Pharmacological study

Male Wistar rats (6–8 weeks with Mean±SD weight of 200±20g) were housed in individual cages in a controlled room on 12h and off 12h with free access to food and water. The experimental protocol of the work with animals was by the National Institutes of Health (NIH) guide and was approved by the Ethical Committee of the University Research Center (EC/KNRC/94-17).

A total of 126 rats were randomly assigned into 9 groups for MWM and 9 groups for passive avoidance test (PAT) each of 7 animals. All injections were done intraperitoneally (i.p) between 8 AM and 10 AM, and the experiments were performed during daylight between 8 AM and 4 PM to prevent the effects of nighttime rhythm on the experiments. Animals were acclimated to the laboratory condition 1 hour before the test. Experimental groups were 1: Control: No treatment; 2:Normal saline (NS); 3: Solvent (10% dimethyl sulfoxide [DMSO] in NS); 4: Physostigmine (Phys) 0.3 mg/kg; 5:Piracetam (Pir) 200 mg/kg; 6–8: TSPE 100 mg/kg, 200 mg/kg, and 400 mg/kg; 9:Scopolamine (Sco) 1 mg/kg. Animals received 1 mL/kg, i.p of drugs (7 consecutive days). Sco was used for amnesia induction and was injected (i.p) on the 7th day, 30 min after the last dose of the drug (Sco induces memory dys-functions similar to those observed in dementia). Sco group just got 1mg/kg scopolamine and the experiment was performed 30 min later. The groups 2 and 3 were considered negative controls, while the groups 4 and 5 were considered positive controls.

In PAT, which was used to evaluate long spatial memory, a plexiglass box was used consisting of two equal dark and light partitions (20cm, 20cm, 20cm) separated by a remote door. The dark part is covered by a black roof, on the bottom of which steel bars are installed at one-centimeter intervals and connected to the shock device by a communication cable. This device can generate an electric current of 0.5 mA for two seconds with a frequency of 50Hz in these rods, which causes an electric shock to the animal’s hands and feet. During the learning stage, animals were placed in the light partition and allowed to enter the dark side after 10s. Animals with a delay of more than 120s were removed from the experiment. After 2h, during the training phase, the animals were again placed in the light partition and the door was opened after 10s for the animals to enter the dark side and the door was immediately closed. An electrical shock (0.5mA, 50Hz, 2s) was delivered to the animal’s foot, and the animal was immediately given back to the cage. The experiment was repeated after 5 min. When the animals remained on the light side for at least 5 consecutive minutes, the experiment was stopped. After 24h, during the retrieval phase, the animal was directed to the light side, the door was opened after 10s and initial avoidance and crossing latency to the dark part were recorded ( Harandi et al., 2015).

This test is used to evaluate short spatial memory. A black circular pond (60×160cm) was filled with water (24±2°C) and divided into four separated quadrants. Animals were released to swim into the water and their performance was recorded using a computerized camera system. In the target quadrant (TQ), a square platform (10cm in diameter) was placed under the water and during the learning stage, the animals were again allowed to swim via three blocks each of four consecutive 60s trials with 30 min intervals (each trial was started from a different quadrant). After finding the platform, animals could rest on it for 30s and then were transferred to the cage for another 30s until the next trial. The animals were guided to the platform if did not find it after 60s. The time spent in the TQ and the distance moved to find the platform was recorded. A single probe trial was done after 2h to find the plant effect on memory retrieval. The animals were freed from the quadrant as opposed to TQ into the water to swim without the platform for 60s. The crossing number, time remaining in the TQ and the distance traveled to discover the platform was documented. After 2 min, the platform was transferred above the water level and allowed the animal to find it it has become one of the most frequently used laboratory tools in behavioral neuroscience ( D’Hooge & De Deyn, 2001). Many methodological variations of the MWM task have been and are being used by research groups in many different applications. However, researchers have become increasingly aware that MWM performance is influenced by factors such as apparatus or training procedure as well as by the characteristics of the experimental animals (sex, species/strain, age) nutritional state, exposure to stress or infection.