Reduced vesicular storage of dopamine exacerbates methamphetamine-induced neurodegeneration and astrogliosis

Abstract

The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5–10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.

The vesicular monoamine transporter 2 (VMAT2) packages catecholamine and indolamine neurotransmitters into synaptic vesicles for exocytotic release (Fon et al. 1997; Eiden 2000). In this capacity, VMAT2 controls the loading of the vesicle and thereby determines quantal size, receptor sensitivity, and synaptic plasticity (Gasnier 2000; Pothos et al. 2000; Sulzer and Pothos 2000). VMAT2 is also critical to neuronal health by removing neurotransmitter from the cytosol before it can be oxidized. When VMAT2 expression or function is reduced, DA neurons are particularly susceptible to damage from mishandled DA and exogenous toxicants (Takahashi et al. 1997; Gainetdinov et al. 1998; Fumagalli et al. 1999; Caudle et al. 2007; Vergo et al. 2007).

In dopamine (DA) neurons, VMAT2 is a target for amphetamine and its analogs such as methamphetamine (METH). METH is a dimethyl substituted phenylethylamine with high abuse potential and a trend of increased illicit manufacture and use in the United States. The neurotoxic consequences of METH manifest as a loss of the neurotransmitter DA, as well as long-term decreases in the rate limiting enzyme for DA synthesis, tyrosine hydroxylase (TH), and the dopamine transporter (DAT), the terminator of DA synaptic signaling (Ricaurte et al. 1980; Wagner et al. 1980b; Seiden 1985). Dopamine itself is thought to be required for the toxic actions of METH and is postulated to be a major contributor of oxidative stress through the formation of quinones and cysteinyl catechols (Wagner et al. 1983; Axt et al. 1990; Cubells et al. 1994; Giovanni et al. 1995; LaVoie and Hastings 1999b; Sulzer and Zecca 2000; Miyazaki et al. 2006; Thomas et al. 2008). It is known that oxidative and nitrative stress are important for these toxic effects, as administration of antioxidants or spin trapping reagents decrease the magnitude of DA, TH, and DAT loss (Wagner et al. 1986; Cadet et al. 1994; Cappon et al. 1996; Yamamoto and Zhu 1998; LaVoie and Hastings 1999a; Ali et al. 2005; Yamamoto and Bankson 2005). Finally, the damage to striatal DA terminals after METH administration has been shown to culminate in neurodegeneration (Ricaurte et al. 1982; Bowyer et al. 1994; O’Callaghan and Miller 1994).

It is also recognized that neuroinflammation is important to the neurotoxicity of METH. The mechanisms of glial perturbation are unclear, but both astroglia and microglia are activated after the administration of METH (Hess et al. 1990; O’Callaghan and Miller 1994; Sheng et al. 1994; Escubedo et al. 1998; LaVoie et al. 2004). Glial activation leads to an increase in inflammatory signaling molecules like TNF-alpha and the formation of reactive molecules such as nitric oxide, superoxide, and peroxynitrite (Aschner 1998; Aschner et al. 1999; Ladenheim et al. 2000; Flora et al. 2002; Block and Hong 2005). In addition, microglial activation has been shown to occur after administration of neurotoxic amphetamines analogues and this is thought to be a critical marker of exposure to this class of chemicals (Guilarte et al. 2003; Thomas et al. 2004a,b).

While glia are thought to be important in determining the extent of neuronal damage after insult, it is apparent that damage to the DA neurons is a necessary antecedent to astroglial activation (astrogliosis) in some cases (Gao et al. 2003). Animals that do not express DAT, a molecular gateway into the DA terminals for neurotoxicants, do not exhibit astrogliosis after METH or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration (Gainetdinov et al. 1997; Fumagalli et al. 1998; Miller et al. 1999). Interestingly, mice with increased DAT or decreased VMAT2 display potentiated astrogliosis when given MPTP, suggesting that VMAT2 can modulate glial responses by mitigating neuronal damage (Gainetdinov et al. 1998; Donovan et al. 1999; Richardson et al. 2006). Furthermore, a recent report from our laboratory suggests that VMAT2 expression is inversely proportional to gliosis after METH exposure (Guillot et al. 2008).

There is evidence that the expression level of VMAT2 modulates the ability of METH to cause DA loss in animals and autophagy in culture (Fumagalli et al. 1999; Larsen et al. 2002; Johnson-Davis et al. 2004; Guillot et al. 2008). While we and others have reported on the effects of METH administration in VMAT2 heterozygote knockout animals, early postnatal lethality of VMAT2 knockout animals has prevented examination of the effects of severely reduced VMAT2 levels on the susceptibility of DA neurons to neurotoxic damage (Fon et al. 1997; Fumagalli et al. 1999). With our recent development of VMAT2 LO mice, which express 5–10% of VMAT2 found in wild-type animals, we now possess an excellent model to study assess the contribution of drastically reduced vesicular storage of DA on the neurotoxicity of METH (Mooslehner et al. 2001; Patel et al. 2003; Caudle et al. 2007; Colebrooke et al. 2007).

