Address correspondence and reprint requests to Dr R. Mark Wightman, Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290, USA. E-mail: email@example.com
The present experiment examined the effect of the dopamine transporter blocker nomifensine on subsecond fluctuations in dopamine concentrations, or dopamine transients, in the nucleus accumbens and olfactory tubercle. Extracellular dopamine was measured in real time using fast-scan cyclic voltammetry at micron-dimension carbon fibers in freely-moving rats. Dopamine transients occurred spontaneously throughout the ventral striatum in the absence of apparent sensory input or change in behavioral response. The frequency of dopamine transients increased at the presentation of salient stimuli to the rat (food, novel odors and unexpected noises). Administration of 7 mg/kg nomifensine amplified spontaneous dopamine transients by increasing both amplitude and duration, consistent with its known action at the dopamine transporter and emphasizing the dopaminergic origin of the signals. Moreover, nomifensine increased the frequency of detected dopamine transients, both during baseline conditions and at the presentation of stimuli, but more profoundly in the nucleus accumbens than in the olfactory tubercle. This difference was not explained by nomifensine effects on the kinetics of dopamine release and uptake, as its effects on electrically-evoked dopamine signals were similar in both regions. These findings demonstrate the heterogeneity of dopamine transients in the ventral striatum and establish that nomifensine elevates the tone of rapid dopamine signals in the brain.
Dopamine transients have been linked to specific aspects of sensorimotor processing, such as interaction with another rat and cues and operant responses associated with cocaine and sucrose administration (Robinson et al. 2001, 2002; Phillips et al. 2003; Roitman et al. 2004). However, dopamine transients also occur spontaneously in the absence of overt sensory input or specific behaviors (Robinson et al. 2002; Cheer et al. 2004). The functional significance of this spontaneous transmission is not known, but may underlie a fundamental aspect of dopamine signaling. The present experiment was designed to explore this aspect of dopamine transmission and how it is modified by the presentation of salient stimuli and the administration of a dopamine uptake blocker. Stimuli (food, novel odors and unexpected noise) that have previously been linked to changes in dopamine transmission via microdialysis measurements (e.g. Bassareo and Di Chiara 1999; Guion and Kirstein 2001; Storozheva et al. 2003) were presented while rapid dopamine transmission was recorded in the two major nuclei of the ventral striatum (Heimer et al. 1995), the nucleus accumbens (NAc) and the olfactory tubercle (OT). Nomifensine was administered to produce stimulant-induced locomotor behavior (Costall et al. 1975) at a dose (7 mg/kg) that has previously been used to characterize dopamine release and uptake kinetics in the NAc (Garris et al. 2003). The resulting data offer insight onto the occurrence and regulation of rapid dopamine signals in the ventral striatum during behavior.
Materials and methods
Fourteen male Sprague-Dawley rats (285 ± 11 g at surgery) were singly housed in a vivarium on a 12 : 12 h light : dark cycle with food and water ad libitum. This strain has been previously used for the characterization of drug effects on fast dopamine transmission in awake rats (e.g. Garris et al. 2003). Experiments were conducted during the dark phase of the cycle. Rats were habituated to the tether and experiment cage on a separate day prior to the experiment. All procedures were approved by the Institutional Animal Care and Use Committee at the University of North Carolina in accordance with the Public Health Service policy on Humane Care and Use of Laboratory Animals.
Surgical procedures were carried out as previously described (Robinson et al. 2002). In brief, rats were anesthetized with ketamine (80–100 mg/kg) and xylazine (12–20 mg/kg) and positioned in a stereotaxic frame. A guide cannula (Bioanalytical Systems, West Lafayette, IN, USA) was positioned above the ventral striatum at 1.2 mm anterior and 2 mm lateral to bregma and extended 2.5 mm below the skull surface. A reference electrode (Ag/AgCl, ∼ 5 mm length) was placed in the contralateral cortex extending to the dorsal caudate. Finally, a bipolar stimulating electrode (Plastics One, Roanoke, VA, USA) was positioned ipsilateral to the guide cannula at the medial forebrain bundle (4.0 mm posterior and 1.4 mm lateral to bregma, 8.5–9.5 mm ventral from the skull surface) in order to stimulate dopaminergic fibers. Rats were allowed 3–6 days to recover.
