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

Arbetet stöddes av Elizabethtown College (R.J.W, M.L., och L.M.), av en NSF Graduate Fellowship (R.J.W.) och av Yale School of Medicine (N.A.).

Alla experiment utfördes enligt National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals och godkändes av både Elizabethtown College och Yale University Institutional Animal Care and Use Committee (IACUC). Detta protokoll är specifikt för den sövda råtta beredningen av att använda CIS-FSCV.
1. Presurgical preparat
<ol><li>Beredning av elektrodlösning<ol>
<li>För att göra elektrodfyllningslösningen, förbered en lösning av 4 M kaliumacetat med 140 mM k...

<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 ger en unik möjlighet att undersöka VTA-receptormekanismer som ligger till grund för fasisk DA-frisättning. Det finns två kritiska steg för att säkerställa en korrekt inspelning. För det första måste en stabil baslinjeinspelning uppnås, med liten drift i den framkallade DA-signalen. Ett viktigt sätt att öka sannolikheten för att etablera en stabil inspelning är att se till att elektroden har haft gott om tid att cykla vid både 60 Hz och 10 Hz (vanligtvis 15 min vid 60 Hz och 10 min vid 10 Hz). N...

Phasic dopamin (DA) frigörare från ventrala tegmental området (VTA) till kärnan accumbens (NAc) spelar en viktig roll i belöningsrelaterade beteenden. VTA DA-nervceller växlar från en tonic-liknande avfyrning (3-8 Hz) till en burst-liknande bränning (>14 Hz)1, som producerar fasisk DA-utgåva i NAc. VTA uttrycker en mängd somatodendritiska receptorer som är väl positionerade för att kontrollera övergången från tonic till burst-firing2,3,4,5. Att identifiera vilka av dessa receptorer, och deras respektive ingångar, kontrollfasisk DA-frisättning kommer att fördjupa vår förståelse för hur de belöningsrelaterade kretsarna är organiserade. Syftet med den metod som beskrivs här, kombinerad infusion och stimulering med snabbskanning cyklisk voltammetri (CIS-FSCV), är att snabbt och robust bedöma funktionaliteten hos VTA-receptorer vid körning av fasisk DA-frisättning.
Termen kombinerad infusion och stimulering (CIS) avser farmakologiskt manipulerande receptorer på en grupp neuroner (här VTA) och stimulera dessa nervceller att studera receptorns funktion. I den sövda råttan stimulerar vi elektriskt VTA att framkalla en stor fasisk DA-signal (1-2 μM) i NAc-kärnan, mätt med snabbskanning cyklisk voltammetri (FSCV). Infusioner av farmakologiska läkemedel (dvs. receptoragonister/antagonister) vid stimuleringsstället kan användas för att mäta funktionen hos VTA-receptorer genom att observera den efterföljande förändringen i framkallad fasisk DA-frisättning. FSCV är ett elektrokemiskt tillvägagångssätt som har både hög rumslig (50-100 μm) och temporal (10 Hz) upplösning, och är väl lämpad för att mäta belöningsrelaterade, fasa DA-händelser6,7. Denna upplösning är finare än andra in vivo neurokemiska mätningar, såsom mikrodialys. Således, tillsammans, CIS-FSCV är väl lämpad att bedöma VTA receptor reglering av fasa dopamin release.
Ett vanligt sätt att undersöka VTA-receptorfunktionen är genom att använda en kombination av elektrofysiologiska metoder som adresserar hur dessa receptorer ändrar avfyrningshastigheten för nervceller1,8. Dessa studier är mycket värdefulla för att förstå vilka receptorer som är involverade i att driva DA-avfyrning vid aktivering. Dessa studier kan dock bara föreslå vad som kan hända nedströms vid axonterminalen (dvs. frisättning av en neurotransmittor). CIS-FSCV bygger på dessa elektrofysiologiska studier genom att svara på hur effekten av VTA burst-firing, fasisk DA-frisättning, regleras av receptorer som finns på VTA-dendriter och cellkroppar. Cis-FSCV är således väl lämpad att bygga vidare på dessa elektrofysiologiska studier. Som ett exempel kan nikotinisk receptoraktivering inducera burst-firing i VTA9, och CIS-FSCV i den sövda råttan användes för att visa att nikotin acetylkolinreceptor (nAChR) aktivering i VTA också kontrollerar fasisk DA-frisättning i NAc10,11.
Mekanistisk undersökning av phasic DA reglering studeras också ofta med hjälp av skiva preparat tillsammans med bad tillämpning av droger. Dessa studier fokuserar ofta på den presynaptiska regleringen av phasic DA-frisättning från dopaminterminaler, eftersom cellkropparna ofta avlägsnas från skiva12. Dessa preparat är värdefulla för att studera presynaptiska receptoreffekter på dopaminterminaler, medan CIS-FSCV är bättre lämpad att studera somatodendritiska receptoreffekter på dopaminneuroner, liksom presynaptiska ingångar till VTA. Denna skillnad är viktig, eftersom somatodendritisk receptor aktivering i VTA kan ha en annan effekt än NAc presynaptisk receptor aktivering. Faktum är att blockering av dopaminerg presynaptiska nAChRs i NAc kan höja fasasisk dopaminfrisättning under burst-firing13, medan motsatsen gäller vid VTA somatodendritc nAChRs10,11.
CIS-FSCV är ett idealiskt tillvägagångssätt för att studera VTA-receptorernas förmåga att reglera fasisk DA-frisättning. Viktigt är att detta tillvägagångssätt kan utföras i en intakt råtta, antingen sövd eller fri rörelse. Detta tillvägagångssätt är lämpligt för akuta studier, för att studera receptorfunktionen i dess utgångstillstånd10,14 samt långsiktiga studier som kan bedöma funktionella förändringar i en receptor efter läkemedelsexponering eller beteendemanipulation11,15.

