Abstract
Dopamine transporter (DAT) inhibitors are expected to decrease dopamine (DA) clearance from the extracellular space of the brain. However, mazindol and cocaine have been reported to “anomalously” increase DA clearance rate. To better understand in vivo DAT activity both in the absence and presence of DAT inhibitors, clearance of exogenously applied DA was measured in dorsal striata of urethane-anesthetized rats using high-speed chronoamperometry. As higher amounts of DA were ejected, DA signal amplitudes, but not time courses, increased. Clearance rates increased until near maximal rates of 0.3 to 0.5 μM/s were attained. Provided baseline clearance rates were relatively low (< 0.1 μM/s), local application of either nomifensine or cocaine markedly increased exogenous DA signal amplitudes and time courses. Relative to the low baseline group, locally applied nomifensine decreased clearance rate when baseline clearance was high (∼0.4 μM/s). However, even when baseline clearance rates were high, systemic injection of nomifensine, mazindol, GBR 12909, or benztropine increased DA signal amplitudes to a greater extent than time courses, consistent with the observed increases in clearance rates. In contrast, despite low baseline clearance rates, systemic injection of cocaine, WIN 35,428, ord-amphetamine preferentially increased DA signal time course, consistent with the observed decreases in clearance rates. Our results emphasize that as extracellular DA concentrations increase, DAT velocity increases to a maximum, partially explaining the ability of DAT inhibitors to increase DA clearance rates. However, by itself, kinetic activation is not sufficient to explain the ability of certain systemically administered DAT inhibitors to anomalously increase DA clearance.
The dopamine transporter (DAT) plays an important role in terminating dopaminergic neurotransmission and in setting overall dopaminergic tone in the central nervous system. The activity of the DAT in intact brain can be assessed in real time using in vivo electrochemical recording to measure the clearance of extracellular dopamine (DA; Ewing and Wightman, 1984; Stamford et al., 1984; Cass et al., 1992; Ng et al., 1992; Suaud-Chagny et al., 1995). It has been established that clearance of both stimulation-evoked endogenous DA and locally applied exogenous DA can reliably reflect DAT activity (Wightman et al., 1988;Cass et al., 1993b). Alterations in DAT activity and DA clearance are associated with changes in the amplitudes and/or time courses of the voltammetric DA signals (May et al., 1988; Cass et al., 1993b;Suaud-Chagny et al., 1995). Also, the slope of the initial, pseudolinear portion of the declining DA signal, which takes into account changes in both signal amplitude and time course, has been used as a quantitative measure of DA clearance rate (Stamford et al., 1984;Wightman et al., 1988; Ng et al., 1992).
The activity of the DAT is inhibited by many of the psychomotor stimulants with high abuse liability, such as cocaine andd-amphetamine, and by other clinically used drugs, such as benztropine and mazindol. Cocaine, d-amphetamine, benztropine, and mazindol–as well as a number of other agents such as nomifensine, 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-[3-phenylpropyl]piperazine (GBR 12909), and (−)2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (CFT; WIN 35,428)–all bind to rat striatal DATs with relatively high affinity and inhibit the accumulation of [3H]DA (Javitch et al., 1984; Dubocovich and Zahniser, 1985; Andersen, 1989;Carroll et al., 1992; Boja et al., 1995). Most DAT inhibitors block the DAT-mediated inward translocation, or uptake, of DA (Horn, 1990;Sonders et al., 1997). However, the precise mechanism(s) by which they inhibit DAT activity may differ (Meiergerd and Schenk, 1994; Xu and Reith, 1997). In contrast, the primary effect ofd-amphetamine is to reverse the DAT so that it predominately translocates DA in an outward direction (Parker and Cubeddu, 1986;Sulzer et al., 1993). However, regardless of the mechanism involved, it is well established that inhibition of DAT activity uniformly results in elevated extracellular DA concentrations and psychomotor stimulation (Nomikos et al., 1990; Kuczenski et al., 1991). Thus, exposure to DAT inhibitors would be expected to decrease the rate of DA clearance; this has often been observed (Wightman and Zimmerman, 1990; Cass et al., 1993a). However, in some instances, exposure to DAT inhibitors such as mazindol or cocaine “anomalously” increases DA clearance rate (Stamford et al., 1986; Ng et al., 1992; Cass et al., 1993a).
