Introduction

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Although the neurotoxic potential of methamphetamine (METH) was discovered more than two decades ago (Seiden et al., 1976; Kogan et al., 1976), the mechanisms underlying METH-induced dopamine (DA) neurotoxicity remain unclear. Endogenous formation of the known DA neurotoxin 6-hydroxydopamine (6-OHDA) has been postulated to play a role (Seiden and Vosmer, 1984), but efforts to identify 6-OHDA in the brain of METH-treated animals have not always been successful (Rollema et al., 1986; but see Axt et al., 1990; Marek et al., 1990a; Karoum et al., 1993). Attempts to identify toxic amphetamine (AMPH) metabolites also have failed to directly implicate a specific metabolite (Matsuda et al., 1989; Elayan et al., 1992; Johnson et al., 1992; Zhao et al., 1992). On the basis of findings with N-methyl-D-aspartate receptor antagonists, glutamate-mediated excitotoxicity was proposed (Sonsalla et al., 1989, 1991), but it now appears that the DA neuroprotective effects of N-methyl-D-aspartate antagonists may be largely related to their hypothermic action (Bowyer et al., 1994; O'Callaghan and Miller, 1994; Albers and Sonsalla, 1995). An extensive series of pharmacological and toxicological studies has strongly implicated endogenous DA in METH-induced DA neurotoxicity (Gibb et al., 1994; Cubells et al., 1994). However, much of the data supporting this hypothesis is confounded by drug effects on core temperature, which strongly influences METH neurotoxicity (Bowyer et al., 1992, 1994; O'Callaghan and Miller, 1994; Albers and Sonsalla, 1995; Ali et al., 1996). Supporting the potential role of DA in METH-induced neurotoxicity are recent findings of increased formation of DA-derived reactive oxygen species (ROS) in the setting of METH neurotoxicity (Cubells et al., 1994; Huang et al., 1997; Fumagalli et al., 1998). However, somewhat at odds with the notion that DA and/or DA-derived ROS mediate METH neurotoxicity is the observation that animals with marked depletions of brain DA are as vulnerable to METH-induced DA neurotoxicity as animals with normal DA brain levels (Wagner et al., 1983; Albers and Sonsalla, 1995).

Elucidation of the mechanisms underlying METH-induced DA neurotoxicity is important because it could provide clues regarding the mechanism of cell death in Parkinson's disease, where DA-derived ROS also are suspected to play a role (Graham, 1978; Olanow and Tatton, 1999). Difficulty identifying the mechanisms of METH neurotoxicity can, to some extent, be attributed to the unavailability of a fully validated in vitro model. Although DA cells in culture have been available for a number of years (Park and Mytilineou, 1992) and indeed, have been fruitfully used to study some aspects of METH neurotoxicity (Bennett et al., 1993, 1998; Cubells et al., 1994), there are some core features of METH-induced DA neurotoxicity that have not been established in the cell culture system. For instance, it is not known if DA uptake blockers, whose neuroprotective effects in intact animals are well established (Marek et al., 1990b), protect DA neurons in culture from METH neurotoxicity. Also, the selectivity and specificity of METH toxicity in cultured DA cells has not been fully explored.

The present study was undertaken as part of an effort to develop an alternative in vitro model that might be useful for studying the molecular mechanisms of METH-induced DA neurotoxicity. Key features of METH-induced DA neurotoxicity that we sought to reproduce in vitro included: 1) DA nerve terminal damage associated with a decrease in V_max of [³H]DA uptake, 2) time and dose dependence, 3) specificity, 4) selectivity, 5) protection by DA uptake blockers, and 6) temperature dependence. We now describe a synaptosomal model system that appears to fulfill most of these criteria, and may prove useful for further delineation of the mechanisms underlying METH-induced DA neurotoxicity.

