Introduction

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Amphetamine and some of its analogs have toxic potential toward brain monoamine-containing neurons. For example, p-chloroamphetamine (Sanders-Bush et al., 1972), fenfluramine (FEN) (Harvey and McMaster, 1977; Schuster et al., 1986; Appel et al., 1990; McCann et al., 1994), methylenedioxyamphetamine (Ricaurte et al., 1985; Stone et al., 1986), and its N-methylated analog 3,4-methylenedioxymethamphetamine (MDMA) (Stone et al., 1986; Schmidt, 1987; O'Hearn et al., 1988) have neurotoxic potential toward brain serotonin (5-HT) neurons. Interestingly, when amphetamine action is prolonged by inhibiting its metabolism (Fuller and Hemrick-Luecke, 1980; Steranka, 1982; Ricaurte et al., 1984) by frequent repeated dosing (Kogan et al., 1976; Seiden et al., 1976; Wagner et al., 1980) or by continuous administration (Steranka and Sanders-Bush, 1980; Ricaurte et al., 1984), the neurotoxic potential of amphetamine and methamphetamine (METH) toward brain dopamine (DA) neurons becomes apparent. Typically, amphetamine damages DA but not 5-HT neurons (Wagner et al., 1980), whereas METH generally damages both DA and 5-HT neurons (Hotchkiss and Gibb, 1980; Ricaurte et al., 1980; Fuller, 1985).

Despite extensive investigation, the mechanisms by which amphetamine analogs damage brain DA and/or 5-HT neurons remain unknown (Gibb et al., 1994; Lew et al., 1997), although plasma membrane monoamine transporters (Fuller and Hemrick-Luecke; 1982; Steranka, 1982; Schmidt, 1987; Marek et al., 1990; Fumagalli et al., 1998) and temperature (Bowyer et al., 1992, 1994; Miller and O'Callaghan, 1994; Albers and Sonsalla, 1995; Farfel and Seiden, 1995; Ali et al., 1996; Colado et al., 1998; Yuan et al., 2001) play important roles. It is also unknown whether all amphetamine analogs share a common neurotoxic mechanism or whether there are individual drug differences. Furthermore, we do not know whether the mechanism by which a particular amphetamine analog (e.g., METH) damages brain DA and 5-HT neurons is the same or different. In fact, based upon findings with 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glucose transport and phosphorylation that also produces hypothermia (Shiraishi and Mager, 1980), we recently suggested that although METH-induced DA neurotoxicity was highly susceptible to thermoregulatory influence, METH-induced 5-HT neurotoxicity might be more vulnerable to glucoprivation (Callahan et al., 1998). This suggestion stemmed from the observation that, under some conditions, the toxic effects of METH on rat brain 5-HT neurons were exacerbated by 2-DG.

Recent findings cast doubt on this view. Specifically, Hervias and colleagues (2000) find that 2-DG attenuates, rather than potentiates, the 5-HT neurotoxic effect of MDMA, a congener of METH. Whether this difference is drug related (i.e., METH versus MDMA) or some other factor is at work is unclear, but nevertheless is important to determine, because findings derived from these studies may have important implications for the hypothesis that perturbations in energy metabolism play a role in amphetamine neurotoxicity (Callahan et al., 1998; Burrows et al., 2000).

The notion that a local impairment in energy metabolism plays a role in amphetamine neurotoxicity derives support from several recent observations. First, increased tissue levels of lactate, suggesting of increased metabolic demand, are observed after repeated doses of METH (Stephans et al., 1998). Second, toxic doses of METH have also been reported to decrease levels of striatal ATP (Chan et al., 1994). Third, substituted amphetamine-induced DA neurotoxicity is attenuated by nicotinamide (Huang et al., 1997; Stephans et al., 1998; Wan et al., 1999), which increases neuronal ATP concentrations, and is potentiated by malonate, a metabolic inhibitor (Albers et al., 1996; Nixdorf et al., 2001). Fourth, 2-DG has been found to enhance amphetamine-induced DA neurotoxicity (Chan et al., 1994), although not in all studies (Callahan et al., 1998; Hervias et al., 2000). Recent findings with nicotinamide in METH-treated animals also do not appear to generalize to MDMA-treated animals (Hervias et al., 2000). Nevertheless, collectively, the available evidence suggests that 5-HT neurotoxicity induced by some amphetamine derivatives may be associated with local alterations in cellular energy metabolism, a view further supported by the recent finding that MDMA produces glycogenolysis (Darvesh et al., 2002).

