Behavioral manifestations in the chronic MPD
The present study examined and compared the cognitive and motor skills in the chronic MPD with severe nigrostriatal neurodegeneration shortly after and beyond 8 weeks of Parkinson's syndrome development. In association with marked loss of nigrostriatal dopaminergic cells and DA content, we observed that the chronic MPD showed significant deficits in (1) gait consistency pattern; (2) the learning capacity when challenged with the cued version of water maze; (3) balance and motor coordination skills on the challenging beam and walking grid; (4) the spontaneous and amphetamine-stimulated locomotor behaviors in the open field. Noticeably, these diminished behavioral responses lasted for at least 8–12 weeks after MPTP/probenecid treatment. The inhibited behavioral manifestations were apparently associated with the severe loss of nigrostriatal transmission, since the function of the hippocampus remained unaffected as shown by their unaltered ability to learn the spatial reference version of the water maze.
One of the clinical features of advanced PD is truncal rigidity that creates difficulty for affected patients to maintain an upright posture and a normal gait; thus, while walking they tend to take small steps in a hasty pace, which would result in frequent falls . Totally replicating the PD-liked postural and gait deficits in quadrapedal rodents is not expected to be physiologically feasible. At best, a gait analysis technique that measures and compares the variability of stride length and stepping pattern may be applied to rodent models of PD . Indeed, as demonstrated in the present study, the gait analysis revealed that the severe chronic MPD has a relatively shorter stepping length and a less consistent gait pattern certainty than the non-Parkinsonian mice. Therefore, this technique does provide a reliable approach for assessing the experimental outcomes and for predicting the severity of neurological lesions comparable to the clinical gait variability as reported in human PD [18, 19].
Human studies through several neuropsychological tests have reported that PD patients without dementia have a reduced ability for acquiring cue-based probability learning tasks with no impairment in the process of recollective memory; these findings suggest that neostriatum plays an important role not only for learning and performing motor skills but is also essential for processing the gradual, incremental learning of associations known as habitual learning [20, 21]. In the rodent literature, the cued version of the MWM is a stimulus-response or habitual task, which requires an intact network of the basal ganglia in order for successfully acquiring the task . Therefore, lesions present in the basal ganglia would suppress the learning ability of rodents to associate a unique cue located on the platform as a visual sign with a safe landing place in the water maze. In the present study and to our knowledge for the first time, we tested and confirmed that a deficit in the cued version of the MWM could be demonstrated not only shortly after MPTP/probenecid treatment but could also be associated with long-term and severe nigrostriatal neurodegeneration in the chronic MPD . Considering that various attempts for behaviorally characterizing different MPTP mouse models of PD have produced inconsistent results so far, the findings of this study indicate that the cued version of the MWM would be a useful test for assessing short-term and long-term habitual learning deficit associated with MPTP-induced lesions in the basal ganglia of mice.
As mentioned earlier, the spatial reference version of the MWM requires the rodents to use various cues from the environment in the close vicinity of the maze for locating the escape platform and this skill is dependent on the integral function of the hippocampus. Several dissociation studies have clearly distinguished the two cognitive learning systems independently involving the hippocampus and the basal ganglia [22, 23]. When MPTP was administered centrally or through nasal inhalation, rats developed short-term impairment in their learning of the cued version but not the spatial reference version of the MWM task [24–26]. This notion is substantiated by the observations of the current study, in which we detected no change in the performance of the spatial reference version of the MWM in mice 1 week or 8 weeks after MPTP/probenecid treatment. Hence, there is no evidence suggesting that chronic MPD mice have developed learning deficits linked to the hippocampus, and we have not found published reports describing a direct toxic effect of MPTP on the hippocampus that would dysregulate the cognitive function.
In this study, we strove to determine whether the order of administering the two different versions of MWM to the same animal would impact the outcome of learning the respective task. We therefore randomly altered the sequence for conducting the two versions of MWM in the chronic MPD. The results were consistent regardless which order of the task that we used first, because it is apparent that they are functionally regulated by two distinctive neuronal network systems. Furthermore, we performed a control experiment illustrating that the chronic MPD mice did not have physical impairment that would hinder their ability for swimming, since the amount of time for them to swim across the maze without visual cues was indifferent from that of the control mice.
The extrapyramidal system of the basal ganglia has long been recognized for its control and refinement of motor performance and coordination [27, 28]. We examined the short-term and long-term motor and balancing skills in the chronic MPD animals by monitoring their walk on a challenging beam and a wired grid. The challenging beam task has been successfully used for demonstrating motor deficit in a transgenic mouse model of PD that overexpresses α-synuclein . The grid walk task has been widely used for testing animal models for spinal injury, ischemia and traumatic brain injury [29, 49]. Both tests confirmed that the chronic MPD mice exhibited motor and balance deficits not only within 2 weeks but also persisted for at least 10 weeks after the MPTP/probenecid treatment when compared to the control animals. The prolonged motor dysfunction displayed by the chronic MPD mice appears to resemble the movement disability and imbalance in the human PD.