	Materials and Methods

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Drugs and Chemicals. [³H]DA hydrochloride, [³H]glutamic acid, and [³H]WIN 35,428 were obtained from New England Nuclear (Boston, MA). (+)-METH hydrochloride, [³H]METH hydrochloride, AMPH sulfate, and cocaine hydrochloride were obtained from the National Institute on Drug Abuse. Bupropion hydrochloride and chelerythrine chloride were purchased from Research Biochemicals International (Natick, MA). DA hydrochloride and 6-hydroxydopamine (6-OHDA) hydrobromide were purchased from the Sigma Chemical Co. (St. Louis, MO), methylphenidate hydrochloride from Ciba Geigy (Basel, Switzerland), and 1-methyl-4-phenyl-pyridinium (MPP⁺) iodide was purchased from Aldrich Chemicals Co. (Milwaukee, WI). Bisindolylmaleimide I (BIS) hydrochloride was purchased from Calbiochem (San Diego, CA).

Animals. Male Sprague-Dawley rats (Harlan Co., Indianapolis, IN) weighing 300 to 400 g were used. Animals were housed individually in clear acrylic cages in a temperature-controlled room (20 ± 1^oC). Experimental protocols were approved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions. The facility for housing and care of the animals is accredited by the American Association for the Accreditation of Laboratory Animal Care

Synaptosomal Preparation. Isolated nerve terminals (synaptosomes) were prepared from the striata of rats. Striatal tissue was placed in 50 volumes (w/v) of 0.32 M sucrose, homogenized with a glass-Teflon pestle, and centrifuged at 2000g for 10 min. The synaptosome-rich supernatant was retained and stored on ice until use. Synaptosomes (~2 mg of protein) were incubated at 37°C for various periods of time (10-80 min) after the addition of various concentrations of METH or vehicle (saline) in a total volume of 15 ml of Krebs- phosphate buffer (pH 7.4), containing 136 mM NaCl, 4.8 mM KCl, 1.2 mM Mg²⁺, 1.4 mM Ca²⁺, 10 mM glucose, 1 mM ascorbate, 140 µM EDTA, and 120 µM pargyline. The incubation was terminated by centrifugation at 10,000g for 20 min at 4°C. After removal of the supernatant by gentle aspiration, synaptosomes were resuspended in 5 ml of 0.32 M sucrose, washed once by means of recentrifugation (10,000g for 20 min at 4°C), then resuspended in 650 µl of 0.32 M sucrose, and stored on ice until use.

[³H]DA Uptake. A 25-µl aliquot of the synaptosomal suspension was added to assay tubes containing 10 nM [³H]DA and a range of unlabeled dopamine concentrations (3 nM-10 µM) in a total volume of 0.5 ml of Krebs-phosphate buffer. All constituents were added to assay tubes on ice before the incubation was begun in an oscillating water bath set at 37°C. The assay was terminated 5 min later by placing the tubes over ice, then filtering the tube contents with a cell harvester (Brandel, Gaithersburg, MD) and Whatman GF/B filters. Filters were washed (three times with 5 ml of ice-cold 0.9% NaCl) to remove excess [³H]DA, placed into scintillation vials containing 5 ml of scintillation fluid (Liquiscint; National Diagnostics, Atlanta, GA), and allowed to equilibrate at room temperature overnight. Radioactivity was counted at ~48% efficiency on a Packard 1500 scintillation counter with on board quench correction. The resulting uptake inhibition of cold saturation data was analyzed with the nonlinear computer fitting program (Kell-Rdligand/ligand) to estimate V_max (maximum uptake rate = transporter density) and K_m (inverse of transmitter affinity for transporter) values. Protein concentrations were estimated by the method of Lowry et al. (1951). DA uptake was assayed at 37°C (total uptake) and at 4°C (nonspecific uptake), and the difference between uptake at 37^oC and 4°C was defined as specific uptake. This specific uptake was then expressed in picomoles per milligram of protein per 5-min incubation. Values are reported as the mean ± S.E. Although some graphs present data as percentage of control, all analyses were performed with values expressed as picomoles/5 min/mg protein (for V_max) and molarity (for K_m).

