Behavioral tests

Step-through passive avoidance (PA) apparatus (UgoBasile, Comerio, Italy) is divided into two chambers: white lighted chamber and a black dark one. The grid floor of the dark chamber can be programmed to deliver an electric shock of the required intensity whenever stepped on. The two chambers are separated by an automatically operated sliding gate. Each rat was subjected to two sessions; acquisition session to acclimatize (training) in the first day and retention session (test) after 24 h from training. During the training session, rats were gently placed individually in the lighted chamber. When a rat stepped through the dark compartment, placing its 4 paws on the grid floor, the sliding door closed, and an electric shock of 1 mA was delivered for 2 s. Twenty-four hours later, rats were re-placed gently in the light chamber and their latency to step through the dark chamber was recorded and considered as a passive avoidance behavior to evaluate their memory acquisition after being exposed to an electrical shock. This test evaluates the contextual fear for assessing memory changes. The cut-off time was set to 3 min in both the training and the retention sessions (i.e., both were of equal total time); only one trial for each rat was included in each of the acquisition and retention sessions (one trial each day). Besides, no electric shocks were delivered during test sessions [23].

The apparatus consisted of a circular water pool (120 cm diameter, 60 cm in height), containing water to a depth of 15.5 cm. The water temperature was maintained at 24 ± 1 °C and was rendered opaque by addition of milk powder. The pool was virtually divided into four quadrants, i.e., North (N), South (S), East (E), and West (W). A transparent platform (10 cm diameter) was hidden 1.5 cm below the surface of water and placed at the midpoint of the fourth quadrant “SW.” The test was conducted as previously described [31, 32]. The test trial ends by either finding the platform or continuing for a maximum of 90 s. Briefly, each rat was trained to acclimatize for 4 consecutive days. Each rat was given a series of daily trials using a semi-random set of start locations. Semi-random start position sets were used such that the four positions are used, with the restriction that one trial each day for each of the four positions (each of the four start positions were used once each day) which were described previously [32]. During the first four training days, the rats were placed into the maze pool to reach the hidden platform using four semi-random set of start locations as mentioned previously (day 1: N, E, SE, NW), (day 2: SE, N, NW, E), (day 3: NW, SE, E, N), and (day 4: E, NW, N, SE). These set of start locations are designed so that rats will not be able to learn a specific order of right or left turns to locate the platform [32]. The time allowed for the rats to reach the platform is 60 s, then, rats were allowed to sit on the platform for 30 s. Those who failed to find the platform in 60 s were guided to the platform and could sit on the platform for 30 s. Each rat was subjected to four trials every day for four consecutive days as previously described [33].

On the fifth day, a probe trial was performed to evaluate the extent of memory consolidation as previously reported [34]. On the fifth day, the platform was removed, and the rats were placed and released at (NE) opposite to the site where the platform had been located (SW). The single trial consisted of a 90-s swim in the pool without the platform. The time spent in the target quadrant indicated the degree of memory consolidation after learning and the percentage of time spent in the former platform was calculated for the probe trial. All data were recorded with a video system.

The Y-maze apparatus consists of a black wood maze with 3 similar opaque arms (40 cm length, 15 cm height, and 8 cm width) intersected at 120° and were labeled as A, B, or C. The animal is positioned in the start arm B and permitted to acclimatized and explore the 3 arms for 5 min. Afterwards, rats were put at the starting area to begin the experiment. A spontaneous alternation was recorded for 5 min and counts begin when 4 paws of the rat are inside the arm and the rat had entered the three different arms sequentially. Spontaneous alteration behavior was defined as the entry into all three arms on consecutive choices in overlapping triplet sets (e.g., ABC, BCA, CAB) [35].

The total number of alternations and total arm entries (TAE) were documented, and the spontaneous alternation percentage (SAP) was computed from it according to the following formula: “the number of alternations” divided by “the total possible alternations (i.e., the total number of arm entries minus 2)” and multiplied by 100, i.e., SAP = [(number of alternations)/(TAE − 2)] × 100 [36]. Pearson’s correlation analysis [37] was performed of SAP to TAE made, to exclude the influence of hyper- or hypodynamic locomotion on the apparent cognitive endpoint [38].

Activity monitor (Opto-Varimex-Mini Model B, Columbus Instruments, OH, USA) was used to evaluate the locomotor activity of animals based on the traditional infrared photocell principle (68 × 68 × 45 cm) equipped with 15 infrared (IR) beams (wavelength = 875 nm and diameter = 0.32 cm), spaced 2.65 cm apart, and scan rate = 160 Hz. The principle of measurement depends on the emittance of evenly spaced infrared light beams, where beam interruptions caused by movements of the animal are sensed and counted. Prior to starting the test, each rat was gently placed in the activity monitor chamber for 5 min to acclimatize. Then, locomotion of each animal was calculated as the number of movements per 5 min [39].