Fig 1.
Experimental timeline and effects of M5 modulation on parkinsonian forepaw deficits.
A) A schematic showing the timeline and design of this study. A total of 22 C57BL6/J mice were included in this study, they arrived and were habituated to STAT caloric supplement and mush food daily for one week before surgery. Following this, a unilateral right medial forebrain bundle (MFB) 6-OHDA lesion was performed. Mice were given 3 weeks for recovery, during which they were given STAT caloric supplement and mush food daily. At three weeks after surgery mice underwent baseline motor testing with cylinder test and an adapted version of the forehand adjusted steps test for mice to confirm lesion. Unlesioned mice were excluded from further study (forehand percent intact score above 40%). Mice then underwent a battery of motor testing to study the effects of M5 modulation on PD motor deficits which included cylinder test, forepaw adjusting steps test, and Erasmus ladder testing. In a within-subjects, counterbalanced design, mice were M5 PAM, VU0238429 (100 mg/kg, i.p.), or M5 NAM ML375 (30 mg/kg, i.p.) to understand their effects on baseline motor deficits resulting from lesion. Thereafter, mice were divided into sub-cohorts for further participation in a study on M5 modulation effects on L-DOPA motor efficacy and M5 modulation effects on L-DOPA-induced dyskinesia. In the L-DOPA-induced dyskinesia experiment, mice were given daily, chronic L-DOPA treatment in an ascending dose paradigm where each dose was given on 4 days, (1.5, 3.0, and 6.0 mg/kg). For each dose step on the 3rd and 4th day, in a within-subjects, counterbalanced design mice received vehicle + L-DOPA or M5 PAM + L-DOPA. L-DOPA-induced dyskinesia was scored using the validated rodent Abnormal involuntary movements scale (AIMs) beginning 20 min after injection. Mice were video recorded in a 3-D neurobehavioral apparatus designed to show all body angles, and data were later scored using the AIMs scale for 1 min every 20 min for 140 min. For the effects of M5 modulation on L-DOPA efficacy, we again used cylinder test, forepaw adjusting steps, and the Erasmus ladder to understand how M5 modulation affected L-DOPA-mediated behavior. In a within-subjects, counterbalanced design, mice were given 1.5 mg/kg L-DOPA, 3.0 mg/kg L-DOPA, L-DOPA(1.5 mg/kg) + M5 PAM, and L-DOPA(1.5 mg/kg) + M5 NAM. All behavior for these experiments was run 1 hour after injection of L-DOPA and PAM or NAM, as this corresponds to peak brain plasma expression of both L-DOPA and the M5 compounds. B) Shows forepaw asymmetry in the cylinder test resulting from 6-OHDA lesion at baseline. Data were analyzed via paired-samples t-test. *** p < 0.001. C) Shows forepaw stepping from forehand adjusting steps test. Data were analyzed via two-way repeated-measures ANOVA with within-subject factors of treatment (3: Baseline (off treatment), PAM, NAM), and side (2: left(lesioned) paw, right (intact) paw). **** p < 0.0001. Overall there was no effect of treatment on the akinetic motor deficit in the left (lesioned) forepaw. D) Shows forepaw stepping from forehand adjusting steps test on vehicle + L-DOPA, PAM (100 mg/kg) + L-DOPA, or NAM (30 mg/kg) + L-DOPA. As in C, a two-way repeated measures ANOVA was conducted, which revealed that there were no effects of treatment but there was an effect of lesion, with the left (lesioned) forepaw taking significantly less steps despite treatment. **** p < 0.0001. E) Tyrosine hyroxlase positive (TH+) neurons in the substantia nigra pars compacta (SNc) were averaged by animal over each hemisphere, and then were submitted to an independent samples t-test to compare total number of TH+ neurons in the left (unlesioned) vs. right (lesioned) hemispheres. As expected, there was a significant reduction in TH+ neurons in the lesioned SNc. ** p < 0.01. F) Shows representative images of TH staining the SNc in an unlesioned animal (top) and in a 6-OHDA lesioned animal (bottom). Overall, TH+ neurons are severely reduced on the lesioned side.
Table 1.
Erasmus ladder deficits in 6-OHDA lesioned mice vs. controls.
Fig 2.
Differences between age-matched control and 6-OHDA lesioned mice in Erasmus ladder.
For all data included in this section, we used age-matched C57BL6/J mice (n = 10) as comparators to our 6-OHDA lesioned C67BL6/J mice (n = 22). This is the first description of Eramsus ladder deficits in 6-OHDA lesioned mice. All data were analyzed using two-tailed independent-samples t-tests, alpha = 0.05. Overall, we observed that 6-OHDA lesioned mice show bradykinesia in multiple aspects of the Erasmus ladder, A –I. A) There is no change in overall trial duration between 6-OHDA lesioned and control mice. B) 6-OHDA lesioned mice take more time to complete backward steps on the high rung. C) 6-OHDA lesioned mice take more short steps on the high rung than controls, which recapitulates aspects of human gait in Parkinson’s disease, as human PD patients show short, shuffling steps. D) 6-OHDA lesioned mice take more time to complete long steps on the high rung. E) 6-OHDA lesioned mice take more time to complete long steps on the high rung. E) 6-OHDA lesioned mice take longer to make high rung jumps than controls, F) 6-OHDA lesioned mice take longer to complete High to low rung shortsteps G) 6-OHDA lesioned mice take longer to complete high to low rung longsteps, H) 6-OHDA lesioned mice take longer to complete low to high rung longsteps. I) 6-OHDA lesioned mice take longer to complete low to high rung jumps. J) 6-OHDA lesioned mice take a higher percentage of high rung short steps than control mice, showing a clear parkinsonian phenotype. * p < 0.05, ** p < 0.01, *** p < 0.001.
