Methamphetamine-induced deficits of brain monoaminergic neuronal markers: distal axotomy or neuronal plasticity

Neuroscience 122 (2003) 499 –513

METHAMPHETAMINE-INDUCED DEFICITS OF BRAIN MONOAMINERGIC NEURONAL MARKERS: DISTAL AXOTOMY OR NEURONAL PLASTICITY T. R. GUILARTE,* M. K. NIHEI, J. L. McGLOTHAN AND A. S. HOWARD

markers, glial cells, peripheral benzodiazepine receptor, neurodegeneration.

Molecular Neurotoxicology Laboratory, Department of Environmental Health Sciences, 615 North Wolfe Street, Room W2001, The Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, USA

Methamphetamine (METH) is a drug of abuse with increasing popularity in the United States and other industrialized nations (Kozel, 1997). Besides its addictive properties, there is concern about METH neurotoxic potential since its use has been shown to produce protracted deficits in brain monoaminergic (i.e. dopamine and serotonin) axonal markers. Long-term deficits in the levels of dopamine (DA) and serotonin (5-HT; Ricaurte et al., 1980, 1982, 1984; Axt and Molliver, 1991; O’Dell et al., 1991; Fukumura et al., 1998), their synthesizing enzymes tyrosine hydroxylase (TH) and tryptophan hydroxylase (Ricaurte et al., 1982; Broening et al., 1997; Fukumura et al., 1998; Cappon et al., 2000), and dopamine transporters (DAT) and serotonin transporters (5-HTT) have been documented in experimental animals (Wagner et al., 1980; Brunswick et al., 1992; Eisch et al., 1992). Further, human studies have documented decreased levels of DAT in the brain of chronic METH abusers in vivo (McCann et al., 1998; Volkow et al., 2001) and in DA, TH, and DAT in postmortem tissue (Wilson et al., 1996a). It has been suggested that the loss of dopaminergic axonal markers following METH administration is the result of degeneration of neuronal terminals (Ricaurte et al., 1982, 1984; Broening et al., 1997; Fukumura et al., 1998), typically with sparing of nerve cell bodies (Ricaurte et al., 1982; Brunswick et al., 1992; but see Sonsalla et al., 1996; Hirata and Cadet, 1997). A limited number of studies provide evidence of silver-positive staining of nerve fibers in the striatum of METH-exposed rats (Ricaurte et al., 1982, 1984; Bowyer et al., 1994; but see Bowyer and Holson, 1995, 1996). Based on these findings along with studies demonstrating the loss of DA, TH and DAT following METH administration, it has been proposed that METH produces an irreversible distal axotomy of dopaminergic terminals. This hypothesis needs close scrutiny because if chronic METH abusers express an irreversible loss of dopaminergic terminals in the striatum, it could increase their risk for Parkinsonism with advancing age. On the other hand, the possibility has been raised in a number of reports that METH-induced deficits in dopaminergic axonal markers may occur independent of terminal degeneration (Lorez, 1981; Bowyer et al., 1994; Bowyer and Holson 1995; Bowyer et al., 1998; Wilson et al., 1996a; O’Callaghan and Miller, 2002). Further, relative to the marked deficits in brain dopaminergic neuronal mark-

Abstract—We examined the effects of methamphetamine (METH) on monoaminergic (i.e. dopamine and serotonin) axonal markers and glial cell activation in the rat brain. Our findings indicate that the loss of dopamine transporters (DAT), serotonin transporters (5-HTT), vesicular monoamine transporter type-2 (VMAT-2) and glial cell activation induced by METH in the striatum and in the central gray are consistent with a degenerative process. Our novel finding of METH effects on monoaminergic neurons in the central gray may have important implications on METH-induced hyperthermia. In other brain regions examined, DAT and 5-HTT deficits after METH administration were present in the absence of lasting changes in VMAT-2 levels or glial cell activation. Brain regions exhibiting protracted deficits in DAT and/or 5-HTT and VMAT-2 levels also expressed increased levels of [3H]-RPK11195 binding to peripheral benzodiazepine receptors, a quantitative marker of glial cell activation. Immunohistochemical assessment of microglia and astrocytes confirmed the PBR results. Microglia activation was more pronounced than astrocytosis in affected regions in most METH-exposed brains with the exception of a small number of rats that were most severely affected by METH based on loss of body weight. In these rats, both microglia and astrocytes were highly activated and expressed a distinct regional pattern suggestive of widespread brain injury. The reason for the pattern of glial cell activation in this group of rats is not currently known but it may be associated with METH-induced hyperthermia. In summary, our findings suggest two neurotoxic endpoints in the brain of METH-exposed animals. Brain regions exhibiting DAT and 5-HTT deficits that co-localize with decreased VMAT-2 levels and glial cell activation may represent monoaminergic terminal degeneration. However, the DAT and 5-HTT deficits in brain regions lacking a deficit in VMAT-2 and glial cell activation may reflect drug-induced modulation of these plasma membrane proteins. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: methamphetamine, rat brain, monoaminergic *Corresponding author. Tel: ⫹1-410-955-2485; fax: ⫹1-410-9556222. E-mail address: [email protected] (T. R. Guilarte). Abbreviations: ANOVA, analysis of variance; DA, dopamine; GFAP, glial fibrillary acidic protein; METH, methamphetamine; PB, phosphate buffer; PBR, peripheral benzodiazepine receptor; PKC, protein kinase C; TBST, Tris-buffered saline with Tween 20; TH, tyrosine hydroxylase; VMAT-2, vesicular monoamine transporter type-2; 5-HT, serotonin; 5-HTT, serotonin transporter.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00476-7

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ers induced by METH, levels of the vesicular monoamine transporter type 2 (VMAT-2) are either unaltered (Wilson et al., 1996a) or less severely affected (Frey et al., 1997; Villemagne et al., 1998). In addition, glial cell activation in METH-exposed rat brain is less robust and regionally restricted compared with the magnitude and global deficits in dopaminergic and serotonergic neuronal markers (O’Callaghan and Miller 1993; Broening et al., 1997; Fukumura et al., 1998; Cappon et al., 2000). Therefore, it is possible that alternative mechanisms, besides axonal degeneration, are operational in decreasing dopaminergic and serotonergic phenotypic markers in the METH-exposed brain. The present study examined the regional and temporal changes in brain DAT, 5-HTT and VMAT-2 levels following METH administration in conjunction with several markers of glial cell activation. Our findings indicate that METHinduced deficits in DAT and 5-HTT are not always associated with VMAT-2 deficits or the activation of microglia and astrocytes. These findings indicate that caution should be taken in making conclusions of axonal terminal degeneration based on the analysis of phenotypic proteins unless markers of degeneration are also used.

