Research reported in this publication was supported by an NSF Graduate Research Fellowship (RJW) and by the National Institute on Drug Abuse of the National Institutes of Health and FDA Center for Tobacco Products (CTP) under Award Number P50DA036151(EJN and NAA). The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

All experiments were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Yale University Institutional Animal Care and Use Committee (IACUC).
1) Pre-surgical Preparations
<ol>
	<li>Oral solution preparation
	<ol>
		<li>Create solutions of 10% sucrose and 0.005% L-menthol in deionized water (pH 7.4). Store these solutions in closed containers, at RT, and remake every 2 weeks, to prevent degradation.</li>
	</ol>
	</li>
	<li>Electrode calibration solutions
	<ol>
		<li>Make Tris buffer (pH 7.4) at any time prior to calibration. The solution consists of 12.0 mM Tris-HCl, 140 mM NaCl, 3.25 mM KCl, 1.20 mM CaCl2, 1.25 mM NaH2PO4, 1.20 mM MgCl2, and 2.00 mM Na2SO4.</li>
		<li>Create a stock solution of 10 mM dopamine (DA; dopamine hydrochloride) in 0.1 M perchloric acid. The perchloric acid helps prevent oxidation of dopamine. Store this stock solution at -20 &#176;C and use for up to 6 months. Make several aliquots of the DA stock solution for storage to use each aliquot only once.</li>
		<li>From the 10 mM DA stock, dilute solutions in Tris buffer to concentrations of 1 &#956;M, 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM.</li>
		<li>For pH calibration, prepare solutions of Tris buffer with the pH of 7.2, 7.3, 7.5, and 7.6. This will provide solutions that mimic pH shifts that may occur during the in vivo experiment.</li>
	</ol>
	</li>
	<li>Electrode fabrication
	<ol>
		<li>By vacuum suction, aspirate a T-650 carbon fiber (7 &#956;m diameter) into a borosilicate glass capillary (length 10 mm, outside diameter 0.6 mm, inside diameter 0.4 mm).</li>
		<li>Place glass capillary filled with carbon fiber into a vertical electrode puller. For initial parameters, set the heater to 55.0 and turn off the magnet. However, further optimization may be necessary as both heater and magnetic settings vary from lab to lab. 
		Note: The electrode should be at least 5.5 cm long (including taper) so that it can easily fit into the micromanipulator and be inserted at least 6.9 mm below dura in the rat brain. If the electrode is too short, recordings from ventral portion of the nucleus accumbens will not be achievable. On the other hand, the total electrode length should not exceed 6.5 cm otherwise it will be too long to fit into the micromanipulator.</li>
		<li>Using a light microscope, cut the exposed carbon-fiber so that it extends 75-100 &#956;m beyond the end of the glass. Verify that the electrode has a very tight, barely discernible, seal between the glass electrode and carbon fiber, which will produce minimal noise during subsequent recording. Discard the electrode if there are any noticeable cracks in the glass or if the seal is loose, which is indicative of a poor seal. For more detailed instructions see [12] and [13].</li>
	</ol>
	</li>
	<li>Assembling the electrode into the micromanipulator
	<ol>
		<li>Obtain a 3 in, 30G&#160;wire that is insulated along half the length of the wire and use a small brush to apply a thin layer of silver paint directly to the wire. Immediately after applying silver paint, insert the wire into the hole on the top of the electrode to provide a physical and electrical connection to the carbon fiber. Under a microscope, ensure that the silver wire makes contact with the carbon fiber.</li>
		<li>Secure the silver wire and electrode to the micromanipulator using heat shrink.</li>
		<li>Place the prepared carbon-fiber electrode into purified isopropyl alcohol (IPA) for at least 10 min to clean the electrode surface and to increase subsequent sensitivity to dopamine. After soaking in IPA, rinse the carbon fiber with tap water to remove the IPA.</li>
	</ol>
	</li>
	<li>Reference electrode fabrication
	<ol>
		<li>Take a 13 mm silver wire containing an approximately 6 mm Ag/Ag Cl mesh (7 mm silver wire alone, 6 mm mesh, 0.4 mm diameter), cut the silver portion down to approximately half its original length and solder the silver portion of the wire to a gold pin.</li>
		<li>Apply epoxy over the solder on the pin.</li>
