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

Il lavoro è stato sostenuto dall'Elizabethtown College (R.J.W, M.L. e L.M.), da una NSF Graduate Fellowship (R.J.W.) e dalla Yale School of Medicine (N.A.).

Tutti gli esperimenti sono stati condotti secondo la Guida del National Institutes of Health (NIH) per la cura e l'uso degli animali da laboratorio e sono stati approvati sia dall'Elizabethtown College che dal Yale University Institutional Animal Care and Use Committee (IACUC). Questo protocollo è specifico per la preparazione di ratti anestetizzati dell'utilizzo di CIS-FSCV.
1. Preparazioni prechirurgiche
<ol><li>Preparazione della soluzione di elettrodi<ol>
<li>Per realizzare la soluzione...

<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. 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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. 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CIS-FSCV offre un'opportunità unica per studiare i meccanismi del recettore VTA alla base del rilascio di DA fasico. Ci sono due passaggi critici per garantire una corretta registrazione. In primo luogo, è necessario ottenere una registrazione di base stabile, con poca deriva nel segnale DA evocato. Un modo importante per aumentare la probabilità di stabilire una registrazione stabile è assicurarsi che l'elettrodo abbia avuto tutto il tempo di pedalare sia a 60 Hz che a 10 Hz (in genere 15 minuti a 60 Hz e 10 minuti ...

Il rilascio di dopamina fasica (DA) dall'area tegmentale ventrale (VTA) al nucleo accumbens (NAc) svolge un ruolo vitale nei comportamenti legati alla ricompensa. I neuroni VTA DA passano da un fuoco tonico (3-8 Hz) a un fuoco a raffica (>14 Hz)1, che produce il rilascio di DA fasico nel NAc. Il VTA esprime una varietà di recettori somatodendritici che sono ben posizionati per controllare il passaggio da tonico a scoppio2,3,4,5. Identificare quali di questi recettori e i loro rispettivi input controllano il rilascio di DA fasico approfondirà la nostra comprensione di come è organizzato il circuito correlato alla ricompensa. Lo scopo della metodologia qui descritta, combinata di infusione e stimolazione con voltammetria ciclica a scansione rapida (CIS-FSCV), è quello di valutare rapidamente e in modo robusto la funzionalità dei recettori VTA nel guidare il rilascio di DA fasico.
Il termine infusione e stimolazione combinata (CIS) si riferisce alla manipolazione farmacologica dei recettori su un gruppo di neuroni (qui il VTA) e alla stimolazione di quei neuroni a studiare la funzione del recettore. Nel ratto anestetizzato, stimoliamo elettricamente il VTA per evocare un grande segnale DA fasico (1-2 μM) nel nucleo NAc, misurato mediante voltammetria ciclica a scansione rapida (FSCV). Infusioni di farmaci farmacologici (cioè agonisti/antagonisti del recettore) nel sito di stimolazione possono essere utilizzate per misurare la funzione dei recettori VTA osservando il successivo cambiamento nel rilascio di DA fasico evocato. FSCV è un approccio elettrochimico che gode di un'elevata risoluzione spaziale (50-100 μm) e temporale (10 Hz) ed è adatto per misurare eventi DA fasici correlati alla ricompensa6,7. Questa risoluzione è più fine di altre misurazioni neurochimiche in vivo, come la microdialisi. Pertanto, insieme, CIS-FSCV è adatto per valutare la regolazione del recettore VTA del rilascio di dopamina fasica.
Un modo comune per studiare la funzione del recettore VTA è utilizzando una combinazione di approcci elettrofisiologici che affrontano il modo in cui tali recettori alterano la velocità di attivazione dei neuroni1,8. Questi studi sono molto preziosi per capire quali recettori sono coinvolti nel guidare l'attivazione di DA al momento dell'attivazione. Tuttavia, questi studi possono solo suggerire cosa potrebbe accadere a valle del terminale assonale (cioè il rilascio di un neurotrasmettitore). CIS-FSCV si basa su questi studi elettrofisiologici rispondendo a come l'output del rilascio di DA fasico del VTA è regolato dai recettori situati sui dendriti VTA e sui corpi cellulari. Pertanto, CIS-FSCV è adatto per costruire su questi studi di elettrofisiologia. Ad esempio, l'attivazione del recettore nicotinico può indurre scoppi nel VTA9e CIS-FSCV nel ratto anestetizzato è stato usato per dimostrare che l'attivazione del recettore nicotinico dell'acetilcolina (nAChR) nel VTA controlla anche il rilascio di DA fasico nel NAc10,11.
L'esame meccanicistico della regolazione da DA fasica è anche comunemente studiato utilizzando preparazioni a fette insieme all'applicazione del bagno di farmaci. Questi studi si concentrano spesso sulla regolazione presinaptica del rilascio di DA fasico dai terminali della dopamina, poiché i corpi cellulari vengono spesso rimossi dalla fetta12. Questi preparati sono preziosi per studiare gli effetti dei recettori presinaptici sui terminali della dopamina, mentre CIS-FSCV è più adatto per studiare gli effetti del recettore somatodendritico sui neuroni della dopamina, così come gli input presinaptici al VTA. Questa distinzione è importante, perché l'attivazione del recettore somatodendritico nel VTA può avere un effetto diverso rispetto all'attivazione del recettore presinaptico NAc. In effetti, il blocco dei nAChR presinaptici dopaminergici nel NAc può elevare il rilascio di dopamina fasica durante la raffica13, mentre il contrario è vero a VTA somatodendritc nAChRs10,11.
CIS-FSCV è un approccio ideale per studiare la capacità dei recettori VTA di regolare il rilascio di DA fasico. È importante sottolineare che questo approccio può essere eseguito in un ratto intatto, anestetizzato o in movimento libero. Questo approccio è adatto per studi acuti, per studiare la funzione del recettore nel suo stato basale10,14 e studi a lungo termine in grado di valutare i cambiamenti funzionali in un recettore dopo l'esposizione al farmaco o la manipolazione comportamentale11,15.

