Striatal parvalbumin interneurons are activated in a mouse model of cerebellar dystonia

ABSTRACT Dystonia is thought to arise from abnormalities in the motor loop of the basal ganglia; however, there is an ongoing debate regarding cerebellar involvement. We adopted an established cerebellar dystonia mouse model by injecting ouabain to examine the contribution of the cerebellum. Initially, we examined whether the entopeduncular nucleus (EPN), substantia nigra pars reticulata (SNr), globus pallidus externus (GPe) and striatal neurons were activated in the model. Next, we examined whether administration of a dopamine D1 receptor agonist and dopamine D2 receptor antagonist or selective ablation of striatal parvalbumin (PV, encoded by Pvalb)-expressing interneurons could modulate the involuntary movements of the mice. The cerebellar dystonia mice had a higher number of cells positive for c-fos (encoded by Fos) in the EPN, SNr and GPe, as well as a higher positive ratio of c-fos in striatal PV interneurons, than those in control mice. Furthermore, systemic administration of combined D1 receptor agonist and D2 receptor antagonist and selective ablation of striatal PV interneurons relieved the involuntary movements of the mice. Abnormalities in the motor loop of the basal ganglia could be crucially involved in cerebellar dystonia, and modulating PV interneurons might provide a novel treatment strategy.

Comparisons of the area of dorsolateral striatum between the control and cerebellar dystonia mice (n = 12 in each group).not significant; Mann-Whitney U test

Fig. S2 .
Fig. S2.Comparisons of the density of parvalbumin interneurons in dorsolateral striatumComparisons of the density of parvalbumin interneurons in the dorsolateral striatum between the control and cerebellar dystonia mice (n = 12 in each group).not significant; Mann-Whitney U test

Fig. S4 .
Fig. S4.Immunohistochemistry of the STN in the control and cerebellar dystonia mice A, B: Schema of brain slices and immunostaining of the subthalamic nucleus (STN) using c-fos (green), NeuN (red), and DAPI (blue).The white dotted circles show the outline of the STN.C: Comparisons of the density of c-fos-positive cells in the STN (n = 12 in each group).n.s., not significant; Mann-Whitney U test Abbreviations: STN, subthalamic nucleus; DAPI, 4′,6-diamidino-2-phenylindole

Fig. S5 .
Fig. S5.Comparisons between the positive ratio of FoxP2-and PV-positive cells among c-fos-positive cells in the globus pallidus externus of the cerebellar dystonia mice.A: Immunostaining of the globus pallidus externus (GPe) of the cerebellar dystonia mice using c-fos (green) and parvalbumin (PV) (red).B: Immunostaining of the GPe of the cerebellar dystonia mice using c-fos (green) and FoxP2 (red).C: Comparisons between the positive ratio of FoxP2-and PV-positive cells among c-fos-positive cells in the GPe of the cerebellar dystonia mice (n = 8 in each group).***p < 0.001; Mann-Whitney U test Abbreviations: PV, parvalbumin; DAPI, 4′,6-diamidino-2-phenylindole

Fig
Fig. S6.The result of the preliminary experiment of drug administration.Comparisons of dystonia rating scale scores at 24, 48, and 72 postoperative hours among the following four groups of drug administration: "D1 agonist and D2 antagonist," "D1 agonist and saline," "D2 antagonist and saline," and "saline and saline" (n = 6 in each group).not significant; Mann-Whitney U test Abbreviations: D1 ago, dopamine D1 agonist; D2 anta, dopamine D2 antagonist

Fig. S7 .
Fig. S7.The c-fos-positive ratio of striatal PV interneurons in the drug-treated mouse model of cerebellar dystonia.

Fig. S8 .
Fig. S8.Changes in the dystonia rating scale of cerebellar dystonia mice treated with second immunotoxin.Comparisons of dystonia rating scale scores at 24, 48, and 72 postoperative hours between cerebellar dystonia mice with selectively ablated dorsolateral parvalbumin interneurons using a second immunotoxin and control (n = 8 in each group).*p <0.05; **p < 0.01; n.s., not significant; Mann-Whitney U test

Fig. S9 .
Fig. S9.Time course of the methodology

Fig. S10 .
Fig. S10.Negative control of immunohistochemistryNegative control of immunohistochemistry was detected using fluorescein, cyanine 3, and their merge.