Methamphetamine Changes NMDA and AMPA ... - Wiley Online Library

Methamphetamine Changes NMDA and AMPA Glutamate Receptor Subunit Levels in the Rat Striatum and Frontal Cortex Patr´ıcia F. Simoes, ˜ a Ana P. Silva,a,b Frederico C. Pereira,a,b Elsa Marques,b Nuno Milhazes,c Fernanda Borges,c Carlos F. Ribeiro,a,b and Tice R. Macedoa,b a

b

Biomedical Institute for Research in Light and Image, Azinhaga de Santa Comba, Coimbra, Portugal

Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Coimbra, Azinhaga de Santa Comba, Coimbra, Portugal c

Organic Chemistry Department, Faculty of Pharmacy, University of Porto, Porto, Portugal

Methamphetamine (METH) is a powerful psychostimulant whose noxious effects depend largely on the pattern of abuse. METH-induced glutamate release may overactivate N-methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (NMDAR and AMPAR, respectively) causing excitotoxicity. In the brain, these receptors are also known for their essential role in mediating memory consolidation. Therefore, we assessed glial fibrillary acidic protein (GFAP) expression as a marker for astrogliosis and neurodegeneration by using Fluoro-Jade C (F-J C) staining. Moreover, we investigated the effect of two METH regimens on NMDAR NR1 and NR2A and on AMPAR GluR2 subunit expression in the rat striatum and frontal cortex 24 h after drug treatment. Adult Sprague-Dawley rats were injected subcutaneously (s.c.) on six consecutive days with saline (control and acute groups) or with an increasing dose of METH (10, 15, 15, 20, 20, 25 mg/kg/day; ED group). On the seventh day, both METH groups were given a “bolus” of 30 mg/kg METH, whereas controls received saline. We evaluated the expression levels of GFAP by both Western blot and immunohistochemical assays and concluded that there was no difference from control levels. In addition, neither drug regimen resulted in neurodegeneration within 24 h of last METH administration. In the frontal cortex of the acute group, NR1 expression level was decreased, and both NR2A and GluR2 were increased. Also, in the striatum of the acute group, the expression level of GluR2 was significantly increased, and both GluR2 and NR2A levels were augmented in the striatum of the ED group. Taken together, these results suggest a protective mechanism by decreasing permeability and/or functionality of AMPAR and NMDAR to counteract METH-induced glutamate overflow in the brain. Moreover, these results may explain, in part, the mnemonic deficits and psychotic behavior associated with METH abuse. Key words: methamphetamine; GluR2; NR1; NR2A; striatum; frontal cortex

Introduction

Address for correspondence: Tice R. Macedo, M.D., Ph.D. Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Coimbra, Subunit 1 Polo 3, Azinhaga de Santa Comba, Celas, 3000–354 Coimbra, Portugal. Voice: +351-239-480207; fax: +351-239-480065. [email protected]

Ann. N.Y. Acad. Sci. 1139: 232–241 (2008). doi: 10.1196/annals.1432.028

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Methamphetamine (METH) is a powerful psychostimulant that has a major impact on the cortical and striatolimbic brain regions, namely, the frontal cortex, the striatum and the hippocampus. It is known that METH 2008 New York Academy of Sciences. 232

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Sim˜ oes et al.: METH’s Effects on NMDA and AMPA Receptors in Brain

abusers suffer long-term mnemonic deficits, paranoia, decision-making inability, and motor impairment, deficits that are also described in laboratory animals.1,2 METH administration has been shown to produce dopaminergic and serotonergic dysfunction.3,4 In addition to having an impact on monoaminergic systems, METH also evokes a delayed overflow of glutamate (Glu) in the brain. Excessive Ca2+ entry through Glu ionotropic receptors N methyl-D-aspartate (NMDA) and α-amino-3hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA), may then occur and cause excitotoxic cell death.4,5 On the other hand, Ca2+ influx through NMDA and AMPA receptors (NMDARs and AMPARs, respectively) mediates not only excitotoxicity, but also long-term potentiation (LTP), one of the mechanisms thought to explain memory consolidation.6 NMDA and AMPA receptors’ functionality and/or permeability to Ca2+ ions depend on the assembly of specific subunits that form the receptors’ channels. NMDARs are heteromeric ion channels comprising at least one NR1 and two NR2 (A-D) subunits.7 The NR1 subunit is essential to form a functional receptor, and NR2 subunits, of which NR2A is one of the most commonly found subunits in the adult brain, modulate electrophysiological properties, such as receptor pharmacology, kinetics, and Ca2+ permeability.8 Moreover, AMPARs are cationselective tetrameric hetero-oligomers formed by the combination of four subunits, GluR1–4.9 The GluR2 subunit, when present, forms AMPARs impermeable to Ca2+ ions.10,11 In recent work from our group, NR1, NR2A and GluR2 subunits were shown to be altered by METH in the rat hippocampus 24 h after two different drug schedules.1 In the present work, we further use the same METH dosing paradigms, acute versus subchronic with escalating doses (ED), on the density of NR1, NR2A, and GluR2 subunits in the striatum and frontal cortex of rats 24 h after the last drug treatment. Under the same experimental conditions we also evaluated the presence of astrogliosis and/or neurodegeneration.

