Aspartame decreases evoked extracellular

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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 utilizing 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 6OHDA 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 Aspartame (APM, L-aspartyl-L-phenylalanine methyl ester) is a commonly used artificial sweetener used in numerous foods and beverages. APM was discovered over 40 years ago and is nearly 200 times as sweet as sucrose with virtually no calories (Coulombe and Sharma, 1986). Upon ingestion, it can either be hydrolyzed or undergo desterification to yield phenylalanine (Phe), aspartic acid (Asp), and methanol (Matthews, 1984; Burgert et al., 1991). APM received approval for safe consumer use by the Food and Drug Administration (FDA) close to 30 years ago; however, numerous studies Abbreviations: Aspartame, L-aspartyl-L-phenylalanine methyl ester; Dopamine, DA; 6-OHDA, 6-hydroxydopamine. * Corresponding author. Tel.: þ1 740 826 8225; fax: þ1 740 826 8229. E-mail address: [email protected] (B.P. Bergstrom). 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.09.009

have challenged the safety of APM consumption. Although there has been a wide range of adverse effects reported with APM consumption, most have been neurological in nature and are most likely due to its ability to increase levels of Phe in the brain (Fernstrom et al., 1983; Maher and Wurtman, 1987; Perego et al., 1988; Romano et al., 1990; Sharma and Coulombe, 1987; Torii et al., 1986). Elevated Phe levels have long been a concern due to its potential negative effects on neurological function. This is most clearly demonstrated in individuals with phenylketonuria (PKU) where there is an absence or decrease in the enzyme required to hydrolyze Phe. Indeed, in individuals with persistently high Phe levels normal central nervous system (CNS) development and function is diminished and can lead to mental retardation (Mackey and Berlin, 1992). Similarly, consumption of APM increases the ratio of Phe to other large neutral amino acids in both rat and human brains (Romano

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et al., 1990). The effect of APM may also be augmented due to dietary consumption of carbohydrates (Wurtman, 1983). The increased Phe can potentially alter the way these other critical amino acids cross the bloodebrain barrier (BBB) to support normal CNS function as they compete for the same carrier system. The large neutral amino acid (LNAA) carrier system supplies important precursors for neurotransmitter synthesis (Koeppe et al., 1991). Each substrate competes for the same binding site and therefore results in a high-affinity, low-capacity transport system (Aragon et al., 1982). Phe, tyrosine (Tyr), and tryptophan (Trp) all rely on this carrier system for entry into the brain to support the synthesis of norephinephrine (NE), dopamine (DA), and serotonin (5-HT). An alteration in these precursors has the potential to impact neurotransmitter concentrations in the brain. These findings are complicated by conflicting reports concerning the observed effect APM has in affecting these neurotransmitter concentrations in the brain. The role APM plays in altering some biogenic amines remains unclear. Several studies show no changes in catecholamine tissue content (Fernstrom et al., 1983; Freeman et al., 1990; Torii et al., 1986; Perego et al., 1988). However, other studies report increases in catecholamines (Coulombe and Sharma, 1986) and serotonin (Goerss et al., 2000). An increase in these levels has been linked to an increase in Tyr levels as it can be converted from Phe. However, Tyr levels are already above the Km for the tyrosine hydroxylase under normal conditions (Cooper et al., 2003). Therefore, any increase above the basal Tyr levels would not be able to enhance catecholamine synthesis. Still other studies have shown a decrease in DA tissue content under chronic exposure to APM (Sharma and Coulombe, 1987) and a trend for a decline in DA content after acute APM treatment (Goerss et al., 2000). Since virtually all previous investigations have looked at this question at the tissue content level, utilizing a method that would provide a real-time measurement of DA may provide further insight as to the impact APM has upon this important group of neurotransmitters. In the following study, in vivo voltammetry was used to assess the role of APM in altering evoked extracellular DA levels. To the best of our knowledge, this was the first study to utilize this spatially and temporally resolved technique in order to characterize the impact a single high dose of APM (500 mg/kg; i.p.) has upon dopaminergic signaling. Previous investigations have indicated that this dose of APM is able to increase Phe levels (Maher and Wurtman, 1987; Perego et al., 1988; Romano et al., 1990; Sharma and Coulombe, 1987; Torii et al., 1986). In the following set of experiments we hypothesized that if APM administration increases Phe levels there will be a subsequent decline in evoked extracellular DA levels due to a diminished capacity to support DA synthesis. We further hypothesized that this effect would manifest itself as an impairment in DA release due to limited Tyr precursor availability as a result of Phe obstruction of the LNAA carrier system. As a comparison model we elected to administer APM to intact and 6-OHDA partially lesioned animals, an animal model of Parkinson’s Disease (PD). The