EXPERIMENTAL PROCEDURES Animal protocol and drug treatment Adult male Sprague-Dawley rats (225–250 g; Harlan, Indianapolis, IN, USA) were used in all experiments. Animals were individually housed in wire-bottom cages at 22 °C under a 12-h light/dark cycle. METH was dissolved in saline and administered according to the following schedule. Animals were given four i.p. injections of either METH (15 mg/kg as free base) or 0.9% saline vehicle at 2 h intervals. METH was generously supplied and administered to rats in Dr. George Ricaurte’s laboratory (Johns Hopkins Bayview Campus, Baltimore, MD, USA). Animals were killed at 3, 5, or 14 days after METH administration either by decapitation to obtain freshfrozen brain tissue or fixed by transcardiac perfusion for immunohistochemistry. Saline-injected control animals from all ages were combined as a single group. The majority of METH-exposed animals gained weight at the same rate as controls, with the exception of approximately 10% which had lower body weights at the time of killing. Approximately 10% of the animals injected with METH lost weight by the time of killing. All animal studies were reviewed and approved by the Johns Hopkins University Animal Care and Use Committee. In addition, all efforts were made to minimize the number of animals used and their suffering.

Quantitative receptor autoradiography Fresh frozen whole brains were sectioned at 20 ␮m on a freezing microtome (Hacker, Fairfield, NJ, USA) in the coronal plane, thawmounted on poly-L-lysine-coated slides (Sigma, St. Louis, MO, USA) and stored at ⫺20 °C. [3H]-Paroxetine binding was used to determine levels of 5-HTT in brain slices. Slides were warmed to room temperature and pre-washed in 50 mM Tris–HCl, 300 mM NaCl buffer (pH 7.4) for 15 min. Slides were then incubated at room temperature for 90 min in buffer with 1 nM [3H]-Paroxetine (specific activity: 25 Ci/ mmol; NEN Life Science Products, Boston, MA, USA) to determine total binding. Non-specific binding was determined in adjacent brain slices in the presence of 10 ␮M fluoxetine hydrochloride. The slides were then washed twice in buffer at 4 °C for 4 h each and dipped twice for approximately 5 min each in dH2O at

4 °C. Slides were dried under a stream of cool air and apposed to [3H]-Hyperfilm (Amersham, Piscataway, NJ, USA) for 5 weeks at room temperature. [3H]-WIN 35,428 binding was used to determine DA transporter levels in brain slices. Slides were warmed to room temperature and pre-washed for 20 min in 50 mM Tris–HCl, 120 mM NaCl buffer (pH 7.4) at 4 °C. The slides were then incubated at 4 °C for 90 min in buffer with 5 nM [3H]-WIN 35,428 (specific activity: 86 Ci/mmol; NEN Life Science Products) to determine total binding. Non-specific binding was determined in adjacent brain slices by including 10 ␮M GBR-12909 dihydrochloride. The slides were then washed twice for a minute each in 4 °C buffer and then dipped in dH2O at 4 °C. Slides were dried completely under a stream of cool air and apposed to [3H]-Hyperfilm (Amersham) for 4 – 6 weeks at room temperature. [3H]-Dihydrotetrabenazine ([3H]-DTBZ) binding was used to determine levels of VMAT-2 in brain slices. Slides were prewashed in 20 mM HEPES, 300 mM sucrose buffer (pH 8.0) for 15 min at room temperature. Slides were incubated at room temperature for 60 min in buffer with 7.5 nM [3H]-DTBZ (specific activity: 158 Ci/mmol; Amersham; and provided by Dr. George Ricaurte, Johns Hopkins University) to determine total binding. Non-specific binding was determined in adjacent brain slices with 2 ␮M nonradioactive DTBZ. Slides were then rinsed three times at room temperature in 40 mM HEPES, 32 mM sucrose buffer (pH 8.0) for 5 min each. They were rinsed in dH2O at 4 °C and dried under a stream of cool air. Slides were apposed to [3H]-Hyperfilm (Amersham) for 7 days at room temperature. [3H]-R-PK11195 binding was used to determine peripheral benzodiazepine receptor (PBR) levels in brain slices. Slides were dried for 30 min at 37 °C and then pre-washed for 5 min at room temperature in 50 mM Tris–HCl buffer (pH 7.4). The slides were incubated at room temperature for 30 min in buffer containing 1 nM [3H]-R-PK11195 (specific activity: 85.5 Ci/mmol; custom synthesized by NEN Life Science Products) to determine total binding. Non-specific binding was determined in adjacent brain slices in the presence of 10 ␮M R,S-PK11195. The slides were washed twice for 3 min each in 4 °C buffer and dipped twice in dH2O at 4 °C. Slides were dried under a stream of cool air and apposed to [3H]-Hyperfilm (Amersham) for 12 days at room temperature. In all receptor autoradiography studies, reference standards (tritium microscales, Amersham), were included with each film to ensure linearity of optical density and to allow quantitative analysis of the images. Images from autoradiograms were captured using an image analysis system (Loats Associates, Inc., Westminster, MD, USA) and densitometric analysis was performed using NIH Image.

Immunohistochemistry Rats were anesthetized with urethane (1.5 g/kg, i.p.) and transcardially perfused with 500 mL of cold (4 °C) 0.1 M phosphate buffer (PB, pH 7.4) with 1% lidocaine, followed by 500 mL of cold (4 °C) 4% paraformaldehyde with 0.01% glutaraldehyde in 0.1 M PB. Whole brains were post-fixed in 4% paraformaldehyde for 90 min at room temperature and then cryoprotected for at least 3 days in 20% sucrose in 0.1 M PB. Coronal sections were cut at a thickness of 30 ␮m on a freezing microtome and stored free-floating in 0.1 M PB at 4 °C before use. All brain sections were processed free-floating and were initially rinsed in fresh 0.1 M PB. Adjacent sections from each brain were processed for immunohistochemistry using an anti-rabbit polyclonal antibody directed against glial fibrillary acidic protein (GFAP; Vector Laboratories, Burlingame, CA, USA), or using the mouse anti-rat Integrin ␣M␤2 monoclonal antibody to assess microglia [Mac-1, CD11b/CD18] (Chemicon, Temecula, CA, USA). Selected sections were incubated in 3% hydrogen peroxide and 1% Triton X-100 in 0.1 M PB for 20 min, then rinsed thoroughly in 0.1 M PB 2⫻5 min and 2⫻10 min.