The purpose of the present studies was to further assess the potential role of energy metabolism in substituted amphetamine neurotoxicity. In particular, the present studies examined if glucoprivation induced by 2-DG potentiated the 5-HT neurotoxic effects of FEN and MDMA. This was done by giving MDMA or FEN alone and in combination with doses of 2-DG known to produce glucoprivation (Breier et al., 1993; Takahashi et al., 1997) as well as neuroprotection in neurodegenerative model systems that involve cellular energy perturbations (Lee et al., 1999; Tariq et al., 1999).

	Materials and Methods

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Drugs and Chemicals

Racemic MDMA, as the hydrochloride salt, was obtained from the National Institute of Drug Abuse (Bethesda, MD). Racemic FEN, also as the hydrochloride salt, was purchased from the Sigma-Aldrich (St. Louis, MO). 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and 2-DG were also purchased from the Sigma-Aldrich. [³H]Paroxetine was synthesized by PerkinElmer Life Sciences (Boston, MA).

Animals

Male Sprague-Dawley rats (Harlan, Indianapolis, IN) 8 weeks of age and weighing 200-225 g at the beginning of the study were used. Rats were housed individually in transparent plastic cages. Food and water were provided ad libitum. Animals were maintained in a temperature-controlled room (22 ± 1°C) on a 12-h light/dark cycle (light from 6:00 AM to 6:00 PM), except as otherwise dictated by the experimental protocol. All animal care and experimental manipulations were approved by the Institutional Animal Care and Use Committee at the Johns Hopkins University School of Medicine and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The facility for housing and care of the animals is accredited by the American Association for the Assessment and Accreditation of Laboratory Animal Care.

Drug Treatment

Experiment 1. Rats (n = 6-9 rats/group) were divided into four treatment groups as follows: 1) saline; 2) FEN alone (10 mg/kg, every 2 h × 4, i.p.); 3) 2-DG alone (330 mg/kg, every 2 h × 6, s.c.); 4) 2-DG/FEN (same doses as above, respectively). 2-DG was given 30 min before FEN, then at 2 h intervals for a total dose of 1980 mg/kg. This particular dosage regimen of 2-DG was selected because 1) it is known to decrease cellular glucose utilization (Schneider et al., 1997; Takahasshi et al., 1997), 2) comparable dosage regimens of 2-DG have been widely used to produce glucoprivation in rats, and 3) similar dosage regimens of 2-DG have been shown to produce clear effects in other models of neuronal injury in which energy utilization is strongly suspected (Lee et al., 1999; Tariq et al., 1999). As well summarized by Breier and colleagues (1993), 2-DG is a nonmetabolizable analog of glucose that is transported across the blood-brain barrier into brain tissue, where it is phosphorylated to 2-deoxy-D-glucose-6-phosphate but not further metabolized, resulting in the accumulation of 2-deoxy-D-glucose-6-phosphate and the inhibition of conversion of glucose-6-phosphate to fructose-6-phosphate. This, in turn, blocks glycolysis and glucose metabolism. Animals were maintained at normal (22°C) ambient temperature throughout the experiment. Rat rectal temperature was measured with a Bat-12 thermometer coupled to a RET-2 rat rectal probe (Physitemp, Inc., Clifton, NJ) at baseline and then every hour after 2-DG administration for the duration of the experiment. Two weeks later, the animals were killed for measurement of indexes of 5-HT neurotoxicity, here defined as a prolonged loss or reduction of 5-HT axonal markers.

