Aspartame decreases evoked extracellular dopamine levels in the rat brain: An in vivo voltammetry study
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Neuropharmacology 53 (2007) 967e974
www.elsevier.com/locate/neuropharm
Aspartame decreases evoked extracellular dopamine levels
in the rat brain: An in vivo voltammetry study
Brian P. Bergstrom*, Deirdre R. Cummings, Tricia A. Skaggs
Department of Biology, Neuroscience Program, Muskingum College, New Concord, OH 43762, USA
Received 13 August 2007; received in revised form 19 September 2007; accepted 20 September 2007
Abstract
Conflicting reports exist concerning the effect aspartame (APM, L-aspartyl-L-phenylalanine methyl ester) has upon brain biogenic amines. In
the following study, in vivo voltammetry was utilized to measure evoked extracellular dopamine (DA) levels in the striatum of rats in order to
assess APM’s effect. Time-course experiments revealed a significant decline in evoked extracellular DA levels within 1 h of a single systemic
dose (500 mg/kg i.p.) when compared to vehicle-injected controls. The effect was frequency dependent and showed a significant decrease uti-
lizing high frequency stimulation parameters (50 and 60 Hz). In order to further determine APM’s potential to alter evoked extracellular DA
levels, extended stimulation periods were employed to deplete releasable stores both before and after APM administration in intact and 6-
OHDA partially lesioned animals. The extended stimulation periods were applied at 60 Hz for 2,5,10 and 20 s durations. APM decreased
DA levels under these conditions in both intact and 6-OHDA partially lesioned animals by an average of 34% and 51%, respectively. Kinetic
analysis performed on frequency series indicated that the diminished DA levels corresponded to a significant reduction in DA release. These
findings suggest that APM has a relatively potent effect of decreasing evoked extracellular DA levels when administered systemically under
the conditions specified.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Dopamine; Voltammetry; Aspartame; Striatum; Release; Uptake
1. Introduction have challenged the safety of APM consumption. Although
there has been a wide range of adverse effects reported with
Aspartame (APM, L-aspartyl-L-phenylalanine methyl ester) APM consumption, most have been neurological in nature
is a commonly used artificial sweetener used in numerous and are most likely due to its ability to increase levels of
foods and beverages. APM was discovered over 40 years Phe in the brain (Fernstrom et al., 1983; Maher and Wurtman,
ago and is nearly 200 times as sweet as sucrose with virtually 1987; Perego et al., 1988; Romano et al., 1990; Sharma and
no calories (Coulombe and Sharma, 1986). Upon ingestion, it Coulombe, 1987; Torii et al., 1986).
can either be hydrolyzed or undergo desterification to yield Elevated Phe levels have long been a concern due to its po-
phenylalanine (Phe), aspartic acid (Asp), and methanol (Mat- tential negative effects on neurological function. This is most
thews, 1984; Burgert et al., 1991). APM received approval for clearly demonstrated in individuals with phenylketonuria
safe consumer use by the Food and Drug Administration (PKU) where there is an absence or decrease in the enzyme re-
(FDA) close to 30 years ago; however, numerous studies quired to hydrolyze Phe. Indeed, in individuals with persis-
tently high Phe levels normal central nervous system (CNS)
development and function is diminished and can lead to men-
Abbreviations: Aspartame, L-aspartyl-L-phenylalanine methyl ester; Dopa-
mine, DA; 6-OHDA, 6-hydroxydopamine.
tal retardation (Mackey and Berlin, 1992). Similarly, con-
* Corresponding author. Tel.: þ1 740 826 8225; fax: þ1 740 826 8229. sumption of APM increases the ratio of Phe to other large
E-mail address: brianb@muskingum.edu (B.P. Bergstrom). neutral amino acids in both rat and human brains (Romano
0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2007.09.009968 B.P. Bergstrom et al. / Neuropharmacology 53 (2007) 967e974
et al., 1990). The effect of APM may also be augmented due to lesioned animals would be most sensitive to changes affecting
dietary consumption of carbohydrates (Wurtman, 1983). The the DA synthesis pathway since they exhibit enhanced rates of
increased Phe can potentially alter the way these other critical synthesis.
amino acids cross the bloodebrain barrier (BBB) to support
normal CNS function as they compete for the same carrier 2. Methods
system.