Single carbon fibers (6-µm diameter) were pulled and sealed in glass capillaries and secured in micromanipulators. The exposed fiber was trimmed to extend 160 ± 10 µm from the glass seal to form a cylindrical microelectrode (Cahill et al. 1996). The micromanipulator locked into the guide cannula and allowed ventral placement of the electrode into the brain at 75-µm increments. Voltammetric recordings were made at the carbon-fiber electrode every 100 ms by applying a triangular waveform (−0.4 to 1.0 V vs. Ag/AgCl reference, 300 V/s). The electrode was held at − 0.4 V between scans. Voltammetric parameters, stimulation parameters and data acquisition were controlled by a computer using locally written LabVIEW instrumentation (Michael et al. 1999; National Instruments, Austin, TX, USA). The electrochemical data underwent one pass of two-dimensional smoothing. Electrodes were calibrated for dopamine concentration after each experiment in an in vitro flow-cell system (Logman et al. 2000).
A high-throughput algorithm within the LabVIEW collection and analysis program was used to statistically target dopamine concentration transients as previously described by Robinson et al. (2003). Briefly, a template of the background-subtracted cyclic voltammogram for dopamine was selected for each animal from the electrochemical response during electrical stimulation of dopamine neurons. Each background-subtracted cyclic voltammogram collected during the experiment was then compared with the template, identifying as targets those voltammograms with high correspondence to dopamine as determined by the inverse of the mean squared error and the correlation coefficient. Criteria necessary to positively identify an electrochemical signal as dopamine included peaks at ∼ + 0.6 and ∼ − 0.2 V versus Ag/AgCl, their relative amplitude, the absence of extraneous peaks and a minimum of at least two consecutive scans (Heien et al. 2003). Duration of dopamine transients was determined from the cyclic voltammograms examined on a scan-by-scan basis and calculated as total duration from the first identified dopamine scan to the last. The peak amplitude of each dopamine transient ([DA]max) was measured from the current versus time plot at the dopamine oxidation potential and converted to concentration using post-experiment in vitro calibration of the electrode. The baseline measurement for calculating [DA]max was taken from the 10–20 scans used for background subtraction.
Rats were assigned to one of three experimental groups (brain region + drug): NAc + saline (n = 4), NAc + nomifensine (n = 5) and OT + nomifensine (n = 5). Rat chow was removed from each animal's cage approximately 22 h before the start of the experiment to increase the salience of the food stimulus. On the morning of the experiment, each rat was tethered in the experiment chamber by a cable secured to the stimulating electrode which allowed full access to the chamber. After at least 1 h habituation, a fresh carbon-fiber electrode was inserted into the ventral striatum at a position where a robust dopamine signal was observed immediately following mild electrical stimulation of the medial forebrain bundle. An effort was made to use the smallest stimulation sufficient to produce a reliable signal, resulting in consistent within-subject but variable between-subject stimulation parameters (12–24 rectangular pulses, 30–60 Hz, 125 µA, 2 ms/phase, biphasic). These stimulations typically produced no movement but were quickly followed by sniffing. The stimulations continued every 4 min throughout the experiment to monitor the effect of nomifensine. Voltammetric recordings were made continuously throughout the experiment for a total of 54 min.
In our previous study (Robinson et al. 2002) we found that the dopamine response to repeated presentations of the same rat could habituate. Therefore, to ward against habituation in the present study we used a variety of stimuli. Four stimuli were presented to the rat in randomized order: food (one piece of fruit-flavored cereal), lemon odor, banana odor and a brief noise sufficient to produce a startle response. The food was presented by hand, as the rats were previously trained to take the cereal from the experimenter's hand. The odors were presented by dipping the absorbent end of a cotton applicator into either lemon or banana extract and holding it before the rat's nose for 5 s. During the habituation period, the experimenter's hand was repeatedly inserted into the cage to habituate the rat to that aspect of the presentation. The noise was produced by popping 3-cm diameter compartments of bubble-wrap outside the Faraday cage and the startle response was operationally defined as a quick head or body movement simultaneous to the noise. The stimuli were presented to each rat 4 min apart. Next, either 7 mg/kg nomifensine or an equivalent volume of saline was administered i.p. to the rat. After 15 min (allowing for the absorption and distribution of the drug to the brain), each stimulus was presented a second time, again 4 min apart and in random order.