The goal of the present studies was to better understand the kinetics of in vivo DAT activity, both in the absence and presence of DAT inhibitors. In these experiments we used high-speed chronoamperometry in dorsal striata of urethane-anesthetized rats to measure changes in exogenous DA clearance. This method measures primarily DA uptake in the absence of any direct contributions from released endogenous DA (Gratton et al., 1988; Cass et al., 1993b; Zahniser et al., 1998). We first determined the quantitative relationship between extracellular DA concentrations and DA clearance rates. We then investigated how DATs respond to changes in extracellular DA concentrations induced by DAT inhibitors, administered either locally or systemically, and how the basal activity of DAT influences the subsequent effects produced by DAT inhibitors.
Materials and Methods
Animals.
Male Sprague-Dawley rats (200–380 g; SASCO, Omaha, NE) were used. Groups of four to six animals were housed under a 12-h light/dark cycle with food and water freely available. All animal use procedures were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Colorado Health Sciences Center.
In Vivo Electrochemical Measurements.
The electrochemical recording electrodes each contained a single carbon-fiber sealed in a glass capillary (fiber diameter 30–33 μm; exposed length 90–160 μm) coated at high temperature with Nafion (5% solution, 6–8 coats at 200°C, Aldrich Chemical Co., Milwaukee, WI; see Zahniser et al., 1998). The sensitivities and linearities were determined by generating calibration curves at 22°C for each recording electrode in stock 0.1 M PBS solutions (pH 4). Responses of electrodes were linear for 2 to 10 μM and for 8 to 40 μM (reduced gain) increments of DA (r2 ≥ 0.997). The electrodes showed good sensitivity to DA but were relatively insensitive to ascorbic acid, with an average selectively ratio of DA to ascorbic acid of 2409 ± 383 to 1 (n = 41). They were also relatively insensitive to 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid.
Assemblies consisted of an recording electrode and either single-, double-, or quadruple-barrel micropipettes with outer tip diameters of 10 to 15 μm each. The electrode and the micropipette(s) were mounted together with sticky wax, with the tips separated by 280 to 320 μm. The single-barrel micropipettes contained a 200 μM DA solution (154 mM NaCl and 100 μM ascorbic acid, pH 7.4). Double-barrel micropipettes contained either 200 or 800 μM DA solution in one pipette and either 800 μM or 3.2 mM drug solution (154 mM NaCl, pH 7.4), respectively, in the other pipette. The quadruple-barrel micropipettes contained 200, 400, 600, or 800 μM DA solutions in each pipette.
Rats were anesthetized with urethane (1.25–1.5 g/kg i.p.) and placed in a stereotaxic frame. Body temperature was maintained at 37°C with a heating pad coupled to a rectal thermometer. The skull and dura overlying the striatal recording sites were removed bilaterally. Ag/AgCl reference electrodes were implanted into brain regions remote from recording sites and were cemented into place using dental acrylic. The electrode/micropipette assembly was lowered into the dorsal striatum using the following coordinates calculated from bregma (Paxinos and Watson, 1986): anterior-posterior + 1.5 mm, medial-lateral ± 2.2 mm, dorsal-ventral −3.8 to −5.4 mm. Once in position, a calibrated volume of DA was applied by pressure ejection (12.5–250 nl, 5–40 psi for 0.1–4 s) at 5-min intervals until reproducible responses (variation in signal amplitudes of <±10%) were obtained; this usually occurred within three or four applications. The volume applied was determined and controlled using a stereomicroscope fitted with a reticule in one eyepiece to measure the movement of the meniscus in the micropipette (Friedemann and Gerhardt, 1992). For the extracellular DA concentration experiments, two to three signals were recorded and averaged for each different amount of DA ejected in each animal. The conditions for the local drug application experiments were based on the studies of Cass et al. (1993b). Once two reproducible DA signals were obtained, drug solution was applied at 4 times the concentration and twice the volume of the DA solution 30 to 60 s before the next application of DA. The drug solution was applied slowly over a 30-s period so as to minimize disturbances to the DA signal. Recording continued at 5-min intervals for an additional 30 min. For the systemic drug injection experiments, saline or drug was injected (i.p.) after two reproducible baseline signals were obtained; DA signals were recorded at 5-min intervals for 1 h postinjection.