The large neutral amino acid (LNAA) carrier system sup- 2.1. Overall experimental design
plies important precursors for neurotransmitter synthesis
All experiments were designed to assess APM’s effect on evoked extracel-
(Koeppe et al., 1991). Each substrate competes for the same lular DA levels. Since there are no comparable studies that have looked at DA
binding site and therefore results in a high-affinity, low-capac- levels after APM administration using a real-time method, it was necessary to
ity transport system (Aragon et al., 1982). Phe, tyrosine (Tyr), conduct three separate groups of experimentation. First, a time-course series
and tryptophan (Trp) all rely on this carrier system for entry was performed to determine when the optimal effect of APM, if any, occurred.
into the brain to support the synthesis of norephinephrine The second set of experiments was a frequency response series that was used
for mathematical modeling and for selection of stimulation parameters for the
(NE), dopamine (DA), and serotonin (5-HT). An alteration final portion of the study. The final group employed the use of 6-OHDA par-
in these precursors has the potential to impact neurotransmitter tially lesioned animals as well as intact animals and extended stimulation pe-
concentrations in the brain. These findings are complicated by riods to deplete releasable DA stores and activate synthesis in order to best
conflicting reports concerning the observed effect APM has in assess APM’s role in altering DA levels.
affecting these neurotransmitter concentrations in the brain.
The role APM plays in altering some biogenic amines re- 2.2. Animals
mains unclear. Several studies show no changes in catechol-
Adult male SpragueeDawley rats (100e124 g) were purchased from Har-
amine tissue content (Fernstrom et al., 1983; Freeman et al., lan (Indianapolis, IN) and typically weighed between 200 and 400 g by the
1990; Torii et al., 1986; Perego et al., 1988). However, other time of experimentation. All rats were provided standard conditions of light-
studies report increases in catecholamines (Coulombe and ing, temperature and humidity with food and water provided ad libitum. All
Sharma, 1986) and serotonin (Goerss et al., 2000). An increase animal care and experimental protocols were in accordance with NIH guide-
in these levels has been linked to an increase in Tyr levels as it lines (Publication No. 8023) and approved by the Animal Care and Human
Subjects Committee of Muskingum College.
can be converted from Phe. However, Tyr levels are already
above the Km for the tyrosine hydroxylase under normal con-
2.3. Lesion procedure
ditions (Cooper et al., 2003). Therefore, any increase above
the basal Tyr levels would not be able to enhance catechol- The 6-OHDA unilateral graded lesion procedure utilized in the extended
amine synthesis. Still other studies have shown a decrease in stimulation experiments is described in detail elsewhere (Bergstrom et al.,
DA tissue content under chronic exposure to APM (Sharma 2001). Rats were anesthetized with Equithesin (6 ml/kg i.p.) and immobilized
and Coulombe, 1987) and a trend for a decline in DA content in a stereotaxic apparatus (David Kopf Instruments, Tajunga, CA). Body tem-
perature was maintained by Deltaphase Isothermal Pads (Braintree Scientific,
after acute APM treatment (Goerss et al., 2000). Since virtu- Braintree, MA) throughout the entire procedure. After drilling a hole over the
ally all previous investigations have looked at this question lateral substantia nigra, 7 mg of 6-OHDA was suspended in a volume of 2 ml
at the tissue content level, utilizing a method that would pro- and injected over a period of 10 min. The stereotaxic coordinates for the le-
vide a real-time measurement of DA may provide further in- sioning procedure were 5.4 AP, þ3.0 ML and 8.2 DV and referenced
sight as to the impact APM has upon this important group from bregma (Paxinos and Watson, 1986). 6-0HDA was dissolved in a 0.9%
sodium chloride solution that contained 100 mM ascorbic acid. Only the right
of neurotransmitters. brain was lesioned and corresponded to the same side voltammetric recordings
In the following study, in vivo voltammetry was used to as- would take place.
