Male Swiss albino mice (30–50 g; from the animal facility of the Federal University of Santa Catarina, Florianópolis, Brazil) and Sprague–Dawley rats (240–250 g; Charles River Laboratories, L’Arbresle, France) were used. Animals were housed in standard cages with free access to food and water, and were maintained under controlled standard conditions (12 h dark/light cycles starting at 7:30 a.m., 22°C temperature, and 66% humidity). All manipulations were carried out between 0900 and 1600 h. Procedures in this study were performed in accordance with relevant guidance from the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80-23), the Guide for the Care and Use of Laboratory Animals (Clark et al., 1997), and European Union directives (2010/63/EU). The ethics committees of the relevant institutions (CEUA/UFSC and CEEA/UB) approved the protocol. Efforts were made to minimize suffering and reduce the number of animals used in the experiments.

Reserpine (Sigma-Aldrich, St. Louis, MO, United States) was dissolved in 0.1% acetic acid for subcutaneous (s.c.) administration. GUO (Sigma-Aldrich) was dissolved in saline (NaCl 0.9%) containing 0.5% methylcellulose for oral (p.o.) administration. The 6-hydroxydopamine (6-OHDA; Sigma–Aldrich) was dissolved in a saline solution containing 0.05% ascorbic acid. DL-serine 2-(2,3,4-trihydroxybenzyl) hydrazide hydrochloride (benserazide; Sigma-Aldrich) and 3,4-Dihydroxy-L-phenylalanine (L-DOPA; Abcam Biochemicals, Cambridge, United Kingdom) were dissolved in saline for intraperitoneal (i.p.) administration.

Mice were administered reserpine (1 mg/kg, s.c.) or vehicle (0.1% acetic acid solution) twice at an interval of 48 h. GUO (3, 5, 7.5, or 10 mg/kg; p.o.) was administrated 20 min before behavioral testing and 24 h after the last injection of reserpine (Figure 1A). To quantify the occurrence of oral dyskinesia, mice were placed individually in a glass cylinder (13 cm diameter) and hand-operated counters were used to count TJM frequency. Mirrors were placed under the floor and behind the back wall of the cylinder to allow observation when the animal faced away from the observer. TJMs were defined as rapid vertical deflections of the lower jaw that resembled chewing, but were not directed at any particular stimulus (Salamone et al., 1998). If TJM occurred during a period of grooming, they were discounted. The incidence of these oral movements was measured continuously for 10 min.

After treatment with reserpine alone or reserpine plus GUO (Figure 1A), catalepsy behavior was assessed by placing the forepaws of mice on a horizontal bar (6 mm diameter) positioned at 4.5 cm above the bench surface. The duration of catalepsy, which was defined as an immobile posture, was measured while the animal kept both forepaws on the bar, with a cut-off maximum of 180 s. Three trials were carried out and the results were analyzed using the mean value of the three trials, as adapted from Santos et al. (2013).

The spontaneous locomotor activity of mice after reserpine or reserpine plus GUO treatment was tested in the open-field test. The apparatus consisted of an acrylic box measuring 45 cm × 45 cm × 45 cm, with each mouse placed in the center and recorded for 10 min with a video camera system. The distance traveled by each animal was analyzed using Bonther Activity Monitoring software (Bonther, Co., Brazil).

The spontaneous locomotor activity of rats was tested in an open-field Plexiglas^® arena box measuring 1 m × 1 m × 1 m. Each rat was placed in the center and recorded for 5 min, as described above.

Experimental hemiparkinsonism was induced in rats by unilateral injection of 6-OHDA in the medial forebrain bundle, as previously described (Fernández-Dueñas et al., 2015). Rats were stereotaxically injected with 6-OHDA (8 μg of 6-OHDA in 4 μL of saline containing 0.05% ascorbic acid) at anterior–posterior (AP; -2.2 mm), medial–lateral (ML; -1.5 mm), and dorsal–ventral (DV; -7.8 mm) locations with respect to the bregma (Paxinos and Watson, 2007). To minimize damage to noradrenergic neurons, rats were pretreated with desipramine hydrochloride (10 mg/kg, i.p.) 20 min before surgery.