All in vivo chronoamperometric measurements were made using a high-speed electrochemical recording system (IVEC-10; Medical Systems Corp., Greenvale, NY). Square-wave pulses of 0.00 to +0.55 V, with respect to the reference electrode, were applied to the recording electrode for 100 ms and repeated 5 times/s. The resulting oxidation and subsequent reduction currents were digitally integrated during the last 80 ms of each 100-ms pulse. Changes in extracellular DA signals were expressed quantitatively in terms of the DA calibration curves (see Zahniser et al., 1998).
Data and Statistical Analysis.
Data are presented as mean values ± S.E.M. N equals the number of animals, except for the local drug application experiments in which N equals the number of electrode/micropipette assembly placements. Three parameters were calculated from the DA oxidation currents: 1) maximal signal amplitude; 2) signal time course (T80), which is the time for the signal to rise to its maximum and to decay by 80%; and 3) clearance rate, which is the slope of the initial pseudolinear portion (between the T20 and T60 time points) of the decaying signal. The validity and reliability of these parameters to reflect changes in DA clearance have been demonstrated in a number of studies (Stamford et al., 1984; May et al., 1988; Wightman et al., 1988; Ng et al., 1992;Cass et al., 1993b; Suaud-Chagny et al., 1995). The amplitude reflects changes in extracellular DA concentration. The T80 reflects the time for the signal to return essentially to baseline and takes into account changes in the “tail” of the decay curve where the DA concentrations are lower; this parameter is often preferentially affected by DAT inhibitors. The clearance rate is an initial rate, taking into account changes in both amplitude and time course.
The concentration-clearance curve was fit to the equation for a rectangular hyperbola using InPlot software (GraphPad, San Diego, CA). Statistical analyses were carried out using SYSTAT software (SPSS, Inc., Chicago, IL). All p values <.05 were considered statistically significant. One-factor ANOVAs were used to compare groups in the local drug application experiments, and Student’st tests with Bonferroni’s correction were used for post hoc analysis. Two-factor ANOVAs with repeated measures were used to test for significant concentration × volume and group/dose × time interactions. When significant interactions were observed, one-factor ANOVAs were used to test for significant effects of volume or time. In the case of volume, Student’st tests with Bonferroni’s correction were used for post hoc analyses. Significant effects involving dose were further analyzed using Tukey-Kramer post hoc tests to determine which doses produced significantly different effects. For these analyses, data were collapsed across time, as indicated in the figure legends.
Drugs.
Nomifensine maleate, mazindol, and GBR 12909 dihydrochloride were purchased from Research Biochemicals International (Natick, MA). Benztropine mesylate and d-amphetamine sulfate were purchased from Sigma Chemical Co. (St. Louis, MO). CFT methyl ester tartrate and (−) cocaine HCl were obtained from National Institute on Drug Abuse (Research Triangle Park, NC).
Results
Effect of Extracellular DA Concentration on In Vivo DA Clearance Rate.
The relationship between the extracellular concentration of exogenously applied DA and the in vivo clearance rate of DA was investigated in the medial dorsal striata of urethane-anesthetized rats. Different extracellular concentrations were achieved by locally pressure-ejecting different amounts (picomoles) of DA from the electrochemical electrode/micropipette assemblies. Two different approaches were compared: 1) ejecting four different volumes (25, 50, 75, or 100 nl) of DA from a single micropipette barrel and 2) ejecting the same four volumes of DA from quadruple micropipette barrels, each of which contained a different concentration of DA (200, 400, 600, or 800 μM). Oxidation and reduction currents were measured using high-speed chronoamperometry. For simplicity, only data from oxidation signals are reported here. The low micromolar concentrations of DA transiently achieved in our experiments (Fig.1) are substantially higher than the low nanomolar steady-state endogenous DA levels found using in vivo microdialysis (Kuczenski et al., 1991; Kuczenski and Segal, 1992). However, the DA concentrations tested here may be physiologically relevant for several reasons. First, electrical stimulation like that used in brain stimulation reward studies (single 500-ms trains of pulses applied to the ventral tegmental area) results in transient DA concentrations as high as 4 μM (Gratton et al., 1988). Second, the velocity of the DAT did change in response to these DA concentrations (vide infra), suggesting that they may be in the range normally encountered by the DAT.