lesioned animals would be most sensitive to changes affecting the DA synthesis pathway since they exhibit enhanced rates of synthesis. 2. Methods 2.1. Overall experimental design All experiments were designed to assess APM’s effect on evoked extracellular DA levels. Since there are no comparable studies that have looked at DA levels after APM administration using a real-time method, it was necessary to conduct three separate groups of experimentation. First, a time-course series was performed to determine when the optimal effect of APM, if any, occurred. The second set of experiments was a frequency response series that was used for mathematical modeling and for selection of stimulation parameters for the final portion of the study. The final group employed the use of 6-OHDA partially lesioned animals as well as intact animals and extended stimulation periods to deplete releasable DA stores and activate synthesis in order to best assess APM’s role in altering DA levels.

2.2. Animals Adult male SpragueeDawley rats (100e124 g) were purchased from Harlan (Indianapolis, IN) and typically weighed between 200 and 400 g by the time of experimentation. All rats were provided standard conditions of lighting, temperature and humidity with food and water provided ad libitum. All animal care and experimental protocols were in accordance with NIH guidelines (Publication No. 8023) and approved by the Animal Care and Human Subjects Committee of Muskingum College.

2.3. Lesion procedure The 6-OHDA unilateral graded lesion procedure utilized in the extended stimulation experiments is described in detail elsewhere (Bergstrom et al., 2001). Rats were anesthetized with Equithesin (6 ml/kg i.p.) and immobilized in a stereotaxic apparatus (David Kopf Instruments, Tajunga, CA). Body temperature was maintained by Deltaphase Isothermal Pads (Braintree Scientific, Braintree, MA) throughout the entire procedure. After drilling a hole over the lateral substantia nigra, 7 mg of 6-OHDA was suspended in a volume of 2 ml and injected over a period of 10 min. The stereotaxic coordinates for the lesioning procedure were 5.4 AP, þ3.0 ML and 8.2 DV and referenced 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 brain was lesioned and corresponded to the same side voltammetric recordings would take place.

2.4. Voltammetry procedures All voltammetric recordings were electrically evoked in the striatum of anesthetized rats utilizing fast-scan cyclic voltammetry at carbon-fiber microelectrodes. A period of 2e5 weeks elapsed after the lesioning procedure before the extended stimulation voltammetry experiments occurred. Urethane anesthesia (1.5 g/kg i.p.) was used in all voltammetry experiments. Separate holes were drilled through the skull at locations ipsilateral to the lesion (right side for intact animals) in order to accommodate a stimulating, reference and 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 until an optimal DA signal was obtained and remained unchanged thereafter. The reference electrode consisted of a chloridized silver wire that was placed in contralateral superficial cortex. The carbon-fiber microelectrodes were placed in a micromanipulator and lowered into the striatum (þ1.2 AP, þ1.4 ML, 4.3 to 4.7 DV). In voltammetry experiments performed on the partial 6-OHDA lesioned animals the mediolateral coordinates for microelectrode placement varied due to the process of searching for a region that appeared lesioned (þ1.4 to 1.8 ML). In both frequency series and extended stimulation experiments, pre-APM values were obtained, followed by injection

B.P. Bergstrom et al. / Neuropharmacology 53 (2007) 967e974 of APM (500 mg/kg, i.p) and a period of 1 h transpired before post-APM recordings were taken.