T. R. Guilarte et al. / Neuroscience 122 (2003) 499 –513 Sections intended for use with GFAP were blocked using 4% goat sera in 50% avidin solution (Blocking kit; Vector Laboratories) and 0.1% Triton X-100 for 45 min. After 2⫻5 min and 2⫻10 min rinses in 0.1 M PB, sections were incubated in the primary antibody solution (primary antibody in 50% biotin with 0.1% Triton X-100 in 0.1 M PB). The GFAP primary antibody was diluted 1:10,000 and incubated overnight at 4 °C. Control experiments included omission of primary antibody or incubation with rabbit IgG. Sections were rinsed 2⫻5 min and 2⫻10 min in 0.1 M PB before incubation in secondary antibody solution (1:5000 biotinylated goat-anti-rabbit antisera in 0.1 M PB with 0.1% Triton X-100) for 60 min at room temperature. After rinsing 2⫻5 min and 2⫻10 min in 0.1 M PB, sections were incubated in avidin-biotin complex (ABC Elite kit; Vector Laboratories), rinsed for 2⫻5 min and 2⫻10 min in 0.1 M PB. Immunoreactivity was visualized by incubating sections in 0.05% diaminobenzidine and 0.01% hydrogen peroxide in distilled, deionized water for approximately 6 min, and rinsing in distilled, deionized water. Sections were mounted onto poly-Llysine coated microscope slides and coverslipped with a nonaqueous mounting fixative (DPX; Electron Microscopy Services, Ft. Washington, PA, USA) following progressive dehydration in a series of ethyl alcohol rinses (70%, 90%, 95%, 100%) and xylene. Sections intended for processing with the monoclonal antibody (Mac-1) were handled in the same manner with the exception of the following steps. Before incubation in the primary antibody solution sections were incubated in 2% horse sera (Vector Laboratories) 50% avidin solution (Blocking kit; Vector Laboratories) and 0.1% Triton X-100 in 0.1 M PB for 45 min. The primary antibody solution contained monoclonal antibody at 1:1000, 1.5% horse sera and 0.1% Triton X-100 in 0.1 M PB. Sections were incubated overnight in primary antibody solution at 4 °C. Biotinylated anti-mouse IgG (H⫹L), rat adsorbed, affinity purified (Vector Laboratories) secondary antibody was used at 1:5000, in 0.1 M PB with 0.1% Triton X-100 and incubated for 60 min at room temperature. The remaining steps followed the polyclonal procedure.

GFAP Western immunoblots Corpus striatum was homogenized in 50 mM Tris–HCl (pH 7.5), 0.25 M sucrose, 2 mM EDTA, 0.5 mM EGTA (Sigma, St. Louis, MO, USA), with protease inhibitors (Complete, Mini; Roche, Indianapolis, IN, USA). Protein analysis was performed by the method of Bradford. After dissolution in SDS-sample buffer (0.5 M Tris– HCl, 10% (w/w) SDS, 1% glycerol, 0.5% ␤-mercaptoethanol and 0.05% bromophenol blue) and heating to 100 °C for 3 min, 20 ␮g of total protein was added to each lane and resolved by 7.5%, 0.75 mm SDS-PAGE. Blots were Ponceau stained for 15 min and imaged using NIH image. Blots with equal protein loading were used for further analysis. Following electrophoretic transfer to nitrocellulose membrane, blots were rinsed with distilled deionized water and Tris-buffered saline with Tween 20 (0.1%; TBST) and incubated for 1 h in 5% bovine serum albumin in TBST. Following two washes with TBST, blots were incubated overnight in primary antibodies against GFAP (1:2000 dilution). Presence of the primary antibody was detected using chemiluminescence and ECLHyperfilm (Amersham). The optical density of each band was quantified after correction for film background. A protein gradient was run in order to determine that the amount of protein used was within the linear range of the film signal. Within a film, optical density values for control animals were averaged and designated as 100%. Optical densities of bands for METH treatment groups were calculated as the percent change from control.

Statistical analysis One-way analysis of variance (ANOVA) was used for statistical analysis. If ANOVA statistics reached significance at the P⬍0.05

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level, then Scheffe’s post hoc test for multiple means comparisons was used.

RESULTS Effects of METH administration on monoaminergic neuronal proteins Binding of [3H]-paroxetine to 5-HTT. 5-HTT levels were measured using [3H]-paroxetine quantitative autoradiography at 3, 5, and 14 days after METH administration. Due to the widespread distribution of 5-HTT in the CNS, three different levels of the brain were sampled. Brain sections at the level of the striatum, hippocampus and dorsal raphe were selected to examine 5-HTT expression on serotonergic terminal fields and cell bodies. Fig. 1 depicts representative brain levels of [3H]-paroxetine binding to 5-HTT in brain slices from control and METH-exposed rats 3 days after exposure. Table 1 provides a quantitative assessment of regional [3H]-paroxetine binding levels in control and METH-exposed rats. Following METH administration, [3H]-paroxetine binding was dramatically decreased (in the range of 65–92%) in brain structures receiving serotonergic innervation (i.e. terminal fields) while regions containing cell bodies (i.e. dorsal and medial raphe) did not appear to be affected. Robust decreases in [3H]-paroxetine binding were measured at 3, 5 and 14 days after METH exposure. A small but significant increase in [3H]-paroxetine binding was measured at 3 days after METH in the medial raphe (Table 1). In some brain areas, such as in layers I and IV of the somatosensory cortex, [3H]-paroxetine binding was not different from control values at 5 or 14 days after exposure (Table 1). Binding of [3H]-WIN 35,428 to DA transporters. Levels of DAT were measured using [3H]-WIN 35,428 autoradiography. We performed [3H]-WIN 35,428 autoradiography at the level of the striatum, hippocampus and dorsal raphe using adjacent sections to those used for 5-HTT and VMAT-2 autoradiography. However, specific binding of [3H]-WIN 35,428 to DAT at the level of the hippocampus and dorsal raphe was too low to reliably measure. Therefore, only corpus striatum values are provided. We measured [3H]-WIN 35,428 binding in the anterior and posterior aspects of the striatum and the striatum was subdivided into nucleus accumbens, dorsal and lateral aspects. In general, there were significant reductions in [3H]-WIN 35,428 binding in the nucleus accumbens and dorsal and ventral striatum at 3 days after METH treatment (Table 2). No significant differences from control values were present in the posterior nucleus accumbens or striatum at 5 days after METH exposure. However, [3H]-WIN 35,428 binding decreased at 14 days such that binding in all striatal regions examined was significantly reduced at this time (Table 2). Further, it appears that DAT levels in the ventral aspects of the anterior striatum are more affected than the dorsal striatum (see Table 2). Binding of [3H]-dihydrotetrabenazine to VMAT2. VMAT-2 levels were measured in adjacent brain slices from the same animals used for [3H]-paroxetine and [3H]-

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Fig. 1. Quantitative autoradiography of [3H]-paroxetine binding to 5-HTT at 3 days after METH administration. Autoradiograms from control (A, C, E) and METH (B, D, F) animals at the level of the striatum/nucleus accumbens (A, B), dorsal hippocampus (C, D) and dorsal raphe (E, F). Pseudocolors represent different levels of binding with white–red being the highest level of binding, yellow– green representing intermediate levels, and blue depicting low levels of binding.

