Introduction

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Abuse of the amphetamine analog methamphetamine (METH) is a serious world-wide health concern because of its widespread availability and neurotoxic potential. Deleterious effects of high-dose METH treatment can include long-lasting reductions in dopamine (DA) and 5-hydroxytryptamine (5HT) content in extrapyramidal and limbic systems (Wagner et al., 1980; Schmidt and Gibb, 1985) as well as decreases in the activities of tyrosine hydroxylase and tryptophan hydroxylase (Hotchkiss and Gibb, 1980; Haughey et al., 1999).

In contrast to the persistent deficits induced by METH, this laboratory reported recently that a single injection of METH rapidly decreases DA transporter (DAT) function as assessed by measuring DA uptake into striatal synaptosomes prepared 1 h after drug treatment, a decrease that is fully reversed 24 h later (Fleckenstein et al., 1997b). This transient decrement is likely distinct from the long-term loss of DAT sites after repeated administrations of METH (Wagner et al., 1980; Nakayama et al., 1993), as evidenced by findings that binding of the DAT ligand WIN-35428 is not altered by this acute treatment (Kokoshka et al., 1998). In contrast, multiple administrations of METH cause a greater acute reduction in DAT activity that is only partially recovered 24 h after treatment (Kokoshka et al., 1998). In this case, maximal binding of WIN-35428 is decreased 1 h after multiple administrations of METH (Kokoshka et al., 1998), although this decrease is not as great as the reduction in DAT activity. The amount of DAT protein, as determined by Western blot analysis, is not altered by multiple METH injections (Kokoshka et al., 1998). The effect of neither a single nor multiple administrations of METH is attributable to the direct actions of residual drug introduced by the original drug treatments (Fleckenstein et al., 1997b; Kokoshka et al., 1998). Factors(s) contributing to the decrease in DAT function after a single or multiple administrations of METH have yet to be determined.

METH administration increases extracellular levels of DA (Nash and Yamamoto, 1992; Melega et al., 1995) and may redistribute cytosolic DA as well (Cubells et al., 1994; Jones et al., 1998). Auto-oxidation of DA causes the formation of reactive oxygen species (Graham, 1978; Chiueh et al., 1993; Zhang and Dryhurst, 1993), which in turn may decrease DAT function (Berman et al., 1996; Fleckenstein et al., 1997a). Accordingly, METH-induced oxygen radical formation in rat striatum has been described (Giovanni et al., 1995; Fleckenstein et al., 1997c; Yamamoto and Zhu, 1998). In particular, METH may cause the generation of reactive species such as superoxide or peroxynitrite, which are capable of oxidizing DA to highly reactive DA quinones (for discussion, see LaVoie and Hastings, 1999). Hence, DA and oxygen radicals may be two factors contributing to the rapid and transient decrease in DAT function caused by METH treatment. Hyperthermia may also contribute to this transient deficit because it facilitates the formation of reactive oxygen species after METH treatment (Fleckenstein et al., 1997c; LaVoie and Hastings, 1999).

METH also influences 5HT neurons, resulting in increased extracellular concentrations of striatal 5HT (Kuczenski et al., 1995; Segal and Kuczenski, 1997). Others have shown that 5HT can mediate reactive oxygen species generation (Wrona et al., 1986; Matuszak et al., 1997). In addition, it has been demonstrated that multiple administrations of METH can increase extracellular glutamate levels in the striatum (Nash and Yamamoto, 1992; Abekawa et al., 1994), and N-methyl-D-aspartate (NMDA) receptors are necessary for some D1-mediated effects (Wagstaff et al., 1997; Huang et al., 1998; Keefe and Ganguly, 1998). Hence, METH-induced changes in 5HT and glutamatergic systems may contribute to the acute diminution in DAT function observed after METH treatment.

The purpose of this study was to investigate the possibility that hyperthermia, DA, oxygen radicals, and other factors contribute to the rapid and profound diminution of striatal DAT activity induced by METH treatment. Results reveal similarities and differences among factors contributing to the rapid effects of a single, non-neurotoxic METH treatment and multiple injections of METH administered at doses demonstrated to cause long-term deficits. Interestingly, similar factors contribute to the rapid decrease in DAT activity and the long-term DA deficits induced by multiple METH injections, suggesting a link between these phenomena. These findings may have important implications regarding the mechanisms that underlie the long-term changes caused by a neurotoxic regimen of METH.