[³H]Glutamate Uptake. [³H]Glutamate uptake studies were performed exactly the same as the [³H]DA uptake studies with a 10 nM final concentration of [³H]glutamate and various concentrations of cold agent for displacement.

[³H]WIN 35,428 Binding. [³H]WIN 35,428-binding assays were carried out as the uptake measurements described above, with the following modifications. Samples were incubated with a final concentration of 3.6 nM [³H]WIN 35,428. Cold saturation experiments were performed with unlabeled WIN 35,428 concentrations ranging from 3 to 1000 nM. Nonspecific uptake was measured in the presence of 100 µM cocaine.

[³H]METH Measurements. METH remaining in the synaptosomal fraction after it had been washed once was determined with radioisotope and radioimmunoassay (RIA) methods. For the former, crude striatal synaptosomes were incubated at 37°C in Krebs-phosphate buffer containing 10 µM [³H]METH (specific activity 23.5 Ci/mmol) for 60 min. Synaptosomes were then washed once and resuspended in 650 µl of 0.32 M sucrose. A 100-µl aliquot of the synaptosomes was added to the scintillation vial and counted with 10 ml of Liquiscint scintillation fluid. The amount of residual METH in the tissue was calculated by comparing counts per second with those of a known standard, and the concentration was expressed in micromolar.

Statistics. Data were analyzed by one-way ANOVA, followed by Duncan's multiple range post hoc comparisons where appropriate. Comparisons between two groups were conducted using Student's t test. Results were considered significant when P was <.05, with a two-tailed test. Data analysis was performed with the Statistical Program for the Social Sciences (SPSS for Windows, release 6; SPSS, Inc., Chicago, IL).

	Results

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Striatal synaptosomes exposed to METH (10 µM) for various periods of time (10, 20, 40, 60, and 80 min) showed a time-related reduced capacity to take up [³H]DA compared with controls (Fig. 1). A 60-min incubation with METH was used in all subsequent experiments because this length of incubation with METH produced significant decreases in [³H]DA uptake compared with control tissue, while retaining substantial DA uptake capacity in control synaptosomes. In addition to being time-dependent, the effect of METH on [³H]DA uptake was concentration-dependent, with higher concentrations of METH producing greater decreases in [³H]DA uptake (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of incubation time on [3H]DA uptake by striatal synaptosomes previously incubated with METH (10 µM) and washed once. Synaptosomes were incubated with either METH or vehicle (control) for various times (10-80 min) at 37°C. METH-treated synaptosomes () showed greater reductions in [3H]DA uptake over time compared with controls (). Values represent mean ± S.E. from three experiments, with samples run in triplicate. *, significant difference from control (P < .05).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of various concentrations of METH on [3H]DA uptake. Synaptosomes were incubated with various concentrations of METH (107-103 M) for 60 min at 37°C. METH produced concentration-dependent reductions in DAT function. Data shown represent the mean ± S.E. from three experiments, with samples run in triplicate. *, significant difference from control (P < .05).

Eadie-Hofstee analysis showed that METH exposure decreased [³H]DA uptake by reducing the maximum number of [³H]DA transporters (DATs; V_max), without significantly altering DAT affinity (K_m) (Fig. 3A). This noncompetitive inhibitory effect of METH contrasted with a competitive inhibitory effect (change in K_m, no change in V_max) observed when METH was added to the synaptosomal suspension at a concentration of 0.1 µM after the wash procedure (Fig. 3B).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of METH incubation on Km and Vmax of [3H]DA uptake in striatal synaptosomes (left). Synaptosomes were assayed for [3H]DA uptake after exposure to 10 µM METH for 60 min at 37°C, as described in Materials and Methods. The Eadie-Hofstee plot represents data from one of three experiments, with samples run in triplicate. The Km values were 79.3 ± 11.0 and 74.3 ± 18.9 nM for synaptosomes treated with vehicle and METH, respectively. The Vmax values for control and METH-treated synaptosomes were 1.03 ± 0.10 and 0.74 ± 0.03 pmol/5 min/tube, respectively; these values differed significantly (P < .05). The right graph shows the effect of METH added to the synaptosomal suspension at a concentration of 0.1 µM after the wash procedure. As before, the Eadie-Hofstee plot represents data from one of three experiments, with samples run in triplicate. The Vmax values for uptake in the absence and presence of METH were 2.64 ± 0.33 and 2.75 ± 0.18 pmol/5 min/tube, respectively. The Km values were 77.4 ± 18.3 and 126 ± 13.6 nM, respectively; these values differed significantly (P < .05).