Experiment 2. This experiment was carried out identically to the first, with the exception that the animals were treated in a warm environment to avert drug-induced hypothermia. Specifically, animals were moved into an elevated (28 ± 1°C) temperature-controlled room from a normal (22 ± 1°C) temperature-controlled room 60 min before drug administration and were moved back after the last rectal temperature was obtained.

Experiments 3 and 4. These experiments paralleled experiments 1 and 2, except that MDMA was used instead of FEN. As before, ambient temperature was maintained at either 22 ± 1°C or elevated to 28 ± 1°C to prevent drug-induced hypothermia. Moreover, in these studies, the number of animals per group was increased in anticipation of increased lethality at the higher ambient temperature (n = 6-12 rats/group).

Determination of the Concentration of Monoamines and Metabolites

Two weeks after treatment, rats were killed by decapitation, and their brains were regionally dissected and analyzed for their content of monoamines and their major metabolites, as previously described (Yuan et al., 2001).

Measurement of 5-HT Transporter Density

The density of 5-HT transporters (5-HTTs) in various brain regions was determined using recently described methods (Yuan et al., 2001).

Data Analysis

Temperature data were analyzed by two-way analysis of variance (ANOVA) for repeated measures, with treatment as the between-subjects factor and time as the within-subjects factor. When appropriate, group means at individual time points were compared by a one-way ANOVA, and post hoc comparisons were performed by Duncan's multiple range test. Overall temperature comparisons were also carried out by determining area under the curve (AUC) under the various treatment conditions. AUC was approximated by summation of fixed width (1 h) midpoint rectangles, with height (T°C) corresponding to each temperature measurement. Regional brain 5-HT, 5-HIAA, and 5-HTT data were analyzed by one-way ANOVA, followed by Duncan's multiple range post hoc comparisons. Results were considered significant when P was less than 0.05 using a two-tailed test. Data analysis was done using the statistical program for the social sciences (SPSS for Windows, Release 10.5; SPSS, Inc., Chicago, IL).

	Results

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Experiment 1: 2-DG and Fenfluramine at 22 ± 1°C

Long-Term Effects on 5-HT, 5-HIAA, and 5-HTT. At 22 ± 1°C, FEN produced significant long-lasting reductions of 5-HT and 5-HIAA in the cortex, striatum, hypothalamus, and hippocampus (Fig. 1). 2-DG alone was without significant long-term effects on 5-HT neuronal markers. When given with FEN, 2-DG did not potentiate FEN-induced 5-HT and 5-HIAA deficits in any brain region examined. To the contrary, at least in some regions, 2-DG tended to ameliorate the long-term effects of FEN on regional brain 5-HT and/or 5-HIAA (see cortical 5-HT; cortical, hippocampal, and hypothalamic 5-HIAA). Similar effects were observed on 5-HTT (Fig. 2). In particular, 2-DG did not exacerbate FEN-induced 5-HTT deficits. Indeed, in the hippocampus, 2-DG afforded partial protection (Fig. 2, left panel).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effect of 2-DG on FEN-induced 5-HT and 5-HIAA deficits at 22°C. Shown are the long-term effects of 2-DG (330 mg/kg, s.c., every 2 h × 6), given alone or in combination with a neurotoxic dose regimen of FEN (10 mg/kg, i.p., every 2 h × 4; given 30 min after the first four injections of 2-DG), on regional 5-HT (left panel) (expressed as nanograms per milligram wet weight tissue) and regional 5-HIAA (right panel) (expressed as nanograms per milligram wet weight tissue) measured 2 weeks after treatment. Values represent the mean ± S.E.M, n = 6-9 rats/group. COR, cortex; STR, striatum; HYP, hypothalamus; HPC, hippocampus; 1, designates significant difference from CON; 2, designates significant difference from FEN; 3, designates significant difference from 2-DG; 4, designates significant difference from 2-DG/FEN.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effects of 2-DG on FEN-induced 5-HTT deficits at 22°C (left panel) and 28°C (right panel). Shown are the long-term effects of 2-DG (330 mg/kg, s.c., every 2 h × 6), given alone or in combination with a neurotoxic dose regimen of FEN (10 mg/kg, i.p., every 2 h × 4; given 30 min after the first four injections of 2-DG), on 5-HTT in the cortex and hippocampus measured 2 weeks after treatment. Values represent the mean ± S.E.M, n = 6-9 rats/group. 1, designates significant difference from CON; 2, designates significant difference from FEN; 3, designates significant difference from 2-DG; 4, designates significant difference from 2-DG/FEN.