Materials and methods

Animals

VMAT2 deficient (VMAT2 LO) mice were generated as described elsewhere (Mooslehner et al. 2001; Caudle et al. 2007). Briefly, the mouse VMAT2 locus was cloned from the 129/Sv genomic library and a 2.2-kb PvuII fragment from the third intron of the VMAT2 gene was then cloned into the blunt-ended NotI site of this construct. The targeting vector was introduced into 129/Ola CGR 8.8 embryonic stem (ES) cells and injected into blastocysts of C57BL/6 mice. Highly chimeric males were bred with C57BL/6 females and genotype was confirmed by Southern blot. Male and female VMAT2 wild-type (WT), heterozygous (HT, which express approximately 50–60% of WT VMAT2 levels), and LO (express 5–10% of WT VMAT2 levels) mice 8–12 weeks of age were used in these studies in accordance with the Animal Care and Use Policies of Emory University. Mice received chow and water ad lib on a 12 : 12 light cycle (lights on at 7:00 AM).

Administration of drugs

(+)-Methamphetamine (Sigma, St Louis, MO, USA) was dissolved in 0.9% saline and administered subcutaneously in a volume of 100 μL. Mice were given a neurotoxic regimen of four doses of 15 mg/kg (free base) METH, two hours apart and killed by decapitation 48 h after the last dose (Fumagalli et al. 1999).

Chemicals

All chemicals were purchased from Sigma–Aldrich, unless otherwise noted.

Temperature

Core body temperature was monitored 30 min before and 1 h after each injection of saline or METH. Temperature was taken rectally by use of a digital thermometer (VWR International, Westchester, PA, USA) lubricated with AstroGlide (BioFilm, Inc., Vista, CA, USA).

Western blotting

Western blots were used to quantify the amount of DAT, tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), glucose transporter 5 (GLUT5), and α-tubulin present in samples of striatal tissue and were performed as previously described (Caudle et al. 2007; Guillot et al. 2008). Briefly, mice were killed by decapitation, the brains were removed and striatal tissue samples homogenized in 0.32 M sucrose with 4 mM HEPES, pH 7.4, supplemented with protease inhibitors (1 μg/mL of each: aprotinin, leupeptin, pepstatin) using a Polytron (Kinematic, Luzern, Germany). The homogenate was centrifuged at 1000 g for 10 min at 4°C and the resulting supernant centrifuged for 1 h at 20 000 g at 4°C. The pellet was resuspended in the homogenization buffer and protein was quantified using the BCA protein assay (BCA Protein Assay kit; Pierce, Rockford, IL, USA). Ten micrograms of protein from each sample was subjected to polyacrylamide gel electrophoresis (NuPAGE, 10% BisTris, 1 mm thick, 12-well gels, Invitrogen, Carlsbad, CA, USA) then electrophoretically transferred to polyvinylidene difluoride membranes (Invitrolon 0.45 μm PVDF, Invitrogen). Blots were incubated in 7.5% non-fat dry milk (Carnation, Glendale, CA, USA) in Tris-buffered saline (TBS) for 1 h at 23°C. Membranes were then incubated overnight with an antibody to DAT (1 : 5000; Chemicon, Temecula, CA, USA). Primary antibody binding was detected using a goat anti-rat horseradish peroxidase (HRP) secondary antibody (1 : 10 000; Jackson Immuno Research, West Grove, PA, USA) and enhanced chemiluminescence (SuperSignal; Pierce, Rockford, IL, USA). Luminescence was captured with a Fluorochem 8800 (Alpha Innotech, San Leandro, CA, USA) imaging system. Densitometric analysis was performed and calibrated to co-blotted dilutional standards of pooled striata from all control samples. Membranes were sequentially stripped for 15 min at 23°C with stripping buffer (Restore; Pierce) and reprobed with antibodies against TH (rabbit polyclonal, 1 : 1000; Chemicon) followed by goat anti-rabbit HRP-conjugated secondary antibody (1 : 10 000; Jackson Immuno Research). This sequence was repeated with GFAP (rabbit polyclonal, 1 : 5000; Sigma), and GLUT5 (rabbit polyclonal, 1 : 5000; Chemicon). Each blot was then stripped a final time and probed for α-tubulin (mouse monoclonal, 1 : 10 000; Sigma; goat anti-mouse HRP-conjugated secondary, 1 : 10 000, Jackson Immuno Research) and density from each blot was used to ensure equal protein loading across samples.

Protein carbonyl detection

Protein carbonyl levels in striatal tissue were determined using Oxyblots. Striatal samples were homogenized in 0.32 M sucrose with 4 mM HEPES, pH 7.4 supplemented with 1 μL/mL leupeptin, aprotinin, and pepstatin. The homogenate was centrifuged for 10 min at 1000 g at 4°C. The supernatant was removed and centrifuged for 60 min at 21 000 g at 4°C. The pellet was resuspended in the homogenization buffer and protein concentrations were determined using the BCA protein assay (BCA Protein Assay kit; Pierce).