The amount of residual drug in synaptosomes previously incubated with METH (10 µM) and washed once was estimated with [³H]METH and an RIA procedure (see Materials and Methods). Both methods yielded estimates of ~0.1 to 0.2 µM residual METH. As noted above, addition of 0.1 µM METH to the final synaptosomal suspension produced changes in K_m but not in V_max of [³H]DA uptake (Fig. 3B).

To assess the specificity of the observed effects, studies were carried out with other DA neurotoxins (AMPH, MPP⁺, and 6-OHDA), as well as with fenfluramine, a selective serotonin neurotoxin (Schuster et al., 1986). Similar effects to those obtained with METH were obtained with AMPH, MPP⁺ and 6-OHDA, but not fenfluramine (Fig. 4).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Effect of incubation with other DA neurotoxins [amphetamine (10 µM), MPP+(10 µM), and 6-OHDA (1 µM)] and the serotonin neurotoxin fenfluramine on [3H]DA uptake by striatal synaptosomes washed once after a 60-min exposure period. METH and the other neurotoxins were incubated under identical conditions. The concentrations of 6-OHDA and MPP+ tested were selected on the basis of previous studies (Michel and Hefti, 1990; Park and Mytilineou, 1992), and the concentrations of other amphetamines tested were the same as that of METH. Amphetamine (10 µM), MPP+ (10 µM), and 6-OHDA (1 µM) produced significant reductions in [3H]DA uptake, whereas fenfluramine (10 µM) produced no effect. Data shown represent the mean ± S.E. from one of three experiments, with samples run in triplicate. *, significant difference from control (P < .05).

To assess the selectivity of METH's effects, uptake of [³H]glutamate by striatal synaptosomes previously incubated with METH (10 µM; 60 min) was measured. METH had no effect on [³H]glutamate uptake (Fig. 5), a finding that is consistent with the lack of an effect of METH on glutamate decarboxylase activity (Hotchkiss et al., 1979).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Lack of an effect of METH incubation on [3H]glutamate uptake. Striatal synaptosomes were assayed for [3H]DA and [3H]glutamate uptake after exposure to 10 µM METH for 60 min at 37°C as described in Materials and Methods. METH produced a significant reduction in [3H]DA uptake (56.4 ± 2.7% of control uptake) but not in [3H]glutamate uptake (98.6 ± 2.8% of control). Data represent the mean ± S.E. *, significant difference from control (P < .05). The dotted horizontal line indicates the control (100%).

Next, the effect of DA uptake blockers was tested because these are known to block the toxic effects of METH in the intact animal (Marek et al., 1990b; G. A. Ricaurte, B. Callahan, and J. Yuan, unpublished observations). Bupropion (10 µM), methylphenidate (10 µM), or cocaine (10 µM) were added to the incubation medium at concentrations known to completely inhibit DA uptake. All DAT inhibitors tested blocked the effect of METH on synaptosomal [³H]DA uptake (Fig. 6).

View larger version (61K): [in this window] [in a new window]   Fig. 6.   Effect of DA uptake blockers on METH-induced decreases in [3H]DA uptake. DA uptake blockers bupropion (BUP), methylphenidate (MP), and cocaine (COC), at a concentration of 10 µM, were added to the incubation with or without 10 µM METH. The values shown are means ± S.E. of three separate experiments, with samples run in triplicate. Uptake blockers by themselves produced slight but nonsignificant increases in [3H]DA uptake [BUP (121 ± 11% contol), MP (121 ± 18% control), and COC (116 ± 5% control)], and completely blocked METH-induced decreases in DA uptake [BUP + METH (110 ± 23% control), MP + METH (121 ± 18% control), and COC + METH (120 ± 4% control)]. *, significant difference from control (P < .05). The dotted horizontal line indicates the control (100%).

