Introduction

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The nigrostriatal DA pathway is an important component of basal ganglia circuitry, which has been postulated to play a critical role in the initiation and maintenance of motor behaviors (Graybiel, 1995). Loss of nigrostriatal DA function leads to the clinical manifestations of Parkinson's disease (PD) in humans, which include rigidity, tremor and bradykinesia (Hornykiewicz and Kish, 1987). In both human PD and in animal models of the disease, replenishment of midbrain DA levels, especially within the striatum, has been shown to ameliorate many of the symptoms of the illness. However, the failure of DA replacement strategies to completely restore normal motor function in individuals affected with PD has led several investigators to reconsider the current model of DA and its role within the basal ganglia. In particular, there has been a renewed focus on the role of DA in "extrastriatal" nuclei within the basal ganglia, such as the subthalamic nucleus, globus pallidus and SN. A growing body of literature supports the concept that DA signaling within the SN may modulate basal ganglia output, thereby contributing to various motor behaviors (Robertson and Robertson, 1989; Hudson et al., 1993; Tseng et al., 1997). Thus, while the importance of DA within the striatum remains unequivocal under both normal and pathophysiological conditions, there is evidence to suggest that investigations of DA regulation within other areas of the basal ganglia would enhance our current understanding of movement and movement disorders.

The high-affinity DAT is a primary regulator of DA signaling at mammalian synapses and is known to be localized to both the somatodendritic and axonal processes of midbrain DA neurons (Ciliax et al., 1995; Nirenberg et al., 1996b). In particular, DAT has been shown to exist on cell bodies of the SNc, as well as on dopaminergic dendrites within the SNr (Coulter et al., 1995; Nirenberg et al., 1996b). The DAT, SERT and NET are related members of a superfamily of Na⁺- and Cl-dependent neurotransmitter transporters, which are important targets for both drugs of abuse and antidepressant compounds (Giros and Caron, 1993; Amara and Kuhar, 1993). Blockade or reversal of DAT-mediated transport results in an elevation of extracellular DA levels and has been shown to underlie the neurobiological actions of several major psychomotor stimulants, including cocaine and amphetamine (Giros et al., 1996). The contribution of DAT to DA release and clearance has been most clearly established within terminal regions, such as the striatum and nucleus accumbens, which are enriched in DAT relative to NET and SERT. However, anatomic studies suggest that DAT may also play a role in mediating somatodendritic DA clearance and have led some investigators to postulate that DAT reversal may represent a primary means of somatodendritic DA release (Nirenberg et al., 1996b). Given the potential importance of DA released within the SN to influence motor behaviors, as well as the known role of DAT in central DA regulation, functional studies pertaining to the actions of DAT within the SN are clearly of interest.

Despite the anatomical evidence that DAT plays a major role in DA clearance, biochemical studies have suggested that SERT and/or NET may also contribute to DA uptake in the SN (Kelly et al., 1985; Simon and Ghetti, 1993). However, these studies rely on prolonged incubation of dissociated tissue with [³ H]DA and may therefore be less applicable to physiological conditions. In contrast, in vivo voltammetric recording techniques have been developed that allow for fast (<1 sec) and sensitive (nM) detection of DA release and uptake (Kawagoe et al., 1993). In anesthetized animals, DA uptake can be assessed by locally applying small (pmol) amounts of DA from a micropipette attached 250 to 300 µm from a carbon fiber electrode. The parameters of the DA electrochemical signal, detected by high-speed chronoamperometry, are then used to describe DA uptake. Additionally, a variety of pharmacological manipulations have been shown to modulate the DA signal in a manner consistent with loss of DAT activity (Cass et al., 1993a; Cass et al., 1993b; Cass and Gerhardt, 1995). Recently, using this approach in the SN of anesthetized rats, we have shown that DAT-mediated DA clearance can be modulated by uptake inhibitors and 6-hydroxydopamine lesions of the nigrostriatal pathway (Hoffman and Gerhardt, 1998). Although such recordings in the intact animal are clearly feasible, there are technical issues, such as localization of probe placement, which make these studies more demanding. In contrast, the brain slice preparation allows for both ease of access to discrete anatomic sites such as the SN, as well as controlled delivery of drugs to tissue.

In the present study, we used high-speed chronoamperometric recording techniques in rat brain slices to address several questions regarding somatodendritic DA regulation. First, we compared the properties of DA uptake and clearance in the SN with those seen in the striatum to determine if base-line clearance properties differed between the two regions. Next, SN slices were superfused with DAT, NET or SERT uptake inhibitors to determine which transporter(s) were responsible for DA clearance in this brain region. The effects of DAT inhibitors in the SN were compared with the effects of these same drugs in the striatum. Finally, we compared stimulus-evoked release between the SN and striatum using KCl-evoked depolarization and d-amphetamine-evoked DA release.