DA signals with progressively larger amplitudes resulted as the extracellular DA concentration was increased (Fig. 1A). Signal amplitudes increased in a linear/curvilinear fashion to values as high as 20 μM. In general, similar amplitudes were observed when the same number of picomoles of DA were ejected, regardless of whether different volumes or concentrations were used to achieve a certain number of picomoles. However, the signal amplitudes were significantly higher when 20 pmol of DA resulted from ejection of 100 nl of 200 μM DA, as opposed to 25 nl of 800 μM DA, the most extreme difference tested (Fig. 1A). In contrast, over the range of amounts of DA ejected, the time courses of the DA signals, or the T80values, remained relatively constant (Fig. 1B). The T80 values were approximately twice as long with 800 μM DA (45–50 s), as compared with 200 μM DA (25–28 s). However, this apparent difference was not statistically significant. Clearance rates increased as higher amounts of DA were ejected (Fig.1C). Clearance rate takes into account changes in both signal amplitude and time course in the pseudolinear, decaying portion (T20-T60) of the DA signal. Clearance rates increased in a curvilinear saturating fashion, with the exception of the clearance rates from the 200 μM DA concentration, which appeared to increase in a more linear fashion.
The maximal DA signal amplitude indicates the extracellular concentration of exogenous DA achieved in these experiments. Because DAT activity obeys Michaelis-Menten kinetics (Nicholson, 1995), the apparent in vivo transporter affinity (KT) for exogenous DA and maximal velocity (Vmax) of the DAT can be determined by plotting the clearance rate as a function of signal amplitude. The data from Fig. 1, A and C, are plotted in this manner in Fig. 2A. Three out of the four curves appeared to be approaching an asymptote of 0.4 to 0.5 μM/s. To better define the maximal velocity, a second set of experiments was conducted using a wider range of volumes (25–250 nl) with electrode/single-barrel micropipette assemblies containing 200 μM DA (Fig. 2B). The DA clearance rate again appeared to plateau at ∼0.45 μM/s. Fitting these data to a rectangular hyperbolic curve revealed aKT of 12 μM and aVmax of 0.7 μM/s (r2 = 0.994).
Effects of Locally-Applied DAT Inhibitors on In Vivo DA Clearance Rate.
The relationship between the initial baseline exogenous DA clearance rate in medial dorsal striata of urethane-anesthetized rats and the change in this rate induced by local application of a DAT inhibitor was next examined. First, reproducible baseline DA signals were obtained in response to locally applied DA (either 200 or 800 μM). Three baseline response groups were defined by their initial baseline clearance rates. The “low” and “medium” groups corresponded to application of 200 μM DA, whereas the “high” group corresponded to application of 800 μM DA. The low and medium clearance rates observed with the 200 μM DA likely reflect locally lower and higher densities of DATs, respectively. The mean initial baseline clearance rate of the high group was 0.30 ± 0.04 μM/s, ∼20-fold higher than that of the low group and 5-fold higher than that of the medium group. The baseline signal amplitudes showed a similar profile, with the high group having a mean amplitude of 7.9 ± 0.44 μM; this amplitude was ∼40-foldhigher than that of the low group and 8-fold higher than that of the medium group. In contrast, the mean T80 values ranged from 37 to 42 s and did not differ among the three groups. Each of these three groups was then randomly subdivided into two groups for the local nomifensine or cocaine application experiments (Table1).
After two reproducible baseline DA signals and 30 to 60 s before the next DA ejection, either the DAT inhibitor nomifensine or cocaine was pressure-ejected, at a 4-fold higher concentration and 2-fold higher volume than DA, from the second barrel of the micropipette (Cass et al., 1993b). Consistent with inhibition of DAT, nomifensine increased both DA signal amplitudes and T80values (Fig. 3). The increased amplitudes induced by nomifensine were inversely related to the baseline clearance responses (Fig. 3A). Significant maximal increases of 500% and 100% above baseline were induced in the low and medium baseline response groups, respectively, whereas no significant change was induced in the high group. In the 30 min after nomifensine application, amplitudes in the low group were significantly greater than those in either the medium or high groups. The T80 values were also increased above baseline, indicating significant prolongation of the signal time courses, in the low and high groups (Fig. 3B). The low group again showed the most pronounced change. Local application of nomifensine did not significantly change the clearance rates from baseline in any of the groups (Fig. 3C). However, comparison of nomifensine-induced changes in clearance rates in the low and high groups revealed a significant difference, with the clearance rate being slower in the high group.
