BALB/c mice (n = 24) were separated into 4 groups based on body weight distribution. Dyskinesia was induced using 10 mg/Kg BW of dopaminergic D₂ blocker (Haloperidol i.p. 28 days) in n = 12 animals [13] [14] . Subsequently, n = 6 animals were treated with 100 mg/Kg BW (i.p. 7 days) VD₃ (−D₂/+VDR) while the remaining set of n = 6 animals were untreated (−D₂). A separate group of n = 6 animals received VD₃ for 28 days (100 mg/Kg BW intraperitoneal; +VDR) while the control (n = 6) received normal saline for 28 days (NS; intraperitoneal).

At the end of the treatment phase (Day 28 for Vehicle, −D₂, +VDR; Day 35 for −D₂/+VDR), the animals were examined in various tests for motor function. All animals were familiarized with the behavioral testing tools during the treatment phase and were moved to the testing area 72 hours before the commencement of the tests.

Abnormal Involuntary Movements (AIMs): Involuntary movements were accessed in AIMs (Orolingual and Limb AIMs) tests for dyskinesia. The values were recorded as amplitude of movement and basic movement using the methods of Cenci and co-workers [15] . Three experienced scientists assessed each animal independently using the grading scale of 1 - 4. The average score was adopted in each case for the amplitude and basic movement score at 0, 15 and 30 minutes respectively.

Parallel Bar Test: Motor coordination was accessed on two raised 1m long (1 mm) parallel bars (3 cm apart) mounted on the 60 cm high wooden frame. The animal was placed at the 0.5 m mark (center of the raised bars) following which we determined the duration taken by the mice to make a 90˚ turn (latency of turning; LOT) [16] .

Rotarod: The test involved three trials of 3 minutes each (T₁, T₂ and T₃) separated by an inter trial time of 90 minutes. The time spent on the Rotarod in T₁, T₂ and T₃ was determined and averaged to calculate the latency of fall (LOF).

Cylinder Test: Each mouse was placed in a 500 ml transparent beaker (cylinder) and was allowed to explore the walls of the cylinder with the forelimbs while standing on the two hind limbs [15] . The number of times an animal explored the wall of the cylinder with the forelimb was counted to determine the average score of climbing attempts for each group.

Bar Test: the magnitude of motor impairment was also measured in the bar test [15] . The forelimbs of the animal were placed on a raised wooden bar for 3 minutes. The time taken by the animal to move the limb off the raised bar was measured for the treatment and the control groups.

Electrophysiological recordings of extracellular calcium hyperpolarization currents were obtained from the basal nucleus (CPu), Motor cortex (M1; L4 - L6), Hippocampus (CA1) and the Prefrontal cortex (PFC) using chronically implanted wire electrodes. Thirty minutes before the implant, animals received 2 mg/Kg i.p. meloxicam and were deeply anesthetized using 100 mg/Kg Ketamine and 5 mg/Kg Diazepam (i.p.) to keep the mice immobile but awake for basal motor functions (corneal reflexes and diaphragmatic movement) [17] [18] . Using a stereotaxic frame, the scalp was removed above the bregma to expose the cranium. Periosteal tissue was removed using hydrogen peroxide solution and a cotton bud. The M1, CPu, PFC and Hipp were located using a calibrated grid to determine the position and depth (electrode length) relative to the bregma [M1 (AP: +3.34 mm ML: +3 mm DV: +2.5 mm), CPu coordinates (AP: +2.28 mm ML: +3 mm DV: +6 mm), Hipp (AP: −4.4 mm ML: +2 mm DV: +3 mm), PFC (AP: +2.2 mm ML: +1 mm DV: +2.5 mm)]. A dental drill was used to make holes in the cranium following which insulated wire electrodes were inserted to the appropriate depth. The ground electrode was placed on the cranium of the contralateral side. Subsequently, the implant was covered by orthodontic resin to hold the electrodes in position during the recording procedure. The terminal wires of the electrodes were connected to the amplifier through small head-sockets in preparation for immobile awake recordings. The data from the amplifier (Spiker Box; Backyard Brains, Michigan, USA) was captured on the Audacity software v4.2 and analyzed in Sig View v2.1 (Signal Labs, USA) to determine the extracellular summation epoch neural activity (calcium signals) expressed as Frequency (Hz) per unit time (Figure 1(g) and Figure 2(a)).

