neurotoxicity and/or an impairment in the recovery pro- cess of the nigrostriatal ..... controls injected either with saline or with DSP-4 (data not shown). As shown ...
Journal of Neurochemistry Lippincott Williams & Wilkins, Philadelphia © 1999 International Society for Neurochemistry
Effects of Pretreatment with N-(2-Chloroethyl)-N-Ethyl-2Bromobenzylamine (DSP-4) on Methamphetamine Pharmacokinetics and Striatal Dopamine Losses *†Francesco Fornai, *‡Filippo S. Giorgi, †Maria G. Alessandrı`, §Mario Giusiani, and *Giovanni U. Corsini *Department of Neuroscience, Section of Pharmacology; §Department of Public Health, University of Pisa; †IRCCS Stella Maris-INPE; and ‡S.S.S.U.P. S. Anna, Pisa, Italy
Abstract: We recently demonstrated that pretreatment with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) exacerbates experimental parkinsonism induced by methamphetamine. The mechanism responsible for this effect remains to be elucidated. In this study, we investigated whether the exacerbation of chronic dopamine loss in DSP-4-pretreated animals is due to an impairment in the recovery of dopamine levels once the neurotoxic insult is generated or to an increased efficacy of the effects induced by methamphetamine. We administered different doses of methamphetamine either to DSP-4-pretreated or to intact Swiss–Webster mice and evaluated the methamphetamine-induced striatal dopamine loss at early and prolonged intervals. As a further step, we evaluated the striatal pharmacokinetics of methamphetamine, together with its early biochemical effects. We found that previous damage to norepinephrine terminals produced by DSP-4 did not modify the recovery of striatal dopamine levels occurring during several weeks after methamphetamine. By contrast, pretreatment with DSP-4 exacerbated early biochemical effects of methamphetamine, which were already detectable 1 h after methamphetamine administration. In addition, in norepinephrine-depleted animals, the clearance of striatal methamphetamine is prolonged, although the striatal concentration peak observed at 1 h is unmodified. These findings, together with the lack of a methamphetamine enhancement when DSP-4 was injected 12 h after methamphetamine administration, suggest that in norepinephrine-depleted animals, a more pronounced acute neuronal sensitivity to methamphetamine occurs. Key Words: Dopamine—DSP-4 —Locus coeruleus—Methamphetamine—Norepinephrine—Recovery. J. Neurochem. 72, 777–784 (1999).
relationship between norepinephrine (NE) loss [produced either via intraperitoneal N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) or through focal microinfusions of 6-hydroxydopamine] and dopamine (DA) nigrostriatal degeneration, providing evidence that NE depletion worsens the toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the nigrostriatal DA pathway in different animal species (Mavridis et al., 1991; Marien et al., 1993; Bing et al., 1994; Fornai et al., 1997). Similarly, a deleterious effect of a previous DSP-4 administration has been demonstrated for the nigrostriatal damage induced by methamphetamine (Fornai et al., 1995b, 1996a). Despite the evidence provided in these studies, the mechanism by which this exacerbation of DA toxicity in DSP-4-pretreated animals occurs is not clear. In particular, it needs to be elucidated whether this exacerbation is achieved via an enhancement of methamphetamine neurotoxicity and/or an impairment in the recovery process of the nigrostriatal DA pathway once the lesion has been produced. If the latter effect plays a significant role, then it would be expected that to worsen chronic striatal DA loss, NE terminals might be damaged immediately after methamphetamine administration. Similarly, an impaired recovery of striatal DA levels in DSP-4-pretreated compared with intact mice should be detected at prolonged intervals after methamphetamine administration. On the other hand, following the hypothesis of an increased toxicity [which appears to be the case in MPTPtreated, locus coeruleus (LC)-lesioned animals (Fornai et al., 1997)], the early effects of methamphetamine on
Neuropathological and biochemical studies carried out in parkinsonian patients have documented a severe impairment of the central noradrenergic system in combination with the loss of nigrostriatal dopaminergic neurons (Hornykiewicz and Kish, 1986; Alvord and Forno, 1992). Recent experimental findings suggest a causal
Received July 8, 1998; revised manuscript received September 18, 1998; accepted September 18, 1998. Address correspondence and reprint requests to Dr. F. Fornai at Unita` di Ricerca, IRCCS Stella Maris-INPE, Viale del Tirreno 347b, 56018, Calambrone, Pisa, Italy. Abbreviations used: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; DSP-4, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine; LC, locus coeruleus; NE, norepinephrine.