	Materials and Methods

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Drugs and drug solutions. DA, cocaine hydrochloride and d-amphetamine sulfate were purchased from Sigma Chemical (St. Louis, MO). DMI and nomifensine maleate were purchased from Research Biochemicals (Natick, MA). Citalopram was generously provided by Dr. Alan Frazer. Uptake inhibitors were prepared as fresh 100× stocks dissolved in distilled H₂O and were bath-applied to the slices by a Razel pump (Model A99).

Slice preparation. All animal protocols were approved by the University of Colorado Institutional Animal Care and Use Committee. Young Sprague-Dawley rats (50-75 g) were anesthetized with halothane and decapitated. The brains were rapidly removed and chilled in ice-cold aCSF (126 mM NaCl, 2.9 mM KCl, 1.5 mM MgCl₂, 2.5 mM CaCl₂, 1.4 mM NaH₂PO₄, 10 mM glucose and 25 mM NaHCO₃, 200 µM ascorbate), which was continuously bubbled with a 95% O₂/5% CO₂ mixture. After blocking the tissue, the brains were mounted in a chilled, aCSF-filled chamber, and coronal sections (300 µm thickness) were cut using a vibratome Series 1000 (Technical Products International, St. Louis, MO). Sections containing the brain regions of interest, with respect to bregma, were as follows: striatum, AP 0.26 to +1.7 mm; SN, AP 6.04 to 4.8 mm; hippocampus AP 4.52 to 3.14 mm; dorsal raphe, 8.0 to 7.3 mm (Paxinos and Watson, 1986). Slices were incubated in oxygenated aCSF at 22°C for at least 1 hr before electrochemical recordings. During the recordings, slices were continuously superfused with oxygenated aCSF solution at a rate of 2 ml/min. All recordings were carried out at 32° to 33°C.

Electrochemical recordings. High-speed chronoamperometric measurements were carried out using an IVEC-10 system (Medical Systems, Greenvale, NY). Square-wave pulses of 0.00 to +0.55 V, with respect to a Ag/AgCl reference electrode, were applied to the working electrode for 100 msec and repeated 5 times per sec (Gratton et al., 1989). The resulting oxidation current (measured during the 0 to +0.55 V step) and reduction current (measured during the +0.55 V to 0 step) were digitally integrated during the last 80 msec of each pulse. Single carbon fiber electrodes (AVCO Specialty Materials, Lowell MA; 100 µm length × 30 µm o.d.) were coated with Nafion using the high temperature coating procedure previously described (Hebert et al., 1996). All electrodes were calibrated using 2 to 10 µM increments of DA before each experiment and were both linear (r²  .997) and selective (500:1) for DA vs. either DOPAC or ascorbate. Based on the measured detection limit of the electrodes, DA signals had to achieve amplitudes of 20 ± 2 nM (n = 30) to obtain a signal-to-noise of ratio of >3. Extracellular changes in DA concentration were expressed quantitatively based on the pre-experiment DA calibration curves (Gerhardt et al., 1988). In a few experiments, electrodes were calibrated with serotonin (5-HT), using the "delayed pulse" protocol previously described (Luthman et al., 1997).

DA clearance experiments. DA was locally applied at 5-min intervals to establish a stable base-line (10% variation in the measured signal parameters). After three such base-line signals were obtained, uptake inhibitors were added to the bath at the desired concentration; subsequent DA applications were performed every 5-min. When drug effects were seen, maximal effects were observed 10-min after drug application. Washout of drug effects within 20 to 30 min was observed in many, but not all, cases.

DA release experiments. Local pressure applications of either d-amphetamine or KCl were performed in multiple sites in several slices, to obtain a large number of signals for analysis. Although the pressure x time parameters varied between experiments (15-20 psi, 2-4 sec), our experience utilizing these single-barrel pipettes and pressure × time parameters suggests that volume outputs are likely to be 50 to 200 nl. Identification of the endogenous signal as "DA-like" was based on the red/ox ratio of the electrochemical signal (Gratton et al., 1989; Gerhardt, 1995). Typical in situ red/ox ratios were between 1 and 1.4 for DA, 0.05 and 0.1 for 5-HT and 0 for ascorbate.