	Experimental Procedures

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Materials. (±)-METH hydrochloride and ()-cocaine hydrochloride were generously supplied by the National Institute on Drug Abuse (Rockville, MD). ()-Eticlopride hydrochloride and (+)-MK-801 hydrogen maleate were purchased from Research Biochemicals International (Natick, MA). Pargyline hydrochloride, -methyl-p-tyrosine methyl ester hydrochloride (MT), p-chlorophenylalanine methyl ester hydrochloride (pCPA), N-t-butyl--phenylnitrone (PBN), and (+)-SCH-23390 hydrochloride were purchased from Sigma (St. Louis, MO). Analytical reference materials for METH determination were obtained from Radian Corporation (Austin, TX). [7,8-³H]DA (46 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Drugs were administered as indicated in the legends of the appropriate figures, and doses were calculated as the respective free bases.

Animals. Male Sprague-Dawley rats (250-350 g; Simonsen Laboratories, Gilroy, CA) were maintained under conditions of controlled temperature and lighting, with food and water provided ad libitum. On the day of the experiment, rats were housed in groups (6-9 rats/group) in plastic cages and were maintained in an ambient temperature of 24°C (room temperature). Upon treatment with METH or saline, some cages were placed in a cool environment (ambient temperature 6°C) or placed over heating pads (ambient temperature 28.5°C) to manipulate body temperatures. Core (rectal) body temperatures were recorded using a digital rectal thermometer (Physiotemp Instruments, Clifton, NJ) in all experiments in which ambient temperature was manipulated (see figure legends). For experiments in which rats received multiple administrations of METH, rectal temperatures were recorded immediately before the first METH or saline administration (t = 0 h) and every hour thereafter (t = 0-7 h). For experiments in which rats received a single administration of METH, rectal temperatures were recorded immediately before treatment (t = 0 h), then immediately before decapitation (t = 1 h). All procedures were conducted in accordance with approved National Institutes of Health guidelines. Dosing paradigms used in these studies were selected because these were originally used to characterize the rapid DAT inhibition phenomena (Fleckenstein et al., 1997b; Kokoshka et al., 1998).

Synaptosomal [³H]DA Uptake. Uptake of [³H]DA was determined according to the method described by Fleckenstein et al. (1997b). Fresh striatal tissue was homogenized in ice-cold 0.32 M sucrose and centrifuged (800g for 12 min; 4°C). The supernatant (S1) was then centrifuged (22,000g for 15 min; 4°C), and the resulting pellet (P2) was resuspended in ice-cold 0.32 M sucrose. Assays were conducted in modified Krebs' buffer (126 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 16 mM sodium phosphate, 1.4 mM MgSO₄, 11 mM dextrose, 1 mM ascorbic acid; pH 7.4). Each assay tube contained synaptosomal tissue (i.e., resuspended P2 obtained from 1.5 mg of original wet weight striatal tissue) and 1 µM pargyline. Nonspecific values were determined in the presence of 1 mM cocaine. After preincubation of assay tubes for 10 min at 37°C, assays were initiated by the addition of [³H]DA (0.5 nM final concentration). Samples were incubated at 37°C for 3 min, then filtered through Whatman GF/B filters soaked previously in 0.05% polyethylenimine. Filters were washed rapidly 3 times with 3 ml of ice-cold 0.32 M sucrose using a Brandel filtering manifold. Radioactivity trapped in filters was counted using a liquid scintillation counter. Remaining resuspended P2 samples were assayed for protein concentrations according to the method of Lowry et al. (1951).

DA and 5HT Content Determination. After appropriate treatments, animals were decapitated, and striatal tissue was immediately removed and frozen on aluminum foil placed over dry ice. Tissue was obtained from the striatum contralateral to that used for synaptosomal [³H]DA uptake. Samples were stored at 70°C until assayed. Monoamine levels were determined in tissue homogenates using HPLC, with electrochemical detection using the method of Chapin et al. (1986). Briefly, on the day of the assay, tissue samples (approximately 10 mg of striatal tissue) were thawed in 500 µl of ice-cold tissue buffer [0.1 M phosphate-citrate buffer (pH 2.5) containing 15% methanol], sonicated for 3 to 5 s, and then centrifuged (22,000g for 15 min at 4°C). Tissue pellets were retained and dissolved in 1 N NaOH, and protein content was determined according to the method of Lowry et al. (1951). The supernatant (S1) was then centrifuged (22,000g for 10 min at 4°C), and the resulting supernatant (S2) was injected onto an HPLC system equipped with a Partisphere C₁₈ reverse-phase analytical column (5-µm spheres; 110 × 4.6 mm) and a reverse-phase guard column (Whatman Inc., Clifton, NJ). The mobile phase consisted of 0.05 M sodium phosphate, 0.03 M citrate buffer (pH 2.8) containing 0.1 M EDTA, 0.035% sodium octyl sulfate, and 25% methanol. Monoamines were detected with an amperometric electrochemical detector with the working electrode potential set at +0.73 V relative to an Ag⁺/AgCl reference electrode.