Abbreviations used in the figures and tables ACg Aq BLA C CG Ctx DHA DR Ent. Ctx FrPam FrpaSS Hipp Inf. Coll. LH LSN LV

anterior cingulate cortex cerebral aqueduct basal lateral amygdaloid nucleus control central gray cerebral cortex dorsal hypothalamic area dorsal raphe entorhinal cortex fronto-parietal motor area fronto-parietal somatosensory cortex hippocampus inferior colliculus lateral hypothalamus lateral septal nucleus lateral ventricle

MR MT NAc NAcC NAcS PV Str Str. Ctx Sup. Coll. Thal 3D 3V 5D 14D

medial raphe medial thalamus nucleus accumbens nucleus accumbens core nucleus accumbens shell paraventricular thalamic nucleus striatum striate cortex superior colliculus thalamus 3 days after METH exposure third ventricle 5 days after METH exposure 14 days after METH exposure

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Table 1. Effects of METH administration on [3H]-paroxetine binding to 5-HT transporters in the rat brain Brain region

Control

METH (15 mg/kg) 3 Days

Level of striatum/nucleus accumbens Striatum, anterior 66.8⫾7.3 Striatum, posterior 73.5⫾8.0 NAcC, anterior 116.8⫾16.0 NAcC, posterior 130.3⫾13.3 NAcS, anterior 212.5⫾20.5 NAcS, posterior 210.3⫾20.3 ACg 68.5⫾5.2 FrPam (I) 127.3⫾12.6 FrPam (IV) 81.5⫾5.0 FrPaSS (I) 119.0⫾7.8 FrPaSS (IV) 125.7⫾6.5 LSN 266.3⫾20.4 MFB 375.3⫾26.5 Level of hippocampus FrPam 66.9⫾4.9 FrPaSS 78.6⫾3.2 Hipp, CA3 170.0⫾20.0 DLT 184.0⫾15.0 PV 284.5⫾22.6 MT 263.0⫾25.2 DLH 290.0⫾13.1 BLA 334.4⫾14.7 LH 314.7⫾25.4 Level of dorsal raphe Str Ctx 70.6⫾5.7 Ent. Ctx 130.0⫾12.3 Sup. Coll. 324.0⫾18.1 CG 312.6⫾10.8 Raphe, dorsal 531.3⫾21.1 Raphe, medial 427.8⫾19.2

5 Days

14 Days

16.2⫾6.1* 25.8⫾3.9* 16.4⫾7.2* 30.2⫾5.1* 27.8⫾6.7* 37.6⫾8.4* 5.0⫾2.7* 21.8⫾2.7* 22.6⫾2.3* 19.4⫾3.8* 24.8⫾2.7* 90.0⫾14.6* 38.4⫾8.7*

41.8⫾10.0 38.2⫾8.4* 55.8⫾16.4* 63.2⫾17.0* 111.8⫾35.5* 89.4⫾19.2* 32.2⫾7.6* 64.0⫾15.3* 48.0⫾9.2 65.2⫾15.8 94.8⫾24.3 178.2⫾25.5 266.0⫾90.0

15.5⫾8.2* 18.8⫾6.0* 18.0⫾12.5* 37.8⫾13.6* 41.8⫾23.7* 76.5⫾25.4* 22.8⫾4.0* 63.3⫾24.5 46.5⫾16.0 76.5⫾31.8 72.0⫾27.0 129.0⫾36.6* 172.0⫾64.3

12.3⫾6.2* 15.5⫾4.6* 23.1⫾6.8* 17.3⫾5.3* 74.6⫾14.9* 38.5⫾5.4* 25.9⫾7.2* 37.1⫾5.8* 326.1⫾30.7

25.9⫾11.4* 32.5⫾11.8* 60.0⫾24.0* 49.1⫾22.1* 130.0⫾48.7* 77.4⫾32.6* 104.4⫾36.1* 171.7⫾54.2* 278.0⫾27.3

12.0⫾2.7* 8.7⫾3.6* 34.1⫾9.7* 10.9⫾4.5* 70.0⫾16.8* 33.3⫾11.3* 44.3⫾18.7* 125.4⫾58.0* 212.8⫾11.4*

12.3⫾2.3* 10.7⫾5.4* 14.4⫾4.4* 33.0⫾5.6* 510.0⫾9.1 [494.8⫾5.3]*

29.5⫾9.2* 29.3⫾11.4* 142.3⫾47.2* 156.0⫾31.8* 499.0⫾20.4 386.5⫾26.8

21.9⫾4.9* 22.4⫾5.8* 45.7⫾16.3* 100.4⫾32.3* 484.2⫾37.9 330.4⫾32.6

(I) (IV) ⫽ Cortical layers. [ ]. ⫽ Significantly increased from control. Values are in fmol/mg tissue. Each value is the mean⫾S.E.M. of four to six rats. * P⬍0.05 relative to control.

WIN 35,428 autoradiography. Fig. 2 shows representative [3H]-DTBZ autoradiograms at the level of the striatum/ nucleus accumbens at 3, 5, and 14 days after METH exposure. Table 3 provides the analysis of VMAT-2 levels in control and METH-exposed rats. The data show that VMAT-2 levels are significantly decreased in some but not all brain areas exhibiting deficits in [3H]-paroxetine or [3H]-

WIN 35,428 binding to 5-HTT and DAT, respectively. Levels of [3H]-paroxetine and [3H]-WIN 35,428 binding were markedly decreased in virtually all brain regions examined at 3 days after METH exposure. On the other hand, [3H]DTBZ binding levels were decreased only in the striatum, nucleus accumbens, CA3 region of the hippocampus, basolateral amygdaloid nucleus and dorsal central gray at

Table 2. Effects of METH administration on [3H]-WIN 35,428 binding to DA transporters in the rat brain Brain region

Control

Level of striatum/nucleus accumbens - anterior NAc 19.8⫾0.9 Striatum, dorsal 34.8⫾1.8 Striatum, ventral 29.5⫾1.8 Level of striatum/nucleus accumbens - posterior NAc 15.0⫾0.9 Striatum, dorsal 26.0⫾3.6 Striatum, ventral 23.0⫾3.4

METH (15 mg/kg) 3 Days

5 Days

14 Days

6.8⫾0.9* 7.4⫾1.3* 5.4⫾1.0*

14.5⫾1.8 21.3⫾3.4* 10.8⫾2.5*

8.5⫾1.3* 16.3⫾4.4* 8.5⫾2.2*

6.2⫾1.0* 9.2⫾1.6* 7.0⫾2.1*

12.4⫾1.1 20.0⫾2.8 12.4⫾2.8

7.3⫾1.3* 9.3⫾2.3* 7.0⫾1.9*

Values are in fmol/mg tissue. Each value is the mean⫾S.E.M. of four to five different rats. * P⬍0.05 relative to control.