Three weeks later the extent of dopamine deafferentation was checked by assessing the rotating behavioral response to L-DOPA administration. In brief, rats were injected with L-DOPA (50 mg/kg, i.p.) in the presence of benserazide hydrochloride (25 mg/kg, i.p.), an inhibitor of DOPA decarboxylase that minimizes peripheral metabolization of L-DOPA, and the number of full contralateral turns were recorded during a 2 h period. Dopamine deafferentation was considered successful in animals made at least 200 net contralateral rotations.

Thereafter, animals were housed for 3 weeks before being used in the behavioral analyses. GUO was administered orally in a vehicle (0.5% methylcellulose and 2% DMSO) 40 min before benserazide (25 mg/kg; i.p.). Subsequently, L-DOPA (6 mg/kg; i.p.) was delivered after 20 min. The animals were then placed in the rotametry chambers, as previously described (Hodgson et al., 2009), and the number of contralateral rotations was recorded over a 2 h period.

L-DOPA-induced dyskinesia were triggered in hemiparkinsonian rats by twice daily administration of L-DOPA (6 mg/kg, i.p.) plus benserazide hydrochloride (15 mg/kg, i.p) for 22 consecutive days. L-DOPA-induced abnormal involuntary movements (AIMs) were scored by a blinded experimenter following a previously described rat dyskinesia scale (Winkler et al., 2002). In brief, rats were injected with L-DOPA, placed in individual transparent plastic cages, and observed every 20 min for 220 min. Three AIM subtypes were monitored (i.e., axial, forelimb, and orolingual) and their respective severity scored from 0 to 4, as previously described (Winkler et al., 2002). Enhanced manifestations of otherwise normal behaviors, such as rearing, sniffing, grooming, and gnawing, were not included. AIM ratings were performed on treatment days 1, 7, 14, and 22 during the chronic L-DOPA administration phase. We calculated integrated AIM scores for each animal and behavioral session using the sum of all three AIM subtypes. AIM was also expressed as an area under the curve (AUC) analysis.

Data are represented as means ± SEM. Comparisons among experimental and control groups were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test when comparing GUO treatments or Tukey’s post hoc test when comparing to internal control within the behavioral test, if any. Statistical significance was accepted when P < 0.05.

Reserpine administration to mice was performed to evaluate the ability of GUO to counteract reserpine-mediated changes (Figure 1A). We first determined the change in spontaneous locomotor activity. While it was significantly reduced by reserpine administration, acute GUO treatment (3, 5, 7.5, or 10 mg/kg, p.o.) was unable to reverse the change [F_(4,43) = 0.2241, P = 0.9234] (Figure 1B).

Next, we assessed the ability of GUO to reduce reserpine-induced TJMs, a parameter known to ameliorate by antiparkinsonian drugs (Collins-Praino et al., 2011). Reserpine-administered animals did, indeed, show a significant increase in TJMs that was partially blocked by GUO administration (Figure 1C). One-way ANOVA revealed significant differences between the GUO-treated reserpinized mice [F_(4,23) = 5.603, P = 0.0027], with a significant reduction in reserpine-mediated TJMs observed at 7.5 mg/kg GUO (P < 0.01) (Figure 1C). Interestingly, treatment with 10 mg/kg of GUO was unable to preclude reserpine-induced TJMs, thus resulting in a U-shaped dose-dependent GUO activity (Figure 1C).

Finally, reserpine-induced catalepsy was assessed as an experimental model of akinesia and bradykinesia (Duty and Jenner, 2011). Reserpine treatment induced a cataleptic state, as measured by the latency time of mice to move forepaws from the bar in the bar test, and acute GUO treatment significantly attenuated this increase in latency time [F_(4,40) = 3.518, P = 0.0149] (Figure 1D). GUO induced significant reductions in reserpine-mediated catalepsy at doses of 7.5 mg/kg (P < 0.01) and 10 mg/kg (P < 0.05) (Figure 1D).