METH Determination. After appropriate treatments, animals were decapitated, and whole brains (without striatum, cerebellum, and brainstem) were removed immediately and frozen on aluminum foil placed over dry ice. Tissue samples were stored at 70°C until assayed. Concentrations of METH were determined according to a modification of a method described by Wilkins et al. (1989).

Data Analysis. Statistical analyses between two groups were conducted using a two-tailed, unpaired Student's t test. Analyses among multigroup data were conducted using ANOVA, followed by a Fisher's least-significant difference test. Differences among groups were considered significant if the probability of error was less than 5%. The data represent mean ± 1 S.E.

	Results

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Role of Hyperthermia in the Acute Effect of Multiple METH Administrations on DAT Activity. Multiple administrations of METH (4 × 10 mg/kg s.c.; 2-h intervals) to rats typically increases core body temperature by approximately 2-4°C when measured during and immediately after treatment. The role of hyperthermia in the acute decrease in DAT function was investigated by preventing this METH-induced increase in body temperature. On administration of METH, some rats were exposed to an ambient temperature of 6°C for the duration of the experiment (to maintain normothermic body temperature), whereas other METH-treated rats remained exposed to room temperature (24°C) to allow hyperthermia to occur. As shown in Fig. 1A, prevention of METH-induced hyperthermia attenuated the rapid decrease in [³H]DA uptake induced by multiple administrations of METH. Corresponding rat core body temperatures are shown in Fig. 1B. In a separate experiment, the effects of increasing core body temperature per se on [³H]DA uptake were assessed; exposure to an ambient temperature of 28.5°C for 7 h (the duration of the multiple METH-injection paradigm) did not alter DAT function (116 ± 7 versus 116 ± 5 dpm/µg for control and hyperthermic rats, respectively). To rule out the possibility that the prevention of hyperthermia attenuated the METH-induced reduction in DAT activity attributable to altering drug pharmacokinetics, METH brain content was measured in whole brain minus striata, brainstem, and cerebellum 1 h after treatment. METH levels were not statistically different in the rats that were maintained normothermic (20.6 ± 1.8 versus 19.1 ± 1.0 ng/mg of tissue for normothermic versus hyperthermic rats, respectively).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   A, effect of core body temperature on the decrease in [3H]DA uptake in rat striatal synaptosomes induced by multiple administrations of METH. Rats were maintained in an ambient temperature of 24°C before treatment. On receiving METH (4 × 10 mg/kg s.c.; 2-h intervals) or saline (1 ml/kg s.c.; 2-h intervals), rats were exposed to 6 or 24°C ambient temperature for the duration of the experiment. Rats were decapitated 1 h after the last METH or saline administration. *, values different from respective saline-treatment control group. #, value different from the METH-treatment/24°C group. B, time course of core body temperatures. Inverted arrows represent time points of METH or saline administrations. *, values different from saline-treated controls (P < .05).

