Characterization of Extracellular Dopamine Clearance in the Medial
Prefrontal Cortex: Role of Monoamine Uptake and Monoamine
Hollie K. Wayment,1James O. Schenk,1,2,3and Barbara A. Sorg3,4
Departments of1Chemistry and2Biochemistry and Biophysics, and3Program in Neuroscience and4Department of
Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, Washington
In vitro rotating disk electrode (RDE) voltammetry and in vivo
microdialysis were used to characterize dopamine clearance in
the rat medial prefrontal cortex (mPFC). RDE studies indicate
that inhibition by cocaine, specific inhibitors of the dopamine
transporter (DAT) and norepinephrine transporter (NET), and
low Na?produced a 50–70% decrease in the velocity of do-
pamine clearance. Addition of the monoamine (MAO) inhibitors,
L-deprenyl, clorgyline, pargyline, or in vivo nialamide produced
30–50% inhibition. Combined effects of uptake inhibitors with
L-deprenyl on dopamine clearance were additive (up to 99%
inhibition), suggesting that at least two mechanisms may con-
tribute to dopamine clearance. Dopamine measured extracel-
lularly 5 min after exogenous dopamine addition to incubation
mixtures revealed that most conditions of DAT/NET inhibition
did not produce elevated dopamine levels above controls. In-
hibition of MAO produced elevated dopamine levels only after
long-term, but not short-term, incubation in vitro. Short-term
incubation of L-deprenyl combined with DAT and NET uptake
inhibitors increased dopamine above control levels, consistent
with more than one mechanism of dopamine clearance. Local
infusion of pargyline (100 or 300 ?M) into the mPFC or striatum via
microdialysis produced more pronounced and immediate in-
creases in mPFC dopamine levels compared with striatum. Fur-
thermore, dopamine elevation in the mPFC was not accompanied
dihydroxyphenylacetic acid and homovanillic acid, as found in the
striatum. These findings may have revealed a unique mechanism
of mPFC dopamine clearance and therefore contribute to the
understanding of multiple behaviors that involve mPFC dopamine
transmission, such as schizophrenia, drug abuse, and working
Key words: dopamine; cocaine; dopamine transporter; me-
dial prefrontal cortex; monoamine oxidase; rotating disk elec-
Altered function of the medial prefrontal cortex (mPFC) has
been implicated in multiple processes and behavioral disorders,
including schizophrenia (Weinberger, 1995), drug abuse (Goe-
ders and Smith, 1983; Isaac et al., 1989; Piazza et al., 1991; Schenk
et al., 1991; Duvauchelle et al., 1992; McGregor and Roberts, 1995;
Wolf et al., 1995; McGregor et al., 1996; Wise et al., 1996; Prasad
et al., 1999), depression (Baxter et al., 1989; Tanda et al., 1994;
Drevets, 1999; Juckel et al., 1999; Merriam et al., 1999; Rajkowska
et al., 1999), and attention deficit hyperactivity disorder (Boix et
al., 1998; Ernst et al., 1998; Puumala and Sirvio, 1998), as well as
normal cognitive processes, including working memory function
(Williams and Goldman-Rakic, 1995; Murphy et al., 1996; Cai and
Arnsten, 1997; Jentsch et al., 1997a,b; Zahrt et al., 1997; Seamans
et al., 1998; Wang, 1999) and decision making (Eslinger and
Damasio, 1985; Damasio, 1995). Several of these studies have
focused in particular on altered dopaminergic functioning within
Despite the importance of prefrontal cortical dopamine in
modulating cognition and behavior, little is known regarding
processes that regulate extracellular clearance of dopamine in the
mPFC. Inhibitors of dopamine transport demonstrate a weak
effect on mPFC extracellular dopamine levels in vivo, including
cocaine (Moghaddam and Bunney, 1989), amphetamine (Sorg et
al., 1997; Pehek, 1999), nomifensine, and GBR 12909 (Cass and
Gerhardt, 1995). The decreased responsiveness to dopamine up-
take inhibitors may be explained partially by the lower terminal
density and decreased number of dopamine transporters per
terminal relative to striatal regions (Sesack et al., 1998). However,
in vitro studies have shown that, in contrast to the striatum/
nucleus accumbens, dopamine uptake inhibitors only partially
diminish dopamine uptake in the PFC (Hadfield and Nugent,
1983; Izenwasser et al., 1990; Elsworth et al., 1993; Wheeler et al.,
1993). These findings suggest that an additional mechanism may
contribute importantly to regulating clearance of extracellular
dopamine in the mPFC.
Few studies have focused on measuring the kinetics of dopa-
mine clearance in the mPFC. Garris et al. (1993) and Garris and
Wightman (1994) have used in vivo voltammetry to examine
clearance within the mPFC. Their findings suggest that dopamine
clearance occurs over a large tissue volume because of the more
restricted distribution of dopamine transporter (DAT) in this
region, and they enhance the notion of volume transmission and
Received April 5, 2000; revised Sept. 22, 2000; accepted Oct. 13, 2000.
This work was supported by Public Health Service Grants DA07384 (J.O.S.) and
DA11787 (B.A.S). J.O.S. is also a recipient of an Independent Scientist Award (KO2
DA00184). We are grateful to Dr. Dale Edmondson (Emory University School of
Medicine) for helpful discussions and to Dr. Weiran Wu and Na Li for assistance
with microdialysis studies.
Correspondence should be addressed to Dr. Barbara A. Sorg, Program in Neu-
roscience, Department of Veterinary and Comparative Anatomy, Pharmacology
and Physiology, Washington State University, Pullman, WA 99164-6520. E-mail:
Dr. Wayment’s present address: Ischemia Technologies, 6830 North Broadway,
Suite A, Denver, CO 80142.