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striatal DA and its metabolites should be investigated in DSP-4-pretreated compared with intact animals. Also, we evaluated whether in DSP-4-pretreated animals alterations in the pharmacokinetics of striatal methamphetamine occur. This point might be particularly crucial because NE varicosities represent a major target site of systemically administered methamphetamine (Kuczenski and Segal, 1992; for review, see Seiden et al., 1993). To address these critical issues, in this study we treated Swiss–Webster mice with the NE neurotoxin DSP-4, which induces a selective pattern of NE loss involving NE terminals arising from the LC (Jonsson et al., 1981; Grzanna et al., 1989; Fornai et al., 1996b) similar to that occurring in Parkinson’s disease. We administered methamphetamine at different doses, starting with a low dose that, by itself, does not produce a loss of DA terminals. Mice were administered methamphetamine either 12 h before or 3 days after DSP-4. In these animals we studied the combined phenomena of DSP-4induced enhancement of methamphetamine-induced striatal DA depletion at different intervals, together with the kinetics of methamphetamine after treatment. MATERIALS AND METHODS Animals Male Swiss–Webster mice (Morini, Italy), 8 weeks old, weighing 24 –26 g, received food and water ad libitum; as methamphetamine toxicity partly depends on room temperature, mice were kept under strictly environmentally controlled conditions (12-h light/dark cycle with lights on between 700 and 1900 h; room temperature, 21°C). Given the high variability of methamphetamine toxicity, which also depends partly on housing conditions (for review, see Seiden and Ricaurte, 1987), the number of animals per cage (n ⫽ 10), and the size of the cages (38 ⫻ 22 cm wide and 15 cm high) were kept uniform throughout the study. Animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experiments described in this article were approved by the School of Medicine Ethical Committee on Experimental Studies at the University of Pisa.
Experimental design In the first set of experiments, mice were treated intraperitoneally with a single dose of DSP-4 hydrochloride (Sigma Chemical Co., St. Louis, MO, U.S.A.; 50 mg/kg); 3 days after DSP-4, a single, a double, or a triple dose of methamphetamine (Sigma; 5 mg/kg, one, two, or three doses 2 h apart, respectively) was administered intraperitoneally. The dose of methamphetamine was selected based on previous studies (Fornai et al., 1995b, 1996a) to produce a subthreshold effect (single injection), an intermediate degree of striatal DA deficit (double injection), or an extensive striatal DA loss (triple injection). Control groups received saline, methamphetamine, or DSP-4 alone, at the same doses and times used for the groups given the combined treatments. Three days after the DSP-4 injection, a group of animals treated with DSP-4, together with a group of saline-injected controls, was killed to assay regional cerebral monoamine levels at the time of methamphetamine administration. Seven days after methamphetamine injection (10 days after DSP-4), the remaining animals were killed. The second set of experiments was performed to investigate whether the enhancement of methamphetamine-induced striatal
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DA loss observed in DSP-4-pretreated animals at 7 days after methamphetamine might result from an impaired recovery of striatal DA levels during this interval. This experiment was carried out by injecting DSP-4 12 h after methamphetamine administration, when methamphetamine had been completely cleared from the striatum (see Results). The dosages of methamphetamine used in these experiments (5 mg/kg ⫻ 1 and ⫻ 2, respectively) correspond to those for which an enhancement of DA toxicity in NE-lesioned animals was observed (see Results). Mice were killed 7 days after methamphetamine injection (6 21 days after DSP-4). In another set of experiments, a further analysis of the potential effects of NE lesions on the recovery of striatal DA was carried out. These experiments were performed chronically by injecting methamphetamine at a dose (5 mg/kg ⫻ 2 or ⫻ 3) that by itself induced a significant decrease in striatal DA levels. Mice were killed 7 days or 1, 2, or 3 months after methamphetamine administration, to check for possible differences