The entire experiment was videotaped with a camera mounted in the Faraday cage. The electrochemical and behavioral data were synchronized with a video character generator (Chemistry Electronics Facility, University of North Carolina) that superimposed the episode and electrochemical scan number on the video record. As we had previously found that 7 mg/kg nomifensine produced largely locomotion rather than focused stereotypical movements (Garris et al. 2003), we measured general locomotion throughout the experiment by counting each time the rat crossed between chamber quadrants and reared. The video record was also used to verify presentation times of the various stimuli and the presence of a startle response of the rat to the noise stimulus.
Rats were given a lethal dose of urethane and perfused transcardially with saline, followed by 10% formalin. The brains were removed, frozen, sectioned and stained with thionin. The 6-µm carbon fiber was too small to produce damage detectable with a light microscope. However, the dorsal part of the glass capillary left a detectable tract and the total distance inserted was known from the micromanipulator, allowing reconstruction of the tip location. The reconstructed recording sites are shown in Fig. 1.
Statistics were calculated with SAS software (SAS Institute Inc., Cary, NC, USA) and GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). As the frequency data for naturally-occurring dopamine transients were count data with occasional bins of zero counts, the frequencies across experimental phases were fit with a Poisson distribution regression model with repeated measures and main effects and pairwise contrasts were tested with Wald statistics (Robinson et al. 2002). Both naturally-occurring and electrically-evoked dopamine transients were quantitated by measuring [DA]max and the duration of the signal. As the data for naturally-occurring transients were skewed toward the detection limits of the voltammetric method, these parameters were analyzed by fitting a gamma distribution regression model with repeated measures and testing contrasts with Wald statistics. Locomotion scores and the characteristics of electrically-evoked dopamine signals were analyzed across experimental phases with two-way anovas with repeated measures followed by Dunnett's multiple comparison tests or one-way anovas when indicated. Correlations between behavioral scores and dopamine signals were calculated as Spearman correlation coefficients. Data are presented as mean ± SEM.
Naturally-occurring dopamine transients
Representative dopamine transients before and after nomifensine administration are shown in Fig. 2. The 5-s traces show the current at the oxidation potential of dopamine over time, with each circle indicating a measurement taken every 100 ms. Changes in current at that potential could be due to either oxidation of dopamine or interferences such as oxidation of another species, electrical noise or a change in local pH (Robinson et al. 2003). The transients depicted here, denoted by the open circles, were verified to be due to dopamine oxidation by inspection of the cyclic voltammograms. Figure 2 shows dopamine transients in the NAc occurring during baseline conditions. Pre-drug (Fig. 2 top), the rat was sniffing the cage floor during the entire 5-s trace. After nomifensine (Fig. 2 bottom), the rat was crossing the middle of the cage while sniffing the floor. In neither example did the behavior of the rat change after the dopamine transient. The dopamine transient was more robust after the administration of nomifensine.
The frequency of dopamine transients was measured during baseline conditions as well as at the presentation of various stimuli. Rats (n = 4) were tested with saline injections to demonstrate the effectiveness of the stimuli in increasing the frequency of dopamine transients in the NAc both pre- and post-injection. Figure 3 (top) shows the frequency of the dopamine transients in 1-min bins. The frequency of dopamine transients was calculated as the number of transients per min per rat; data are presented as the mean frequency ± SEM of all rats in the group. Over the 54 min of data collection, 138 transients were detected in these rats (35 ± 5 transients per rat). The baseline frequency of dopamine transients detected in these rats was 0.6/min. Pre-injection, the frequency of transients during a total of 18 baseline bins before saline administration was compared with that during a total of four bins containing stimulus presentations; post-injection, a total of 18 baseline bins starting 10 min after saline administration were compared with the four bins containing stimulus presentations. Indeed, the frequency of dopamine signals increased 90% at the presentation of stimuli both before and after saline injection (main effect of stimulus presentation, Z = 26.87, p < 0.0001). Importantly, these data justified the use of within-subject comparisons of pre- versus post-injection frequencies for the nomifensine groups.