Table 2.
Erasmus ladder motor improvements with M5 PAM and NAM.
Fig 3.
M5 receptor modulation alleviates Erasmus ladder deficits in 6-OHDA lesioned mice.
All Erasmus ladder data were analyzed using a repeated-measures ANOVA with factor of treatment (3: no treatment baseline, PAM, NAM). Dunnett tests were used as necessary (PAM and NAM treatment were only compared with Baseline). Mice were injected with M5 PAM VU0238429 (100 mg/kg, i.p.) or M5NAM ML375 (30 mg/kg, i.p.) one hour before behavioral testing. A) There was a decrease in overall trial duration for mice treated with M5 PAM, suggesting the M5 PAM alleviates bradykinesia in the Erasmus ladder, *** p < 0.001 B) Mice treated witih M5 PAM take less time to complete high rung long steps C) Mice treated with M5 PAM show a reduction in the time it takes to complete high rung jumps. D) Mice treated with M5 PAM take less time to complete high to low rung short steps E) Mice treated with M5 PAM take a smaller percentage of short steps on the high rung, suggesting that M5 PAM improves stride length F) Mice treated with M5 PAM show a higher percentage of high rung long steps. Overall data in this figure suggest that M5 PAM alleviates Parkinsonian-like deficits in stride length and bradykinesia in our 6-OHDA lesioned model. G) Mice with more remaining DA neurons show a decreased percentage of short steps on the High rung when treated with M5 PAM, one-tailed Pearson correlation. DA neurons remaining are calculated by tyrosine hydroxylase percent intact, which is calculated by the right (lesioned) side divided by the intact side, multiplied by 100. H) Mice with more remaining DA neurons show decreased time to complete high rung jumps when treated with M5 PAM. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 4.
M5 PAM does not potentiate the effects of L-DOPA.
All data were analyzed with a repeated-measures ANOVA with factor of treatment. Tukey post-hocs were conducted as appropriate to understand significant differences. Comparisons were only made between treatment groups and no treatment baseline A) Trial duration was not affected by L-DOPA. B) The percentage of high rung short steps was reduced by 3 mg/kg of L-DOPA, suggesting an improvement in stride length. C) Time to complete high rung long steps was not significantly reduced by L-DOPA. D) Percentage of high rung longsteps was a increased in mice with 3.0 mg/kg of L-DOPA, again suugesting an improvement in stride length with L-DOPA. E) Overall trial duration was not affected when PAM or NAM were added to L-DOPA. F) Time to complete high rung short steps was not affected by co-treatment with PAM or NAM. G) Time to complete short steps was unaffected by L-DOPA co-treatment with M5 PAM and NAM. H) Percentage of high rung longsteps was not significantly changed by L-DOPA co-treatment with M5 PAM or NAM. Overall, this suggests that M5 PAM does not affect L-DOPA’s motor efficacy. * p < 0.05.
Fig 5.
M5 receptor modulation does not affect L-DOPA-induced dyskinesia and does not cause dyskinesia.
Using a within-subjects design, mice were given chronic, daily L-DOPA treatment in an ascending dose paradigm (four days of each dose, 1.5, 3.0, and 6.0 mg/kg). In a within-subjects, counterbalanced design, mice received either vehicle + L-DOPA or M5 PAM VU0238429 + L-DOPA. Beginning 20 min after drug administration, animals were video recorded in a 3D Neurobehavioral Chamber (3D NC, see supplement) for one min every 20 min for 140 min. Videos were scored by a trained rater who was blind to condition using the validated rodent abnormal involuntary movements scale (AIMs) of L-DOPA-induced dyskinesia (LID). Data were analyzed for each time point using Wilcoxon Signed Ranked Test for each time point and for the overall sum of Axial, limb and orolingual (ALO) behavior. A) Shows the timeline of our experiments following initial motor testing on M5 modulators. B) Shows the median ALO sums for each time point for both vehicle + L-DOPA 1.5 mg/kg and M5 PAM, VU0238429 in black. The inset graph shows the overall sum of ALO behaviors during the 140 min of ratings. AIMs data were analyzed by non-parametric Kruskal Wallis ANOVA with Dunn’s post-hoc tests employed as appropriate. Overall, there were no significant differences in overall ALO behaviors, nor were there differences at any specific time point. C) ALO AIMs data are shown for every 20 min for 140 min for Vehicle + L-DOPA 3.0 and M5 PAM + L-DOPA 3.0. The inset graph represents the overall sum of ALO behaviors. Overall, there were no significant differences in overall ALO sums nor were their differences in timecourse of ALO behavior. D) Shows ALO AIMs on vehicle + L-DOPA 6.0 or M5 PAM + L-DOPA 6.0. Again, mirroring other doses, there were no significant differences in overall ALO AIMs scores nor in timecourse of ALO behavior. E) We compared ALO AIMs scores for peak plasma concentrations of M5 PAM alone or L-DOPA alone (1.5, 3.0, 6.0 mg/kg) using a Friedman ANOVA with test. Overall, we found that PAM did not produce robust dyskinesia, in contrast to L-DOPA alone. Data were analyzed using Friedman ANOVA with Dunn post-hoc tests as appropriate, * p < 0.05, *** p < 0.001.