Methods Animals and METH Treatments Male, 8-week-old Sprague-Dawley rats (Charles River Laboratories Inc., Barcelona, Spain), weighing between 250–300 g were housed one per cage under controlled environmental conditions (12 h light/dark schedule, at room temperature of 21 ± 1◦ C) with food and water supplied ad libitum. A group of animals (the ED group) received a subchronic administration, being injected for 7 consecutive days with increasing doses of METH (10, 15, 15, 20, 20, 25, 30 mg/kg, s.c.). The total daily doses were given in three injections per day at 6-h intervals (8:00, 14:00 and 20:00) except for the last dose of 30 mg/kg METH, given on the seventh day, which was administrated in a single injection “bolus” (at 14:00). A second group of animals (the acute group) was injected with saline, 0.9% NaCl, for six consecutive days, three times per day, receiving the bolus of 30 mg/kg METH on the seventh day. Finally, a third group of animals (the control group) received saline throughout the 7 days of treatment. All animals survived these dosing regimens. All procedures involving experimental animals were performed in accordance with European Community guidelines (86/609/EEC). All efforts were made to minimize animal suffering and to reduce the number of animals used. METH HCl was synthesized in the Organic Chemistry Department, University of Porto, Portugal. Perfusion and Histologic Processing A different set of animals was anesthetized with 45 mg/kg pentobarbital 24 h after the last METH/saline administration and intracardially perfused with 250 mL of a saline solution (0.9% NaCl, 4% sucrose, pH 7.4) followed by 250 mL of 4% paraformaldehyde solution (4% paraformaldehyde, 0.9% NaCl, 4% sucrose, pH 7.4). Brains were post-fixed for 24 h and 20-μm-thick coronal sections from the

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beginning to the end of the striatum and frontal cortex were cut in a cryostat and collected in a sodium phosphate buffer (1 × PBS, 25% sucrose, pH 7.3). Tissue sections were mounted onto gelatine-coated slides, dried at room temperature, and stored at –20◦ C until processed. Fluoro-Jade C Staining and GFAP Immunohistochemistry Neurodegeneration was assessed by FluoroJade C (F-J C) staining as it shows degenerating neurons, dendrites, axons, and terminals.12 In brief: the slides with the striatum and frontal cortex slices were immersed 1 × 5 min in a basic alcohol solution (0.1% NaOH in 80% of absolute ethanol), 1 × 2 min in 70% ethanol, and 1 × 2 min in distilled water. Slides were transferred to 0.06% potassium permanganate for 10 min, under constant shaking, rinsed in distilled water 1 × 2 min, and incubated for 10 min in 0.0001% F-J C (Histo-Chem Inc., Jefferson, Arkansas, USA) freshly prepared. After 3 × 1 min rinse in distilled water, the slices were air-dried on a 50◦ C slide warmer for 5 min, dehydrated in xylene, and cover-slipped with DPX (Sigma-Aldrich, Sintra, Portugal). Finally, the staining was examined under a Zeiss Axioskop 2 plus microscope (PG-HITEC, Mem Martins, Portugal) equipped with an EBQ100 isolated fluorescent lamp, and analysis was performed using the Axiovision Release 4.2 software. In order to determine whether METH evoked an astrocytic response, immunohistochemical tests for GFAP were performed, as this protein is a sensitive marker of astrogliosis and consequently of a neurotoxic condition.13 Slides were rinsed 1 × 5 min in PBS (in g/L: 8 NaCl, 0.2 KCl, 2.9 Na 2 HPO 4 .12H 2 O, 0.2 KH 2 PO 4 , pH 7.4) and then 3 × 5 min in TBS (0.05 M Trizma base buffer containing 150 mM of NaCl, pH 7.2). The slices were blocked with TBS containing 0.2% Triton X-100 and 10% normal goat serum for 45 min. A monoclonal antibody for GFAP (1:500, mouse anti-glial fibrillary acidic protein, Cy3