sess the role of APM in altering evoked extracellular DA
levels. To the best of our knowledge, this was the first study 2.4. Voltammetry procedures
to utilize this spatially and temporally resolved technique in
order to characterize the impact a single high dose of APM All voltammetric recordings were electrically evoked in the striatum of
(500 mg/kg; i.p.) has upon dopaminergic signaling. Previous anesthetized rats utilizing fast-scan cyclic voltammetry at carbon-fiber micro-
electrodes. A period of 2e5 weeks elapsed after the lesioning procedure be-
investigations have indicated that this dose of APM is able fore the extended stimulation voltammetry experiments occurred. Urethane
to increase Phe levels (Maher and Wurtman, 1987; Perego anesthesia (1.5 g/kg i.p.) was used in all voltammetry experiments. Separate
et al., 1988; Romano et al., 1990; Sharma and Coulombe, holes were drilled through the skull at locations ipsilateral to the lesion (right
1987; Torii et al., 1986). In the following set of experiments side for intact animals) in order to accommodate a stimulating, reference and
we hypothesized that if APM administration increases Phe carbon-fiber microelectrode. Bipolar stimulating electrodes were placed dorsal
to the medial forebrain bundle (4.6 AP, þ1.4 ML and 7.0 DV) and lowered
levels there will be a subsequent decline in evoked extracellu- until an optimal DA signal was obtained and remained unchanged thereafter.
lar DA levels due to a diminished capacity to support DA syn- The reference electrode consisted of a chloridized silver wire that was placed
thesis. We further hypothesized that this effect would manifest in contralateral superficial cortex. The carbon-fiber microelectrodes were
itself as an impairment in DA release due to limited Tyr pre- placed in a micromanipulator and lowered into the striatum (þ1.2 AP,
cursor availability as a result of Phe obstruction of the þ1.4 ML, 4.3 to 4.7 DV). In voltammetry experiments performed on the
partial 6-OHDA lesioned animals the mediolateral coordinates for microelec-
LNAA carrier system. As a comparison model we elected to trode placement varied due to the process of searching for a region that ap-
administer APM to intact and 6-OHDA partially lesioned an- peared lesioned (þ1.4 to 1.8 ML). In both frequency series and extended
imals, an animal model of Parkinson’s Disease (PD). The stimulation experiments, pre-APM values were obtained, followed by injectionB.P. Bergstrom et al. / Neuropharmacology 53 (2007) 967e974 969
of APM (500 mg/kg, i.p) and a period of 1 h transpired before post-APM HPLC-EC (ESA Coulochem III, Chelmsford, MA) using a millibore, reverse
recordings were taken. phase column (MD-150X3.2, ESA). The mobile phase is commercially avail-
able (MDTM, ESA). Each tissue size was determined by protein content (Bio-
2.5. Electrochemistry Rad, Hercules, CA).
All microelectrodes were locally constructed using a method developed by 2.9. Statistical analysis
Cahill et al. (1996). Carbon fibers (r ¼ 2.5 mm) extended 20e100 mm beyond
the tip of insulating glass. An EI400 bipotentiostat performed all electrochem- The data are expressed as individual values and as the mean ± SEM. Sta-
istry (Ensman Instruments, Bloomington, IN) and was computer controlled tistical analysis of averaged effects was performed by SPSS Inc. (Chicago, IL)
(Michael et al., 1999). Microelectrode potential moved from 0.4 to 1.0 V and used t-test or ANOVA with repeated measures. The significance level was
and back at a scan rate of 300 V/s using a silver/silver chloride reference elec- set at p < 0.05 for all comparisons.
trode. This allowed DA levels to be monitored every 100 ms (Bergstrom et al.,
2001). Based upon the characteristics of each microelectrode, DA peak oxida-
2.10. Reagents and drugs
tion typically occurred at 0.6e0.8 V and was converted to concentration based
on post-calibration using a flow-cell. A buffer that consisted of 150 mM NaCl
and 25 mM HEPES at a pH of 7.4 was used for flow-cell calibrations (Wu All reagents and drugs were used as received and purchased from Sigma
et al., 2001a). Background subtracted cyclic voltammograms were used to de- Chemical Company (St. Louis, MO). Aqueous solutions were prepared in
termine that the individual responses were a result of current changes due to MilliporeÔ deionized water (Millipore, Billerica, MA). APM was suspended
DA (Michael et al., 1998). in 2.0e2.5 ml of water, prepared immediately before injection and adminis-
tered systemically at a dose of 500 mg/kg, i.p.