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Fig. 2. Quantitative autoradiography of [3H]-dihydrotetrabenazine binding to VMAT-2. Autoradiograms are representative of VMAT-2 distribution at the level of the striatum/nucleus accumbens in control (A), 3 days (B), 5 days (C), and 14 days (D) after METH exposure. Pseudocolors represent different levels of binding with red being the highest level of binding, yellow– green representing intermediate levels, and blue depicting low levels of binding.

this time point. At 5 days after METH exposure, the striatum was the only brain region expressing decreased levels of binding (Table 3). By 14 days after METH exposure, the striatum and central gray exhibited the greatest decrements in [3H]-DTBZ binding relative to controls. It should be noted that other brain structures such as the nucleus accumbens, frontoparietal motor cortex, CA3 region of the hippocampus and the entorhinal cortex also exhibited reduced levels of [3H]-DTBZ binding in the order of 20 –27% at 14 days. However, the differences did not reach statistical significance (see Table 3). Glial responses following METH administration PBR Levels in METH-exposed rat brain. PBR levels have been used as a quantitative index of glial cell activation following chemical-induced brain injury (Guilarte et al., 1995; Kuhlmann and Guilarte, 1999, 2000). Brain levels of PBR, as assessed by the PBR specific ligand [3H]-RPK11195, are present at very low concentrations in the brain neuropil. Following brain insult, high levels of [3H]-RPK11195 binding are expressed in brain areas exhibiting degeneration as a result of increased PBR expression in microglia and astrocytes (Kuhlmann and Guilarte, 2000). [3H]-R-PK11195 binding to PBR in the brain of METHexposed rats showed two distinct patterns, possibly representative of different degrees of brain insult. In the majority of METH-exposed brains, increased levels of [3H]-RPK11195 binding were transient and only occurred in the

striatum and central gray (Table 4). [3H]-R-PK11195 levels were significantly increased at 3 days (central gray) or 5 days (striatum) but not at 14 days after METH administration. In the most severely affected METH-exposed rats, based on body weight loss, a different pattern of [3H]-RPK11195 binding was observed. In these animals, increased levels of [3H]-R-PK11195 binding were also present in thalamic areas, hippocampus and in the dorsal raphe/central gray region (Fig. 3). In general, the regional pattern of increased [3H]-R-PK11195 binding correlates with the distinct pattern of microglia activation and astrocytosis observed in these severely affected animals (see below). Mac-1 immunohistochemistry. We visualized the state of microglia activation using Mac-1 immunohistochemistry. This antibody is known to recognize “resting” as well as “activated” microglia in the CNS. Microglia in the “resting state” express a highly branched and ramified morphology throughout the brain neuropil (Fig. 4A). The majority of control brains examined exhibited this type of microglia morphology observed. However, occasional cells expressing slight activation represented by thickening of branches and darker staining were also present (Fig. 4A). In the majority of brains from METH-exposed rats, there was significant microglia activation in the striatum and central gray areas at 3 and 5 days after exposure (Fig. 4B). In some animals, microglia activation was also present in

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Table 3. Effects of METH administration on [3H]-dihydrotetrabenazine binding to VMAT-2 in the rat brain VMAT-Z Brain region

METH (15 mg/kg) Control

Level of striatum/nucleus accumbens Striatum, anterior 315.8⫾20.5 Striatum, posterior 324.5⫾16.9 NAcC, anterior 263.8⫾10.8 NAcC, posterior 287.3⫾8.5 NAcS, anterior 303.3⫾11.8 NAcS, posterior 277.3⫾15.7 ACg 40.0⫾3.5 FrPam 34.8⫾0.5 FrPaSS 29.5⫾1.9 Level of hippocampus FrPaM 30.5⫾2.2 FrPaSS 27.5⫾0.6 Hipp, CA3 36.0⫾1.2 PV 166.5⫾4.6 DHA 147.5⫾8.7 BLA 88.8⫾8.1 Level of dorsal raphe Str Ctx 27.5⫾0.9 Ent. Ctx 41.3⫾2.1 Sup. Coll. 50.5⫾6.9 CG 121.0⫾5.4 CG, dorsal 62.5⫾1.2 Raphe, dorsal 246.8⫾5.4 Raphe, medial 100.0⫾16.9

3 Days

5 Days

14 Days

247.0⫾10.2 249.0⫾8.0* 201.8⫾5.8* 195.0⫾7.2* 276.0⫾5.8 267.0⫾3.7 32.2⫾1.9 31.4⫾1.6 27.2⫾1.9

256.8⫾19.6 255.6⫾17.6* 244.0⫾19.1 252.3⫾29.7 284.0⫾13.3 289.8⫾5.1 45.6⫾5.1 41.0⫾3.9 37.6⫾3.3

227.0⫾18.3* 215.8⫾10.8* 214.8⫾15.7 223.8⫾24.5 280.8⫾17.1 262.5⫾15.6 37.5⫾3.4 35.8⫾3.5 34.0⫾5.1

22.8⫾0.7 21.8⫾1.0 26.8⫾1.6* 143.0⫾14.5 122.8⫾3.8 52.0⫾3.7*

26.8⫾2.3 25.0⫾2.4 30.0⫾2.0 168.6⫾9.7 125.8⫾6.4 71.2⫾8.0

23.7⫾3.5 25.3⫾3.2 27.3⫾2.2 157.7⫾6.4 122.7⫾3.2 76.7⫾13.2

25.8⫾2.7 32.0⫾2.6 35.0⫾2.6 98.6⫾5.3 41.6⫾3.6* 198.4⫾18.2 101.0⫾12.9

28.2⫾1.4 33.2⫾3.3 51.4⫾6.1 96.8⫾6.9 48.4⫾6.3 196.6⫾6.7 95.6⫾12.6

25.7⫾0.9 30.3⫾1.8 35.3⫾1.3 86.3⫾9.0* 40.8⫾2.1* 209.8⫾16.6 83.3⫾5.1

Values are in fmol/mg tissue. Each value is the mean⫾S.E.M. of four to six different rats. * P⬍0.05 relative to control.

thalamic areas. “Activated” microglia are characterized by thickening and retraction of branches and darker-stained cell bodies (Fig. 4B). At 14 days after METH exposure, microglia in most brain areas appeared to have reverted to a “resting state” with the possible exception of the striatum.

