Departments of
Pharmacology (N.R.Z., G.A.L., G.A.G.) and
Psychiatry
(G.A.G.),
Neuroscience Training Program (N.R.Z., G.A.G.), and
Rocky
Mountain Center for Sensor Technology (N.R.Z., G.A.G.), University of
Colorado Health Sciences Center, Denver, Colorado
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, or
d-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.
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Introduction |
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 and d-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-
-carbomethoxy-3--(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 of d-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.
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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's t 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's
t 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).
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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.
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Fig. 1.
Exogenous DA signal amplitudes (A) and clearance
rates (C), but not signal time courses (T80 values; B),
varied with the amount of DA locally applied into medial dorsal
striatum of urethane-anesthetized rats. Signals produced by pressure
ejection of DA (200, 400, 600, or 800 µM) at 5-min intervals were
measured using high-speed chronoamperometry. The same four volumes (25, 50, 75, and 100 nl) of each DA concentration were ejected, resulting in
the picomoles of DA ejected that are plotted on the
Y-axis. Mean values ± S.E.M.,
N = 4 to 8 rats. Two-factor ANOVAs with repeated
measures (volume) were used for statistical analysis. A,
concentration × volume interaction (F = 2.8;
df = 9,57; p < .01);
one-factor ANOVA, effect of volume for the 200 (p < .001), 400 (p < .001), 600 (p < .01), and 800 (p < .001)
µM DA concentrations. Post hoc paired t tests and
Bonferroni's correction, amplitudes at the lowest and highest volumes
for the 200, 400, and 800 µM DA concentrations (p
values < .0125). B, main effect of volume (p < .05). C, main effect of volume (p < .001).
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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 T80
values, 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 a
KT of 12 µM and a
Vmax of 0.7 µM/s (r2 = 0.994).
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Fig. 2.
DA clearance rates in rat striatum increased as
higher extracellular exogenous DA concentrations were achieved. Mean
values ± S.E.M. A, data are from Fig. 1, A and C, in which
increasing volumes (25-100 nl) were ejected from quadruple-barrel
micropipette assemblies containing 200, 400, 600, or 800 µM DA. B,
data are from experiments in which higher volumes (25-250 nl) of 200 µM DA were ejected from single-barrel micropipette assemblies.
N = 4 rats.
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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 (Table
1).
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TABLE 1
Baseline DA signal parameters for each of the groups of rats
subsequently used in the local drug application experiments
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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 T80 values (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.
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Fig. 3.
Effects produced by locally applied nomifensine in
rat striatum depended on the magnitude of the baseline DA clearance
rates. Exogenous DA signals were generated by ejecting 12.5 to 75 nl of
either 200 or 800 µM DA. The initial baseline clearance rates, shown
in Table 1, were used to distinguish three groups with "low",
"medium", or "high" baseline clearance. Nomifensine (4-fold
higher concentration than DA; arrow) was pressure ejected at twice the
volume of DA. Changes in DA signal amplitude (A), T80 (B),
and clearance rate (C) are expressed as a percentage of baseline signal
parameters (Table 1). Mean values ± S.E.M., N = 4 to 9 electrode placements (3-5 rats). Two-factor ANOVAs with
repeated measures (time) were used for statistical analysis. A,
group × time interaction (F = 11.0;
df = 16,136; p < .001);
one-factor ANOVA, effect of time in the low (p < .001) and medium (p < .05) groups. Main effect of
group (F = 25.0; df = 2,17;
p < .001); post hoc Tukey-Kramer after nomifensine
application (0-30 min) in the low versus medium (p < .01) and low versus high (p < .001) groups. B,
group × time interaction (F = 2.0;
df = 16,136; p < .05);
one-factor ANOVA, effect of time in the low and high groups
(p values < .001). Main effect of group
(F = 4.4; df = 2,17;
p < .05); however, post hoc Tukey-Kramer showed
only a trend after nomifensine in the low versus medium
(p = .053) and low versus high
(p = .055) groups. C, main effect of group
(F = 4.3; df = 2,17;
p < .05); post hoc Tukey-Kramer after nomifensine
application in the low versus high groups (p < .05).