2.6. Stimulation parameters
3. Results
Electrical stimulation was performed using bipolar stimulating electrodes
(Plastics One, MS 303/2, Roanoke, VA) with tips locally untwisted such 3.1. Time-course
that they were separated by approximately 1.0 mm. Time-course experiments
and extended stimulation experiments were performed with pulse trains deliv-
ered at 60 Hz. The frequency series ranged between 10 and 60 Hz at intervals
Systemic APM administration decreased evoked extracellu-
of 10. Biphasic (300 mA, 2 ms) stimulus pulses were computer generated and lar DA over the course of 2.5 h (Fig. 1). Vehicle-injected
applied at various train durations. Time-course experiments utilized 2 s (120- controls showed a stable signal for the duration of the entire
pulse) stimulus train durations; whereas, extended stimulation experiments time-course experiments. Recordings from APM injected ani-
used 1 s (60-pulse), 2 s (120-pulse), 5 s (300-pulse), 10 s (600-pulse), and 20 s mals initially showed only a modest drop, however, after
(1200-pulse) stimulus train durations. Frequency series applied the stimulus
over a 2 s period. Therefore, pulse number varied according to the frequency
approximately 60 min levels stabilized to show a drop of 25%.
(10 Hz (20-pulse), 20 Hz (40-pulse), 30 Hz (60-pulse), 40 Hz (80-pulse), The largest decline occurred just past one hour at 35%.
50 Hz (100-pulse), 60 Hz (120-pulse). A period of 5 min elapsed between Statistical analysis revealed these declines to be significantly
each individual stimulation during the extended stimulation experiments. Elec- different over time for the APM treatment when compared
trical pulses were maintained at a constant-current and passed through an opti- to vehicle-injected control (ANOVA, F15,75 ¼ 3.001,
cal isolator (NL 800A, Harvard Apparatus, Holliston, MA).
p < 0.001). In agreement with previous studies, the 60 min
2.7. Kinetic analysis
120
The kinetic analysis used on the 60 and 20 Hz frequency response data
used a mathematical model listed in Eq. (1). The model connects the rate of
change of electrically evoked DA to the counteraction of dopamine release
100
and uptake (Wightman et al., 1988):
% Max [DA]EC
d½DA=dt ¼ ½DAp f Vmax =ðKm =½DAÞ þ 1Þ ð1Þ
80
where [DA]p is a release term describing the concentration of dopamine
evoked by each stimulus pulse and f is the stimulation frequency. Both Vmax
and Km are MichaeliseMenten uptake terms; Vmax is correlated to the number
of DA transporters (DAT) and their turnover. Km is inversely related to the af- 60
finity that DA has for the DAT. All voltammetric curve fitting used non-linear
regression based upon a simplex minimization algorithm (Wu et al., 2001b). APM
Control
40
2.8. HPLC-EC 0 20 40 60 80 100 120 140 160
Time (Min)
Lesioned animals underwent tissue content analysis for DA. Upon comple-
tion of the extended stimulation voltammetry experiments, the brain was Fig. 1. Time-course of the effects of systemically administered APM (500 mg/
removed and chilled in an ice-cold 150 mM NaCl solution. After kg) on evoked DA concentrations. Evoked extracellular DA concentrations
approximately 7e10 min the brain was placed in a chilled aluminum block were monitored for 150 min at 10-min intervals in the striatum utilizing
(Braintree Scientific, Braintree, MA) and sliced into 1 mm coronal sections 60 Hz stimulations delivered at 2 s durations. All data are expressed as a per-
using razor blades. The slice containing the striatum was further dissected cent of maximum extracellular DA ([DA]EC). The arrow at time zero repre-
into four equal tissue sections for both the lesioned side (right) and the intact sents the time of the i.p. APM administration or vehicle (2.0 ml Millipore
side (left). During later analysis, the intact side of each respective animal was water). All data represented are mean values ± SEM. Filled circles (C) repre-
used as a same animal control to determine the degree of lesion. All samples sent animals injected with APM (n ¼ 4) and open circles (B) represent vehi-
were frozen at 80 C until assay. Tissue DA content was determined by cle-injected controls (n ¼ 3).970 B.P. Bergstrom et al. / Neuropharmacology 53 (2007) 967e974
time frame we observed in producing a stabilized decrease in represented a drop in DA levels by approximately 25%. In
DA levels also coincides with studies showing significant peak contrast to evoked release in intact animals, substantial de-
increases in Phe levels after APM (Fernstrom et al., 1983; Ro- creases in evoked extracellular DA were seen across all stim-
mano et al., 1990; Hjelle et al., 1992). ulation durations in lesioned animals.
