Figure 1.
αMT Induces Severe DA Depletion in the Striatum of DAT-KO Mice
(A) Tissue levels of DA in the striatum of saline-treated control WT and DAT-KO mice (n = 7 per group). Striatal levels of DA were significantly lower in DAT-KO versus WT mice (p < 0.05, Student's t-test).
(B) Dynamics of the effect of αMT (250 mg/kg IP) on striatal tissue DA in WT and DAT-KO mice (n = 5–8 per group). DA levels were significantly lower versus control values at all the time points after αMT treatment in DAT-KO mice and 2–24 hours after treatment in WT mice (p < 0.05, one-way ANOVA followed by Dunnet's multiple comparison test). The magnitude of the effect was significantly different between genotypes from 1 to 16 h after αMT injection (p < 0.05, two-tailed Mann-Whitney U test).
(C) Tissue levels of NE in the frontal cortex of saline-treated WT and DAT-KO mice (n = 7 per group).
(D) Dynamics of the effect of αMT (250 mg/kg IP) on tissue levels of NE in the frontal cortex of WT and DAT-KO mice (n = 5–8 per group). NE levels were significantly lower versus control values at time points 2–16 after αMT treatment in DAT-KO mice and at 4–16 hours after treatment in WT mice (p < 0.05, one-way ANOVA followed by Dunnet's multiple comparison test). The magnitude of the effect was not different between genotypes at any time point after αMT injection (p > 0.05, two-tailed Mann-Whitney U test).
(E) Effect of αMT on extracellular DA levels in the striatum of WT mice, measured using in vivo microdialysis. Data are presented as a percentage of the average level of DA measured in at least three samples collected before the drug administration. (Saline, n = 5; αMT, n = 7). αMT significantly decreased DA levels 60–180 min after treatment (p < 0.05, two-tailed Mann-Whitney U test versus respective time points in saline-treated controls).
(F) Effect of αMT on extracellular levels of DA in the striatum of DAT-KO mice, measured by using in vivo microdialysis in freely moving mice. Data are presented as a percentage of the average level of DA measured in at least three samples collected before drug administration. (Saline, n = 4; αMT, n = 6). αMT significantly decreased DA levels 20–180 min after treatment (p < 0.05, two-tailed Mann-Whitney U test versus respective time points in saline-treated controls). Analysis of area under curve values for 120-min periods after drug administration revealed significant difference between DAT-KO and WT groups (p < 0.05, two-tailed Mann-Whitney U test). Note also that the basal extracellular levels of DA in DAT-KO mice were significantly higher than in WT mice (predrug concentrations of DA in dialysates were: WT, 76 ± 17 fmol/20 μl; DAT-KO, 340 ± 63 fmol/20 μl).
Figure 2.
Display of Behavioral Phenotypes of DDD Mice
(A) Akinesia and rigidity of DDD mice. Photos were taken 30 min after treatment of DAT-KO mice with αMT (250 mg/kg IP).
(B) Ptosis in DDD mice. Photos were taken 3 h after treatment of DAT-KO mice with αMT (250 mg/kg IP). Lower panel: Mouse on the left side is saline-treated DAT-KO mouse, whereas the mouse on the right side is αMT-treated DAT-KO mouse.
Figure 3.
αMT-Induced Impairment in Motor Control in DAT-KO Mice
Dynamics of locomotor activity following systemic administration of αMT (250 mg/kg IP) and saline (30 min after placement in the locomotor activity chamber) in WT (A) and DAT-KO (B) mice (n = 6–8 per group). Analysis of total distance traveled for 210 min after drug administration revealed significant effect of αMT treatment (p < 0.05; Student's t-test) in DAT-KO but not WT mice (WT-saline, 516 ± 50 cm/210 min; WT-αMT, 505 ± 98 cm/210 min; DAT-KO–saline, 18,489 ± 4,795 cm/210 min; DAT-KO–αMT, 448 ± 75 cm/210 min). αMT (injected at time 0) induced profound alterations in the akinesia (C), catalepsy (D), grasping (E), bracing (F) tremor (G), and ptosis (H) tests, but did not affect the righting reflex (I) in DAT-KO mice. Behavioral tests were performed as described in Materials and Methods. At all the time points, DAT-KO mice were significantly different versus respective values (data not shown) of saline-treated DAT-KO controls (p < 0.05; Student's t-test n = 6 per group) in these tests with exception of 15-min time point for ptosis (H) and all time points for righting reflex test (I). In WT mice only the akinesia test (C) revealed minor, yet significant, effect (1.5–4 h after αMT treatment) versus values (data not shown) of the respective saline treated WT controls (p < 0.05; Student's t-test; n = 6 per group). No significant alterations in any other test at any time point examined (D–I) was noted in αMT-treated versus saline treated (data not shown) WT mice. Locomotor activity is restored in DAT-KO mice 16–24h after αMT (250 mg/kg IP) treatment (J).
Figure 4.