As indicated in the previous section, the most severely affected METH-exposed rats (based on body weight loss) expressed a different pattern and degree of microglia activation. Microglia in these animals displayed a morphological profile representative of a highly activated and phago-

Table 4. Effects of METH administration on [3H]-R-PK11195 binding to PBR in the rat brain Brain region

Level of striatum/nucleus accumbens Striatum NAc Level of hippocampus Hipp Hipp, dentate Hipp, CA3 PV MT DLH Level of dorsal raphe Str Ctx Ent. Ctx Inf. Coll. Dorsal and central grey Raphe, dorsal

Control

METH (15 mg/kg) 3 Days

5 Days

14 Days

20.4⫾2.2 22.4⫾2.6

22.5⫾2.5 22.4⫾1.9

30.9⫾1.8* 27.8⫾2.1

25.2⫾0.5 23.6⫾2.5

28.3⫾1.7 32.5⫾1.1 38.6⫾3.8 39.4⫾2.2 23.3⫾1.6 44.0⫾1.2

34.6⫾5.6 44.7⫾7.3 35.7⫾1.6 41.7⫾2.1 23.6⫾1.6 48.0⫾5.2

28.0⫾3.6 40.9⫾2.1 43.8⫾5.9 46.9⫾4.3 27.6⫾3.0 54.9⫾4.3

25.9⫾6.0 34.5⫾4.5 37.2⫾6.2 40.5⫾2.2 23.1⫾2.5 47.4⫾3.0

22.7⫾1.1 24.1⫾1.3 28.5⫾2.7 32.8⫾1.2 39.7⫾1.2

25.4⫾2.8 30.3⫾3.0 30.2⫾2.4 49.0⫾3.9* 48.8⫾4.4

28.0⫾1.7 30.9⫾2.4 33.3⫾2.0 43.2⫾3.7 45.2⫾3.5

21.8⫾4.7 20.9⫾4.8 25.2⫾4.1 36.2⫾7.1 41.3⫾8.5

Values are in fmol/mg tissue. Each value is the mean⫾S.E.M. of four to five different rats. * P⬍0.05 relative to control.

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Fig. 3. Quantitative autoradiography of [3H]-R-PK11195 binding to PBR in the rat brain. Autoradiograms from a control (A, C, E) and most severely affected METH-exposed (B, D, F) rat represent the distribution of PBR at the level of the striatum (A, B), dorsal hippocampus (C, D), and dorsal raphe (E, F). Pseudocolors are representative of receptor levels with red being the highest level of binding, yellow– green representing intermediate levels, and blue depicting low levels of binding. PBR are normally found in high concentrations in the lining of the LV and 3V, choroid plexus and Aq (see panels A, C, E). Increased levels of binding can be observed in the striatum, hippocampus, thalamus and dorsal raphe/central gray.

cytic state. Microglia were darkly stained, with thickening and shortening of branches and in many cases had complete retraction of branches expressing a round morphology (Fig. 4B). Further, there was an increase in the number of microglia and they were arranged in large clusters (Fig. 5). These highly activated microglia were prominent in the striatum, thalamus, dorsal raphe/central gray area, hippocampus, and inferior colliculus (Fig. 5). The pattern of highly activated microglia in this small number of METHexposed rats appeared to be similar to the pattern of increased [3H]-R-PK11195 binding measured in the same brain regions (Fig. 3). Importantly, even in these severely affected METH-exposed rats, microglia were not activated in the substantia nigra indicating that dopaminergic cell bodies are not affected by METH (data not shown).

GFAP immunohistochemistry and Western blot as an index of astrocyte activation. GFAP immunohistochemistry has been extensively used to assess activation of astrocytes in injured brain tissue (Norton et al., 1992; O’Callaghan and Miller, 1993; O’Callaghan et al., 1995). We used GFAP immunohistochemistry and GFAP Western blot to assess the degree of astrocyte activation in METH-exposed rats. In general, GFAP immunohistochemistry was unremarkable in most brain regions examined with the exception of the striatum where increased GFAP staining was present in the ventral aspects of the striatum. In this brain region, astrocytes have thicker processes and darker-stained cell bodies and branches (Fig. 4D). This was different from the highly ramified, thin and faint GFAP staining observed in astrocytes from control brains (Fig.

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Fig. 4. Immunohistochemistry of microglia and astrocytes. Top panels are representative of Mac-1 immunostaining of microglia in the “resting” state from a control rat (A) and “activated” state (B) from the striatum of a METH-exposed rat. Resting microglia express small cell bodies with highly ramified processes. Microglia activation is represented by retraction and thickening of processes and darker staining of cell bodies. Highly activated microglia have a complete loss of processes and express a round morphology (see arrows in panel B). Bottom panels are representative of GFAP immunostaining of astrocytes in control (C) and METH (D) striatum. Activated astrocytes express darker staining and thickening of processes relative those expressed in control animals. Bar⫽50 ␮m.

4C). GFAP Western blots of striatal tissue showed an apparent increase as percent of controls in GFAP levels in METH-exposed rats at 3 (53.2%), 5 (46.6%) and 14 (26.3%) days after exposure (see Fig. 6). However, statistical analysis did not show a significant treatment effect (ANOVA; F3,16⫽2.14; P⫽0.136). The lack of statistical significance may be related to the fact that increased activation of astrocytes in the striatum of METH rats observed by immunohistochemistry was regionally selective and Western immunoblots used the entire striatum including non-affected regions, thus “diluting” the overall effect. Rats most severely affected by METH, based on body weight loss, also expressed a high degree of GFAP staining in brain regions previously noted to have highly activated microglia and increased levels of [3H]-R-PK11195 binding to PBR. These brain regions included the striatum, thalamus, hippocampus, dorsal raphe/central gray and inferior colliculus (data not shown).

DISCUSSION The major findings of this study are: 1) a METH exposure protocol that produces global and lasting deficits in the levels of the plasma membrane transporters DAT and 5-HTT in the rat brain, fails to produce similar global reductions in VMAT-2 levels or marked activation of micro-

glia and astrocytes. The striatum and central gray are brain regions, among those examined, exhibiting significant deficits in DAT and/or 5-HTT and also in VMAT-2 levels and glial cell activation. However, deficits in VMAT-2 levels in these brain regions were less severe in magnitude than those measured for DAT and 5-HTT. Taken together, these data indicate that the striatum and central gray are brain regions in which some degree of terminal degeneration may be present. 2) To our knowledge this is the first report indicating monoaminergic terminal damage in the central gray (also known as the periaqueductal gray). The central gray is important in temperature regulation (Widdowson et al., 1983; Tanaka et al., 2002), aggressive/defensive behavior (Bandler, 1982; Lovick, 1994), pain modulation (Deakin and Dostrovsky, 1978) and behavioral sensitization to drugs of abuse (Miczek et al., 1999). It receives innervation from the periventricular dopaminergic system (Lindblom et al., 2002), serotonergic input from the raphe nucleus (Lovick, 1994) and expresses neurons containing opioid peptides (Finley et al., 1981; Widdowson et al., 1983) with high expression of opiate receptors (Law et al., 1979). Immunocytochemical studies provide evidence of a connection between monoaminergic and opioid peptides in the central gray (Khachaturian and Watson, 1982). Injection of ␤-en-

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Fig. 5. Immunohistochemistry of microglia using Mac-1 antibody in the most severely affected METH-exposed rats. Control (A, C, E, G) and METH (B, D, F, H) panels are at the level of striatum (A, B), dorsal raphe/central gray (C, D), inferior colliculus (E, F), and thalamus (G, H). Note the highly activated state of microglia in this subset of METH-exposed rats expressing darker staining, round morphology and clustering of microglia. Lower left box in panels (A) and (B) are sampled from the ventral aspects of the striatum. See greater staining in activated microglia of panel (B). Bar⫽500 ␮m.