Like nomifensine, local application of cocaine increased DA signal amplitudes and time courses, but, as expected, the effects of cocaine were more transient than those of nomifensine (Fig.4). Amplitudes and T80 values were significantly increased above baseline in all three groups by exposure to cocaine (Fig. 4, A and B). Again, the magnitudes of the cocaine-induced increases were greatest in the low baseline clearance group; and the effects produced in the low and high groups were significantly different. However, clearance rates were not significantly altered (Fig. 4C). Thus, local application of nomifensine or cocaine increased both DA signal amplitudes and time courses; and the increases were greatest when the initial baseline DA signal clearance rates were low. The drug-induced changes in clearance rate were less consistent, but there was a trend for clearance rates to increase in the low group and decrease in the high group.
The Effects of Systemically Injected DAT Inhibitors on In Vivo DA Clearance Rate.
The effect of systemic (i.p.) administration of DAT inhibitors on exogenous DA clearance rate in medial dorsal striata of urethane-anesthetized rats was also explored. First, stable baseline signals were obtained in response to pressure-ejection of 200 μM DA (25–250 nl) from electrode/single-barrel micropipette assemblies (Table 2). The baseline amplitudes ranged from 1.6 to 5.9 μM, T80 values ranged from 16 to 41 s and clearance rates ranged from 0.10 to 0.48 μM/s. Subsequently, each animal received an i.p. injection of either saline or drug; we continued to eject DA once every 5 min and to monitor the resulting signals for the next 60 min. After saline injection, the amplitudes and T80 values of the exogenous DA signal slowly, but significantly, declined by 15 to 20% from baseline, whereas the clearance rates remained relatively constant. Because the control and drug experiments were interspersed, the results of all the saline experiments were combined and are shown with each of the drugs (Figs. 5-11).
Initially the effects of nomifensine, mazindol, GBR 12909, and benztropine were investigated. Nomifensine was tested at two doses. In both groups the baseline clearance rates were relatively high (0.4–0.5 μM/s; Table 2). Administration of 3 mg/kg of nomifensine produced a significant 15% increase from baseline in the T80 value, whereas administration of 10 mg/kg produced a significant 50% increase in amplitude (Fig. 5). However, both doses of nomifensine produced a significant increase in DA signal amplitude when compared with saline (Fig. 5A). Although the higher dose of nomifensine increased mean clearance rate by 60%, this effect was not statistically significant (Fig. 5C). The baseline clearance rate of the mazindol group was also relatively high, 0.4 μM/s (Table 2). The predominant effect of mazindol (3 mg/kg) was to increase both amplitude and clearance rate by approximately 25 to 30% (Fig.6). The mazindol-induced increase in amplitude was significantly different from saline, whereas the increase in clearance rate was significantly different from baseline. The initial baseline clearance rates for the GBR 12909 and benztropine groups were 2-fold lower (0.2 μM/s; Table 2). However, once again, both GBR 12909 (Fig. 7) and benztropine (Fig. 8) produced persistent, significant increases in DA signal amplitude and clearance rate. GBR 12909 (10 mg/kg) increased all three parameters measured relative to baseline (Fig. 7), whereas benztropine (10 mg/kg) increased only amplitude and clearance rate (Fig. 8). Furthermore, the increased amplitudes and clearance rates resulting from administration of either GBR 12909 or benztropine were significantly different from saline. To summarize, this group of drugs–nomifensine, mazindol, GBR 12909, and benztropine–increased DA signal amplitudes with minimal changes in T80 values. These results are consistent with the increased rates of exogenous DA clearance, which were observed whether or not the initial baseline clearance rates were close to maximal.