L-DOPA and Nonselective DA Agonists Are Effective in Restoring Locomotion in DDD Mice
DAT-KO mice were placed in the locomotor activity chamber and 30 min later were treated with αMT (250 mg/kg IP) and 1 h after αMT were challenged with single or multiple doses of a drug (interval between treatments is 1 h). L-DOPA itself (A) or in combination with carbidopa (B–D) effectively restored locomotion in DDD mice, as revealed by the significant effect of L-DOPA at doses 100 and 200 mg/kg IP, or combinations of L-DOPA/carbidopa at doses 20/20, 50/20, and 50/50 mg/kg, IP (analysis of total distance traveled for 1 h after each dose of the drug; p < 0.05, two-tailed Mann-Whitney U test versus respective values in saline-treated DDD mice; data not shown). Nonselective DA receptor agonists, apomorphine (E) at doses 2 and 3 mg/kg SC, and pergolide (F) at doses 5, 10, and 20 mg/kg IP, induced locomotion in DDD mice (analysis of total distance traveled for 1 h after each dose of the drug; p < 0.05, two-tailed Mann-Whitney U test, versus respective values in saline-treated DDD mice; data not shown). D2 DA receptor agonists bromocriptine (G), quinpirole (H), and D1 DA receptor agonist (+)-SKF81297 (I) were not effective, but the combinations of D1 and D2 DA agonists (+)-SKF81297 plus quinpirole at doses 5/1 and 10/5 mg/kg IP, induced significant locomotion in DDD mice (analysis of total distance traveled for 1 h after each treatment; p < 0.05, two-tailed Mann-Whitney U test versus respective values in saline-treated DDD mice; data not shown). Experiments were performed in 6–12 mice per group.
Table 1.
Treatments That Were Not Effective in Restoring Forward Locomotion in DDD Mice
Figure 5.
Amphetamine Derivatives at High Doses Are Effective in Reversing Abnormal Motor Behaviors of DDD Mice
DAT-KO mice were placed in the locomotor activity chamber and 30 min later were treated with αMT (250 mg/kg IP), and 1 h after αMT were challenged with single or multiple doses of drugs (in cumulative dosing experiments, the interval between treatments was 1 h). Grasping (A), catalepsy (B), and akinesia (C) tests were performed as described in Materials and Methods 1 h after each dose (the only exception is (±)-MDMA at 80 mg/kg IP where measurements were performed 2 h after the drug administration). An asterisk indicates p < 0.05 versus respective values of saline-treated DDD mice (one-way ANOVA followed by Dunnet's multiple comparison test). Experiments were performed in 6–16 mice per group. d-AMPH indicates d-amphetamine; METH, d-methamphetamine; and 4-chloro-AMPH, 4-chloro-amphetamine.
Figure 6.
(+)-MDMA Induces Forward Locomotion in DDD Mice
(A–C) DAT-KO mice were placed in the locomotor activity chamber and 30 min later were treated with αMT (250 mg/kg IP) and 1 h after αMT were challenged with single (B and C) or multiple doses (A) of a drug (interval between treatments is 1 h) (n = 10–16 per group). Repeated treatment with (+)-MDMA (30 and 60 mg/kg IP) induces forward locomotion in DDD mice (A). Analysis of total distance traveled for 1 h after 60 mg/kg IP of (+)-MDMA reveals significant effect of treatment versus respective period in saline-treated controls (p<0.05, two-tailed Mann-Whitney U test, data not shown). Dynamics (B) and dose-response (C) of locomotor effect of (+)-MDMA in DDD mice are shown. Pretreatment with D1 and D2 DA antagonists (SCH23390, 0.1 mg/kg SC plus raclopride, 2 mg/kg IP) 30 min before 100 mg/kg IP (+)-MDMA) did not affect locomotor action of (+)-MDMA (C).
(D) (+)-MDMA (100 mg/kg IP) fails to affect DA dynamics in the striatum of DDD mice as measured by in vivo microdialysis. Data are presented as a percentage of the average level of DA measured in at least three samples collected before αMT administration (n = 4). Analysis of area under curve values for 120-min periods after (+)-MDMA administration revealed no significant difference in comparison with respective values in control group (Figure 1F; p > 0.05, two-tailed Mann-Whitney U test).
(E and F) (+)-MDMA (E) as well as d-amphetamine and d-methamphetamine (F) at moderate doses potentiate locomotor-stimulating effect of subthreshold dose of L-DOPA/carbidopa (10/10 mg/kg IP). DAT-KO mice were treated with αMT as described above (A–C) and 45 min after αMT were injected with amphetamines. L-DOPA/carbidopa was injected 15 min after amphetamines, and distance traveled for 2h was measured (n = 6–15 per group). Note, that no forward locomotion was observed after these doses of (+)-MDMA, d-amphetamine and d-methamphetamine without L-DOPA/carbidopa, whereas L-DOPA/carbidopa (presented as drug dose 0) induced only a modest but significant (p < 0.05) increase in locomotion over saline-treated controls (data not shown).
Single asterisk indicates p < 0.05; double asterisks indicate p < 0.01; and triple asterisks indicate p < 0.001 versus saline-treated controls (C) or L-DOPA/carbidopa-treated (10/10 mg/kg IP) group (E and F) (two-tailed Mann-Whitney U test). d-AMPH, d-amphetamine; METH, d-methamphetamine.
Figure 7.
Nomifensine, but Not GBR12909, Is Effective in Reversing Abnormal Motor Behaviors of DDD Mice
DAT-KO mice were placed in the locomotor activity chamber and 30 min later were treated with αMT (250 mg/kg IP). Mice were challenged 1 h later with two doses (10 and 30 mg/kg IP) of each drug or saline with a 1 h interval between treatments (n = 9–15 per group). Grasping (A) and akinesia (B) tests were performed as described in Materials and Methods 1 h after each dose. A single asterisk indicates p < 0.05, double asterisks indicate p < 0.01, and triple asterisks indicate p < 0.001 versus respective values of saline-treated DDD mice (one-way ANOVA followed by Dunnet's multiple comparison test). Note that no significant differences between GBR12909-treated mice and saline-treated controls in both tests were found, whereas nomifensine-treated mice were significantly different from GBR12909-treated mice (p < 0.05) in both experimental paradigms and doses tested.