CONSEQUENCES OF DOPAMINERGIC AGONIST ASSOCIATED WITH BDNF TREATMENT ON BEHAVIORAL ALTERATIONS AND CYTOLOGICAL CHANGES IN AN EXPERIMENTAL MODEL OF PARKINSON DISEASE DUE TO MANGANESE EXPOSURE.

Parkinson disease (PD) is characterized by dopaminergic (DA) neuronal loss and motor alterations; in this way, an effective therapy should protect and regenerate the DA neurons and striatal innervation. Conclusion:-Our findings provide evidence that the continuous D3 receptor activation associated with the BDNF gene non-viral transfection to the SNc DA neurons induce a significant and persistent motor behavior recovery in a bilateral PD rat model. The behavioral recovery is in association with the increased TH + neurons of a well-reinnervated striatum, evidenced by the dendritic spines recovery of the striatal medium-sized spiny neurons. The pharmacological effect is improbable to explain the functional recovery since the motor behavior improvement continued two months after treatment was finished, suggesting a trophic effect. This combined treatment appears to be a favorable approach for DA cells recovering in this PD experimental model, which in our opinion is a middle PD stage because we found 66.65% cell death, and, since it has been reported that D3 receptors decrease with the PD progression (Szabolcs et al. 2012; Rangel-Barajas et al. 2015), thus this kind of treatment should be given at initial or middle stages of the disease. Moreover, we assure that the inhalation of MnCl 2 /Mn(OAc) 3 mixture is an appropriate PD model, since it provides similar behavioral and morphological changes to those observed in PD patients contributing as a

Parkinson disease (PD) is characterized by dopaminergic (DA) neuronal loss and motor alterations; in this way, an effective therapy should protect and regenerate the DA neurons and striatal innervation. Brain-derived neurotrophic factor (BDNF) is a cell survival mediator and can improve neuronal death, and the activation of DA D3 receptors seems to protect the DA neurons. Thus, there is a synergistic relationship between BDNF and D3 receptors; which would be a neuroprotective therapy. PD animal models, although exhibit some of the features, no one mimics the alterations observed in the disease. PD-Manganese (Mn) inhalation model was used in this report since it is bilateral, non-invasive and progressive. The rats were exposed to Mn 1 hour 3 times a week, and the motor tests were conducted to measure the performance as well as the progressive damage at 3 and 6 months of exposure. Subsequently, the D3 agonist treatment (7-OH-DPAT) and BDNF gene transfection to DA neurons were co-administered, and then we evaluated its effect measuring the animals' performance, and if the recovery was associated with DA neuronal and striatal dendritic spines preservation. The results showed that the animals presented PD-like motor alterations, and great DA neurons and striatal dendritic spines loss. The behavior recovery was associated with the DA neurons recuperation and with the number of dendritic spines of the striatal neurons. Thus, the BDNF overexpression in DA neurons related to the D3 receptors activation seems to be a promising approach for restoring motor alterations and DA neurons in PD.
On the other hand, the DA D3 receptor activation also demonstrates trophic effects by increasing the subventricular zone, and neostriatum neurogenesis in adult rat brain via the progenitor cells fast amplification (Van Kampen et al. 2003;Van Kampen and Eckman, 2006); thus, the D3 receptor activation stimulates mitogenesis (Chio et al. 1994;Pilon et al. 1994;Griffon et al. 1997) and increases SNc neuronal dendrites arborization (Collo et al. 2008 (Guillin et al. 2001). The activation of these receptors by specific agonists protects neurons from MPTP-induced degeneration. The effect apparently is exerted through BDNF, since blocking BDNF action eliminates such protection (Du et al. 2005). This result suggests that there is an interaction between D3 receptors and BDNF, which would regulate the expression of the D3 receptors, and those would exert their protective effect, and probably its neurogenic effect (Merlo et al. 2011) trough BDNF.
It seems that BDNF facilitates, in part, the DA D3 receptors trophic effect activation (Du et al. 2005) increasing the number of D3 receptors (Guillin et al. 2001;Sokoloff et al. 2002). So, there is the possibility that BDNF potentiates the trophic effect of the activation of dopamine D3 receptors in the adult brain.