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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.
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Fig. 4.
Effects produced by locally applied cocaine
(arrow) in rat striatum also depended on the magnitude of the baseline
DA clearance rates. Changes in DA signal amplitude (A), T80
(B), and clearance rate (C) are expressed as a percentage of baseline
signal parameters (Table 1). See Fig. 3 for experimental details. Mean
values ± S.E.M., N = 5 to 9 electrode
placements (3-5 rats). Because of the transient effects produced by
cocaine, only data up to 10 min after cocaine application were included
in the statistical analysis (two-factor ANOVAs with repeated measures
for time). A, group × time interaction (F = 4.0; df = 8,72; p < .001);
one-factor ANOVA, effect of time in all three groups (low,
p < .05; medium and high, p
values < .001). Main effect of group (F = 4.5; df = 2,18; p < .05); post
hoc Tukey-Kramer after cocaine application (0-10 min) in the low
versus high groups (p < .05). B, group × time interaction (F = 4.1; df = 8,72; p < .001); one-factor ANOVA, effect of time
in all three groups (low, p < .05; medium and
high, p values < .001). Main effect of group
(F = 17.8; df = 2,17;
p < .001; 1 outlier not included); post hoc
Tukey-Kramer after cocaine application in the low versus medium
(p < .01) and low versus high
(p < .001) groups. C, no significant
differences.
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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).
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TABLE 2
Baseline DA signal parameters for each group of rats subsequently used
in the systemic drug administration experiments
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Fig. 5.
Effects of systemically administered nomifensine (3 and 10 mg/kg) on DA signal amplitudes (A), T80 values (B),
and clearance rates (C). After stable baseline signals were obtained in
response to pressure ejection of DA (200 µM; 25-250 nl) at 5-min
intervals into the medial dorsal striata of urethane-anesthetized rats,
either saline or nomifensine, at the doses indicated, was injected i.p.
(arrow). Data are expressed as a percentage of the baseline values
(Table 2). Mean values ± S.E.M., N = 8 rats
(saline; these data are also shown in Figs. 6-11) and
N = 3 rats (both doses of nomifensine). Two-factor
ANOVAs with repeated measures (time) were used for statistical
analysis. A, dose × time interaction (F = 4.0; df = 28,154; p < .001);
one-factor ANOVA, effect of time in the saline and 10 mg/kg nomifensine
groups (p values < .05). Main effect of dose
(F = 9.6; df = 2,11;
p < .01); post hoc Tukey-Kramer after drug
injection (0-60 min) in the saline versus 10 mg/kg nomifensine groups
(p < .01). There was also a trend for the saline
versus 5 mg/kg nomifensine groups to differ (p = .051). B, dose × time interaction (F = 2.4;
df = 28,154; p < .001);
one-factor ANOVA, effect of time in the saline (p < .05) and 3 mg/kg nomifensine (p < .01) groups.
C, dose × time interaction (F = 1.9;
df = 28,154; p < .05);
however, one-factor ANOVA revealed no significant effects of time in
any group.
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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.
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Fig. 6.
Effects of systemically administered mazindol (3 mg/kg) on DA signal amplitudes (A), T80 values (B), and
clearance rates (C). Data are expressed as a percentage of the baseline
values (Table 2). See Fig. 5 for experimental details and statistical
analyses of the saline group data. Mean values ± S.E.M.,
N = 4 rats (mazindol). Two-factor ANOVAs with
repeated measures (time) were used for statistical analysis. A,
dose × time interaction (F = 4.4;
df = 14,140; p < .001). Main
effect of dose (F = 8.4; df = 1,10; p < .05) after drug injection (0-60 min) in
the saline versus mazindol groups. B, no significant differences. C,
dose × time interaction (F = 2.1;
df = 14,140; p < .05);
one-factor ANOVA, effect of time in the mazindol group
(p < .01).
