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NEUROPHARMACOLOGY
Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland
Received April 25, 2005; accepted June 1, 2005.
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Abstract |
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). Numerous studies in rodents and nonhuman primates indicate that MDMA administration produces long-lasting decreases in markers of serotonergic nerve terminals (for review, see Sprague et al., 1998; Green et al., 2003; Lyles and Cadet, 2003). The MDMA-induced changes in 5-HT markers include depletions of tissue 5-HT levels, reductions in 5-HT transporter (SERT) binding and function (Commins et al., 1987; Schmidt, 1987), and loss of tryptophan hydroxylase activity. Immunohistochemical analysis of 5-HT shows an apparent loss of 5-HT nerve terminals. The spectrum of decrements in serotonergic markers produced by MDMA administration is typically described as neurotoxicity.
More than 60 studies report that MDMA administration produces a long-lasting depletion of brain 5-HT (Green et al., 2003), but fewer studies have systematically examined the effect of MDMA administration on validated markers of neurotoxicity in the rat. The results from some studies examining markers of toxicity are not consistent with the hypothesis that MDMA-induced 5-HT depletion is accompanied by axotomy. One well established index of neurotoxicity is glial fibrillary acidic protein (GFAP), which is a structural protein that occurs in astroglia and not in neurons. O'Callaghan and associates carefully validated the use of GFAP to detect neuronal degeneration following administration of various drugs, including substituted amphetamines (O'Callaghan and Miller, 1993, 2002). Indeed, recent data supports the hypothesis that neuronal degeneration is accompanied by an increased expression of GFAP (O'Callaghan and Sriram, 2005). Their work and that of others (Bai et al., 2001; Pubill et al., 2003) shows that MDMA administration regimens that deplete 5-HT do not result in increased GFAP expression in the rat, indicating that neuronal degeneration has not occurred.
Recently, Thomas et al. (2004) suggested that neurotoxic amphetamines, in contrast to non-neurotoxic amphetamines, activate both microglia and astroglia. Perhaps this notion holds true for mice, but not necessarily for rats. Although Swiss-Webster mice may be an exception (Achat-Mendes et al., 2005), it is generally well accepted that rats and mice are differentially affected by high-dose MDMA treatment, with rats exhibiting depletions of brain tissue 5-HT and mice exhibiting depletions of brain tissue DA (Green et al., 2003). To complicate matters further, when rats are treated with doses of MDMA or methamphetamine (METH) that deplete tissue 5-HT, activation of microglia and astroglia occurs only in the METH-treated rats (Pubill et al., 2003). Thus, 5-HT depletion and glial activation can occur independently. Although one could argue that the neurotoxicity markers used in the Pubill study were not sensitive enough to detect degeneration of nerve terminals, this possibility seems unlikely since these markers have been shown to detect degeneration of nerve terminals induced by METH and 5,7-dihydroxytryptamine (5,7-DHT) in other studies (O'Callaghan and Miller, 1993, 2002).
In reviewing the literature on the serotonergic neurotoxicity of substituted amphetamines, we noted that few studies measured the effect of these agents on the expression of the SERT protein, in contrast to the level of SERT binding. In our first study, we determined the effect of D-fenfluramine and para-chloroamphetamine, administered according to a neurotoxic injection regimen, on SERT protein expression measured with Western blots (Rothman et al., 2003). Surprisingly, despite pronounced decreases in tissue 5-HT levels and radioligand binding to SERT, there were no changes in SERT protein expression measured 2 days or 2 weeks after drug administration. Consistent with previous reports, the substituted amphetamine D-fenfluramine also failed to affect expression of GFAP (Bendotti et al., 1994; O'Callaghan and Miller, 1994; Stewart and Slikker, 1999).
In light of our results with D-fenfluramine and para-chloroamphetamine, we administered MDMA according to a neurotoxic regimen and measured SERT and GFAP protein expression 2 weeks later (Wang et al., 2004). As a control, we administered the neurotoxin 5,7-DHT via the intracerebroventricular (i.c.v.) route in a separate group of animals. We hypothesized that both MDMA and 5,7-DHT would decrease SERT expression and increase GFAP expression. Interestingly, it was observed that MDMA had no effect on either SERT or GFAP expression, despite large decreases in tissue 5-HT. 5,7-DHT decreased SERT protein expression and increased GFAP consistent with the profile of neurotoxic injury.
One hypothesis to explain how MDMA can decrease SERT binding and function, but not decrease SERT protein expression, is that MDMA alters the distribution of SERT between the plasmalemma and endosome compartments. Presumably, plasmalemmal transporter will be functional, whereas endosomal transporter might not be functional. In the present study, we determined the effect of MDMA on the expression of SERT protein in subcellular fractions of brain tissue collected 2 weeks after MDMA administration. Additionally, the effects of MDMA on markers for astroglia, microglia, and neurons were evaluated at 3 days and 2 weeks after drug treatment. Using a new and more sensitive affinity-purified anti-SERT antibody, the present findings indicate that MDMA treatment does not decrease SERT protein expression in endosomes or plasma membranes. Furthermore, MDMA does not alter the expression of glial markers in a pattern consistent with neurotoxicity.