in the recovery of DA levels following the striatal depletion produced by methamphetamine. In these experiments, mice were given methamphetamine either 12 h before or 3 days after DSP-4 administration. These groups of animals were compared with mice treated either with saline or with methamphetamine alone, which were killed after the same intervals. The last series of experiments was performed acutely, after methamphetamine administration, to evaluate the effects of DSP-4 on the pharmacokinetics of striatal methamphetamine and on methamphetamine-induced acute striatal DA depletion as well. Given the reversible inhibitory activity of methamphetamine on monoamine oxidase (Suzuki et al., 1980), levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in acute studies. Animals were treated intraperitoneally with a single dose either of DSP-4 hydrochloride (50 mg/kg) or of saline. Three days later, all animals but controls were administered a single intraperitoneal injection of methamphetamine (5 mg/kg). Animals were killed at three intervals (1, 2, and 4 h) after methamphetamine, to evaluate striatal methamphetamine levels. The same animals were studied to evaluate at the same intervals the acute effects of methamphetamine on striatal DA and DOPAC levels in intact or in DSP-4-pretreated animals. In each series of experiments, all injections were carried out using a constant volume (10 ml/kg); mice were killed by cervical dislocation, the brains were immediately removed, and the striatum was dissected and assayed for NE, DA, and metabolites.
Assay of catecholamines The striatum, the frontal cortex, and the hippocampus were sonicated in 0.6 ml of ice-cold 0.1 M perchloric acid containing 10 ng/ml 3,4-dihydroxybenzylamine (Sigma) as the internal standard. An aliquot of the homogenate (50 l) was assayed for protein (Lowry et al., 1951). After centrifugation at 8,000 g for 10 min, 20 l of the clear supernatant was injected into an HPLC system where NE, DA, and metabolites were analyzed as previously described (Fornai et al., 1995a).
Assay of methamphetamine The striatum was sonicated in 0.2 ml of ice-cold 0.1 M perchloric acid. An aliquot of the homogenate (50 l) was assayed for protein (Lowry et al., 1951). After centrifugation at 8,000 g for 10 min, 100 l of the clear supernatant was assayed for methamphetamine, using a fluorescence polarization immunoassay (ADx; Abbott Laboratories, Abbott Park, IL, U.S.A.). To exclude cross-reactivity in the immu-
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TABLE 1. NE and DA levels within discrete brain areas 3 days after DSP-4 NE
Hippocampus Frontal cortex Striatum
DA
Controls
DSP-4
Controls
DSP-4
4.08 ⫾ 0.27 3.87 ⫾ 0.19 0.95 ⫾ 0.08
1.18 ⫾ 0.22a 0.47 ⫾ 0.11a 0.69 ⫾ 0.11
0.33 ⫾ 0.05 0.41 ⫾ 0.03 116.38 ⫾ 4.35
0.38 ⫾ 0.04 0.47 ⫾ 0.05 122.60 ⫾ 6.71
NE and DA levels were measured in male Swiss–Webster mice 3 days after administration of either saline or DSP-4 (50 mg/kg) to produce a selective lesioning of NE terminals arising from the LC. Results were obtained from 12 animals per group and are expressed as mean ⫾ SEM values, in ng/mg of protein. Differences among groups were evaluated using ANOVA with Scheffe´’s post hoc analysis: ap ⬍ 0.05 compared with controls.
noassay, striata from animals administered either saline or DSP-4 were analyzed. No cross-reactivity was detected in these treatment groups. The specificity of the methamphetamine assay was further validated performing a mass spectrum of the molecule in the striatal homogenate. Gas chromatography–mass spectrometry was performed carrying out an extraction from the homogenate as previously described (Moeller et al., 1993), and it confirmed the structural identity of the molecule identified as methamphetamine in the previous enzymatic quantitative assay. In particular, gas chromatography–mass spectrometry was performed in the scan mode, which allowed the detection of methamphetamine excluding other amphetamine derivatives and gave a mass spectrum overlapping with that of a standard solution of methamphetamine. To strengthen further the identity of methamphetamine, we carried out selected ion monitoring, using three individual ions (91, 160, and 204) that appeared with a retention time of 6.37 min, and compared it with selected ion monitoring for amphetamine (91, 118, and 190; retention time, 5.77 min), which was not found in the homogenate.