Role of DA in the Acute Effect of Multiple METH Administrations on DAT Activity. The role of DA in the reduction of DAT function induced by multiple administrations of METH was assessed by depleting striatal DA levels using the tyrosine hydroxylase inhibitor MT before METH treatment. MT (150 mg/kg i.p.) was administered 5 and 1 h before, and 3 h after, the first injection of METH. Striatal DA levels were greatly reduced by MT pretreatment (147.9 ± 16.6 versus 24.0 ± 4.0 pg/µg of protein for nonpretreated versus pretreated rats, respectively). Because pretreatment with MT prevents METH-induced hyperthermia, some of the pretreated rats were exposed to a warmer ambient temperature (28.5°C) during METH treatment to maintain hyperthermia. The remaining groups were exposed to an ambient environment of 24°C. As demonstrated in Fig. 2A, pretreatment with MT attenuated the METH-induced decrease in DAT activity. In addition, maintaining hyperthermia in some of these rats diminished, but did not prevent, the attenuation by MT pretreatment. Corresponding rat core body temperatures are shown in Fig. 2B.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   A, effect of MT pretreatment, with and without hyperthermia reinstated, on the decrease in [3H]DA uptake in rat striatal synaptosomes induced by multiple administrations of METH. Rats received MT (150 mg/kg i.p.) or saline (1 ml/kg i.p.) 5 and 1 h before and 3 h after the first METH or saline injection. Rats then received METH (4 × 10 mg/kg s.c.; 2-h intervals) or saline (4 × 1 ml/kg s.c.; 2-h intervals). Rats were maintained in an ambient temperature of 24°C before METH treatment. Upon METH treatment, rats were exposed to an ambient temperature of 24 or 28.5°C. Rats were decapitated 1 h after the last METH or saline administration. *, values different from respective saline-treatment control group. #, value different from the other METH-treatment groups. ¥, value different from the saline/24°C/METH group (P < .05). B, time course of core body temperatures. Downward arrows represent time points of METH or saline administrations. *, values different from the saline/saline (24°C) group on treatment. #, value different from the saline/METH (24°C) group (P < .05).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Effect of SCH-23390 (A), eticlopride (B), or combined pretreatment (C) on the decrease in [3H]DA uptake in rat striatal synaptosomes induced by multiple administrations of METH. Rats received SCH-23390 (SCH; 0.5 mg/kg i.p.), eticlopride (etic; 0.5 mg/kg i.p.), the two drugs in combination, or saline (1 ml/kg i.p.) 15 min before each administration of METH (4 × 10 mg/kg s.c.; 2-h intervals) or saline (1 ml/kg s.c.; 2-h intervals). Rats were maintained in an ambient temperature of 24°C before METH treatment. Upon METH or saline treatment, rats were exposed to an ambient temperature of 24 or 28.5°C. Rats were decapitated 1 h after the last METH administration. *, values different from respective saline-treatment control. #, values different from other METH-treatment groups. ¥, values different from the saline/24°C/METH group (P < .05).

Role of Hyperthermia in the Acute Effect of a Single METH Administration on DAT Activity. The role of METH-induced hyperthermia in the acute decrease in DAT activity induced by single administration of METH was investigated. Upon METH treatment, some rats were exposed to an ambient temperature of 6°C to maintain normothermic body temperature, whereas other METH-treated rats remained exposed to room temperature (24°C). As shown in Fig. 4A, prevention of METH-induced hyperthermia had no effect on the diminution of striatal synaptosomal [³H]DA uptake induced by a single administration of METH. Corresponding rat core body temperatures (at 0 and 1 h) are shown within the columns of Fig. 4A.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   A, effect of core body temperature on the decrease in [3H]DA uptake in rat striatal synaptosomes induced by a single administration of METH. Rats were maintained in an ambient temperature of 24°C before treatment. Rats received METH (15 mg/kg s.c.) or saline (1 ml/kg s.c.) and were exposed to 6 or 24°C ambient temperature. The two mean temperatures recorded for each group are indicated in the appropriate column (i.e., t = 0 h  t = 1 h) in degrees Centigrade. S.E. for each mean was less than 0.25°C. The mean temperature recorded at t = 1 h for the METH-treated/24°C ambient temperature group (39.5°C) was significantly higher than the other mean temperatures at t = 1 h (P < .05). B, effect of MT pretreatment on the decrease in [3H]DA uptake in rat striatal synaptosomes induced by a single administration of METH. Rats received MT (150 mg/kg; i.p) or saline (1 ml/kg i.p.) 5 and 1 h before METH (15 mg/kg s.c.) or saline (1 ml/kg s.c.). Rats were decapitated 1 h after METH administration. *, values different from the respective saline-treatment control group (P < .05).