2.5. Electrochemistry All microelectrodes were locally constructed using a method developed by 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 electrochemistry (Ensman Instruments, Bloomington, IN) and was computer controlled (Michael et al., 1999). Microelectrode potential moved from 0.4 to 1.0 V and back at a scan rate of 300 V/s using a silver/silver chloride reference electrode. This allowed DA levels to be monitored every 100 ms (Bergstrom et al., 2001). Based upon the characteristics of each microelectrode, DA peak oxidation 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 et al., 2001a). Background subtracted cyclic voltammograms were used to determine that the individual responses were a result of current changes due to DA (Michael et al., 1998).

2.6. Stimulation parameters

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HPLC-EC (ESA Coulochem III, Chelmsford, MA) using a millibore, reverse phase column (MD-150X3.2, ESA). The mobile phase is commercially available (MDTM, ESA). Each tissue size was determined by protein content (BioRad, Hercules, CA).

2.9. Statistical analysis The data are expressed as individual values and as the mean ± SEM. Statistical analysis of averaged effects was performed by SPSS Inc. (Chicago, IL) and used t-test or ANOVA with repeated measures. The significance level was set at p < 0.05 for all comparisons.

2.10. Reagents and drugs All reagents and drugs were used as received and purchased from Sigma Chemical Company (St. Louis, MO). Aqueous solutions were prepared in MilliporeÔ deionized water (Millipore, Billerica, MA). APM was suspended in 2.0e2.5 ml of water, prepared immediately before injection and administered systemically at a dose of 500 mg/kg, i.p.

3. Results

Electrical stimulation was performed using bipolar stimulating electrodes (Plastics One, MS 303/2, Roanoke, VA) with tips locally untwisted such that they were separated by approximately 1.0 mm. Time-course experiments and extended stimulation experiments were performed with pulse trains delivered at 60 Hz. The frequency series ranged between 10 and 60 Hz at intervals of 10. Biphasic (300 mA, 2 ms) stimulus pulses were computer generated and applied at various train durations. Time-course experiments utilized 2 s (120pulse) stimulus train durations; whereas, extended stimulation experiments used 1 s (60-pulse), 2 s (120-pulse), 5 s (300-pulse), 10 s (600-pulse), and 20 s (1200-pulse) stimulus train durations. Frequency series applied the stimulus over a 2 s period. Therefore, pulse number varied according to the frequency (10 Hz (20-pulse), 20 Hz (40-pulse), 30 Hz (60-pulse), 40 Hz (80-pulse), 50 Hz (100-pulse), 60 Hz (120-pulse). A period of 5 min elapsed between each individual stimulation during the extended stimulation experiments. Electrical pulses were maintained at a constant-current and passed through an optical isolator (NL 800A, Harvard Apparatus, Holliston, MA).

3.1. Time-course Systemic APM administration decreased evoked extracellular DA over the course of 2.5 h (Fig. 1). Vehicle-injected controls showed a stable signal for the duration of the entire time-course experiments. Recordings from APM injected animals initially showed only a modest drop, however, after approximately 60 min levels stabilized to show a drop of 25%. The largest decline occurred just past one hour at 35%. Statistical analysis revealed these declines to be significantly different over time for the APM treatment when compared to vehicle-injected control (ANOVA, F15,75 ¼ 3.001, 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 and uptake (Wightman et al., 1988): ð1Þ

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 affinity that DA has for the DAT. All voltammetric curve fitting used non-linear regression based upon a simplex minimization algorithm (Wu et al., 2001b).

2.8. HPLC-EC Lesioned animals underwent tissue content analysis for DA. Upon completion of the extended stimulation voltammetry experiments, the brain was removed and chilled in an ice-cold 150 mM NaCl solution. After approximately 7e10 min the brain was placed in a chilled aluminum block (Braintree Scientific, Braintree, MA) and sliced into 1 mm coronal sections using razor blades. The slice containing the striatum was further dissected into four equal tissue sections for both the lesioned side (right) and the intact side (left). During later analysis, the intact side of each respective animal was used as a same animal control to determine the degree of lesion. All samples were frozen at 80 C until assay. Tissue DA content was determined by

% Max [DA]EC

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Time (Min) Fig. 1. Time-course of the effects of systemically administered APM (500 mg/ kg) on evoked DA concentrations. Evoked extracellular DA concentrations were monitored for 150 min at 10-min intervals in the striatum utilizing 60 Hz stimulations delivered at 2 s durations. All data are expressed as a percent of maximum extracellular DA ([DA]EC). The arrow at time zero represents the time of the i.p. APM administration or vehicle (2.0 ml Millipore water). All data represented are mean values ± SEM. Filled circles (C) represent animals injected with APM (n ¼ 4) and open circles (B) represent vehicle-injected controls (n ¼ 3).


