**2. Methods**

Male CD1 mice (n = 15) with an initial weight of 33 ± 3 g were used; the mice were maintained in 12:12 light/dark cycles, with free access to water and food (except on the days of motor test evaluation). Mice were divided into three groups (see **Table 2**).

### **2.1 Melatonin pretreatment**

The pretreated group was administered, melatonin 10 mg/kg mixed with Nestlé® Cerelac in 0.3 ml water orally in the afternoon (2:30 p.m.); the animals received the dose for 30 days before the MnCl2/Mn(OAc)3 inhalation.

### **2.2 Motor evaluation**

On day 23 of melatonin administration, the animals of the three groups were trained in the beam walking test and the reaching task.

### *2.2.1 Beam walking test*

This test evaluates the mice's ability to cross a narrow beam (3 mm wide) to reach an enclosed safety platform [64]. A wooden device with two pedestals was used, to which a 1 m long wooden beam with a 15° inclination and 3 mm width was attached (**Figure 1**), where the animals had to walk from the bottom of the device until they reached their home box. During training, animals were placed at the beam start with no inclination and trained over 4r days (four trials per day). Once the animals crossed the beam in a ~20 s interval, they received two more consecutive trials with the inclined beam. The time it took for the animals to cross the beam (total time) [27] was recorded with a stopwatch; a maximum time of 16 s was expected, and if at the end of this time the mouse did not cross, the activity was terminated. The parameters evaluated by this test are motor impairment in slowness, balance, and muscle stiffness, in addition to the alternate use of the limbs [28].

### *2.2.2 Single pellet reaching task*

Besides the beam-walking training, the mice were taught the single pellet reaching task. Each mouse was placed inside an acrylic box 19.5 cm long, 8 cm wide, and 20 cm high, with a 1-cm wide vertical slit in the front of the box. A 0.2 cm thick plastic shelf (8.3 cm long and 3.8 cm wide) through which the animal had to reach a pellet with its preferred forelimb and eat it (**Figure 2**). A total of 20-milligram food pellets were


**Table 2.** *Group distribution.*

*Melatonin Pretreatment Effect in a Parkinson Disease Experimental Model Induced… DOI: http://dx.doi.org/10.5772/intechopen.106001*

**Figure 1.** *The beam walking test dimensions. The home box is located at one end of the beam.*

placed in indentation spaced 1 cm away from the slit and centered on its edges. Before training and testing, the mice were food-deprived for 24 h. Afterward, they received a restricted diet of ~10 g/kg body weight-adjusted to keep their weight constant. Each animal reached 20 pellets each day during the testing period. Every time the animal took the pellet and brought it to its mouth, it was scored as a success, and when it could not hold it or fell off, the reach was scored as a miss [65]; each animal was given 20 opportunities. The qualitative evaluation comprised the "reaching performance" analysis, the postural shift, impairments in limb extension, aim, supination-pronation of the paw during grasping, and the pellet release into the mouth. The following sequence of movements of the forelimbs was considered [27]: 1. The mouse is placed facing the box slit; 2. Raises the forelimb adjusting its posture to project the limb toward the food pellet, and 3. Holds the pellet, returns its forelimb to the mouth, and introduces it.

This test helps determine the motor deterioration in terms of slowness in the sequence of movements, tremors, and a decrease in strength and precision [28]. The evaluations of both tests were carried out once a week for 5 months.

The inclusion/exclusion criteria of the mice trained to continue with the experiment were those animals that took less than 17 s to perform the test in the case of the beam test. Only those mice that obtained 16 or more successful executions were used for the single pellet reaching task.

#### **2.3 Manganese inhalation**

Mn inhalation was performed as described previously by our group [66]. Ten mice were placed in an acrylic compartment inhaling the mixture of 0.04 M MnCl2 and 0.02 M Mn(OAc)3 (Sigma Aldrich Co., Mexico), 1 h twice a week for 5 months. Five control mice inhaled deionized water for the same time. Inhalations were performed in closed acrylic boxes (35 cm wide × 44 cm long and 20 cm high) connected to an ultra-nebulizer (Ultra Neb DeVilbiss, IL, USA) with 10 l/min continuous flux. The ultra-nebulizer produces drops in a 0.5–5 μ range. A vapor trap was placed on the opposing side with a sodium bicarbonate solution to precipitate the residual Mn. During inhalation, mice were constantly monitored for respiration rate, depth, and regularity. The exposure system's temperature, oxygen level, and Mn concentration were continuously examined.

#### **Figure 2.** *The single pellet reaching apparatus. The figure shows the dimensions of the reaching box.*

After 40 inhalations, when significant motor alterations were observed, mice were anesthetized and sacrificed using a lethal dose of sodium pentobarbital. The animals were perfused via aorta with phosphate buffer saline (0.1 M pH 7.4) containing 2% glutaraldehyde and 2% paraformaldehyde. The brain was removed and placed in a fixative solution for 2 h and processed for tyrosine hydroxylase (TH) immunocytochemistry and Golgi impregnation method.