Effects on Core Temperature. At 22 ± 1°C, FEN produced significant hypothermia (Fig. 3). The same was true for 2-DG. The hypothermic effect of 2-DG, when given in combination with FEN, was greater than that produced by either drug alone, with mean core temperature dropping below 32°C. Given previous results (Bowyer et al., 1994; Miller and O'Callaghan, 1994; Albers and Sonsalla, 1995; Ali et al., 1996; Farfel and Seiden, 1995; Colado et al., 1998;Yuan et al., 2001), it seems possible that the expected exacerbation of FEN-induced 5-HT neurotoxicity secondary to 2-DG-induced glucoprivation might be masked by possible neuroprotective effects due to more pronounced hypothermia (Fig. 3).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of 2-DG and FEN on rat core temperature at an ambient temperature of 22°C. Shown are the effects of 2-DG (330 mg/kg, s.c., every 2 h × 6), given alone or in combination with a neurotoxic dose regimen of FEN (10 mg/kg, i.p., every 2 h × 4; given 30 min after the first four injections of 2-DG). Note marked lowering of core temperature in rats treated with 2-DG in combination with FEN. Values represent the mean ± S.E.M, n = 6-9 rats/group. 1, designates significant difference from CON; 2, designates significant difference from FEN; 3, designates significant difference from 2-DG; 4, designates significant difference from 2-DG/FEN.

Experiment 2: 2-DG and Fenfluramine at 28 ± 1°C

Effects on Core Temperature. To determine whether the failure of 2-DG to exacerbate FEN-induced 5-HT deficits was due to a confounding effect of temperature (hypothermia), experiment 1 was repeated at a higher ambient temperature (28 ± 1°C), a strategy previously successfully used by several groups to avert or minimize drug-induced hypothermia (Callahan et al., 1998; Colado et al., 1998; Yuan et al., 2001). As shown in Fig. 4, even at the higher ambient temperature, 2-DG, when given alone, produced mild hypothermic effects. In contrast, FEN, when given alone at the higher ambient temperature (28 ± 1°C), produced significant hyperthermia. When 2-DG was given in combination with FEN, hyperthermic effects predominated (Fig. 4), as evidenced by the fact that the temperature curve for FEN alone was comparable to that of FEN plus 2-DG, although AUC measurements revealed that a slight, but statistically insignificant, temperature difference remained (Fig. 4).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of 2-DG and FEN on rat core temperature at an ambient temperature of 28°C. Shown are the effects of 2-DG (330 mg/kg, s.c., every 2 h × 6), given alone or in combination with a neurotoxic dose regimen of FEN (10 mg/kg, i.p., every 2 h × 4; given 30 min after the first four injections of 2-DG). Note that the higher ambient temperature largely prevented 2-DG-induced hypothermia (i.e., core temperature of rats given 2-DG in combination with FEN was not significantly different from that in the FEN group). Values represent the mean ± S.E.M, n = 6-9 rats/group. 1, designates significant difference from CON; 2, designates significant difference from FEN; 3, designates significant difference from 2-DG; 4, designates significant difference from 2-DG/FEN.