The gradual loss of nigrostriatal neurons and depletion of the neurotransmitter, DA have been considered as the basis of locomotor deficit in Parkinson's disorder, which can be demonstrated in laboratory animals by monitoring their ambulatory movement in the open-field. In the present study, we measured both the spontaneous and the amphetamine-stimulated movement in the chronic MPD and we found that these animals exhibited a long-term deficit in horizontal movement whether the striatal DA system was unstimulated or stimulated. These observations support the concept that the level of DA in the striatal terminal vesicles is considerably depleted as demonstrated in the chronic MPD [7, 8]; thus the normal release and stimulated release of the stored vesicular DA through the indirect action of amphetamine are both diminished. Recently, similar abnormalities in amphetamine-challenged horizontal movement in the open field that correlate with increasing levels of striatal DA depletion in an acute MPTP mouse model of PD have also been reported implicating that the amphetamine-stimulated assessment of movement in PD models can be validated .
Following an acute single injection with a moderate dose of amphetamine, it is known that the drug is taken up by the terminal dopaminergic transporter before it triggers the release of stored DA from vesicles. Nevertheless, the amphetamine effect is not specific to the dopaminergic neurons. It can be taken up by other monoamine terminal transporters in the same fashion and the involvement of norepinephrine and serotonin release may further modify the ambulation of the animal . It is also possible that amphetamine exerts its effects on locomotion via the terminal DA2 autoreceptors . MPTP is shown to reduce the DA2 receptor mRNA expression  and mice with knockout DA2 receptors have exhibited reduced spontaneous locomotion [34, 35]. Taken together, the reduced spontaneous and amphetamine-stimulated ambulatory movement detected in the chronic MPD could be contributed by either or both the depletion of DA content in the storage vesicles and a down-regulation of the DA2 receptors. The latter possibility involving the DA2 receptors requires further investigation in the chronic MPD mice.
Effect of endurance exercise on behavioral deficits in the chronic MPD
Following the investigation of behavioral deficits in the chronic MPD, we further studied the effect of endurance exercise on their behavioral deficits. Although continuous exercise for 12 weeks after MPTP/probenecid treatment did not significantly reverse the neurological deficit in the nigrostriatal dopaminergic system in this severe animal model of Parkinsonism, we are gratified to detect that endurance exercise training significantly invigorated the spontaneous movement and restored the gait pattern consistency and balance in the chronic MPD. However, exercise training did not have the same positive impact on the motor coordination and cognitive learning deficit that are also associated with the severe nigrostriatal neuron degeneration.
Behavioral assessment of exercise effect has been reported in several animal models of Parkinsonism. In the rat model of Parkinsonism induced by 6-hydroxydopamine, either voluntary running or treadmill paced exercise attenuated DA loss in the striatum with or without significant recovery of behavioral deficits [36–38]. The neuronal recovery in 6-hydroxydopamine-treated rats triggered by exercise is associated with an increase of the striatal glial cell line-derived neurotrophic factor . In an acute mouse model of Parkinsonism induced by MPTP, treadmill exercise ameliorates behavioral deficits and reverses several striatal dopaminergic indices including the loss of DA, TH-immunoreactivity, and DA transporter levels when compared to the sedentary Parkinsonian animals . In another study, high-intensity treadmill exercise in acute MPTP-treated mice leads to behavioral recovery; however, the striatal expression of DA transporter is down-regulated and the expression of TH is not changed . Differences in the exercise results obtained from animal models of Parkinsonism could be due to experimental variables such as the age and species of the animal, the method and severity of the induced nigrostriatal lesion, and the type and intensity of the exercise regimen.
The chronic MPD that is induced by MPTP/probenecid and used in this study affords a neurodegenerative model for PD. With 10 doses of MPTP (25 mg/kg) plus probenecid (250 mg/kg), the chronic MPD attains a severe level of neurodegeneration showing neurochemical, histological, behavioral and pathological characteristics resembling that of PD at advanced stages . Offering exercise training to the severe chronic MPD animals does not appear to produce neuroprotective or neuroregenerative effect to the dopaminergic neurons. It is not expected that exercise or other pharmacotherapeutic approaches would deliver such promises in the severe chronic MPD or advanced PD, in which the majority of neurons and neurotransmitters are already irreparably lost. However, it is so encouraging to learn based on the observations of this study that exercise training can effectively reverse certain behavioral deficits, like impaired movement, imbalance and inconsistent gait pattern that are associated with the Parkinsonian syndrome in spite of the existing severe loss of dopaminergic neurons and neurotransmitter. It is not clear why other behavioral abnormalities, like the impaired cognitive learning and motor coordination skills, are not recovered by exercise training. It is possible that intact nigrostriatal system and adequate level of striatal DA are obligatory for maintaining these behaviors, like in the case of open field study with amphetamine that its pharmacological action is dependent on the undamaged DA tract for eliciting movement hyperactivity. Additional studies are necessary for exploring and substantiating such a hypothesis.