Annals of the New York Academy of Sciences

conjugated; Sigma-Aldrich) was prepared in TBS containing 0.2% Triton X-100 and 10% normal goat serum and the slices were incubated for 72 h at 4◦ C in a humid atmosphere. Sections were then rinsed 3 × 10 min in TBS and 1 × 10 min in distilled water, dehydrated (1 × 2 min in 70%, 80% and 100% ethanol), passed through xylol, cover-slipped with Vectashield Hardset mounting medium, and observed under the previously mentioned fluorescence microscope. Western Blot Analysis A set of animals was sacrificed 24 h after the last administration. The brains were quickly removed, and the striatum and frontal cortex were dissected on ice and finally stored at –70◦ C until Western blot analysis. Total tissue was homogenized in lysis buffer (50 mM TrisHCl pH 7.4, 0.5% Triton X-100) containing protease inhibitor cocktail [1 mM PMSF, 1 mM DTT, 5 μg/mL CLAP (5 μg/mL chymostatin, 5 μg/mL leupeptin, 5 μg/mL antiparin, 25 mg/mL pepstatin A); Sigma-Aldrich]. Protein concentration was determined by using the BCA method (Pierce Perbio, Rockford, IL, USA), and 25 μg of protein was loaded on the electrophoresis gel for NR1, NR2A, and GluR2 analysis and 2 μg for GFAP analysis. Samples were separated by electrophoresis on 10% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Buckinghamshire, UK) and blocked with 5% nonfat dry milk in Tris-buffered saline for 2 h. The blots were probed with rabbit polyclonal anti-NR1 (1:400; Upstate Cell Signalling Solutions, Hampshire, UK), rabbit polyclonal anti-NR2A (1:200; Upstate Cell Signalling Solutions), rabbit polyclonal anti-GluR2 (1:600, Upstate Cell Signalling Solutions), and rabbit polyclonal anti-GFAP (1:500; Sigma-Aldrich) overnight at 4◦ C. Samples from the three experimental conditions were always loaded in the same gel, and the results were expressed as percentage of control. To confirm equal protein loading and sample transfer, blots


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were re-probed with mouse monoclonal anti-αtubulin (1:3000; Sigma-Aldrich) or with mouse monoclonal anti-β-actin (1:10000; SigmaAldrich) overnight at 4◦ C. Immunoreactive bands were visualized by the addition of alkaline phosphatase conjugated antibodies directed against rabbit and mouse IgG (1:20000 and 1:10000, respectively; Amersham), an enhanced chemifluorescence detection reagent (Amersham), and using a Versa Doc 3000 imaging system (Bio-Rad, Richmond, CA, USA). Densitometric analyses were performed using the BioRad Quantity One software. Statistical Analysis Results are expressed as means ± SEM. Western blot results were converted to percentage of control for each gel. This approach normalizes differences in the development of chromagen solution between blots. Each blot contained all experimental groups and the two brain regions, allowing standardization across all blots, and these data were averaged. The differences between groups were determined by ANOVA followed by Bonferroni’s multiple comparison test. Results METH Treatment Did Not Cause Neurodegeneration or Astrogliosis in the Rat Striatum and Frontal Cortex Several in vivo studies, using different METH schedules, reported early signs of neurodegeneration and astrogliosis in the rodent brain.4 Therefore, we investigated whether our dosing schedules might lead to neurodegeneration within 24 h. F-J C staining showed that none of the METH administration paradigms evoked F-J C–positive cells at 24 h following last administration in any of the studied brain regions (data not shown). The occurrence of astrogliosis is also well documented in METH studies, and it is a measurable parameter of the neurotoxic condition.13 Indeed, we have recently

Figure 1. Changes in GFAP protein level in the frontal cortex of METH-treated animals (acute and escalating doses) 24 h after last administration. Above the bars, representative Western blots of tissue lysates are shown. Band densities are relative to the respective β-actin band, used as a control of protein loading. Each blot contained all experimental groups and results were expressed as percentage of control. Data are represented as average ± SEM (n = 6 animals per group). The differences between groups were determined by ANOVA followed by Bonferroni’s multiple comparison test.