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Materials and Methods |
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Experimental Design. Two large groups of animals (n = 28 rats per group) were used in these experiments; one group was sacrificed 3 days after drug treatments, whereas the other group was sacrificed 2 weeks after drug treatments. Within each large group of rats, there were four drug treatment conditions: 1) i.p. MDMA treatment (n = 7), 2) i.p. saline treatment (n = 7), 3) i.c.v. 5,7-DHT treatment (n = 7), and 4) i.c.v. vehicle treatment (n = 7). Post-mortem tissue levels of 5-HT were measured in the frontal cortices of all animals as the primary endpoint marker to determine the level of 5-HT depletion produced by drug treatment. We did not perform extensive radioligand binding experiments in this study because it is known that SERT binding decreases in tandem with tissue 5-HT (Green et al., 2003). Based on the post-mortem tissue data, we chose four rats from the drug-treated groups that showed similar decreases in tissue 5-HT to generate a uniform sample to assay with Western blots. The main purpose of including the 5,7-DHT treatment groups was to include a known neurotoxin as a positive control.
Drug Treatments. For MDMA-HCl treatments, rats received i.p. injections of 7.5 mg/kg every 2 h for three doses. Control rats received i.p. injections of 1 ml/kg saline on the same schedule. Colonic temperature was measured in all rats that received i.p. injections. A temperature probe (Physitemp Instruments, Inc., Clifton, NJ) was inserted into the rectum immediately after each i.p. injection, and temperature was recorded in °C. For 5,7-DHT treatments, rats were pretreated with nomifensine (15 mg/kg i.p.) 30 min before receiving a single i.c.v. injection of 150 µgin10 µl of vehicle (0.1% ascorbic acid in saline). Control rats received a single i.c.v. injection of 10 µl of vehicle. Rats were sacrificed 3 days or 2 weeks later. After sacrifice, the frontal cortex, caudate, and hippocampus were dissected. Tissue was kept frozen at –70°C until the time of assay by high-performance liquid chromatography with electrochemical detection or Western blot.
We developed the MDMA dose and injection schedule according to the principles of "effect scaling", rather than "interspecies scaling" (Ricaurte et al., 2000) because the latter approach is hampered by significant limitations (de la Torre and Farre, 2004). Effect scaling involves the determination of drug doses required to evoke equivalent pharmacological responses in rats versus humans. Doses of MDMA (
1.5 mg/kg) that are discriminated readily by rats (Glennon and Higgs, 1992; Baker and Makhay, 1996) are identical to those misused recreationally by humans (Cole and Sumnall, 2003). We therefore administered a dose of MDMA that is 5 times higher than the discriminated dose three times in 6 h, an effective dose 15 times higher than the dose typically taken by a human. We chose a 2-week sacrifice time, since this time point is commonly used in the literature as an appropriate time to measure long-term effects of MDMA. The 3-day sacrifice time was chosen to test for the possibility of MDMA-induced transient increases in glial markers.
Measurement of 5-HT. Rat cortical tissue levels of norepinephrine, 3,4-dihydroxyphenylacetic acid, DA, homovanillic acid, 5-HT, and 5-hydroxyindole acetic acid were measured using high-pressure liquid chromatography with electrochemical detection as previously described (Baumann et al., 1998). All data were acquired and analyzed using a Millennium software system (Millipore Corporation, Billerica, MA).
SERT Binding. SERT binding assays and preparation of membranes from whole rat brain minus caudate proceeded with minor modifications of published procedures (Nandi et al., 2004). The assay conditions and procedures were as described except for the following changes: assay buffer (10 mM HEPES, 100 mM NaCl, pH 74) and incubation time (2 h at 25°C). We used [125I]RTI-55 [3
-(4'-125iodophenyl)tropan-2-carboxylic acid methyl ester] and a selective DA transporter blocker [100 nM 4-(2-benzhydryloxy-ethyl)-1-(4-nitrobenzyl)piperidine oxalate] to block DA transporter binding (Greiner et al., 2003).
