bipolar-stimulating electrode guide cannula until a stimulation produces phasic dopamine release at the carbon fiber microelectrode within the dorsal striatum. Once a dopamine release has been observed, lower the carbon fiber microelectrode until it is at least six millimeters below the dura into the most dorsal part of the nucleus accumbens core.When the electrode is in position, stimulate the VTA and record the peak amplitude of the dopamine peak, then lower or raise the carbon fiber microelectrode to the site that produces the greatest dopamine release, making sure that the peak of the dopamine response is represented as a clear oxidation peak at 0.6 volts and a reduction peak at minus 0.2 volts. Once the carbon fiber and stimulating electrode guide cannula location has been optimized, stimulate the animal every three minutes for 20 to 30 minutes. After achieving a stable baseline, gently lower the internal cannula by hand into the guide cannula pre-fitted into the bipolar stimulator and obtain an additional two to three baseline recordings to confirm that the cannula insertion did not cause a change in the evoked signal.Once a baseline response has been established, use a syringe pump and a microsyringe to infuse 0.5 microliters of the drug solution of interest into the VTA over a two-minute period. After the infusion, leave the internal cannula in place for at least one minute prior to removal, continuing to record the response every three minutes to measure the post-infusion effects. As demonstrated in these representative color plots, saline infusion does not alter the stimulated phasic dopamine release.The infusion of NMDA produces a robust increase in stimulated phasic dopamine release, while the NMDA receptor competitive antagonist, AP5, produces a robust decrease. Both the non-competitive nicotinic acetylcholine receptor antagonist mecamylamine and the non-selective competitive muscarinic acetylcholine receptor antagonist scopolamine produce a robust decrease in the stimulated phasic dopamine release. In this graph, a summary of the effects of the tested drugs over time can be observed.The infusion cannula must be carefully inserted into the bipolar stimulating electrode. Otherwise, excessive damage can occur, leading to artifactual changes in phasic dopamine release. This approach has allowed investigators to more finely address the specific receptor mechanisms by which phasic dopamine release is regulated in vivo.

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

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode 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

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

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

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

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

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

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

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

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

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

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 ...