Data was presented as mean ± SEM; analyzed using ANOVA and Tukeys Post-Hoc test. Statistical Significance was set as P < 0.05^*, P < 0.01^**, P < 0.001^***.

The global AIMs score for Limb (Li) and Orolingual (Ol) AIMs were assessed from 0 - 30 minutes after the animals were pretreated with 10 mg/Kg Haloperidol (intraperitoneal). Basic and amplitude scores were allotted by three independent scientists on a scale of 1 - 4 at 0, 15 and 30 minutes. Subsequently, the basic and amplitude scores were converted to the global AIMs score (Basic X Amplitude) for the −D₂ group after 28 days of haloperidol treatment to confirm dyskinesia, and −D₂/+VDR treatments 7 days after VD₃RA intervention (Day 35).

Li AIMs: Abnormal involuntary movement was observed in the limbs of the haloperidol treated mice after 28 days (−D₂). The frequency and threshold of such movements increased between 0 - 15 minutes after haloperidol treatment and decreased sharply between 15 - 30 minutes. After 7 days of VD₃RA intervention (−D₂/+VDR), the AIMs score decreased significantly when compared with the −D₂ at 0 - 15 and 15 - 30 minutes (Figure 3(a)).

Ol AIMs: Haloperidol treatment (−D₂) caused abnormal involuntary movements of the Orolingual area and was characterized by uncontrolled chewy mouth movements and tongue protrusion. The threshold (AIMs score) was at its peak shortly after haloperidol was given intraperitoneally (0 - 15 minutes) and decreased significantly thereafter (15 - 30 minutes). Similar to the observations in Li AIMs, VD₃RA intervention also reduced the severity of Ol AIMs when the −D₂/+VDR treatment was compared with the −D₂ group. Lower AIMs scores were recorded in this group throughout the duration of the test (from 0 - 15 and 15 - 30 minutes) (Figure 3(b)).

Thus, VD₃RA treatment after haloperidol induced dyskinesia significantly reduced the threshold of Li and Ol AIMs seen as a decline in the AIMs score from 0 - 30 minutes post haloperidol treatment. It is important to mention that the mice were also assessed for Axial AIMs (Ax) but showed no significant change in ipsilateral and contralateral turns when compared with the control (untreated animals).

After establishing that dyskinesia was induced in the animals, the dyskinesia models (−D₂ and −D₂/+VDR) were compared with the VD3RA treated (+VDR) and untreated control groups (NS) in motor function tests.

Rotarod: After haloperidol induced dyskinesia (−D₂), the LOF decreased significantly on the treadmill when compared with the control (P < 0.05). Subsequent VD₃RA intervention (−D₂/+VDR) caused an increase in the LOF (motor function) when compared with the −D₂ treatment and the control (P < 0.05). However, VD₃RA treatment (28 days; +VDR), without prior induced dyskinesia, significantly increased the LOF (motor function); when this treatment was compared with the control and the −D₂/+VDR (P < 0.01) (Figure 1(a)).

Cylinder Test: Motor activity was measured as a function of climbing attempts score. Haloperidol induced dyskinesia (−D₂) caused a decrease in motor activity which was seen as a decline in climbing attempts score versus the control (Figure 1(b)). VD₃RA intervention in dyskinetic mice (−D₂/+VDR) increased the climbing attempts to match control scores as no significance was observed between the −D₂/+VDR and the control (untreated) mice. Similar to our observations in the Rotarod test, VD₃RA treatment (+VDR) without prior induced dyskinesia significantly increased the climbing attempts (motor function) when compared with the control and −D₂/+VDR (P < 0.05) (Figure 1(b)).

Parallel Bar Test: All groups were compared with the untreated control (standard) such that a significant increase or decrease in LOT score was considered a decline in motor function. LOT scores decreased after haloperidol induced dyskinesia when the −D₂ treatment was compared with the control, thus suggesting a decline in motor coordination (P < 0.05). Subsequent VD₃RA intervention (−D₂/+VDR) significantly increased the LOT when compared with the control (P < 0.05) and −D₂ (P < 0.001). This outcome suggests a decline in motor coordination when compared with the control as the animals were characterized by freezing of movements on the raised bars. VD₃RA treatment (+VDR), in non-dyskinetic control mice, caused a decline in LOT when compared with the control (P < 0.05) and was not significant versus −D₂ treatment (Figure 1(c)); thus suggesting a decline in motor coordination when compared with the control.