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Fig. 7.
Effects of systemically administered GBR 12909 (10 mg/kg) on DA signal amplitudes (A), T80 values (B), and
clearance rates (C). Data are expressed as a percentage of the baseline
values (Table 2). See Fig. 5 for experimental details and statistical
analyses of the saline group data. Mean values ± S.E.M.,
N = 3 rats (GBR 12909). Two-factor ANOVAs with
repeated measures (time) were used for statistical analysis. A,
dose × time interaction (F = 10.9;
df = 14,140; p < .001);
one-factor ANOVA, effect of time in the GBR 12909 group
(p < .001). Main effect of dose
(F = 7.6; df = 1,10;
p < .05) after drug injection (0-60 min) in the
saline versus GBR 12909 groups. B, dose × time interaction
(F = 3.6; df = 14,140;
p < .001); one-factor ANOVA, effect of time in the
GBR 12909 group (p < .05). C, dose × time
interaction (F = 4.0; df = 14,140; p < .001); one-factor ANOVA, effect of
time in the GBR 12909 group (p < .01). Main effect
of dose (F = 8.7; df = 1,9;
p < .05; 1 outlier not included) after drug
injection in the saline versus GBR 12909 groups.
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Fig. 8.
Effects of systemically administered benztropine (10 mg/kg) on DA signal amplitudes (A), T80 values (B), and
clearance rates (C). Data are expressed as a percentage of the baseline
values (Table 2). See Fig. 5 for experimental details and statistical
analyses of the saline group data. Mean values ± S.E.M.,
N = 4 rats (benztropine). Two-factor ANOVAs with
repeated measures (time) were used for statistical analysis. A,
dose × time interaction (F = 10.5;
df = 14,140; p < .001);
one-factor ANOVA, effect of time in the benztropine group
(p < .001). Main effect of dose
(F = 30.8; df = 1,10;
p < .001) after drug injection (0-60 min) in the
saline versus benztropine groups. B, no significant differences. C,
dose × time interaction (F = 8.0;
df = 14,140; p < .001);
one-factor ANOVA, effect of time in the benztropine group
(p < .001). Main effect of dose
(F = 19.8; df = 1,10;
p < .01) after drug injection (0-60 min) in the
saline versus benztropine groups.
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In a second set of experiments, cocaine, CFT, and
d-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/kg
d-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 of
d-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.
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Fig. 9.
Effects of systemically administered cocaine (20 and
30 mg/kg) on DA signal amplitudes (A), T80 values (B), and
clearance rates (C). Data are expressed as a percentage of the baseline
values (Table 2). See Fig. 5 for experimental details and statistical
analyses of the saline group data. Mean values ± S.E.M.,
N = 4 rats (both doses of cocaine). Two-factor
ANOVAs with repeated measures (time) were used for statistical
analysis. A, no significant differences. B, dose × time
interaction (F = 5.7; df = 28,182; p < .001); one-factor ANOVA, effect of
time in the 20 (p < .01) and 30 (p < .001) mg/kg cocaine groups. Main effect of
dose (F = 5.8; df = 2,13;
p < .05); post hoc Tukey-Kramer, after drug
injection (0-60 min) in the saline versus 30 mg/kg cocaine groups
(p < .05). C, trend for a significant dose × time interaction (F = 1.5; df = 28,182; p = .070). Main effect of dose
(F = 4.5; df = 2,12;
p < .05; 1 outlier not included); post hoc
Tukey-Kramer after drug injection in the saline versus 30 mg/kg cocaine
groups (p < .05).
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Fig. 10.