		<li>Dip the Ag/Ag Cl mesh end into a copolymer of 5% polytetrafluroethylene and perfluoro-3,6-dioxa-4-methyl-7octene-sulfonic acid 5 times, with 30 min&#160;air drying periods between each dip.</li>
		<li>Allow the coated Ag/Ag Cl mesh to air dry O/N.</li>
		<li>Cure in oven at 120 &#186;C for 1 hr.</li>
	</ol>
	</li>
</ol>
&#160;2) Combined Intraoral Surgery and Intracranial Cannulation Surgery (Approximately 75 min)
<ol>
	<li>Oral catheter fabrication
	<ol>
		<li>Make the oral catheter, and sterilize by ethylene oxide sterilization, at least 10 days prior to surgery.</li>
		<li>Cut PE 100 tubing into 6 cm long segments.</li>
		<li>Using a soldering iron, melt one end of the PE tubing and immediately press against a solid surface to cool the PE tubing. The result should create a small, flat disc at one end of the PE tubing and should not be flanged. Use a needle (18 G) or push pin to create a hole in the center of this disc.</li>
		<li>On the other end of the PE tubing, snugly insert the blunt end of a 18 G needle into the tube.</li>
		<li>Using a standard paper hole puncher, punch several circular pieces of a polytetrafluroethylene sheet (3.18 mm thick, 6 mm in diameter) to create polytetrafluroethylene washers.</li>
		<li>Create a hole in the center of the washer. Take the needle attached to the PE tubing and push it completely through the washer. Once the needle is through, slide the washer to the bottom of the PE tubing so that it is flush against the washer.</li>
	</ol>
	</li>
	<li>Oral surgery
	<ol>
		<li>After sterilizing the surgical tools, cannula (trimmed to 2.5 mm below the pedestal before sterilization), and reference electrode, anesthetize the rat with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).
		<ol>
			<li>After 5 min, apply a noxious stimulus test (foot or tail pinch) to assess the level of anesthesia. If the subject shows any physical reaction to the noxious stimulus test (flinch response, increase in respiratory or heart rate), administer a ketamine boost of 10% to 15% of the original dose and repeat the noxious stimulus protocol above.</li>
		</ol>
		</li>
		<li>After the rat is fully anesthetized and shows no reaction to the noxious stimulus test, use animal hair clippers to shave the rat&#39;s fur from the eyes to about 2 mm behind the ears. Then place the rat on a thin water recirculating heating pad to maintain body temperature during surgery. Apply eye lubricant. Administer the non-steroidal anti-inflammatory (NSAID) drug, Carprofen (5 mg/kg), by intraperitoneal injection prior to performing any incisions and prior to inserting intraoral cannulas. Use aseptic technique at all times.</li>
		<li>Apply betadine followed by 70% ethanol to clean the skin. Repeat 3 times.</li>
		<li>Place the rat on its back, and open the mouth using a sterile cotton swab. Find the first upper molar.</li>
		<li>Insert the needle into the tissue between the cheek and the first upper molar until the needle exits just behind and medial to the ears.</li>
		<li>Pierce the needle through the skin until the PE tubing exits the skin and is accessible.</li>
		<li>Pull up on the cannula (gripping by the cannula itself and not the needle) and secure the polytetrafluroethylene washer into the molars so that the catheter remains in place. 
		Note: Cutting the washer into a trapezoid shape and securing it against the molars with the flat side facing the cheek makes this step easier.</li>
		<li>Take another polytetrafluroethylene washer and slide it over the exposed needle, down the plastic tubing, and abut against the incision.</li>
		<li>To secure the polytetrafluroethylene washer, use sterilized tape to create a triangular &#34;flag&#34; so that the washer does not slide up. Secure the washer against the rat&#39;s skin and the sterilized tape.</li>
		<li>Cut the exposed catheter to allow 2-4 cm of tubing to be exposed above the rat&#39;s head.</li>
		<li>Repeat steps 2.2.1 through 2.2.10 for the other side of the mouth.</li>
	</ol>
	</li>
	<li>Intracranial surgery for voltammetry
	<ol>
		<li>Place the rat in the stereotaxic frame and clean the animal&#8217;s scalp using a two stage scrub (a betadine scrub followed by a 70% ethanol scrub, perform with a 3 cycle repetition).</li>
		<li>Use sterilized needle nose tweezers and surgical scissors to cut away a 15 x 15 mm section of scalp tissue.</li>
		<li>Gently clean the surface of the skull using sterilized cotton tip applicators. Further clean the skull by applying 2 to 3 drops of 3% hydrogen peroxide.</li>
