Research ArticleNEUROPHARMACOLOGY

Effect of Glucoprivation on Serotonin Neurotoxicity Induced by Substituted Amphetamines

Jie Yuan, Branden J. Cord, Una D. McCann, Brian T. Callahan and George A. Ricaurte
Journal of Pharmacology and Experimental Therapeutics November 2002, 303 (2) 831-839; DOI: https://doi.org/10.1124/jpet.102.041277

Abstract

The present studies were conducted to further explore the potential role of metabolic compromise in substituted amphetamine-induced serotonin (5-HT) neurotoxicity. To this end, we examined the glucoprivic effects of 2-deoxy-d-glucose (2-DG) on the 5-HT neurotoxic effects of fenfluramine (FEN) and methylenedioxymethamphetamine (MDMA). Rats were treated with either FEN or MDMA, alone and in combination, with doses of 2-DG known to produce glucoprivic effects at either 22 ± 1 or 28 ± 1°C. At 22 ± 1°C, FEN produced hypothermia, MDMA induced hyperthermia, and both drugs produced significant long-term reductions in regional brain 5-HT neuronal markers. 2-DG did not enhance 5-HT neurotoxicity induced by either FEN or MDMA; indeed, in some instances, it afforded partial neuroprotection. Although 2-DG afforded partial protection from both FEN and MDMA-induced 5-HT neurotoxic changes, it also caused significant hypothermia, raising the possibility that protection was due to a lowered temperature. Increasing the ambient temperature to 28 ± 1°C largely eliminated drug-induced hypothermia and eliminated the neuroprotective effects of 2-DG. Thus, even without the confounding effect of temperature, 2-DG still did not potentiate FEN or MDMA-induced 5-HT neurotoxicity. These findings suggest that the role of metabolic compromise in amphetamine-induced 5-HT neurotoxicity merits further study.

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

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 byBreier 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

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).

Figure 1
Figure 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.

Figure 2
Figure 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).

Figure 3
Figure 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).

Figure 4
Figure 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).

Figure 5
Figure 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.

Figure 6
Figure 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.

Figure 7
Figure 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.

Figure 8
Figure 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.

Figure 9
Figure 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

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.

Footnotes

  • This work was supported by United States Public Health Service Awards DA-09487, DA-05707, DA-05938, and DA-10217.

  • The Department of Neurology at Johns Hopkins University School of Medicine is the laboratory of origin for this research.

  • DOI: 10.1124/jpet.102.041277

  • Abbreviations:
    FEN
    fenfluramine
    MDMA
    3,4- methylenedioxymethamphetamine
    5-HT
    serotonin
    METH
    methamphetamine
    DA
    dopamine
    2-DG
    2-deoxy-d-glucose
    5-HIAA
    5-hydroxyindoleacetic acid
    5-HTT
    5-HT transporter
    ANOVA
    analysis of variance
    AUC
    area under the curve
    • Received July 9, 2002.
    • Accepted July 25, 2002.