Western Blot Analysis. Table 1 summarizes the antibodies used in this study. Preliminary experiments were conducted to determine the optimal amount of protein to load on the gel for each antibody. Neuronal markers (synapsin-1), glial markers (GFAP and HSP32), and SERT protein levels were assayed in crude homogenates. Tissues were homogenized by sonication in ice-cold radioimmunoprecipitation assay buffer (1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 1 mM sodium orthovanadate in phosphate-buffered saline buffer, pH 7.4). Protein concentrations were determined using Pierce BCA Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL), and homogenates were diluted to a concentration of 2 mg/ml with 2x SDS-polyacrylamide gel electrophoresis loading buffer (Invitrogen, Carlsbad, CA). Samples were boiled for 6 min. Sample protein was separated on 8 to 16% polyacrylamide minigels (Invitrogen). Proteins separated by electrophoresis were transferred to Immobilon-polyvinylidene difluoride membranes (Millipore Corporation) using a semi-dry apparatus (Bio-Rad, Hercules, CA). Nonspecific binding to membranes was prevented by blocking for 60 min at room temperature in TBS solution containing 5% nonfat dry milk. Membranes were then probed with various antibodies (Table 1). Membranes were rinsed three times with TBS and then incubated with 1:5000 dilution of horseradish peroxidase-labeled secondary antibody in TBS solution containing 0.25% nonfat dry milk for 90 min at room temperature. After washing three more times, antibody complex was visualized by chemiluminescence using a kit from Pierce Biotechnology.
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To determine the intra-assay variation for the SERT protein assay, a sample of rat caudate was run in nine lanes of the same gel. Changes in the immunoreactivity values were expressed relative to average value (defined as 100 value), resulting in a mean ± S.D. = 100.00 ± 2.67.
In separate experiments, levels of immunoreactive SERT protein were measured in subcellular fractions prepared from the 2-week group. All of these procedures used reagents precooled at 4°C. Caudate, cortex, and hippocampus were dissected out into 10 volumes of ice-cold 10 mM Tris/HCl pH 7.4 buffer and homogenized by a polytron. The homogenate (designated H1) was centrifuged for 20 min at 20,000g. The pellet was saved (designated P1), and the supernatant (designated S1) was centrifuged at 200,000g for 60 min to yield a pellet (designated P2) and a supernatant (designated S2). The subcellular fractions were stored at –80°C. The samples were then processed for Western blot analysis as described above.
Data Analyses and Statistics. Western blots were quantitated using standard methods (Jayanthi et al., 2002). Pooled control and treated samples were run side by side. The density of the SERT band in the treated sample was divided by the density of the SERT band in the control sample and multiplied by 100 to yield a percent of control. This was done in at least three independent experiments. Statistical significance of the SERT binding, 5-HT tissue data, and the Western blot data were determined by the Student's t test.
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Results |
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). To confirm this, we treated 16 µgof homogenate protein with peptide-N-glycosidase F for 4 h at 37°C and observed a substantial reduction in the 70-kDA band (data not shown). Importantly, immunohistochemical studies with the anti-SERT antibody labeled anatomically defined serotonergic cell bodies and nerve terminals (Fig. 1B) (Jacobs and Azmitia, 1992).
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Effect of MDMA and 5,7-DHT on Glial and Neuronal Markers. We determined the effect of drug treatments on both glial and neuronal markers using a crude homogenate prepared from frontal cortex, caudate, and hippocampus. Microglial activation has been monitored using HSP32 (Acarin et al., 2002) and the peripheral benzodiazepine receptor (PBR) (Casellas et al., 2002), although both can also be expressed in astroglia (Kuhlmann and Guilarte, 2000; Bechtold and Brown, 2003). MDMA administration increased HSP32 expression about 30% in cortex and caudate in the 3-day group, but not in the 2-week group. MDMA and had no effect on HSP expression in the hippocampus (Fig. 2A). In contrast, 5,7-DHT increased HSP32 expression by 50 to 70% in the cortex and caudate in both the 3-day and 2-week groups and increased HSP32 expression in the hippocampus in the 2-week group (Fig. 2B). As reported in Fig. 3, MDMA administration failed to change GFAP expression in any brain region in either the 3-day and 2-week groups. Administration of 5,7-DHT increased GFAP expression by about 40% in the cortex and hippocampus in the 2nd week, but not the 3-day group. In striatum, 5,7-DHT increased GFAP expression in the 3-day and 2-week groups. Neither MDMA nor 5,7-DHT altered PBR (Fig. 4) or synapsin-1 expression (Fig. 5).
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) and endosomes (EEA1; Mu et al., 1995). Fractions H1 and P1 are enriched with plasma membranes and almost undetectable levels of endosomes. Fraction S2 is highly enriched with endosomes and no detectable plasma membranes.