		<li>Use a stereotaxic drill to make 3 small (1 mm diameter) holes in the skull for subsequent insertion of screws, 1 hole for the guide cannula, 1 hole for the stimulating electrode, and 1 hole for the reference electrode.</li>
		<li>For recordings in the NAc core, position the guide cannula at 1.2 mm anterior and 1.4 mm lateral to Bregma. Lower the cannula apparatus until the plastic base is flush with the skull.</li>
		<li>Place the sterilized reference electrode in the hemisphere contralateral to the guide cannula. Lower the reference approximately 2 mm below the dura.</li>
		<li>Position the stimulating electrode at 5.2 mm posterior and 1.0 mm lateral to Bregma and lowered 8.0 mm below dura to target the ventral tegmental area (VTA).</li>
		<li>Use a sterilized small surgical screwdriver to secure screws to the skull. Then, insert the reference electrode, stimulating electrode, and guide cannula. Finally, secure all components using cranioplastic cement.</li>
		<li>For the first 48 hours of post-operative care, administer the non-steroidal anti-inflammatory drug (NSAID) Carprofen by subcutaneous injection (5 mg/kg). Allow at least 1 week for complete surgical recovery prior to performing voltammetry recording.
		<ol>
			<li>During post-operative care, flush oral catheters daily with water. Provide food saturated in water for 2 days to aid the animal in chewing and eating.&#160; Provide daily monitoring for potential signs of stress of pain (due to mild infection or dehydration) and provide appropriate interventions as necessary (including administration of fluids, topical administration of antiseptics, and or NSAID Carprofen administration at 5 mg/kg).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3) Voltammetric Recordings in Awake, Freely Moving Rat
<ol>
	<li>Lowering the electrode and acquiring a DA training set.
	<ol>
		<li>On the day of the experiment, check the patency of the oral cannula by infusing 200 &#956;l of water and observing orofacial responses (to verify that the rat is tasting the water).</li>
		<li>Attach the FSCV headstage to the rat by inserting the stimulating electrode (on the headstage) into the bipolar stimulating electrode cannula (on the rat). Thus, the headstage (miniaturized current-to-voltage converter) connects directly to the bipolar stimulating electrode.</li>
		<li>Remove the dummy cannula from the cannula apparatus and apply two drops of 50% heparin solution into the cannula. Then gently insert a dummy cannula (slightly longer that the dummy cannula that was previously removed) to clear any blood clots or glial scarring that may have occurred, which will break carbon fiber electrodes that will subsequently be inserted during the experiment. Extend the cannula used for clearing clots 3-4 mm below the skull (at least 2 mm above the recording site).</li>
		<li>Insert the micromanipulator, with electrode, into the guide cannula and place the omega ring in a &#34;locked&#34; position.</li>
		<li>Connect the reference electrode wire from the headstage to the appropriate pin. Then connect the working electrode wire from the micromanipulator to the appropriate wire on the headstage.</li>
		<li>Lower the electrode to approximately 4-4.5 mm below skull.</li>
		<li>Use a potentiostat to apply a triangular waveform (400 V/s, -0.4 to +1.3 V). Apply the waveform to the electrode at 60 Hz for at least 10 min, then change the waveform application frequency to 10 Hz for subsequent experimental recordings. 
		Note: The 60 Hz waveform application step produces a stable carbon fiber surface and will help prevent electrical drift</li>
		<li>Apply electrical stimulation to the VTA using various frequencies (10 to 60 Hz) pulses (10 to 30 pulses) and currents (50 to 150 &#956;A) all with a pulse width of 2 ms/phase, to evoke different concentrations of DA release and pH shifts. Acquire at least 5 different concentrations of DA release and 5 different pH shifts. Wait (2 - 3 min&#160;minimum) between stimulations to allow for vesicular reloading[14]. 
		Note: It is important to obtain stimulations that produce DA concentrations across the entire range that will be collected during the subsequent experiment since they will be used as a training set for Principal Component Analysis (see section 5.1).</li>