<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

Phasic dopamin (DA) frigörare från ventrala tegmental området (VTA) till kärnan accumbens spelar en avgörande roll i belöning bearbetning och förstärkning lärande. Att förstå hur de olika neuronala ingångarna i VTA-kontrollens fasa DA-utgåva kan ge en bättre bild av kretsarna som styr belöningsbehandling och förstärkningsinlärning. Här beskriver vi en metod som kombinerar intra-VTA cannula infusioner av farmakologiska agonister och antagonister med stimulering-framkallad fasa DA release (kombinerad infusion och stimulering, eller CIS) mätt med in vivo snabb-scan cyklisk voltammetry (FSCV). Med hjälp av CIS-FSCV hos sövda råttor kan ett fasiskt DA-svar framkallas genom att elektriskt stimulera VTA med en bipolär elektrod utrustad med en kanyl medan du registrerar i kärnan accumbens kärna. Farmakologiska agonister eller antagonister kan infunderas direkt på stimuleringsstället för att undersöka specifika VTA-receptorers roller för att driva fasisk DA-frisättning. En stor fördel med CIS-FSCV är att VTA-receptorfunktionen kan studeras in vivo, med utgångspunkt i in vitro-studier.

CIS-FSCV användes för att studera funktionen hos VTA N-metyl-D-aspartatreceptorer (NMDAR), nikotinacetylkolinreceptorer (nAChRs) och muscariniska acetylkolinreceptorer (mAChRs) för att driva fasisk DA-frisättning i NAc-kärnan. Figur 2 visar representativa data för en negativ kontroll, infusion av 0, 9% saltlösning, före (baslinje) och 9 min postinfusion (saltlösning). Figur 2 visar en färgplotter med potential på y-axe...

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

Målet med detta protokoll är att direkt manipulera ventrala tegmentala områdesreceptorer för att studera deras bidrag till subsekund dopamin frisättning.

Kombinerad infusion och stimulering med snabbskanning cyklisk voltammetri (CIS-FSCV) för att bedöma ventral tegmental område receptorreglering av phasic dopamin

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

Kombinera transkraniell magnetisk stimulering och fMRI för att undersöka nätverket standardläge

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 ...

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

TMS: Använda Theta-Burst protokollet Utforska Mechasnism av plasticitet hos personer med Fragile X-syndrom och autism

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

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

Presynaptisk dopamin Dynamics i striatum hjärnan skivor med Fast-scan Cykliska Voltammetry

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

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

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

Spänning polarisering cyklisk voltammetri, &amp; Electrical Impedance spektroskopi för neurala gränssnitt

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

Encelliga Mätning av dopaminfrisättning med samtidig Spänning-klämma och amperometri

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

Omfattande Profilering av dopamin förordning i substantia nigra och ventrala tegmentumområdet

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

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

Laser Capture Microdissection - En demonstration av Isolering av Individuell dopaminneuroner och hela Ventrala tegmentområdet

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

Beredning av Oblique ryggmärgs skivor för ventrala Root Stimulering

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

Använda enzymbaserade biosensorer för att mäta tonic och phasic glutamat i Alzheimers musmodeller

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

Modellering Snabbsök cyklisk voltammetrydata från elektriskt stimulerade data om dopaminöverföring via QNsim1.0

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