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. 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 – bypassing reward-directed behavior and voluntary drinking behavior – allowing for direct examination of DA responses to tastant stimuli.
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 µm) 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.
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
2) Combined Intraoral Surgery and Intracranial Cannulation Surgery (Approximately 75 min)
3) Voltammetric Recordings in Awake, Freely Moving Rat
4) Electrode Calibration
5) Data Analysis
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 µA, 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 plot is DA concentration versus time trace. Concentration versus time was determined using the current versus time trace at the oxidation potential of dopamine (indicated by dashed white line) which was converted to concentration, following post-calibration of the electrode. This DA concentration versus time trace was used as part of the training set for PCA.
After the training set was acquired, 25 infusions of sucrose (10%, 200 µl per infusion) were given every 1-3 min. Examples of sucrose modulation of phasic DA release are shown in Figures 2 and 3. In Figure 2, the 6.5 sec infusion (indicated by the red bar) produced an elevation in current (indicated by the white, downward facing arrow) at the oxidation potential of DA (indicated by the white dashed line). Transforming the data into a concentration versus time plot after PCA verified that the increased current in responses to the tastant is the result of an increase in DA concentration. Another tastant, menthol (0.005%, 200 µl per infusion) was also infused during FSCV recordings in the NAc core. Figure 3 shows DA concentration versus time example traces for intra-oral sucrose and menthol, revealing increased DA concentration after infusion of either tastant. One should expect to see some trial to trial variability in phasic DA release to tastants (Figure 2 and 3A). However, we do not typically see any non-specific trends towards an increase or decrease in phasic DA release over multiple trials. Ranges from 10-100 nM DA can be expected in a typical experiment in a single animal.
Figure 1. Representative color plot (top) and concentration versus time plot (bottom) of electrically evoked DA release in the NAc core. Red bar indicates stimulation time. Please click here to view a larger version of this figure.
Figure 2. Representative color plot (top) and DA concentration versus time trace (bottom) during a single infusion of sucrose. Red bar indicates 6.5 sec infusion. White downward facing arrow indicates in elevation in current (represented in false color) at the oxidation potential of DA. Please click here to view a larger version of this figure.
Figure 3. Representative DA concentration versus time traces during (A) 10% sucrose and (B) 0.005% menthol by intraoral administration. Red bar indicates 6.5 sec intraoral infusion. Please click here to view a larger version of this figure.
Intraoral tastant delivery combined with FSCV permits analysis of “real-time” 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 days after recovery is also important, since the soft food given to the animal during the first 2 or 3 days post-surgery could clog the catheter. Second, the quality of the electrode is critical for increasing signal-to-noise ratio in a freely moving rat. In some instances, electrodes that visually appear to be of high quality and good condition may still give noisy signals. One way to troubleshoot this issue is to take baseline recordings in the dorsal striatum (4 mm below dura). If the electrode produces too much noise, the electrode can be removed and replaced with a new electrode prior to performing the experiment in the NAc core recording site (6-7 mm below dura). A third critical step is finding a suitable recording location in the targeted brain region, where spontaneous DA release events (“transients”) are observed at a sufficient frequency (at least 1 per min) with sufficient increases in DA concentration (at least 20 nM to 40 nM). This optimization will ensure that spontaneous phasic DA release events can be detected at specific location, prior to flavorant administration. Lowering the electrode in increments equivalent to the size of the exposed carbon fiber (50-100 µm) maximizes the chances of finding a recording site with detectable transients.
One important caveat of FSCV is the inability to determine absolute analyte concentration at a point in time, since FSCV is a differential technique that measures relative concentration changes (compared to a stable baseline) during each voltammetry recording file. However, the differential technique also allows for quantification of sub-second fluctuations in dopamine concentration in response to both rewarding and aversive tastants 5. In the context of nicotine addiction, tobacco product additives, such as menthol and oral sweeteners, have been shown to influence smoking behavior 18 and have been recently been banned in cigarettes (except for menthol) by the Family Smoking Prevention and Tobacco Control Act. Thus, intraoral delivery in combination with FSCV provides an important methodological tool that can be used to examine how tobacco product flavorants can alter DA signaling, and potentially the reinforcing properties of nicotine.
