The open field test is a common measure of exploratory behavior and general activity [19]. As illustrated in Figure 1, the numbers of grids crossed in the testing apparatus was not different among groups (p > 0.05). The activities of rearing/leaning (p < 0.05) and grooming (p < 0.05) were significantly decreased in the group treated with 200 mg kg⁻¹ d⁻¹ NP. However, the groups treated with NP at 50 and 100 mg kg⁻¹ d⁻¹ showed no significant difference in rearing/leaning (p > 0.05) and grooming activity (p > 0.05) as compared with the control group. The numbers of crossing and rearing are usually used as measures of locomotor activity, and also measures of exploration and anxiety as well. A low frequency of these behaviors indicates decreased locomotion and exploration and/or a higher level of anxiety. Grooming behavior is a displacement response and is expected to be displayed in a novel environment in an open field test. Our results indicated that a high dose of NP exposure decreases locomotion and exploration of mice.

In the acquisition trial, the latencies for entry into the dark compartment did not differ significantly among groups (p > 0.05) (Figure 2). In the 72-hour-retention trial, the latencies in mice treated with NP at 200 mg kg⁻¹ d⁻¹ were significantly decreased (p < 0.05), but the decrease was not significant in the groups at 50 and 100 mg kg⁻¹ d⁻¹ compared with the control group (p > 0.05).

The Morris water maze test was used to reveal the effects of NP on spatial learning and memory. The mean latency to find the platform declined progressively during the training days in all groups (Figure 3A). Significant differences were found in mean latencies between training days (p < 0.01) and between treatments (p < 0.01), but there was no interaction between the factors day and treatment (p > 0.05). The escape latencies were significantly higher in NP (200 mg kg⁻¹ d⁻¹) treated mice than in the control mice (Day 3: p < 0.05; Day 4: p < 0.01). As there was no significant difference between the groups on Day 1, mice of every group started at the same level of performance. The mean swimming speeds of differently treated mice during the training days are presented in Figure 3B. Mice also showed significant differences in swimming speeds between training days (p < 0.01) and between treatments (p < 0.01), while no interaction between the factors day and treatment (p > 0.05) were observed. Swimming speeds of NP (200 mg kg⁻¹ d⁻¹) treated mice were significantly decreased compared to the control group (Day 1–Day 4: p < 0.05). This indicates that the motor problem of mice may contribute to the latency differences during the training days. This shows that NP could impair the locomotor activity of mice, leading to extended latency to the platform.

On Day 5, latency, swimming speed, the number of times crossing over the platform site and the time spent in four quadrants were measured for all groups. The NP-treated mice (at the dose of 200 mg kg⁻¹ d⁻¹) had longer latencies (p < 0.05) and a slower swimming speed (p < 0.05) compared with the control (Figures 3C and 3D). In the probe trial, the NP (200 mg kg⁻¹ d⁻¹) treated group made fewer platform crossings than the control group (p < 0.05) (Figure 3E). Both 50 mg kg⁻¹ d⁻¹ NP-treated and control mice spent most of their time in the target quadrant where the platform had been located during the training (Figure 3F).On the other hand, the groups treated with 100 and 200 mg kg⁻¹ d⁻¹ NP showed a decreased spatial preference for the target quadrant as compared with the control. All results revealed that NP at a higher dose (200 mg kg⁻¹ d⁻¹) could impair the ability of spatial learning and memory as well as the locomotor activity of mice.

The open field test was performed as described by Lu et al. [31]. The open field apparatus was a circular arena (50 cm in diameter, 30 cm in height) illuminated with a single 40 W bulb (3000 lux) placed 2.8 m above the bottom center. The bottom of the arena was divided into 21 equal-area grid cells by white lines. Tests were performed in the breeding room from 8:30 to 16:00 h. The individual mice were placed in the middle of the chamber for each trial. After 1-min adaptation, the behavior of each mouse was recorded for 5 min by two observers 1 m away from the open-field area. Between trials, the mice were returned to their home cage in the same room and the open field was wiped clean with a slightly damp cloth. The behavioral parameters recorded were: (1) ambulation: the number of grids crossed in the arena during the observation period; (2) rearing: the number of times the mouse stood on its hind legs; (3) leaning: the number of times the mouse placed one or two forelimbs on the wall of the arena; (4) grooming: the number of times the mouse “washed” itself by licking, wiping, combing, or scratching any part of the body.

The trough-shaped step-through passive avoidance apparatus consisted of an illuminated chamber (11.5 cm × 9.5 cm × 11 cm) attached to a darkened chamber (23.5 cm × 9.5 cm × 11 cm) containing a metal floor that could deliver footshocks. The two compartments were separated by a guillotine door. The illuminated chamber was lit with a 25 W bulb. The following test was performed as described by Lu et al. [31]. Before training, the mice were placed in the dimly lit room containing the apparatus for 0.5 h to acclimatize to the new environment. During training, each mouse was placed into the illuminated chamber, facing away from the door to the dark chamber to acclimatize for 1 min. When the mouse turned its body fully away from the dark chamber, the door was raised; when the mouse next turned fully toward the darkened chamber, the timer was started. An initial time measure spanned from the time that the mouse faced the opened darkened chamber to the time that the mouse fully entered, with all four paws, the dark chamber. As soon as the mouse entered the dark chamber the door was slid back into place, triggering a mild footshock (0.3 mA, 50 Hz, 5 s). The mouse was then immediately removed from the chamber and returned to its home cage. The retention test was conducted 24 h later with the mouse again being placed in the illuminated chamber and subjected to the same protocol described above in the absence of footshock. The upper time limit was set at 300 s.