shown that a single-dose of METH (30 mg/kg) induced astrogliosis in rat hippocampus 24 h after injection.1 Thus, we further sought to determine whether there were any signs of reactive astroglia in the studied brain regions at 24 h after treatment. For that, we measured the immunoreactivity of GFAP, a specific biomarker of astrocytes.13 However, the acute administration did not change GFAP expression levels either in the striatum (n = 6, 104.7 ± 8.7% of control) or in the frontal cortex, even though there is a clear tendency to increased values of GFAP in the latter (n = 6, 121.8 ± 8.1% of control, P > 0.05) (Fig. 1). The escalating doses treatment also resulted in unchanged GFAP values in the striatum (n = 6, 112.1 ± 11.3% of control). Again, there was a tendency towards an increase in GFAP values in the frontal cortex (n = 6, 121.8 ± 12.3% of control, P > 0.05) for the ED group (Fig. 1). In accordance,

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Figure 2. Changes in the protein levels of NR1 and NR2A subunits in the striatum (A) and frontal cortex (B) of METH-treated animals (acute and escalating doses) 24 h after the last administration. Above the bars, representative Western blots of tissue lysates are shown for each receptor subunit. Bands densities are relative to the respective α-tubulin band, used as a control of protein loading. Each blot contained all experimental groups and results were expressed as percentage of control. Data are represented as average ± SEM (n = 6 animals per group). The differences between groups were determined by ANOVA followed by Bonferroni’s multiple comparison test. ∗ P < 0.05, ∗∗ P < 0.01, significantly different as compared to controls (salinetreated animals); ## P < 0.01, ### P < 0.001, significantly different between the two METH groups (acute versus escalating doses).

immunohistochemical analysis of the two brain regions previously described did not reveal any significant astrogliosis with either of the METH administration paradigms (data not shown). METH Altered NMDAR and AMPAR Subunits in the Rat Striatum and Frontal Cortex NMDARs and AMPARs undergo subunitspecific regulation under physiological or pathologic conditions. In the present study, we investigated METH effects on NMDARs’ NR1 and NR2A subunits, since the first is essential to form a functional receptor and the second is one of the most predominant NR2 subunits in the adult brain, regulating important physiological features of the receptor. Also, we chose to study the AMPAR GluR2 subunit since its

presence in the receptor channel dramatically reduces the permeability to Ca2+ ions, providing neuroprotection under excitotoxic conditions in the brain. As revealed by Western blot analysis, in the striatum any of the METH paradigms altered NR1 protein density levels (n = 6, 91.7 ± 9.7% of control, acute treatment and 83.4 ± 9.6% of control, ED treatment). However, the ED administration significantly raised NR2A density in the striatum (n = 6, 145.3 ± 8.3% of control, ∗∗ P < 0.01), whereas the acute administration did not alter this subunit value (n = 6, 86.3 ± 13.0% of control) (Fig. 2A). Regarding the frontal cortex, the acute treatment resulted in diminished levels of the NR1 subunit (n = 6, 80.0 ± 7.4% of control, ∗ P < 0.05) and although the ED treatment had a similar effect on NR1 subunit expression, it

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Discussion

Figure 3. Changes in the protein levels of the GluR2 subunit in the striatum and frontal cortex of METH-treated animals (acute and escalating doses) 24 h after the last administration. Above the bars, representative Western blots of tissue lysates are shown. Band densities are relative to the respective α-tubulin band, used as a control of protein loading. Each blot contained all experimental groups and results were expressed as percentage of control. Data are represented as average ± SEM (n = 6 animals per group). The differences between groups were determined by ANOVA followed by Bonferroni’s multiple comparison test. ∗ P < 0.05, ∗∗ P < 0.01, significantly different as compared to controls (salinetreated animals).

failed to reach significance (n = 6, 81.3 ± 8.6% of control). In addition, the acute treatment raised NR2A levels (n = 6, 132.1 ± 13.7% of control, ∗ P < 0.05), but the ED treatment did not change these subunit levels (n = 6, 103.7 ± 4.7% of control) (Fig. 2B). Both METH treatments led to increased levels of the GluR2 subunit in the striatum as follows: 138.8 ± 10.7% of control ∗∗ P < 0.01 (n = 6) in the acute group and 129.2 ± 10.2% of control ∗ P < 0.05 (n = 6) in the ED group. Also, in the frontal cortex, the acute METH administration caused an increase in GluR2 expression levels (n = 6, 130.9 ± 7.4% of control ∗∗ P < 0.01), but this subunit was not significantly altered in the ED group (n = 6, 116.3 ± 9.5% of control) (Fig. 3).

