Methamphetamine causes degeneration of dopamine cell bodies and terminals of the nigrostriatal pathway evidenced by silver staining

Abstract

Methamphetamine is a widely abused illicit drug. Recent epidemiological studies showed that methamphetamine increases the risk for developing Parkinson's disease (PD) in agreement with animal studies showing dopaminergic neurotoxicity. We examined the effect of repeated low and medium doses vs single high dose of methamphetamine on degeneration of dopaminergic terminals and cell bodies. Mice were given methamphetamine in one of the following paradigms: three injections of 5 or 10 mg/kg at 3 h intervals or a single 30 mg/kg injection. The integrity of dopaminergic fibers and cell bodies was assessed at different time points after methamphetamine by tyrosine hydroxylase immunohistochemistry and silver staining. The 3 × 10 protocol yielded the highest loss of striatal dopaminergic terminals, followed by the 3 × 5 and 1 × 30. Some degenerating axons could be followed from the striatum to the substantia nigra pars compacta (SNpc). All protocols induced similar significant degeneration of dopaminergic neurons in the SNpc, evidenced by amino-cupric-silver-stained dopaminergic neurons. These neurons died by necrosis and apoptosis. Methamphetamine also killed striatal neurons. By using D1-Tmt/D2-GFP BAC transgenic mice, we observed that degenerating striatal neurons were equally distributed between direct and indirect medium spiny neurons. Despite the reduced number of dopaminergic neurons in the SNpc at 30 days after treatment, there was a partial time-dependent recovery of dopamine terminals beginning 3 days after treatment. Locomotor activity and motor coordination were robustly decreased 1-3 days after treatment, but recovered at later times along with dopaminergic terminals. These data provide direct evidence that methamphetamine causes long-lasting loss/degeneration of dopaminergic cell bodies in the SNpc, along with destruction of dopaminergic terminals in the striatum.

Figure 1

Single administration of methamphetamine (1 × 30) induced less dopaminergic terminal degeneration in the striatum than multiple administration treatments (3 × 5 or 3 × 10). (a) Methamphetamine produced hyperthermia after each injection, with three peaks in the multiple administration regimens, and a single and more sustained peak of hyperthermia after single administration of (1 × 30 mg/kg). Data represent mean±SEM, n=10–14 per group. Arrows indicate drug injection. (b and c) Histograms show the proportional stained area of tyrosine hydroxylase (TH) and amino-cupric-silver staining in the striatum. Data represent mean±SEM, n=4–6 per group. (d) Photomicrographs of striatal sections from mice 1 and 3 days after treatment with saline (sal) or methamphetamine (3 × 5), (3 × 10), or (1 × 30), stained for TH (top), A-Cu-Ag (middle), or TH/A-Cu-Ag (bottom). *p<0.05, **p<0.001 vs sal, ^#p<0.05, ^##p<0.001 vs (1 × 30), ^δp<0.05, ^δδp<0.001 vs (3 × 10), ^Δp<0.05, ^ΔΔp<0.001 vs 1 day. Statistical analysis was performed by two-way analysis of variance and post hoc Newman–Keuls analysis. Bar indicates 500 μm (d).

Figure 2

Methamphetamine produced degeneration of striatal neurons. (a) Photomicrographs of tyrosine hydroxylase/amino-cupric-silver (TH/A-Cu-Ag)-stained sections of the striatum of mice treated with saline (sal) or methamphetamine (3 × 5), (3 × 10), or (1 × 30) 1 day or 3 days after the treatment, at high magnification. (b) Histogram showing the number of A-Cu-Ag-positive cells in the striatum counted by stereology in sections of mice treated with saline or methamphetamine (3 × 5), (3 × 10), or (1 × 30) 1 day or 3 days after the treatment (mean±SEM, n=4–6 per group). Treatment with methamphetamine (3 × 10) or (1 × 30) induced the appearance of A-Cu-Ag-stained neurons in the striatum. Data represent mean±SEM, n=4–6 per group, *p<0.05 vs sal. (c) Stereoinvestigator drawings of the striatum of mice treated with saline or methamphetamine (3 × 5), (3 × 10), or (1 × 30), showing the distribution of A-Cu-Ag-stained neurons at different rostrocaudal levels at 1 day (stars) and 3 days (dots) after treatment. (d) Striatal neurons in D1-Tmt/D2-GFP BAC transgenic mice did not coexpress GFP and Tmt proteins. (e) Among the degenerating striatal cells, about one-third were direct pathway medium spiny neurons (MSNs), another third indirect pathway MSN, and the remaining third were other types of striatal neurons (n=5). (f) Photomicrographs of Tmt/A-Cu-Ag-, GFP/A-Cu-Ag-, or A-Cu-Ag-positive neurons in the striatum of D1-Tmt/D2-GFP BAC transgenic mice 1 day after treatment with methamphetamine (1 × 30). Statistical analysis was performed by two-way analysis of variance and post hoc Newman–Keuls analysis. Bar indicates 30 μm (a), 25 μm (d), 500 μm (c), or 10 μm (f). BAC, bacterial artificial chromosome; GFP, green fluorescent protein; Tmt, tomato red.