The averaged evoked extracellular DA concentrations also
3.2. Frequency response showed declines (Fig. 4). These declines were statistically sig-
nificant ( p < 0.05) at 20, 5, and 2 s in the intact animals (Panel
Frequency series demonstrated that the APM induced de- A). The animals that underwent the lesion surgery showed sig-
pletion of evoked extracellular DA was frequency dependent nificant declines ( p < 0.05) in DA tissue content by an average
(Fig. 2). Panel A illustrates that the lower frequencies (10 of 52%. These lesioned animals showed significant evoked ex-
and 20 Hz) produced levels that were virtually identical. In tracellular DA declines ( p < 0.05) at all stimulus durations ex-
contrast, the higher frequency stimulation produced significant cept 1 s (Panel B). Overall, intact animals showed average
declines at 50 Hz ( p < 0.05) and 60 Hz responses ( p < 0.02). declines in DA levels by approximately 1/3, whereas lesions
These declines were 50% and 40%, respectively. Similar to dropped by 1/2.
previous studies, all voltammetric recordings in the striatal re-
gions sampled produced steady-state signals between 10 and 3.4. Kinetic analysis of aspartame effect
30 Hz and peak-shaped release dominated signals at frequen-
cies between 40 and 60 Hz (Wightman et al., 1988; Kawagoe APM altered dopaminergic neurotransmission by diminish-
et al., 1992; Garris and Wightman, 1994). Although declines ing DA release in the striatum. We performed kinetic analysis
were observed at 30 and 40 Hz they were not significantly dif- on the data obtained using 2 s stimulation at 20 and 60 Hz.
ferent. Panel B demonstrates that the ratio of post-APM to pre- APM produced a significant ( p < 0.05) drop in [DA]p at
APM decreases with an increase in stimulation frequency and 60 Hz frequencies (Table 1). No significant changes were ob-
is strongly correlated (r2 ¼ 0.86). Taken together, these results served for [DA]p at 20 Hz frequencies. Additionally, Vmax was
suggest that the effect of APM is limited to those frequencies not significantly changed in 60 or 20 Hz data. These findings
that are dominated by DA release because they place a greater provide further evidence that the declines in the 60 Hz evoked
demand on DA synthesis. extracellular DA levels are a result of a decrease in DA release
rates without any significant changes to DA uptake rates.
3.3. Extended stimulus duration
4. Discussion
APM administration induced DA declines during the ex-
tended stimulus duration experiments. Fig. 3 illustrates repre- This study described the effect of a single high dose of sys-
sentative voltammetric traces from both an intact and lesioned temically administered APM on evoked extracellular DA
animal. The result of the lesion procedure performed on the levels in the striatum. By using a spatially and temporally re-
animal illustrated in column C of Fig. 3 represented a 79% de- solved technique such as in vivo voltammetry we have charac-
cline in striatal DA content. The intact animal seen in column terized changes in dopaminergic signaling induced by APM
B showed the most sensitivity to the 10 and 20 s durations and that have not been reported before. The first portion of the
A B 120
1.8
Post-Aspartame/Pre-Aspartame (%)
Pre-APM
1.6 Post-APM
**
100
1.4
1.2 * 80
[DA]EC µM
1.0
0.8 60
0.6
40
0.4
0.2 20
0.0
0 10 20 30 40 50 60 70 0 20 40 60
Frequency (Hz) Frequency (Hz)
Fig. 2. Effects of APM during frequency series. (A) All voltammetric recordings were made in the striatum and utilized 2 s stimulus durations delivered at the
corresponding frequency. Filled circles (C) represent the mean values SEM prior to APM administration (500 mg/kg, i.p.) and open circles (B) represent
post-APM values. (B) Ratio of post-APM to pre-APM against stimulation frequency (r2 ¼ 0.86). The asterisks represent significant differences (*p < 0.05;
**p < 0.02), (n ¼ 4).B.P. Bergstrom et al. / Neuropharmacology 53 (2007) 967e974 971
A B C decline in DA release prevented normal DA levels to be main-
Pre-APM tained over a wide variety of demanding stimulation periods.