Several PD experimental models display many of the distinctive features of the disease; however, none resembles the chronic neurodegenerative features of human PD (Betarbet et al. 2002).
When choosing an animal model for PD, one must consider the amount of similarity or discrepancy between the anatomy, physiology, and behavior between humans and animals. The existing models have been useful for understanding the etiology of the disease and compromise resources for proving new treatments (Potashkin et al. 2010). However, the loss of the nigrostriatal DA pathway that has been replicated in animals, either unilaterally or bilaterally, using a variety of selective toxins or by genetic manipulations, is rapid and not progressive, and for those derived through genetic manipulations relevant to human PD, the loss, although more progressive, may be limited in extent or may not even occur at all (Emborg, 2004). In summary, after six months of Mn mixture inhalation, striatal DA content decreased 71%, SNc showed a significant reduction in the number of THimmunopositive neurons, the animals displayed akinesia, postural instability, and action tremor; these alterations were improved with L-DOPA treatment. Our data provided evidence that MnCl 2 /Mn(OAc) 3 mixture inhalation produces similar morphological, neurochemical and behavioral alterations to those observed in PD, suggesting a useful experimental model for the study of this neurodegenerative disease. Additionally, Mn inhalation is progressive and bilateral, which makes it more reliable.

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Based on this background, and taking into account that BDNF itself also induces neurogenesis from stem cells in the adult brain (Benraiss et al. 2001), we decided to explore whether continuous and chronic administration of the D3 agonist 7-OH-DPAT, associated with BDNF gene transfection to DA neurons recovers the altered motor behavior induced by the inhalation of MnCl 2 /Mn(OAc) 3  Thus, the aim of this study was to characterize the motor, and cytological alterations induced by Mn mixture inhalation as PD experimental model and determine if the co-treatment (BDNF gene transfection and D3 agonist (7-OH-DPAT)) improves the motor recovery and cell death by means of open field test determining: walking distance (ambulation), rearing and walking speed, freezing time, rotarod performance and bradykinesia and counting the number of TH neurons in the SNc and the number of dendritic spines on striatal medium-sized spiny neurons.

Materials and Methods:-Ethics Statement:
The experimental protocol was carried out by the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996; Guide for Care and Use of Laboratory Animals certificated by the Secretaria de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA) (NOM-062-ZOO-1999, Mexico) and approved by the Institutional Animal Care Committee of UNAM. All efforts were made to minimize the number of animals used and their suffering. Anesthetics (sodium pentobarbital anesthesia, 35 mg/kg, i.p.) was used when appropriate under the direction of a veterinarian to relieve potential pain and distress and was given before euthanasia with the transcardial perfusion with 0.9% saline, followed by 300 ml paraformaldehyde 4%.

Experimental Protocol:
Twenty-four male Wistar rats weighing 180  10 g were housed in groups of four in hanging plastic cages under controlled light conditions (12/12 h light/dark regime) and fed with Purina Rat Chow (Mexico) and water ad libitum. Body weight was verified daily. The animals were divided into two groups: the first consisted of 18 rats that inhaled the mixture of MnCl 2 , 0.04M and Mn(OAc) 3 , 0.02M three times a week for six months to induce the PD model, and the other six were exposed to deionized water under the same conditions (control group) this group was used exclusively for cytological comparison.
