D3 receptor-deficient mice and MPTP-treated mice
The D3 receptor-deficient mice genotype was confirmed by PCR. Knockout mice displayed a 300-bp band, and the DNA samples isolated from wild-type mice displayed a 200-bp band using forward primer (D3F: 5′GCTCACCACTAGGTAGTTG3′) and reverse primer (D3R: (5′ACCTCTGAGCCAGATAAGC3′) (data not shown). The D3 receptor-knockout mice appeared to be healthy and had no gross physical abnormalities. The mutant mice were fertile, their litter sizes were normal, and there was no obvious sex bias in their offspring. For all subsequent studies, male D3 mutant mice were used, with male wild-type littermates as controls.
The MPTP-induced neurotoxicity mouse model was established by MPTP dose grouping. After the fourth MPTP injection and in all subsequent experiments, animals that received 15 mg/kg per injection displayed significant bradykinesia based on the open-field test and rotarod test. No significant behavioral change was observed among the mice that received 5 mg/kg MPTP per injection or the mice injected with an equal volume of saline (data not shown). Thus, 15 mg/kg/day was established as the adaptive dose for MPTP-induced neurotoxicity.
D3 receptor-deficient mice displayed no change in the time taken for the “T-turn” in the pole test
The pole test was used to examine behavioral changes in the wild-type and D3 receptor-deficient mice treated with MPTP. After three training sessions, the time taken (mean±SEM) from the head-upward position on the pole until the mouse turned its head completely downward, was recorded in the four cohorts: wild-type injected with saline, wild-type injected with MPTP, D3 receptor-deficient injected with saline, and D3 receptor-deficient injected with MPTP. The pre-injection locomotor activity baseline results were 4.1 ± 1.1, 5.2 ± 1.0, 2.2 ± 0.4, and 2.0 ± 0.4 s, respectively (Figure 1). When wild-type mice were injected with MPTP, the “T-turn” times after the fourth injection, after the eighth injection, 4 days after the final injection and 8 days after the final injection, were 26.7 ± 4.9, 35.5 ± 6.7, 30.4 ± 6.1, and 32.4 ± 6.4 s, respectively. These times were significantly longer than those of the wild-type mice injected with saline (p < 0.01 at each time point; Figure 1). These data suggest that MPTP treatment changed the locomotor activity of wild-type mice. Interestingly, when D3 receptor-deficient mice were injected with an equivalent dose of MPTP, the “T-turn” times at the four aforementioned time points were 1.4 ± 0.1, 3.3 ± 1.3, 4.3 ± 1.5, and 2.8 ± 0.7 s, respectively. These times were close to those of the wild-type and D3 receptor-deficient mice injected with saline (Figure 1). The above data suggest that the D3 receptor might weakly participate in PD-related dyskinesia and that D3 receptor-deficient mice are protected from MPTP-induced locomotor activity changes.
Total time taken in the pole test
The total time taken in the pole test was defined as the duration from when the animals were positioned head-upward near the top of the pole until they turned completely downward and landed on the floor. The average total times (mean±SEM) of the four cohorts (wild-type injected with saline, wild-type injected with MPTP, D3 receptor-deficient injected with saline, and D3 receptor-deficient injected with MPTP) after training were 12.4 ± 1.5, 17.5 ± 1.9, 12.5 ± 1.5, and 11.0 ± 1.5 s, respectively (Figure 2). When wild-type mice were injected with MPTP, the average total times at the four time points (after the fourth injection, after the eighth injection, 4 days after the final injection and 8 days after the final injection) were 33.5 ± 5.4, 41.8 ± 7.0, 36.6 ± 6.2 and 39.8 ± 6.6 s, respectively. These times were significantly longer than those of the wild-type mice injected with saline (p < 0.01 at each time point; Figure 2). When D3 receptor-deficient mice were injected with MPTP, the average total times at the aforementioned four time points were 11.0 ± 1.4, 14.0 ± 4.3, 14.5 ± 2.3 and 22.2 ± 5.9 s, respectively (Figure 2). The average total time taken by the D3 receptor-deficient mice injected with saline was slightly longer than that of wild-type mice at the following three time points: after the eighth injection (19.4 ± 6.7 s), 4 days after the final injection (21.7 ± 6.8) and 8 days after the final injection (22.5 ± 7.7). The p value at each time point was < 0.05, which might suggest that the D3 receptor-knockout genotype altered locomotor activity (Figure 2). With the exception of the time point 8 days after the final injection (22.2 ± 5.9 s), the average total time taken by the D3 receptor-deficient mice injected with MPTP was close to that of wild-type mice injected with saline (p = 0.04; Figure 2).
