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Vol. 288, Issue 2, 879-887, February 1999
Neuroscience Training Program (M.A.H., G.A.G.), Departments of Psychiatry (G.A.G.) and Pharmacology (G.A.G.), and the Rocky Mountain Center for Sensor Technology (M.A.H., G.A.G.), University of Colorado Health Sciences Center, Denver, Colorado
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Abstract |
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 Top
 Abstract
 Introduction
Materials and methods
Results
Discussion
References
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Age-related changes in the capacity, rate, and modulation of dopamine (DA) uptake within the striatum and the nucleus accumbens core of Fischer 344 rats were investigated using in vivo electrochemical recordings coupled with local drug application techniques. Equimolar amounts of DA were pressure ejected into the striatum and the nucleus accumbens of 6-, 12-, 18-, and 24-month old rats. The DA ejections produced larger DA signal amplitudes in the older rats, suggesting age-related differences in the capacity to clear extracellular DA. Within the striatum, the capacity and rate of DA uptake were reduced by 50% in the aged groups (18 and 24months) compared with the younger rats (6 and 12 months). In the nucleus accumbens, significant reductions in DA uptake capacity and rate were observed in the 24-month group. In both brain regions and in all age groups studied, the rate of DA uptake was found to be concentration-dependent until a maximal rate was reached. The maximum rate of DA transport was significantly reduced in both the striatum and the nucleus accumbens of aged rats (18 and 24 months versus 6 and 12 months). The ability of nomifensine, an inhibitor of the DA transporter, to modulate DA signal amplitudes in the striatum and the nucleus accumbens was also decreased with age (24 months versus 6 months). Taken together, these findings demonstrate substantial age-related deficits in DA uptake processes within the striatum and the nucleus accumbens, consistent with the hypothesis that DA uptake may be slowed in aged animals to compensate for reductions in DA release.
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Introduction |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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The
progressive development of motor and cognitive dysfunction during
senescence may be associated with alterations in neurotransmitter function in the central nervous system. In particular, age-related diminutions in dopamine (DA) neurotransmission within the basal ganglia
of humans and animals are believed to contribute to changes in the
execution and coordination of voluntary movements (Finch et al., 1981
).
Age-related alterations in both pre- and postsynaptic indices involved
in dopaminergic (DAergic) neurotransmission have been well documented
(Joseph et al. 1978; Goldman-Rakic and Brown, 1981; Rose et al. 1986;
Friedemann and Gerhardt, 1992; Hebert and Gerhardt, 1998).
The major regulator of DAergic neurotransmission is the reuptake of
released DA via the dopamine transporter (DAT) (Giros et al., 1996).
This high-affinity uptake process is sodium-, chloride-, and
temperature-dependent; it is also saturable and has an affinity for
monoamine substrates of approximately 0.1 to 2 µM (Meiergerd and
Schenk, 1994; Lenhard et al. 1998). Not surprisingly, this important
regulatory mechanism has also been shown to be altered with age.
Several studies have shown a progressive age-related decline in the
number of DA transporters (Zelnik et al., 1986) and a sharp decline in
DAT mRNA in both rats and humans (Bannon and Whitty, 1997). As the
status of this integral membrane protein during aging may have
important implications for the proper functioning of the DAergic
system, the actual dynamic performance of the DA uptake mechanism is of
vital importance, because it regulates the amount of DA available for
neurotransmission. It has been shown that the affinity of ligand
binding to the rat and human DA transporter decreases with age (Shimizu
and Prasad, 1991; Volkow et al., 1994). In addition, previous studies
from our laboratory involving measures of stimulus-evoked release of DA
in aged animals have yielded data implicating age-related changes in
DAT function (Friedemann and Gerhardt, 1992; Hebert and Gerhardt,
1998). The purpose of this study was to systematically study changes in
DAT function that may occur with age.
The in vivo activity of the DA transporter can be assessed in various
brain regions by monitoring the disappearance of locally ejected DA
with high-speed electrochemical recordings (Cass et al. 1993; Gerhardt
et al. 1996). This technique allows the precise measurement of DA
concentration (nanomolar) within a discrete brain region at a fast
sampling rate (5 Hz). The peak response confers information regarding a
brain region's ability (capacity) to clear the exogenous DA (Cass and
Gerhardt, 1995). The duration of the electrochemical signal indicates
the rate at which the removal is occurring. Direct electrochemical
measurements of the presence and successive uptake of exogenously
applied DA eliminates contributions from the release of endogenous DA
and confines the processes being examined to those responsible for
clearing DA from the extracellular space (Cass et al. 1993). The
electrochemical recordings are reproducible and the resulting kinetic
parameters permit quantitative comparison of extracellular DA
regulation between age groups and brain regions. This technique also
provides in vivo assessment of the efficacy of drugs that modulate the DA uptake process (Cass et al., 1993).