The effect of temperature on METH-induced decreases in [³H]DA uptake also was evaluated because METH-induced DA neurotoxicity in animals is highly temperature-dependent (Bowyer et al., 1994; O'Callaghan and Miller, 1994; Albers and Sonsalla, 1995; Ali et al.,1996). At the higher temperature (40°C), 10 µM METH reduced [³H]DA uptake to a significantly greater extent than at 37°C. Conversely, at the lower temperature (34°C), 10 µM METH did not reduce [³H]DA uptake to the same extent as at 37°C. In fact, at 34°C, incubation with METH did not lead to a significant reduction in [³H]DA uptake (Fig. 7).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effect of temperature on METH-induced reductions in [3H]DA uptake. Synaptosomes were incubated with either vehicle or 10 µM METH at various temperatures (34, 37, and 40°C) for 60 min, washed once, and assayed for [3H]DA uptake. At 34°C, [3H]DA uptake of METH-treated synaptosomes was 81.1 ± 6.9% of control values, with no significant difference between the control and METH-treated groups. At 37°C, [3H]DA uptake of METH-treated synaptosomes was 63.8 ± 6.6% of control values, with a significant difference between control and METH-treated groups (P < .05). At 40°C, [3H]DA uptake of METH-treated synaptosomes was 32.4 ± 4.8% of control values, with a greater significant difference between the control and METH-treated groups (P < .01). Data shown represent the mean ± S.E. from one of three experiments, with samples run in triplicate. *, significant difference from control (P < .05). **, significant difference from the 37°C group (P < .01).

Given that DA has been strongly implicated in METH neurotoxicity (Gibb et al., 1994; Cubells et al., 1994), the effect of DA on METH-induced decreases in [³H]DA uptake was examined by first loading synaptosomes with unlabeled DA, then incubating them with METH (10 µM) in the presence and absence of 1 µM DA. Addition of DA to the medium during the 60-min incubation with METH attenuated METH-induced decrements in V_max of [³H]DA uptake (Fig. 8).

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Effect of DA on METH-induced reductions of [3H]DA uptake. Striatal synaptosomes were first loaded with unlabeled DA, then incubated for 60 min with vehicle or 10 µM METH in the absence or presence of DA (1 µM) in the incubation medium. METH produced a 57.4 ± 5.0% reduction in the absence of external DA and a smaller reduction, 16.7 ± 14.8%, in the presence of external DA. Data shown represent the mean ± S.E. from three experiments, with samples run in triplicate. *, value significantly different from control (P < .05).

Because protein kinase C (PKC) activation can cause decreases in DA uptake (Zhang et al., 1997), the effects of the PKC inhibitors chelerythrine and BIS were tested at several concentrations (0.1-1 µM). Chelerythrine did not block METH-induced decreases in [³H]DA uptake (Fig. 9A). BIS also failed to block METH-induced decreases in [³H]DA at 0.1 µM (Fig. 9B), a concentration that is 10-fold higher than its IC₅₀ for inhibiting PKC in synaptosomal suspensions (Batchelor and Schenk, 1998). At 1 µM, BIS did appear to attenuate METH-induced decreases in [³H]DA uptake (Fig. 9B). However, at this higher concentration, BIS alone produced an inhibitory effect on [³H]DA uptake.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Effect of the PKC inhibitors, chelerythrine (A) and BIS (B), on METH-induced decreases of [3H]DA uptake. Various concentrations (0.1-1 µM) of chelerythrine and BIS were added before the 60-min incubation with vehicle or 10 µM METH at 37°C. Data shown represent the mean ± S.E. from one of three experiments, with samples run in triplicate. *, value significantly different from control (P < .05).