In a second set of experiments, cocaine, CFT, andd-amphetamine were studied. In these experiments, only one group of animals, the one that subsequently received the 20 mg/kg dose of cocaine, had a relatively high initial baseline clearance rate (0.3 μM/s; Table 2). All other groups had baseline clearance rates of 0.1 to 0.2 μM/s. Administration of cocaine (20 and 30 mg/kg) produced a dose-related increase in the T80 values, relative to baseline, but no significant changes in either amplitude or clearance rate (Fig. 9). As expected from its pharmacokinetic profile, the effects of cocaine were more transient than those of the other drugs tested. Compared with saline, only the higher dose resulted in a statistically significant 50% increase in time course and 30% decrease in clearance rate (Fig. 9, B and C). The effect of the cocaine congener CFT (3 and 10 mg/kg), relative to baseline, was also to alter the signal amplitudes minimally but to increase the T80 values and to decrease the clearance rates significantly at both doses tested (Fig.10). The effect of the 10 mg/kg dose of CFT to increase T80 was significantly different from saline, as was the effect of both doses to decrease clearance rate. Lastly, three doses of d-amphetamine (1, 5, and 10 mg/kg) were tested. It should be noted that although amphetamine releases DA, our method eliminates any direct contribution of the endogenous DA to the signal by rezeroing the baseline before each ejection of DA. Only the highest dose of d-amphetamine increased (25%) DA signal amplitude, whereas the 5-mg/kg dose, like saline, produced a significant decrease in amplitude relative to baseline (Fig. 11A). The change in amplitude induced by the 10-mg/kg dose of d-amphetamine differed significantly from that induced by either saline or 5-mg/kgd-amphetamine. On the other hand, relative to baseline, all three doses of d-amphetamine tested produced significant increases of 50 to 250% in the T80 values and decreases of 30 to 70% in the clearance rate (Fig. 11, B and C). The effect of d-amphetamine on T80 was somewhat more dose-related in that the increase induced by the 10-mg/kg dose was significantly different from that produced by saline or the 1- and 5-mg/kg doses. The effects on T80 and clearance rate produced by both the 5- and 10-mg/kg doses ofd-amphetamine differed significantly from saline. Thus, the predominate effect of cocaine, CFT, and d-amphetamine was to increase the duration of the locally applied DA signals rather than to alter signal amplitude. These results are consistent with the fact that these drugs decreased exogenous DA clearance rate, despite the fact that the initial baseline clearance rates were well below the maximal rate.
Discussion
In the medial dorsal striata of urethane-anesthetized rats, we observed that exogenous DA signal amplitudes and clearance rates, but not signal time courses (T80 values), increased with increasing amounts of DA ejected. Thus, the velocity of DAT in vivo accelerated as extracellular DA concentrations increased until rates of 0.4 to 0.5 μM/s were attained. When baseline clearance rates were relatively low (<0.1 μM/s), local application of the DAT inhibitors nomifensine or cocaine markedly increased both DA signal amplitudes and times courses. Clearance rates were either increased or unaltered, presumably due to the balance between the higher activity of the uninhibited DATs in response to increasing extracellular DA concentrations and the inhibitory effects of nomifensine or cocaine. Decreased clearance rates, reflected as prolongation of signal time courses with little change in amplitude, were observed only when the baseline clearance rate was high (∼0.4 μM/s). In contrast, the effects of systemically administered DAT inhibitors were not readily explained by differences in baseline DA clearance rates. Even when baseline clearance rates were high, systemic injection of nomifensine, mazindol, GBR 12909, or benztropine increased DA signal amplitudes to a greater extent than time courses. These results are consistent with the observed increases in clearance rates. On the other hand, systemic administration of cocaine, CFT, or d-amphetamine preferentially prolonged the duration of the DA signals, consistent with the observed decreases in clearance rate. Reduced clearance rates were observed even when the baseline clearance rates were low. Taken together, these data suggest that the mechanisms by which these two groups of DAT inhibitors, at least when administered systemically, affect transporter activity are not identical. Furthermore, our results emphasize that increased DA clearance rates can accompany DAT inhibition.
Two methods were used to increase exogenous extracellular DA concentrations and resulted in similar maximal DAT clearance rates. Different DA concentrations (200–800 μM) were ejected from quadruple-barrel micropipettes, or a wider range of volumes of 200 μM DA was ejected from a single-barrel micropipette. With increasing amounts of DA, higher amplitudes were observed. Generally, similar amplitudes were detected for a given amount of DA ejected with both methods. However, at the extremes, a significantly higher amplitude was observed with the larger volume ejected, e.g., 100 nl of 200 μM DA versus 25 nl of 800 μM DA (20 pmol DA; Fig. 1A). This difference may reflect the different sized spheres of DA achieved in the brain microenvironment (Nicholson, 1985) and the 300-μm distance between the ejection micropipette and the electrochemical electrode. In contrast with the greater signal amplitudes, the signal time courses were unchanged as DA concentrations increased. These results indicate that clearance rate increased. It is well known that in vitro DAT activity obeys Michaelis-Menten kinetics (Nicholson, 1995). Thus, it is not surprising that, as extracellular DA increased, in vivo DA clearance rates increased in a saturable fashion until an apparent maximal value of 0.4 to 0.5 μM/s was reached. A maximal rate of ∼0.45 μM/s has also been observed when clearance of K+-evoked endogenous DA was measured in dorsal striatum of Fischer 344 rats (M.A. Hebert and G.A.G., unpublished observations). Therefore, clearance rates for exogenous and endogenous DA appear comparable in dorsal striatum.
