Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on May 10, 2006; DOI: 10.1124/jpet.105.099200
0022-3565/06/3182-676-682$20.00
JPET 318:676-682, 2006
NEUROPHARMACOLOGY
Methamphetamine Administration Reduces Hippocampal Vesicular Monoamine Transporter-2 Uptake
Kristi S. Rau,
Elisabeth Birdsall,
Trent J. Volz,
James A. Riordan,
Anthony J. Baucum, II,
Brian P. Adair,
Rebecca Bitter,
James W. Gibb,
Glen R. Hanson, and
Annette E. Fleckenstein
Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah
Received for publication
January 13, 2006
Accepted
May 8, 2006.
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Abstract
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Repeated high-dose injections of methamphetamine (METH) rapidly decrease dopamine uptake by the vesicular monoamine transporter-2 (VMAT-2) associated with dopaminergic nerve terminals, as assessed in nonmembrane-associated vesicles purified from striata of treated rats. The purpose of this study was to determine whether METH similarly affects vesicular uptake in the hippocampus; a region innervated by both serotonergic and noradrenergic neurons and profoundly affected by METH treatment. Results revealed that repeated high-dose METH administrations rapidly (within 1 h) reduced hippocampal vesicular dopamine uptake, as assessed in vesicles purified from treated rats. This reduction was likely associated with serotonergic nerve terminals because METH did not further reduce vesicular monoamine uptake in para-chloroamphetamine-lesioned animals. Pretreatment with the serotonin transporter inhibitor fluoxetine blocked both this acute effect on VMAT-2 and the decrease in serotonin content observed 7 days after METH treatment. In contrast, there was no conclusive evidence that METH affected vesicular dopamine uptake in noradrenergic neurons or caused persistent noradrenergic deficits. These findings suggest a link between METH-induced alterations in serotonergic hippocampal vesicular uptake and the persistent hippocampal serotonergic deficits induced by the stimulant.
Repeated high-dose methamphetamine (METH) administrations produce persistent striatal and hippocampal serotonergic deficits including reduced activity of the serotonin-synthesizing enzyme tryptophan hydroxylase (TPH; Hotchkiss and Gibb, 1980
; Bakhit et al., 1981; Ricaurte et al., 1983) and reduced serotonin and 5-hydroxyindoleacetic acid concentrations (Hotchkiss and Gibb, 1980; Bakhit et al., 1981; Green et al., 1992). Dopamine contributes to these deficits as evidenced by findings that METH-induced decreases in striatal and hippocampal TPH activity are prevented by coadministration of the dopamine receptor antagonist haloperidol (Hotchkiss and Gibb, 1980). Furthermore, pretreatment with the dopamine synthesis inhibitor 
-methyl-p-tyrosine prevents the METH-induced reduction in TPH activity in both the striatum and hippocampus (Hotchkiss and Gibb, 1980); a deficit that is prevented in the striatum, as assessed 18 h after treatment when dopamine content is replenished by administering L-dihydroxyphenylalanine (Schmidt et al., 1985; effects in the hippocampus were not investigated). Pretreatment with serotonin transporter (SERT) inhibitors prevent METH-induced decreases in striatal and/or hippocampal TPH activity, and serotonin content (Hotchkiss and Gibb, 1980; Ricaurte et al., 1983; Schmidt and Gibb, 1985). These findings have led to the hypothesis that dopamine uptake into serotonergic terminals may contribute to these deficits (Seiden and Sabol, 1995; but also see Fleckenstein et al., 1997a) because accumulation of cytoplasmic dopamine can promote reactive species formation (i.e., in striatal dopaminergic neurons; for review, see Sulzer and Zecca, 2000). These SERT inhibitor studies have also led to the speculation that cytoplasmic accumulation of serotonin-derived toxins may contribute to this phenomenon (Wrona and Dryhurst, 2001).
In contrast to serotonergic neurons, effects of METH on noradrenergic neurons are less understood. Seiden et al. (1976) reported that METH causes persistent noradrenergic deficits in the midbrain and frontal cortex of rhesus monkeys. However, other studies using this species did not demonstrate such deficits (Finnegan et al., 1982; Preston et al., 1985). Likewise, investigations using rats, guinea pigs, or mice have not reported long-lasting noradrenergic deficits in any brain region (Wagner et al., 1979, 1980). The lack of consistent findings may be due to dose and/or frequency of METH administration or possibly to reduced sensitivity of noradrenergic neurons to METH.