The effect of nomifensine on spontaneous dopamine transients was measured in both the NAc and OT. Figures 3 (middle and bottom) show the frequency of the dopamine transients in 1-min bins; 398 and 343 transients were detected over the 54 min of data collection in the NAc and the OT, respectively (n = 5 rats for each group; 80 ± 27 and 69 ± 22 transients per rat). The frequency of dopamine transients detected before nomifensine was 0.4/min in the NAc and 0.7/min in the OT. Transients were grouped into bins for comparison as described for the saline group above. Statistical analysis revealed a significant main effect of stimulus presentation (Z = 84.36, p < 0.0001) and a significant interaction between drug and region on dopamine transients (Z = 2.13, p < 0.05). Pre-drug, the frequency of dopamine transients significantly increased at the presentation of stimuli, by 120% in the NAc and by 40% in the OT (p < 0.0001), with no significant differences between brain regions. Post-drug, the increase in frequency at stimulus presentations was still apparent but to a lesser degree, by 30% in both brain regions (p < 0.0001). Nomifensine administration increased the overall frequency of transients in both brain regions but to a greater extent in the NAc (570%, p < 0.0001) than the OT (130%, p < 0.05). As two of the recordings from the NAc group were in the shell, additional analyses were performed to compare the frequency of dopamine transients in the core (n = 3) versus shell (n =2). While the main effects of stimulus presentation and nomifensine were still significant (p < 0.0001), there was no difference between NAc core and shell (p > 0.73).
The various stimuli appeared to be effective triggers of dopamine transients, as shown in Table 1. Under control conditions (before drug injection), dopamine concentration transients were detected within 5 s of 50% of the presentations; post-injection, transients were detected within 5 s of 63% of the presentations. Statistical analysis was not conducted on these data, however, as the experiment was not designed to compare the efficacy of these different stimuli. The timing of the dopamine transients around the presentation of the various stimuli was such that they clustered around the onset of the presentation (data not shown), similar to the timing of transients previously reported (Robinson et al. 2002).
Table 1. Number of stimulus presentations closely temporally associated with dopamine concentration transients (within 5 s)
Each stimulus was presented to each rat once during control conditions and once after drug administration.
Nomifensine affected the apparent kinetics of spontaneous dopamine transients in the ventral striatum, amplifying the signals by increasing the peak concentration as well as duration. Figure 4 shows the distribution of the amplitude of dopamine transients before and starting 10 min after nomifensine administration. This distribution shifted toward larger amplitudes in both NAc and OT, increasing the [DA]max of the spontaneous signals. Statistical analysis yielded a significant interaction between drug and region (Z = 4.79, p < 0.0001) but no effect of experimental phase, so subsequent analysis discarded phase from the statistical model. Pairwise comparisons confirmed that nomifensine significantly increased the amplitude of dopamine transients in both the NAc (Z = 3.79, p < 0.001) and OT (Z = 2.21, p < 0.05). Moreover, [DA]max was not different between the NAc and OT either pre- or post-drug.
Nomifensine also shifted the distribution of the duration of naturally-occurring dopamine transients toward longer signals (Fig. 5). As with amplitude, the analysis of duration revealed a significant interaction between drug and region (Z = 3.80, p < 0.0001) but no effect of experimental phase, so subsequent analysis discarded phase from the model. Pairwise comparisons showed that nomifensine extended the length of the dopamine transients in both brain regions (NAc, Z = 3.83, p < 0.0001; OT, Z = 7.30, p < 0.0001). While the duration of spontaneous dopamine transients was not different between the NAc and OT in the pre-drug state, nomifensine had a greater effect on transients in the OT than the NAc (Z = 3.06, p < 0.01).
Electrically-evoked dopamine release
Nomifensine increased the [DA]max of electrically-stimulated signals obtained during the behavioral studies described above by 2.5-fold in both the NAc and OT. These results are similar to the increases previously reported in the NAc following the same dose of nomifensine (Garris et al. 2003). The maximal effect of nomifensine on stimulated release of dopamine in both regions occurred at about 13 min post-injection (Fig. 6 top), while saline had no effect. Statistical analysis of the last pre-drug stimulation and all the post-drug stimulations revealed a significant interaction between group and time (F = 1.82, p < 0.05). Indeed, nomifensine induced a significant increase in [DA]max within 3 min in the OT and within 5 min in the NAc (OT, F = 9.77, p < 0.001; NAc, F = 2.82, p < 0.05), while saline had no effect.