Copyright © 2001 Society for Neuroscience 0270-6474/01/210035-10$15.00/0
The Journal of Neuroscience, January 1, 2001, 21(1):35–44
the possible paracrine function of cortical dopamine. Cass and
Gerhardt (1995) also have examined dopamine clearance in dif-
ferent regions of the mPFC using in vivo voltammetry. However,
no studies to date have defined the contribution of metabolism to
the kinetics of clearance, and some previous mPFC clearance
studies are confounded by the factor of diffusion occurring in vivo.
The present study used rotating disk electrode (RDE) voltamme-
try in vitro to characterize the kinetics of dopamine clearance in
the mPFC and to determine regulatory processes that contribute
to dopamine clearance. In vivo microdialysis in the mPFC and
striatum was also performed to examine the effect of the mono-
amine (MAO) inhibitor, pargyline, on extracellular dopamine
and metabolite levels.
MATERIALS AND METHODS
Animals and housing. Experiments were conducted according to the
National Institutes of Health Guide for the Care and Use of Laboratory
Animals, and experimental protocols were approved by the University
animal care and use committee. Male Sprague Dawley rats weighing
250–300 gm were group-housed (three to four per cage) in a
temperature- and humidity-controlled environment with ad libitum ac-
cess to food and water. Animals were maintained on a 12 hr light/dark
schedule, with lights on at 7 A.M.
Drugs. Cocaine-hydrochloride was a gift from the National Institute on
Drug Abuse. Dopamine, pargyline, clorgyline, desmethylimipramine
(DMI), and nialamide were purchased from Sigma (St. Louis, MO), and
GBR 12909, fluoxetine, and L-deprenyl were purchased from Research
Biochemical Inc. (Natick, MA). All drugs used for RDE experiments
were dissolved in distilled water and diluted to a final concentration in
the incubation buffer. Solutions were made fresh each day and stored on
RDE voltammetry and HPLC. Unanesthetized rats were decapitated, and
their brains were rapidly removed. The mPFC was dissected and weighed.
The tissue was immediately chopped on an ice-cold glass plate and placed
into 500 ?l of physiological buffer composed of (in mM): 124 NaCl, 1.80
KCl, 1.30 MgSO4, 1.24 KH2PO4, 2.50 CaCl2, 26 NaHCO3, 10 glucose,
saturated with 95% O2–5% CO2gas mixture) as described (Meiergerd and
Schenk, 1995; Earles et al., 1998; Earles and Schenk, 1999) and maintained
at 37°C in a temperature-controlled chamber. Composition of the buffer in
the low Na?condition consisted of replacement of 124 mM NaCl with
choline chloride. The tissue was disrupted by repetitive pipetting and
washed by the repeated addition and removal of 250 ?l fresh buffer seven
times. The RDE (Pine Instruments, Grove City, PA) was lowered into the
chamber and rotated at 2000 rpm, and a potential of 450 mV relative to a
Ag/AgCl reference electrode was applied with a Bioanalytical Systems
LC4B potentiostat (W. Lafayette, IN) with a 20 msec time constant,
defined as 5 ? resistance ? capacitance. The tissue was incubated for 20
min until a stable baseline was reached. After the incubation period,
dopamine was added to produce a final concentration of 2.0 ?M, and its
clearance was monitored on a Nicolet 310 digital oscilloscope. The concen-
tration of dopamine used was based on a previous in vitro RDE study in the
mPFC that demonstrated this concentration to be near Vmaxfor dopamine
clearance (Meiergerd et al., 1997). The initial rate of dopamine disappear-
ance was estimated as described previously (Meiergerd and Schenk, 1995;
Earles et al., 1998). The velocity of clearance was expressed as picomoles
per second ? grams of wet weight. This value was determined by calculat-
ing the slope of the line, as determined by time points taken every 20 msec,
and converting from current to dopamine concentration by using a standard
calibration curve prepared each day. The standard curve was generated
using 0.1, 0.25, 0.5, 1.0, and 2.0 ?M dopamine. At a cumulative concentra-
tion of 1.85 ?M dopamine, clearance was followed for 25 sec and is reported
as the “buffer” sample throughout. The supernatant was prepared as de-
scribed above for tissue preparation, but after washing seven times, the
tissue was rapidly centrifuged and the supernatant removed for analysis of
dopamine clearance, as described above. To determine dopamine clearance
(in picomoles per second ? grams of wet weight) for the buffer and
supernatant, the mean tissue weight of all samples (33.4 mg) was used.
The concentrations of DAT/norepinephrine transporter (NET) uptake
inhibitors were chosen on the basis of the Kior IC50of each inhibitor and
were 10–100 times higher than the Kior IC50for each particular neuro-
transmitter. For DMI, both 0.1 and 100 ?M doses were used, with the
former inhibiting only NET and the latter concentration inhibiting both
NET and DAT. A 0.1 ?M concentration of fluoxetine, which inhibits
serotonin uptake, was chosen. Activity for NET may have been partially
inhibited at this concentration of fluoxetine, the Kiof which has been
reported as ?0.2 ?M (Burke and Preskorn, 1995). For GBR 12909, a 3.0
?M solution was chosen, which is ?50 times higher than the IC50for
dopamine and approximately at the Kifor norepinephrine uptake (Heik-
kila and Manzino, 1984).