Effects of systemically administered CFT (3 and 10 mg/kg) on DA signal amplitudes (A), T80 values (B), and
clearance rates (C). Data are expressed as a percentage of the baseline
values (Table 2). See Fig. 5 for experimental details and statistical
analyses of the saline group data. Mean values ± S.E.M.,
N = 4 rats (both doses of CFT). Two-factor ANOVAs
with repeated measures (time) were used for statistical analysis. A,
dose × time interaction (F = 2.1;
df = 28,168; p < .01);
one-factor ANOVA, effect of time in the 3 mg/kg CFT group
(p < .01). B, dose × time interaction
(F = 6.0; df = 28,168;
p < .001); one-factor ANOVA, effect of time in
both CFT groups (p values < .01). Main effect of
dose (F = 7.2; df = 2,12;
p < .01); post hoc Tukey-Kramer after drug
injection (0-60 min) in the saline versus 10 mg/kg CFT groups
(p < .05; trend for the saline versus 3 mg/kg CFT
groups to differ, p = .077). C, dose × time
interaction (F = 4.2; df = 28,168; p < .001); one-factor ANOVA, effect of
time in both CFT groups (p values < .001). Main
effect of dose (F = 7.4; df = 2,12; p < .01); post hoc Tukey-Kramer after drug
injection in the saline versus 3 mg/kg CFT groups and the saline versus
10 mg/kg CFT groups (p values < .05).
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Fig. 11.
Effects of systemically administered
d-amphetamine (1, 5, and 10 mg/kg) on DA signal
amplitudes (A), T80 values (B), and clearance rates (C).
Data are expressed as a percentage of the baseline values (Table 2).
See Fig. 5 for experimental details and statistical analyses of the
saline group data. Mean values ± S.E.M., N = 3 to 4 rats (each dose of d-amphetamine). Two-factor
ANOVAs with repeated measures (time) were used for statistical
analysis. A, dose × time interaction (F = 1.8; df = 42,196; p < .01);
one-factor ANOVA, effect of time in the 5 mg/kg
d-amphetamine group (p < .05). Main
effect of dose (F = 5.0; df = 3,13; p < .05; 1 outlier not included); post hoc
Tukey-Kramer after drug injection (0-60 min) in the saline versus 10 mg/kg d-amphetamine groups and the 5 versus 10 mg/kg
d-amphetamine groups (p values < .05). B, dose × time interaction (F = 5.3;
df = 42,196; p < .001);
one-factor ANOVA, effect of time in the 1 (p < .001), 5 (p < .001), and 10 (p < .05) mg/kg d-amphetamine groups. Main effect of dose
(F = 22.6; df = 3,13;
p < .001; 1 outlier not included); post hoc
Tukey-Kramer after drug injection in the saline versus 5 mg/kg
d-amphetamine groups (p < .05), the
saline versus 10 mg/kg d-amphetamine groups
(p < .001), the 1 versus 10 mg/kg
d-amphetamine groups (p < .001),
and the 5 versus 10 mg/kg d-amphetamine groups
(p < .05). C, dose × time interaction
(F = 5.5; df = 42,196;
p < .001); one-factor ANOVA, effect of time in the
1 (p < .05), 5 (p < .001) and
10 (p < .001) mg/kg d-amphetamine
groups. Main effect of dose (F = 11.6;
df = 3,14; p < .001); post hoc
Tukey-Kramer after drug injection in the saline versus 5 mg/kg
d-amphetamine groups and the saline versus 10 mg/kg
d-amphetamine groups (p values < .01).
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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 of
Vmax 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 a
Vmax of 0.7 µM/s. Nicholson (1995)
proposed that concentrations measured in vivo should be corrected by
the extracellular volume fraction, 
= 0.21 (Rice and Nicholson,
1991). Multiplying our Vmax value by
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 in
Nicholson, 1995). However, the KT
value we derived (2.5 µM, corrected by ) 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 a
KT of 0.22 µM and a
Vmax 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 our KT value is inaccurate, given that the
clearance rate was measured from the pseudolinear portion of the
signals where the DA concentration is well above
KT.
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.
We thank Dr. Shelly Dickinson for help with the statistical analyses.
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Abstract
Introduction
Pharmacological profile of a specific serotonin uptake inhibitor with antidepressant activity.
Prog Neuro-Psychopharmacol Biol Psychiatry
6:
277-295[Medline].