Our investigations have focused on DA uptake within the striatum and
the core of the nucleus accumbens because of their distinct role in
motor activity (Beninger, 1983) and because each system appears to be
differentially affected by the aging process (Friedemann, 1992;
Friedemann and Gerhardt, 1992). More important, reports have suggested
differences in the status and function of high-affinity DA uptake
systems within these two regions. The differences observed include
regional variation in the density of DATs (Marshall et al., 1990),
differences in the molecular features of the DAT in the two areas
(Amara and Kuhar, 1993), and a reduced capacity of DA neurons in the
nucleus accumbens to clear DA from the extracellular space as compared
with those in the striatum (Stamford et al. 1988). Additionally,
reports indicate that DA uptake inhibitors produce differential effects
in the two regions, both in vitro (Izenwasser and Cox, 1990) and in
vivo (Cass and Gerhardt, 1995).
To determine age-related and region-specific differences in the
capacity to clear DA, we compared the amplitudes of electrochemical signals generated by applying a range of amounts of exogenous DA in
young (6-month), middle-aged (12-month), and aged (18- and 24-month)
Fischer 344 (F344) rats. Next, we compared the decay portion of
equivalent amplitude signals to assess age-related and region-specific
differences in the rate of DA uptake. Finally, we measured the
abilities of nomifensine, a combined DAT and norepinephrine transporter
(NET) inhibitor, desipramine, a selective NET inhibitor, and
citalopram, a selective inhibitor of the serotonin transporter (SERT),
to alter the capacity of DA uptake in the striatum and the nucleus
accumbens of young (6-month) versus aged (24-month) rats. These uptake
inhibitors have been used to investigate age-related differences in the
stimulation of motor behavior (Hebert and Gerhardt, 1998) and their
ability to inhibit monoamine uptake in vivo has been demonstrated with
in vivo electrochemistry (Cass and Gerhardt, 1995; Hoffman and
Gerhardt, 1998).
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Materials and Methods |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Animals. Young adult (4-6 months, n = 28), middle-aged (10-12 months, n = 24), and aged (18-20 months, n = 30; 24-26 months, n = 32) male F344 rats obtained from the National Institute on Aging (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were used for all experiments. Protocols for animal use were approved by the Institutional Animal Care and Use Committee. Animals were housed according to approved guidelines under a 12 h light/dark cycle with food and water available ad libitum. All recordings were conducted in naïve rats during the light cycle.
In Vivo Electrochemistry. Rats were anesthetized with urethane (1.0-1.5 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. Electrochemical reference electrodes (Ag/AgCl) were implanted into brain regions remote from recording sites and were cemented into place with dental acrylic.
Electrode/micropipette assemblies used for the in vivo recordings consisted of a working electrode and a single-barrel (1 mm o.d., 0.58 mm i.d. glass, A-M Systems, Inc., Everett, WA) or a triple-barrel micropipette (1.2 mm o.d., 0.68 mm i.d. glass, World Precision Instruments, Inc., Sarasota, FL). The tips of the micropipettes had outer diameters of 10 to 15 µm and they were positioned 300 ± 10 µm from the tips of the electrodes. Single-barrel micropipettes contained 200 to 400 µM DA in 0.9% NaCl with 100 µM ascorbic acid added as an antioxidant. Triple-barrel pipettes contained 200 to 400 µM DA solution in one or two barrels and drug solutions (800 µM nomifensine maleate, 800 µM desipramine, and/or 800 µM citalopram) in the other barrel(s). All locally applied drugs were prepared in saline and adjusted to pH 7.4. High-temperature Nafion-coated (5% solution, 4-7 coats at 200°C, Aldrich Chemical Co., Milwaukee, WI) single carbon-fiber electrodes (fiber diameter 30 µm; exposed length 100-150 µm) were used as recording electrodes (Hebert et al., 1996). The sensitivities and linearities of all recording electrodes were determined by generating calibration curves for each recording electrode in stock
solutions of DA in 0.1 M phosphate-buffered saline at 22°C. All
electrodes showed linearities for increments of DA ranging from 2 to 14 µM (r2 
0.997) and selectivities
greater than 500:1 for DA versus 3,4-dihydroxyphenylacetic acid (DOPAC)
and ascorbic acid. Extracellular changes in DA were expressed
quantitatively in terms of the DA calibration curves (Gerhardt, 1995).