Role of DA in the Acute Effect of a Single METH Administration on DAT Activity. Results presented in Fig. 4B demonstrate that depletion of striatal DA levels, by pretreatment with MT (150 mg/kg i.p.; 5 and 1 h before METH), failed to prevent the decrease in synaptosomal [³H]DA uptake induced by a single injection of METH. The lack of attenuation by this pretreatment regimen was apparent despite the depletion of striatal DA content levels by 72% (134.3 ± 10.9 versus 37.9 ± 3.47 pg/µg of protein for nonpretreated versus pretreated rats, respectively). Neither eticlopride nor SCH-23390 prevented the decrease in DAT function caused by a single METH injection (15 mg/kg s.c.; Table 1).

Contribution of the Single-Injection Effect to the Multiple-Injection Effect of METH on DAT. As discussed in the Introduction, the decrease in DAT function induced by a single METH injection fully recovers 24 h after treatment (Fleckenstein et al., 1997b), whereas the decrease induced by multiple administrations only partially recovers 24 h after the final injection (Kokoshka et al., 1998). Because the diminution of DAT function observed after a single administration of METH is fully reversible (Fleckenstein et al., 1997b), yet insensitive to hyperthermia and DA levels (Fig. 4), this effect may constitute one component of the total decrease observed 1 h after multiple administrations of METH (i.e., the hyperthermia- and DA-insensitive, reversible component; Figs. 1 and 2). Furthermore, it may be that the hyperthermia-sensitive portion of the multiple-injection effect does not recover 24 h after treatment and is associated with the residual decrease in DAT activity observed 24 h after multiple administrations of METH. These two possibilities were assessed. Rats received multiple administrations of METH and were sacrificed 1 or 24 h after treatment. Of these two METH-treated groups (i.e., 1- and 24-h groups), some rats were maintained in an ambient temperature of 6°C during treatment (to maintain normothermic body temperature), whereas the others remained exposed to room temperature (24°C). As shown in Fig. 5, the METH-induced decrease in striatal synaptosomal [³H]DA uptake partially recovered 24 h after treatment in the hyperthermic animals. In contrast, DAT activity was completely recovered 24 h after treatment in rats that were maintained normothermic.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Time-response effect of multiple administrations of METH, with and without hyperthermia, on [3H]DA uptake in rat striatal synaptosomes. Rats were maintained in an ambient temperature of 24°C before treatment. On receiving METH (4 × 10 mg/kg s.c.; 2-h intervals), rats were exposed to 6 or 24°C ambient temperature for the duration of treatment. METH-treated rats were decapitated 1 and 24 h after the final METH injection. Control rats received saline (4 × 1 ml/kg s.c.; 2-h intervals) and were decapitated 1 h after the final saline injection. *, values different from the saline-treatment control group. #, values different from the 1 h group of similar ambient temperature (P < .05).

Role of 5HT and NMDA Receptors in the Acute Effect of Multiple METH Administrations on DAT Activity. Findings that prevention of hyperthermia, DA depletion, or DA antagonists did not fully prevent the decrease in DAT activity caused by multiple METH administrations (nor attenuate the decrease caused by a single administration) demonstrate that other factors contribute to the decrease in DAT function caused by METH treatment. Accordingly, the role of NMDA receptors and 5HT was assessed because both glutamate and 5HT, as with DA, are released after METH treatment (Nash and Yamamoto, 1992; Abekawa et al., 1994; Kuczenski et al., 1995; Segal and Kuczenski, 1997). Moreover, each has been implicated in the regulation of DAT function. In one experiment, rats received multiple injections of METH (four injections, 10 mg/kg s.c.), and the noncompetitive NMDA antagonist MK-801 (dizocilpine, 0.5 mg/kg i.p.) was administered 15 min before each METH injection. Because pretreatment with MK-801 attenuates METH-induced hyperthermia, some of the pretreated rats were exposed to a warmer ambient temperature (28.5°C) upon METH treatment to maintain hyperthermia to the extent observed in nonpretreated rats. The remaining groups were exposed to an ambient temperature of 24°C. As shown in Fig. 6, pretreatment with MK-801 attenuated the METH-induced decrease in DAT activity. However, this attenuation was no longer significant in the pretreated rats that were maintained hyperthermic (body temperatures averaging 39.7°C).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of MK-801 pretreatment on the decrease in [3H]DA uptake in rat striatal synaptosomes induced by multiple administrations of METH. Rats received MK-801 (0.5 mg/kg i.p.) or saline (1 ml/kg i.p.) 15 min before each administration of METH (4 × 10 mg/kg s.c.; 2-h intervals) or saline (1 ml/kg s.c.; 2-h intervals). Rats were maintained in an ambient temperature of 24°C before METH treatment. Upon METH or saline treatment, rats were exposed to an ambient temperature of 24 or 28.5°C. Rats were decapitated 1 h after the last METH administration. *, values different from the saline-pretreatment/saline-treatment group. #, value different from other METH-treatment groups (P < .05).