Signal parameters. For each individual signal, the following parameters were analyzed: (1) peak amplitude of the obtained signal; (2) T₈₀, the time for the signal to rise and decay by 80% from peak amplitude; and (3) clearance rate (T_c, in µM/sec), defined by the change in DA concentration between the T₂₀ and T₆₀ time points (e.g., the slope of the linear portion of the decay curve). These parameters were selected based on previous work from our laboratory that has characterized the effects of uptake inhibitors on these portions of the electrochemical signal (Cass et al., 1993a, 1993b; Cass and Gerhardt, 1995) (Hoffman and Gerhardt, 1998). In addition, these parameters were chosen because they are known to primarily reflect DA uptake, rather than metabolism or diffusion (Cass et al., 1993b). The red/ox ratio, calculated by dividing the reduction current by the oxidation current at the peak of the response, was also analyzed for each signal. For the d-amphetamine-evoked release experiments, the rise time of the signal to peak amplitude was also analyzed.

Statistical analysis. Linear regression analysis was performed using Prism version 2.01 (GraphPAD Software, San Diego, CA). For the experiments with the uptake inhibitors, base-line parameters were defined at each site by averaging three reproducible signals to yield a single value at a given recording site. Changes in these parameters after drug application were expressed as a percentage change from the base-line and were analyzed using a two-tailed Student's t test (hypothetical mean of 100%). Comparisons among the striatum, cortex and SN were performed using a one-way ANOVA followed by Tukey-Kramer post-hoc comparisons. All other comparisons were performed using a two-tailed Student's t test. In all tests, P < .05 was defined as statistically significant. Statistical analyses were performed using SigmaStat 2.0 (Jandel Scientific Software, San Rafael, CA) or Instat 2.04 (GraphPAD Software).

	Results

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Comparisons of Base-line DA Clearance Properties Among Different Brain Regions

Previous studies utilizing high-speed chronoamperometric recordings in acutely anesthetized rats have shown that DA clearance can differ dramatically among various brain regions, and we have hypothesized that these differences relate to the distribution and activity of DAT sites (Cass and Gerhardt, 1995; Hoffman and Gerhardt, 1998). To confirm this hypothesis in the slice preparation, we collected a large number of electrochemical signals from three brain regionscortex, striatum and SNknown to differ in the number and/or activity of DAT sites. The clearance rates and time courses of signals of equivalent amplitude were then used to compare DA uptake in the various areas.

Substantia nigra versus striatum. To compare properties of DA clearance between the SN and striatum, we examined clearance rates (T_c) and time courses (T₈₀) of electrochemical signals over several amplitude ranges in each brain region. A total of 181 signals from the striatum and 284 signals from the SN were used in the analysis. Red/ox ratios for locally applied DA signals averaged 1.18 ± 0.02. There was considerable variability in the other observed parameters of the electrochemical signals, perhaps owing to the exact location of the recording electrode from experiment to experiment. To facilitate the analysis, signals were arbitrarily grouped into the following amplitude ranges (in µM): <.5, 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 3, 3 to 4 and 4 to 8. The resulting clearance rates and time courses were then plotted against the amplitude ranges. The results of this analysis are shown in figure 1. As shown in figure 1A, in both the SN and striatum, increases in signal amplitude resulted in linear increases in the observed rate of DA clearance (r² = .995 and .963 for the SN and striatum, respectively). However, the slope of the line was significantly (P < .05) lower in the SN, relative to the striatum, indicating a reduced clearance rate over the entire amplitude range. Similarly, for all signal amplitudes tested, the time courses of the electrochemical signals were significantly (P < .05) longer in the SN, relative to the striatum (fig. 1B).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Comparison of DA clearance parameters in striatal () and SN () slices. Signals between 0.2 and 8 µM were sorted into amplitude ranges to simplify the analysis. All data points represent the mean ± S.E.M. of at least 10 data points per range. A, Clearance rates were linear with respect to amplitude over this range in both brain regions (r2 = .96 and r2 = .99 for the striatum and SN, respectively). However, rates of clearance were significantly lower in the SN than in the striatum (slope difference, P < .05). B, T80 values for the same amplitude range. The elevations (intercepts) of the lines were significantly (P < .05) different between the SN and striatum, indicating that time courses were slower in the SN over the tested range of amplitudes.

Striatum vs. cortex. As an additional comparison of DAT function among brain regions, we compared signals of equivalent amplitude in the striatum and cortex. A total of 42 signals (amplitude range, 0.2-10 µM) were collected from regions of the cortex overlying the striatum. Subsequently, an equal number of signals, over the same amplitude range, were selected from the striatum and SN to yield the same (mean) amplitude value. The results are shown in table 1. For signals of equivalent mean amplitude, the time course was significantly (P < .05) prolonged, and the clearance rate was significantly (P < .05) reduced in the cortex and SN, relative to the striatum. Additional experiments, in which the same recording assembly was used in both the striatum and cortex of the same slices, revealed that these differences were not due to variability in the electrode-pipette assembly (data not shown).