		<li>Lower the electrode into the NAc core (6.3 mm ventral to dura). Subsequently lower the electrode in 100 &#956;m increments within the NAc core until transient DA release events are observed at frequencies of at least 1 per min.</li>
		<li>Connect the tubing (inner diameter 1/32&#34;, outer diameter 3/32&#34;) to a 20 ml syringe, attached to a syringe pump. On the other end of the tube, use a beveled 23 G needle to connect the tubing to the rat&#39;s cannula. Ensure that the tubing is filled completely with the desired tastant and verify that no bubbles exist in the tubing. 
		Note: Typically, administer a small amount of tastant into the mouth of the rat prior to recording to ensure that the first infusion for recording delivers the full infusion amount and not any residual water or air in the tubing. Moreover, this allows the animal to have some prior experience with the tastant since novel tastants, even appetitive ones, can be aversive initially due to its novelty.</li>
		<li>For data collection, infuse 200 &#956;l of tastant (6.5 sec using a pump and previously described tubing). Infuse the tastant every 1-3 min. Infuse a total of 25 times per tastant. Each infusion delivers 200 &#956;l of solution.</li>
		<li>If multiple tastants are being delivered in a single experiment, flush the oral catheter with water after collecting data for one tastant and wait at least 20 min&#160;before infusing the new tastant.</li>
		<li>After the DA recording session, prepare for histological verification by creating a small lesion at the recording site. To preserve the carbon fiber electrode for subsequent calibration, first retract the electrode and remove the micromanipulator. Then use a new micromanipulator with a glass microelectrode and a tungsten wire (extending 100&#160;&#956;m beyond the glass tip) to the same depth (below dura) as the original carbon fiber microelectrode.</li>
		<li>For histological verification, create a small lesion by applying 3 V at the tungsten wire and increasing the voltage at 1 V increments, every 10 sec, until the 10 V setting is reached.</li>
		<li>If a standard concentration curve has already been created using multiple carbon fiber electrodes, perform the lesion step above (3.1.14) by applying the potential directly the carbon fiber electrode. 
		Note: This lesion method will damage the carbon fiber and prevent subsequent calibration with that specific electrode.</li>
		<li>Euthanize the animal using a lethal injection pentobarbital (150 mg/kg, i.p.).</li>
		<li>Dislodge the headcap with the cannulae using pliers by pulling directly upward on the headcap. Do not twist the headcap since this will cause unnecessary damage to the brain and make it challenging to identify the location of the electrode during histology.
		<ol>
			<li>Remove the brain and place in 4% formalin for 3 days followed by 30% sucrose for 1 week. Create 30 &#956;m slices using a cryostat, mount on cover slips and examine slices under a light microscope to identify the location of the carbon fiber or tungsten lesion. 
			Note: Perfusion is optional for 3.1.17.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4) Electrode Calibration
<ol>
	<li>Using the flow cell to create a current-concentration conversion factor.
	<ol>
		<li>In order to determine electrode sensitivity, calibrate electrodes after each experiment using a flow &#34;t-cell&#34; apparatus. Lower the electrode into the &#34;t-cell&#34; while Tris buffer, pH solutions, or DA solutions are washed over the electrode surface (at a rate of 2 ml/min). Use a six-position air solenoid actuator to switch back and forth between buffer and pH or DA solution.</li>
		<li>For each calibration, collect voltammetric data during 5 sec application of Tris buffer (baseline) followed by 5 sec application of pH or DA solution, followed by 20 sec application of Tris buffer (30 sec total file). Repeat each run 3 times for each calibration standard concentration, counterbalancing between different concentrations of each standard.</li>
		<li>For each sample collection, measure the peak DA or pH-evoked current. Average the peak current across each trial to obtain a concentration versus peak current relationship. For more detailed instructions see [12] and [13].</li>
	</ol>
	</li>
</ol>
5) Data Analysis
<ol>
	<li>Using a training set in principal component analysis (PCA) 