Protein carbonyl levels were determined using the Oxyblot Protein Detection Kit (Chemicon) in the aforementioned homogenization buffer. Protein carbonyls are derivatized to 2, 4-dinitrophenylhydrazone (DNP) by reaction with concentrated 2,4-dinitrophenylhydrazine. DNP-derivatized protein samples were analyzed by dot blots, using a primary antibody against DNP (1 : 1500; Sigma) and an HRP-conjugated goat anti-rabbit secondary (1 : 10 000; Jackson Immuno Research).

Immunohistochemistry

Tissue staining was performed as previously described (Caudle et al. 2007). Briefly, animals were perfused transcardially with phosphate-buffered saline (pH 7.4), followed by 4% paraformaldehyde. Brains were then removed, cryoprotected in 30% sucrose for 48 h, frozen, and cut to a thickness of 40 μm on a freezing-sliding microtome (Microm, Kalamazoo, MI, USA). Sections were incubated with antibodies against DAT (rat monoclonal, 1 : 750; Chemicon), TH (rabbit polyclonal, 1 : 2,000; Chemicon), or GFAP (rabbit polyclonal, 1 : 1000; Sigma) overnight at 4°C and then incubated in a biotinylated secondary antibody (goat anti-rat or goat anti-rabbit 1 : 200, Jackson Immuno Research) for 1 h at 23°C. Sections were washed and incubated 1 h at 23°C in avidin-biotin-HRP conjugate solution (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA). Visualization was performed using 0.03% 3, 3′-diaminobenzidine (SigmaFast diaminobenzidine, Sigma) for 3 min at 23°C. For microglial visualization, sections were incubated with biotinylated isolectin B4 (1 : 1000; Invitrogen) overnight at 4°C and then incubated 1 h at 23°C in avidin–biotin-HRP conjugate solution (Vectastain ABC kit, Vector Laboratories). Visualization was performed using DAB as above for 25 min at 23°C. Sections were mounted on Superfrost slides (Brain Research Laboratories, Cambridge, MA, USA) dehydrated with increasing ethanol concentrations and xylenes, then coverslipped using Permount (Fisher Scientific, Hampton, NH, USA). Sections were viewed using a light microscope (Olympus, Albertslund, Denmark).

Silver staining

Silver staining (FD Neurosilver kit; FD NeuroTechnologies, Ellicott City, MD, USA) for degenerating neurons was performed according to the manufacturer’s protocol. Mice were transcardially perfused with 4% paraformaldehyde, brains removed and placed in 4% paraformaldehyde for 48 h. Brains were then cryoprotected in 30% sucrose for 48 h, then frozen and 40 μm sections through the striatum were cut on a freezing-sliding microtome. After processing and mounting, slides were coverslipped using Permount (Fisher Scientific) and sections were viewed using a light microscope (Olympus).

Neurotransmitter and metabolite detection

HPLC-EC analysis of DA and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was performed as previously described (Caudle et al. 2006; Hatcher et al. 2007; Guillot et al. 2008). Briefly, dissected striata were sonicated in 0.1 M perchloric acid containing 347 μM sodium bisulfite and 134 μM EDTA. Homogenates were centrifuged at 15 000 g for 10 min at 4°C, the supernatant was removed, and filtered through a 0.22 μm filter by centrifugation at 15 000 g for 10 min at 4°C. The supernatants were then analyzed for levels of DA, DOPAC, HVA using HPLC with a coulometric electrode detector (Waters, Inc., Milford, MA, USA). Quantification was made by reference to calibration curves made with individual standards.

Primary culture

The methods used to generate substantia nigra (SN) neuron-glia co-cultures were described in detail previously (Cardozo 1993; Smeyne and Smeyne 2002), with slight modifications. Briefly, brains from postnatal days 0–6 (P0–6) VMAT2 WT, HT, LO and mice were removed and placed in dissociation media (DM) containing 90 mM sodium sulfate, 30 mM potassium sulfate, 0.25 mM calcium chloride, 5.8 mM magnesium chloride, 10 mM glucose, 1 mM HEPES, pH 7.4. Under a dissecting microscope, a 0.8–1.0 mm coronal section of the mesencephalon was made using a scalpel, and the regions containing SN were isolated. The SN was dissected, placed in fresh DM, minced into small pieces. The SN was digested in DM containing papain and DNAase (Worthington Biochemical, Freehold, NJ, USA) and incubated at 37°C (2 times for 30 min). The tissue was rinsed twice with DM, and once with plating media (PM) containing 1 mg/mL bovine serum albumin, 1 mg/mL ovalbumin, 20 mM glucose, 10 mM HEPES, 0.5 mL concentrated media additives dissolved in basal medium Eagle. The SN pieces were then triturated and the cell suspension was layered over PM with 100 mg/mL bovine serum albumin and 100 mg/mL ovomucoid albumin. The cell suspension was then centrifuged for 8 min at 1400 g. The supernatant was removed, and the pellet was resuspended in PM with 2% rat serum (RS). Cells were counted using Trypan Blue, and plated at 350 000 cells/cm² in Lab-Tek (TM) four-well Permanox chamber slides that were previously coated with 20 μg/mL laminin and 200 μg/mL poly-D-lysine (Collaborative Biomedical Products, Bedford, MA, USA) at 1 : 1 (vol:vol).