It was reported previously that multiple high-dose injections of METH rapidly decreased dopamine uptake through the vesicular monoamine transporter-2 (VMAT-2) within striatal dopaminergic terminals, as assessed in nonmembrane-associated vesicles prepared from treated rats (Brown et al., 2000); an effect likely contributing to its consequent persistent deficits (for review, see Fleckenstein and Hanson, 2003; Hanson et al., 2004). Effects of this drug on VMAT-2 associated with hippocampal serotonergic and noradrenergic neurons have not been described. VMAT-2 is a principal regulator of cytoplasmic monoamine concentrations, and as noted above, aberrant accumulation of monoamines and/or related toxins can be cytotoxic. Therefore, the purpose of this study was to investigate the impact of METH on the activity of VMAT-2 associated with serotonergic and noradrenergic neurons. The hippocampus was selected for study, because it 1) is a target of METH toxicity and 2) has a considerable density of serotonergic and noradrenergic terminals. Results revealed that repeated high-dose METH administrations rapidly (within 1 h) reduced hippocampal vesicular monoamine uptake, a reduction likely associated with serotonergic terminals because METH did not further reduce vesicular uptake in para-chloroamphetamine (PCA)-lesioned animals. Pretreatment with the serotonin transporter inhibitor fluoxetine blocked both this acute effect on VMAT-2 and the decrease in whole-tissue serotonin content observed 7 days after METH treatment, suggesting an association between the acute and persistent serotonergic deficits caused by the stimulant. In contrast, METH treatment did not cause persistent noradrenergic deficits nor was there conclusive evidence that METH affected vesicular uptake in noradrenergic neurons. These findings suggest a link between METH-induced alterations in hippocampal vesicular uptake and the persistent hippocampal serotonergic deficits induced by the stimulant.
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Materials and Methods
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Animals. Male Sprague-Dawley rats weighing 270 to 380 g (Charles River, Wilmington, MA) were maintained under conditions of controlled lighting and temperature. Rats were maintained in a warm environment to ensure hyperthermia in METH-treated rats. Food and water were available ad libitum. Animals were euthanized by decapitation. All procedures were conducted in accordance with the National Institutes of Health Guidelines and approved by the Institutional Animal Care and Use Committee at the University of Utah.
Drugs and Chemicals. (±)-Methamphetamine hydrochloride was supplied by Research Triangle Institute (Research Triangle Park, NC). PCA, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), desipramine hydrochloride, fluoxetine hydrochloride, yohimbine hydrochloride, and methiothepin mesylate salt were purchased from Sigma-Aldrich (St. Louis, MO). CP93129 was purchased from Tocris Cookson Inc. (Ellisville, MO). 3,4-[ring-2,5,6-3H]Dihydroxyphenylethylamine (dopamine; 39.3, 59.7, and 60 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Citalopram hydrobromide was generously supplied by H. Lundbeck and Company (Copenhagen, Denmark). Doses were calculated as the respective free bases.
Preparation of Synaptic Vesicles. The hippocampi from treated rats were quickly dissected and homogenized in ice-cold 0.32 M sucrose and then centrifuged (800g for 12 min; 4°C). The resulting supernatant was centrifuged (22,000g for 15 min; 4°C). The synaptosomes, contained in the pellet from the second spin, were lysed using deionized water, and osmolarity was restored by the addition of HEPES and potassium tartrate (final concentration 25 and 100 mM, respectively; pH 7.5). To remove lysed synaptosomal membranes, samples were centrifuged at 20,000g for 20 min (4°C). MgSO4 (final concentration 1 mM) was added to the supernatant that was subsequently centrifuged at 100,000g for 45 min (4°C). The resulting pellet was resuspended in wash buffer (assay buffer containing 2 mM MgSO4 substituted for the ATP-Mg2+; pH 7.5) at a concentration of 150 mg/ml (original tissue wet weight).
