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Fig. 7. Pharmacokinetics of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and MPP⁺ following sporadic injections and continuous pump infusions. (a) Levels of MPTP and MPP⁺ at different time points in osmotic minipumps used for continuous MPTP administration. (b-d) Striatal MPP⁺ levels produced by continuous infusion of MPTP (30 mg MPTP/kg daily; b), by four separate injections of MPTP (4 × 20 mg MPTP/kg, 2 h apart; c), and by a single injection of MPTP (30 mg/kg; d). Asterisks indicate statistically significant differences (P < 0.05) to the initial value (a and b) or peak concentration (c and d) as judged by the ANOVA test.

Fig. 8. Neurotoxicity caused by discontinuous 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administrations. (a) Tyrosine hydroxylase (TH) immunostaining of substantia nigra from control mice, mice treated with a single MPTP dose (30 mg/kg), and mice treated with four separate MPTP doses (20 mg/kg each, 2 h apart; scale bars for all panels, 720 m m). (b and c) Low- and high-magnification views of fluoro Jade B staining of degenerating neurons in the S. nigra of a mouse injected with four doses of MPTP. (Scale bars, 560 m m in b and 90 m m in c). (d) Number of TH-positive cells in the substantia nigra of control mice (C), mice treated with a single MPTP dose (30 mg/kg), and mice treated with four separate MPTP doses (20 mg/kg each, 2 h apart; n = 10 mice). Mice were killed 7 days (this figure). Analysis 30 days after injections (n = 10; data not shown) exhibited no recovery. (e) Tyrosine hydroxylase (TH) immunostaining of the striatum from control mice, mice treated with a single MPTP dose (30 mg/kg), and mice treated with four separate MPTP doses (20 mg/kg each, 2 h apart; scale bars for all panels, 230 m m). (f-i) Dopamine, DOPAC, homovanillic acid (HVA), and serotonin levels in the striatum (all in ng/mg protein) of control mice (C) and mice treated with a single MPTP (30 mg/kg) or with four separate MPTP doses (20 mg/kg each, 2 h apart)(n = 10 mice). Mice were killed 7 days (this figure). Analysis 30 days after injections (n = 10; data not shown) revealed no recovery of catecholamine levels in this time interval. In d and f-i, single and double asterisks indicate statistically significant [P < 0.05] differences of the sample compared to control mice only (single asterisks) or both to control mice and mice treated with the single MPTP administration.

Fig. 9. Neurotoxicity induced by continuous 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administrations. (a) Representative sections of the substantia nigra from mice that were continuously treated for 28 days with control pump infusions or with infusions of 1, 5, or 30 mg MPTP/kg bodyweight daily were stained for GFAP (scale bars, 470 m m). (b) TH staining of sections from the ventral striatum from mice that were continuously treated for 28 days with saline infusions (control) or with infusions of 1 mg, 5 mg or 30 mg MPTP/kg body weight daily. (Scale bars,640 m m.)

Fig. 10. Neuronal degeneration in the substantia nigra induced by continuous MPTP administration. (a and b) Hematoxylin and eosin (H&E)-stained sections of the substantia nigra from control mice (a) and mice treated with 30 mg/kg MPTP infusion for 28 days (b; scale bars, 120 m m). (c) Quantitation of the density of neurons as measured by H&E staining in mice continuously treated for 28 days with control infusions or 1, 5, and 30 mg/kg MPTP infusions (single asterisk, significantly different from control; double asterisk, significantly different from all other conditions; n = 10). (d) Fuoro-Jade B staining of neurons in the substantia nigra of mice treated with 30 mg/kg MPTP infusions (scale bar, 20 m m).

Fig. 11. Deletion of a -synuclein protects against dopaminergic neuronal cell death. Staining of neurons for tyrosine hydroxylase (TH) in the substantia nigra of wild-type (WT) and a -synuclein KO mice that were treated with control infusions or 30 mg/kg daily MPTP infusions for 28 days (scale bars, 560 m m). Detail shows immunogold labeled a -synuclein in the peripheral area of a neuronal inclusion (Scale bar, 0.1 m m).

Fig. 12. Neurotoxic effects and neuronal inclusions in the locus coeruleus caused by continuous MPTP administration. (a) Dopamine b -hydroxylase immunostaining of the locus coeruleus from control and MPTP-treated mice (28 day infusion at 30 mg MPTP/kg daily; scale bar, 400 m m). (b) Cell counts of dopamine b -hyroxylase positive cells in the locus coeruleus from control mice (C), mice treaded with a single MPTP injection (30 mg/kg) or four separate injections (4 × 20 mg/kg; mice were analyzed 30 days after the injection), and mice treated with continuous pump infusions (PI) of 1, 5, and 30 mg MPTP/kg body weight daily for 28 days (n = 10). (c) Fluoro-Jade B staining of degenerating neurons in locus coeruleus from a mouse treated continuously with MPTP (scale bar, 7 m m). (d) Norepinephrine levels in various brain areas (PC, piriform cortex; FC, frontal cortex; H, hippocampus; A, amygdala) from control mice or mice treated with continuous pump infusions of MPTP (30 mg/kg daily) for 28 days. (e) Clustered ubiquitin and a -synuclein immunostaining in the locus coeruleus after continuous MPTP treatment (scale bars, 30 m m). (f) representative neuronal inclusion body in MPTP-treated locus coeruleus (scale bar, 0.18 m m), which is absent from controls (data not shown). *, P < 0.05 compared with controls; **, P < 0.05 compared with control, sporadic, and continuous low dose of MPTP.

Movie 1. Control mouse. The movie shows the spontaneous motor activity in open field of a mouse receiving control infusions (i.p. pump delivering saline solution).

Movie 2. MPTP mouse. The movie shows the dramatic decrease of spontaneous motor activity in open field of a mouse receiving continuous infusions of MPTP (i.p. pump delivering MPTP at the dose of 30 mg/kg, daily).

Movie 3. MPTP+Apo mouse. The movie shows the recovery of spontaneous motor activity occurring when a mouse receiving continuous infusions of MPTP (i.p. pump delivering MPTP at the dose of 30 mg/kg daily, as in Movie 2) is administered with the mixed dopamine agonist apomorphine (Apo, s.c., at the dose of 5 mg/kg, daily).