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

O trabalho foi apoiado pela Elizabethtown College (R.J.W, M.L., e L.M.), por uma Bolsa de Pós-Graduação da NSF (R.J.W.) e pela Yale School of Medicine (N.A.).

Todos os experimentos foram realizados de acordo com o Guia nacional de Cuidados e Uso de Animais de Laboratório (NIH) e aprovados pelo Elizabethtown College e pelo Comitê Institucional de Cuidados e Uso de Animais da Universidade de Yale (IACUC). Este protocolo é específico para a preparação anestésima de ratos de utilização do CIS-FSCV.
1. Preparações pré-úrgicas
<ol><li>Preparação da solução de eletrodos<ol>
<li>Para fazer a solução de enchimento de eletrodo, prepare um...

<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. 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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. 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O CIS-FSCV oferece uma oportunidade única para investigar os mecanismos receptores VTA subjacentes à liberação da DA fásica. Há duas etapas críticas para garantir uma gravação adequada. Em primeiro lugar, uma gravação de linha de base estável deve ser alcançada, com pouca deriva no sinal de DA evocado. Uma maneira importante de aumentar a probabilidade de estabelecer um registro estável é garantir que o eletrodo tenha tido tempo de sobra para pedalar tanto a 60 Hz quanto a 10 Hz (tipicamente 15 min a 60 Hz...

A liberação de dopamina phasic (DA) da área tegmental ventral (VTA) para o núcleo accumbens (NAc) desempenha um papel vital em comportamentos relacionados à recompensa. Os neurônios VTA DA mudam de um disparo tônico (3-8 Hz) para um disparo em forma de estouro (>14 Hz)1, que produz a liberação da DA phasic no NAc. O VTA expressa uma variedade de receptores somatodendritic que estão bem posicionados para controlar o interruptor de tônica para estouro2,3,4,5. Identificar quais desses receptores, e seus respectivos insumos, controlarão a liberação da DA phasic aprofundará nossa compreensão de como os circuitos relacionados à recompensa são organizados. O objetivo da metodologia aqui descrita, infusão combinada e estimulação com voltametria cíclica de varredura rápida (CIS-FSCV), é avaliar de forma rápida e robusta a funcionalidade dos receptores VTA na liberação da DA fásica.
O termo infusão e estimulação combinada (CEI) refere-se a receptores farmacologicamente manipuladores em um grupo de neurônios (aqui o VTA) e estimular esses neurônios a estudar a função do receptor. No rato anestesiado, estimulamos eletricamente o VTA para evocar um grande sinal da fásico (1-2 μM) no núcleo NAc, medido pela voltammetry cíclica de varredura rápida (FSCV). Infusões de fármacos farmacológicos (ou seja, agonistas receptores/antagonistas) no local de estimulação podem ser usadas para medir a função dos receptores VTA observando a mudança subsequente na liberação da FAsica evocada. FSCV é uma abordagem eletroquímica que desfruta tanto de alta resolução espacial (50-100 μm) quanto temporal (10 Hz), e é adequada para medir eventos da DA relacionados à recompensa6,7. Esta resolução é mais fina do que outras medidas neuroquímicas in vivo, como a microdiálise. Assim, em conjunto, o CIS-FSCV é adequado para avaliar a regulação do receptor VTA da liberação de dopamina fásica.
Uma maneira comum de investigar a função do receptor de VTA é usando uma combinação de abordagens eletrofisiológicas que abordam como esses receptores alteram a taxa de disparo dos neurônios1,8. Esses estudos são altamente valiosos para entender quais receptores estão envolvidos na condução de disparos da promotoria após a ativação. No entanto, esses estudos só podem sugerir o que pode acontecer a jusante no terminal de axônio (ou seja, liberação de um neurotransmissor). O CIS-FSCV baseia-se nesses estudos eletrofisiológicos, respondendo como a saída de disparo de VTA, liberação de DA fásica, é regulada por receptores localizados em dendritos de VTA e corpos celulares. Assim, o CIS-FSCV é adequado para se basear nesses estudos de eletrofisiologia. Como exemplo, a ativação do receptor nicotínico pode induzir o disparo de explosão no VTA9, e o CIS-FSCV no rato anestesiado foi usado para mostrar que a ativação do receptor de acetilcolina nicotínica (nAChR) no VTA também controla a liberação da Fásica no NAc10,11.
O exame mecanicista da regulação da da afásica também é comumente estudado usando preparações de fatias juntamente com a aplicação de banho de drogas. Esses estudos muitas vezes se concentram na regulação pré-sináptica da liberação da DA afásica dos terminais de dopamina, uma vez que os corpos celulares são frequentemente removidos da fatia12. Essas preparações são valiosas para estudar efeitos de receptores pré-sinápticos nos terminais de dopamina, enquanto o CIS-FSCV é mais adequado para estudar efeitos do receptor somatodendritico em neurônios de dopamina, bem como entradas pré-sinápticas no VTA. Essa distinção é importante, pois a ativação do receptor somatodendritic no VTA pode ter um efeito diferente da ativação do receptor pré-sináptico NAc. De fato, bloquear nAChRs pré-sinápticas dopaminérgicos no NAc pode elevar a liberação de dopamina afásica durante o estouro13, enquanto o oposto é verdadeiro no VTA somatodendritc nAChRs10,11.
CIS-FSCV é uma abordagem ideal para estudar a capacidade dos receptores VTA de regular a liberação da DA fásica. É importante ressaltar que essa abordagem pode ser realizada em um rato intacto, seja anestesiado ou em movimento livre. Esta abordagem é adequada para estudos agudos, para estudar a função receptora em seu estado de base10,14, bem como estudos de longo prazo que possam avaliar alterações funcionais em um receptor após exposição a medicamentos ou manipulação comportamental11,15.

