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Novel Object Recognition Test

The novel object recognition (NOR) test was used to determine working and spatial memory. In this study 75cm × 50cm × 30cm transparent box was used. Three days prior to the habituation sessions, the rats were exposed to the box to familiarize with the environment.

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On the test day, they were exposed to identical objects to acclimatize with for 5min thereafter, the rats were then returned into their home cage with food and water. Thirty minutes later, the probe test was conducted, each rat was placed inside the box with one of the object replaced by a novel one for 5 min. It is important that (a) the objects have a “neutral” shape in terms of its significance to the animal, (b) be devoid of any marked characteristics, such as odor and movement, for instance (Li et al., 2011).

The total time spent exploring the two objects was recorded. “Object exploration” is defined as directing the nose and vibrissae to the object at a distance of less than 2 cm, as if “smelling” it with caution, while bumping, turning around or sitting upon the object were not considered. Discrimination ratio, which is the difference in exploration time, expressed as the ratio of total exploration time with both objects in the choice phase (this ratio allows to adjust for individual or group differences in the total amount of exploration time) was calculated as percentage memory index as shown below:

  • Time spent with new object X 100
  • Memory index (%) = Total time spent with both old and new object
    (Ogundele et al., 2014).

Morris Water Maze

The Morris water maze (MWM) is another apparatus designed to examine the memory impairment in rodents, thus it is highly specific for hippocampal function. This study was carried out as earlier described by Barnhart et al (2015).

Each trial began by placing the rat on the platform for 20 s in a bid to allow orientation to extramaze cues found around the apparatus. After orientation, rats were gently lowered into the pool by facing the wall at one of 3 positions (i.e., each at the center of the wall of a different quadrant not housing the platform). After the rat was released into the pool, the observer had to retreat away from the pool to a constant position within the room, such that he served as an additional distal visual cue.

Maximum swim time was set at 60 s. Any rat that locates the platform before 60 s was removed from the pool immediately, while the rats that are unable to locate the platform after 60 s of swimming were gently guided to the platform and allowed to re-orient to the distal visual cues for an additional 20 s before being eventually removed from the pool. After removal from the pool, each rat was manually dried with a terrycloth towel and placed in a plastic cage with wood shavings for at least 5 min before returning to the home cage. Each rat was trained twice a day for 2 days with an inter-trial interval of approximately 30 min. Training was conducted at roughly the same time every day in bid to minimize variability in performance due to time of day (Morris, 1984).

To examine spatial reference memory, a probe test was administered 24 h after the last training session. During the probe test, the platform was removed from the pool and the rat was allowed to swim freely for 1 min. The rationale of this task was to determine number of time the initial location of the platform was crossed/visited.