Cells were maintained in an incubator at 37°C, 5% CO₂, and fed with PM containing 2% RS. At 24 h post-plating, the cultures were fed with complete feeding media (containing 0.6 mM glutamax, 12 mM glucose, 100 mg/ml transferrin, 60 μM sodium selenite, 2 mM putrescine, 25 mg/mL insulin, 0.02 mL concentrated media additives, and 2% rat serum). After 7 days in culture, neurons were treated with either METH (100 μM, dissolved in feeding media) or feeding media (controls). Seventy-two hours after administration, cultures were rinsed two times for 5 min with TBS, fixed for 10–15 min in 4% buffered paraformaldehyde, and rinsed three times with TBS. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol for 10 min. Cells were permeabilized with 0.1% Triton X-100, 5% goat serum in TBS for 10 min. Dopaminergic neurons were visualized with a rabbit polyclonal antibody directed against polyclonal tyrosine hydroxylase (1 : 500; Chemicon). Cultures were incubated with primary antibody overnight at 4°C. Cultures were then rinsed two times with TBS followed by application of secondary antibody (goat anti-rabbit, 1 : 200; Jackson Immuno Research) and amplification with avidin–biotin-HRP (Vector Laboratories). Final visualization of the immunoreactive neurons was made using 0.03% DAB for 3 min and then washed three times with TBS. All TH-positive cells from each culture were counted on a light microscope at magnification of 200× (Olympus). The tyrosine hydroxylase (TH) positive processes were quantified by counting the TH processes from a total of 30 neurons from four random fields in a manner previously described (Radad et al. 2008). The total number of processes counted from 30 neurons was divided by the number of neurons counted and values are expressed as number of TH processes per neuron. The data reflect two separate experiments and the data were combined to yield a final sample size of 4 per treatment per genotype.

Detection of oxidative stress in cultures was accomplished by adding 2, 7-dichlorofluorescein diacetate (DCF; Invitrogen) (5 μM) to cultures 72 h after treatment with 100 μM METH. The cultures were then placed at 37°C for 1 h. Cells were washed twice, coverslipped, and viewed using an epifluorescence microscope (Olympus).

Statistics

All statistical analysis was performed on raw data for each treatment group by one-way ANOVA or Student’s t-test using Prism 4.0 (GraphPad, San Diego, CA). Post hoc analysis was performed using Student–Newman–Keuls (SNK) test. Statistical significance is reported at the p < 0.05 level.

Results

Effect of METH on striatal dopamine and metabolites

Forty-eight hours after the last dose of METH, striatal DA was reduced 79% in WT animals compared with saline controls, from 20.29 ± 0.71 (all values in units of ng/mg tissue) to 4.29 ± 0.68. Heterozygous (HT) mice exhibited an 84% decrease in DA, 13.47 ± 0.41 to 2.16 ± 0.37. VMAT2 LO animals decreased from 3.18 ± 0.12 to 0.57 ± 0.11, an 82% loss of DA (Fig. 1a). DOPAC/DA ratio for WT animals was 0.08 and increased to 0.15 after METH (88%), in HT animals the ratio increased from 0.12 to 0.22 (88%) and in LO mice from 0.24 to 0.39 (63%; Fig. 1b). HVA/DA ratio in WT animals was 0.10 and METH raised it to 0.37 (3.7-fold), HT increased from 0.13 to 0.50 (3.8-fold), and LO animals went from 0.303 to 0.971 (3.20-fold; Fig. 1c). After METH administration, DOPAC in WT animals decreased from 1.630 ± 0.041 to 0.567 ± 0.030, 1.588 ± 0.064 to 0.419 ± 0.035 in HT animals, and 0.760 ± 0.037 to 0.200 ± 0.023 in LO animals. HVA was also decreased after METH: 1.997 ± 0.110 to 1.375 ± 0.062 in WT, 1.793 ± 0.050 to 0.943 ± 0.053 in HT, and 0.955 ± 0.046 to 0.495 ± 0.048 in LO animals.

Fig. 1.

HPLC analysis of dopamine, DOPAC, and HVA in the striatum 48 h after 4 × 15 mg/kg METH. METH caused depletion of tissue dopamine levels in each genotype: WT by 79%, HT by 84% and LO by 82% (a). DOPAC/DA ratio was increased by METH 88% in WT, 88% in HT, and 63% in LO (b). METH led to an elevation of HVA/DA ratio by 3.7-fold in WT, 3.7-fold in HT, and 3.2-fold in LO (c). Dissimilar letters above bars indicate a significant difference of at least p < 0.05 between groups (n = 6–8 per treatment group).