<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

A liberação de dopamina phasic (DA) da área tegmental ventral (VTA) para o núcleo accumbens desempenha um papel fundamental no processamento de recompensas e aprendizado de reforço. Entender como as diversas entradas neuronais na liberação da DA de controle VTA podem fornecer uma melhor imagem dos circuitos que controlam o processamento de recompensas e o aprendizado reforçado. Aqui, descrevemos um método que combina infusões de cânulas intra-VTA de agonistas farmacológicos e antagonistas com a liberação de DA fásica evocada por estimulação (infusão e estimulação combinada, ou CIS) medida pela voltametria cíclica in vivo de varredura rápida (FSCV). Usando CIS-FSCV em ratos anestesiados, uma resposta da DA fásica pode ser evocada estimulando eletricamente o VTA com um eletrodo bipolar equipado com uma cânula durante a gravação no núcleo do núcleo accumbens. Agonistas ou antagonistas farmacológicos podem ser infundidos diretamente no local de estimulação para investigar os papéis específicos dos receptores de VTA na liberação da DA fásica. Um grande benefício do CIS-FSCV é que a função receptorA VTA pode ser estudada in vivo, com base em estudos in vitro.

CIS-FSCV foi utilizado para estudar a função de receptores de pós-metila-d-aspartato (NMDAR), receptores de acetilcolina nicotínica (nAChRs) e receptores de acetilcolina muscarínica (mAChRs) na liberação da DA fásica no núcleo NAc. A Figura 2 mostra dados representativos para um controle negativo, infusão de 0,9% salina, antes (linha de base) e 9 min de pós-infusão (soro fisiológico). A Figura 2 mostra um enredo de c...

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

O objetivo deste protocolo é manipular diretamente os receptores da área tegmental ventral para estudar sua contribuição para a liberação de dopamina subsegundo.

Infusão e Estimulação Combinada com Voltammetry Cíclica de Varredura Rápida (CIS-FSCV) para avaliar a regulação do receptor da área tegmental ventral da dopamina fástica

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

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Neurociência

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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

Profiling abrangente do Regulamento dopamina em substância negra e área tegmental ventral

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

Captura Laser Microdissection - uma demonstração do Isolamento de Pessoa dopamina Neurônios e região ventral tegmental Entire

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

A Preparação de Oblique Spinal Cord Fatias de Estimulação raiz ventral

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

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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

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