References

  1. ↵
    1. Albers DS,
    2. Sonsalla PK
    (1995) Methamphetamine-induced hyperthermia and dopaminergic neurotoxicity in mice: pharmacological profile of protective and nonprotective agents. J Pharmacol Exp Ther 275:1104–1114.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Albers DS,
    2. Zeevalk GD,
    3. Sonsalla PK
    (1996) Damage to dopaminergic nerve terminals in mice by combined treatment of intrastriatal malonate with systemic methamphetamine or MPTP. Brain Res 718:217–220.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Ali SF,
    2. Newport GD,
    3. Slikker W, Jr
    (1996) Methamphetamine-induced dopaminergic toxicity in mice. Role of environmental temperature and pharmacological agents. Ann N Y Acad Sci 801:187–198.
    OpenUrlPubMed
  4. ↵
    1. Appel NM,
    2. Mitchell WM,
    3. Contrera JF,
    4. De Souza EB
    (1990) Effects of high-dose fenfluramine treatment on monoamine uptake sites in rat brain: assessment using quantitative autoradiography. Synapse 6:33–44.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Bowyer JF,
    2. Davies DL,
    3. Schmued L,
    4. Broening HW,
    5. Newport GD,
    6. Slikker W, Jr,
    7. Holson RR
    (1994) Further studies of the role of hyperthermia in methamphetamine neurotoxicity. J Pharmacol Exp Ther 268:1571.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Bowyer JF,
    2. Tank AW,
    3. Newport GD,
    4. Slikker W, Jr,
    5. Ali SF,
    6. Holson RR
    (1992) The influence of environmental temperature on the transient effects of methamphetamine on dopamine levels and dopamine release in rat striatum. J Pharmacol Exp Ther 260:817–824.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Breier A,
    2. Crane AM,
    3. Kennedy C,
    4. Sokoloff L
    (1993) The effects of pharmacologic doses of 2-deoxy-d-glucose on local cerebral blood flow in the awake, unrestrained rat. Brain Res 618:277–282.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Burrows KB,
    2. Nixdorf WL,
    3. Yamamoto BK
    (2000) Central administration of methamphetamine synergizes with metabolic inhibition to deplete striatal monoamines. J Pharmacol Exp Ther 292:853–860.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Callahan BT,
    2. Yuan J,
    3. Stover G,
    4. Hatzidimitriou G,
    5. Ricaurte GA
    (1998) Effects of 2-deoxy-d-Glucose on methamphetamine-induced dopamine and serotonin neurotoxicity. J Neurochem 70:190–197.
    OpenUrlPubMed
  10. ↵
    1. Chan P,
    2. DiMonte DA,
    3. Luo JJ,
    4. DeLanney LE,
    5. Irwin I,
    6. Langston JW
    (1994) Rapid ATP loss caused by methamphetamine in the mouse striatum: relationship between energy impairment and dopaminergic neurotoxicity. J Neurochem 62:2484–2487.
    OpenUrlPubMed
  11. ↵
    1. Colado MI,
    2. Granados R,
    3. O'Shea E,
    4. Esteban B,
    5. Green AR
    (1998) Role of hyperthermia in the protective action of clomethiazole against MDMA (“ecstasy”)-induced neurodegeneration, comparison with the novel NMDA channel blocker AR-R15896AR. Br J Pharm 124:479–484.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Darvesh AS,
    2. Shankaran M,
    3. Gudelsky GA
    (2002) 3,4-Methylenedioxymethamphetamine produces glycogenolysis and increases the extracellular concentration of glucose in the rat brain. J Pharmacol Exp Ther 301:138–144.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Farfel GM,
    2. Seiden LS
    (1995) Role of hypothermia in the mechanism of protection against serotonergic toxicity. II. Experiments with methamphetamine, p-chloroamphetamine, fenfluramine, dizocilpine, and dextrorphan. J Pharmacol Exp Ther 272:868–875.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Fuller RW
    (1985) Persistent effects of amphetamine, p-chloroamphetamine and related compounds on central dopamine and serotonin neurons in rodents. Psychopharmacol Bull 21:528–532.
    OpenUrlPubMed
  15. ↵
    1. Fuller RW,
    2. Hemrick-Luecke S
    (1980) Long-lasting depletion of striatal dopamine by a single injection of amphetamine in iprindole-treated rats. Science (Wash DC) 209:305–307.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Fuller RW,
    2. Hemrick-Luecke SK
    (1982) Further studies on the long-term depletion of striatal dopamine in iprindole-treated rats by amphetamine. Neuropharmacology 21:433–438.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Fumagalli F,
    2. Gainetdinov RR,
    3. Valenzano KJ,
    4. Caron MG