Effects of Uptake Inhibitors on DA Clearance

The results outlined above suggest that DA clearance differs across brain regions in a manner consistent with known differences in DAT density. However, to more conclusively demonstrate the role of DAT in mediating the cessation of the DA electrochemical signal, we used uptake inhibitors which are known to interfere with DAT function. In these experiments, DA was locally applied at 5-min intervals to obtain a minimum of three reproducible base-line electrochemical signals. DA clearance was then monitored at 5-min intervals during and after bath application of uptake inhibitors. Changes after drug application were represented as a percentage change from the base-line, which was defined as 100%. Average base-line signals were 2 µM in amplitude in all experiments, since we have previously found that DA signals in this concentration range are consistently modulated by uptake inhibitors (Cass and Gerhardt, 1995; Hoffman and Gerhardt, 1998).

Cocaine. Although cocaine has similar affinity for all three major amine transporters, it is believed that most of its neurobiological effects are related to inhibition of DAT function (Cass et al., 1993a; Giros et al., 1996). We therefore compared the actions of this drug on DA electrochemical signals in both the striatum and SN. A representative experiment in a striatal slice is shown in figure 2. After establishing a stable base-line signal, cocaine (50 µM) was superfused onto the slice for 10-min. This resulted in a potentiation of both the amplitude and time course (T₈₀) of the signal, which gradually returned to base-line after cessation of drug delivery. Figure 3A shows a summary of the effects of cocaine on the observed signal parameters in both the SN and striatum. In both brain regions, cocaine significantly potentiated both the amplitude and time course of the electrochemical signal. The clearance rate parameter (T_c) was not significantly affected by the drug.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Representative effect of cocaine on DA clearance in a striatal slice. Local application of DA at 5-min intervals produced reproducible base-line signals, as shown. Cocaine (50 µM) was applied for a 10-min period, indicated by the solid bar. The last signal in the trace shows a partial washout of the drug effect. The inset plot shows the last base-line signal and 10-min post-cocaine signal on an expanded time scale, to highlight the differences in the time course after drug application.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Summary of the effects of cocaine and nomifensine on measured DA clearance parameters in brain slices. The black bars represent drug effects in the SN, and the striped bars represent drug effects in the striatum. Since base-line clearance parameters differed between the two regions, all data represent the percentage change from the average of three reproducible base-line signals (mean ± S.E.M.). A, In both the SN (n = 11) and striatum (n = 18), cocaine significantly potentiated the amplitude and time course (T80) of the electrochemical signal. B, Nomifensine also potentiated the amplitude and time course of the signal in the SN (n = 14) and striatum (n = 14). Significant differences are shown at the *P < .05, ** P < .01 and *** P < .001 level.

Nomifensine. The effects of cocaine on the DA electrochemical signal are consistent with the previously reported effects of this drug on DAT activity (Cass et al., 1993a; Cass et al., 1993b). To extend this finding, we used the DAT/NET inhibitor nomifensine in both the SN and striatum. Figure 4 shows a representative effect of nomifensine (5 µM) on the DA signal in both the striatum and SN. Similar to cocaine, nomifensine potentiated both the amplitude and time course of the electrochemical signal in both brain regions. A summary of the effects of nomifensine is shown in figure 3B.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Representative effects of nomifensine on DA clearance in A, a striatal slice and B, a SN slice. In both cases, the solid black line shows the last of three reproducible base-line signals taken before drug application. The broken line shows the effect of a 10-min bath application of nomifensine (5 µM). Note that, although the base-line signals are of similar amplitude in the two brain regions, the time courses are different.