Before Mn inhalation, rotarod performance and open field were tested as initial parameters (control condition) of motor coordination, postural balance and muscle rigidity of each rat to track their performance during the experiment. The rats were first evaluated in the rotarod and trained to remain on the rod at 5 and 10 rpm for two min (as described by Razgado-Hernandez et al. (2015)), discarding those animals that following three consecutive days were incapable to stay on the rod. We evaluate the performance of the motor activity in the rotarod (coordination of the legs), in the open field (distance traveled, bradykinesia (walking speed) rearing and freezing), the evaluations were repeated at three and six months while the animals were exposed to Mn mixture. After six months of Mn mixture inhalation, the experimental group (n = 18) was divided into subgroups for treatments. One group (n= 6) was treated with continuous administration of D3 agonist (7-OH-DPAT) + BDNF gene transfection; the other group (n= 6) was used as a positive control with saline solution treatment. Also, after six months of Mn mixture inhalation, the remaining rats (n= 6) were kept for five months without treatment to analyze the number of SNc dopaminergic cells to ensure that there was no cell recovery by stopping Mn inhalation (-recovery group‖). No treatment was given to the control group; these animals were maintained for histological comparison.
The D3 receptor agonist 7-OH-DPAT (Sigma, St., Louis, MO) was administered through a micro-diffusion pump (Alzet micro-osmotic pump, model 2006) that was surgically implanted subcutaneously, the pump was activated for 3 months (1mg/day), one week after the pump implantation, transfection of the BNDF (single-shot bilateral and intracerebrally in the SNc) was performed. Three months post-implantation motor performance was again tested to determine treatment effects. Motor tests were repeated two months after the pump was removed to determine if the effects were permanent.
At the end of the treatment phase, the rats were sacrificed by transcardial perfusion. The brain tissue containing the SNc and the striatum were obtained, from which 50μm sections were taken and processed with the 832 immunocytochemistry technique for tyrosine hydroxylase (TH) (for the counting of the dopaminergic neurons) and Golgi stain (for the counting of dendritic spines in striatal medium-size spiny neurons) (Figure 1). Motor Tests:-Rotarod Mn effect on motor coordination was examined by training the rats to remain on a rotarod. It has been established that rodents motor performance, the rotarod allows for the evaluation of the loss or recovery of nigrostriatal innervation in PD rodent models (Diaz et al. 1997;Rozas et al. 1998). As previously described (Razgado-Hernandez et al. 2015), the rotarod contains four-lane rotating rod (diameter 7.5 cm) and infrared beams to detect the moment of fall. The body of the animal was placed perpendicular to the rotating axis and the head against the direction of the rotation; the rat must, therefore, move forward to stay on the rod. The rats were trained twice on the rotarod at the constant speed of 5 rpm and 10 rpm for two min during three consecutive days before the first evaluation. In the evaluating phase, the rats were positioned on the rod, and their coordination was evaluated at different speeds ( Open Field Test The rats were placed in a square compartment of 83 cm per side. A camcorder recorded the activity for 20 minutes. The obtained videos were analyzed offline to determine 1) the walking distance during 20 min. 2) the total distance traveled during the recording period 3) the dwell time in any area of the field, rearing and walking speed. With these parameters, the relationship between motion and immobility during the recording period and the speed of movement were calculated (Prut and Belzung, 2003). The coordinates of the rat position in the arena were estimated frame by frame from the videos to get the movements' spatiotemporal sequence. Bradykinesia was calculated by the decrease in walking speed judged by the time taken to move from one corner of the arena to the next one, with constant speed. The area was cleaned with a water/alcohol (70%) solution before every behavioral testing to avoid probable bias due to odors and residues left by rats tested earlier. All experiments were carried out from 11:00 to 15:00 p.m.

Rotarod
The rotarod test was divided into three stages: pre-exposure, three and six months of inhalation to determine progressive damage (Fig. 2A). The results show that there was a progressive decrease in the permanence in the rod directly proportional to the months of exposure. Between the pre-exposure stage vs. three months, there was an evident decrease in the rod permanence at revolutions 20 and 25, decreasing by 46% and 8%, respectively. However, at six months of Mn exposure, the decrease in the rod permanence was overwhelming in the five revolutions evaluated (5, 10, 15, 20 and 25 rpm) the rod permanence loss was between 70 and 90%. Figure 2A shows the performance in the test.