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Discussion |
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In our previous study (Wang et al., 2004), we reported that administration of MDMA at a dose that depletes 50% of brain 5-HT fails to decrease the expression of SERT protein or increase expression of GFAP, an accepted marker for neurotoxicity. In contrast, administration of 5,7-DHT, a known neurotoxin, decreases SERT expression and increases GFAP expression. A central question raised by our previous work is how can MDMA administration decrease SERT binding and function without changing SERT protein expression? In view of data demonstrating the importance of intracellular trafficking of SERT (Blakely and Bauman, 2000), we hypothesized that administration of MDMA altered the distribution of SERT between the plasmalemma and endosomal compartments. It is assumed that SERT inserted into plasma membranes will be functional, whereas SERT within endosomes may not be functional in vivo and not accessible to radioligands used to label SERT in vitro. In the present study, we used a more sensitive affinity-purified antibody to extend our previous findings and to test the "altered intracellular trafficking" hypothesis.
The new antibody we used worked quite well for both Western blots and immunohistochemistry (Fig. 1). The antibody detected bands with molecular weights of 70 and 50 kDA, which are the glycosylated and deglycosylated forms of SERT, respectively (Yamamoto et al., 1998). The SERT antibody could be used to clearly identify serotonergic nerve terminals and fibers in immunohistochemical studies. We believe that these studies validate the selectivity and sensitivity of this antibody. The fractionation procedure we used effectively generated fractions enriched with plasma membranes without detectable levels of endosomal membranes (Fig. 7, H1 and P1) and a fraction enriched with endosomal membranes without detectable levels of plasma membranes (Fig. 7, S2). Interestingly, the fractionation procedure also physically separated the 70- and 50-kDa forms of SERT, indicating that plasma membranes are enriched with the deglycosylated SERT and that endosomes are enriched with the glycosylated SERT. Two weeks after MDMA administration, in the presence of a 68% depletion of cortical 5-HT, there were no changes in SERT protein expression in any brain region, in either endosomes or plasma membranes. These data appear to rule out changes in transporter trafficking as an explanation for why MDMA reduces SERT binding and function without altering SERT protein expression.
At the present time, it is clear that our investigations to date have failed to determine why MDMA treatment causes a decrease in SERT binding and function but not a decrease in SERT protein expression. We can only speculate that MDMA must ultimately relocate SERT into a compartment where it is nonfunctional in vivo and inaccessible to radioligand binding in vitro. A possible mechanism that remains to be explored is alterations in the activity of p38 mitogen-activated protein kinase (Samuvel et al., 2005). Inhibition of p38 mitogen-activated protein kinase decreases the activity of its substrate, phospho-mitogen-activated protein kinase-activated protein kinase 2, which leads to decreased SERT activity. Thus, it is possible that MDMA produces long-term changes in this regulatory pathway, resulting in long-term decreases in SERT function. Another possibility is that administration of MDMA results in less plasma membrane SERT being located in lipid rafts. Since SERT proteins not associated with lipid rafts are not functional, this also could provide a mechanism to reduce SERT activity but not SERT protein levels (Magnani et al., 2004).
Using our more sensitive anti-SERT antibody, we confirmed and extended our previous findings (Wang et al., 2004). Our previous work measured expression of SERT and GFAP 2 weeks after MDMA administration and failed to see any changes. Although the 2-week time point is generally accepted as being relevant to detecting neurotoxic effects of MDMA, it was possible that changes consistent with neurotoxicity had occurred earlier and had normalized by 2 weeks. Thus, in the present study, we measured all end-points at 3 days and 2 weeks after MDMA administration. We also assessed additional markers of neurotoxicity, including synapsin-1 to detect signs of neuronal degeneration (Monnet-Tschudi et al., 1995), HSP32 to detect signs of neuronal stress and microglial activation (Acarin et al., 2002; Bechtold and Brown, 2003), and the PBR to detect signs of microglial activation (Pubill et al., 2003). It must be mentioned that HSP32 (Bechtold and Brown, 2003) and the PBR (Kuhlmann and Guilarte, 2000) can be expressed on astroglia in addition to microglia. Thus, the results obtained with these markers must be interpreted in concert with changes in GFAP.
The results, detailed in Figs. 2, 3, 4, 5, 6, indicate that administration of MDMA failed to produce astrogliosis in either the 3-day or the 2-week group or alter expression of the PBR, indicating no evidence of microgliosis. The transient increase in HSP32 expression is most likely related to the occurrence of hyperthermia in the MDMA-treated rats, which is known to increase HSP32 expression (Bechtold and Brown, 2003). As noted under the Results section, administration of MDMA according to the dosing regimen employed here caused a sustained hyperthermic response that lasted at least 4 h postinjection. Importantly, MDMA treatment did not alter expression of the SERT protein in either the 3-day or 2-week groups. The data obtained with synapsin-1 were not revealing, since 5,7-DHT treatment failed to decrease expression of synapsin-1. The simplest explanation of this is that the proportion of synapsin-1 in the degenerating 5-HT nerve terminals was too small a fraction of the total to be detected. The findings with MDMA contrast sharply with the effects of the known neurotoxin 5,7-DHT. Administration of 5,7-DHT decreased SERT expression, increased GFAP expression, and increased HSP32 expression in both the 3-day and 2-week groups. Interestingly, 5,7-DHT decreased SERT expression at 2 weeks but not 3 days, indicating that some time must elapse for the lesion to fully develop. Viewed collectively, these data support the hypothesis that 5-HT depletion induced by MDMA might not be due to axotomy.