	Note: During a voltammetry experiment, output is produced as current, which is the result of dopamine oxidation and reduction, pH shifts, and electrochemical noise. PCA allows separation of these factors so that one can isolate the desired components (DA and pH) and remove additional components that might distort the desired signals (for a more detailed description, see 15, 16).
	<ol>
		<li>Assign a calibration factor to the output current (approximately 5-10 nA/&#956;M) after PCA to allow concentration values of the analytes to be determined.</li>
		<li>Use the training set obtained during an experiment to &#34;train&#34; the PCA model to separate out DA, pH, and other factors. PCA takes the training set cyclic voltammograms (collected in section 3.1.8) and determines which essential characteristics of the voltammogram can be used to identify each analyte collected during in vivo voltammetry recordings.</li>
		<li>To determine the number of principal components to keep, use a Malinowski F-test. Typically, keep only 2 or 3 principal components which normally explain about 95-99% of the variance in the data. For further discussion on implementing the Malinowski F-test, see17. 
		Note: Once the number of components are determined, the PCA analysis effectively will project the measured voltammograms from DA and pH training sets onto the principal components that have been retained. The result is a data set that has been filtered so that only DA (or pH) data remains and the residual data that does not resemble DA or pH is removed (See Representative Results for expected output). For a further detailed explanation and step by step instruction for using PCA for voltammetric analysis see Materials sheet and16, 17.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Roitman, M. 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=Dopamine+operates+as+a+subsecond+modulator+of+food+seeking.">Dopamine operates as a subsecond modulator of food seeking.</a> J Neurosci. 24 (6), 1265-1271 (2004).</li><li>Aragona, B. 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=Regional+specificity+in+the+real-time+development+of+phasic+dopamine+transmission+patterns+during+acquisition+of+a+cue-cocaine+association+in+rats.">Regional specificity in the real-time development of phasic dopamine transmission patterns during acquisition of a cue-cocaine association in rats.</a> European Journal of Neuroscience. 30 (10), 1889-1899 (2009).</li><li>Wheeler, 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=Cocaine+cues+drive+opposing+context-dependent+shifts+in+reward+processing+and+emotional+state.">Cocaine cues drive opposing context-dependent shifts in reward processing and emotional state.</a> Biol Psychiatry. 69 (11), 1067-1074 (2011).</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> Front Biosci (Elite Ed). 5, 982-999 (2013).</li><li>Roitman, M. 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=Real-time+chemical+responses+in+the+nucleus+accumbens+differentiate+rewarding+and+aversive+stimuli).">Real-time chemical responses in the nucleus accumbens differentiate rewarding and aversive stimuli).</a> Nature Neuroscience. 11 (12), 1376-1377 (2008).</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=Phasic+dopamine+release+evoked+by+abused+substances+requires+cannabinoid+receptor+activation.">Phasic dopamine release evoked by abused substances requires cannabinoid receptor activation.</a> J Neurosci. 27 (4), 791-795 (2007).</li><li>Owesson-White, C. 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=Neural+encoding+of+cocaine-seeking+behavior+is+coincident+with+phasic+dopamine+release+in+the+accumbens+core+and+shell.">Neural encoding of cocaine-seeking behavior is coincident with phasic dopamine release in the accumbens core and shell.</a> Eur J Neurosci. 30 (6), 1117-1127 (2009).</li><li>Phillips, P. E., 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=Subsecond+dopamine+release+promotes+cocaine+seeking.">Subsecond dopamine release promotes cocaine seeking.</a> Nature. 422 (6932), 614-618 (2003).</li><li>Chen, 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=Levels+of+mint+and+wintergreen+flavorants:+smokeless+tobacco+products+vs.+confectionery+products.">Levels of mint and wintergreen flavorants: smokeless tobacco products vs. confectionery products.</a> Food Chem Toxicol. 48 (2), 755-763 (2010).