Figure 3

Some degenerating nigrostriatal axons are observed in sagital sections of methamphetamine-treated animals. (a) Representative photomicrographs of sagital sections of the brain of a mouse that received methamphetamine (3 × 5) stained for amino-cupric-silver (A-Cu-Ag) or tyrosine hydroxylase (TH) 1 day after drug delivery. (b) Note that some degenerating A-Cu-Ag axons can be observed in the nigrostriatal pathway among many intact TH-stained axons. Bar indicates 500 μm (a) and 30 μm (b).

Figure 4

Methamphetamine induces degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc). (a–d) Left, histograms show the number of tyrosine hydroxylase (TH)- (b), TH/amino-cupric-silver (A-Cu-Ag)- (c), A-Cu-Ag-positive cells (d), and the accumulated mean of each of them (a) in the SNpc, counted by stereology in sections of mice treated with saline (sal) or methamphetamine (3 × 5), (3 × 10), or (1 × 30) 1 day or 3 days after the treatment (mean±SEM, n=4–6 per group). ^*,**p<0.05, 0.001 vs sal. Statistical analysis was performed by Student's t-test. Right, high magnification photomicrographs of nigral sections stained for TH/A-Cu-Ag illustrating TH-stained neurons in saline animals (e), and neurons seen in animals treated with methamphetamine 3 × 5, 3 × 10, or 1 × 30 (f–i). (f) Neurons that have only faint remaining TH staining. (g) Degenerating dopaminergic neurons stained with TH and A-Cu-Ag. (h) Degenerating neurons that do not express TH. (i) A-Cu-Ag-stained apoptotic bodies. Bars indicate 10 μm. (j) Degeneration of nigrostriatal neurons may be caused by different death pathways. Photomicrographs of neurons in the SNpc of mice show normal neurons in a saline control (upper row), and degenerating neurons 1 day after treatment with methamphetamine (lower row). Positive FluoroJade staining, eosinophilic necrotic red neurons stained with hematoxylin and eosin (H&E), and Nissl-stained apoptotic bodies were observed in mice after methamphetamine (3 × 5) treatment but not in saline animals. Bar indicates 10 μm.

Figure 5

Time course of methamphetamine (3 × 5) effects: neurotoxicity in the striatum, substantia nigra pars compacta (SNpc), and motor behavior. (a) Photomicrographs of tyrosine hydroxylase- (TH) or amino-cupric-silver- (A-Cu-Ag) stained sections of the striatum of mice treated with saline (sal) or methamphetamine (3 × 5) 3 h, 12 h, 1 day, 3 days, 7 days, and 30 days after the treatment. Bar indicates 500 μm. (b) Ultrastrutural evidence of nitration of a striatal dendrite at 3 h after methamphetamine (3 h, upper middle) and, at 1 day after methamphetamine, nitration (lower middle), a degenerating terminal (lower left), and degenerating TH-immunoreactive (TH-ir) axon (lower right) in the striatum. Bar indicates 0.5 μm. (c) Histograms show the proportional stained area of TH and amino-cupric-silver staining in the striatum. Data represent mean±SEM, n=4–6 per group. (d and e) Histograms show the number of TH- (d), TH/A-Cu-Ag- (e, left), A-Cu-Ag-positive cells (e, middle), and the accumulated mean of each (e, right) in the SNpc, counted by stereology in sections of mice treated with saline or methamphetamine (3 × 5), 3 h, 12 h, 1 day, 3 days, 7 days and 1 month after the treatment (mean±SEM, n=4–6 per group). (f) Methamphetamine induced a decrease in horizontal locomotor activity 1 and 3 days after treatment with methamphetamine (3 × 5), with return to control levels at 7 days after treatment (f, left). Methamphetamine (3 × 5) also induced a decrease in the vertical motor activity of the animals at 1 day after treatment, returning to control levels at 3 days, and remaining stable 7 days after the treatment (f, middle). Methamphetamine (3 × 5) impaired motor coordination, as animals showed reduced latency time on an accelerating rotarod 1 day after treatment with the drug. Motor coordination returned to normal at 3 and 7 days after treatment with methamphetamine (f, right). *p<0.05, **p<0.001 vs sal. Statistical analysis was performed by Student's t-test.