Role of Reactive Oxygen Species in the Acute Effect of METH on DAT Activity. The role of reactive oxygen species in the rapid diminution of DAT activity after multiple administrations of METH was assessed by administering the spin-trapping reagent PBN before METH treatment. PBN (150 mg/kg i.p.) was administered 20 min before each METH injection. As shown in Fig. 7, pretreatment with PBN attenuated the METH-induced decrease in DAT activity. PBN pretreatment did not diminish METH-induced hyperthermia. In addition, PBN did not alter the decrease in DAT function induced by a single METH injection (Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effect of PBN pretreatment on the decrease in [3H]DA uptake in rat striatal synaptosomes induced by multiple administrations of METH. Rats received PBN (150 mg/kg i.p.) or vehicle (1 ml/kg i.p.) 20 min before each administration of METH. Rats received METH (4 × 10 mg/kg s.c.; 2-h intervals) or saline (1 ml/kg s.c.; 2-h intervals). Rats were maintained in an ambient temperature of 24°C and were decapitated 1 h after the last METH administration. *, values different from respective saline treatment control group. #, value different from the vehicle pretreatment/METH-treatment group (P < .05).

	Discussion

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There are several differences in the acute effects of a single injection versus multiple injections of METH on DATs. For example, results presented in this study demonstrate that multiple administrations of METH characteristically induce a rapid decrease in DAT activity approximately 2 times greater in magnitude than that of a single high-dose injection. In addition, it has been reported that binding of the DAT ligand WIN-35428 is decreased after multiple administrations of METH but not after a single administration (Kokoshka et al., 1998). Moreover, DAT function is only partially recovered 24 h after multiple administrations (Kokoshka et al., 1998), whereas it is completely recovered 24 h after a single administration of METH (Fleckenstein et al., 1997b). This study was undertaken to extend these comparisons by investigating mechanisms responsible for the rapid changes in DAT function induced by these two dosing regimens.

Results reveal that the mechanisms underlying the acute decrease in DAT function by multiple administrations of METH differ in part from those underlying the decrease observed after a single administration. For example, prevention of METH-induced hyperthermia partially blocked the effect of multiple administrations, but not of a single administration, of METH (Figs. 1 and 4A), suggesting that hyperthermia contributes only to the effect induced by multiple administrations of METH. Also, DA depletion attenuated the decrease induced by multiple administrations, but not of a single administration, of METH (Figs. 2 and 4B). These data suggest that the additional decrease in DAT activity induced by multiple administrations (versus a single injection) is DA- and hyperthermia-dependent. The finding that neither DA depletion nor prevention of hyperthermia completely prevented the multiple-injection effect underscores the fact that there are multiple components contributing to this phenomenon.

Results presented in Fig. 5 further indicate that there are at least two components to the decrease in DAT activity induced by multiple administrations of METH. As noted above, one component of this decrease is insensitive to hyperthermia. This decrement recovers by 24 h and may be attributable to the same mechanism(s) that induce a decrease in DAT function by a single administration of METH (a reversible, hyperthermia-independent phenomenon). In contrast, the additional decrease in DAT function observed after multiple METH administrations appears to constitute a second component that is not initiated by a single administration of METH. As shown in Figs. 1, 2, and 5, this second component in the decrease in DAT activity is hyperthermia- and DA-sensitive. The results shown in Fig. 5 also indicate that this second component is not reversed 24 h later.

The mechanism by which METH-induced hyperthermia contributes to the decrease in DAT function caused by multiple METH injections remains speculative. Because hyperthermia in vivo does not alter synaptosomal [³H]DA uptake ex vivo per se, it appears that hyperthermia has a facilitative role in mediating the DAT effect induced by multiple administrations of METH. This may occur because METH-induced hyperthermia promotes oxygen radical formation (Fleckenstein et al., 1997c; LaVoie and Hastings, 1999), which in turn may induce alterations in the DAT that have functional consequences (Berman et al., 1996; Fleckenstein et al., 1997a). Accordingly, results presented in Fig. 7 demonstrate that pretreatment with the antioxidant PBN attenuates the decrease in transporter function caused by multiple METH injections, indicating that oxygen radicals contribute to the decrement caused by multiple METH administrations.

