Apomorphine

Three days after apomorphine screening (Figure 2A), 45 successful hemiparkinsonian rats received intraperitoneal injections of L-DOPA plus benserazide (12/6 mg/kg) once daily. L-DOPA-induced AIMs were recorded every other day. Rats were observed for 1 min every 35 min intervals for a total of 140 min following L-DOPA treatment. AIMs were evaluated using the validated AIMs scale. Orofacial, limb, and axial dyskinesia were graded from score 0 to 4: 0 = absent; 1 = occasional, present during less than half min; 2 = frequent, present during more than half min; 3 = continuous but interrupted by strong sensory distraction; 4 = continuous, not interrupted by strong sensory distraction (Winkler et al., 2002 (link); Lundblad et al., 2004 (link)). The total of axial, limb and orofacial dyskinesia was also named ALO dyskinesia and the maximum ALO dyskinesia score in each session was 48. Rotation (contralateral turns) test was performed as before (Breger et al., 2013 (link)) at day 1, 4, 12, 16, and 20, and only turns of completed 360° were counted.
From day 10, 42 rats with complete dyskinesia (ALO AIMs score = 12 at 70 min after L-DOPA) were divided into 3 groups: LID group, LID + PD98059 group, and LID + vehicle group. There was cannula implantation for each rat in the latter two groups. A volume of 3 μl PD98059 solution (0.4 μg/μl) or vehicle was infused into the right lateral ventricle over 6 min through the cannula connected to the microinjection system half hour before L-DOPA administration from day 10 to 21 (Figure 2A). The infusion needle was left in place for 2 min at the end of infusion for drug diffusion. Normal rats with sham operation and hemiparkinsonian rats were chosen as normal group and PD group, respectively. There were 14 rats in each group.
The stepping test was carried out 15 min before (pre-) and after (post-) L-DOPA treatment as previously described (Pinna et al., 2007 (link), 2010 (link)) on days 2, 10, and 18. Rats were moved on the surface of the table for 0.7 m in 4 s by the experimenter. The number of adjusting steps of left and right forelimbs in the forward directions was counted.
All behavioral tests were performed by a blinded examiner.

To test apomorphine-induced turning behavior the animals received in the neck a subcutaneous injection of 0,05 mg/kg apomorphine hydrochloride (Sigma) dissolved in 1% ascorbic acid 0,9% NaCl, and placed on metal testing bowls (MED-RSS, Med Associates) for 45 minutes. The number of contralateral rotations was digitally recorded, and 6-OHDA-lesioned animals presenting more than 100 rotations in 45 minutes were considered to have a nearly complete lesion (n = 28), whereas the ones presenting less than 100 rotations were classified as having an incomplete lesion (n = 13). Sham animals (n = 19) showed no turning behavior.

The unilateral 6-OHDA lesions of the MFB in rats were carried out as previously described (Carvalho et al., 2013 (link)). Rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) followed by the injection of desipramine (25 mg/kg, i.p.) to protect noradrenergic neurons. Then the rats were placed on a stereotaxic frame (SN-2N; Narishige, Tokyo, Japan) and 6-OHDA (12 μg/4 μl) was injected into the left MFB (AP –4.4 mm, ML –1.2 mm, DV –7.8 mm; Paxinos and Watson, 1998 ). After the injection, the glass pipette was left in place for 5 min to allow diffusion. Two weeks later, rats were subcutaneously injected with apomorphine (0.05 mg/kg, s.c.) to verify the effectiveness of 6-OHDA lesion (Wang et al., 2009 (link)) and those showing more than 20 contralateral turns per 5 min were selected for further experiments. All rats used in the following experiments turned consistently toward right of >30 turns per 5 min.

Figure 1 shows the timeline of the experiments. Four weeks after 6-OHDA injection, the lesioned rats which passed the apomorphine-induced contralateral rotation test were divided into three groups according to the number of pulses received: sham-iTBS, 1 block-iTBS (one iTBS block) and 3 block-iTBS (three blocks of iTBS). All three groups were subjected to behavioral tests, histology and immunohistochemistry at 30 or 80 min post-stimulation. In the specific case of behavioral tests including the object-place recognition (OPR) and hole-board (HB) test, the first trial of the two tests started at 30 or 80 min after real (different blocks) or sham-iTBS. Besides, hippocampal local field potential (LFP) was recorded in all three groups for 3 h, including 1 h pre-stimulation baseline and 2 h post-stimulation recordings 4 weeks after 6-OHDA injection.

Adult Male Sprague-Dawley rats (Experimental Animal Center of Xi’an Jiaotong University, Xi’an, China) weighing 270–320 g were used in this study (n = 130 rats). Room temperature was kept constant at 22 ± 2°C and the light/dark cycle was 12 h/12 h. Rats had free access to water and food. All experimental procedures were conducted according to the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8,023, revised 1978), and approved by the Ethics Committee for Animal Experimentation of Xi’an Jiaotong University. All efforts were made to minimize the number of animals used and their suffering.
Desipramine hydrochloride, 6-OHDA hydrochloride, and apomorphine hydrochloride were purchased from Sigma–Aldrich (Sigma–Aldrich, St. Louis, MO, USA). 6-OHDA was dissolved in artificial cerebrospinal fluid containing 0.02% ascorbic acid; apomorphine was prepared in saline with 0.02% ascorbic acid; desipramine was dissolved in saline. These drugs were prepared on the day of use.