It was demonstrated that in addition to DA and 5-HT overflow, METH also induced increased Glu extracellular levels in the CNS. Excitotoxic neurodegeneration can then result from activation of the Glu ionotropic receptors, in particular NMDARs and AMPARs.4,5 These receptors are known for mediating not only excitotoxicity, but also longterm potentiation, and they are therefore crucial in mnemonic processes.14,15 Functional NMDARs are made up of at least one obligatory NR1 and two NR2(A-D) subunits. The NR2A subunit is one of the most predominant in the adult mammalian brain16 and modulates the electrophysiological properties of the receptor, such as pharmacology, kinetics, and Ca2+ permeability (see the Introduction).8 The assembly of different subunits form NMDARs with different electrophysiological and pharmacologic properties. Therefore, several physiological and pathophysiological conditions are known to alter NMDARs in terms of subunit composition/expression, phosphorylation, or anchoring,17–19 including administration of additive substances.20–22 Concerning AMPARs, these are cation-selective tetrameric hetero-oligomers formed by the combination of four subunits, GluR1–4, which mediate the majority of the fast excitatory synaptic transmission in the CNS.23 Likewise, AMPARs’ subunit composition is also regulated under different noxious brain conditions and the downregulation of the GluR2 subunit is associated with increased excitotoxic cell death.24,25 However, information about METH’s impact on Glu ionotropic receptors subunits is scarce. Our results show that both METH schedules decreased NR1 protein density in the frontal cortex in comparison with the rats treated with saline. However, the decrease evoked by ED did not reach significance. In contrast, METH failed to change NR1 expression in the striatum. On the other hand, in NR2A density the two drug schedules had different effects in the frontal cortex and in the striatum. In

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the striatum, the ED group administration led to a great increase in NR2A expression levels, while with the acute treatment group the subunit levels were not different from those of the control group. In contrast, in the frontal cortex the ED group presented NR2A levels equal to those of the control, whereas the acute group showed a significant increase in NR2A density. It is noteworthy that the alterations induced by both METH regimens observed in the frontal cortex were similar to those observed in the hippocampus, except that NR1 decrease was significant with the ED treatment in the hippocampus,1 whereas it only reached significance with the acute treatment in the frontal cortex. The regional variation of the effect of the treatments on the receptor subunits may reflect different long- versus short-term neuroadaptations to METH across brain regions. NMDARs containing the NR2A subunit are located mainly at the synapse,26 and biophysical analysis indicates that heteromers containing NR2A subunits produce NMDA channels with shorter opening times and shorter-lasting series of opening/closing than heteromers containing other NR2 subunits.27,28 Therefore, and as pointed out by Ali et al.29 in 2005, a decrease in NMDARs containing NR2A might lead to increased vulnerability of cells to excitotoxicity. Thus, our results suggest that METH exposure, depending on the administration schedule, either reduces NMDAR functionality by decreasing NR1 subunit expression or increases NMDAR desensitizing rate by increasing NR2A expression in the brain. In agreement with our results, many authors have also observed a downregulation and/or reduced expression of NR1 subunit after different excitotoxic insults.30–32 Our data also showed an increase in GluR2 expression levels in the striatum and in frontal cortex of rats acutely treated with METH and in the striatum of rats treated with the escalating doses. As previously obtained in the hippocampus,1 in the rats treated with the escalating doses of METH there was a tendency for the GluR2 levels to increase in the frontal cortex. This