Finally, to determine whether decreases in DA uptake were associated with reductions in the number of DA terminals, [³H]WIN 35,428 binding was measured. METH-induced decreases in the V_max of [³H]DA uptake were not associated with comparable decreases in the B_max of [³H]WIN 35,428 binding (Fig. 10A). This was also the case in synaptosomes incubated with MPP⁺ and studied in a manner identical with METH (Fig. 10B) and in synaptosomes prepared from intact animals treated with a known DA neurotoxic dose of METH (45 mg/kg s.c.; Fukumura et al., 1998) and sacrificed 1 h after METH administration (Fig. 10C).

View larger version (23K): [in this window] [in a new window]   Fig. 10.   Comparison of [3H]DA uptake and [3H]WIN binding sites of striatal synaptosomes after the in vitro exposure to either 10 µM METH (A) or 10 µM MPP+ (B) for 60 min at 37°C. Synaptosomes were assayed for [3H]DA uptake and [3H]WIN binding as described in Materials and Methods. After METH treatment, a significant decrease in [3H]DA uptake (54.4 ± 2.7% of control) was observed, with no significant reductions in [3H]WIN 35,428 binding (92.2 ± 4.2% of control). After MPP+ treatment, [3H]DA uptake was reduced to 40.0 ± 1.2% of control values with no significant reductions in [3H]WIN binding (80.2 ± 3.9% of control). Effect of a single injection of METH (40 mg/kg s.c.) in an intact animal on [3H]DA uptake and [3H]WIN 35,428 binding sites (C). Rats received either METH (40 mg/kg), or saline vehicle 1 h before decapitation. Crude synaptosomes harvested from the rats were assayed with 500 nM [3H]DA or 30 nM [3H]WIN 35,428 (final concentrations). A significant decrease of [3H]DA uptake (58.3 ± 19.6% of control) was observed, with no significant reductions found in [3H]WIN 35,428 binding (97.1 ± 0.9% of control). Data shown represent the mean ± S.E. *, value significantly different from control (P < .05).

	Discussion

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The present study sought to develop a valid in vitro model of METH-induced DA neurotoxicity. Ideally, the effects of METH in vitro should parallel the well established characteristics of METH-induced DA neurotoxicity observed in vivo. In particular, the effects of METH would be anticipated to be 1) associated with reductions in DA terminal markers, including the V_max of DA uptake; 2) dose- and time-dependent; 3) selective; 4) specific; 5) blocked by DA uptake inhibitors; and 6) temperature-dependent. To a considerable degree, the synaptosomal model described herein meets these various criteria, each of which is discussed in turn below.

Prolonged incubation of striatal synaptosomes with METH led to a time- and dose-dependent decrease in [³H]DA uptake, with kinetic studies showing that the decrease in [³H]DA uptake was due to a decrease in V_max, without any change in K_m. This is in keeping with findings in intact animals treated with neurotoxic doses of METH (Wagner et al., 1980). In both instances, METH decreases the V_max of DA uptake without altering its K_m. This noncompetitive nature of METH-induced changes in DAT function suggests that the observed effects are not due to residual drug, at least not residual drug interferring with DA uptake in a competitive fashion, in which case a change in K_m but not in V_max would be observed. The kinetic results do not, however, exclude the possibility that residual drug (or a metabolite) irreversibly bound to the DAT might be responsible for the decrease in DA uptake. Although this is theoretically possible, it should be recognized that irreversible binding of METH (or a metabolite) to the DAT has, to our knowledge, never been reported.