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Fig. 6. Representative Western immunoblots of GFAP in corpus striatum (top panel). Graph represents quantitative results of GFAP levels in METH-exposed rats relative to controls. Each value is the mean⫾S.E.M. of five different animals. One-way ANOVA for treatment effects did not reach statistical significance (F3,16⫽2.14; P⫽0.136).

dorphin, an endogenous opioid, into the central gray produces hyperthermia (Widdowson et al., 1983). Importantly, the time course and the degree of hyperthermia induced by injection of opioids into the central gray is similar to those produced by METH administration (compare Fukumura et al., 1998 and Widdowson et al., 1983). If opioid peptides are co-localized within monoaminergic neurons, or their secretion modulated by monoaminergic innervation, then the massive release of DA induced by METH may also promote the release of opioid peptides endogenous to the central gray. Consistent with this hypothesis, previous studies have shown that activation of DA receptors increases the basal secretion of ␤-endorphin in the brain (Nohtomi et al., 1989) and in other organ systems (Stratakis et al., 1996). Thus, it is possible that the hyperthemia induced by METH administration may be the result of DA-mediated release of opioid peptides in the central gray. Future studies should directly investigate this intriguing possibility. 3) We have performed extensive regional and temporal assessment of METH-induced changes in 5-HTT and VMAT-2 levels beyond the corpus striatum, the brain region most commonly studied in the majority of previous studies on METH neurotoxicity. Our findings clearly indicate a disconnect between METH-induced changes in DAT/5-HTT and VMAT-2 levels suggesting that different neurotoxic mechanism may be operational in altering plasma membrane and vesicular transporters. Further, it appears that temporally, there is a transient increase in the levels of DAT and 5-HTT in some brain regions at 5 days after METH administration (see Tables 1 and 2). This transient increase in plasma membrane transporter levels at 5 days may be an attempt to compensate for the nearly complete loss of these transporters measured at 3 days after exposure. 4) Lastly, assessment of microglia and astrocyte activation was performed using immunohistochemical methods and PBR autoradiography as indirect indices of METH-induced neurodegeneration. To our knowledge, this is the first report of immunohistochemical assessment of

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microglia activation in METH-exposed animals. Our findings indicate that microglia activation followed a regional pattern similar to METH-induced deficits in VMAT-2 levels. On the other hand, brain regions that exhibited lasting deficits in DAT and 5-HTT but not in VMAT-2 levels did not express increased levels of PBR or activation of microglia and astrocytes. These findings raised the question whether METH-induced deficits in DAT and/or 5-HTT in the rat brain reflect a distal axotomy of monoaminergic terminals, long-term modulation of phenotypic axonal proteins or a combination of both. The protracted loss of dopaminergic and/or serotonergic terminal markers induced by METH can persist for long periods of time in rodents (Friedman et al., 1998; Cass and Manning, 1999; Cass, 2000) non-human primates (Melega et al., 1997; Harvey et al., 2000) and in humans (McCann et al., 1998; Volkow et al., 2001). However, full or partial recovery has been observed after several months following cessation of METH exposure calling into question whether there is “frank” degeneration of neuronal terminals (Friedman et al., 1998; Cass and Manning, 1999; Cass, 2000; Melega et al., 1997; Harvey et al., 2000; Volkow et al., 2001). It is possible that the recovery of dopaminergic neuronal markers in the striatum after cessation of METHexposure may result from “sprouting” of dopaminergic terminals. However, Bowyer et al. (1998) have shown that levels of TH mRNA and protein are not changed in the substantia nigra of rats exposed to the METH metabolite amphetamine despite recovery of striatal DA levels at 4 months following exposure. The notion that METH produces an irreversible distal axotomy of dopaminergic terminals arises from a limited number of studies using silver staining as an index of terminal degeneration in the striatum of exposed animals (Ricaurte et al., 1982, 1984; see O’Callaghan and Miller, 2002). Since DA, TH and DAT are also decreased by METH in the striatum, it has been assumed that the silver staining is associated with degeneration of dopaminergic terminals. An electron microscopy study by Ryan et al. (1990), using amphetamine-exposed animals showed that while some of the ultrastructural changes detected by silver staining in the striatum included swollen and abnormal processes that labeled for TH, other degenerating profiles resembled darkened glial processes and myelinated fibers which are not dopaminergic. Thus, silver positive staining in the amphetamine-exposed rat brain is not always associated with dopaminergic terminals. Nevertheless, these findings along with studies demonstrating slight gliosis in the striatum of METH-exposed animals (O’Callaghan and Miller, 1993; Broening et al., 1997; Fukumura et al., 1998; Fumagalli et al., 1998) suggests that some of the METHinduced loss of dopaminergic neuronal proteins may be associated with terminal degeneration. The present study also supports the notion that a degenerative process may be active in selective regions of the corpus striatum and in the central gray. Accumulating experimental evidence also supports alternative mechanism(s) that may account for some of the METH-induced deficits in DAT and 5-HTT. 1) Clinically,