Electrodes were used to record from one animal and then discarded.
High-speed chronoamperometric electrochemical measurements were
continuously made and averaged to 1 Hz with an IVEC-10 system (Medical
Systems Corp., Greenvale, NY). Square-wave pulses of 0.00 to +0.55 V,
with respect to a Ag/AgCl reference electrode, were applied to the
working electrode for 0.10 s and repeated 5 times/s (Gerhardt,
1995). The resulting oxidation and subsequent reduction currents were
digitally integrated during the last 80 ms of the 100-ms pulses. The
detection limits of the electrodes averaged 25 ± 5 nM
(n = 107), with a signal-to-noise ratio of 3.0.
All coordinates used for recordings were based on the rat atlas of
Paxinos and Watson (Paxinos et al., 1985; Paxinos and Watson, 1986).
With the incisor bar positioned at 
2.3 mm, electrode assemblies were
positioned in the striatum (1.0-1.7 mm anterior to bregma, 2.0-2.2 mm
lateral from midline, 3.5-5.0 mm below the surface of the brain) or in
the core of the nucleus accumbens (1.5-1.7 mm anterior to bregma,
2.0-2.2 mm lateral from midline, 6.5-8.0 mm below the surface of the
brain). Once the electrode/micropipette recording assembly was
positioned at each recording site, a baseline signal was recorded for
approximately 5 to 10 min before DA was ejected. Baseline signals
obtained from each recording position were considered to be the
theoretical zero response. DA (2-100 pM) was applied by pressure
ejection (10-40 psi for 0.5-6 s) with a Picospritzer II pressure
ejection system (General Valve Corp., Fairfield, NJ). The volume
applied was determined and controlled with a stereomicroscope fitted
with a reticule in one eyepiece to measure the movement of the meniscus
in the micropipette (Friedemann and Gerhardt, 1992).
For experiments designed to compare DA uptake in the striatum and the
nucleus accumbens of different ages of animals, various volumes of DA
solution (10-250 nl containing 2-100 pmol DA) were applied at each
individual site before lowering to a new site. Characteristics of the
resulting signals were used to quantitate DA uptake capacity and rate
(see Data Analysis). Specific volumes of DA were pressure
ejected to determine the capacity of DAergic terminals to clear
exogenous DA and the rate at which DA uptake occurs in these regions.
After completing recordings resulting from various concentrations of
DA, the electrode assembly was lowered 250 to 500 µm and allowed to
equilibrate at the new site for 5 to 10 min.
For experiments involving the local application of uptake inhibitors, a
specific amount of DA was applied by pressure ejection at 5-min
intervals to obtain reproducible responses (usually two to three
applications). Once the DA signals were stable (
10% variation), the
drug solution (nomifensine, desipramine or citalopram) was applied
slowly over 10 to 20 s, approximately 30 s before the next
application of DA. The volume of drug solution applied was two to three
times the volume of DA solution applied to "saturate" the uptake
sites. Because the concentration of drug was twice the concentration of
the DA solution, the amount (in picomoles) of drug applied was four to
six times the amount (in picomoles) of DA applied. After the effects of
locally applied drugs were recorded, the electrode assembly was lowered
250 to 500 µm and allowed to equilibrate at the new site for 5 to 10 min. At the end of the experiments, animals were euthanized and their
brains were removed for histological confirmation of electrode
placements. Only recordings in which the electrodes were clearly
positioned in the striatum or the core of the nucleus accumbens were
included in Results.