Post-APM
The dose of APM in the following study was chosen due to
its ability to increase Phe levels. Numerous reports demon-
strate the ability of APM to increase plasma Phe concentra-
0.07 µM
1 second tions along with brain Phe and Tyr levels at doses equal or
4S greater than the 500 mg/kg chosen for this study (Maher and
Wurtman, 1987; Perego et al., 1988; Romano et al., 1990;
Sharma and Coulombe, 1987; Torii et al., 1986). The increases
in Phe levels are also seen at lower APM doses (Fernstrom
et al., 1983). In general, dosages in the range of 500e
1000 mg/kg have routinely been used in animal studies involv-
ing APM. Even though the intraperitoneal injection route is
2 second unlikely to be used by humans, it was chosen due to the nature
0.45 µM
of the voltammetry experiments, which would make oral ad-
4S ministration difficult. Furthermore, the intraperitoneal injec-
tion of APM has been utilized in previous APM studies
(Goerss et al., 2000; Kiritsy and Maher, 1986; Yokogoshi
and Wurtman, 1986).
Dosing of APM in rats is of particular importance because
they metabolize the APM at a much greater rate than humans.
5 second Due to the different rates of APM metabolism between rats
0.7 µM
and humans, a conversion factor can be used to estimate equiv-
5S alent dosages. Although different reports exist as to an appro-
priate conversion factor, most are around 5 (Fernstrom, 1989;
Hjelle et al., 1992) and up to 60 (Wurtman and Maher, 1987).
At the lower conversions a dose of 500 mg/kg that is adminis-
tered to rats would not exceed the recommended daily allow-
ance of 50 mg/kg set for humans by the FDA (1984). However,
10 second
at a conversion factor of 60 the dosing of 500 mg/kg in rats
could potentially be attainable in humans consuming large
0.9 µM
quantities of APM containing foods and beverages within
6S
a short period of time.
The frequency dependent effect that APM exhibits on di-
minishing the releasable pool of DA is a unique result. As
seen from the results in Fig. 2, APM only produces a signifi-
cant decline in DA levels at relatively high stimulation fre-
quencies and pulse number. In contrast, frequency response
20 second studies from our laboratory have shown that synthesis inhibi-
0.9 µM
tors such as alpha-methyl-para-tyrosine (ampt) and NSD-1015
9S diminish evoked extracellular DA levels at all stimulation fre-
quencies (unpublished). This finding may be a result of the de-
cline being linked to a decrease in the precursor Tyr instead of
Fig. 3. APM induced alterations of dopaminergic neurotransmission during ex-
tended stimulation experiments in representative animals. (A) Stimulus dura- a potent inhibition of the enzymes necessary for DA synthesis.
tion. (B) Representative intact animal recordings. (C) Representative lesioned The kinetic analysis performed on frequency series data
animal recordings. Each circle in panels B and C represents the concentration provided important insights into how APM decreased evoked
of DA at 100 ms intervals. The solid line under each recording represents the extracellular DA levels in the striatum. As seen in Table 1,
time and duration of the stimulus train. All recordings represent 60 Hz re-
a significant drop in [DA]p was seen at the 60 Hz frequency
sponses in the striatum. Filled circles (C) represent recordings prior to
APM administration (500 mg/kg, i.p.) and open circles (B) represent post- yet failed to decline at 20 Hz. This may have an important
APM values. The average decline for both intact and lesioned animals repre- consequence for maintaining dopaminergic tone due to the dy-
sented for all stimulation parameters was 24% and 92%, respectively. namics of phasic and tonic DA signaling in the brain. Indeed,
the 20 Hz response may serve to represent the slow and irreg-
study identified that APM diminished DA levels within 1 h of ular firing rates observed in tonic signaling (Schultz, 1998;
administration. The second set of experiments determined that Grace, 2000; Garris and Rebec, 2002; Venton et al., 2003).
the effect was frequency dependent and through kinetic anal- Whereas the 60 Hz is more representative of phasic firing
ysis linked to a subsequent decline in DA release. The final that is characterized by a short concentration spike that is pro-
portion of the study demonstrated that the APM induced duced in addition to these basal levels by synchronous burst972 B.P. Bergstrom et al. / Neuropharmacology 53 (2007) 967e974
A 5
Pre-APM
B 5
Post-APM
4 4
[DA]EC µM
[DA]EC µM
3 3
*
2 2
*
1 * 1
* * *
*
0 0
20 10 5 2 1 20 10 5 2 1
Stimulus Duration(s) Stimulus Duration(s)
Fig. 4. Changes in evoked extracellular dopamine after systemic APM administration during extended stimulation experiments. (A) Intact (n ¼ 4). (B) Lesion
(n ¼ 7). Both sets of data represent the mean values SEM of voltammetric responses taken in the striatum. The black bars represent pre-APM values and the
grey bars represent post-APM values (*p < 0.05). Lesioned animals were denervated by an average of 52%.