</li><li>Manning, K. C., Kelly, K. J., Comello, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavoured+cigarettes,+sensation+seeking+and+adolescents'+perceptions+of+cigarette+brands.">Flavoured cigarettes, sensation seeking and adolescents' perceptions of cigarette brands.</a> Tob Control. 18 (6), 459-465 (2009).</li><li>Rainey, C. L., Conder, P. A., Goodpaster, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+characterization+of+dissolvable+tobacco+products+promoted+to+reduce+harm.">Chemical characterization of dissolvable tobacco products promoted to reduce harm.</a> J Agric Food Chem. 59 (6), 2745-2751 (2011).</li><li>Phillips, P. E., 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=Real-time+measurements+of+phasic+changes+in+extracellular+dopamine+concentration+in+freely+moving+rats+by+fast-scan+cyclic+voltammetry.">Real-time measurements of phasic changes in extracellular dopamine concentration in freely moving rats by fast-scan cyclic voltammetry.</a> Methods Mol Med. 79, 443-464 (2003).</li><li>Robinson, D. 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=.">.</a> Rapid Dopamine Release in Freely Moving Rats. Electrochemical Methods for Neuroscience. , (2007).</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> J Neurosci. 24 (7), 1754-1759 (2004).</li><li>Heien, M. L., Johnson, M. 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=Resolving+neurotransmitters+detected+by+fast-scan+cyclic+voltammetry.">Resolving neurotransmitters detected by fast-scan cyclic voltammetry.</a> Anal Chem. 76 (19), 5697-5704 (2004).</li><li>Keithley, R. B., Heien, 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=Multivariate+concentration+determination+using+principal+component+regression+with+residual+analysis.">Multivariate concentration determination using principal component regression with residual analysis.</a> Trends Analyt Chem. 28 (9), 1127-1136 (2009).</li><li>Keithley, R. B., Carelli, R. 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=Rank+estimation+and+the+multivariate+analysis+of+in+vivo+fast-scan+cyclic+voltammetric+data.">Rank estimation and the multivariate analysis of in vivo fast-scan cyclic voltammetric data.</a> Anal Chem. 82 (13), 5541-5551 (2010).</li><li>Rising, J. P., 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=Healthy+Young+Adults:+description+and+use+of+an+innovative+health+insurance+program.">Healthy Young Adults: description and use of an innovative health insurance program.</a> J Adolesc Health. 41 (4), 350-356 (2007).</li><li>Day, J. 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=Associative+learning+mediates+dynamic+shifts+in+dopamine+signaling+in+the+nucleus+accumbens.">Associative learning mediates dynamic shifts in dopamine signaling in the nucleus accumbens.</a> Nature Neuroscience. 10 (8), 1020-1028 (2007).</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=Catecholamines+in+the+bed+nucleus+of+the+stria+terminalis+reciprocally+respond+to+reward+and+aversion.">Catecholamines in the bed nucleus of the stria terminalis reciprocally respond to reward and aversion.</a> Biol Psychiatry. 71 (4), 327-334 (2012).</li><li>Wheeler, R. A., Carelli, 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=The+neuroscience+of+pleasure.+Focus+on+'Ventral+pallidum+firing+codes+hedonic+reward:+when+a+bad+taste+turns+good.">The neuroscience of pleasure. Focus on 'Ventral pallidum firing codes hedonic reward: when a bad taste turns good.</a> J Neurophysiol. 96 (5), 2175-2176 (2006).</li><li>Bunin, M. 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=Release+and+uptake+rates+of+5-hydroxytryptamine+in+the+dorsal+raphe+and+substantia+nigra+reticulata+of+the+rat+brain.">Release and uptake rates of 5-hydroxytryptamine in the dorsal raphe and substantia nigra reticulata of the rat brain.</a> J Neurochem. 70 (3), 1077-1087 (1998).</li><li>Zimmerman, J. B., 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=Simultaneous+electrochemical+measurements+of+oxygen+and+dopamine+in+vivo.">Simultaneous electrochemical measurements of oxygen and dopamine in vivo.</a> Anal Chem. 63 (1), 24-28 (1991).</li><li>Levy, 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=A+novel+procedure+for+evaluating+the+reinforcing+properties+of+tastants+in+laboratory+rats:+operant+intraoral+self-administration.">A novel procedure for evaluating the reinforcing properties of tastants in laboratory rats: operant intraoral self-administration.</a> J Vis Exp. (84), e50956 (2014).</li></ol>