Assuming that the observed changes in synaptosomal DA uptake are not due to residual drug, a key question is whether the observed changes reflect loss of DA nerve endings, or an alteration of function of the DAT that is not associated with DA terminal destruction. The finding that the density of [³H]WIN 35,428 binding sites is not reduced would seem to suggest that DA nerve ending loss has not occurred. However, in our in vitro paradigm, there is neither a mechanism nor sufficient time for clearing damaged nerve endings, as there is in vivo where all DA terminal markers, including [³H]WIN 35,428 binding, are decreased on a long-term basis after toxic doses of METH (Villemagne et al., 1998). Moreover, decreases in [³H]DA uptake were not accompanied by decreases in [³H]WIN 35,428 binding in the intact animal examined 1 h after treatment with a neurotoxic dose of METH, a condition simulating the present in vitro model. Nor were decreases in [³H]WIN 35,428 observed after incubation of synaptosomes with the documented DA neurotoxin MPP⁺. Finally, it should be noted that the procedures used herein to wash and harvest the synaptosomes do not eliminate membrane fragments (Gray and Whittaker, 1962).

Apart from the possible role of residual drug and the preservation of [³H]WIN 35,428-binding sites, there are a number of features of the present model that make it attractive for studying the mechanisms of METH-induced DA neurotoxicity in vitro. In addition to being time- and dose-dependent, METH's effects are reproduced by compounds with known DA neurotoxic activity (AMPH, MPP⁺, 6-OHDA), but not by an AMPH analog that lacks DA neurotoxic potential (fenfluramine), nor by compounds that interact with the DAT but are not neurotoxic (bupropion, cocaine, and methylphenidate) (Marek et al., 1990b). Furthermore, the observed effects are selective for DA uptake (glutamate uptake was unaffected), completely blocked by DAT inhibitors, and temperature-dependent. In all of these respects, the in vitro effects closely parallel what is known to occur in vivo, suggesting that the synaptosomal system described herein may have utility for elucidating the molecular mechanisms of METH-induced DA neurotoxicity.

Several mechanisms by which METH may damage DA neurons have been proposed over the past decade (see the Introduction). Of these, the one that has received most support is the hypothesis that METH toxicity is mediated by DA, perhaps through the production of DA-derived ROS (De Vito and Wagner, 1989; Cubells et al., 1994; Cadet et al., 1994; Huang et al., 1997; Fumagalli et al., 1998; Yamamoto et al., 1998). Given these findings, the effect of DA was tested by first loading striatal synaptosomes with DA, then incubating the synaptosomal suspension with METH in the presence and absence of additional DA (1 µM). Notably, addition of DA attenuated rather than exacerbated METH's effects on the V_max of DA uptake. This could be taken to indicate that the in vitro findings do not accurately reflect what occurs in vivo. Alternatively, these results may provide an initial indication that DA is not as crucial for the expression of METH toxicity as is currently suspected.

The process underlying METH-induced reductions in the V_max of DA uptake remains to be fully elucidated. ROS and/or DA quinones could be involved (Berman et al. 1996; Fleckenstein et al., 1997a,b), as could activation of PKC (Zhang et al., 1997) or production of arachidonic acid (Zhang and Reith, 1996). Contrary to the finding of Berman et al. (1996) however, we observed that DA decreased rather than increased METH-induced reductions in the V_max of DA uptake, a difference that is probably related to the lower concentration of DA tested in the present study. With PKC activation, the PKC inhibitor chelerythrine did not block METH-induced decreases in [³H]DA uptake at any of the concentrations tested, and the PKC inhibitor BIS produced an effect, but only at a concentration that is 100-fold higher than its IC₅₀ to inhibit PKC in synaptosomal suspensions (Batchelor and Schenk, 1998). Thus, a role for PKC in an in vitro model remains to be established.

In summary, the present results indicate that there are a number of parallels between the DA neurotoxic effects of METH in vivo and its effects on striatal synaptosomes in vitro, including time and dose dependence, specificity and selectivity, sensitivity to DA uptake blockers, and temperature dependence. Two features of the model system that require further study are the possible role of residual drug and the preservation of [³H]WIN35,428-binding sites, neither of which is observed in long-term in vivo studies. Although each of these apparent shortcomings of the model may be more apparent than real, they need to be resolved before the system can be regarded as a valid in vitro model of METH-induced DA neurotoxicity. Finally, even after these issues are resolved, it is important to recognize that the in vitro model here characterized may only be useful for studying certain, probably early, phases of METH-induced DA axonal injury.