Data Analysis. For each individual electrochemical signal, two parameters were analyzed: 1) the peak amplitude resulting from DA pressure ejection; and 2) the clearance rate (TC in µM/s), defined by the change in DA concentration between the T20 and T60 time points (e.g., the slope of a pseudolinear portion of the decay curve). The peak amplitude is a measure of extracellular DA. The decay slope of the signal indicates the rate at which the DA is removed from the extracellular space. For purposes of analysis, peak amplitude and clearance values for each signal were grouped and averaged according to concentration (for capacity analysis) or signal amplitude (for rate analysis). The relative capacity of each brain region to clear DA was determined by correlating the amplitude of the resulting signal with the moles of DA applied by pressure ejection. The rate of DA uptake was determined by plotting the amplitude of each signal against the Tc for that signal. A total of 128 to 357 signals recorded in the striatum and 52 to 92 responses in the core of the nucleus accumbens for each age group were used to analyze properties of DA uptake. Linear regression analyses were performed with Prism v2.01 (GraphPad Software, Inc., San Diego, CA). The capacity and rate data were analyzed by a repeated-measures analysis of variance (ANOVA) followed by Tukey-Kramer post hoc comparisons. For experiments involving the local application of uptake inhibitors, baseline parameters were defined at each recording site by averaging the reproducible signals (two to three) preceding drug application; the first DA signal after the drug ejection was taken as the modulated response. Changes in amplitude and Tc after the application of uptake inhibitors were analyzed by repeated-measures ANOVA followed by Tukey-Kramer post hoc comparisons. For statistical comparisons of the electrochemical responses between the striatum and the nucleus accumbens in each age group, a two-way ANOVA (age × region) was performed. In all tests, p < .05 was defined as statistically significant. Statistical analyses were performed with Instat 2.04 and StatMate (GraphPad Software).
Materials. Nomifensine maleate and desipramine were purchased from Research Biochemicals International (Natick, MA). DA, DOPAC, ascorbic acid, and urethane were purchased from Sigma Chemical Co. (St. Louis, MO). Citalopram was generously provided by Dr. Alan Frazer (University of Texas Health Sciences Center, San Antonio). All other reagents were research grade.
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Results |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Age-Related Changes in Signal Amplitudes: DA Uptake Capacity. To investigate age-related changes in DA uptake, equal amounts of exogenous DA were locally applied in rats of different ages and the resulting signal amplitudes were compared. Application of exogenous DA into the striatum and the nucleus accumbens in all age groups of F344 rats produced reproducible electrochemical signals. Typical electrochemical signals detected after application of 30 pmol of DA within the striatum and the nucleus accumbens of young (6-month), middle-aged (12-month) and aged (18- and 24-month) rats are presented in Fig. 1. Using this amount of locally applied DA, the recorded responses within the striatum were significantly (F(3,75) = 59, all p values < .001) larger as a function of age, indicating reduced DA uptake. Significant age-dependent deficits in the ability to clear exogenous DA within the striatum were measured as early as 12 months and became quite pronounced (2-3 fold greater) at 18 and 24 months. Within the nucleus accumbens, the amplitude of the electrochemical signals remained unchanged until 24 months, at which age modest, but significantly increased, amplitudes were measured (F(3,61) = 11, p < .001).
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Age-Related Changes in Tc Values: DA Uptake Rates. Because DAergic neurotransmission is not only regulated by the amount but also the duration of extracellular DA, we investigated possible age-dependent alterations in the rate of DA clearance from the extracellular space. The amplitude comparisons illustrated in Fig. 1 suggest significant age- and region-related differences in the relative numbers of functional DA transporters, which in all likelihood affect the DA uptake rate. Specific information regarding the rate of DA uptake was obtained from the decay portion of the electrochemical current traces. The clearance rate, Tc, defined as the micromolar change in DA concentration per second, is calculated from a pseudolinear portion of the decay curve (slope of line between T20 and T60). As seen in Fig. 4, comparison of equivalent amplitude signals recorded from the striatum of young (6-month) and aged (24-month) rats indicated significant differences (unpaired, two-tailed Student's t test, p < .001) in the Tcs, which suggested age-related alterations in the rate of DA uptake between the young and aged rats.
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Age-Related Differences in DA Uptake Inhibition: Efficacy of Nomifensine. Just as the function and capacity of the DAT regulates DA neurotransmission, selective drugs that competitively inhibit the transport process can regulate the DA uptake process. To assess the modulation of DA uptake across age groups, we tested the degree of uptake inhibition produced by nomifensine, desipramine or citalopram in the striatum and the nucleus accumbens of young (6-month) and aged (24-month) rats. Changes in signal amplitudes after drug application were defined as a percentage of change from the baseline, which was defined as 100%.