Bar Test: No significant change was observed in the bar test when the treatment groups were compared with the control. However, empirical data suggests that −D₂, −D₂/+VDR and VD₃R treatments did induce hyperkinesia, thus, causing a reduction in time taken for the mice to remove the limbs from the raised platform (versus the control; Figure 1(d)).

Haloperidol induced dyskinesia (−D₂) caused a decline in motor function (increase in Ol/Li AIMs), abnormal neural activities in the M1 (L4 - L6) and CPu- when the −D₂ was compared with the control. Prominent changes in the neural spike trains involved an increase in M1 activity (Figure 1(f)) and a reduced threshold of CPu outputs when the dyskinetic group (−D₂) was compared with the control (Figure 1(e)). After VD₃RA intervention (−D₂/+VDR), an increase in motor activity (decreased threshold of Ol/Li AIMs; Figure 3(a) and Figure 3(b)) and CPu neural frequency bursts were observed in the VD₃RA intervention group (−D₂/+VDR; Figure 1(f)). In addition, the threshold of the M1 and CPu activities (RMS) for the intervention group (−D₂/+VDR) decreased

versus the control and dyskinetic mice (−D₂; Figure 1(e) and Figure 1(f)). Furthermore, haloperidol induced dyskinesia (−D₂) increased M1 but reduced CPu neural outputs and was associated with a decline in motor function, while VD₃RA treatment (VDR+) of control mice decreased M1 activity and was associated with an increase in motor coordination. However, VD₃RA intervention after haloperidol induced dyskinesia (−D₂/+VDR) decreased M1 activity and increased CPu outputs―when compared with −D₂―leading to an improvement in motor function. From these findings, we deduced loss of CPu burst frequencies was associated with abnormal movement in dyskinetic and VD₃RA (only) treated mice. By contrast, VD₃RA intervention reduced abnormal movements (dyskinesia) by increasing CPu burst frequencies in vivo. From these findings, both M1 and CPu neural outputs were affected in dyskinesia and subsequent VD₃RA intervention.

The effect of haloperidol induced dyskinesia was further investigated on memory-related brain centers (PFC and Hipp). Unilateral electrode recordings in immobile awake mice showed a resting state PFC activity and Hippocampal CA1 burst in the control. Haloperidol induced dyskinesia increased the PFC frequency in immobile awake animals and reduced the CA1 burst activity when compared with the control (Figure 2(c)). This treatment was also

characterized by an irregular RMS threshold for PFC and CA1 versus the control (Figure 2(b)). However, VD₃RA intervention in dyskinetic mice (−D₂/+VDR) reduced the PFC neural frequency (reduced RMS threshold) while restoring the CA1 burst pattern (increased RMS threshold). Thus we deduced that VD₃RA intervention can significantly restore memory alterations associated with dyskinesia by increasing CA1 burst and reducing PFC activity (opposite for dyskinetic mice). Interestingly, VD₃RA treatment of control animals (+VDR), however, increased PFC activity and reduced CA1 bursts similar to the effect of −D₂. Due to movement impairments, memory function was not assessed in these animals in behavioral tests (Figure 2(b) and Figure 2(c)).

After confirming the role of haloperidol in −D₂ induced dyskinesia, and the effect of VD₃RA in ameliorating the dyskinesia-linked movement disorders, we examined the general effects of prolonged D₂R blockade and VD₃RA intervention on motor function in these animals using arrays of motor function tests.