It should be noted that at the concentrations of L-deprenyl and clor-
gyline used (100 ?M), both MAO A and B would be expected to be
inhibited. However, these agents as well as pargyline were examined
because of previous work demonstrating an inhibitory effect of clorgyline
(Lai et al., 1980; Fang and Yu, 1994) and L-deprenyl on dopamine uptake
(Knoll, 1978; Lai et al., 1980; Zsilla et al., 1986; Knoll, 1992; Okuda et al.,
1992; Fang and Yu, 1994), whereas pargyline has been reported not to
influence dopamine uptake in the striatum (Fang and Yu, 1994).
After most treatments, at the end of a 5 min incubation, a 100 ?l
sample was removed while the electrode was still rotating. Samples were
placed into 20 ?l of 0.1 M perchloric acid solution and immediately
centrifuged to remove the tissue. The supernatant, which will be referred
to as “aqueous phase” throughout, was collected and added to 50 ?l
HPLC mobile phase containing 1 ? 10?7M isoproterenol and stored at
?80°C until assayed by HPLC for dopamine and its metabolites, 3,4-
dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and
3-methoxytyramine (3-MT), as well as for NE. The results are reported
as concentration of dopamine in micromolar taken from the original 100
?l sample removed at the end of the 5 min incubation. Determination of
dopamine and metabolites, DOPAC, HVA, and 3-MT, and NE present
in the aqueous phase was conducted by HPLC analyses according to
Kalivas et al. (1988).
In vivo microdialysis. In vivo microdialysis was conducted in awake,
unrestrained rats as described by Sorg et al. (1997). Animals were
implanted with a chronic guide cannula into the mPFC ?1 week before
microdialysis experiments [for mPFC: anteroposterior (AP) ? ?3.2 mm
from bregma, mediolateral (ML) ? 0.7 mm, dorsoventral (DV) ? ?1.5
mm from skull; for striatum: AP ? ?1.0 mm from bregma, ML ? 2.2
mm, DV ? ?4.0 mm from skull, according to Paxinos and Watson
(1998)]. Microdialysis probes with an active membrane region of 3 mm
(mPFC) or 2 mm (striatum) were prepared as described (Sorg et al.,
1997). Just before use, pargyline was diluted to its final concentration in
artificial CSF (aCSF), which consisted of (in mM): 5.0 glucose, 5 KCl, 120
NaCl, 1.2 CaCl2, 1.2 MgCl2, 0.23 sodium phosphate, pH 7.4. Probes were
implanted the evening before the experiment, and on the next day, a
minimum of 3–4 hr was allowed for a stable baseline to be obtained.
After this, baseline samples were collected, and pargyline (either 100 or
300 ?M given to separate animals) was infused for a 60 min period and
then replaced with aCSF for the remainder of the experiment. HPLC
analyses of dopamine, DOPAC, and HVA were conducted as described
by Sorg et al. (1997). Microdialysis probe placements within the mPFC
and striatum were verified by cresyl violet staining of coronal brain
sections (see Fig. 1, B and C, respectively).
Data analyses and statistical testing. The percentage reduction in do-
pamine clearance was determined by subtracting the mean value for
supernatant and buffer conditions, which were not significantly different
from each other, from the values obtained for control and each treatment
condition, dividing by the mean of the controls, and multiplying by 100.
The differences between means of the transport velocities (see Figs. 2–4),
dopamine, its metabolites, and NE measured from sampled aqueous
phase (see Fig. 5) were tested using a one-way ANOVA followed by a
post hoc Fisher’s test. Each neurotransmitter and metabolite was ana-
lyzed separately with an ANOVA (see Fig. 5). The microdialysis data
(see Figs. 6, 7) were analyzed using a one-way repeated measures
ANOVA followed by a Fisher’s test to determine significant increases
above the last baseline sample. All data were considered statistically
significant at p ? 0.05.
Figure 1A shows the brain area that was dissected for all exper-
iments presented in Figures 2-5. This region included all mPFC
regions anterior to 2.2 mm from bregma (Paxinos and Watson,
1998). Figure 1, B and C, shows photomicrographs of coronal
brain sections demonstrating typical microdialysis probe place-
ments in the mPFC and striatum for the data shown in Figures 6
and 7. In the mPFC, probes spanned the deeper layers of the
cortex within the prelimbic and infralimbic cortices.
36 J. Neurosci., January 1, 2001, 21(1):35–44Wayment et al. • Dopamine Clearance in the Prefrontal Cortex
General characteristics of clearance of extracellular
dopamine in the mPFC
Figures 2–4 show graphs of the mean raw data taken from control
and each of the various treatment groups presented along with a
summary bar graph of the mean ? SEM of dopamine clearance
velocities. Clearance in the mPFC was found to be slower than
that observed in tissues from the striatum or nucleus accumbens
[compare with McElvain and Schenk (1992); Povlock and Schenk
(1997), respectively] and generally consisted of a single linear
phase. However, under some treatment conditions, two phases
were observed and are described further in the results for Figure
4. For the summary bar graph presentation, all velocities that
showed a single linear clearance phase were determined by cal-
culating the slope from 5 to 25 sec, whereas those exhibiting
biphasic profiles were calculated for the second phase (linear
portion) from 20 to 30 sec. The slightly later time period of 20–30
sec was chosen for the graphs shown in Figure 4 because of the
longer delay for the second phase of the velocity profile to appear.
The buffer and supernatant conditions also exhibited biphasic
profiles, and for these two conditions velocities were determined
by calculating the slope from 15 to 25 sec.