Data analysis For catecholamine assays, a standard curve was prepared using known amounts of DA, NE, and metabolites (Sigma), dissolved in 0.1 M perchloric acid containing a constant amount (10 pg/l) of the internal standard 3,4-dihydroxybenzylamine, as used for tissue samples. The standard curve for each compound (DA, NE, or its metabolite) was calculated using regression analysis of the ratios of the peak areas (compound area/3,4-dihydroxybenzylamine area) for various concentrations of each compound recorded at the reducing electrode. An analogous regression analysis was performed using different concentrations for the methamphetamine assay. The standard curve was prepared using known amounts of methamphetamine dissolved in 0.1 M perchloric acid. The decrease in the polarization for various amounts of the external standard was measured, and a regression curve was determined. For NE, DA, and DOPAC levels, results are expressed as mean ⫾ SEM values of 10 –12 animals per group. For methamphetamine levels, results are expressed as mean ⫾ SEM values of 10 animals per group. The effects of the combined treatments on striatal catecholamine levels were statistically evaluated using ANOVA with Scheffe´’s post hoc analysis, whereas effects on striatal methamphetamine levels (two experimental groups) were analyzed using two-tailed t test for unpaired data. The null hypothesis was rejected when p ⬍ 0.05.
RESULTS DSP-4 induces a specific pattern of NE depletion Three days after administration, DSP-4 (50 mg/kg, i.p.) induced a marked NE loss that was detected in the hippocampus and the frontal cortex but not in the striatum. In particular, the frontal cortex and the hippocampus were markedly affected with a subtotal NE loss. By contrast, DA levels in the striatum, the frontal cortex, and the hippocampus were unchanged (Table 1). Pretreatment with DSP-4 enhances long-lasting striatal DA depletion only after moderate doses of methamphetamine As shown in Fig. 1A, a single dose of methamphetamine (5 mg/kg, i.p.) did not modify striatal DA levels 7 days after treatment. By contrast, in animals administered DSP-4 (10 days before the assay) and then given this dose of methamphetamine (7 days before the assay), there was a significant reduction in striatal DA levels compared with controls (83.25 ⫾ 5.74 and 109.14 ⫾ 6.30 ng/mg of protein, respectively). As already measured 3 days after DSP-4 administration, there was no change 10 days after treatment in striatal DA levels of mice treated only with DSP-4 (Fig. 1A). A double dose of methamphetamine produced a significant degree of striatal DA depletion when administered to intact animals (73.26 ⫾ 5.81 compared with 114.09 ⫾ 8.73 ng/mg of protein for controls; Fig. 1B); by contrast, the same dose of methamphetamine produced a profound striatal DA loss in LC-lesioned animals (35.54 ⫾ 4.10 ng/mg of protein; Fig. 1B). In contrast, when methamphetamine was administered in a triple dose, a massive loss of striatal DA was measured, and this reduction was similar in saline- and in DSP-4-pretreated mice (8.58 ⫾ 1.70 and 10.51 ⫾ 2.41 ng/mg of protein, respectively; Fig. 1C). DSP-4 administered 12 h after methamphetamine does not modify striatal DA loss Injection of DSP-4 (50 mg/kg) after a single methamphetamine administration, when methamphetamine had been completely cleared from the striatum, 12 h after injection, did not produce any decrease in striatal DA levels 7 days after administration, compared with controls. Despite the marked effects of DSP-4 on cerebral J. Neurochem., Vol. 72, No. 2, 1999
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F. FORNAI ET AL. NE levels (corresponding to what we observed 3 days after DSP-4 injection; Table 1), the levels of striatal DA did not differ from those observed in animals injected with methamphetamine alone or in controls (Table 2). Similarly, when a double dose of methamphetamine was administered, we did not find any differences in chronic striatal DA levels between DSP-4-pretreated and intact mice; indeed, both groups of animals showed an intermediate degree of striatal DA depletion. Effects of pretreatment with DSP-4 on recovery of striatal DA levels As shown in Fig. 2A, there was a significant decrease in striatal DA levels in animals administered a double dose of methamphetamine; this effect disappeared at 60 days, when there was no longer a statistical difference between methamphetamine-treated animals and controls (Fig. 2A). In the DSP-4-pretreated group, despite the more marked striatal DA depletion observed at 7 days, a similar trend was observed in the recovery process, although at 90 days, striatal DA levels were still significantly lower than controls (Fig. 2A). In mice administered DSP-4 12 h after methamphetamine injection, striatal DA levels were similar to those in mice given methamphetamine alone, and again there was no difference in the recovery process for striatal DA levels at any interval compared with the group injected with methamphetamine alone (Fig. 2A). No modifications in striatal DA levels were observed at any interval in mice treated with DSP-4 alone (data not shown). When a triple dose of methamphetamine was administered (Fig. 2B), a massive striatal DA deficit was detected at 7 days in all groups of animals. No statistical differences were detected in the recovery process, which occurred for all treatment groups to a similar extent, leading only to a partial recovery of striatal DA compared with control values.