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time frame we observed in producing a stabilized decrease in DA levels also coincides with studies showing significant peak increases in Phe levels after APM (Fernstrom et al., 1983; Romano et al., 1990; Hjelle et al., 1992). 3.2. Frequency response Frequency series demonstrated that the APM induced depletion of evoked extracellular DA was frequency dependent (Fig. 2). Panel A illustrates that the lower frequencies (10 and 20 Hz) produced levels that were virtually identical. In contrast, the higher frequency stimulation produced significant declines at 50 Hz ( p < 0.05) and 60 Hz responses ( p < 0.02). These declines were 50% and 40%, respectively. Similar to previous studies, all voltammetric recordings in the striatal regions sampled produced steady-state signals between 10 and 30 Hz and peak-shaped release dominated signals at frequencies between 40 and 60 Hz (Wightman et al., 1988; Kawagoe et al., 1992; Garris and Wightman, 1994). Although declines were observed at 30 and 40 Hz they were not significantly different. Panel B demonstrates that the ratio of post-APM to preAPM decreases with an increase in stimulation frequency and is strongly correlated (r2 ¼ 0.86). Taken together, these results suggest that the effect of APM is limited to those frequencies that are dominated by DA release because they place a greater demand on DA synthesis.

represented a drop in DA levels by approximately 25%. In contrast to evoked release in intact animals, substantial decreases in evoked extracellular DA were seen across all stimulation durations in lesioned animals. The averaged evoked extracellular DA concentrations also showed declines (Fig. 4). These declines were statistically significant ( p < 0.05) at 20, 5, and 2 s in the intact animals (Panel A). The animals that underwent the lesion surgery showed significant declines ( p < 0.05) in DA tissue content by an average of 52%. These lesioned animals showed significant evoked extracellular DA declines ( p < 0.05) at all stimulus durations except 1 s (Panel B). Overall, intact animals showed average declines in DA levels by approximately 1/3, whereas lesions dropped by 1/2. 3.4. Kinetic analysis of aspartame effect APM altered dopaminergic neurotransmission by diminishing DA release in the striatum. We performed kinetic analysis on the data obtained using 2 s stimulation at 20 and 60 Hz. APM produced a significant ( p < 0.05) drop in [DA]p at 60 Hz frequencies (Table 1). No significant changes were observed for [DA]p at 20 Hz frequencies. Additionally, Vmax was not significantly changed in 60 or 20 Hz data. These findings provide further evidence that the declines in the 60 Hz evoked 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 extended stimulus duration experiments. Fig. 3 illustrates representative voltammetric traces from both an intact and lesioned animal. The result of the lesion procedure performed on the animal illustrated in column C of Fig. 3 represented a 79% decline in striatal DA content. The intact animal seen in column B showed the most sensitivity to the 10 and 20 s durations and

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This study described the effect of a single high dose of systemically administered APM on evoked extracellular DA levels in the striatum. By using a spatially and temporally resolved technique such as in vivo voltammetry we have characterized changes in dopaminergic signaling induced by APM that have not been reported before. The first portion of the

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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).