The maximal in vivo DAT clearance rate derived here is also in good agreement with those reported from a variety of in vitro and in vivo techniques in rat striatal tissue. The range ofVmax values summarized from the literature is 0.1 to 0.8 μM/s (see Table 1 in Nicholson, 1995). Curve fitting of the concentration-clearance relationship in Fig. 2B revealed a KT of 12 μM and aVmax of 0.7 μM/s. Nicholson (1995)proposed that concentrations measured in vivo should be corrected by the extracellular volume fraction, α = 0.21 (Rice and Nicholson, 1991). Multiplying our Vmax value by α yields a maximal rate of 0.14 μM/s. The affinity of DAT for DA in striatum is generally reported to be 0.1 to 0.4 μM (see Table 1 inNicholson, 1995). However, the KTvalue we derived (2.5 μM, corrected by α) was at least 6-fold higher. The urethane anesthesia is unlikely to be the explanation for the apparent lower affinity. For example, Jones et al. (1995b), using fast-scan cyclic voltammetry to monitor the disappearance of electrically-evoked DA release in striatum of urethane-anesthetized rats and nonlinear regression of the Michaelis-Menten equation to determine kinetic parameters for DA uptake, reported aKT of 0.22 μM and aVmax of 3.8 μM/s. Furthermore,Garris et al. (1997) reported that the kinetic constants for striatal DAT were similar in urethane-anesthetized and unanesthetized, freely moving rats. It is possible that ourKT value is inaccurate, given that the clearance rate was measured from the pseudolinear portion of the signals where the DA concentration is well aboveKT.
DAT inhibitors increase in vivo extracellular DA concentrations (Nomikos et al., 1990; Kuczenski et al., 1991), and higher concentrations of DA result in acceleration of DAT velocity (vide supra). Therefore, when subsaturating concentrations of both DA and DAT inhibitors are present, one would predict that the rate of uptake by the DATs that are unoccupied by inhibitor would be accelerated. With local application of drugs, it is difficult to predict the concentrations achieved. In addition, the drugs are applied only transiently, which does not allow for steady state to develop. Nonetheless, this method has been useful to demonstrate that DAT inhibitors alter DA signals, whereas other drugs do not (Cass et al., 1993b). When baseline DA clearance rates were low (< 0.02 μM/s), we observed marked, but transient, increases in both DA signal amplitudes and time courses in response to local application of nomifensine or cocaine. With nomifensine, clearance rate was also enhanced. This effect is reminiscent of the “anomalous” DAT inhibitor-induced increase in clearance rate previously reported for mazindol and cocaine (Stamford et al., 1986; Ng et al., 1992; Cass et al., 1993a). However, when baseline clearance rates were high (0.4 μM/s), local application of nomifensine reduced DA clearance rate, presumably because any noninhibited DATs were already translocating DA at close to maximal rates.
Our results with systemically administered DAT inhibitors suggest, however, that factors in addition to differences in baseline DA clearance rates contribute to the observation that DAT inhibitors can increase DA clearance rates. Each drug was administered at behaviorally active doses, and significant inhibition of striatal DAT activity resulted. Systemic injection of nomifensine, mazindol, GBR 12909, and benztropine preferentially increased signal amplitudes and clearance rates. No decrease in clearance rates occurred even when the baseline clearance rates were high (0.3–0.5 μM/s), as was the case for nomifensine and mazindol. In contrast, systemic administration of cocaine, CFT, and d-amphetamine preferentially decreased clearance rates and thereby increased the signal time courses. These results were observed even though the majority of the baseline clearance rates were somewhat lower, 0.1 to 0.2 μM/s. The most striking effects were produced by d-amphetamine, which dose-dependently diminished clearance rate. Interestingly, benztropine produced different effects from cocaine and CFT, even though all three compounds are tropane derivatives. It is tempting to speculate that these different effects may play a role in the higher abuse potential of drugs like cocaine and d-amphetamine (Foltin and Fischman, 1991). However, previously, both mazindol and cocaine have been reported to increase DA clearance rate (Stamford et al., 1986; Ng et al., 1992; Cass et al., 1993a). Furthermore, Suaud-Chagny and colleagues (1995), measuring clearance of electrically-stimulated DA release in striatum, observed more marked increases in signal amplitude than time course (T50) after systemic administration of nomifensine and cocaine, but the opposite relationship with GBR 12909 and mazindol. In any case, our results emphasize that increases in DA clearance rate may occur in response to DAT inhibitors.