The value of dopamine clearance in buffer (used in preparing
the standard curve) was not significantly different from that ob-
served in the supernatant preparation. By 30 sec after dopamine
addition to the incubation mixture containing the supernatant,
the amount of dopamine cleared was 2.5% of the total dopamine
added, and this disappearance may have been caused by the sum
of oxidation of dopamine by the electrode, oxidation by dissolved
O2, auto-oxidation of dopamine, or changes in the residual cur-
rent of the electrode. The dopamine clearance profile for the
buffer and supernatant are shown throughout Figures 2–4 for
purposes of comparison.
Monoamine uptake inhibitors
Figure 2A–G demonstrates the effect of low Na?and inhibitors
of dopamine, NE, and serotonin (5-HT) uptake on the velocity of
dopamine clearance in the mPFC. The data are presented in
summary form (bar graph) in Figure 2H. The results indicate that
?50–70% of the dopamine clearance rate is dependent on the
function of the Na?/Cl?-dependent transporters DAT and NET.
Low Na?buffer (26 mM NaCl vs 150 mM for the control condi-
tion) reduced dopamine velocity by 69%. DMI added to a final
concentration of 0.1 ?M to specifically inhibit NET reduced
dopamine clearance velocity by 46%, whereas a higher dose that
inhibits both NET and DAT inhibited dopamine clearance veloc-
ity by 65%. Although the higher concentration of DMI did not
significantly alter dopamine clearance velocity as compared with
the 0.1 ?M dose, the results suggest possible additive effects of
uptake inhibitors acting at DAT and NET. The serotonin uptake
inhibitor fluoxetine, given at 0.1 ?M, did not significantly reduce
the velocity of dopamine clearance. Addition of 3 ?M GBR 12909,
a DAT inhibitor, reduced dopamine clearance by ?70%. The
effects of GBR 12909 and DMI were not additive, because the
combination of the same concentrations of GBR 12909, DMI,
and fluoxetine inhibited dopamine clearance velocity by only
55%, and this combination was equally as effective as cocaine,
which inhibits all three monoamine transporters. This may have
been caused partially by the concentration of GBR 12909 used,
which was close to the IC50reported for norepinephrine in rat
cortical tissue slices (Heikkila and Manzino, 1984).
Monoamine oxidase inhibitors
Figure 3A–F shows the results of MAO inhibitors on the velocity
of dopamine clearance in the mPFC. The propargylamines, par-
gyline, clorgyline, and L-deprenyl, all reduced dopamine clear-
ance velocity by 30–50%. To determine whether a structurally
different MAO inhibitor also attenuated the velocity of dopamine
striatum sampled in these studies. A, The portion of mPFC tissue re-
moved for dopamine uptake studies. Shaded area represents the most
caudal region used and includes all tissue rostral to the area shown.
Diagram is taken from Paxinos and Watson (1998) at ?2.2 mm from
bregma. B, Photomicrograph of a coronal brain section demonstrating
microdialysis probe placement in the mPFC. The tip of the 3 mm probe
is indicated by a white arrow. C, Photomicrograph of a coronal brain
section demonstrating microdialysis probe placement in the striatum. The
tip of the 2 mm probe is indicated by a white arrow.
Coronal brain sections illustrating regions of the mPFC and
presence of low Na?or monoamine uptake inhibitors. A–G, Mean raw
clearance profiles for dopamine. In some cases, velocity profiles were not
collected from all animals for the entire period, and the lines are trun-
cated (B, D, F). H, Mean ? SEM of dopamine clearance velocities shown
in A–G. Mean values obtained in buffer and supernatant alone are shown
for comparison. n ? 16 for control; n ? 3–5 for all other groups.1p ? 0.05,
comparing with control condition;
mined with a one-way ANOVA followed by a Fisher’s test. Sup, Super-
natant; Low Na?, 26 mM Na?; Cocaine, 100 ?M; GBR 12909, 3.0 ?M; DMI,
0.1 or 100 ?M (summary bar graph only); Fluox, 0.1 ?M fluoxetine;
GBR/DMI/Flu, combination of GBR 12909 (3.0 ?M), DMI (0.1 ?M), and
fluoxetine (0.1 ?M).
Velocity of 2.0 ?M dopamine clearance in mPFC tissue in the
2p ? 0.05, comparing with buffer
3p ? 0.05, comparing with supernatant condition, as deter-
Wayment et al. • Dopamine Clearance in the Prefrontal CortexJ. Neurosci., January 1, 2001, 21(1):35–44 37
clearance, nialamide was administered to rats systemically. Nial-
amide was administered systemically because previous in vitro
experiments using RDE demonstrated that nialamide nonspecifi-
cally alters electrode responses (J. O. Schenk, unpublished obser-
vations). This dose of nialamide (75 mg/kg, i.p.) 5 hr before rats
were killed (Hovevey-Sion et al., 1989) resulted in a ?30%
inhibition of dopamine clearance rate.
Inhibition of DAT/NET uptake and MAO activity
The time-dependent effects of DAT/NET and MAO inhibition
were examined by adding these inhibitors simultaneously with
dopamine to the incubation media (t ? 0 sec). For Figure 4, all
inhibitor additions to the incubation were given simultaneously
with exogenous dopamine addition at t ? 0 sec. Figure 4A–F
shows that a biphasic clearance profile was observed in each case
when these inhibitors were added at t ? 0 sec. The bar graph in
Figure 4G summarizes the means ? SEM of dopamine clearance
occurring from 20 to 30 sec. The early component of the biphasic
response demonstrated inhibition of dopamine clearance that
lasted for several seconds. This initial inhibition lasted signifi-
cantly longer under conditions in which cocaine ? L-deprenyl
were added compared with all other conditions.