may reflect the tolerant effect associated with chronic abuse. As previously mentioned, AMPARs assembled from GluR2 subunits are not permeable to Ca2+ ions. On the other hand, the GluR2 subunit is important to the anchoring of the receptor. Therefore, we can speculate that a rise in this subunit density may reflect greater incorporation of Ca2+ -impermeable AMPARs into the cell membrane or the arresting of the receptor in the cytoplasm.33 In any case, our overall data suggest an early mechanism set to prevent METH-induced excessive Ca2+ entry through Glu ionotropic receptors. In agreement with our results, a previous study using repeated cocaine administration to rats reported a decreased NR1 mRNA in the striatum and increased GluR2 mRNA in the prefrontal cortex following a posterior acute injection of the drug.34 In a model of chronic nicotine self-administration in rats, Glu ionotropic receptors NR2A and NR2B subunits were found to be upregulated within the prefrontal cortex and the GluR2 subunit increased in the ventral tegmental area (VTA).21 However, the authors did not find a concomitant decrease of NR1 subunit expression. Moreover, chronic ethanol exposure was also shown to increase most of the NMDAR and AMPAR subunits in rat cortical neuronal cultures,35 which likely reflects a neuroadaptation to the inhibitory effect of ethanol on these receptors. Also, an acute exposure of cultured brain neurons to amphetamine (AMPH) was associated with a GluR2-mediated decreased in Ca2+ influx,36 and in vitro studies developed in our laboratory suggest that hippocampal neurons exposed to different concentration of METH for 24 h (range between 0.02–0.08 mM) were visibly less permeable to divalent cations upon NMDAR and AMPAR activation, as assessed by cobalt staining (data not shown).1 It has been suggested that METH causes loss of dopaminergic and serotonergic terminal markers and upregulation of pro-necrotic/ pro-apoptotic factors.5 Furthermore, many studies have reported neurodegeneration in

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several brain areas after different METH treatments from 24 to 72 h following treatment. Indeed a single 30 mg/kg METH dose, when administered to mice, induced apoptosis in dopamine projection neurons in the mice striatum, 24 h upon treatment.37 Cell death was also observed in the cortex, striatum, and hippocampus of mice 3 days after administration of 40 mg/kg i.p. METH.38 In addition, it was recently reported that an acute treatment with 40 mg/kg METH induced an upregulation of activated pro-necrotic calpain-1 and proapoptotic caspase-3 events, previous to neurodegeneration, in rat cortical neurons 48 h upon drug exposure.39 On the contrary, in the present work, neither the acute nor the escalating dose paradigms led to F-J C–positive cells in the rat striatum and frontal cortex within 24 h of METH administration (data not shown). These observations reproduce what we have reported for the hippocampus.1 The lack of neurodegeneration in our models is compatible with the onset of an early neuroprotective mechanism suggested by the altered Glu ionotropic receptor subunit expression levels. In addition, the METH regimens used in the present study were neither lethal nor caused severe hyperthermia and/or seizures to the animals, which may have contributed to the absence of neurodegeneration.40 However, several variables, such as animal species, drug doses and schedules, administration pathway, and ambient temperature must all be considered when evaluating METH effects as they greatly influence the results. Also, later time analysis upon exposure to an elevated dose of METH may be necessary in order to observe cell death, since the putative early neuroprotective mechanisms might have delayed neurodegeneration. In addition, we did not observe astrogliosis, even though there is a tendency to increased GFAP density, especially in the frontal cortex, as assessed by Western blot. But we earlier observed a GFAP increase at 24 h in hippocampus.1 Previous studies have shown evidences of reactive astroglia after METH exposure in the rodent striatum and cortex, but mostly at later times

after drug treatment. Increased GFAP density was observed in the rat striatum, 3 days post treatment with single 30 mg/kg or 40 mg/kg s.c. METH doses41 and 10 days after a single 40 mg/kg s.c. dose of METH.42 Moreover, GFAP immunoreactivity was augmented in the mice striatum, 48 and 72 h following a single 30 mg/kg i.p METH dose, but failed to change GFAP levels at 24 h.43,44 As with METH’s effects on NMDAR and AMPAR, we observed different METH effects on GFAP expression across brain regions. Conclusion The acute and binge-like METH administration used in the present work significantly altered Glu ionotropic receptor subunit expression levels in the striatum and frontal cortex of rats 24 h after treatment, and the alterations observed in each brain region were dependent on the METH paradigm. Our results suggest that NMDAR functionality is diminished in the brain of rats treated with high doses of METH and that this is not prevented by a subchronic administration previous to the bolus. In the same way, AMPAR GluR2 subunit levels were either significantly elevated or tending to increase, which suggests a greater production of Ca2+ -impermeable receptors. These data correlate with an early mechanism set by cells to counteract METH-induced Glu overflow and consequent excitotoxicity in the brain. Acknowledgments

This work was supported in part by GAI project 23/06 and by IPDT, Portugal. We thank Paula Canas, a Ph.D. student, for helping us with the immunohistochemistry studies. We also would like to kindly acknowledge Dr. Jo˜ao Malva for his important suggestions and discussions of the present work. Conflicts of Interest

The authors declare no conflicts of interest.

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