The local application of nomifensine 30 s before pressure ejection of DA in 6-month-old rats greatly increased the amplitude and time course of the electrochemical responses (Fig. 7); by itself, nomifensine produced no detectable change in the electrochemical response (data not shown). Nomifensine modulated DA signal amplitudes within both the striatum and the nucleus accumbens of 6-month rats (Fig. 8) by 200% compared with control signals, indicating that local application of this drug greatly diminished the capacity of these regions to clear exogenous DA and permitted DA to remain in the extracellular space for a longer time. In 24-month-old rats, nomifensine-modulated signal amplitudes and Tc values in the striatum (Fig. 8A) and the core of the nucleus accumbens (Fig. 8B) to a much lesser degree than in the 6-month group (unpaired, two-tailed Student's t test, p < .001, Tc data not shown). The differences in the ability of nomifensine to modulate signal amplitudes in the aged rats indicate age-related differences in the efficacy of this DAT inhibitor. Desipramine and citalopram produced no noticeable modulation of DA signal amplitude in the young or aged rats (Fig. 8), indicating that the NET and SERT were not involved in DA uptake measured in these experiments.
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Discussion |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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The purpose of this study was to compare the regulation of extracellular DA levels in the striatum and the nucleus accumbens core of four age groups of F344 rats. We have found marked age-related differences in DA uptake processes in both brain regions. First, we observed age-related reductions in the capacity to clear exogenous DA over a wide range of concentrations in both regions. Second, the rate of DA uptake was found to be significantly decreased with age. Finally, we observed that modulation of DA uptake resulting from addition of the DAT inhibitor nomifensine was significantly reduced in aged rats.
Signal amplitudes resulting from the application of exogenous DA
provide information regarding the capacity of the surrounding region to
clear DA and are thought to relate to the number of functional DA
transporter sites in that region (Cass et al. 1993; Cass and Gerhardt,
1995; Hoffman and Gerhardt, 1998). The relative number of functional DA
transporters can be thought of as the in vivo
"Bmax" in that region for that age
group. A comparison of the DA uptake capacity across age groups
demonstrated that the ability to remove exogenous DA was significantly
reduced in older rats, indicating possible age-related reductions in
DAT Bmax. Within the striatum, signal
amplitudes recorded in aged (18- and 24-month) rats were almost twice
those recorded in the younger (6- and 12-month) animals, suggesting a
40 to 50% reduction in the Bmax of
the aged rats. Likewise, differences in DA uptake capacity within the
nucleus accumbens (6- versus 24-month) suggest age-related
decreases in the Bmax in mesolimbic
DAergic terminals. Dramatic regional-differences in DAT
Bmax were also noted when comparing
the uptake capacity of the striatum to that of the nucleus accumbens,
with the nucleus accumbens being less efficient in clearing
extracellular DA. The power of in vivo electrochemistry lies with its
ability to measure functional events in a dynamic manner. This aspect,
combined with the short exposure times of exogenous DA to the DATs
within a certain brain region, limits direct comparison of our in vivo
Bmax (DA uptake capacity) indicies with previously reported in vitro values. However, we suggest that
relative in vivo Bmax values are
complimentary to those measured with in vitro radioligand binding or
uptake studies. In fact, the results of this study coincide with
reported age-related deficits and region-specific differences in
striatal and accumbens DAT Bmax values
in a variety of systems (Zelnik et al. 1986; Shimizu and Prasad, 1991).
Similar to information regarding the
Bmax of DAT in various regions and
groups, analysis of the Tc of similar
amplitude signals provided important information regarding uptake rate
(V) and apparent maximum rate
(Vmax) of DA uptake from the
electrochemical measures. Comparison of the
Tc indicated significant age-related
alterations in the lifetime of DA in the extracellular space, which
suggested that age may alter the rate (V) of DA uptake. DA
was cleared approximately 40% slower in the striatum of aged (18- and
24-month) rats as compared with young (6- and 12-month) animals. Within
the nucleus accumbens, the V did not change with age until
24 months of age, at which point it was 40% slower than all other age
groups. The region-specific and age-related differences we have
observed in DA uptake are generally consistent with previous in vivo
studies from our laboratory and others. Our results further confirm the finding that DA uptake is more efficient in the striatum than in the
nucleus accumbens and in the young rat versus the aged rat (Friedemann,
1992; Cass et al., 1993; Suaud-Chagny et al., 1995). Because DA
autoreceptors have been shown to modulate DA uptake (Cass and Gerhardt,
1994) age-related differences in autoreceptor-modulation of uptake may
be involved in the observed differences in DA uptake between age
groups. The consequence of age-related reductions in DA uptake rate
(V) and capacity (Bmax)
could be that DA remains in the extracellular space longer, which would
in turn result in altered postsynaptic signaling in the aged rat.