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Fig. 3. APM induced alterations of dopaminergic neurotransmission during extended stimulation experiments in representative animals. (A) Stimulus duration. (B) Representative intact animal recordings. (C) Representative lesioned animal recordings. Each circle in panels B and C represents the concentration of DA at 100 ms intervals. The solid line under each recording represents the time and duration of the stimulus train. All recordings represent 60 Hz responses in the striatum. Filled circles (C) represent recordings prior to APM administration (500 mg/kg, i.p.) and open circles (B) represent postAPM values. The average decline for both intact and lesioned animals represented for all stimulation parameters was 24% and 92%, respectively.

study identified that APM diminished DA levels within 1 h of administration. The second set of experiments determined that the effect was frequency dependent and through kinetic analysis linked to a subsequent decline in DA release. The final portion of the study demonstrated that the APM induced

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decline in DA release prevented normal DA levels to be maintained over a wide variety of demanding stimulation periods. The dose of APM in the following study was chosen due to its ability to increase Phe levels. Numerous reports demonstrate the ability of APM to increase plasma Phe concentrations along with brain Phe and Tyr levels at doses equal or 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 involving APM. Even though the intraperitoneal injection route is unlikely to be used by humans, it was chosen due to the nature of the voltammetry experiments, which would make oral administration difficult. Furthermore, the intraperitoneal injection 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. Due to the different rates of APM metabolism between rats and humans, a conversion factor can be used to estimate equivalent dosages. Although different reports exist as to an appropriate 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 administered to rats would not exceed the recommended daily allowance of 50 mg/kg set for humans by the FDA (1984). However, at a conversion factor of 60 the dosing of 500 mg/kg in rats could potentially be attainable in humans consuming large quantities of APM containing foods and beverages within a short period of time. The frequency dependent effect that APM exhibits on diminishing the releasable pool of DA is a unique result. As seen from the results in Fig. 2, APM only produces a significant decline in DA levels at relatively high stimulation frequencies and pulse number. In contrast, frequency response studies from our laboratory have shown that synthesis inhibitors such as alpha-methyl-para-tyrosine (ampt) and NSD-1015 diminish evoked extracellular DA levels at all stimulation frequencies (unpublished). This finding may be a result of the decline being linked to a decrease in the precursor Tyr instead of a potent inhibition of the enzymes necessary for DA synthesis. The kinetic analysis performed on frequency series data provided important insights into how APM decreased evoked extracellular DA levels in the striatum. As seen in Table 1, a significant drop in [DA]p was seen at the 60 Hz frequency yet failed to decline at 20 Hz. This may have an important consequence for maintaining dopaminergic tone due to the dynamics of phasic and tonic DA signaling in the brain. Indeed, the 20 Hz response may serve to represent the slow and irregular firing rates observed in tonic signaling (Schultz, 1998; Grace, 2000; Garris and Rebec, 2002; Venton et al., 2003). Whereas the 60 Hz is more representative of phasic firing that is characterized by a short concentration spike that is produced in addition to these basal levels by synchronous burst


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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; Robinson et al., 2001; Garris and Rebec, 2002; Phillips et al., 2003; Venton et al., 2003). Therefore, the evoked steady-state signals mirror dopaminergic tone and evoked peak-shaped signals mirror the DA concentration spikes associated with phasic signaling. These characteristics of DA signaling and the findings of this study suggest APM’s effect is likely limited to phasic dopaminergic signaling. The extended stimulation experiments were implemented in order to diminish DA stores and activate synthesis. Utilizing this type of design would make it easier to identify changes in extracellular DA that were dependent on DA synthesis. In addition, alterations in DA tissue content following synthesis inhibition occur more rapidly when coupled to electrical stimulation (Kuhr et al., 1986). By using prolonged stimulation periods with limited periods of rest, DA stores begin to be depleted and synthesis is activated. In support of this rationale, it has been reported that newly synthesized DA is preferentially released (McMillen et al., 1980; Herdon et al., 1985). Therefore, animals undergoing this protocol would be more dependent on the precursor Tyr in an attempt to replenish stores of DA and sustain normal release rates. Using these findings as our basis, we postulated that a substance that would

Table 1 APM effects on DA release and uptake at 60 and 20 Hz responses Analysis parameters Pre-APM 60 Hz

Post-APM 60 Hz

Pre-APM 20 Hz

Post-APM 20 Hz

[DA]p (mM) 0.035 0.013a 0.010 0.002b 0.009 0.003 0.006 0.001 Vmax (mM/s) 2.043 1.043 1.080 0.355 0.559 0.236 0.248 0.048 ([DA]p), DA release; Vmax, uptake. The MichaeliseMenten parameter related to DA release ([DA]p) and uptake (Vmax) was calculated from Eq. (1) using the animals from the frequency response series. Data are the mean SEM (n ¼ 4). Km was fixed at 0.2 mM during all curve fitting. r2 was typically >0.9 for all curve fitting. Values with different superscripts differ significantly ( p < 0.05).