METH greatly decreases dopaminergic terminal markers in VMAT2 LO animals

Previous studies have demonstrated that DAT, a sensitive marker for terminal damage, is reduced after METH administration (Wagner et al. 1980a; Ricaurte et al. 1982). In wild-type VMAT2 animals, DAT was decreased by 40% at 48 h after METH. The loss is potentiated in HT (−57%) and LO animals (−74%; Fig. 2a). Immunohistochemical images of the striatum reinforce the DAT loss seen by western blotting (Fig. 3a).

Fig. 2.

Western blot analysis of DAT, TH, GFAP, and GLUT5 in the striatum 48 h after 4 × 15 mg/kg METH. DAT was decreased by METH 40% in WT, 57% in HT, and 74% in LO (a). TH was non-significantly reduced 18% in WT after METH, but HT lost 45% while LO saw a 78% loss of TH (b). GFAP increased after METH by 45% in WT, 68% in HT and 92% in LO (c). GLUT5 was elevated by METH by approximately 25% in each genotype (d). Dissimilar letters above bars indicate a significant difference of at least p < 0.05 between groups (n = 6–8 per treatment group).

Fig. 3.

Dopamine terminal marker immunohistochemistry 48 h after 4 × 15 mg/kg METH. METH decreased DAT and TH expression by varying amounts depending on VMAT2 expression. Dopamine transporter in the striatum (a). Scale bar is 500 μM. Tyrosine hydroxylase in the striatum (b). Scale bar is 500 μM.

Another critical marker of dopaminergic terminal integrity, TH, can also be lost after METH (Ricaurte et al. 1982; O’Callaghan and Miller 1994). TH was not significantly decreased in WT animals exposed to METH at this time point (−18%). However, there were significant TH losses in HT animals (−45%) and more so in LO animals (−78%; Fig. 2b). This exaggerated loss of TH after METH may be indicative of terminal degeneration. Immunohistochemical staining for TH reveals the significant loss of TH immunoreactivity in the striatum of HT and LO animals (Fig. 3b).

METH leads to increased astrogliosis, but does not alter microgliosis in VMAT2 LO animals

Astrogliosis was measured by observing GFAP expression after 48 h after the final METH administration. Wild-type animals show a 45% increase in GFAP, whereas HT animals (+68%) and LO animals (+92%) experience larger GFAP increases in the striatum following METH administration (Fig. 2c). This step-wise increase in GFAP expression was supported by immunohistochemical staining (Fig. 4a).

Fig. 4.

Gliosis markers in the striaturo by immunohistochemistry 48 h after 4 × 15 mg/kg METH. GFAP expression in the striatum (a). GFAP was increased by METH with a magnitude correlated to VMAT2 expression. Scale bar is 100 μM. IB4 staining in the striatum (b). Activated microglia, stained with IB4, were present in the striatum of each genotype after METH. Scale bar is 200 μM.

Microgliosis was measured by immunoblotting for glucose transporter 5 (GLUT5), a specific marker for microglia in the brain. GLUT5 expression increases after METH by the same proportion (+25%) in each genotype (Fig. 2d). However, a microglial stain for isolectin B4 (IB4) suggests microgliosis was worse in HT and LO animals compared to WT after METH (Fig. 4b).

METH leads to increased protein carbonyl production

Free radical generation can lead to oxidative stress and one consequence of this is an oxidation of carbons on protein residue side chains. Oxidation of these carbons leads to the formation of carbonyls that can be derivatized to hydrazones and detected in tissue samples by immunoblot. Forty-eight hours after the final dose of METH, protein carbonyls were elevated in WT animals by 26%, in HT animals by 45%, and LO animals exhibited a 94% increase (Fig. 5). This indicates that more oxidative stress was present in the HT and LO animals after METH.

Fig. 5.

Protein carbonyl analysis of striatal tissue by oxyblot 48 h after 4 × 15 mg/kg METH. METH caused an increase in protein carbonyls by 26% in WT, 45% in HT, and 94% in LO. Dissimilar letters above bars indicate a significant difference of at least p < 0.05 between groups (n = 6–8 per treatment group).

METH induces degeneration according to VMAT2 expression

Silver staining for degenerating neurons demonstrates that there is increased vulnerability to METH in neurons with decreased VMAT2 expression. There is a small amount of argyrophilic deposits in the striatum of WT animals 48 h after the last METH injection, reminiscent of the first observations of silver deposits in rat striatum after neurotoxic doses of METH (Ricaurte et al. 1982). However, HT animals show greatly increased deposits and LO animals show massive degeneration in the striatum (Fig. 6).

Fig. 6.