Literature Review. Many studies demonstrate that MDMA administration produces long-term reductions in markers of the serotonergic nerve terminal (Green et al., 2003). Although Swiss-Webster mice may be an exception (Achat-Mendes et al., 2005), it is generally well accepted that rats and mice are differentially affected by high-dose MDMA treatment, with rats exhibiting depletions of brain tissue 5-HT and mice exhibiting depletions of brain tissue DA (Green et al., 2003). The effects of MDMA on 5-HT markers in rats include depletions in tissue 5-HT levels, decreases in SERT binding, and reduction of tryptophan hydroxylase activity (Sprague et al., 1998; Ricaurte et al., 2000; Lyles and Cadet, 2003). Evidence that these changes result from death of nerve terminals (axotomy) is based primarily on immunohistochemical analysis of 5-HT (O'Callaghan and Miller, 2002). However, the use of semiquantitative immunohistochemical methods with intact nerve terminals possessing considerably less 5-HT could produce misleading results if not supported by other neurotoxicity markers. Thus, there is clearly a need to test the hypothesis that MDMA produces axotomy using additional measures of neurotoxicity.
Several published studies examined the effect of MDMA administration on validated markers of neurotoxicity. O'Callaghan and associate determined the effect of MDMA treatment on expression of GFAP in rats (O'Callaghan and Miller, 1993, 2002). They observed no change in GFAP expression except at exceedingly high doses (at least 75 mg/kg twice daily for 4 days). Moreover, this group reported that when MDMA and 5,7-DHT produce a similar decrease in 5-HT depletion (about 70%), only 5,7-DHT increased GFAP (O'Callaghan and Miller, 1993). Pubill et al. (2003) administered MDMA (20 mg/kg b.i.d. x 4 days) and assessed SERT binding and glial activation 3 and 7 days post-treatment. Despite the expected decreases in SERT binding, there were no changes in several measures of glial responses. For example, MDMA administration failed to increase [3H]PK-11195 binding, which labels the PBR, or induce expression of major histocompatibility complex class II (OX-6), both markers for microglial activation. Similarly, MDMA administration failed to induce astroglial activation, as indicated by Western blot measurement of GFAP and HSP27 expression. In support of these findings, Bai et al. (2001) reported that MDMA administration (10 mg/kg) failed to increase GFAP expression. In contrast, Aguirre et al. (1999) reported that a single dose of MDMA (20 mg/kg i.p.) increased GFAP expression in the hippocampus. The reasons for these discrepant results are unclear but may be related to the route of MDMA administration or specific strain of rats tested in each study. Most of the studies cited above administered MDMA via the s.c. route (O'Callaghan and Miller, 1993, 2002; Bai et al., 2001; Pubill et al., 2003), whereas Aguirre et al. (1999) administered MDMA via the i.p. route. MDMA administration to female C57BL6/J mice increased striatal GFAP expression (O'Callaghan and Miller, 1994; Miller and O'Callaghan, 1995). However, MDMA persistently depletes dopamine in the mouse, and the observed increase in GFAP expression could reflect dopaminergic axotomy. Taken together, the available findings indicate that MDMA-induced 5-HT depletion in the rat can occur without significant changes in GFAP expression.
Although MDMA administration fails to increase glial markers of neurotoxicity, it is well known that MDMA administration, even a single relatively low dose, results in neuronal damage as assessed by silver staining (Commins et al., 1987; Jensen et al., 1993). Similar results are obtained with the newer stain, Fluoro-Jade B (Schmued and Hopkins, 2000). As reviewed in detail elsewhere (Jensen et al., 1993), much of the staining is not associated with serotonergic nerve terminals, indicating that MDMA can produce some degree of nonserotonergic brain damage. This is not surprising, since MDMA could damage nonserotonergic neurons via its hyperthermic and sympathomimetic effects (Cole and Sumnall, 2003; Green et al., 2003).