The authors have nothing to disclose.

Intraoral tastant delivery combined with FSCV permits analysis of &#8220;real-time&#8221; DA responses to oral flavorants. There are three critical steps in the protocol that are required for successful DA measurements. First, proper implantation of the oral catheter is critical for delivery of flavorants. Ensuring that the catheter is inserted behind the first molar and wedged into place prevents the catheter from losing patency and prevents accidental removal by the animal. Flushing the catheter during the first few da...

Erratum: Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

Phasic DA release plays an important role in mediating reward-directed behavior [1-3]. However, isolating and studying how a primary reward alters phasic DA release is often complicated by co-occurring behavioral or cognitive processes that are also capable of altering phasic DA release - such as decision making processes or reward-directed motor behavior to acquire the reward. In the current work, we isolate phasic DA responses to tastants through the use of in vivo fast-scan cyclic voltammetry (FSCV) combined with tastant delivery through intraoral cannulae. This technique bypasses choice and action and allows us to examine extracellular DA release during direct infusion of a tastant to the palate of a rat.
FSCV is an electrochemical technique with high temporal and spatial resolution, permitting measurements of DA release in a discrete local area (approximately 100 &#181;m) on a sub-second scale (10 Hz resolution). The combination of intra-oral delivery and FSCV provides the advantage of observing rapid, 'real-time' DA responses to a tastant, which cannot be examined using conventional microdialysis methods. Furthermore, phasic DA release in response to either rewards or reward-associated cues occurs at concentrations between 20-100 nM, which is above the 10-20 nM detection threshold for FSCV [4]. The high spatial resolution of FSCV also permits recording from sub-regions of small brain areas, such as the nucleus accumbens core (NAc). Thus, FSCV combined with intraoral infusions of tastants is an ideal model for studying how tastants or other stimuli alter phasic DA release in an awake, behaving animals. Indeed, these techniques have permitted experiments investigating how rewarding and aversive tastants alter extracellular DA release [5]. 
Over the last decade, FSCV analyses have been successfully combined with intravenous drug delivery [6] and rat self-administration paradigms [7, 8] to identify the role of phasic DA mechanisms in drug addiction models. In addition, combined intraoral delivery with FSCV has been used to examine how tastant cues paired with cocaine availability modulate phasic DA release and behavioral responses that reflect emotional affect [3]. This combined intraoral and FSCV methodology can also be powerfully utilized to examine phasic DA responses to flavorants, such as menthol and oral sweeteners, that are added to cigarettes and dissolvable tobacco products [9-11]. Although many of the flavorants added to tobacco products are appetitive [9-11], it is unknown if these flavorants increase phasic DA release in a manner consistent with a rewarding hedonic valence. Indeed, flavorants added to cigarettes and to dissolvable tobacco products may have direct effects on the DA reward system and may act through dopaminergic mechanisms to influence the rewarding valence of cigarettes and other tobacco products. Thus, intraoral delivery combined with FSCV can provide new understanding on how flavorants modulate rapid DA release. The use of combined intra-oral and in vivo FSCV methodology, and the data obtained from such studies, can also facilitate future studies to determine how flavorants and nicotine interact to alter DA signaling and to potentially modulate nicotine reinforcement. Further, the data gained from such studies can be used to inform regulatory decisions about tobacco product flavorants.

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examination of rapid dopamine dynamics with fast scan cyclic voltammetry during intra-oral tastant administration in awake rats

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique with high spatial and temporal (sub-second) resolution that has been utilized to examine phasic DA release in several types of preparations. In vitro experiments in single-cells and brain slices and in vivo experiments in anesthetized rodents have been used to identify mechanisms that mediate dopamine release and uptake under normal conditions and in disease models. Over the last 20 years, in vivo FSCV experiments in awake, freely moving rodents have also provided insight of dopaminergic mechanisms in reward processing and reward learning. One major advantage of the awake, freely moving preparation is the ability to examine rapid DA fluctuations that are time-locked to specific behavioral events or to reward or cue presentation. However, one limitation of combined behavior and voltammetry experiments is the difficulty of dissociating DA effects that are specific to primary rewarding or aversive stimuli from co-occurring DA fluctuations that mediate reward-directed or other motor behaviors. Here, we describe a combined method using in vivo FSCV and intra-oral infusion in an awake rat to directly investigate DA responses to oral tastants. In these experiments, oral tastants are infused directly to the palate of the rat &#8211; bypassing reward-directed behavior and voluntary drinking behavior &#8211; allowing for direct examination of DA responses to tastant stimuli.

FSCV combined with intraoral catheter implantation was used to examine how sucrose, an appetitive tastant, modulates phasic DA release in the NAc core. Prior to tastant infusion, electrical stimulation (150 &#181;A, 60 Hz, 24 pulses; indicated by the red bar) of the VTA produces robust increases in phasic DA release in the NAc (Figure 1). Figure 1 shows a color plot with potential on the y-axis, time on the x-axis, and current (represented as false color) on the z-axis. Below the color p...

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Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats.

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. ...

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Research

JoVE Journal

Behavior

12.7K Views.  Yale University. Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats.

Video: Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

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

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

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

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 ...

Neuroscience

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

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

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

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

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

Biology

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

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

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

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

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

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

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

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors&#39; roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

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

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

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

Monitoring Dopamine Release in a Rat Model with VTA Dopaminergic Neuron Receptor Manipulation

Source: Wickham, R. J., et al. <a href="https://app.jove.com/v/60886">Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine.</a> J. Vis. Exp. (2020).
This video demonstrates the implantation of electrodes and cannulas in an anesthetized rat to measure dopamine release in the nucleus accumbens using a potentiostat and the infusion of drugs to modulate dopaminergic activity in the ventral tegmental area.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

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