Figure 4A demonstrates that inhibition by cocaine added at t ?
0 sec was greater than that observed for cocaine when it was
added 30 sec before dopamine addition, although these values
were not significantly different from each other ( p ? 0.23) (com-
pare with Fig. 2B,H). There was a trend for cocaine given at t ?
0 sec to cause a lower rate of dopamine clearance than the buffer
and supernatant conditions, but the value from cocaine at t ? 0
sec shown in the summary bar graph was not statistically different
from those of the buffer and supernatant conditions (Fig. 4G).
L-deprenyl addition to the incubation at t ? 0 sec also resulted in
a biphasic profile and only partially inhibited the clearance veloc-
ity of dopamine. This value, shown in the summary bar graph, was
significantly reduced from control, although significantly higher
than the values for the buffer and supernatant conditions. Inhibi-
tion by L-deprenyl added at t ? 0 sec was not different from that
produced by L-deprenyl present in the incubation mixture for 20
min before dopamine addition ( p ? 0.61) (compare with Fig.
Cocaine and other DAT and NET inhibitors were combined
with the MAO inhibitor L-deprenyl to determine whether the
partial inhibitory effects on dopamine clearance velocity de-
scribed above were additive when assessed over the 20–30 sec
range. Figure 4C–E,G demonstrates that additive effects of
L-deprenyl occurred when combined with these uptake inhibitors,
with percentage inhibitionranging
L-deprenyl) to 99% (GBR ? L-deprenyl), with a 94% inhibition by
cocaine ? L-deprenyl (refer to summary bar graph in Fig. 2H for
effects of DMI and GBR 12909 given alone).
from 83% (DMI
For most of the RDE experiments, samples of aqueous phase
from brain homogenates were collected 5 min after the addition
of dopamine to determine the levels of dopamine and its metab-
olites, DOPAC, HVA, and 3-MT. Norepinephrine was also mea-
sured, because dopamine is taken up into NE terminals via NET.
The relatively longer time point of 5 min was chosen to increase
presence of MAO inhibitors. A–E, Mean raw clearance profiles for
dopamine. D, Nialamide (75 mg/kg, i.p.) was administered in vivo 5 hr
before rats were killed. F, Mean ? SEM of dopamine clearance velocities
shown in A–E. Mean values obtained in buffer and supernatant alone are
shown for comparison. n ? 16 for control; n ? 3–5 for all other groups.
1p ? 0.05, comparing with control condition;2p ? 0.05, comparing with
determined with a one-way ANOVA followed by a Fisher’s test. Sup,
Supernatant; Parg, 100 ?M pargyline; Clorg, 100 ?M clorgyline; Depr, 100
?M deprenyl; Nial, 75 mg/kg (i.p.) nialamide in vivo.
Velocity of 2.0 ?M dopamine clearance in mPFC tissue in the
3p ? 0.05, comparing with supernatant condition, as
presence of cocaine, L-deprenyl, or the combination of L-deprenyl with
GBR 12909 (3.0 ?M), DMI (0.1 ?M), or cocaine (100 ?M). A–C, Mean raw
clearance profiles for dopamine. For A–E, all components were added
simultaneously with 2.0 ?M dopamine. G, Mean ? SEM of dopamine
clearance velocities shown in A–F. Mean values obtained in buffer and
supernatant alone are shown for comparison. n ? 16 for control; n ? 3–5
for all other groups.1p ? 0.05, comparing with control condition;2p ? 0.05,
comparing with buffer condition;3p ? 0.05, comparing with supernatant
condition, as determined with a one-way ANOVA followed by a Fisher’s
test. Sup, Supernatant; Cocaine, 100 ?M; Depr, 0.1 ?M deprenyl;
GBR?Depr, DMI?Depr, and Cocaine?Depr were added to the incubation
in the same concentrations as used singly.
Velocity of 2.0 ?M dopamine clearance in mPFC tissue in the
38 J. Neurosci., January 1, 2001, 21(1):35–44Wayment et al. • Dopamine Clearance in the Prefrontal Cortex
the possibility that metabolic pathways of dopamine may be
traced after the various treatment conditions. Before the addition
of 2.0 ?M dopamine, basal concentrations from separate experi-
ments were (in micromolar per original sample removed) as
follows: dopamine, 0.0048 ? 0.0003; DOPAC, 0.0085 ? 0.0012;
HVA, 0.0102 ? 0.0026; 3-MT, 0.0328 ? 0.0078; NE, 0.0058 ?
0.0008 (n ? 4).
Figure 5A corresponds to the aqueous phase taken from exper-
iments shown in Figure 2A–G in which dopamine uptake inhib-
itors were added to the incubation medium. Five minutes after
the addition of dopamine, the level of dopamine on the outside
(aqueous phase) of controls was 0.627 ?M. The level of dopamine
remaining in the aqueous phase of the supernatant condition was
significantly increased, whereas the metabolites, DOPAC and
HVA, were significantly decreased compared with the control
condition. Dopamine, metabolites, and NE values for all condi-
tions were compared with both the control and supernatant con-
ditions, and significant differences are indicated in Figure 5.