The animals were analyzed at three months (when D3 agonist infusion pump life was terminated) and two months (no pump) after a total of five months to determine whether the recovery was progressive and permanent and compared to animals that did not receive treatment. We observed that at three months of treatment the animals still did not fully recover the motor ability to remain in the rotarod, an activity that was determined by the permanence time in the rod; but two months later (five-month total) and without treatment the animals recovered their activity, being significant the recovery in all revolutions evaluated, this was comparable to their activity in the pre-exposure stage (Fig. 2B).
835 Figure 2:-Motor coordination (rotarod test). A Mn mixture inhalation reduced the time spent on the rod; after three months of inhalation the exposed rats spent less time on the rod, but after six months, the motor deterioration was more evident, the animals fall from the rod almost immediately. In contrast, in B it is observed that 7-OH-DPAT/BDNF-transfection co-treatment improve the time on the rod, mainly after five months post treatment. Oneway ANOVA *P< 0.05 compared to control evaluation (Pre Mn exposure); @ P< 0.05 five months post treatment vs. three months post treatment, Mn saline-treated and Mn -recovery‖ groups. (Tukey multiple comparison posttests). The data are given as the mean ± SEM (n= 6 rats/group).

Open Field
Motor activity progressive deterioration of the Mn-exposed group during its active phase was determined from the distance-traveled log. Mn-exposed animals decreased their exploratory activity directly proportional to the exposure time (three and six months) compared to the pre-exposure stage. Before Mn inhalation, the animals traveled time mean was of 5875 cm in 20 min (this value was taken as 100%), at three months of exposure a significant decrease was observed, on average, each animal traveled 3250 cm which is equivalent to a 45% reduction; however, at six months, the decrease was more evident, corresponding to 66%, traveling 1985.83 cm in 20 min (Fig. 3A). It was determined that motor activity recovery is trophic because it persisted even when the animals were no longer cotreated. At the end of three months, co-treatment showed a significant tendency of the animals to recover, two months after (without pump), the animals significantly recovered their exploratory activity (Fig. 3A). We also determined the speed of walking to assess bradykinesia, ambulation, and frequency of rearing during the first ten minutes and the freezing behavior. As seen in figure 3B Mn mixture inhalation, after three and six months induced bradykinesia, which was reverted with the co-treatment. Also, the rearing decrease (Fig. 3C) and freezing behavior increase (Fig. 3D), both behaviors statistically improved with the co-treatment (Figs. 3C and 3D).
836 Figure 3:-Open Field Performance. Mn-mixture inhalation reduced the ambulatory activity evaluated by the distance traveled (A), induced bradykinesia (B), reduced the rearing frequency (C) and increased the freezing time (D) in the open field. It is evident that with the co-treatment, the animals significantly improve their behavior being more evident after five months post-treatment. One-way ANOVA *P< 0.05 compared to control evaluation (Pre Mn exposure); @ P< 0.05 three and five months post-treatment vs. Mn saline-treated and Mn -recovery‖ groups. (Tukey multiple comparison posttests). The data are given as the mean ± SEM (n= 6 rats/group).
Cytological Analysis:-TH + Nigral Cells Recovery Bilateral counting of TH + neurons was performed throughout the SNc to determine if the co-treatment regained or maintained the number of neurons. These data were compared with the control, saline-treated and Mn-exposed without treatment (-recovery‖) groups. After Mn mixture exposure, there was a significant decrease in the number of TH+ neurons of 66.65%. When counting the remaining neurons in the tissue of animals with the co-treatment of 7-OH-DPAT and BDNF (5 months post-treatment) a 100% recovery was obtained in comparison with the control, saline-treated and -recovery‖ groups, determining that there were no significant differences between control and cotreated groups. This is shown in Figures 4 and 5. Dendritic Spines Dendritic spine count was performed on neostriatal medium-sized spiny neurons to determine if the co-treatment recovers the number of dendritic spines. These data were compared with the control and Mn-exposed saline-treated and Mn-exposed -recovery‖ groups. The Mn model significantly reduced the number of spines of the medium-sized spiny neurons of the striatum (Figs. 6 and 7). The D3 agonist associated with the BDNF gene fully recovered the 838 spines. In contrast, both, Mn-exposed group (-recovery‖) and Mn-exposed saline treated showed a significant dendritic spine loss (Figs. 6 and 7). Our results showed that in the rotarod test the animals that inhaled the Mn mixture and received the co-treatment, partially recovered their performance in the motor activity, remaining longer at the speed of 20 rpm at two months 840 after the co-treatment was applied; these data are consistent with those reported by Hernandez- Chan et al. (2015), where animals at 12 weeks of 7-OH-DPAT and BDNF co-treatment, have improved motor performance in the rotarod test (10, 15 and 20 rpm) compared to the 6-OHDA lesion phase. And with Razgado et al. (2015) whose experimental animals (intrastriatally 6-OHDA lesioned) received the co-treatment 7-OH-DPAT and BDNF, presented recovery in the rotarod permanence time at 6.5 months of co-treatment.