The findings reported here for the rat demonstrate that a dosing regimen of MDMA that produces long-lasting depletion of brain 5-HT fails to alter either expression of SERT or expression of astroglial and microglial markers. The dosing regimen we used, which was rationally chosen based on effect scaling (see Materials and Methods), uses lower doses than those used by some other investigators. However, our dosing regimen produces acute hyperthermia and long-term 5-HT depletions that are comparable with other studies employing much higher doses (Battaglia et al., 1987; Commins et al., 1987; Scanzello et al., 1993). In our experience, administration of three sequential doses of MDMA at 7.5 mg/kg constitutes the upper limit of dosing that can be used without killing 15–20% of rats. The decrease in SERT expression and increase in GFAP expression induced by i.c.v. administration of 5,7-DHT, a known neurotoxin, shows that the Western blot assays can detect changes expected to occur following neurotoxic injury. It is interesting that SERT expression level in 5,7-DHT-treated rats is higher than expected based on the degree of 5-HT depletion. A possible explanation is that, whereas 5-HT in compromised nerve terminals is cleared rapidly via metabolic pathways, large integral membrane proteins are probably cleared much more slowly. It seems unlikely that we are measuring proteolytic fragments of the SERT, since the bands we quantitated occurred at the expected molecular weight.
As noted in several reviews (Sprague et al., 1998; Ricaurte et al., 2000; Lyles and Cadet, 2003), numerous variables affect MDMA responses, including dosage, species, ambient and body temperature, and route of administration (Bai et al., 2001; Esteban et al., 2001). Under the conditions used here, MDMA administration failed to produce changes consistent with axotomy. These findings are similar to those we found with using D-fenfluramine and para-chloroamphetamine (Rothman et al., 2003). The simplest explanation of our data are that, using our dosing regimen, administration of MDMA, D-fenfluramine, or para-chloroamphetamine leads to functional inactivation of SERT, not serotonergic axotomy (Molliver and Molliver, 1990).
In conclusion, this study demonstrates that the MDMA administration to rats does not decrease SERT expression or increase validated markers of neurotoxicity. These data suggest that the putative serotonergic toxicity of substituted amphetamines should be re-examined using immunological methods to measure SERT protein changes, and explanations other than axotomy should be sought to explain the long-lasting changes produced by MDMA on SERT binding and function.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: MDMA, (±)-3,4-methylenedioxymethamphetamine; 5-HT, serotonin; SERT, 5-HT transporter; GFAP, glial fibrillary acidic protein; DA, dopamine; METH, methamphetamine; 5,7-DHT, 5,7-dihydroxytryptamine; [125I]RTI-55, 3-(4'-125iodophenyl)tropan-2-carboxylic acid methyl ester; HSP32, heat shock protein 32; TBS, Tris-buffered saline; PBR, peripheral benzodiazepine receptor; PK-11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide.
Address correspondence to: Dr. Richard B. Rothman, Clinical Psychopharmacology Section, Triad Technology Center, 333 Cassell Drive, Suite 4500, Baltimore, MD 21224-6825. E-mail: rrothman{at}intra.nida.nih.gov
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Acarin L, Paris J, Gonzalez B, and Castellano B (2002) Glial expression of small heat shock proteins following an excitotoxic lesion in the immature rat brain. Glia 38: 1–14.[CrossRef][Medline]
Achat-Mendes C, Ali SF, and Itzhak Y (2005) Differential effects of amphetamine-induced neurotoxicity on appetitive and aversive Pavlovian conditioning in mice. Neuropsychopharmacology 30: 1128–1137.[CrossRef][Medline]
Aguirre N, Barrionuevo M, Ramirez MJ, Del Rio J, and Lasheras B (1999) Alphalipoic acid prevents 3,4-methylenedioxy-methamphetamine (MDMA)-induced neurotoxicity. Neuroreport 10: 3675–3680.[Medline]
Bai F, Jones DC, Lau SS, and Monks TJ (2001) Serotonergic neurotoxicity of 3,4-(+/-)-methylenedioxyamphetamine and 3,4-(+/-)-methylenedioxymethamphetamine (ecstasy) is potentiated by inhibition of gamma-glutamyl transpeptidase. Chem Res Toxicol 14: 863–870.[CrossRef][Medline]
Baker LE and Makhay MM (1996) Effects of (+)-fenfluramine on 3,4-methylenedioxymethamphetamine (MDMA) discrimination in rats. Pharmacol Biochem Behav 53: 455–461.[CrossRef][Medline]
Banken JA (2004) Drug abuse trends among youth in the United States. Ann NY Acad Sci 1025: 465–471.
Battaglia G, Yeh SY, O'Hearn E, Molliver ME, Kuhar MJ, and De Souza EB (1987) 3,4-Methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine destroy serotonin terminals in rat brain: quantification of neurodegeneration by measurement of [3H]paroxetine-labeled serotonin uptake sites. J Pharmacol Exp Ther 242: 911–916.
Baumann MH, Ayestas MA, and Rothman RB (1998) Functional consequences of central serotonin depletion produced by repeated fenfluramine administration in rats. J Neurosci 18: 9069–9077.
