Investigating the role of striatal dopamine receptor 2 in motor coordination and balance: Insights into the pathogenesis of DYT1 dystonia

Abstract

DYT1 or DYT-TOR1A dystonia is early-onset, generalized dystonia. Most DYT1 dystonia patients have a heterozygous trinucleotide GAG deletion in DYT1 or TOR1A gene, with a loss of a glutamic acid residue of the protein torsinA. DYT1 dystonia patients show reduced striatal dopamine D2 receptor (D2R) binding activity. We previously reported reduced striatal D2R proteins and impaired corticostriatal plasticity in Dyt1 ΔGAG heterozygous knock-in (Dyt1 KI) mice. It remains unclear how the D2R reduction contributes to the pathogenesis of DYT1 dystonia. Recent knockout studies indicate that D2R on cholinergic interneurons (Chls) has a significant role in corticostriatal plasticity, while D2R on medium spiny neurons (MSNs) plays a minor role. To determine how reduced D2Rs on ChIs and MSNs affect motor performance, we generated ChI- or MSN-specific D2R conditional knockout mice (Drd2 ChKO or Drd2 sKO). The striatal ChIs in the Drd2 ChKO mice showed an increased firing frequency and impaired quinpirole-induced inhibition, suggesting a reduced D2R function on the ChIs. Drd2 ChKO mice had an age-dependent deficient performance on the beam-walking test similar to the Dyt1 KI mice. The Drd2 sKO mice, conversely, had a deficit on the rotarod but not the beam-walking test. Our findings suggest that D2Rs on Chls and MSNs have critical roles in motor control and balance. The similarity of the beam-walking deficit between the Drd2 ChKO and Dyt1 KI mice supports our earlier notion that D2R reduction on striatal ChIs contributes to the pathophysiology and the motor symptoms of DYT1 dystonia.

Keywords: Cholinergic interneurons; Corticostriatal plasticity; DYT1/DYT-TOR1A dystonia; Dopamine D2 receptor; Medium spiny neurons.

Figure 1

Schematic diagram of the generation of Drd2 ChKO mice. Chat-cre+/−Drd2 loxP+/− (ChDHet) mice were crossed with Drd2 loxP−/− mice to generate ① Chat-cre+/−Drd2 loxP−/− (Drd2 ChKO), ② ChDHet, ③ Drd2 loxP−/−, and ④ Drd2 loxP+/− mice. PCR-based tail DNA genotyping of Chat-cre and Drd2 loxP loci (top left).

Figure 2

Electrophysiological characterization of D2R function in Drd2 ChKO mice. (A) Representative recording traces before and after the bath application of the D2R agonist quinpirole (10 μM, 90 seconds). The yellow area indicates the 30 seconds used to determine spontaneous firing property before drug application. The green area indicates 30 seconds used to determine the frequency after 60 seconds of drug application. (B) Increased spontaneous firing frequency of ChIs in Drd2 ChKO and ChDHet compared to control mice before quinpirole treatment. (C) All genotypes showed a reduced firing rate after the quinpirole application. (D) Drd2 ChKO and ChDHet mice showed significantly increased frequency ratios of after/before quinpirole over the control mice. Vertical bars represent means ± standard errors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 3

Striatal protein levels in Drd2 ChKO and Drd2 sKO mice. Representative western blot images and their quantifications of striatal ChAT (A), AChE (B), and their loading controls (GAPDH) in Drd2 ChKO mice. Representative western blot images and their quantifications of striatal D2R (C) and their loading controls (GAPDH) in Drd2 sKO. Vertical bars represent means ± standard errors.

Figure 4

Impaired motor coordination and balance in Drd2 ChKO and ChDHet mice. (A) Timeline of the behavior tests. Lack of motor deficits in Drd2 ChKO and ChDHet mice at 3 months of age on the accelerated rotarod test (B-C) and the beam-walking test (D). Lack of motor deficits in Drd2 ChKO and ChDHet mice at 7 months of age in the rotarod test. 1-6: trial number. 1-3 trials on the first day and 4-6 trials on the second day. (E-F). Drd2 ChKO and ChDHet mice displayed excessive slips on the beam-walking test (G). **p ≤ 0.01.

Figure 5

Impaired motor coordination and balance in Drd2 sKO mice. (A-B) Drd2 sKO mice displayed reduced latency to fall in the rotarod test compared with control and sDHet mice. 1-6: trial number. 1-3 trials on the first day and 4-6 trials on the second day. (C) Lack of motor deficits in the beam-walking test in Drd2 sKO mice. *p ≤ 0.05.

Figure 6

Impaired motor coordination and balance in Dyt1 KI mice. (A-B) Dyt1 KI mice showed motor deficits on the first day but not across all six trials on the accelerated rotarod test. 1-6: trial number. 1-3 trials on the first day and 4-6 trials on the second day. (C) Dyt1 KI mice displayed excessive slips on the beam-walking test. *p ≤ 0.05.