Vesicular [3H]Dopamine Uptake. In the present study, a vesicular [3H]dopamine uptake assay was used as an index of VMAT-2 function. It is noteworthy that this protein transporter lacks monoamine selectivity. Thus, VMAT-2 comparably transports serotonin and norepinephrine (i.e., using membranes prepared from stable Chinese hamster ovary transformants expressing synaptic vesicle amine transporter cDNAs, the Km for serotonin transport was 0.19 µM and the Ki for dopamine and norepinephrine transport were 0.32 and 0.33 µM, respectively (Peter et al., 1994). The uptake of either monoamine could have been selected for use as a general indicator of VMAT-2 function; however, [3H]dopamine was chosen for study because it 1) is a well established assay used by others (Teng et al., 1997; Hogan et al., 2000) and us (Brown et al., 2000); and 2) could be considered a ligand of choice for study of noradrenergic neurons, because the VMAT-2 within these nerve terminals transport dopamine (before conversion by dopamine 
-hydroxylase to norepinephrine within the synaptic vesicles). [3H]Dopamine uptake was evaluated by incubating 100 µl of prepared hippocampal synaptic vesicles at 30°C for 3 min in assay buffer (final concentration 25 mM HEPES, 100 mM potassium tartrate, 1.7 mM ascorbic acid, 0.05 mM EGTA, 0.1 mM EDTA, and 2 mM ATP-Mg2+; pH 7.5) in the presence of [3H]dopamine (40 nM final concentration; for kinetics experiment, 25 nM-10 µM final concentration). Samples were rapidly filtered using a filtering manifold (Brandel Inc., Gaithersburg, MD) through GF/F filters (VWR, West Chester, PA) previously soaked in 0.5% polyethylenimine and washed three times with ice-cold wash buffer. Using a liquid scintillation counter, the radioactivity trapped in filters was counted. Nonspecific values were determined by measuring vesicular [3H]dopamine uptake at 4°C in wash buffer. All proteins were determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).
Whole-Tissue Serotonin and Norepinephrine Content. Hippocampal tissue was sonicated in 1 ml of ice-cold tissue buffer [0.05 M sodium phosphate and 0.03 M citric acid with 5% methanol (v/v); pH 2.5] and centrifuged at 22,000g for 15 min (4°C). The pellet was retained for protein determination using the Lowry Protein Assay (Lowry et al., 1951), and the supernatant was centrifuged four more times at 22,000g for 10 min (4°C). The resulting supernatant (50 µl) was then loaded directly onto a high-performance liquid chromatography system coupled to an electrochemical detector (+0.73 V compared with a Ag/AgCl reference electrode) for separation and quantification of serotonin and norepinephrine (mobile phase consisting of 0.05 M sodium phosphate, 0.03 M citric acid, 0.16 mM EDTA, 0.035% sodium octyl sulfate, and 5% methanol, pH 2.86; Whatman 25-cm C18 column).
Data Analysis. Comparisons between three or more groups were statistically analyzed using one-way analysis of variance followed by Fisher's protected least significant difference post hoc comparison. Comparisons between two groups were made using paired or unpaired two-tailed t test. Differences were considered significant if the probability of error was less than 5%.
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Results
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Results presented in Fig. 1 demonstrate that treatment with the serotonergic and noradrenergic neurotoxins PCA and DSP-4, respectively, caused persistent decreases in vesicular dopamine uptake, as assessed 7 days after DSP-4 or 14 days after the first PCA treatment in vesicles purified from rat hippocampi. Combined administration of these neurotoxins caused a further reduction in VMAT-2 function, confirming the existence of two populations of the transporter (i.e., within noradrenergic and serotonergic neurons, respectively). The efficacies of the toxic lesions were confirmed by findings that PCA treatment decreased whole-tissue serotonin content (13.6 ± 1.3 versus 2.6 ± 0.8 pg serotonin/µg protein for Sal/Sal- versus PCA/Sal-treated animals, respectively; n = 5-6), and DSP-4 decreased whole-tissue norepinephrine content (9.1 ± 1 versus 4.8 ± 1.5 pg norepinephrine/µg protein for Sal/Sal- versus Sal/DSP-4-treated animals, respectively; n = 5-6).