Plotting the Tc against a range of
signal amplitudes indicated that there is a maximal uptake velocity
after which the uptake rate is no longer concentration-dependent, an
apparent "Vmax". This in vivo
Vmax was significantly reduced by 50%
in both the striatum and the nucleus accumbens of aged (18- and
24-month) rats as compared with younger (6- and 12-month) animals.
Regional comparisons of uptake rates indicated a 40 to 50% decrease in both concentration-dependent rate of uptake and the maximum rate of
uptake within the nucleus accumbens as compared with the striatum. The
age-related and region-specific differences in the maximum rate of
uptake that we measured were of the same magnitude as those
demonstrated in situ and in vitro (Shimizu and Prasad, 1991). In
reconciling the functional consequences of maximal uptake rates, it is
important to consider that anything above 12 µM in the striatum and
10 µM in the nucleus accumbens probably does not represent normal
physiological conditions in any age group (Friedemann and Gerhardt,
1992; Hebert et al., 1996; Hebert and Gerhardt, 1998). The
determination that an in vivo Vmax can
be measured in both regions of all age groups may provide information
regarding the regulation of the DAT protein in the plasma membrane and
its turnover. In addition, our Tc data
from the electrochemical measures can be used to determine in situ
apparent maximum rate or in vivo Vmax
values that can be compared with other measures of uptake.
In addition to conferring information regarding the capacity and rate
of DA uptake, the reproducibility of electrochemical recordings permits
investigation of pharmacological modulation of DA uptake. Although DAT
is the primary mechanism by which DA is removed from the extracellular
space, DA uptake has been reported to occur in situ via other monoamine
transporters such as the NET and SERT (Izenwasser et al., 1990).
Whereas the relative abundance of serotoninergic and noradrenergic
content within the striatum and the nucleus accumbens is less than 10%
(Schenk et al., 1983), it was important to determine the relative
contribution of DAT, NET, and SERT in the DA uptake process across age
groups and regions. This was accomplished by pretreating DAergic
terminals with uptake inhibitors selective for specific transporters.
Only application of nomifensine was found to increase the concentration
and duration of DA within the extracellular space, supporting the
hypothesis that the DA uptake measures are recording the removal of DA
by DA transporters and not by SERTs or NETs.
Nomifensine inhibited DA uptake in both the striatum and the nucleus
accumbens to a larger extent in the young (6-month) rats compared with
the aged (24-month) rats. These results support earlier findings that
the effects of high-affinity uptake inhibitors, such as nomifensine,
are diminished in the striatum of aged rats (24-month) as compared with
young animals (6-month) (Friedemann, 1992; Friedemann and Gerhardt,
1992). The inability of nomifensine to alter uptake in the aged rats
confirms data from a recent study investigating behavioral stimulation
by various monoaminergic uptake inhibitors (Hebert and Gerhardt, 1998)
and suggests age-related changes in the pharmacological properties of
the DAT.
It should be noted that metabolism and diffusion, in addition to DA
uptake processes, contribute to the shape of the recorded electrochemical signals. Although, extrasynaptic catabolism of DA to
homovanillic acid or DOPAC reduces the concentration of DA measured in
the extracellular space, we know that homovanillic acid and DOPAC do
not contribute to the oxidation current measurements and metabolism
appears to play a minor role (Cass et al. 1993). Diffusion of DA in the
extracellular space has been measured and modeled previously by our
laboratory (Gerhardt and Palmer, 1987) and has been considered to play
a minor role in the measured uptake processes. In this study, we
examined the clearance of exogenously applied DA in the frontal cortex,
a region with little DA uptake capacity and where diffusion is
considered to be the primary mechanism of DA removal (Cass and
Gerhardt, 1995; Sesack et al. 1998). The average
Tc values for signals within the
frontal cortex of 6-month rats, measured at or near
Vmax concentrations, averaged
0.014 ± 0.001 µM/sec (n = 4) and those in the
24-month rats averaged 0.017 ± 0.002 µM/s (n = 5; data not shown). Consequently, these data support the hypothesis
that diffusion plays a minor role in the measured DA uptake rates, it
is consistent across age groups, and contributes less than 10% to the
Tc values indicated in this paper.