Effects of SERT and NET inhibitors. The similarity of the effects of cocaine and nomifensine on DA clearance in the striatum and SN would suggest that DAT plays a major role in DA clearance in both brain regions. However, it has also been argued that DA clearance in the SN may be mediated by SERT and NET, which are also inhibited by cocaine and nomifensine, respectively (Hyttel, 1982; Richelson and Pfenning, 1984). To examine this possibility, we tested the effects of the NET inhibitor DMI (200 nM) and the SERT inhibitor citalopram (500 nM). To ensure that inhibitors were effective at these concentrations, we tested the effects of citalopram on 5-HT clearance in the dorsal raphe, a region of high SERT density (Gehlert et al., 1995). In this experiment, 5-HT was substituted for DA in the micropipette, and clearance was monitored as described for DA. As shown in figure 5A, a 10-min bath application of citalopram potentiated the 5-HT electrochemical signal, consistent with an inhibition of SERT function. The effects of DMI on NET-mediated DA clearance were tested in the CA3 region of the hippocampus. Although the DA signal was relatively prolonged in this area, consistent with a low DAT density, DMI effectively potentiated the signal in this region (fig. 5B). The effects of DMI and citalopram were confirmed in 2 or 3 additional slices from each region (data not shown). As shown in figure 6, neither citalopram nor DMI significantly affected the measured DA clearance parameters in the SN.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of SERT and NET inhibition. A, Recording from a slice containing the dorsal raphe. 5-HT (200 µM barrel concentration) was locally applied at 5-min intervals to achieve three stable base-line signals. These signals were averaged together to yield the lighter tracing shown. Following a 10-min application of the SERT inhibitor citalopram (500 nM), the signal was potentiated (darker trace). B, Recording from the CA3 region of a hippocampal slice. Local application of DA at 5-min intervals resulted in 3 reproducible signals, averaged together to yield the lighter trace. The NET inhibitor DMI (200 nM) was applied for 10-min, resulting in a potentiation of the signal (darker trace).

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Summary of the effects of uptake inhibitors in SN slices. Data are represented as in figure 4. Both cocaine (50 µM; n = 11) and nomifensine (5 µM; n = 14) significantly potentiated the amplitude and time course of the electrochemical signal (*** P < .001; ** P < .01). Neither DMI (200 nM; n = 9) nor citalopram (500 nM; n = 7) affected any of the measured signal parameters.

Comparison of DA and 5-HT Red/ox Ratios In Situ

We and others have previously used the red/ox ratio of the chronoamperometric recording to distinguish among various monoamines that oxidize at similar potentials (Gratton et al., 1989; Luthman et al., 1997). In our DA clearance studies, it was noted that in situ red/ox ratios for DA were markedly higher than previously reported and were consistently higher than red/ox ratios observed in vitro. Aware of reports that 5-HT may be the predominant electroactive species detected within the rat SN (Rice et al., 1994; Iravani and Kruk, 1997), we wanted to ensure that this higher red/ox ratio for DA would not preclude discrimination between endogenously released DA and 5-HT in situ. Therefore, we measured the red/ox ratios of exogenously applied 5-HT signals in the dorsal raphe or SN, collected during the course of the clearance experiments described above. A total of 111 signals were analyzed, and the average red/ox ratio of 5-HT signals was found to be 0.08 ± 0.01 (mean ± S.E.M.). In vitro calibrations of electrodes (n = 5) against both DA and 5-HT revealed that these electrodes were more sensitive for 5-HT (68 ± 7 picocoulombs/µM) than for DA (26 ± 1 picocoulombs/µM).

Effects of KCl and d-Amphetamine on DA Release

The results outlined above suggest a common role for DAT-mediated DA clearance in both the striatum and SN. However, it has also been suggested that the DAT may play an important role in DA release under certain circumstances and that DAT-mediated efflux may be one way in which somatodendritic DA release occurs (Nirenberg et al., 1996b). Therefore, we compared the effects of KCl-evoked depolarization and d-amphetamine-evoked efflux on DA release in the SN and striatum.

KCl-evoked release. Previous data have shown that local application of small amounts of KCl from a micropipette produces an electrochemical signal that is identical to that observed after local application of DA (Gratton et al., 1989; Hebert et al., 1996). Using local application of 70 mM KCl (15-20 psi, 2-3 sec in all cases), a total of 103 signals were obtained from striatal slices. The red/ox ratio of these signals (1.50 ± 0.04) was nearly identical to that observed when DA was applied locally within the striatum (1.30 ± 0.03, n = 181), confirming the DA-like nature of the electrochemical signal. Although there was heterogeneity in the amplitude of the responses, the signals were then grouped into amplitude ranges, similar to the DA clearance studies. As shown in figure 7, when the clearance rates of these KCl-evoked DA responses were compared to the clearance rates of locally applied DA signals over the same amplitude range, no significant differences were observed. Similarly, the T₈₀ values between endogenous and exogenous DA signals were not statistically different over the same amplitude range (data not shown). Interestingly, application of KCl in the SN did not produce any detectable electrochemical signals (n = 12).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Comparison of clearance rates for locally applied DA signals (, solid line) and KCl-evoked releases (, broken line). Signals were compared over the same amplitude range; each data point represents at least 10 individual signals. For both the DA applications (r2 = .97) and the KCl-evoked responses (r2 = .98), clearance rates were linear over the observed amplitude range. Note the juxtaposition of the regression lines. Linear regression analysis revealed that the slopes and intercepts of the lines were not significantly different. The inset plot shows representative oxidation current signals produced by local application of DA (solid line) and 70 mM KCl (broken line). Signals were chosen to be of similar amplitude to more directly compare clearance rates and time courses.