There are several mechanisms that could contribute to the different results with the two groups of DAT inhibitors. First, the mechanisms by which the two groups inhibit DAT activity may not be identical. Whether all of the drugs tested are competitive inhibitors of DAT is unclear. However, most evidence suggests that they are (see Xu and Reith, 1997). For example, mazindol, GBR 12935, cocaine, and CFT all bind to the same single site in mouse striatum (Reith and Selmeci, 1992). Likewise, nomifensine and cocaine competitively inhibit uptake of stimulation-evoked DA in striatal slices (Jones et al., 1995a). However, using rotating disk electrode voltammetry and kinetic modeling to measure DA uptake in in vitro rat striatal suspensions, Meiergerd and Schenk (1994) found that cocaine is competitive with GBR 12909 and benztropine, but not with nomifensine or mazindol. A second possibility relates to differences in the abilities of the two drug groups to affect reverse transport of DAT. Eshleman et al. (1994) found a similar grouping of drugs: nomifensine, mazindol, GBR 12935, and benztropine all inhibited spontaneous release of DA via reversal of DAT expressed in COS-7 cells, whereas drugs with abuse potential either had no effect (cocaine and CFT) or enhanced (amphetamine) DA release. Although our method factors out any direct contribution of endogenous DA to the clearance measurement, different endogenous DA concentrations could indirectly impact clearance by altering DAT activity. The rank-order based on the magnitude of the increased extracellular DA concentration observed in striatum of freely-moving rats is amphetamine (5 mg/kg) ≫ cocaine (30 mg/kg) > nomifensine (10 mg/kg) (Kuczenski and Segal, 1992). However, in chloral hydrate-anesthetized rats, the relationship between nomifensine and cocaine is reversed (Church et al., 1987). A third possibility relates to the fact that nomifensine, GBR 12909, and benztropine have 20- to 200-fold lower affinities for the serotonin transporter than for DAT (mazindol has only a 2-fold lower affinity; Hyttel, 1982; Ritz et al., 1987; Andersen, 1989). In contrast, the affinities of cocaine, CFT, and d-amphetamine are similar for the two transporters (Hyttel, 1982; Ritz et al., 1987). Systemic injection of these latter drugs could increase extracellular serotonin in the dorsal raphe nucleus, thereby regulating dorsal raphe neuronal firing. Dorsal raphe neurons regulate DA release in dorsal striatum (De Deurwaerdère et al., 1998). Altered extracellular DA concentrations may, in turn, modulate DAT activity. This mechanism could also explain our different results with locally applied and systemically administered cocaine. These possibilities remain to be tested in future experiments.
Acknowledgment
We thank Dr. Shelly Dickinson for help with the statistical analyses.
Footnotes
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Send reprint requests to: Dr. Nancy R. Zahniser, Department of Pharmacology C-236, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. E-mail:nancy.zahniser{at}uchsc.edu
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↵1 This work was supported by National Institutes of Health Grants DA04216 (N.R.Z.), NS09199, and AG06434 (G.A.G.), and Research Scientist Development Awards DA00174 (N.R.Z.) and MH01245 (G.A.G.). A preliminary report of this work has been presented: Zahniser NR, Larson GA and Gerhardt GA (1996) Soc Neurosci Abstr22:1577.
- Abbreviations:
- CFT
- (−)2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane or WIN 35,428
- DA
- dopamine
- DAT
- dopamine transporter
- GBR 12909
- 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine
- KT
- transporter affinity
- Vmax
- maximal velocity
- Received August 13, 1998.
- Accepted November 24, 1998.
- The American Society for Pharmacology and Experimental Therapeutics