Evidence of neurodegeneration by silver deposition in the striatum 48 h after 4 × 15 mg/kg METH; In WT animals, METH led to noticeable silver deposition. Deposition is increased in HT animals, and is dramatically elevated in LO animals. Scale bar is 100 μM.

VMAT2 expression and the thermal response to METH

METH administration leads to hyperthermia in mice and rats that has been shown to be a determinant of neurotoxicity and death (Miller and O’Callaghan 1994; Albers and Sonsalla 1995; Ali et al. 1995). One hour after the first s.c. injection of 15 mg/kg METH, core body temperature was increased in WT animals from 37.6 ± 0.4°C to 39.3 ± 0.6°C, in HT animals from 37.4 ± 0.3°C to 39.1 ± 0.5°C, and in LO animals from 36.8 ± 0.4°C to 38.2 ± 0.3°C. Basal core temperature and core temperature 1 h after 15 mg/kg METH did not differ significantly between genotypes. However, the HT mice had a paradoxical response to METH starting with the second injection: their core temperature decreased to saline treatment levels and remained non-significantly elevated for the duration of the experiment. The LO mice also experienced a dip in core temperature after the third injection, which rebounded after the last injection. Thus, core temperature was significantly elevated above saline-treated controls for all time points in the WT and LO groups, but only after the first injection in the HT group (Fig. 7). The source of this core temperature variation after METH is unknown, but is most likely regulated by all monoamine systems (dopamine, norepinephrine, serotonin, histamine) affected in this genetic model (Shaw 1971; Katsuyama et al. 1986; Numachi et al. 2007; Ito et al. 2008). It has also been shown that brain hyperthermia can lead to astrogliosis (Miller et al. 1987), however, considering the magnitude of core temperature change was identical in WT and LO mice, the increase of GFAP expression in the striatum of VMAT2 LO animals after METH is not the result of a difference in core temperature.

Fig. 7.

Temperature regulation after saline (SAL) or METH. WT mice showed significantly increased core temperature after each injection of METH (a). HT mice exhibited elevated core temperature after the first injection of METH, which then decreased to SAL control levels after subsequent injections (b). LO mice displayed elevated temperature after each injection of METH (c). * indicates at least p < 0.05 (n = 6–8 per treatment group).

METH induces oxidative stress and leads to process loss in primary cultures

To determine if METH had similar effects on oxidative stress and terminal marker loss ex vivo, we utilized a primary culture model. Oxidative stress was again assayed but by means of a fluorescent marker. DCF, a sensor for peroxides and other oxygen radicals, can be oxidized within the cell and detected by fluorescence. METH exposure for 72 h causes a slight increase in DCF fluorescence in WT cultured neurons. There is a greater increase in HT neurons and a very large increase in neurons from LO animals (Fig. 8). This shows METH itself can cause oxidative stress in DA neurons.

Fig. 8.

METH leads to oxidative stress in primary midbrain culture. Cultures from VMAT2 WT, HT, and LO were treated with 100 μM METH. Cultures were analyzed 72 h after METH administration. DCF fluorescence increases across WT (a), HT (b), and LO (c) cultures. Scale bar is 100 μM.

Neurons in culture send out dendritic processes and the loss of these after METH exposure can be used as a measure of toxicity. The TH immunoreactive processes from DA neurons in all three genotypes look normal after vehicle treatment. When exposed to METH for 72 h, WT and HT neurons exhibit a small amount of loss, whereas neurons from LO animals show an almost complete loss of TH immunoreactive processes (Fig. 9).

Fig. 9.

Cultures from VMAT2 WT, HT and LO were treated with 100 μM METH and were analyzed 72 hours later. (a) TH immunoreactive processes are qualitatively reduced following METH treatment. Scale bar is 50 μM. (b) The number of TH immunoreactive processes per neuron is significantly reduced by METH, and the severity of loss is inversely correlated to VMAT2 expression. Letters above bars, when they do not match, indicate a significant difference of at least p < 0.05 between groups.

Discussion

We have demonstrated that mice deficient in VMAT2 (VMAT2 LO) have more dramatic neurotoxicity in the striatum after METH administration than wild-type controls. Subsequent to METH exposure, DA terminal markers, which are used as measures of neuron terminal integrity, were depleted to a greater extent in VMAT2 LO mice. VMAT2 LO mice also exhibited exaggerated levels of oxidative stress markers (in vivo and in vitro) and reactive astrogliosis after METH. Most importantly, the expression of VMAT2 determined the amount of striatal silver deposition and the loss of TH immunoreactive processes, two measures of neurodegeneration.