Long-Term Effects on 5-HT, 5-HIAA, and 5-HTT. Once drug-induced hypothermia was largely abolished (by raising ambient temperature), the trend toward partial neuroprotection by 2-DG on FEN-induced 5-HT deficits was no longer evident. More importantly, even at the higher ambient temperature (which largely averted hypothermia), there was still no exacerbation of FEN-induced 5-HT neurotoxicity by 2-DG in any brain region examined (Figs. 2, right panel, and 5), suggesting that a potentiating effect of 2-DG was not being masked by changes in temperature. Of note, the long-term effects of FEN on 5-HT axonal markers at 28 ± 1°C were generally more pronounced than those observed after FEN administration at 22 ± 1°C (compare FEN alone groups in Figs. 1, 2, and 5; statistical difference achieved for striatal 5-HT/5-HIAA and hippocampal 5-HT/5-HTT).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Effects of 2-DG on FEN-induced 5-HT and 5-HIAA deficits at 28°C. Shown are the long-term effects of 2-DG (330 mg/kg, s.c., every 2 h × 6), given alone or in combination with a neurotoxic dose regimen of FEN (10 mg/kg, i.p., every 2 h × 4; given 30 min after the first four injections of 2-DG), on regional 5-HT (left panel) (expressed as nanograms per milligrams wet weight tissue) and 5-HIAA (right panel) (expressed as nanograms per milligrams wet weight tissue) measured 2 weeks after treatment. Note lack of exacerbating effect of 2-DG. Values represent the mean ± S.E.M, n = 6-9 rats/group. 1, designates significant difference from CON; 2, designates significant difference from FEN; 3, designates significant difference from 2-DG; 4, designates significant difference from 2-DG/FEN.

Experiment 3: 2-DG and MDMA at 22 ± 1°C

Long-Term Effects on 5-HT and 5-HIAA. In keeping with prior findings, MDMA, when given alone at 22 ± 1°C, produced significant long-lasting decrements in 5-HT and 5-HIAA (Fig. 6). 2-DG alone did not produce any significant long-term effects. When given along with MDMA, 2-DG did not exacerbate MDMA-induced 5-HT neurotoxicity in any region or index examined. Indeed, at 22 ± 1°C, 2-DG afforded complete or partial neuroprotection in some brain regions.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Effect of 2-DG on MDMA-induced 5-HT and 5-HIAA deficits at 22°C. Shown are the long-term effects of 2-DG (330 mg/kg, s.c., every 2 h × 6), given alone or in combination with a neurotoxic dose regimen of MDMA (10 mg/kg, i.p., every 2 h × 4; given 30 min after the first four injections of 2-DG), on regional 5-HT (left panel) (expressed as nanograms per milligrams wet weight tissue) and 5-HIAA (right panel) (expressed as nanograms per milligrams wet weight tissue) measured 2 weeks after treatment. Values represent the mean ± S.E.M, n = 6-7 rats/group. 1, designates significant difference from CON; 2, designates significant difference from MDMA; 3, designates significant difference from 2-DG; 4, designates significant difference from 2-DG/MDMA.

Effects on Core Temperature. At 22 ± 1°C, MDMA produced significant hyperthermia (Fig. 7). By contrast, 2-DG produced hypothermia, both when given alone and when given in combination with MDMA, raising the possibility that the above described neuroprotection was due to hypothermia.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Effect of 2-DG and MDMA on rat core temperature at an ambient temperature of 22°C. Shown are the effects of 2-DG (330 mg/kg, s.c., every 2 h × 6), given alone or in combination with a neurotoxic dose regimen of MDMA (10 mg/kg, i.p., every 2 h × 4; given 30 min after the first four injections of 2-DG), on rat core temperature. Note lower core temperature in rats treated with 2-DG. Values represent the mean ± S.E.M, n = 6-7 rats/group. 1, designates significant difference from CON; 2, designates significant difference from MDMA; 3, designates significant difference from 2-DG; 4, designates significant difference from 2-DG/MDMA.