    (1998) Role of dopamine transporter in methamphetamine-induced neurotoxicity: evidence from mice lacking the transporter. J Neurosci 18:4861.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Cho A,
    2. Segal D
    1. Gibb JW,
    2. Hanson GR,
    3. Johnson M
    (1994) Neurochemical mechanisms of toxicity. in Amphetamine and its Analogs: Neuropsychopharmacol, Toxicology and Abuse, eds Cho A, Segal D (Academic Press, New York), pp 269–289.
  19. ↵
    1. Harvey JA,
    2. McMaster SE
    (1977) Fenfluramine: cumulative neurotoxicity after chronic treatment with low dosages in the rat. Commun Psychopharmacol 1:3–17.
    OpenUrlPubMed
  20. ↵
    1. Hervias I,
    2. Lasheras B,
    3. Aguirre N
    (2000) 2-Deoxy-d-glucose prevents and nicotinamide potentiates 3,4-methylenedioxymethamphetamine-induced serotonin neurotoxicity. J Neurochem 75:982–990.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Hotchkiss AJ,
    2. Gibb JW
    (1980) Long-term effects of multiple doses of methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in rat brain. J Pharmacol Exp Ther 214:257–262.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Huang NK,
    2. Wan FJ,
    3. Tseng CJ,
    4. Tung CS
    (1997) Nicotinamide attenuates methamphetamine-induced striatal dopamine depletion in rats. Neuro Report 8:1883–1885.
    OpenUrlPubMed
  23. ↵
    1. Kogan FJ,
    2. Nichols WK,
    3. Gibb JW
    (1976) Influence of methamphetamine on nigral and striatal tryosine hydroxylase activity and on striatal dopamine levels. Eur J Pharmacol 36:363–371.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Lee J,
    2. Bruce-Keller AJ,
    3. Kruman Y,
    4. Chan SL,
    5. Mattson MP
    (1999) 2-Deoxy-d-glucose protects hippocampal neurons against excitotoxic and oxidative injury: evidence for the involvement of stress proteins. J Neurosci Res 57:48–61.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Kostrzewa RM
    1. Lew R,
    2. Malberg JE,
    3. Ricaurte GA,
    4. Seiden LS
    (1997) Evidence for and mechanism of action of neurotoxicity of amphetamine related compounds. in Highly Selective Neurotoxins: Basic and Clinical Applications, ed Kostrzewa RM (Humana Press, Inc. Totowa, NJ), pp 235–268.
  26. ↵
    1. Malberg JE,
    2. Seiden LS
    (1997) Administration of fenfluramine at different ambient temperatures produces different core temperature and 5-HT neurotoxicity profiles. Brain Res 765:101–107.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Marek GJ,
    2. Vosmer G,
    3. Seiden LS
    (1990) Dopamine uptake inhibitors block long-term neurotoxic effects of methamphetamine upon dopaminergic neurons. Brain Res 513:274–279.
    OpenUrlCrossRefPubMed
  28. ↵
    1. McCann UD,
    2. Hatzidimitriou G,
    3. Ridenour A,
    4. Fisher C,
    5. Yuan J,
    6. Katz J,
    7. Ricaurte GA
    (1994) Dexfenfluramine and serotonin neurotoxicity: further preclinical evidence that clinical caution is indicated. J Pharmacol Exp Ther 269:792–798.
    OpenUrlAbstract/FREE Full Text
    1. Miller KW,
    2. Freeman JJ,
    3. Dingell JV,
    4. Sulser F
    (1970) On the mechanism of amphetamine potentiation by iprindole. Experientia 26:863–864.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Nixdorf WL,
    2. Burrows KB,
    3. Gudelsky GA,
    4. Yamamoto BK
    (2001) Enhancement of 3,4-methylenedioxymethamphetamine neurotoxicity by the energy inhibitor malonate. J Neurochem 77:647–654.
    OpenUrlCrossRefPubMed
  31. ↵
    1. O'Hearn E,
    2. Battaglia G,
    3. De Souza EB,
    4. Kuhar MJ,
    5. Molliver ME
    (1988) Methylenedioxy-amphetamine (MDA) and methylenedioxymethamphetamine (MDMA) cause selective ablation of serotonergic axon terminals in forebrain: immunocytochemical evidence for neurotoxicity. J Neurosci 8:2788–8803.
    OpenUrlAbstract
  32. ↵
    1. Ricaurte GA,
    2. Bryan G,
    3. Strauss L,
    4. Seiden LS,
    5. Schuster CR
    (1985) Hallucinogenic amphetamine selectively destroys brain serotonin nerve terminals. Science (Wash DC) 229:986–988.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Ricaurte GA,
    2. Guillery RW,
    3. Seiden LS,
    4. Schuster CR
    (1984) Nerve terminal degeneration after a single injection of d-amphetamine in iprindole-treated rats: relation to selective long-lasting dopamine depletion. Brain Res 291:378–382.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Ricaurte GA,
    2. Schuster CR,
    3. Seiden LS