Bechtold DA and Brown IR (2003) Induction of Hsp27 and Hsp32 stress proteins and vimentin in glial cells of the rat hippocampus following hyperthermia. Neurochem Res 28: 1163–1173.[CrossRef][Medline]
Bendotti C, Baldessari S, Pende M, Tarizzo G, Miari A, Presti ML, Mennini T, and Samanin R (1994) Does GFAP mRNA and mitochondrial benzodiazepine receptor binding detect serotonergic neuronal degeneration in rat? Brain Res Bull 34: 389–394.[CrossRef][Medline]
Blakely RD and Bauman AL (2000) Biogenic amine transporters: regulation in flux. Curr Opin Neurobiol 10: 328–336.[CrossRef][Medline]
Casellas P, Galiegue S, and Basile AS (2002) Peripheral benzodiazepine receptors and mitochondrial function. Neurochem Int 40: 475–486.[CrossRef][Medline]
Cole JC and Sumnall HR (2003) The pre-clinical behavioural pharmacology of 3,4-methylenedioxymethamphetamine (MDMA). Neurosci Biobehav Rev 27: 199–217.[CrossRef][Medline]
Commins DL, Vosmer G, Virus RM, Woolverton WL, Schuster CR, and Seiden LS (1987) Biochemical and histological evidence that methylenedioxymethylamphetamine (MDMA) is toxic to neurons in the rat brain. J Pharmacol Exp Ther 241: 338–345.
de la Torre R and Farre M (2004) Neurotoxicity of MDMA (ecstasy): the limitations of scaling from animals to humans. Trends Pharmacol Sci 25: 505–508.[CrossRef][Medline]
Esteban B, O'Shea E, Camarero J, Sanchez V, Green AR, and Colado MI (2001) 3,4-Methylenedioxymethamphetamine induces monoamine release, but not toxicity, when administered centrally at a concentration occurring following a peripherally injected neurotoxic dose. Psychopharmacology 154: 251–260.[CrossRef][Medline]
Glennon RA and Higgs R (1992) Investigation of MDMA-related agents in rats trained to discriminate MDMA from saline. Pharmacol Biochem Behav 43: 759–763.[CrossRef][Medline]
Green AR, Mechan AO, Elliott JM, O'Shea E, and Colado MI (2003) The pharmacology and clinical pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"). Pharmacol Rev 55: 463–508.
Greiner E, Prisinzano T, Johnson IE, Dersch CM, Marcus J, Partilla JS, Rothman RB, Jacobson AE, and Rice KC (2003) Structure-activity relationship studies of highly selective inhibitors of the dopamine transporter: N-benzylpiperidine analogues of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine. J Med Chem 46: 1465–1469.[CrossRef][Medline]
Jacobs BL and Azmitia EC (1992) Structure and function of the brain serotonin system. Physiol Rev 72: 165–229.
Jayanthi S, McCoy MT, Ladenheim B, and Cadet JL (2002) Methamphetamine causes coordinate regulation of Src, Cas, Crk and the Jun N-terminal kinase-Jun pathway. Mol Pharmacol 61: 1124–1131.
Jensen KF, Olin J, Haykal-Coates N, O'Callaghan J, Miller DB, and de Olmos JS (1993) Mapping toxicant-induced nervous system damage with a cupric silver stain: a quantitative analysis of neural degeneration induced by 3,4-methylenedioxymethamphetamine. NIDA Res Monogr 136: 133–149; discussion 150–154.[Medline]
Kuhlmann AC and Guilarte TR (2000) Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity. J Neurochem 74: 1694–1704.[CrossRef][Medline]
Lyles J and Cadet JL (2003) Methylenedioxymethamphetamine (MDMA, Ecstasy) neurotoxicity: cellular and molecular mechanisms. Brain Res Brain Res Rev 42: 155–168.[CrossRef][Medline]
Magnani F, Tate CG, Wynne S, Williams C, and Haase J (2004) Partitioning of the serotonin transporter into lipid microdomains modulates transport of serotonin. J Biol Chem 279: 38770–38778.
Miller DB and O'Callaghan JP (1995) The role of temperature, stress and other factors in the neurotoxicity of the substituted amphetamines 3,4-methylenedioxymethamphetamine and fenfluramine. Mol Neurobiol 11: 177–192.[Medline]
Molliver DC and Molliver ME (1990) Anatomic evidence for a neurotoxic effect of (+/-)-fenfluramine upon serotonergic projections in the rat. Brain Res 511: 165–168.[CrossRef][Medline]
Monnet-Tschudi F, Zurich MG, Pithon E, van Melle G, and Honegger P (1995) Microglial responsiveness as a sensitive marker for trimethyltin (TMT) neurotoxicity. Brain Res 690: 8–14.[CrossRef][Medline]
Mu FT, Callaghan JM, Steele-Mortimer O, Stenmark H, Parton RG, Campbell PL, McCluskey J, Yeo JP, Tock EP, and Toh BH (1995) EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif. J Biol Chem 270: 13503–13511.