In addition to promoting reactive oxygen species formation, DA, released by METH, may act via dopaminergic receptors to alter transporter function in response to the stimulant treatment. Consistent with a role for these receptors in affecting METH-induced changes in transporter function, O'Dell et al. (1993) have demonstrated that pretreatment with eticlopride or SCH-23390 attenuates the extracellular overflow of DA induced by multiple METH injections. Accordingly, if this enhanced overflow was attributable to a METH-induced decrease in DAT function that resulted in less reuptake and thereby increased extraneuronal concentrations of the transmitter, then it would be anticipated that the D1 or D2 antagonists would attenuate the METH-induced decrease in transporter function. In fact, results presented in Fig. 3 demonstrate that blockade of either the D1 receptor with SCH-23390 or the D2 receptor with eticlopride attenuates the METH-induced decrease in DAT function. The ability of the D2 antagonist to attenuate this decrease was attributable in part to its ability to attenuate the METH-induced increase in body temperature as evidenced by findings that the antagonist was less effective in METH-treated rats that became hyperthermic. Body temperature did not contribute to the protection afforded by SCH-23390.

Concurrent pretreatment with both eticlopride and SCH-23390 provided no greater attenuation than did treatment with either antagonist alone (compare Fig. 2, A, B, and C). Hence, it appears that D1 and D2 receptors may contribute to the METH-induced decrease in DAT function via a common mechanism. Mechanism(s) whereby D1 and D2 receptors mediate this change remain undetermined, although it is noteworthy that there is evidence for the colocalization of D1 and D2 receptors on striatal neurons (Surmeier et al., 1996; Brismer et al., 1999; Wong et al., 1999). Hence, METH-induced activation of DA receptors on these postsynaptic neurons could lead to an interaction between D1- and D2-mediated pathways that may ultimately influence the presynaptic DAT via biochemical or anatomical feedback mechanisms, as implicated previously for METH-induced neurotoxic changes (O'Dell et al., 1994).

DA depletion, DA receptor antagonists, or prevention of hyperthermia did not fully prevent the METH-induced decrease in DAT function. Similarly, none of these factors altered the decrease in transporter activity observed after a single METH treatment. This suggests that other factor(s) contribute to the METH-mediated disruption of transporter activity. The additional factor(s) do not appear to involve 5HT, as depletion of striatal 5HT did not prevent the METH effect on the transporter. Moreover, the NMDA antagonist-induced attenuation was attributable to its ability to attenuate hyperthermia, suggesting that NMDA receptors are not involved. One possible mechanism yet to be tested is that transporter phosphorylation may be involved because it has been shown that amphetamine in vivo and in vitro can alter protein kinase C activity (Giambalvo, 1992a,b; Iwata et al., 1996, 1997) and that protein kinase C-mediated phosphorylation of the DAT regulates its function (Vaughan et al., 1997; Zhang et al., 1997; Zhu et al., 1997). More recently, Saunders et al. (2000) have demonstrated that amphetamine application causes internalization of human DAT in human embryonic kidney cells. Additional studies are underway to identify contributory factors in the decrease observed after a single administration and to relate this effect to the first component of decrease observed after multiple administrations of METH.

In conclusion, the present study has demonstrated that the mechanisms underlying the diminution in DAT activity caused by multiple administrations of METH differ in part from the mechanisms underlying the diminution induced by a single administration. DA and hyperthermia contribute to one component of the multiple-injection phenomenon. It is noteworthy that this second component of the METH-induced transporter effect depends on the same elements that are involved in the long-term deficits in DA neurons induced by methamphetamine (i.e., DA, activation of DA receptors, hyperthermia, and production of free radicals). The rapid change in DAT occurring 1 h after multiple METH administrations is not caused by a loss of protein (Kokoshka et al., 1998), but it may contribute in some manner to the ultimate neurotoxic properties of METH. The remaining component, as with the effect observed after a single administration, is DA- and hyperthermia-independent. The results presented in this study suggest that the reduction in DAT activity induced by a single administration of METH constitutes one component of the total reduction observed after multiple administrations, although this was not tested directly. These findings may have important implications regarding mechanisms that underlie the long-term monoaminergic changes caused by a neurotoxic regimen of METH.