However, for purposes of clarity, comparison of these values with
those from the supernatant will be discussed here only for dopa-
mine, because several metabolites were significantly higher when
the levels from tissue were compared with those from the super-
The only uptake inhibitor that produced a significant increase
in aqueous phase dopamine levels above control values after the
5 min incubation was GBR 12909, whereas no other conditions of
dopamine, NE, or 5-HT uptake inhibition produced an increase
in aqueous phase dopamine levels. The combination of GBR
12909 with DMI or fluoxetine also did not produce a significant
elevation in aqueous phase dopamine levels. The levels of
DOPAC and HVA measured in the aqueous phase after the 5
min incubation were significantly decreased with GBR 12909
and NE in the aqueous phase from mPFC tissue 5
min after incubation in the presence of dopamine
under low Na?conditions or after addition of
monoamine uptake/MAO inhibitors. Data repre-
sent mean ? SEM of dopamine, DOPAC, HVA,
3-MT, and NE concentrations from the original
(100 ?l) sample taken from the incubation. A,
Monoamine uptake inhibitors (n ? 15 for control;
n ? 3–5 for all other groups). B, MAO inhibitors
(n ? 15 for control; n ? 3–4 for all other groups). C,
Combination of L-deprenyl with uptake inhibitors
GBR 12909 (GBR?Depr), DMI (DMI?Depr), or
cocaine (Cocaine?Depr) (n ? 15 for control; n ?
3–5 for all other groups).
with control condition;2p ? 0.05, comparing with
supernatant condition, as determined with a one-
way ANOVA followed by a Fisher’s test. ND, Not
Concentration of dopamine, metabolites,
1p ? 0.05, comparing
Wayment et al. • Dopamine Clearance in the Prefrontal CortexJ. Neurosci., January 1, 2001, 21(1):35–44 39
addition, whereas 3-MT and NE levels were significantly elevated
after GBR 12909 addition. The only other significant change from
the control condition was an increase in HVA levels in the low
Figure 5B shows dopamine, its metabolites, and NE levels in
the aqueous phase after the addition of MAO inhibitors corre-
sponding to experiments shown in Figures 3A,C–E and 4B.
Dopamine levels were significantly elevated after all conditions of
MAO inhibition, with the exception of the condition in which
L-deprenyl was added at t ? 0 sec and incubated for the 5 min
period. In all conditions, with the exception of L-deprenyl added
simultaneously with dopamine (t ? 0 sec), the MAO inhibitors
significantly reduced DOPAC levels. L-deprenyl added at t ? 0
min attenuated, but did not significantly reduce, DOPAC levels.
Homovanillic acid levels were also significantly reduced after
pargyline and L-deprenyl addition when these drugs were present
during the 20 min baseline stabilization period. The levels of
3-MT in the supernatant were elevated above control values
under all conditions of MAO inhibition. Norepinephrine levels
were not significantly altered by any of the MAO inhibitors.
Figure 5C, corresponding to the aqueous phase taken from
experiments shown in Figure 4C–F, demonstrates that the com-
bination of GBR 12909 ? L-deprenyl, DMI ? L-deprenyl, and
cocaine ? L-deprenyl all significantly increased dopamine levels
above control values and were not different from levels in the
supernatant condition. The levels of DOPAC were significantly
decreased from controls after the combination of L-deprenyl with
either GBR 12909 or DMI. The combination of L-deprenyl with
GBR 12909 also significantly decreased HVA levels. As with the
MAO inhibitors alone, 3-MT levels were also increased when
given in combination with these uptake inhibitors. Norepineph-
rine levels were not determined for the cocaine ? L-deprenyl
group, but no other treatment conditions produced significant
differences in this neurotransmitter.
In vivo microdialysis experiments were conducted in the mPFC
and striatum to determine whether (1) local infusion of the MAO
inhibitor, pargyline, would produce an increase in extracellular
dopamine levels and (2) whether there were differential effects
between these two brain areas in the regulation of dopamine and
metabolites by local pargyline infusion. The results of this exper-
iment are shown in Figures 6A–F (100 ?M pargyline) and 7A–F
(300 ?M pargyline). Infusion with 100 ?M pargyline produced a
significant elevation in extracellular dopamine levels in the mPFC
when infused at a concentration of 100 ?M for a 1 hr period. This
increase was ?300% above baseline values and occurred within
20–40 min after pargyline infusion. A significant increase in
DOPAC levels was found, whereas no significant changes oc-
curred for HVA levels. In contrast, striatal dopamine levels
showed only a trend toward an increase to ?200% above baseline
values, but the trend toward an increase above baseline was
delayed by 20 min and occurred only after significant reduction in
DOPAC levels, which were reduced to ?50% of baseline. Also in
contrast to the mPFC, HVA levels in the striatum were signifi-
cantly reduced to ?40% of baseline.
In the presence of 300 ?M pargyline, dopamine levels in the
mPFC increased by ?5000% above baseline values, and this effect
occurred within 20–40 min after pargyline infusion. Again, no
significant decrease in DOPAC or HVA levels was observed.
Striatal dopamine increased to ?1000% above baseline levels, but
as with the lower dose of pargyline, the effects were delayed by 20
min as compared with the early increase in mPFC dopamine
levels. The levels of DOPAC, although reduced to ?40% of
baseline, did not reach statistical significance because all values
were compared with the last baseline sample, which was already
decreased by 20% before pargyline infusion. HVA levels were
significantly reduced to 30% of baseline levels.