Our open field test results showed that recovery is relative to the co-treatment administration time; at five months, the animals recover their exploratory activity 100%, increase the movement speed through the field, exploring it in greater quantity and presenting few immobility periods, so it is presumed that bradykinesia symptom is eliminated. This data is consistent with the study by Razgado et al. (2015), where rats with 6-OHDA lesion and that received the 7-OH-DPAT and BDNF co-treatment obtained similar results. It is important to mention that the treatment is optimal five months later after started, considering that the co-treatment is only present three months and two months after it is withdrawn, this means that the treatment has a trophic function because its effect remains. Once it was determined that with the co-treatment the motor alterations significantly improve, with the cytological analysis it was analyzed whether this motor recovery was related to the DA system restoration. It is important to mention that the new generation neurons must be functional; it is known that the SNc DA neurons innervate the striatum, which is the main responsible for motor activity, that is why the nigrostriatal pathway recovery is indispensable; the rescue of the striatal dendritic spines is a collateral data of DA system recovery (Yao et al. 2008;Fasano et al. 2013;Toy et al. 2014) and is a necessary condition because it is the place where the DA synaptic contacts take place (Day et al. 2006); we observe that after Mn inhalation the dendritic spine loss is 27.1% and after the co-treatment the recovery is 100%, presenting values very similar to the control group. Therefore, the complete dendritic spines recovery brought about by the D3 receptors activation associated with the BDNF transfection may explain the motor behavior recovery observed in the present work. This fact is in line with the statement that the striatal dopamine grafts efficacy also needs the dendritic spines preservation or recovery (Soderstrom et al. 2010).
The DA neurons regeneration and functionality is attributed to the synergistic relationship between the D3 receptor and BDNF. It seems that BDNF is responsible for the D3 receptors expression during embryonic development and for maintaining their expression in the adult brain (Sokoloff et al. 2002); the D3 receptor is synthesized by the SNc DA neurons (Howells et al. 2000), and is responsible for their neurogenic activity, it has been reported that when the D3 receptor is selectively stimulated, the nigrostriatal circuit is restored (

Conclusion:-
Our findings provide evidence that the continuous D3 receptor activation associated with the BDNF gene non-viral transfection to the SNc DA neurons induce a significant and persistent motor behavior recovery in a bilateral PD rat model. The behavioral recovery is in association with the increased TH + neurons of a well-reinnervated striatum, evidenced by the dendritic spines recovery of the striatal medium-sized spiny neurons. The pharmacological effect is improbable to explain the functional recovery since the motor behavior improvement continued two months after treatment was finished, suggesting a trophic effect. This combined treatment appears to be a favorable approach for DA cells recovering in this PD experimental model, which in our opinion is a middle PD stage because we found 66.65% cell death, and, since it has been reported that D3 receptors decrease with the PD progression (Szabolcs et al. 2012;Rangel-Barajas et al. 2015), thus this kind of treatment should be given at initial or middle stages of the disease. Moreover, we assure that the inhalation of MnCl 2 /Mn(OAc) 3 mixture is an appropriate PD model, since it provides similar behavioral and morphological changes to those observed in PD patients contributing as a 841 convenient experimental model for the study of this neurodegenerative disease and the animals recover with a DA treatment.