FIG. 1. Enhancement of methamphetamine-induced striatal DA loss after DSP-4 administration. Striatal DA levels were measured in male Swiss–Webster mice 7 days after administration of either saline or methamphetamine at different doses: (A) 5 mg/kg ⫻ 1, (B) 5 mg/kg ⫻ 2, and (C) 5 mg/kg ⫻ 3 (always 2 h apart). Three days before methamphetamine or saline administration mice were injected with either saline or DSP-4 (50 mg/kg). Results were obtained from 12 animals per group and are expressed as mean ⫾ SEM (bars) values. Differences among groups were evaluated using ANOVA with Scheffe´’s post hoc analysis: *p ⬍ 0.05 compared with controls, **p ⬍ 0.05 compared with methamphetamine.
TABLE 2. Catecholamine levels 7 days after different doses of methamphetamine given 12 h before DSP-4 Striatum
Controls Methamphetamine Methamphetamine Methamphetamine Methamphetamine
⫻ ⫻ ⫻ ⫻
1 1 ⫹ DSP-4 2 2 ⫹ DSP-4
Frontal cortex
DA
DA
NE
113.30 ⫾ 4.32 115.41 ⫾ 7.43 116.03 ⫾ 6.19 72.87 ⫾ 7.31a 68.34 ⫾ 3.76a
0.37 ⫾ 0.03 0.35 ⫾ 0.02 0.38 ⫾ 0.03 0.32 ⫾ 0.04 0.35 ⫾ 0.03
4.01 ⫾ 0.35 3.85 ⫾ 0.41 0.31 ⫾ 0.02a 4.16 ⫾ 0.38 0.41 ⫾ 0.09a
Striatal DA levels and frontocortical DA and NE levels were measured in male Swiss– Webster mice 7 days after administration of either saline or methamphetamine at different doses: methamphetamine ⫻ 1 (5 mg/kg ⫻ 1) or methamphetamine ⫻ 2 (5 mg/kg ⫻ 2, 2 h apart). Twelve hours after methamphetamine or saline administration mice were injected with either saline or DSP-4 (50 mg/kg) to produce a selective lesioning of NE terminals arising from the LC. Results were obtained from 12 animals per group and are expressed as mean ⫾ SEM values, in ng/mg of protein. Differences among groups were evaluated using ANOVA with Scheffe´’s post hoc analysis: a p ⬍ 0.05 compared with controls.
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mice treated with methamphetamine alone compared with controls. By contrast, in animals injected with DSP-4 plus methamphetamine, a marked acute striatal DA depletion was observed at 1 and 2 h after treatment. Four hours after methamphetamine administration, a significant increase in striatal DA levels was observed in animals treated with methamphetamine alone compared with controls, whereas in DSP-4 plus methamphetaminetreated mice, DA levels were similar to controls. DOPAC levels were similar in the three groups of animals 1 h after administration, but a moderate fall in DOPAC levels was observed at 2 and 4 h after methamphetamine administration, whereas the same phenomenon was significantly more pronounced in animals treated with DSP-4 plus methamphetamine (Table 3).