In addition to age-related reductions in the density of DAT, deficits
in in vivo DA uptake may also involve modifications of the DAT protein
and/or compensatory regulation of the uptake process. Possible
age-related alterations of DAT include phosphorylation or glycosylation
of transporter protein, changes in the membrane potential or fluidity
state of the membrane, or modification of DA transporter sulfhydryl
groups, all of which have been documented and may affect transporter
affinity and, in turn, the rate of uptake (Kuhar et al., 1990;
Meiergerd and Schenk, 1994; Patel et al., 1994). Further studies are
needed to determine whether age-related modifications of the DAT may be
responsible for altered DA uptake, or if aging results in decreased
levels of functional DA transporter protein.
Age-related reductions in DA uptake in the striatum and the nucleus
accumbens do not appear to be due to a reduction in the number of DA
nerve terminals in these areas. Previous studies in the same strain and
age groups of rats have indicated that DA whole tissue levels within
the nigrostriatal and mesolimbic cell body and terminal regions are not
altered with age (Hebert and Gerhardt, 1998). However, the age-related
deficits in DA uptake do occur along a similar time course as deficits
in DA release, as well as declines in locomotor activity (Hebert and
Gerhardt, 1998). The functional implication of the prolonged presence
of DA within the aged rat striatum and/or nucleus accumbens is that DAergic receptor-mediated transmission can remain effective (as in
young animals) in the presence of decreased DA release, thereby serving
as a compensatory mechanism (Rose et al., 1986; Friedemann and
Gerhardt, 1992; Hebert and Gerhardt, 1998). Compelling evidence has
demonstrated the remarkable plasticity of the DAergic system (Zigmond
et al., 1984; Altar et al., 1987; van Horne et al., 1992). The
nigrostriatal system has been suggested to adapt its capacity to
maintain function despite extensive neuronal loss found in parkinsonian
humans and animals (Cass et al., 1995). In Parkinson's disease, not
only is the number of DAT-binding sites reduced as one would suspect
after the loss of DA neurons, but what is possibly more significant is
that the number of DATs per neuron seems to also be reduced compared
with normal aging (Uhl et al., 1994). This would suggest that DA
neurons may undergo adaptive changes by reducing the number of DATs on
their terminals, thus reducing uptake of DA in an attempt to increase
the amount of extracellular DA. Likewise, a down-regulation of
functional DA uptake by reducing the number of DATs available or
modifying the functional capacity of the ones present may help maintain
DAergic transmission in aged animals. Correspondingly, compensatory
modulation of DAT may also render the DA-containing terminals of aged
rats less susceptible to the degenerative influences of neurotoxins
that are incorporated by the high-affinity DA uptake processes
(Gainetdinov et al. 1997). Thus, changes in DAT that occur in aged
animals may represent a form of compensation to maintain DA
neurotransmission in senescence.
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Acknowledgments |
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We gratefully acknowledge the intellectual discussions with Alexander Hoffman, the technical expertise of Scott Robinson, the assistance with statistical evaluation of data provided by Dr. Shelly Dickinson, and the gift of citalopram from Dr. Alan Frazer.
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Footnotes |
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Accepted for publication September 8, 1998.
Received for publication July 10, 1998.
1 This work was supported by grants from U.S. Public Health Services Grants NS09199 and AG06434 and National Institutes of Health Training Grant HDO7408-02. In addition, this work was supported, in part, by a Level II Research Scientist Development Award (MH01245) from the National Institutes of Mental Health to G. Gerhardt.
Send reprint requests to: Greg A. Gerhardt, Ph.D., Department of Psychiatry, Box C268-71, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. E-mail: Greg.Gerhardt{at}uchsc.edu
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Abbreviations |
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DA, dopamine; DAergic, dopaminergic; DAT, dopamine transporter; F344, Fischer 344; NET, norepinephrine transporter; SERT, serotonin transporter; DOPAC, 3,4-dihydroxyphenylacetic acid; Tc, clearance rate; T20, time for signal amplitude to decay by 20%; T60, time for signal amplitude to decay by 60%; ANOVA, analysis of variance.
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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