Experiment 4: 2-DG and MDMA at 28 ± 1°C

Effects on Core Temperature. At 28 ± 1°C, the hypothermic effects of 2-DG in the MDMA-treated animal were largely, although not completely, eliminated (Fig. 8). In addition, MDMA-induced hyperthermia was more pronounced.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effect of 2-DG and MDMA on rat core temperature at an ambient temperature of 28°C. Shown are the effects of 2-DG (330 mg/kg, s.c., every 2 h × 6), given alone or in combination with a neurotoxic dose regimen of MDMA (10 mg/kg, i.p., every 2 h × 4; given 30 min after the first four injections of 2-DG), on rat core temperature. To prevent drug-induced hypothermia, drugs were administered at a higher ambient temperature (28°C). Notably, in the warmer environment, the core temperature of rats given 2-DG in combination with MDMA was only slightly lower than that in the MDMA group. Values represent the mean ± S.E.M, n = 6-12 rats/group. 1, designates significant difference from CON; 2 designates significant difference from MDMA; 3, designates significant difference from 2-DG; 4, designates significant difference from 2-DG/MDMA.

Long-Term Effects on 5-HT and 5-HIAA. Diminution of drug-induced hypothermia abolished the neuroprotective effect of 2-DG in MDMA-treated animals (Figs. 6 and 9), possibly in part because the toxic effects of MDMA at the higher ambient temperature were greater. Notably, even after its hypothermic effects were largely eliminated, 2-DG still did not exacerbate MDMA-induced neurotoxicity.

View larger version (31K): [in this window] [in a new window]   Fig. 9.   Effects of 2-DG on MDMA-induced 5-HT and 5-HIAA deficits at 28°C. Shown are the long-term effects of 2-DG (330 mg/kg, s.c., every 2 h × 6), given alone or in combination with a neurotoxic dose regimen of MDMA (10 mg/kg, i.p., every 2 h × 4; given 30 min after the first four injections of 2-DG), on regional 5-HT (left panel) (expressed as nanograms per milligrams wet weight tissue) and 5-HIAA (right panel) (expressed as nanograms per milligrams wet weight tissue) measured 2 weeks after treatment. Note comparable long-term effects of MDMA on these regional serotonergic markers in rats treated with MDMA, and those treated with 2-DG/MDMA after increasing the ambient temperature. Values represent the mean ± S.E.M, n = 6-12 rats/group. 1, designates significant difference from CON; 2, designates significant difference from MDMA; 3, designates significant difference from 2-DG; 4, designates significant difference from 2-DG/MDMA.

	Discussion

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The principal finding to emerge from the present studies is that glucoprivation by 2-DG does not enhance the 5-HT neurotoxic effects of either FEN or MDMA, even after the potential confounding effects of temperature are controlled. In addition, the present results indicate that the 5-HT neurotoxic effects of FEN and MDMA, like the DA neurotoxic effects of other amphetamine derivatives, are susceptible to thermoregulatory influence because changes in core temperature significantly influenced FEN- and MDMA-induced 5-HT neurotoxicity in various brain regions.

The observation that 2-DG does not exacerbate MDMA-induced 5-HT neurotoxicity confirms the recent findings of Hervias et al. (2000), extends them to FEN, and indicates that the long-term effects of FEN and MDMA on brain 5-HT neurons may differ from those of METH because 2-DG exacerbates METH-induced 5-HT deficits in some brain regions (Callahan et al., 1998) but does not exacerbate FEN- or MDMA-induced 5-HT deficits in any region examined. These findings seem contrary to the notion that the 5-HT neurotoxic effects of substituted amphetamine derivatives involve metabolic energy compromise. If this were the case, 2-DG would be expected to exacerbate long-term 5-HT axonal deficits induced by FEN and MDMA. Possible confounding effects of intercurrent temperature alterations must be considered, however. In particular, since it was not possible to prevent drug-induced temperature alterations on a moment-to-moment basis in the studies described herein, it is possible that transient temperature fluctuations were sufficient to mask potentiating effects of 2-DG glucoprivation. Furthermore, it is possible that it is not feasible to completely separate the influence of temperature from that of metabolism, and therefore, experimental manipulations that led to temperature changes may also have led to changes in brain metabolism.