FIG. 2. Pretreatment with DSP-4 does not impair recovery of striatal DA levels. Striatal DA levels were measured in male Swiss-Webster mice at different intervals (7, 30, 60, and 90 days) after administration of either saline or methamphetamine at different doses: (A) 5 mg/kg ⫻ 2 or (B) 5 mg/kg ⫻ 3. Mice were injected with DSP-4 (50 mg/kg) either 3 days before (DSP-4 ⫹ Methamphetamine) or 12 h after (Methamphetamine ⫹ DSP-4) methamphetamine administration. Results were obtained from 10 animals per group and are expressed as the mean ⫾ SEM (bars) values. Differences among groups were evaluated using ANOVA with Scheffe´’s post hoc analysis: *p ⬍ 0.05 compared with controls, **p ⬍ 0.05 compared with methamphetamine.
Effects of pretreatment with DSP-4 on early effects of methamphetamine on striatal DA and DOPAC levels As shown in Table 3, no differences in DA levels were observed 1 h after methamphetamine administration in
Effects of pretreatment with DSP-4 on striatal methamphetamine pharmacokinetics No cross-reactivity in the fluorescence polarization immunoassay for methamphetamine was observed in controls injected either with saline or with DSP-4 (data not shown). As shown in Fig. 3, no difference was measured in the peak of methamphetamine levels measured 1 h after administration within the striata of saline plus methamphetamine- and DSP-4 plus methamphetamine-injected animals. At 2 h, methamphetamine levels in animals treated with methamphetamine alone were markedly reduced, whereas striatal methamphetamine content in NE-lesioned mice was still elevated. By contrast, in both groups of animals, striatal methamphetamine was markedly reduced to a similar extent at 4 h after administration. DISCUSSION As previously published (Hallman and Jonsson, 1984; Hallman et al., 1984; Fornai et al., 1996b), the neurotoxin DSP-4 (50 mg/kg i.p., 3 days after administration) induces a marked NE loss in selected LC target areas. In animals carrying such a noradrenergic lesion, we confirmed that a low dose of methamphetamine (5 mg/kg) that did not modify striatal DA levels when injected alone produced a long-lasting striatal DA loss, and there
TABLE 3. Striatal DA and DOPAC levels at early intervals after methamphetamine (5 mg/kg ⫻ 1) DA
Controls DSP-4 Methamphetamine DSP-4 ⫹ methamphetamine
DOPAC
1h
2h
4h
1h
2h
4h
91.42 ⫾ 4.08 92.75 ⫾ 3.15 80.57 ⫾ 2.28 40.84 ⫾ 4.36a,b
83.77 ⫾ 3.27 91.26 ⫾ 4.02 91.24 ⫾ 5.99 62.08 ⫾ 4.19a,b
93.58 ⫾ 6.57 95.57 ⫾ 2.75 117.99 ⫾ 5.32a 100.11 ⫾ 3.18
5.44 ⫾ 0.38 4.85 ⫾ 0.35 4.63 ⫾ 0.51 5.01 ⫾ 0.22
5.07 ⫾ 0.17 5.69 ⫾ 0.39 2.94 ⫾ 0.11a 1.11 ⫾ 0.14a,b
6.01 ⫾ 0.54 5.18 ⫾ 0.51 4.08 ⫾ 0.29a 1.63 ⫾ 0.12a,b
Striatal DA and DOPAC levels were measured in male Swiss–Webster mice at early intervals (1, 2, and 4 h) after administration of either saline or methamphetamine (5 mg/kg ⫻ 1). Three days before methamphetamine or saline administration mice were injected with either saline or DSP-4 (50 mg/kg) to produce a selective lesion of NE terminals arising from the LC. Results were obtained from 10 animals per group and are expressed as mean ⫾ SEM values, in ng/mg of protein. Differences among groups were evaluated using ANOVA with Scheffe´’s post hoc analysis: a p ⬍ 0.05 compared with controls, b p ⬍ 0.05 compared with methamphetamine.
