## 2 Materials and Methods
### 6-OHDA Lesion and L-DOPA Administration  
Rats were anesthetized with 2\(-\)3% isoflurane through an animal anesthesia ventilator system (RWD Life Science, Shenzhen, China) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Based on previous PD rat model studies, rats were unilaterally lesioned by injection of 6-OHDA (12 ug/2.4 uL in 0.02% ascorbate in saline (162957, Sigma-Aldrich, St. Louis, MO, USA)) into the medial forebrain bundle (MFB) (from bregma: anterior posterior (AP): \(-\)3.6 mm, medial lateral (ML): \(-\)1.8 mm; dorsal ventral (DV): \(-\)8.2 mm from the skull) using a Hamilton syringe (88000, Hamilton, Reno, NV, USA). The 6-OHDA was injected at a rate of 1 uL/min, and the needle was left in place for an additional 5 min to allow diffusion of 6-OHDA before being slowly retracted. To determine lesion efficacy, turning behavior was recorded 3 weeks later over a 90 min period after injection of apomorphine (0.5 mg/kg by subcutaneous injection (16094, Cayman, Ann Arbor, MI, USA)) [14]. The control rats received a sham lesion using saline.  
Similar to human LID, repeated levodopa therapy induced abnormal involuntary movements (AIMs) (including dystonia, hyperkinesia, and/or stereotypies) in the PD model rats [15]. Starting 3 days after the turning behavior test, PD rats received methyl L-DOPA (6 mg/kg, 5 mg/mL; D9628-5G, Sigma-Aldrich) and peripheral decarboxylase inhibitor, benserazide (B7283-1G, Sigma-Aldrich, 12 mg/kg, 10 mg/mL) using single daily intraperitoneal injections for 21 days. The 6-OHDA-lesioned control rats received saline using the same protocol (Figure 1A). AIMs were rated every 3 days after L-DOPA therapy six times in 180 min [16]. For each AIM category (exhibition of axial, limb, oral-lingual, and locomotor movements), a severity score of \(0-4\) was assigned and summed for each time point. Rats with average AIMs >12 were assigned to the LID group, whereas those with no apparent dyskinesia and average AIM scores \(\leq\)12 constituted the NLID group. After the final AIM rating, the rats were euthanized for analyses. The control rats were fed for the same time without treatment and were euthanized.  
Figure 1: Validation of the levodopa-induced dyskinesia (LID) rat model. (**A**) Experimental timeline. A unilateral Parkinson’s disease (PD) rat model was established by 6-OHDA stereotactic intracerebral injection of the right medial forebrain bundle. Contralateral rotation was induced by apomorphine intraperitoneal injection 3 weeks later. The validated PD rats were administered with L-DOPA and benserazide for 3 weeks to induce a rat model of dyskinesia. Involuntary movements (AIMs) were rated every 3 days during the treatment. (**B**) AIMs of LID rats at various times. At 30 min intervals, AIMs were rated for 60 s for each rat for a total of 180 min. For each AIM category, the scores were summed for each time point. Data are shown as mean \(\pm\) SEM (\(n\) = 15). (**C**) The typical AIM of LID  
rats. The limb AIM was manifested by tremor of the distal extremity and grasp, and contraction of the shoulder muscles, with hemiballismus. The axial AIM is manifested by torsion of the head, neck, and upper torso, or even loss of balance. The locomotive aim was shown as turning in circles to the opposite side of the injury. (**D**,**E**) Western blot of TH in the striatum and substantia nigra (SN) and c-FOS, p-ERK, and ERK in the striatum of control, PD, LID, and (non-LID) NLID rats (\(n=3\)-5). (**F**) Ratio of TH-positive cells in the brain of control, PD, LID, and NLID rats. TH-positive cells were significantly decreased in the striatum and substantia nigra of PD, LID, and NLID rats. (**G**) Immunostaining of TH in the brain of control, PD, LID, and NLID rats. * \(p<0.05\), ** \(p<0.01\), *** \(p<0.001\) vs. sham group; \(\#\)\(p<0.05\), ** \(p<0.01\), *** \(p<0.001\) vs. PD group.