firing of dopaminergic neurons (Schultz, 1998; Grace, 2000; potentially diminish Tyr as a precursor for DA synthesis would
Robinson et al., 2001; Garris and Rebec, 2002; Phillips be sensitive to our experimental design. Furthermore, the 6-
et al., 2003; Venton et al., 2003). Therefore, the evoked OHDA lesioned rat, a common animal model for PD, represents
steady-state signals mirror dopaminergic tone and evoked a condition where the DA system is denervated and exhibits
peak-shaped signals mirror the DA concentration spikes asso- enhanced rates of synthesis (Zigmond et al., 1984; Hefti
ciated with phasic signaling. These characteristics of DA sig- et al., 1985; Altar et al., 1987; Wolf et al., 1989). In turn, these
naling and the findings of this study suggest APM’s effect is animals would likely be more sensitive to changes affecting DA
likely limited to phasic dopaminergic signaling. synthesis than intact animals.
The extended stimulation experiments were implemented The extended stimulation protocols did produce significant
in order to diminish DA stores and activate synthesis. Utilizing declines in evoked extracellular DA levels in both lesioned and
this type of design would make it easier to identify changes in intact animals after APM administration. Although lesioned
extracellular DA that were dependent on DA synthesis. In ad- animals were nearly twice as sensitive to APM when com-
dition, alterations in DA tissue content following synthesis in- pared to intact animals, significant declines were seen at all
hibition occur more rapidly when coupled to electrical but two of the stimulation periods in the intact animals. There
stimulation (Kuhr et al., 1986). By using prolonged stimula- was no significant effect on evoked DA release during the 10 s
tion periods with limited periods of rest, DA stores begin to stimulations; however, there was a tendency towards a de-
be depleted and synthesis is activated. In support of this ratio- crease. This lack of significance may be due to heterogeneity
nale, it has been reported that newly synthesized DA is prefer- of release sites in the striatum (Fallon and Moore, 1978; Ger-
entially released (McMillen et al., 1980; Herdon et al., 1985). fen et al., 1987) which can account for the considerable vari-
Therefore, animals undergoing this protocol would be more ation that can exist in extracellular concentrations (Garris
dependent on the precursor Tyr in an attempt to replenish et al., 1994). No alterations were observed during the 1 s du-
stores of DA and sustain normal release rates. Using these rations for either group as this represents a very modest de-
findings as our basis, we postulated that a substance that would mand on DA synthesis.
The sensitivity of the 6-OHDA lesioned animals to APM
may indicate an increased susceptibility to declines in Tyr
Table 1 levels where previous DA denervation exists. Reports have
APM effects on DA release and uptake at 60 and 20 Hz responses shown that APM produced no motor impairment in individuals
Analysis parameters with PD (Karstaedt and Pincus, 1993). However, Phe levels
Pre-APM Post-APM Pre-APM Post-APM were shown to increase in these individuals and although mo-
60 Hz 60 Hz 20 Hz 20 Hz tor changes were absent they may have been maintained due to
[DA]p (mM) 0.035 0.013a 0.010 0.002b 0.009 0.003 0.006 0.001 the fact that the PD subjects were being treated with levodopa
Vmax (mM/s) 2.043 1.043 1.080 0.355 0.559 0.236 0.248 0.048 and therefore less reliant upon Tyr precursor levels.
([DA]p), DA release; Vmax, uptake. The role of Phe competition for the LNAA carrier system is
The MichaeliseMenten parameter related to DA release ([DA]p) and uptake perhaps a strong link to the decline in evoked extracellular DA
(Vmax) was calculated from Eq. (1) using the animals from the frequency re- levels. Since Tyr and Trp compete with Phe for entrance into
sponse series.
Data are the mean SEM (n ¼ 4). Km was fixed at 0.2 mM during all curve the brain through the LNAA carrier system a decline in these
fitting. r2 was typically >0.9 for all curve fitting. neurotransmitters would seem likely. Phe has a very high-
Values with different superscripts differ significantly ( p < 0.05). affinity for the LNAA carrier system and along with leucineB.P. Bergstrom et al. / Neuropharmacology 53 (2007) 967e974 973
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