Here, we have described the method of combining intraoral tastant infusion with FSCV for the purpose of measuring phasic DA release in response to primary tastant stimuli. It is important to note that while our technique allows the examination of phasic DA release to tastants independent of action or choice, it is also possible that the unexpected nature of the tastant delivery could influence phasic DA release19. Thus, a proper control solution, such as water or artificial saliva, can be used in control experiments to compare and examine the effects of an unexpected reward or stimulus on phasic DA release independent of the tastant of interest. However, the technique is not limited to either measurements of DA release or reward. For example, previous work has examined norepinephrine release during both reward and aversion 20. Since animals do not readily consume aversive tastants intraoral tastant infusion with FSCV is the only technique available for measuring phasic DA release to aversive tastants. However, this technique does not determine whether or not a tastant is aversive or appetitive, rather, simply measures DA release during the consumption of the tastant. In order to determine the hedonic value of a tastant, behavioral tests can be incorporated to observe oro-facial responses during taste reactivity (as described elsewhere 5, 21).
It should also be noted that FSCV is also suitable for measuring other important neurochemicals, such as serotonin22 and oxygen 23, but methods for recording these analytes in awake, freely moving animals are not yet available. Furthermore, with the combined voltammetry and intra-oral infusion method, time-locked dopamine responses to tastant delivery can be analyzed in the absence of other behaviors, such as approach behavior or food retrieval, that might also affect DA release. As an additional application, combined intraoral tastant delivery and FSCV may be ideal for measuring DA signaling to rewarding or aversive events during operant or Pavlovian conditioning. Indeed, a recent JoVE publication by Levy and colleagues combines intra-oral tastant delivery with operant behavior to determine whether intra-oral tastants support the acquisition and maintenance of operant self-administration behavior24. Thus, such methods can also be combined with FSCV to identify phasic DA response to oral tastants during behavior.
The authors have nothing to disclose.
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.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Stimulating electrode | PlasticsOne | MS303/2-A/SPC | (Stimulating electrode) when ordering, request a 22mm cut below pedastal |
Cannula for electrode | BioAnalytical Systems | MD-2251 | |
Glass Capillary | A-M systems | 624503 | |
Carbon Fiber | Thornel | T650 | |
Electrode puller | Narishige International | PE-22 | |
Neurolog stimulus isolator | Digitimer Ltd. | DS4 | |
Heat Shrink | 3M | FP-301 | |
Insulated wires for electrodes | Squires electronics | Custom | (Wire for electrodes) L 3.000 x 1.000s x 1.000s UL1423 30/1 BLU |
Micromanipulator | Univ. of Illinois at Chicago, Engineering Machine Shop | n/a | |
Epoxy | ITW Devcon | 14250 | (Epoxy) "5 minute epoxy" |
copolymer of perfluoro-3,6-dioxa-4-methyl-7octene-sulfonic acid and tetrafluoroethylene | Ion Power | LQ-1105 | "i.e. Nafion" |
Silver Paint | GC Electronics | 22-023 | |
Power Supply | BK Precision | 9110 | |
Tubing for intra-oral catheters | Intramedic | 427426 | (Tubing for intra-oral catheters) PE 100, I.D.=0.86mm; O.D.=1.52mm |
Tubing for intra-oral infusion | Fischer Scientific | 02-587-1A | (Tubing for intra-oral infusion) I.D. 1/32"; O.D. 3/32" |
Syringe pump for flow cell | Pump Systems Inc. | NE 1000 | |
Surgical cement | Dentsply Caulk | 675571 and 675572 | |
Air acuatator | VICI | A60 | (Air actuator) 6 position digital valve interface |
Digital Valve Interface | VICI | DVI | (Digital Valve Interface) 230 VAC |
Quad Headstage | Univ. of N. Carolina, Electronics Facility | n/a | |
UEI Power Supply | Univ. of N. Carolina, Electronics Facility | n/a | |
UEI Breakout Box | Univ. of N. Carolina, Electronics Facility | n/a | |
Power Supply for tastant syringe pump | Med Associates | SG-504 | |
Tastant Syringe Pump | Med Associates | PHM-107 | |
Tungsten Microelectrode | MicroProbes | WE30030.5A3 | |
Silver Wire Reference with AgCl | InVivo Metric | E255A | |
Sucrose | Sigma | 80497 | |
Magnesium Chloride | Sigma | M8266 | |
Sodium Chlroide | Sigma | S7653 | |
Perchloric Acid | Sigma | 244252 | |
Hydrochloric Acid (4M) | Sigma | 54435 | |
Soidum Hydroxide | Sigma | 306576 | |
Hydrogen Peroxide | Sigma | H1009 | |
Dopamine Hydrochloride | Sigma | H8502 | |
TarHeel HDCV Software | University of North Carolina-Chapel Hill | Must request software: click here for link to software request page |