METH is hypothesized to be neurotoxic to DA terminals because it greatly alters the balance of DA compartmentalization generated by vesicular sequestration of the transmitter (Cubells et al. 1994; Sulzer et al. 1995; Larsen et al. 2002; Riddle et al. 2002; Mosharov et al. 2003). METH has been proposed to disrupt this balance by dissipating the pH gradient in vesicles, inhibiting VMAT2 function, promoting DA synthesis and preventing its degradation (Sulzer et al. 1992, 2005; Larsen et al. 2002; Ugarte et al. 2003; Eyerman and Yamamoto 2007; Thomas et al. 2008). These effects combine to yield an elevation in cytosolic DA; some of which can be shunted out of the neuron via the DAT, whereas DA remaining in the cytosol can be oxidized to reactive quinones (Sulzer and Zecca 2000; Sulzer et al. 2005). Dopamine quinones lead to an increase in oxidative stress, are present after METH administration, and are the direct result of increased cytosolic DA levels (LaVoie and Hastings 1999b; Larsen et al. 2002).

Impaired DA compartmentalization in VMAT2 deficient mice leads to decreased tissue levels of DA and higher DA turnover. HPLC analysis showed that VMAT2 LO animals have three-fold higher DOPAC/DA and HVA/DA ratios compared with wild-type animals. VMAT2 LO mice exhibit higher levels of cysteine-conjugated L-DOPA and DOPAC than wild-type animals, indicating the production of catechol quinones (Caudle et al. 2007). VMAT2 LO mice possess only 16% of the DA that wild-type animals have and, while they do not lose a greater proportion of DA after METH, they have signs of severely increased neurotoxicity, such as the potentiated loss of terminal markers (Fig. 1). Tyrosine hydroxylase (TH) activity is higher in VMAT2 LO animals and METH has been shown to increase TH activity, possibly further elevating cytosolic catechols in these mice (Larsen et al. 2002; Caudle et al. 2007). It may also be the case that the spike in cytosolic DA after METH is further enhanced by the reduced amount of the DAT in VMAT2 LO mice, which could reduce non-exocytotic release. These mechanisms leading to elevated cytosolic DA may likely all contribute to the increased neurotoxicity of METH to DA neurons in VMAT2 deficient mice.

DAT and TH have been used in many studies as markers of the integrity of DA neuron terminals after METH administration (Wagner et al. 1980b; Ricaurte et al. 1983; Seiden 1985). In VMAT2 LO mice given METH, the severity of DAT loss is nearly twice that of wild-type mice (Fig. 2a) and the decrease in TH is four-fold higher, indicating an exacerbated impairment of terminal function (Fig. 2b). This shows that VMAT2 expression determines how DA terminal markers change in response to neurotoxic doses of METH and suggests that these terminals would take much longer to recover from this damage.

The presence of reactive astroglia has also been used a marker of neurotoxicity after METH exposure (O’Callaghan and Miller 1994). A reduction in VMAT2 levels contributed to elevated GFAP protein expression in the striatum after METH administration (Figs 2c and 4a). Since functional VMAT2 resides within the neuron terminals, this suggests that METH must access the terminals and inflict neuronal damage before astroglial activation occurs. This hypothesis is supported by other studies showing that, with METH and MPTP, denial of terminal access by genetic deletion of DAT prevents astrogliosis and that VMAT2 heterozygous knockout mice have potentiated GFAP mRNA expression after MPTP administration (Gainetdinov et al. 1997, 1998; Fumagalli et al. 1998).

While the observed increases in GFAP are significant in all genotypes, the magnitude of the elevation is not as great as would be expected from previous studies (O’Callaghan and Miller 1994). This discrepancy may be due to differences in the methods of tissue preparation and GFAP protein measurement (O’Callaghan et al. 1999). Specifically, we utilized a western blot on a membrane enriched fraction to detect GFAP after METH, whereas a larger elevation was observed by subjecting a detergent extraction of total protein to a sensitive ELISA (O’Callaghan and Miller 1994).

Microglia, the macrophagic glial cells, are activated by neuronal injury, including after METH administration (Thomas et al. 2004b). Glucose transporter 5 (GLUT5) is a specific marker of microglia that is elevated by neuronal insult and used to measure microgliosis (Payne et al. 1997; Vannucci et al. 1997; Richardson et al. 2007). VMAT2 expression, however, did not make a significant difference in the severity of microgliosis after METH, with wild-type and VMAT2 LO mice both exhibiting an approximately 25% increase in GLUT5 (Fig. 2d). While the extent of microgliosis did not follow the magnitude of elevation in astroglial activation, the increases in GLUT5 and IB4 staining after METH agree with reports of microglial activation after administration of neurotoxic doses of METH (Escubedo et al. 1998; Guilarte et al. 2003). It is possible that western blot conditions were not optimized for the detection of GLUT5 and this contributed to a the lack of an observed genotype effect after METH (O’Callaghan et al. 1999).

An increase in protein carbonyls, a measure of oxidative stress, has been previously detected in mice after METH, and in untreated VMAT2 deficient mice at 12 months of age (Gluck et al. 2001; Caudle et al. 2007). There is an increase in protein carbonyls in wild-type animals after METH and the magnitude of increase is multiplied with a reduction in VMAT2. Heterozygote animals treated with METH have an approximately two-fold elevation in protein carbonyls, while VMAT2 deficient animals experience a four-fold increase versus wild-type animals. This strongly suggests that the level of VMAT2 expression regulates the amount of oxidative stress produced after METH administration, possibly by its role in mitigating intracellular DA oxidation.