Overall, although GUO did not reverse reserpine-induced locomotor activity depression, it did ameliorate TJM and catalepsy symptoms in reserpinized mice. Therefore, we considered that antiparkinsonian efficacy was shown in this classical pharmacological animal model of acute PD.

After assessing the effects of GUO on reserpinized mice, we evaluated its effectiveness in unilateral 6-OHDA-lesioned rats, a classic animal model of experimental parkinsonism based on toxin-mediated destruction of the dopaminergic nigrostriatal pathway (Schwarting and Huston, 1996). Accordingly, we evaluated the impact of acute GUO treatment on spontaneous locomotor activity and contralateral rotation in 6-OHDA-lesioned rats (Figure 2A). First, we assessed spontaneous locomotor activity of control and hemiparkinsonian animals. While in 6-OHDA-lesioned animals GUO did not have any effect, in saline-lesioned mice the one-way ANOVA analysis revealed a significant GUO-induced increase in spontaneous locomotor activity at 3 mg/kg (P < 0.05) (Figure 2B). Overall, GUO was unable to potentiate spontaneous locomotor activity in 6-OHDA-lesioned rats.

In the hemiparkinsonian animal model, asymmetric motor behavior is observed following dopaminergic treatment (i.e., L-DOPA) because of unilateral dopamine depletion in the nigrostriatal pathway (Duty and Jenner, 2011). Interestingly, when using submaximal doses of L-DOPA, it is possible to potentiate contralateral rotations with other pro-dopaminergic drugs (e.g., A_2AR antagonists) (for review, see Vallano et al., 2011). Therefore, we determined whether GUO could promote contralateral rotations in 6-OHDA-lesioned animals with submaximal (6 mg/kg) L-DOPA dosing in hemiparkinsonian rats (Figure 2C). GUO administration alone, up to 10 mg/kg, did not result in asymmetric turning behavior in 6-OHDA-lesioned rats (data not shown). However, GUO did dose-dependently induce contralateral turning behavior when administrated before the subthreshold dose of L-DOPA (Figure 2C). One-way ANOVA revealed significant differences between GUO treatments [F_(3,36) = 11.65, P < 0.001] (Figure 2C), with GUO inducing significant contralateral rotations at 3 mg/kg (P < 0.05) and 10 mg/kg (P < 0.001) (Figure 2C). Overall, GUO enhanced the effects of L-DOPA with a minimum efficacious oral dose of 3 mg/kg.

Chronic L-DOPA use in PD is associated with the development of LIDs. Therefore, we assessed the potential antidyskinetic activity of GUO after inducing LIDs in 6-OHDA-lesioned rats through chronic L-DOPA administration and monitoring for the emergence of AIMs over time (Figure 3A). AIM severity significantly (P < 0.01) increased after 1 week of L-DOPA treatment (Figure 3A, inset, day 7), thus LID increased during the first 40 min L-DOPA post-injection and remaining elevated for an additional 40 min (Figure 3A, day 7). After 2 weeks of L-DOPA treatment, the increase in LID was higher (P < 0.001) (Figure 3A, inset, day 14) and sustained in time (i.e., 150 min) (Figure 3A, day 14). Finally, after 3 weeks of L-DOPA administration a similar AIMs increase and sustained LID incidences were observed (Figure 3A, day 22), thus reaching a LID plateau. Interestingly, the observed time-course in our LID animal model resembled the so-called peak-dose dyskinesia in PD (Fahn, 2000). Thus, we showed that GUO treatment produced a U-shaped dose-dependent antidyskinetic activity in animals administered with L-DOPA for 3 weeks (Figure 3B). One-way ANOVA revealed significant differences between GUO treatments [F_(5,47) = 11.65, P = 4.866] (Figure 3B), with significantly maximal GUO-induced antidyskinetic activity observed at 5 mg/kg (P < 0.001) and 7.5 mg/kg (P < 0.05) (Figure 3B). Overall, GUO showed antidyskinetic activity in the LID animal model.