The main findings from these studies are as follows. (1) DAT and
NET inhibitors account for only 50–70% of the velocity of do-
pamine clearance in the mPFC; (2) MAO inhibitors attenuate the
velocity of dopamine clearance by ?30–50%; (3) the effects of
DAT/NET uptake inhibitors plus the MAO inhibitor, L-deprenyl,
DOPAC, and HVA levels in the mPFC and striatum after 100 ?M
pargyline infusion. Data represent mean ? SEM of dopamine, DOPAC,
and HVA levels before pargyline infusion, during a 1 hr infusion with 100
?M pargyline in the mPFC (A–C) or striatum (D–F). Pargyline infusion
is indicated by the black bar and was then replaced with aCSF. Samples
collected for the first time point after manual switching of solutions is not
shown because of alterations in aCSF flow rates. Basal levels from mPFC
from all groups (including those shown in Fig. 7) were (in femtomoles per
sample) as follows: for dopamine, 13.0 ? 2.4; for DOPAC, 247 ? 58; for
HVA, 404 ? 57. Basal levels from striatum from all groups (including
those shown in Fig. 7) were (in femtomoles per sample) as follows: for
dopamine, 48.8 ? 9.9; for DOPAC, 7219 ? 1286; for HVA, 6087 ? 1009.
Sample sizes were as follows: mPFC dopamine, n ? 9; DOPAC and HVA,
n ? 7; striatal dopamine, DOPAC, and HVA, n ? 6. *p ? 0.05 compared
with last baseline sample, as determined with a one-way, repeated-
measures ANOVA followed by a Fisher’s test.
In vivo microdialysis measurements of extracellular dopamine,
40 J. Neurosci., January 1, 2001, 21(1):35–44 Wayment et al. • Dopamine Clearance in the Prefrontal Cortex
on dopamine clearance appear to be additive; (4) the effect of
DAT/NET inhibition on the initial rate of dopamine clearance
may be compensated for by another process; and (5) local in vivo
pargyline infusion into the mPFC via microdialysis dramatically
elevates mPFC extracellular dopamine levels with no decreases in
DOPAC or HVA, whereas pargyline-induced dopamine in-
creases in the striatum occur only after infusion of the higher
pargyline dose and are accompanied by decreases in DOPAC or
HVA levels, or both.
Processes of mPFC dopamine clearance in vitro
The RDE findings are most consistent with the possibility that
there are at least two major processes by which dopamine is
cleared from the extracellular space. One is by a Na?-dependent
process, presumably via DAT and NET, and the second is by a
process altered by MAO inhibitors. Several previous in vitro
studies have also reported only a partial (?40–70%) inhibition of
dopamine clearance in the mPFC after cocaine or low Na?
conditions, or both, in contrast to the nucleus accumbens and
striatum, in which cocaine inhibits dopamine clearance by 95%
(Hadfield and Nugent, 1983; Izenwasser et al., 1990; Elsworth
et al., 1993; Wheeler et al., 1993).
The time course of inhibition by L-deprenyl on dopamine
clearance velocity suggests that there is an immediate effect of
MAO inhibitors on dopamine clearance. Several possibilities for
this response are considered.
One explanation for the effect of L-deprenyl is that deprenyl is
converted to L-amphetamine, and this in turn inhibits dopamine
uptake via DAT or NET (Karoum et al., 1982; Tetrud and
Langston, 1989; Okudo et al., 1992). However, all MAO inhibi-
tors tested in the present study decreased the dopamine clearance
rate. Therefore, there may be an alternative process in the mPFC
that is inhibited by all of these agents.
A second possibility is that MAO inhibitors alter the quinpirole
binding site, which in turn may decrease dopamine clearance
velocity. MAO inhibitors have been shown to modulate the
binding of quinpirole (Levant et al., 1993, 1996). If the quinpirole
binding site on D2 or D3 receptors in the mPFC is bound by
MAO, modification of DAT function might be expected, given
that D2 receptors have been shown to regulate DAT activity
(Meiergerd et al., 1993; Cass and Gerhardt, 1994; Batchelor and
Schenk, 1998). However, such an explanation is not consistent
with the partial effects of low Na?or DAT/NET uptake
A third explanation is that these MAO inhibitors may inhibit
other more recently described transporter systems, organic cation
transporter (OCT) 2 or OCT3 (Busch et al., 1998; Wu et al., 1998;
Grundemann et al., 1999). Although both of these transporters
have been reported to transport dopamine and are present in the
brain (Gorboulev et al., 1997), the OCT3 transporter appears
much more abundant in brain tissue than OCT2 and is found in
cortical regions (Wu et al., 1998). The OCT3 transporter medi-
ates the uptake of dopamine, and amphetamine interacts with this
transporter as well (Wu et al., 1998). Thus, interaction with this
newly described transporter may be important in mPFC dopa-
Finally, there is the possibility that different clearance pro-
cesses are present within different heterogenous regions of the
mPFC. Previous work has demonstrated regional effects of DAT
and NET inhibitors (Cass and Gerhardt, 1995), and regional
differences in dopamine clearance may be expected based on
immunohistochemical measures of DAT location (Ciliax et al.,
1995). Our studies examined the entire mPFC and therefore
would not distinguish among clearance processes located
within different mPFC subregions.
Biphasic effects of cocaine and MAO inhibition on
Unexpectedly, when either cocaine or the MAO inhibitor,
L-deprenyl, was added simultaneously with dopamine to the in vitro
incubation mixture, there was a biphasic profile of dopamine clear-
ance rather than a linear clearance profile. The first portion lasted
on the order of seconds and was nearly or completely blocked by
these agents for several seconds. The reason for observing com-
plete inhibition of dopamine clearance for a longer period than
what was observed in the supernatant condition is not clear. It
should be pointed out that no alterations in baseline output were
observed after cocaine or pargyline were added to mPFC tissue in
the absence or presence of exogenous dopamine addition, nor did
DOPAC, and HVA levels in the mPFC and striatum after 300 ?M
pargyline infusion. Data represent mean ? SEM of dopamine, DOPAC,
and HVA levels before pargyline infusion, during a 1 hr infusion with 300
?M pargyline in the mPFC (A–C) or striatum (D–F). Pargyline infusion
is indicated by the black bar and was then replaced with aCSF. Samples
collected for the first time point after manual switching of solutions are
not shown because of alterations in aCSF flow rates. Sample sizes were as
follows: mPFC dopamine and DOPAC, n ? 9; HVA, n ? 8; striatal
dopamine, DOPAC, and HVA, n ? 5. *p ? 0.05 compared with last
baseline sample, as determined with a one-way, repeated-measures
ANOVA followed by a Fisher’s test.