Nomifensine increased the duration of the electrically-evoked dopamine signal threefold in the NAc and 2.3-fold in the OT, with the peak effect at 15–25 min (Fig. 6 middle), whereas saline had no effect. A significant interaction between group and time was demonstrated by analysis of the last pre-drug stimulation and all the post-drug stimulations (F = 2.89, p < 0.001). As with amplitude, nomifensine induced a significant increase in duration within 3 min in the OT and within 5 min in the NAc (OT, F = 12.89, p < 0.0001; NAc, F = 8.03, p < 0.0001), while saline had no effect. A more complete description of nomifensine effects on electrically-stimulated dopamine release and uptake kinetics in the NAc of awake rats was previously reported by Garris et al. (2003).
As the rats were habituated to the experiment chamber for at least 1 h before the experiment, they showed little locomotor activity before drug administration, 2.7 ± 0.2 movements/min. The motor effects of 7 mg/kg nomifensine have been previously described, including the finding that electrical stimulations similar to those used in the present study did not contribute to behavioral scores, even when delivered at 1-min intervals (Garris et al. 2003). Similarly, 7 mg/kg nomifensine increased the locomotor activity of rats approx. ninefold as measured by crossing cage quadrants and rearing (Fig. 6 bottom). The effect was observable within 5 min and peaked at about 10 min. For statistical analysis, the data were grouped into 4-min bins to match the electrically-evoked dopamine data, comparing the last pre- and all post-drug bins. This analysis yielded a significant group by time interaction (F = 6.43, p < 0.0001); while saline had no effect on behavior, nomifensine significantly increased locomotor activity within 10 min (F = 17.29, p < 0.0001).
Locomotor scores from individual rats were tested for correlation to the area (amplitude × ½ width) of the electrically-evoked dopamine transients as well as the number of spontaneous dopamine transients. In saline-treated rats, neither electrically-evoked nor spontaneous dopamine signals correlated with locomotor behavior scores. However, behavioral and neurochemical measures significantly correlated in nomifensine-treated rats, as nomifensine increased all three measures. Specifically, the area of electrically-evoked dopamine signals and locomotor scores were highly correlated in all rats with measurements in the NAc and in four of five rats with measurements in the OT (range of r-values, 0.59–0.82; all p > 0.05). Likewise, in the NAc group behavioral data and the frequency of spontaneous dopamine transients were significantly correlated in four of five rats (range of r-values, 0.35–0.65, all p > 0.05). In contrast, only one of five rats with measurements in the OT showed significantly correlated measurements, possibly because the effect of nomifensine on the rate of dopamine transients was much less in the OT than in the NAc.
Subsecond fluctuations in extracellular dopamine concentrations, or dopamine transients, occurred spontaneously throughout the ventral striatum in the absence of apparent sensory input or change in behavioral response. The frequency of dopamine transients increased at the presentation of salient stimuli to the rat, such as food, odors and unexpected noises. Administration of the dopamine transporter blocker nomifensine increased the number of dopamine transients detected, particularly during baseline conditions but also at the presentation of stimuli. The nomifensine-induced increase in the frequency of spontaneous dopamine signals was much larger in the NAc than the OT, even though the kinetics of dopamine release and uptake were similar in both regions. The locomotor effects of nomifensine correlated with the drug effects on electrically-evoked dopamine release in both the NAc and OT but correlated with the changes in spontaneous dopamine transients only in the NAc. These findings demonstrate the heterogeneity of dopamine transients in the ventral striatum and their sensitivity to pre-synaptic modulation by nomifensine.
While fast-scan cyclic voltammetry has the advantages of subsecond time resolution and micron spatial resolution, it was limited in the past by a lack of sensitivity to dopamine in vivo. Earlier in vivo research used electrical stimulations to activate a population of dopamine neurons in anesthetized rats and then measured the resulting dopamine signals. The effects of drugs on these electrically-evoked signals were systematically studied, providing valuable information on the time course and mechanism of drug effects on dopamine transmission (Garris and Wightman 1995; Bunin and Wightman 1998). Nevertheless, it was thought that interferences such as ascorbate, 3,4-dihydroxyphenylacetic acid (DOPAC), baseline drift and ionic changes would prevent the measurement of the smaller, naturally-occurring fluctuations in extracellular dopamine (Wiedemann et al. 1990; Venton et al. 2003a). Fortunately, such forecasts were incorrect, as advances in instrumentation and data analysis (Michael et al. 1999; Robinson et al. 2003) and explorations of electrochemical processes at the carbon fiber (Bath et al. 2000; Venton et al. 2002; Heien et al. 2003) have led to increased signal-to-noise ratios for fast-scan cyclic voltammetry. Improved sensitivity first allowed experiments to move from anesthetized to freely-moving rats (Garris et al. 2003), as awake rats did not tolerate the larger electrical currents used to stimulate dopamine release in anesthetized rats. The present study builds on these advances by characterizing drug effects on electrically-evoked versus spontaneous dopamine signals in the awake rat.