<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

Il rilascio di dopamina fasica (DA) dall'area tegmentale ventrale (VTA) al nucleo accumbens svolge un ruolo fondamentale nell'elaborazione della ricompensa e nell'apprendimento per rinforzo. Comprendere come i diversi input neuronali nel rilascio di DA fasico di controllo VTA può fornire un quadro migliore dei circuiti che controllano l'elaborazione della ricompensa e l'apprendimento per rinforzo. Qui, descriviamo un metodo che combina infusioni di cannule intra-VTA di agonisti e antagonisti farmacologici con rilascio di DA fasico evocato dalla stimolazione (infusione e stimolazione combinate, o CIS) misurato mediante voltammetria ciclica a scansione rapida in vivo (FSCV). Utilizzando CIS-FSCV in ratti anestetizzati, una risposta DA fasica può essere evocata stimolando elettricamente il VTA con un elettrodo bipolare dotato di una cannula durante la registrazione nel nucleo accumbens. Gli agonisti o gli antagonisti farmacologici possono essere infusi direttamente nel sito di stimolazione per studiare i ruoli specifici dei recettori VTA nel guidare il rilascio di DA fasico. Uno dei principali vantaggi di CIS-FSCV è che la funzione del recettore VTA può essere studiata in vivo, basandosi su studi in vitro.

CIS-FSCV è stato utilizzato per studiare la funzione dei recettori VTA N-metil-D-aspartato (NMDAR), dei recettori nicotinici dell'acetilcolina (nAChR) e dei recettori muscarinici dell'acetilcolina (mAChRs) nel guidare il rilascio di DA fasico nel nucleo NAc. La Figura 2 mostra i dati rappresentativi per un controllo negativo, infusione di soluzione salina allo 0,9%, prima (basale) e postinfusione di 9 minuti (soluzione salina). La Figura...

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

L'obiettivo di questo protocollo è quello di manipolare direttamente i recettori dell'area tegmentale ventrale per studiare il loro contributo al rilascio di dopamina al di sottosecondo.

Infusione e stimolazione combinate con voltammetria ciclica a scansione rapida (CIS-FSCV) per valutare la regolazione del recettore dell'area tegmentale ventrale della dopamina fasica

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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