In vivo microdialysis measuring extracellular dopamine,
Wayment et al. • Dopamine Clearance in the Prefrontal CortexJ. Neurosci., January 1, 2001, 21(1):35–44 41
these drugs cause release of dopamine from mPFC tissue. In
addition, neither pargyline nor cocaine alters sensitivity of the
electrode to dopamine (data not shown). Future studies will need
to directly address the biphasic nature of dopamine clearance in
this brain region.
The second phase of the biphasic profile demonstrated an
additive effect for L-deprenyl with uptake inhibitors, suggesting
that a second process or multiple processes may work either
separately or in tandem with DAT and NET function.
Dopamine and metabolites in vitro
The results from the HPLC analyses of aqueous phase samples
taken 5 min after exogenous dopamine addition indicated that
dopamine levels were significantly elevated only after GBR 12909
addition but, unexpectedly, not in the presence of cocaine or the
combination of GBR 12909, DMI, and fluoxetine. It is unclear
why GBR 12909 alone would produce greater effects than with
the combination of DAT/NET inhibitors. The results suggest that
GBR 12909 may have alternative actions, such as direct inhibition
of the MAO inhibitor-dependent process, or that binding of
cocaine or DMI to NET may activate a process that is blocked by
MAO inhibitors. Any effects of MAO inhibitors on dopamine
clearance dependent on NET activity would not be expected to
occur in brain areas that lack substantial clearance by NET, such
as the nucleus accumbens or striatum. Consistent with the ab-
sence of effects of MAO inhibitors on dopamine clearance in
these latter brain regions, pargyline addition does not alter do-
pamine clearance velocity in the nucleus accumbens or striatum
when tested in the RDE system that has been used in the present
studies (Meiergerd and Schenk, 1994; Povlock and Schenk, 1997).
Dopamine was elevated in the aqueous phase after all condi-
tions of MAO inhibition, with the exception of when L-deprenyl
was added at t ? 0 sec. L-deprenyl incubated over a short-term
period (added at t ? 0 sec) produced immediate partial inhibitory
effects on dopamine clearance yet did not alter extracellular
dopamine levels after the 5 min incubation period, perhaps be-
cause of compensatory DAT and NET activity. However, over
the longer incubation time (20 min or after in vivo administra-
tion), MAO inhibitors, including L-deprenyl, produced elevated
dopamine levels in the aqueous phase, suggesting the possibility
that uptake by DAT and NET may be impaired after longer
incubation with MAO inhibitors. When L-deprenyl (t ? 0 sec) was
combined with DAT/NET uptake inhibitors, extracellular dopa-
mine levels were significantly elevated above controls. Together,
these findings support the results from the RDE studies suggesting
that at least two processes contribute importantly to dopamine
clearance in the mPFC.
Pargyline effects on mPFC and striatal extracellular
dopamine levels in vivo
Microdialysis studies examining MAO inhibitor action on extra-
cellular dopamine levels have used systemic injection of these
drugs, with either increases (Sharp et al., 1986; Butcher et al.,
1990; Okudo et al., 1992) or no changes reported (Kato et al.,
1986; Butcher et al., 1990). In the present study, pargyline con-
centrations prepared for infusion through the microdialysis probe
were higher than those shown to inhibit MAO activity (Cesura
and Pletscher, 1992). However, it was not possible to know the
concentration of parygline reaching the surrounding tissue, and
substantial increases in extracellular dopamine levels occurred in
the mPFC in the absence of decreases in the levels of its metab-
olites, DOPAC and HVA, indicating an effect of pargyline at least
partially independent of its MAO inhibitory action. In contrast,
striatal dopamine levels were elevated by pargyline infusion to a
lesser degree and were accompanied by decreases in DOPAC and
HVA levels. Together, these data suggest that mPFC dopamine is
regulated differently from striatal dopamine and may be caused
by a direct effect of pargyline on mPFC dopamine uptake pro-
cesses, although contribution by long-loop feedback pathways
cannot be ruled out. Future in vivo studies should examine
whether additive effects occur for mPFC dopamine using lower
concentrations of MAO inhibition combined with DAT/NET
In summary, these results demonstrate that more than one
major mechanism appears responsible for dopamine clearance in
the mPFC: (1) clearance occurs by DAT and NET, and (2)
clearance occurs by a second component that is blocked by MAO
inhibitors. The kinetics of dopamine clearance in vitro suggest
that the effects of DAT and NET inhibitors combined with the
MAO inhibitor, L-deprenyl, are additive. This is the first report
describing an important contribution by MAO inhibitors for the
clearance of mPFC dopamine in the kinetic domain. In vivo
microdialysis studies infusing the MAO inhibitor, pargyline, into
the mPFC suggest that pronounced increases in extracellular
dopamine levels occur in the absence of decreases in extracellular
DOPAC or HVA levels, whereas pargyline infusion into the
striatum produces less pronounced increases that appear to be a
consequence of decreased dopamine metabolism. Such findings
may have implications for reinterpreting the role of MAO inhib-
itors in antidepressant action and offer caution against extrapo-
lation of observations across different brain regions when exam-
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