Nandi A, Dersch CM, Kulshrestha M, Ananthan S, and Rothman RB (2004) Identification and characterization of a novel allosteric modulator (SoRI-6238) of the serotonin transporter. Synapse 53: 176–183.[CrossRef][Medline]
O'Callaghan J and Sriram K (2005) Glial fibrillary acidic protein and related glial proteins as biomarkers of neurotoxicity. Expert Opin Drug Saf 4: 433–442.[CrossRef][Medline]
O'Callaghan JP and Miller DB (1993) Quantification of reactive gliosis as an approach to neurotoxicity assessment (Review). NIDA Research Monograph 136: 188–212.[Medline]
O'Callaghan JP and Miller DB (1994) Neurotoxicity profiles of substituted amphetamines in the C57BL/6J mouse. J Pharmacol Exp Ther 270: 741–751.
O'Callaghan JP and Miller DB (2002) Neurotoxic effects of substituted amphetamines in rats and mice: challenges to current dogma, in Handbook of Neurotoxicology (Massaro EJ ed) pp 269–301, Humana Press, Inc., Totowa, NJ.
Papadopoulos V, Lecanu L, Brown RC, Han Z, and Yao ZX (2005) Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis, neuropathology and neurological disorders. Neuroscience, in press.
Pubill D, Canudas AM, Pallas M, Camins A, Camarasa J, and Escubedo E (2003) Different glial response to methamphetamine- and methylenedioxymethamphetamine-induced neurotoxicity. Naunyn-Schmiedeberg's Arch Pharmacol 367: 490–499.[CrossRef][Medline]
Ricaurte GA, Yuan J, and McCann UD (2000) (+/-)3,4-Methylenedioxymethamphetamine (`Ecstasy')-induced serotonin neurotoxicity: studies in animals. Neuropsychobiology 42: 5–10.[CrossRef][Medline]
Rothman RB, Jayanthi S, Wang X, Dersch CM, Cadet JL, Prisinzano T, Rice KC, and Baumann MH (2003) High-dose fenfluramine administration decreases serotonin transporter binding, but not serotonin transporter protein levels, in rat forebrain. Synapse 50: 233–239.[CrossRef][Medline]
Samuvel DJ, Jayanthi LD, Bhat NR, and Ramamoorthy S (2005) A role for p38 mitogen-activated protein kinase in the regulation of the serotonin transporter: evidence for distinct cellular mechanisms involved in transporter surface expression. J Neurosci 25: 29–41.
Scanzello CR, Hatzidimitriou G, Martello AL, Katz JL, and Ricaurte GA (1993) Serotonergic recovery after (+/-)3,4-(methylenedioxy) methamphetamine injury: observations in rats. J Pharmacol Exp Ther 264: 1484–1491.
Schimmel SD, Kent C, Bischoff R, and Vagelos PR (1973) Plasma membranes from cultured muscle cells: isolation procedure and separation of putative plasma-membrane marker enzymes. Proc Natl Acad Sci USA 70: 3195–3199.
Schmidt CJ (1987) Neurotoxicity of the psychedelic amphetamine, methylenedioxymethamphetamine. J Pharmacol Exp Ther 240: 1–7.
Schmued LC and Hopkins KJ (2000) Fluoro-Jade: novel fluorochromes for detecting toxicant-induced neuronal degeneration. Toxicol Pathol 28: 91–99.
Sprague JE, Everman SL, and Nichols DE (1998) An integrated hypothesis for the serotonergic axonal loss induced by 3,4-methylenedioxymethamphetamine. Neurotoxicology 19: 427–441.[Medline]
Stewart CW and Slikker W Jr (1999) Hyperthermia-enhanced serotonin (5-HT) depletion resulting from D-fenfluramine (D-Fen) exposure does not evoke a glial-cell response in the central nervous system of rats. Brain Res 839: 279–282.[CrossRef][Medline]
Thomas DM, Dowgiert J, Geddes TJ, Francescutti-Verbeem D, Liu X, and Kuhn DM (2004) Microglial activation is a pharmacologically specific marker for the neurotoxic amphetamines. Neurosci Lett 367: 349–354.[CrossRef][Medline]
Wang X, Baumann MH, Xu H, and Rothman RB (2004) 3,4-Methylenedioxymethamphetamine (MDMA) administration to rats decreases brain tissue serotonin but not serotonin transporter protein and glial fibrillary acidic protein. Synapse 53: 240–248.[CrossRef][Medline]
Yamamoto H, Fujimiya M, Shirai Y, Nakashita M, Oyasu M, and Saito N (1998) Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain. Neurosci Res 32: 305–312.[CrossRef][Medline]
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