Having confirmed the existence of two populations of VMAT-2, a series of studies focused on the impact of METH on serotonin-associated VMAT-2. Results presented in Fig. 2 demonstrated that multiple administrations of METH acutely decreased vesicular dopamine uptake, as assessed in hippocampal vesicles obtained 1 h after treatment. In contrast, METH was without this acute effect in rats pretreated 7 days earlier with PCA (i.e., no difference was observed when comparing PCA/Sal- with PCA/METH-treated rats). Whole-tissue serotonin tissue content was affected similarly (Table 1). PCA had no effect on whole-tissue norepinephrine tissue content in the hippocampus (Table 1). The effect of METH on VMAT-2 was due to a decrease in Vmax (92.2 ± 16.5 versus 52.2 ± 11.4 fmol/µg protein/3 min for saline- and METH-treated rats, respectively; p 
0.05; n = 3) with no change in transporter affinity (Km for uptake was 198 ± 21 nM after saline treatment versus 271 ± 75 nM after multiple injections of METH; p 0.32; n = 3).
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Fig. 2. Effect of METH on hippocampal vesicular dopamine uptake in animals pretreated with PCA. Rats received two injections of PCA (7.5 mg/kg/injection i.p.; 24-h intervals) or Sal (1 ml/kg/injection i.p.). Seven days later, rats were treated with METH (4 x 7.5 mg/kg/injection s.c.; 2-h intervals) or Sal (1 ml/kg/injection s.c.). Animals were sacrificed 1 h after the last METH or saline injection. Data are expressed as the means + 1 S.E.M. of determinations in six rats. *, vesicular [3H]dopamine uptake significantly different from Sal/Sal-treated animals (p 0.05).
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TABLE 1 Effect of METH on hippocampal serotonin and norepinephrine content in animals pretreated with PCA Rats received two injections of PCA (7.5 mg/kg/injection i.p.; 24-h intervals) or Sal (1 ml/kg/injection i.p.). Seven days later, rats were treated with METH (4 x 7.5 mg/kg/injection s.c.; 2-h intervals) or Sal (1 ml/kg/injection s.c.). Animals were sacrificed 1 h after the last METH or saline injection. Data are expressed as the means ± 1 S.E.M. of determinations in six rats.
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Results presented in Fig. 3 reveal that treatment with the serotonin reuptake inhibitor fluoxetine prevented the acute METH-induced decrease in VMAT-2 function (Fig. 3A). Fluoxetine pretreatment also prevented the persistent serotonin deficits caused by METH treatment as assessed by measuring whole-tissue hippocampal serotonin content 7 days later (Fig. 4A). Neither effect was the result of fluoxetine preventing METH-induced hyperthermia (Figs. 3B and 4B), because body temperature was maintained in METH-treated rats by treating animals in a warm environment. Confirmation of a lack of effect of SERT inhibitors on distribution of VMAT-2 comes from the finding that a single citalopram treatment was without effect on VMAT-2 uptake (Table 2). Furthermore, a single administration of methiothepin (an antagonist of 5HT1, 6, and 7 receptors) or CP93129 (a 5HT1b receptor agonist) did not affect vesicular uptake (Table 2).
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Fig. 3. A and B, effect of fluoxetine on METH-treated animals on hippocampal vesicular dopamine uptake (A) and core body temperature (B). Rats were treated with four injections of METH (7.5 mg/kg/injection s.c.; 2-h intervals) or Sal (1 ml/kg/injection s.c.). Fifteen minutes before each injection, rats were treated with either fluoxetine (serotonin reuptake inhibitor, Flu; 10 mg/kg/injection i.p.) or Sal (1 ml/kg/injection i.p.). Rectal temperatures were taken every hour beginning 30 min before the first METH injection. Arrows indicate when METH was administered with respect to when core body temperatures were taken. Animals were sacrificed 1 h after the last METH or saline injection. Data are expressed as the means and 1 S.E.M. of determinations in 12 rats. *, vesicular [3H]dopamine uptake or core body temperature significantly different from Sal/Sal-treated animals (p 0.05).
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Fig. 4. Effect of fluoxetine pretreatment in METH-treated rats on hippocampal serotonin content (A) and core body temperature (B). Rats were treated with four injections of METH (7.5 mg/kg/injection s.c.; 2-h intervals) or Sal (1 ml/kg/injection s.c.). Fifteen minutes before each injection, rats were treated with either fluoxetine (serotonin reuptake inhibitor, Flu; 10 mg/kg/injection i.p.) or Sal (1 ml/kg/injection i.p.). Rectal temperatures were taken every hour beginning 30 min after the first METH injection. Arrows indicate when METH was administered with respect to when core body temperatures were taken. Animals were sacrificed 7 days after the last METH or saline injection. Data are expressed as the means and 1 S.E.M. of determinations in six rats. *, serotonin content or core body temperature significantly different from Sal/Sal-treated animals (p 0.05).