As alluded to above, failure of 2-DG to exacerbate FEN- and MDMA-induced brain 5-HT neurotoxicity also seems inconsistent with previous observations involving the effects of 2-DG on METH neurotoxicity. In particular, while affording neuroprotection against METH-induced DA neurotoxicity (most likely by inducing hypothermia), 2-DG modestly exacerbated the neurotoxic effects of METH toward brain 5-HT neurons in some brain regions, leading to the suggestion that 5-HT toxicity might be particularly vulnerable to metabolic compromise (Callahan et al., 1998). Although it is conceivable that different amphetamine analogs have different mechanisms of neurotoxic action (and, therefore, interact differently with 2-DG), other explanations are possible (e.g., the interaction between temperature and energy metabolism after various amphetamine analogs may differ). Clearly, additional studies with various amphetamine derivatives and various metabolic inhibitors are needed to clarify the basis for the different observations with METH, MDMA, and FEN in 2-DG-treated animals.

Previous observations with METH (Callahan et al., 1998) also suggested that the neurotoxic effects of amphetamines toward brain 5-HT neurons, in contrast to effects toward DA neurons, may not be strongly influenced by temperature. To the contrary, findings from the present study, like those from others (Malberg and Seiden, 1997; Stewart et al., 1997; Hervias et al., 2000), indicate that FEN produces greater neurotoxic effects at higher ambient temperatures. Conversely, hypothermia, induced by 2-DG, was found to attenuate the neurotoxic effects of FEN and MDMA in this study, at least in some brain regions. Thus, amphetamine-induced 5-HT neurotoxicity, like amphetamine-induced DA neurotoxicity, is susceptible to temperature influence.

It is important to consider other potential effects of 2-DG that may have influenced the present findings. For example, 2-DG could lead to reductions in oxidative stress, which has been postulated to play a role in amphetamine neurotoxicity. Since 2-DG was not neuroprotective independent of temperature effects, however, 2-DG-induced reductions of oxidative stress are unlikely to have confounded the present results. 2-DG has also been reported to influence the basal release of DA from presynaptic terminals. Since DA has been postulated to play a role in amphetamine neurotoxicity, interactions between 2-DG and DA could potentially influence neurotoxicity. The role of DA in amphetamine neurotoxicity, however, has been called into question (Yuan et al., 2001). Furthermore, the lack of a clear temperature-independent protective (or enhancing) effect of 2-DG on neurotoxicity in the present study would suggest that 2-DG/monoaminergic interactions were not an important factor in our findings.

Despite the influence of temperature on FEN- and MDMA- induced 5-HT neurotoxicity, it is important to note that drug-induced hyperthermia is not essential for the development of 5-HT lesions. In particular, at an ambient temperature of 22 ± 1°C, FEN administration was found to produce both hypothermia and significant 5-HT neurotoxicity. Thus, while hyperthermia exacerbates the neurotoxic effects of both FEN and MDMA, it can be dissociated from the 5-HT neurotoxic process.

It is not known whether the toxic effects of substituted amphetamines on 5-HT and DA neurons involve similar mechanisms. It is tempting to speculate that parallel processes in these two neuronal systems underlie their vulnerability to amphetamine neurotoxicity. Ongoing research exploring the structure-activity relationships of the various neurotoxic amphetamine analogs and their relative selectivity (or nonselectivity) toward brain 5-HT and DA neurons may yield important clues to this question. Studies elucidating the apparent immunity of brain noradrenergic neurons to neurotoxic effects of amphetamine may also be helpful in this regard.

In conclusion, the present results indicate that even when potential temperature confounds are eliminated, the 5-HT neurotoxic effects of FEN and MDMA are not exacerbated by the metabolic inhibitor 2-DG. These results would seem to suggest that the 5-HT neurotoxic effects of FEN and MDMA do not involve compromise of brain glucose use. As emphasized above, however, this interpretation must be viewed with caution because temperature and glucose metabolism are closely linked and there could be complex interactions. Additional research is needed to further assess the role of energy metabolism in amphetamine neurotoxicity and to determine whether the toxic effects of different amphetamine analogs toward brain 5-HT and DA neurons involve similar or fundamentally different mechanisms.