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FIG. 3. Striatal methamphetamine levels at different intervals after methamphetamine administration. Striatal methamphetamine levels were measured at 1, 2, and 4 h after methamphetamine (5 mg/kg, single dose) administration. Three days before methamphetamine administration mice were treated with either DSP-4 (DSP-4 ⫹ Methamphetamine) or saline (Methamphetamine). Results were obtained from 10 animals per group and are expressed as mean ⫾ SEM (bars) values. Differences between the two groups were evaluated using two-tailed t test for unpaired data: *p ⬍ 0.05 compared with methamphetamine.
was an enhancement of the DA depletion induced by a double dose of methamphetamine (Fornai et al., 1996a). By contrast, we found in this study that when a triple dose of methamphetamine was administered, no further reduction in amount of the scant, remaining striatal DA was observed in DSP-4-pretreated animals. These data might indicate that the lesion of the noradrenergic system is effective only in worsening the biochemical effects of low to moderate nigrostriatal insults, but it is not important when methamphetamine by itself produces a massive DA loss; in this case, the scant remaining DA levels seem to be resistant to the effects induced by the loss of the noradrenergic innervation. However, an alternative explanation must be considered. In particular, as shown in this article and in previous studies (Fritschy and Grzanna, 1992; Fornai et al., 1996b), DSP-4 produces a regionally heterogeneous NE loss, sparing the scant striatal NE terminals; therefore, it cannot be ruled out that the small striatal DA amount measured in mice administered a triple dose of methamphetamine could derive from the DSP-4-resistant striatal NE terminals. Similarly, despite the fact that the same pattern of NE depletion was observed, we did not find any such enhancement in the present study when DSP-4 was injected 12 h after methamphetamine administration (both at a subthreshold and at an intermediate dose), indicating that to worsen methamphetamine toxicity, DSP-4 needs to be administered before methamphetamine. These latter findings tend to rule out an effect of the noradrenergic system in conditioning the progression of the damage to the nigrostriatal terminals, once dopaminergic neurotoxins have already been cleared from the striatum. J. Neurochem., Vol. 72, No. 2, 1999
Altogether, our findings lend substance to the hypothesis that an intact LC plays a protective role in the interval during which dopaminergic neurotoxins are actually producing their deleterious effects. This conclusion is further strengthened by the fact that in DSP-4pretreated animals, there was no impairment in the trend of recovery of striatal DA levels compared with animals treated with methamphetamine alone at longer intervals. However, the absence of a deleterious effect of DSP-4 administration on the recovery of striatal DA levels does not allow us to rule out an effect of NE on the recovery of striatal DA. First of all, it should be kept in mind that DSP-4 produces only a partial NE loss involving only certain brain regions and completely sparing the striatum. Therefore, the potential role of striatal NE in the recovery of striatal DA levels remains to be elucidated. Furthermore, an extensive lesion of NE axons needs to be achieved to suppress the function of NE neurons, because partial NE lesions might not be sufficient to alter NE release (Abercrombie and Zigmond, 1989); this is further strengthened by the possibility that NE innervation might greatly recover over time following treatment with DSP-4. In particular, a vigorous regenerative sprouting has been demonstrated in surviving NE neurons occurring during the few months after DSP-4 (Fritschy and Grzanna, 1992). In the search for the early mechanisms responsible for the enhancement of methamphetamine toxicity in DSP4-pretreated animals, we carried out a pharmacokinetic study to detect possible differences that could account for the enhancement described here. In this study, we found no differences in the striatal methamphetamine peak measured 1 h after injection, which corresponds to the interval used by other authors to assay striatal methamphetamine (Delle Donne and Sonsalla, 1994). However, a significant difference in the striatal methamphetamine concentration was measured at 2 h after administration: In particular, a marked decrease in striatal methamphetamine levels was observed in animals treated with methamphetamine alone despite a persistent retention of methamphetamine in DSP-4-pretreated mice. We recently described a similar increased retention of 1-methyl-4-phenylpyridinium within the brain of DSP-4 plus MPTP-treated mice (Fornai et al., 1997). This effect in striatal pharmacokinetics could be responsible for a more prolonged action of methamphetamine in the nigrostriatal DA terminals. Indeed, it is likely that pretreatment with DSP-4 might have altered the blood– brain barrier, because an abnormal permeability of this structure has been observed in animals that had undergone a microinfusion of 6-hydroxydopamine within the LC (Harik and McGunigal, 1984). Increased availability of methamphetamine within the brain might explain the early (1- and 2-h) decrease in striatal DA level measured in DSP-4 plus methamphetamine-treated mice; by contrast, in animals treated with a single dose of methamphetamine and possessing an intact noradrenergic system, DA levels did not decrease at any interval. It must be clarified that methamphet-