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TABLE 2 Effect of serotonin or norepinephrine selective drugs on hippocampal VMAT-2 uptake Rats received one injection of methiothepin (5HT1, 6, and 7; 10 mg/kg i.p.), fluoxetine (serotonin reuptake inhibitor; 10 mg/kg i.p.), citalopram (serotonin reuptake inhibitor; 10 mg/kg i.p.), CP93129 (5HT1b agonist; 2 mg/kg i.p.), yohimbine ( 2-adrenergic receptor antagonist; 10 mg/kg i.p.), desipramine (norepinephrine reuptake inhibitor; 10 mg/kg i.p.), or water vehicle (1 ml/kg i.p.). Animals were sacrificed 1 h after injection. Data are expressed as the percentage of the mean control values ± 1 S.E.M. of determinations in six rats. Control values ranged from 14.3 to 17.6 fmol/µg protein.
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A series of studies targeting norepinephrine-associated VMAT-2 were also accomplished. Results presented in Fig. 5 again demonstrate that METH treatment acutely decreased VMAT-2 function, as assessed 1 h after treatment. A METH-induced decrease in vesicular uptake occurred even if rats were pretreated 7 days earlier with DSP-4, a regimen that decreased norepinephrine content by 50% (Table 3; i.e., VMAT-2 function was decreased in DSP-4/METH- versus DSP4/Sal-treated rats). It is noteworthy that fluoxetine was administered 15 min before the DSP-4 to prevent uptake of the toxin into serotonin neurons (Reith et al., 1997). Still, results presented in Table 3 demonstrate that despite fluoxetine pretreatment, DSP-4 decreased serotonin levels by 24% compared with Sal/Sal-treated animals. Furthermore, a single injection of the noradrenergic reuptake inhibitor desipramine was without effect on hippocampal VMAT-2 activity (Table 2). Likewise, a single administration of yohimbine, an -2 adrenergic antagonist, did not alter vesicular monoamine uptake (Table 2).
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TABLE 3 Effect of METH on hippocampal serotonin and norepinephrine content in DSP-4-treated animals Rats received one injection of DSP-4 (50 mg/kg i.p.) or Sal (1 ml/kg i.p.) 15 min after either fluoxetine (10 mg/kg i.p.) or saline (1 ml/kg i.p.) pretreatment. Seven days later, rats were treated with METH (4 x 7.5 mg/kg/injection s.c.; 2-h intervals) or Sal (1 ml/kg/injection s.c.). Animals were sacrificed 1 h after the last METH or saline injection. Data are expressed as the means ± 1 S.E.M. of determinations in five to six rats.
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Finally, the long-term effects of high-dose, multiple METH administrations on hippocampal serotonin and norepinephrine content were investigated. Results revealed that whole-tissue content of serotonin, but not norepinephrine, was reduced 6 days after the last METH administration (Fig. 6, A and B).
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Fig. 6. Long-term effect of METH on hippocampal serotonin (A) and norepinephrine (B) content. Rats received four injections of METH (7.5 mg/kg/injection s.c.; 2-h intervals) or Sal (1 ml/kg/injection s.c.) and were sacrificed 1, 48, or 144 h after the last METH or saline injection. Symbols represent the means, and vertical lines represent 1 S.E.M. of determinations in five to six rats. *, serotonin or norepinephrine content significantly different from Sal/Sal-treated animals (p 0.05).
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Discussion
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VMAT-2 is a membrane transporter located on synaptic vesicles that actively transports monoamines from the cytoplasm into synaptic vesicles where they are stored for subsequent exocytotic release. It is well established that amphetamine analogs, including METH, affect the function of VMAT-2 associated with dopamine neurons. For example, Sulzer and Rayport (1990) demonstrated that amphetamine, and presumably METH, alters VMAT-2 function by disrupting the proton gradient necessary for monoamine transport in midbrain dopaminergic neurons. In addition, our laboratory has reported that multiple, high-dose METH administrations result in a rapid decrease in vesicular dopamine uptake in the striatum, as assessed in vesicles purified from treated rats (Brown et al., 2000; Sandoval et al., 2003).