Total time taken in the beam test
To further confirm whether the D3 receptor-deficient mice could withstand MPTP-induced neurotoxicity, locomotor activity was assessed by the beam test. After training, the average total times taken (mean±SEM) in the beam test by the four cohorts (wild-type injected with saline, wild-type injected with MPTP, D3 receptor-deficient injected with saline, and D3 receptor-deficient injected with MPTP) were 26.0 ± 2.9, 28.1 ± 4.0, 37.9 ± 12.8 and 28.9 ± 4.2 s, respectively (Figure 3). When wild-type mice were injected with MPTP, the average total times at the four time points (after the fourth injection, after the eighth injection, 4 days after the final injection, and 8 days after the final injection) were 46.7 ± 8.8, 66.9 ± 10.4, 71.4 ± 9.8 and 86.0 ± 10.9 s, respectively. These times were significantly longer than those of the wild-type mice injected with saline (Figure 3). When D3 receptor-deficient mice were injected with MPTP, the average total times at the four aforementioned time points were 18.2 ± 4.1, 33.7 ± 7.9, 47.2 ± 9.3 and 46.6 ± 7.8 s, respectively. These times were significantly shorter than those of wild-type mice injected with saline (p < 0.01 at each time point; Figure 3). Similar to the results of the pole test, the average total time taken by the D3 receptor-deficient mice injected with saline was slightly longer than that of wild-type mice at the following two time points: 4 days after the final injection (47.4 ± 11.5 s) and 8 days after the final injection (56.6 ± 14.1 s). Taken together, the above data further suggest that D3 receptor-deficient mice can withstand MPTP-induced locomotor activity changes.
Neurohistological assessment
Because MPTP may induce PD-like neurodegeneration of dopaminergic neurons in the SNpc and because the above data show that D3 receptor-deficient mice can withstand MPTP neurotoxicity, we next evaluated the neurohistological changes in the four cohorts. Tyrosine hydroxylase (TH) is the enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to L-3,4-dihydroxyphenylalanine in the central nervous system. Changes in TH expression are associated with neurodegenerative diseases such as Alzheimer’s disease, PD, and Huntington’s disease. We therefore examined MPTP-induced changes in TH expression. All mice were subjected to neurohistological assessment 8 days after the final injection. Immunohistochemical staining for TH in the SNpc showed that the neuron density and distribution in MPTP-treated wild-type mice were reduced and withered compared with those of the wild-type mice injected with saline. Both D3 receptor-deficient cohorts displayed only slight or no neurological damage (Figure 4, bottom). When these sections were scanned and analyzed by software, the TH-positive cells of the substantia nigra sampled from wild-type mice injected with saline, wild-type mice injected with MPTP, D3 receptor-deficient mice injected with saline, and D3 receptor-deficient mice injected with MPTP comprised 30.3% ± 3.8%, 11.9% ± 3.1%, 23.9% ± 2.1% and 26.0% ± 1.6% of all neurons, respectively (Figure 4, upper). The MPTP-induced neurological damage in D3 receptor-deficient mice was less extensive than it was in wild-type mice, which suggests that D3 receptor deficiency significantly attenuated TH-positive neuron loss in the SNpc.