NE LOSS ENHANCES METHAMPHETAMINE ACUTELY amine-induced early DA depletion in the mouse striatum does not necessarily reflect a neurotoxic effect, and it might well occur as the consequence of a pharmacological, rather than a toxic, mechanism. Nonetheless, a pronounced early DA depletion in DSP-4-pretreated mice indicates that in the absence of LC axons, the nigrostriatal DA neurons possess a different pharmacological sensitivity to methamphetamine compared with control animals. The amount of DA release induced by methamphetamine has been shown to be closely related to the degree of subsequent neurotoxicity in experiments performed using brain dialysis (O’Dell et al., 1991, 1993); similarly, the degree of early DA loss induced by methamphetamine in striatal homogenates is related to chronic toxicity in ex vivo experiments (Chan et al., 1994). In particular, it has been shown that nontoxic doses of methamphetamine do not decrease striatal DA levels even at early intervals, i.e., 1.5 h, after treatment, whereas a higher dose or multiple doses of methamphetamine produced an acute DA depletion followed by chronic striatal DA loss as an expression of dopaminergic neurotoxicity (Chan et al., 1994). In line with this, we found differences in the levels of the DA metabolite DOPAC that might depend on increased efficacy and/or persistence of striatal methamphetamine. It is well known that methamphetamine is an inhibitor of monoamine oxidase (Suzuki et al., 1980), and this is likely to be the reason for the reduction in DOPAC levels observed here at 2 and 4 h in methamphetamine-treated animals compared with controls, confirming previous data (Nicolaou, 1980). Consistently, the decrease in striatal DOPAC level induced by methamphetamine at early intervals was more dramatic in DSP-4-pretreated mice. Such a strong reduction in DA metabolism might explain the paradoxical increase in striatal DA levels we observed at 4 h after methamphetamine administration, in analogy with previous studies (Yamanaka et al., 1983). Alternatively, such a strong reduction in DOPAC levels might be the consequence of a reduced availability of substrate within the DA terminals owing to the dramatic DA release occurring in DSP-4-plus methamphetaminetreated mice. However, this latter hypothesis would not explain the late (4-h) increase in striatal DA content. Moreover, the dose of methamphetamine we used here (5 mg/kg) is far beyond that required to inhibit monoamine oxidase in vivo (Seiden and Ricaurte, 1987). As previously shown for MPTP (Fornai et al., 1997), the mechanism by which the lesion of noradrenergic terminals enhances methamphetamine toxicity seems to depend on early biochemical events. The brain regions where this effect takes place remain to be elucidated because DSP-4 does not affect striatal NE levels. The loss of the NE system in the substantia nigra might be crucial in worsening the neurotoxicity induced by methamphetamine; however, although NEcontaining varicosities arising from the LC are known to project through the substantia nigra pars compacta (Mason and Fibiger, 1979) and we have previously shown a significant loss of nigral NE occurring after DSP-4 in
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Swiss–Webster mice (Fornai et al., 1996b), there is no electron microscopic evidence indicating that NE fibers actually synapse in this area. Similarly, functional studies on the role of nigral NE have never been carried out. In conclusion, our study confirms that pretreatment with DSP-4 worsens methamphetamine-induced experimental parkinsonism. Taken together, the present data indicate that such an NE neurotoxin enhances the chronic striatal DA depletion observed at 7 days after methamphetamine and increases the early biochemical effects of the DA neurotoxin but does not impair the recovery of striatal DA during this interval. An effect of NE axons on the recovery of nigrostriatal DA cannot be definitely ruled out because the degree of NE depletion produced by DSP-4 is not complete and does not occur in certain brain areas. Nonetheless, the results of these experiments specifically indicate that pretreatment with DSP-4 does not attenuate the recovery of striatal DA levels physiologically occurring during the 3 months after treatment. Acknowledgment: Drs. Maria T. Torracca and Irene Bonaccorsi are gratefully acknowledged for their important contribution in the methamphetamine assay.
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