Dyskinetic mice (−D₂) showed a decline in motor function in behavioral tests; Rotarod, Cylinder test, parallel bar test but exhibited no significant change in the raised bar test when compared with the control. The dyskinetic mice recorded a decline in latency of fall (LOF) when compared with the control (P < 0.05; Figure 1(a)). Similarly, these animals scored lower in climbing attempts when assessed in the cylinder test versus control (untreated animals) (P < 0.05; Figure 1(b)). Subsequent analysis of motor coordination on raised parallel bars revealed a decline in motor coordination when compared with the control and was seen as a significant decrease in latency of turning (LOT) (Figure 1(c); P < 0.05). Surprisingly, these animals showed no significant change in motor function when examined for limb removal in a raised bar test and the outcome was rather inconclusive. However, this can be attributed to the abnormal movements associated with the limbs (due to tardive dyskinesia) rather than a swift removal of limbs as a result of motor coordination (Figures 1(a)-(d)).

Although the role of VD₃RA in Parkinsonism has been studied extensively, its importance in dyskinesia remains poorly explored. Other studies have favored experiments on the effect of Vitamins (Vitamin E) in reducing the threshold of tardive dyskinesia [19] . The anti-dyskinetic effect of Vitamin E was attributed mostly to its antioxidant properties and its role in radical detoxification [19] [20] . Similarly, VD₃ deficiency has been linked to the cause and progression of various movement disorders [21] ; including PD. By virtue to its role in radical detoxification, calcium-related signaling and general brain health, VD₃ depicts a potential therapeutic target in reducing dyskinetic symptoms similar to the effects Vitamin E [22] [23] . In support of this hypothesis, 7 days of VD₃RA intervention in the dyskinetic mice reduced Ol and Li AIMs, and was associated with an overall improvement in motor function when compared with the untreated dyskinetic mice (−D₂) (Figure 3(a) and Figure 3(b), Figure 1(a) and Figure 1(b)). In the Rotarod test, the intervention group (−D₂/+VDR) recorded an increase in LOF when compared with the −D₂ (untreated dyskinesia) (P < 0.05; Figure 1(a)). Similarly, these animals recorded an increase in climbing attempts score when compared with the untreated mice (−D₂) (Figure 1(b)). These outcomes suggest that VD₃RA can reduce dyskinesia significantly, similar to what was described for Vitamin E [19] -[21] .

The abnormal involuntary movement observed after prolonged haloperidol treatment (Figure 3(a) and Figure 3(b); Figures 1(a)-(d)) were associated with prominent changes in the M1 and CPu neural outputs in deeply sedated (immobile awake) mice. Haloperidol induced dyskinesia was characterized by an increase in M1 and CPu outputs when compared with the control. This increase in cortical and striatal outputs was attributed to the observed hyperkinesia (AIMs) in this dyskinetic mice; similar to the reports by Lindenbach and Bishop [24] . By contrast, VD₃RA intervention decreased the M1 cortical output , facilitated CPu burst frequencies and was associated with a decrease in global AIMs scores (improved motor function) in the −D₂/+VDR. Thus, these observations, and other reports [25] , suggests that a reduction in CPu burst frequency and an increase in M1 cortical outputs were associated with the observed motor deficits seen in Dopaminergic receptor inhibition.

Haloperidol induced dyskinesia (−D₂) decreased CA1 burst and reduced PFC cortical outputs in resting mice when compared with the control (Figure 2(c)). Subsequent VD₃R intervention (−D₂/+VDR) improved motor function and was associated with a restoration of hippocampal bursts while reducing the PFC cortical outputs when compared with the untreated dyskinetic mice (−D₂; Figure 2(c)). From these observations, we deduced that changes in neural activities in the PFC and CA1 were associated with haloperidol induced dyskinesia when the M1-CPu was compared with the PFC-CA1 outputs (Figure 1(f) and Figure 2(c)).

In addition, haloperidol induced dyskinesia increased M1 and PFC (cortical outputs) but decreased the CA1 and CPu burst frequencies. A similar effect was described by Wang and Goldman-Rakic on the role of D₂R in control of burst activities through its long term depression (LTD) effects on glutamergic systems; often explored in treatment of schizophrenia [26] . However, after VD₃RA intervention (−D₂/+VDR), restoration of CA1 and CPu burst frequencies were observed. This was associated with an increase in motor activity and a decrease in the threshold of abnormal movements when the animals were assessed in behavioral tests for motor function. These observations suggests that D₂R inhibition affects motor and cognitive brain centers sequel to drug induced dyskinesia, through disruption of CA1 and corticostriatal projections to the PFC and M1.