METH administration leads to degeneration of neuronal terminals, as measured by argyrophilic deposits in the striatum of rats and mice with normal levels of VMAT2 (Ricaurte et al. 1982; O’Callaghan and Miller 1994, 2002; O’Callaghan et al. 1995). As expected, VMAT2 wild-type mice exhibited a small but noticeable increase in silver deposition after METH. However, silver deposition is much more evident in heterozygotes and even more pronounced in VMAT2 deficient animals. It must be emphasized that, despite the qualitative nature of the silver stain, METH was vastly more damaging in the striatum of mice with low expression of VMAT2 (Fig. 6). The silver staining data strongly suggest that the changes we are seeing are not regulatory in nature, but rather frank degeneration. Human METH users display reductions in DAT and TH that are generally considered to be due to altered regulation (Wilson et al. 1996). Perhaps some of these changes are indeed degenerative. In addition, users with lower than normal VMAT2 levels (i.e., VMAT2 haplotypes leading to decreased expression) may have impaired vesicular storage and be at greater risk of neurodegenerative changes.

It has been recognized that METH causes an increase in core body temperature and that this contributes to neurotoxicity (Miller and O’Callaghan 1994; Albers and Sonsalla 1995; Ali et al. 1996; Metzger et al. 2000; Ugarte et al. 2003). The increased deleterious effects of METH that occurred in animals expressing reduced amounts of VMAT2 did so without a change in the magnitude of core temperature elevation compared to wild-types. However, heterozygote animals displayed an anomalous thermal response with temperature initially spiking after the first injection and then decreasing to saline control levels with subsequent injections. This response may be due to over-compensation in a signaling pathway in the HT group that is not achieved in VMAT2 deficient animals. In a previous study, using mice of a different knockout line, it was found that there were no significant temperature differences between wild-types and VMAT2 heterozygotes 1 h after 15 mg/kg METH (Fumagalli et al. 1999). There was only one time point that temperature was measured but that observation was statistically identical to that of the VMAT2 heterozygote mice in the present study (39.1 ± 0.5, n = 5, present study vs. 39.4 ± 0.3, n = 7, Fumagalli et al. 1999, p = 0.596). Thus, it is unknown if the temperature variation observed in VMAT2 heterozygotes in the present study also occurred in the strain used by Fumagalli et al. when administered four doses of METH. However, given the nature of the observed neurochemical responses across genotypes in vivo and in vitro in the present study, the thermal response to METH does not appear to be responsible for the increased neurotoxicity of METH in animals with reduced VMAT2.

We utilized primary neuron-glia co-cultures from the midbrains of VMAT2 deficient mice to show that the increase in neurotoxicity is a direct effect on the DA neurons. With addition of METH, oxidative stress, as signaled by DCF fluorescence, increased slightly in wild-type neurons but exhibited an extremely high signal in VMAT2 LO cultures (Fig. 8). Dendritic processes from cultured neurons of each genotype are indistinguishable before and after vehicle treatment, however, after METH treatment, wild-type neurons exhibit some pruning whereas VMAT2 LO neurons exhibit an almost complete loss of TH immunoreactive processes (Fig. 9). These data show that METH is more toxic to DA neurons that are deficient in VMAT2 in vitro as well as in vivo.

In conclusion, we have demonstrated that the spatial control of DA by VMAT2 is a critical factor in METH-induced neurotoxicity and neurodegeneration. These data strongly suggest that this control of DA sequestration is a more important determinant of METH neurotoxicity than the absolute tissue level of neurotransmitter, in agreement with recent findings (Vergo et al. 2007; Guillot et al. 2008; Thomas et al. 2008). Furthermore, disruption of DA storage by METH not only leads to potentiated terminal marker loss but causes massive terminal degeneration in the striatum of VMAT2 LO mice, most likely by the unchecked generation of oxidative stress. This information may be utilized in designing neuroprotective strategies against disorders such as METH abuse and idiopathic Parkinson’s disease which both exhibit mishandling of DA and destruction of neurons in DA rich regions of the brain.

Abbreviations used

DA

dopamine

DAT

dopamine transporter

DCF

dichlorofluorescein

DOPAC

dihydroxyphenylacetic acid

GFAP

glial fibrillary acidic protein

GLUT5

glucose transporter 5

HRP

horseradish peroxidase

HVA

homovanillic acid

IB4

isolectin IB4

METH

methamphetamine

MPTP

1-methyl-4phenyl-1,2,3,6 tetrahydropyridine

S.C.

subcutaneous

TBS

Tris-buffered saline

TH

tyrosine hydroxylase

VMAT2

vesicular monoamine transporter 2