d-Amphetamine-evoked release. Although the DAT normally acts to remove DA from the extracellular space, amphetamine-like drugs are able to promote DA efflux from neurons via a reversal of DAT activity (Sulzer et al., 1993). Given our findings here which suggest that DAT is a major regulator of DA clearance in the SN and striatum, it was of interest to us to compare the actions of d-amphetamine on DA release in these two regions as well. In these experiments, d-amphetamine (2 mM barrel concentration) was locally applied via pressure ejection (15-20 psi, 2-4 sec). Representative signals from the striatum and SN are shown in figure 8. In both brain regions, d-amphetamine application resulted in a slow, prolonged increase in the electrochemical signal, which decayed over several min. In the striatum, the time course of the d-amphetamine-evoked signal was markedly different than that seen after KCl application (fig. 8B). On average, d-amphetamine-evoked signals achieved higher amplitudes in the striatum than in the SN, resulting in longer rise times and overall time courses in the former region. However, the red/ox ratios were found to be DA-like in both regions, confirming the identity of the electroactive species as DA. A summary of all d-amphetamine-evoked signals from both the SN and striatum is shown in table 2.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Representative examples of DA release in midbrain slices. A, Signals from the striatum and SN were obtained by pressure ejection (20 psi × 2 sec in both cases) of a 2 mM d-amphetamine solution. For clarity, only the oxidation current signal is shown, although both signals had red/ox ratios that were DA-like in nature. Note the prolonged time course of the signal in both brain regions and the higher amplitude of the signal in the striatum. B, For comparison, a single KCl-evoked response (solid line) from a striatal slice is superimposed with a d-amphetamine-evoked signal (broken line). Signals were chosen to be of approximately the same peak amplitude; however, note the temporal differences between the responses.

	Discussion

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In the present study, high-speed chronoamperometric recordings were performed in rat brain slices to compare DA clearance and release between the SN and striatum. These data represent the first time that the technique of pressure ejection of DA and chronoamperometric measurements of DA clearance has been applied in brain slices containing the SN and striatum and confirm and extend previous studies performed in anesthetized rats (Hoffman and Gerhardt, 1998; Cass et al., 1993a, 1993b). First, we found that, over a range of DA concentrations, the capacity for DA clearance is significantly lower in the SN, compared to the striatum. Second, the uptake inhibitors cocaine and nomifensine potentiated the amplitude and time course of the DA electrochemical signal to a similar extent in both brain regions. These results, coupled with the finding that selective inhibition of SERT and NET within the SN did not affect DA clearance, strongly support our hypothesis that the DAT is the major contributor to DA clearance within the SN. Third, local application of d-amphetamine promoted DA release in both nigral and striatal slices, although larger mean signal amplitudes were obtained within the striatum. Finally, we report that local applications of KCl produced detectable DA-like signals in striatal slices, but not within the SN.