An association between effects on VMAT-2 and the persistent dopaminergic deficits caused by METH treatment in the striatum is suggested by findings that heterozygous VMAT-2 knockout mice display enhanced dopaminergic deficits following a neurotoxic regimen of METH (Fumagalli et al., 1999). Furthermore, administration of agents that reverse METH-induced alterations in VMAT-2 (i.e., methylphenidate and lobeline) protect against the persistent deficits caused by the stimulant (Sandoval et al., 2003; Eyerman and Yamamoto, 2005). Although considerable effort has focused on investigating the METH-induced changes in vesicular uptake on striatal dopaminergic neurons, effects on VMAT-2 and its relationship to neurotoxicity in other brain regions and on other monoaminergic systems are less understood. Hence, the objective of the present study was to investigate the effects of METH on VMAT-2 function in serotonergic and noradrenergic neurons. The hippocampus was selected for study, because it is 1) profoundly affected by METH treatment (Hotchkiss and Gibb, 1980; Bakhit et al., 1981; Green et al., 1992), 2) innervated by both serotonergic and noradrenergic neurons, and 3) largely devoid of dopaminergic neurons and thus associated VMAT-2 that may confound the interpretation of results.
Results reveal that, as in the striatum, multiple administrations of METH rapidly (within 1 h) decreased hippocampal vesicular dopamine uptake, as assessed in nonmembrane-associated vesicles prepared from treated rats. In contrast, METH was without effect in rats pretreated 7 days earlier with PCA (i.e., no difference was observed when comparing PCA/Sal- with PCA/METH-treated rats). This absence of effect suggests that the PCA lesion already affected and/or damaged the VMAT-2 vulnerable to METH treatment. Based on studies and conclusions involving synaptosomal and vesicular striatal preparations, this effect was not likely due to residual METH introduced by the in vivo administrations (Fleckenstein et al., 1997b; Brown et al., 2000). This effect was due to a decrease in Vmax with no change in transporter affinity. Another possible effect of METH was that treatment caused a redistribution of VMAT-2 protein within the hippocampal nerve terminals as has been reported for striatal dopaminergic VMAT-2 (Riddle et al., 2002). Unfortunately, because of technical difficulties isolating enough hippocampal versus striatal VMAT-2 protein to assess VMAT-2 redistribution, this hypothesis remains to be tested.
It is well documented that METH causes massive release of monoamines (Axt and Molliver, 1991; Rocher and Gardier, 2001; Rothman et al., 2001). Therefore, drug treatments that increase extracellular monoamine concentrations or act presynaptically on monoaminergic nerve terminals may modulate VMAT-2 function. Consistent with this hypothesis, dopamine reuptake inhibitors alter striatal dopaminergic VMAT-2 distribution and function (Sandoval et al., 2002; Rau et al., 2005). Dopamine D2 agonists similarly affect VMAT-2 activity (Truong et al., 2004a,b); however, direct activation of presynaptic 5HT1b receptors had no effect on vesicular dopamine uptake in the hippocampus. Administration of a single injection of either the serotonin reuptake inhibitors fluoxetine or citalopram was without effect as well. These data indicate that neither this serotonin agonist nor serotonin reuptake inhibitors mimic the effects of METH on hippocampal serotonin-associated VMAT-2.
To investigate the effect of METH on noradrenergic neurons, vesicular uptake was assessed in animals pretreated with the noradrenergic neurotoxin DSP-4. Results revealed that METH treatment acutely decreased VMAT-2 function, even in these lesioned rats. Noteworthy, although DSP-4-treated animals were pretreated with fluoxetine, the serotonergic neurons were not completely protected as evidenced by findings that whole-tissue serotonin content in DSP-4 animals was reduced by 24% with respect to control animals. Because data presented in Figs. 2, 3, 4 demonstrate that METH decreased vesicular uptake in serotonergic neurons, it was hypothesized that the acute METH-induced decrease in DSP-4-treated rats presented in Fig. 5 involved VMAT-2 within serotonergic nerve terminals, although an effect on noradrenergic neurons cannot be ruled out. Noteworthy are findings that neither a single injection of the norepinephrine reuptake inhibitor desipramine nor the autoreceptor antagonist yohimbine [i.e., treatments that increase extracellular norepinephrine (Meana et al., 1997; Reith et al., 1997) and thus mimic to some degree the effects of METH] affected hippocampal vesicular uptake. Also important are findings that METH treatment was without persistent effect on whole-tissue norepinephrine concentrations, as assessed 7 days after treatment. These data are consistent with the premise that noradrenergic neurons are relatively resistant to the adverse consequences of METH treatment, findings consistent with those indicating that 1) the plasmalemmal norepinephrine transporter is resistant to some acute effects of METH treatment (Haughey et al., 2000) and 2) METH treatment is without persistent adverse effects on noradrenergic neurons (Wagner et al., 1979, 1980; Finnegan et al., 1982; Preston et al., 1985).