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despite a history of chronic METH abuse in humans, there is no evidence of METH-induced Parkinsonism (Wilson et al., 1996a). It would seem likely that if chronic METH abuse promotes an irreversible distal axotomy of dopaminergic terminals, cases of human Parkinsonism would be apparent as were documented with MPTP (Langston et al., 1983). 2) Previous studies have shown that DAT (Gordon et al., 1996; Kilbourn et al., 1992; Sharpe et al., 1991; Vander Borght et al., 1995; Wiener et al., 1989) and 5-HTT (Whitworth et al., 2001; Zhou et al., 1996) are susceptible to long-term modulation by treatments that alter synaptic DA or 5-HT levels. Unlike DAT and 5-HTT, VMAT-2 levels present in the same dopaminergic or serotonergic terminals are less susceptible to modulation by pharmacological treatments (Frey et al., 2001; Naudon et al., 1994; Vander Borght et al., 1995; Wilson et al., 1996a). Further, VMAT-2 levels decrease following lesions of nigrostriatal neurons (Vander Borght et al., 1995; or in Parkinson’s disease (Lee et al., 2000; Miller et al., 1999; Wilson et al., 1996b) demonstrating the utility of VMAT-2 as a marker of dopaminergic terminal integrity. Previous studies have found a disconnect between DAT and VMAT-2 levels in the METHexposed brain (Frey et al., 1997; Hogan et al., 2000; Wilson et al., 1996a). The only human study in which both DAT and VMAT-2 levels were measured in the striatum of chronic METH users showed marked deficits in DAT (as well as DA and TH) but not in VMAT-2 or dopa decarboxylase levels (Wilson et al., 1996a). In the present study, marked deficits in DAT and 5-HTT were measured in almost all brain regions examined in the METH-exposed brain; however, deficits in VMAT-2 levels were less severe in magnitude and restricted to a limited number of brain regions. These findings strongly suggest that in some brain regions METH-induced deficits in DAT and 5-HTT may be associated with monoaminergic neuronal plasticity in the absence of distal axotomy. If METH-induced deficits in DAT and 5-HTT levels are the result of neuronal adaptation due to the disruptive effects of METH on monoaminergic homeostasis, then it is important to discuss potential mechanism(s) by which such changes may occur. One potential mechanism may be associated with protein inactivation or internalization for recycling or degradation (Fleckenstein et al., 2000). Previous studies show that shortly after METH administration, DAT (Fleckenstein et al., 1997; Kokoshka et al., 1998a) and 5-HTT (Kokoshka et al., 1998b) are inactivated. Inactivation of DAT and 5-HTT may be mediated by the formation of reactive oxygen species and DA quinones from auto-oxidation of the massive METH-induce release of DA. These agents are known to modify cysteinyl residues in the DAT protein, reducing its activity (Berman et al., 1996). This hypothesis is consistent with studies showing that mutations of cysteine residues in the second putative extracellular loop of DAT decreases transporter activity and membrane expression (Wang et al., 1995). Other studies have demonstrated that METH results in swelling of endocytic organelles promoting the internalization and degradation of transporter protein (Cubells et al., 1994). DA quinones have also been shown to form redox-cycling

quinoproteins (Kuhn and Arthur, 1998; Kuhn et al., 1999) allowing the quinone to cycle its redox status extending its biological half-life (Kuhn, 1999). METH-induced formation of DA quinones and protein modification has been demonstrated in the rat striatum (LaVoie and Hastings, 1999). Another potential mechanism for METH-induced modifications of DAT and 5-HTT may be via protein phosphorylation. The cell surface trafficking of members of the Na⫹/Cl⫺ dependent neurotransmitter transporter family, which include DAT and 5-HTT, can be acutely regulated by protein kinase C (PKC) activation (Robinson, 2002). Direct PKC phosphorylation of transporter protein, or an associated protein, results in internalization, endocytosis and targeting to the lysosomal compartment for degradation (Daniels and Amara, 1999) or possibly for recycling to the plasma membrane (Melikian and Buckley, 1999). Previous studies have shown that the METH metabolite amphetamine, increases PKC activity in the striatum and enhances the phosphorylation of endogenous substrates (Giambalvo, 1992a). Further, the effects of amphetamine on PKC were localized to the DA transporter in the plasma membrane (Giambalvo, 1992b). Therefore, it is highly likely that METH induces the inactivation of DAT and 5-HTT via PKC-mediated phosphorylation and/or through a reactive oxygen species pathway. The generation of DAT knockout mice provides the most direct and important insights into the homeostatic control of dopaminergic neurotransmission that may be relevant to psychostimulant abuse such as METH. DAT knockout mice exhibit marked reductions in striatal DA and TH levels but have virtually normal VMAT-2 and dopa decarboxylase levels in the absence of substantial terminal loss in the striatum (Jones et al., 1998; Gainetdinov et al., 1998; Jaber et al., 1999; Gainetdinov and Caron, 2003). These studies clearly demonstrate an important regulatory role of DAT in presynaptic dopaminergic homeostasis. Importantly, the pattern of deficits in dopaminergic phenotypic markers in DAT knockout mice is identical to that observed in human METH abusers (Wilson et al., 1996a). These findings indicate that marked changes in dopaminergic axonal markers are possible when DAT is inactivated in the absence of terminal degeneration. Therefore, it is highly likely that since METH directly interacts with and inactivates DAT, the inactivation of DAT may lead to longterm homeostatic changes similar to those present in DAT knockout mice. Besides METH-induced changes in plasma and vesicular transporters, we also assessed glial responses in similarly treated rats. For the most part, microglia/astrocyte activation was not observed in most regions of the METHexposed brain except in the striatum and central gray. An important observation with glial responses in the present study was in a limited number of rats that were most profoundly affected by METH administration based on body weight loss at the time of killing. The uniqueness of these animals is that glial cell activation was not only prominent in the striatum and central gray, but also in the hippocampus, thalamus, dorsal raphe, and inferior colliculus (Fig. 4). The high degree of microglia and astrocyte

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activation in this subset of METH rats implicates a more global pattern of neuronal injury. The reason for the small number of METH rats expressing this condition is not currently known. However, it is possible that individual differences in the ability of rats to regulate core body temperature following METH administration may explain the differences in response. Only recently in the history of studies on METH neurotoxicity has the role of body temperature been shown to play a potential role in the neurotoxicity of this agent (Bowyer et al., 1994; Bowyer and Holson, 1995; Fukumura et al., 1998; O’Callaghan and Miller, 2002). Therefore, it is possible that in our study, the small number of rats exhibiting widespread glial cell activation in the brain may have reached higher core body temperatures following METH administration. In summary, the present study indicates that METHinduced deficits in the plasma membrane transporters DAT and 5-HTT may represent two distinct neurotoxic endpoints. First, it appears that in the striatum and in the central gray, the loss of DAT and 5-HTT may be representative of axonal terminal degeneration. This is surmised from the fact that these brain structures also exhibited some loss of VMAT-2 and glial cell activation. Secondly, it appears that in other brain regions, lasting deficits in plasma membrane transporters were present in the absence of changes in VMAT-2 levels and glial cell activation. Thus, it is likely that in these brain regions, long-term deficits in plasma membrane transporters are associated with neuronal adaptation. Therefore, the METH-induced loss of plasma membrane transporters cannot always be viewed as an index of axonal terminal integrity unless other independent measures of terminal degeneration are provided. Our results also indicate that METH-induced deficits in VMAT-2 levels are closely associated with the regional pattern of microglia activation measured by immunohistochemical methods or by PBR autoradiography. This finding supports previous studies suggesting that VMAT-2 may be a more reliable marker of monoaminergic terminal integrity than plasma membrane transporters (Frey et al., 2001). Lastly, it is important to indicate that the extent that these different neurotoxic endpoints are operational may be dictated by the METH dose and whether hyperthermia is induced in the animal. Acknowledgements—This work was supported by grants from the National Institute of Environmental Health Sciences (ES07062 to TRG and NIEHS Center grant ES03819). The authors wish to thank Dr. Jim O’Callaghan for reviewing the manuscript and Dr. Brian Schoefield for assistance with microscopy and imaging.

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(Accepted 10 June 2003)