Since the DAT is believed to be a primary regulator of DA clearance in the nigrostriatal pathway, one of the major goals of the present study was to compare DA clearance in both the SN and striatum. Although radioligand binding (Coulter et al., 1995) and immunocytochemical markers (Ciliax et al., 1995; Nirenberg et al., 1996b) have shown that the DAT is present in both brain regions, there remains some controversy as to the functional aspects of DAT-mediated DA uptake in the SN. Data from studies involving [³H]DA uptake in synaptosomes and slices suggest that a large amount of DA may be taken up by serotonergic and/or noradrenergic terminals within the SN (Kelly et al., 1985; Simon and Ghetti, 1993). Given both the low levels of NET within the SN (Gehlert et al., 1995) and the poor affinity of SERT for DA (Hoffman et al., 1991), it seems unlikely that DA clearance would be mediated by other transport systems. In the present study, we report that DA uptake in the SN is mediated by DAT, based on the following lines of evidence. First, direct comparisons of DA clearance in the SN and striatum reveal that, over a range of amplitudes, clearance rates are significantly slower in the SN than in the striatum. These results are consistent with the reported 4- to 10-fold greater number of DAT sites within the striatum (Coulter et al., 1995), although we cannot, at present, directly equate the differences in the electrochemical parameters with reported B_max or V_max values for DAT. In addition, DA clearance was also reduced in the cortex, which contains fewer DAT sites than either the striatum or SN (Coulter et al., 1995; Cass and Gerhardt, 1995). Second, the results obtained with the uptake inhibitors clearly demonstrate the role of DAT in mediating DA clearance within the SN. We report that nomifensine and cocaine potentiated the amplitude and time course of the DA electrochemical signal in both the SN and striatum, in a manner consistent with previous chronoamperometric recordings performed in anesthetized animals (Cass et al., 1993a, 1993b; Hoffman and Gerhardt, 1998). The similarity of the effects of both of these drugs in striatal and nigral slices suggests that a common mechanism (e.g. DAT) is responsible for DA clearance in both regions. To demonstrate that the effects of nomifensine and cocaine were not due to inhibition of other transporters, we also performed experiments with DMI and citalopram, which are potent and selective inhibitors of NET and SERT, respectively (Hyttel, 1982; Richelson and Pfenning, 1984). When applied at concentrations that have previously been shown to be effective in midbrain slices (O'Connor and Kruk, 1991; Cragg et al., 1997b) and were effective in other brain regions in the present study, neither of these agents affected DA clearance within the SN. Although others have reported effects of DMI on DA uptake within the rostral SN, we found no evidence for NET-mediated DA uptake in the SN in the present study (Cragg et al., 1997b). It is possible that species, age or methodological differences are responsible for this discrepancy. However, the data reported here corroborate our recent findings in intact animals(Hoffman and Gerhardt, 1998), and the effects of cocaine and nomifensine are analogous to those seen by Cragg and coworkers using GBR12909 (Cragg et al., 1997b). Thus, we conclude that DAT is the major monoamine transporter responsible for DA clearance within the SN, over the concentration ranges tested.

The DA clearance studies reported here suggest that the DAT is responsible for DA uptake in both the SN and striatum. However, it has also been postulated that DAT may also play a significant role in promoting DA release, since the transporter can operate in a bidirectional manner (Amara and Kuhar, 1993; Eshleman et al., 1994). Recent data regarding the subcellular localization of both DAT and the vesicular monoamine transporter-2 (VMAT2), have led to the proposal that DAT reversal may represent a significant mode of DA release within the SN (Nirenberg et al., 1996a, 1996b). If, as we have hypothesized, DAT represents a primary mechanism for DA uptake within the SN, then agents which can promote DAT reverse transport would also be predicted to promote DA release within the SN. In particular, basic drugs such as d-amphetamine are believed to promote DA release via a disruption of intracellular DA stores, perhaps through alterations in intracellular pH (Sulzer et al., 1993). The DAT-dependence of amphetamine-evoked DA release has been demonstrated in both DAT-expressing cell lines, as well as in DAT knockout mice (Eshleman et al., 1994; Giros et al., 1996). In vivo microdialysis studies have demonstrated that somatodendritic release of DA within the SN is promoted by systemic or local administration of d-amphetamine (Heeringa and Abercrombie, 1995; Hoffman et al., 1997). In the present study, we locally applied d-amphetamine via pressure ejection in the SN and striatum, to demonstrate DAT-dependent DA release. Although a relatively high concentration of d-amphetamine (2 mM) was required to produce release, it should be noted that the achieved tissue concentration produced via pressure ejection is 10 to 100 times less than the barrel concentration of the drug (Nicholson, 1985; Gerhardt and Palmer, 1987). In both brain regions, a slow and prolonged DA-like signal was observed after d-amphetamine application, although the peak amplitude of the signals was higher on average in the striatum. The larger peak response in the striatum is consistent with both the higher tissue DA content and DAT levels within this region, as well as with previous studies which have shown d-amphetamine-evoked release within this region (Heeringa and Abercrombie, 1995; Hebert et al., 1996). The slow time courses of the responses are consistent with the known mechanism(s) of action of amphetamine derivatives, as has previously been observed in hippocampal slices and in the SNr (Su et al., 1990; Iravani and Kruk, 1997).