potentially diminish Tyr as a precursor for DA synthesis would be sensitive to our experimental design. Furthermore, the 6OHDA lesioned rat, a common animal model for PD, represents a condition where the DA system is denervated and exhibits enhanced rates of synthesis (Zigmond et al., 1984; Hefti et al., 1985; Altar et al., 1987; Wolf et al., 1989). In turn, these animals would likely be more sensitive to changes affecting DA synthesis than intact animals. The extended stimulation protocols did produce significant declines in evoked extracellular DA levels in both lesioned and intact animals after APM administration. Although lesioned animals were nearly twice as sensitive to APM when compared to intact animals, significant declines were seen at all but two of the stimulation periods in the intact animals. There was no significant effect on evoked DA release during the 10 s stimulations; however, there was a tendency towards a decrease. This lack of significance may be due to heterogeneity of release sites in the striatum (Fallon and Moore, 1978; Gerfen et al., 1987) which can account for the considerable variation that can exist in extracellular concentrations (Garris et al., 1994). No alterations were observed during the 1 s durations for either group as this represents a very modest demand on DA synthesis. The sensitivity of the 6-OHDA lesioned animals to APM may indicate an increased susceptibility to declines in Tyr levels where previous DA denervation exists. Reports have shown that APM produced no motor impairment in individuals with PD (Karstaedt and Pincus, 1993). However, Phe levels were shown to increase in these individuals and although motor changes were absent they may have been maintained due to the fact that the PD subjects were being treated with levodopa and therefore less reliant upon Tyr precursor levels. The role of Phe competition for the LNAA carrier system is perhaps a strong link to the decline in evoked extracellular DA levels. Since Tyr and Trp compete with Phe for entrance into the brain through the LNAA carrier system a decline in these neurotransmitters would seem likely. Phe has a very highaffinity for the LNAA carrier system and along with leucine


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References

APM Tyr Blood Brain Barrier

Phe

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Fig. 5. Possible modes of action for APM. Cartoon representing different ways Aspartame (APM) may alter DA activity. Abbreviations: Tyr, Tyrosine; Phe, Phenylalanine; LNAA, Large neutral amino acid carrier system; TH, Tyrosine hydroxylase; DA, Dopamine. The dashed line indicates the membrane of a DA neuron.

accounts for more than half of the carrier’s capacity at normal conditions (Smith et al., 1987). Therefore, unless there is a concomitant rise in the other amino acids that rely on the LNAA carrier system, Phe will competitively inhibit their transport into the brain. In addition to inhibiting amino acid transport into the brain, Phe also has been shown to competively inhibit tyrosine hydroxylase (TH) at high concentrations (Ikeda et al., 1967). Since TH is the rate-limiting enzyme for DA synthesis, APM may diminish evoked extracellular DA on two separate levels. These mechanisms of potential APM action are summarized in a cartoon (Fig. 5). Taken together, these findings provide convincing evidence as to why APM and its subsequent increase in Phe may be producing the observed effect in the present study. In conclusion, this work provides a real-time measurement of evoked extracellular DA levels in the striatum before and after the administration of a single high dose of APM. Through a variety of experimental procedures, APM consistently decreased evoked extracellular DA levels. The declines in evoked extracellular DA levels were coupled to diminishing DA release rates and may be limited to effects on phasic DA signaling. This relatively potent effect was more pronounced where previous DA denervation exists. Further study is needed to determine if this effect can be achieved at doses that would likely be consumed by humans. In addition, determining the extent to which the declines in evoked extracellular DA levels are linked to increased brain Phe warrants further investigation. Acknowledgements This research was supported by Muskingum College Professional Development Grants, Symbols of Excellence Fund, and the Summer Fellows Program. We thank Melissa Smith, Kristen Parkinson, and Carie Padro for technical assistance.

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