The finding that fluoxetine pretreatment prevented both the acute effect of METH on VMAT-2 function, and the persistent serotonergic deficits caused by the stimulant, suggests an association between the phenomena. Neither effect was attributable to effects on body temperature, because rats were treated in a warm environment and thus maintained at comparable body temperatures. Mechanisms by which an acute alteration in VMAT-2 activity might contribute to the persistent deficits remain to be elucidated, although a lack of vesicular sequestration capacity is a likely component. As noted above, findings that 1) dopamine contributes to METH-induced serotonergic deficits, 2) dopamine can be taken up by SERT in vivo (Seiden and Sabol, 1995; but also see Fleckenstein et al., 1997a), and 3) pretreatment with SERT inhibitors prevents METH-serotonergic deficits (Hotchkiss and Gibb, 1980; Ricaurte et al., 1983; Schmidt and Gibb, 1985) have led to the hypothesis that dopamine may accumulate in serotonin terminals and promote reactive species formation (i.e., in striatal dopaminergic neurons; for review, see Sulzer and Zecca, 2000). Arguments against this hypothesis include 1) the sparse dopaminergic innervation of the hippocampus, 2) the finding that PCA lesions do not alter striatal dopamine uptake (and thus, little dopamine is transported via the SERT), and 3) the concentration of dopamine required in vitro to cause uptake via SERT may not be attainable in vivo. Noteworthy, there is speculation that cytoplasmic accumulation of serotonin-derived toxins may contribute to this phenomenon (Wrona and Dryhurst, 2001). Alternatively, an intriguing hypothesis stems from findings involving the amphetamine analog methylenedioxymethamphetamine. Specifically, Yamamoto and co-workers (2004) have drawn upon findings that tyrosine, the biosynthetic precursor to dopamine, may be transported into nerve terminals in high concentrations (Morre and Wurtman, 1981). Once inside serotonin terminals, these investigators hypothesize that tyrosine can be nonenzymatically hydroxylated to DOPA and then converted by aromatic amino acid decarboxylase to dopamine. Dopamine may then promote oxidative stress within serotonin neurons. Their data suggest that L-tyrosine contributes to methylenedioxymethamphetamine-induced neurotoxicity to serotonin terminals (Breier et al., 2006) and raise the intriguing possibility that tyrosine-derived reactive species might contribute the METH toxicity in serotonin neurons. Future studies addressing these possibilities are warranted.
In summary, the present study provides insight into the effects of METH on VMAT-2 in nondopaminergic neurons. Specifically, the data demonstrate that METH acutely affects hippocampal serotonin-associated VMAT-2 and that this may contribute to the persistent serotonin deficits caused by the stimulant. In contrast, METH treatment did not cause persistent noradrenergic deficits nor was there conclusive evidence that METH affected vesicular uptake in noradrenergic neurons. Thus, aberrant VMAT-2 function may contribute to accumulation of cytoplasmic monoamine-associated toxins and that this, in turn, contributes to the persistent serotonergic deficits in the hippocampus.
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Footnotes
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This study was supported by a Focused Giving Grant from Johnson and Johnson Pharmaceuticals and by U.S. Public Health Service Grants DA04222, DA00869, DA13367, DA000378, and DA11389.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.105.099200.
ABBREVIATIONS: METH, methamphetamine; TPH, tryptophan hydroxylase; SERT, serotonin transporter; VMAT-2, vesicular monoamine transporter-2; PCA, para-chloroamphetamine; Sal, saline; DSP-4, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride; CP93129, 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridine-5-one dihydrochloride; 5HT, 5-hydroxytryptamine.
Address correspondence to: Dr. Annette E. Fleckenstein, Department of Pharmacology and Toxicology, University of Utah, 30 South 2000 East, Skaggs Hall, Rm. 201, Salt Lake City, UT 84112. E-mail: fleckenstein{at}hsc.utah.edu
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Abstract
Materials and Methods