An interesting difference between the present study and previous voltammetric recordings within the SN pertains to the identity of the predominant neurotransmitter released during stimulation. We report that all of the electrochemical responses in both the striatum and SN were DA-like in nature, as evidenced by red/ox ratios which were similar to those seen when DA was locally applied. This was expected for the striatum, which is known to contain more DA terminals than serotonin terminals (Boja et al., 1992; Coulter et al., 1995). However, it has been reported that 5-HT is the predominant electrochemical species detected in the rat SN after either electrical stimulation or d-amphetamine application (Iravani and Kruk, 1997; Rice et al., 1997). In the present study, we found no evidence for d-amphetamine-evoked 5-HT release within the SN. This difference cannot be attributed to an inability to detect 5-HT, since the electrodes used in these recordings have greater sensitivity for 5-HT than DA. As we have shown, the clear difference in red/ox ratios between DA and 5-HT allows for discrimination between these two compounds in situ. Moreover, the fact that KCl application did not produce detectable 5-HT signals within the SN would seem to indicate that few active 5-HT terminals exist within our slice preparation. Other methodological issues, such as the age of the animals, species of animal used, or recording protocols used, may contribute to the observed differences. Recent data have suggested that the relative incidence of DA and 5-HT release within the SN may be influenced by age and species (Cragg et al., 1997a). The evidence presented here suggests that d-amphetamine is able to promote DA release within both the striatum and SN, in a manner consistent with both DA content and DAT activity in these brain regions.

One issue that we feel merits discussion is our observation of higher in situ red/ox ratios for DA than previously reported. Previously, we have shown that in situ red/ox ratios for DA are between 0.5 and 0.8 and are consistent with ratios obtained in vitro (Gratton et al., 1989; Hebert et al., 1996). In the present study, we found that in situ red/ox ratios for locally applied DA signals were typically 1. It is possible that part of this difference is due to the high-temperature Nafion coating procedure that we have recently begun to employ (Hebert et al., 1996; Hoffman and Gerhardt, 1998). However, since in vitro calibrations with DA never yielded these high ratios, it seems more likely that it is a combination of the microenvironment of the tissue preparation and the surface properties of the electrode which produces this effect. A more detailed explanation of this phenomenon is beyond the scope of the present study, although we suggest that red/ox values 1 may reflect some adsorption of DA molecules to the surface of electrode. Local applications of ascorbate (20 mM barrel concentration) produced signals with red/ox ratios of 0, suggesting that the increase in red/ox ratios observed in situ is not applicable to all electrochemically active compounds (data not shown). Moreover, the mean red/ox ratio of 0.08 for 5-HT in situ that we report here is similar to that previously reported (Luthman et al., 1997). We would emphasize that this higher red/ox ratio does not preclude a distinction between DA and 5-HT signals in situ. Indeed, the higher ratio for DA that we report here appears to enhance the difference in the red/ox ratio between the two compounds. Therefore, we suggest that the red/ox ratio can still be used to identify compounds in situ and that the endogenous signals we report here likely represent DA signals.

In contrast to d-amphetamine, KCl-evoked DA release is believed to reflect the more classical properties of neurotransmitter release, such as Ca⁺⁺ dependence and sensitivity to tetrodotoxin (Elverfors et al., 1997). Electrochemical recordings, performed in the striatum of anesthetized animals, have shown that local application of small volumes of 70 mM KCl produces signals that are identical to signals produced after exogenous application of DA (Gratton et al., 1989; Friedemann and Gerhardt, 1992; Hebert et al., 1996). A comparison between endogenous and exogenous signals had not been performed in brain slices, but is necessary to confirm the validity of the DA clearance protocol in this preparation. We now report that, in striatal slices, local applications of KCl produced electrochemical signals that were similar to those observed after local applications of DA. The inability of locally applied KCl to produce detectable DA signals within the SN may simply be due to the failure of this particular stimulus to sufficiently depolarize the dendritic elements to threshold (Hausser et al., 1995). Within the striatum, the amplitude of the evoked response was variable, perhaps reflecting the heterogeneous distribution of release sites. However, when clearance parameters were compared to locally applied DA signals over the same amplitude range, the KCl-evoked responses were indistinguishable from the exogenous DA signals. Temporally, these signals were much faster than those produced by d-amphetamine application in the striatum, consistent with the differential mechanism of action of these chemical stimuli. Moreover, the red/ox ratios of the KCl-evoked signals were nearly identical to those seen during local application of DA and during d-amphetamine-evoked release. Taken together, the data suggest that endogenous DA release can be elicited by KCl application in striatal slices, and the similarity of the KCl-evoked and locally applied DA signals serves to further validate DA clearance studies in brain slices.

In summary, we have used high-speed chronoamperometric recordings in brain slices to address the functional role of the DAT in the SN and striatum. These data demonstrate the capacity for both DAT-mediated DA release and reuptake by cell bodies within the SN. The clarification of the role of DAT within the SN may be useful in understanding the regulation of somatodendritic DA release, both under normal and pathophysiological conditions.