The baseline rate of dopamine transients was similar in the NAc and the OT before nomifensine administration at ∼ 0.5 transients/min. The behavior of the rats during this time ranged from inactivity to intermittent locomotion around the cage. Consistent with our previous report (Robinson et al. 2002), the transients observed during this time were not associated with any particular behavior, such as rearing or initiating forward movement, nor were they triggered by obvious sensory input. The frequency of dopamine transients increased at the presentation of behaviorally significant stimuli, which replicates and extends our previous report (Robinson et al. 2002). This finding is consistent with a large number of microdialysis reports demonstrating increases in accumbal dopamine at the consumption of food (e.g. Radhakishun et al. 1988; Bassareo and Di Chiara 1999), sniffing of odors (Pfaus et al. 1990; Fibiger et al. 1992; Guion and Kirstein 2001) and exploration of novel objects or environments (Ladurelle et al. 1995; Saigusa et al. 1999; Legault and Wise 2001). In contrast, both increases and decreases in dopamine in the NAc have been reported at the presentation of startling noises, depending on experimental conditions (Humby et al. 1996; Storozheva et al. 2003).
However, a major difference between the present study and microdialysis data is the timing of the dopamine signal. The dopamine transients measured in the present study were localized to the seconds surrounding the presentation of the stimuli, while the increase in dopamine in the microdialysis experiments appeared to persist for tens of minutes, as a result of the time-averaged sampling. It is difficult to directly compare data from the two methods and, indeed, microdialysis and fast-scan cyclic voltammetry probably measure different aspects of dopamine transmission. Fast electrochemical techniques are ideally suited to measuring brief extrasynaptic increases of dopamine that accumulate more rapidly than they can be cleared by the dopamine transporter (Kawagoe et al. 1992; Chergui et al. 1994; Venton et al. 2003b). However, microdialysis is insensitive to these transients except in the presence of a dopamine transporter blocker (Yang et al. 1998), as dopamine must diffuse some distance from functional release sites to the dialysis probe (Bungay et al. 2003).
The dopamine transients measured in this study presumably arise from burst firing of dopamine neurons, although this has not yet been conclusively demonstrated by measuring both firing rate and chemical release from the same neuron in awake rats. However, the frequency at which dopamine transients were detected in the present study and in studies using more sensitive electrochemical parameters (Cheer et al. 2004) is similar to the rates of bursts of ≥ 3 spikes in dopaminergic neurons in awake rats reported by Hyland et al. (2002). Furthermore, the finding that nomifensine enhances the amplitude and duration of dopamine transients strongly supports the dopaminergic origin of these neurochemical signals. These effects of nomifensine on spontaneous dopamine transients are parallel to its effects in the NAc and OT following electrical stimulation of the dopamine fibers at frequencies similar to the frequency of action potentials inside bursts (Garris et al. 1994, 2003; Suaud-Chagny et al. 1995; Fig. 6). In contrast, changes in dopaminergic cell firing after nomifensine administration have not been conducted in awake rats although, in anesthetized rats, dopamine uptake blockers such as nomifensine and cocaine inhibit the firing of dopamine neurons (Einhorn et al. 1988; Mercuri et al. 1992). Nevertheless, while ongoing research attempts to definitively demonstrate the physiological cause of the dopamine transients, an accumulating body of evidence suggests that rapid fluctuations in dopamine concentrations comprise a fundamental aspect of dopamine signaling during behavior (Robinson et al. 2001, 2002; Phillips et al. 2003; Cheer et al. 2004; Roitman et al. 2004).
