Twenty-four hours after the last drug doses, behavioral tests were conducted. These tests were arranged sequentially from least stressful to most stressful and were carried out during the light cycle under top lighting in order to lessen potential circadian variability.

The novel object recognition (NOR) test, used to assess learning and memory, is based on the innate preference for novelty that mice exhibit, where mice that remember the familiar object will take more time exploring a novel object. This test was accomplished by following the procedure described by Antunes and Biala [30]. The test took place over three consecutive days: habituation day, training day, and test day (days 20, 21, and 22, respectively). On habituation day, mice were placed individually into a wooden box (40 cm × 40 cm × 40 cm) and were allowed to explore the environment (without objects) for 10 min. On training day, mice were placed individually in the middle of the box and given 10 min to visually explore two identical objects placed along the diagonal of the box (one in the northwest corner and one in the southeast corner). On testing day, one of the previously explored objects was replaced by a novel object (the diagonal used on the training day was the same as that used on the testing day), and each mouse was given 3 min to explore the objects in the box. Objects that are simply discriminated by mice with the presence of protrusions/intrusions on the surface and have a comparable degree of complexity (texture, shape, color patterning, and brightness, etc.) were selected in order to diminish bias due to object preference [31]. Moreover, objects that are mouse-sized or only slightly larger to encourage exploration were used, and they were made of nonbreakable material, to avoid object damage during experimentation and hence interfere with the continuing testing and to avoid animals' injury. Great attention was directed to the object odors; thus, multiple copies of the sample objects were available and each copy was used once only for the same animal and all objects were cleaned with 70% ethanol after each test to eliminate possible bias due to odors left by previous animals [32]. Exploration was defined as directing the nose to the object at a distance of less than 2 cm, while sitting on the object was not considered exploration. The animals' behavior was video recorded, and the following parameters were calculated:

(1) Discrimination Index. Difference in time exploring the novel and familiar objects divided by the total time spent exploring both objects (this value varies between −1 and +1, with a negative score indicating that the mouse spends more time exploring the familiar object, a zero-score indicating that the mouse has no preference, and a positive score indicating that the mouse spends more time exploring the novel object).

(2) Recognition Index. Time spent by the animal exploring the novel object as a percentage of the total exploration time.

The Morris water maze (MWM) test, used to investigate spatial memory and learning, was performed as described by D'Hooge and De Deyn [33]. A stainless-steel circular tank (150 cm in diameter and 60 cm in height) was filled with water (25°C ± 2°C) to a depth of 35 cm and divided into four quadrants. A black platform (10 cm width, 28 cm height) was positioned inside the target quadrant, and the tank was filled to 2 cm below the top of the platform. The platform was maintained in the same location during the training and testing procedures. During testing, the platform was made invisible to the mice by making the water opaque with a purple-colored nontoxic tempera paint. It is important to ensure that animals can swim adequately before performing the training and testing procedures [34]. This was accomplished by allowing each mouse to swim in the pool to reach a platform that is maintained above the water level. The animal that can swim directly to the visible platform without difficulty was used to perform training and testing procedures. Memory acquisition trials (120 s/trial) were performed over four consecutive days (days 18, 19, 20, and 21), twice a day, with at least 15 min between trials. During training, animals were left to locate the hidden platform in the target quadrant. If the mouse found the hidden platform within 120 s, it was kept there for an additional 20 s before being removed, while if it failed to find the hidden platform during the designated time, the mouse was gently guided to the platform and kept there for 20 s. The mean escape latency was calculated as the time taken by each mouse to find the hidden platform and was used as an index of acquisition or learning. On the fifth day (day 22), mice underwent a probe trial session, in which the platform was removed, and each mouse was given 60 s to explore the pool. The time each mouse spent in the target quadrant in which the hidden platform was previously placed was determined and used as an indicator of retrieval or memory.

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