    (1980) Long-term effects of repeated methyl-amphetamine administration on dopamine and serotonin neurons in the rat brain: a regional study. Brain Res 193:153–163.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Sanders-Bush E,
    2. Bushing JA,
    3. Sulser F
    (1972) Long-term effects of p-chloroamphetamine on tryptophan hydroxylase activity and on the levels of 5-hydroxytryptamine and 5-hydroxyindole acetic acid in brain. Eur J Pharmacol 20:385–388.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Schmidt CJ
    (1987) Neurotoxicity of the psychedelic amphetamine methylenedioxymeth-amphetamine. J Pharmacol Exp Ther 240:1–7.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Schneider JE,
    2. Goldman MD,
    3. Leo NA,
    4. Rosen ME
    (1997) Central vs. peripheral metabolic control of estrous cycles in Syrian hamsters. II. Glucoprivation. Am J Physiol 272:R406–R412.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Schuster CR,
    2. Lewis M,
    3. Seiden LS
    (1986) Fenfluramine: neurotoxicity. Psychopharmacol Bull 22:148–151.
    OpenUrlPubMed
  39. ↵
    1. Seiden LS,
    2. Fischman MW,
    3. Schuster CR
    (1976) Long-term methamphetamine induced changes in brain catecholamines in tolerant rhesus monkeys. Drug Alcohol Depend 1:215–219.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Shiraishi T,
    2. Mager M
    (1980) 2-Deoxy-d-glucose-induced hypothermia: thermoregulatory pathways in rat. Am J Physiol 239:R270–R276.
  41. ↵
    1. Stephans SE,
    2. Whittingham TS,
    3. Douglas AJ,
    4. Lust WD,
    5. Yamamoto BK
    (1998) Substrates of energy metabolism attenuate methamphetamine-induced neurotoxicity in striatum. J Neurochem 71:613–621.
    OpenUrlPubMed
  42. ↵
    1. Steranka LR
    (1982) Long-term decreases in striatal dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid after a single injection of amphetamine in iprindole-treated rats: time course and time-dependent interactions with amfonelic acid. Brain Res 234:123–136.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Steranka LR,
    2. Sanders-Bush E
    (1980) Long-term effects of continuous exposure to amphetamine on brain dopamine concentration and synaptosomal uptake in mice. Eur J Pharmacol 65:439.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Stewart CW,
    2. Bowyer JF,
    3. Slikker W, Jr
    (1997) Elevated environmental temperatures can induce hyperthermia during d-fenfluramine exposure and enhance 5-hydroxytryptamine (5-HT) depletion in the brain. J Pharmacol Exp Ther 283:1144–1150.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Stone DM,
    2. Stahl DC,
    3. Hanson GR,
    4. Gibb JW
    (1986) The effects of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) on monoaminergic systems in the rat brain. Eur J Pharmacol 128:41–48.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Takahashi A,
    2. Ishimaru H,
    3. Ikarashi Y,
    4. Kishi E,
    5. Maruyama Y
    (1997) Hypothalamic cholinergic activity and 2-deoxyglucose-induced hyperglycemia. Brain Res Bull 43:65–68.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Tariq M,
    2. Khan HA,
    3. al Moutaery K,
    4. al Deeb S
    (1999) Protection by 2-deoxy-d-glucose against β,β′-iminodipropionitrile-induced neurobehavioral toxicity in mice. Exp Neurol 158:229–233.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Wagner GC,
    2. Ricaurte GA,
    3. Johanson CE,
    4. Schuster CR,
    5. Seiden LS
    (1980) Amphetamine induces depletion of dopamine and loss of dopamine uptake sites in caudate. Neurology 30:547–550.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Wan FJ,
    2. Lin HC,
    3. Kang BH,
    4. Tseng CJ,
    5. Tung CS
    (1999) d-Amphetamine-induced depletion of energy and dopamine in the rat striatum is attenuated by nicotinamide pretreatment. Brain Res Bull 50:167–171.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Yuan J,
    2. Callahan BT,
    3. McCann UD,
    4. Ricaurte GA
    (2001) Evidence against an essential role of endogenous brain dopamine in methamphetamine-induced dopaminergic neurotoxicity. J Neurochem 77:1338–1347.
    OpenUrlCrossRefPubMed
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Research ArticleNEUROPHARMACOLOGY

Effect of Glucoprivation on Serotonin Neurotoxicity Induced by Substituted Amphetamines

Jie Yuan, Branden J. Cord, Una D. McCann, Brian T. Callahan and George A. Ricaurte
Journal of Pharmacology and Experimental Therapeutics November 1, 2002, 303 (2) 831-839; DOI: https://doi.org/10.1124/jpet.102.041277
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