The histograms of the distributions before and after nomifensine are consistent with the interpretation that the uptake blocker amplifies dopamine signals that were previously undetected, as the distributions appear to be simply shifted towards larger signals. However, as the dopamine signals were often at the detection limit of the analytical technique, the shape of the distribution histograms was not strictly Gaussian. Thus, future studies will be able to measure this more accurately by improving the sensitivity of the technique; while the detection limit in the present study was ∼ 40 nm dopamine, increasing the maximum applied potential from 1.0 to 1.4 V decreases the limit to ∼ 5 nm in anesthetized rats (Heien et al. 2003) and ∼ 13 nm in awake rats (Cheer et al. 2004). On the other hand, if the only effect of nomifensine was to amplify dopamine signals, similar increases in the frequency of naturally-occurring dopamine transients would be expected in both the NAc and OT, as its effect on electrically-stimulated release was very similar between the two nuclei. In spite of this, the number of dopamine transients increased after nomifensine by 570% in the NAc but only by 130% in the OT. One interpretation of this discrepancy is that nomifensine facilitates the occurrence of additional dopamine transients in the NAc by increasing the firing rate of the dopaminergic neurons innervating that nucleus or by an impulse-independent mechanism. In any case, further research using more sensitive detection methods and/or simultaneous electrophysiological recording is required to resolve these issues.
After nomifensine, the transients were likely to occur during ongoing mild stereotypy, as the rats' behavior generally consisted of forward locomotion while sniffing the ground. The increase in locomotor activity correlated well with the effects of nomifensine on electrically-evoked dopamine release in both the NAc and OT, which is a measure of the pre-synaptic effects of nomifensine on the dopamine transporter (Garris et al. 2003). Previous studies have also documented close agreement between the behavioral and pre-synaptic effects of dopamine uptake blockers (Budygin et al. 2000; Garris et al. 2003). In contrast, locomotor activity after nomifensine correlated with the rate of spontaneous dopamine transients only in the NAc. As the frequency of detected transients after nomifensine administration was different between the two nuclei, one interpretation is that the additional dopaminergic activity in the NAc mediated the expression of stereotyped locomotion. This finding is consistent with research indicating that stimulant-induced locomotion is associated with dopamine transmission in the NAc rather than the caudate (Le Moal and Simon 1991; Le Moal 1995) but little is known about the role of the OT in stimulant-induced behaviors.
In control conditions the frequency of dopamine transients in the NAc doubled at the presentation of salient stimuli, whereas after nomifensine administration the rate increased by only 30%. The parsimonious interpretation is that this discrepancy arises from the simple amplification of dopamine transients and, therefore, their detection by nomifensine administration. Regardless of the mechanism, the differential raises the intriguing possibility that the impact of the transients signaling behaviorally relevant stimuli was lessened after nomifensine. While the function of striatal dopamine transients is not yet known, they may be involved in gating neural ensembles in the striatum and connected cortical areas (O'Donnell 2003). The behavioral consequence may be to recognize salient stimuli (Schultz 1998), switch attention toward that stimulus (Redgrave et al. 1999) and facilitate flexible responses (Salamone et al. 1997; Ikemoto and Panksepp 1999). In this case, the functional consequence of an elevation in the tone of dopamine transients, such as that induced by nomifensine in the present study, would be alterations in sensorimotor processing such as those stereotyped behaviors and sensory gating deficits common after stimulant administration (Geyer and Markou 1995; Le Moal 1995). In the present study, the dramatic increase in dopamine transients in the NAc following nomifensine administration was not contingent on the animals' behavior or their environment. Thus, whatever behavior or stimuli that happened to be present might have been inadvertently reinforced. Moreover, new or unexpected stimuli which normally produced a dramatic increase in dopamine signaling would have been less noticed.
In summary, this experiment replicated and extended previous work (Robinson et al. 2002) by establishing that subsecond dopamine signals occur spontaneously throughout the ventral striatum of rats. Moreover, dopamine transients were more likely to occur at the presentation of a variety of salient stimuli. The dopamine uptake blocker nomifensine increased the frequency of detected dopamine transients, both during baseline conditions and at the presentation of stimuli, but more profoundly in the NAc than the OT. These data are among the first to demonstrate a pharmacological effect on naturally-occurring dopamine transients in vivo and establish that nomifensine elevates the tone of rapid dopamine signals in the brain.
The authors thank Dr Regina Carelli for helpful discussion; Megan Austin, Lori Durham, Lisa Gurdin, Matthew Higgins, Shin-Yi Lao, Sophia Papadeas and Elaina Pelky for technical assistance and Chris Wiesen at the University of North Carolina Odum Institute for Research in Social Science for statistical guidance. This work was funded by NIH (R01 DA10900 to RMW).