The Morris water maze test was performed as described by Lu et al. [31]. The experimental apparatus consisted of a circular water tank (100 cm in diameter, 35 cm in height), containing water (23 ± 1 °C) to a depth of 15.5 cm, which was rendered opaque by adding ink. A platform (4.5 cm in diameter, 14.5 cm in height) was submerged 1 cm below the water surface and placed at the midpoint of one quadrant. The pool was located in a test room, which contained various prominent visual cues. Each mouse (about 16-weeks-old) received four training periods per day for 4 consecutive days. Latency to escape from the water maze (finding the submerged escape platform) was calculated for each trial. On Day 5, the probe test was carried out by removing the platform and allowing each mouse to swim freely for 60 s. The times that mice spent swimming in the target quadrant (where the platform was located during hidden platform training), and in the three non-target quadrants (right, left and opposite quadrants), were measured, respectively. For the probe trials, the number of times crossing over the platform site of each mouse was also measured and calculated. All data were recorded with a computerized video system.

For biochemical studies, animals were deeply anesthetized and sacrificed. Brains were promptly dissected, weighed and perfused with 50 mM (pH 7.4) ice-cold phosphate buffered saline solution (PBS). Brains were homogenized in 1/5 (w/v) PBS containing a protease inhibitor cocktail (Sigma-Aldrich, MO, U.S.) using a glass-Teflon homogenizer. Aliquots (2 mL) of this homogenate were directly centrifuged at 8000 × g for 10 min and the supernatant was used to determine brain malondialdehyde (MDA) levels, catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) activities, and protein contents. The remainder homogenate was sonicated four times for 30 s with 20 s intervals using a VWR Bronson Scientific sonicator, centrifuged at 5000 × g for 10 min at 4 °C, and the supernatant was collected and stored at −70 °C for determination of superoxide dismutases (SOD) enzyme activities. Protein concentration was determined by using the BCA assay kit (Pierce Biotechnology Inc., Rockford, IL U.S.) with bovine serum albumin (BSA) used as standard.

Chemicals used in the assay, including xanthine, xanthine oxidase, cytochrome c, BSA and SOD, were purchased from Sigma Chemical Company (St. Louis, MO, U.S.). Total SOD was assayed using the method of Flohe and Otting [32]. The activity was measured by the inhibition of cytochrome c reduction mediated via the superoxide anions that were generated by xanthine-xanthine oxidase and assayed spectrophotometrically at 550 nm. One unit of SOD activity was defined as the amount of SOD required for 50% inhibition of the xanthine and xanthine oxidase system reaction in 1 mL enzyme extraction per milligram of protein.

Catalase activity was assayed by the method of Claiborne et al. [33]. The assay mixture contained 2.4 mL of phosphate buffer (50 mM, pH 7.0), 10 μL of 19 mM hydrogen peroxide (H₂O₂) and 50 μL homogenate. The decrease in absorbance at 240 nm was measured immediately against a blank containing all the components except the homogenate at 10 s intervals for 3 min on a spectrophotometer. The catalase activity was calculated in terms of nmol H₂O₂ consumed min/mg protein.

The activity of GPx was assayed by the method of Mohandas et al. [34]. The assay measures the enzymatic reduction of H₂O₂ by GPx through consumption of reduced GSH that is restored from oxidized glutathione GSSG in a coupled enzymatic reaction by GR. GR reduces GSSG to GSH using NADPH as a reducing agent. Disappearance of NADPH was measured immediately at 340 nm against a blank containing all the components except the enzyme at 10 s intervals for 3 min on a Molecular Devices M2 plate reader (Molecular Devices, Menlo. Park, CA U.S.). One unit of GSH-Px was defined as the amount of enzyme that catalyzed the oxidation of 1.0 mmol of NADPH to NADP⁺ per minute at 25 °C.

GR activity was measured by the method of Mizuno and Ohta [35]. The enzymatic activity was assayed photometrically by measuring NADPH consumption. In the presence of GSSG and NADPH, GR reduces GSSG and oxidizes NADPH, resulting in a decrease of absorbance at 340 nm, which was measured in a M2 plate reader. Quantification was based on the molar extinction coefficient of 6.22 mM⁻¹ cm⁻¹ of NADPH. One unit of GR was defined as the amount of enzyme that reduced 1.0 mmol of GSSG (corresponding to the consumption of 1 mmol of NADPH) per minute at 25 °C.

Chemicals used in the assay, such as n-butanol, thiobarbutiric acid, sodium lauryl sulfate etc., were purchased from Sigma Chemical Company (St. Louis, MO, U.S.). As a marker of lipid peroxidation, the level of MDA was determined as described by Okhawa et al. [36]. The brain homogenate (0.2 mL) was mixed with 0.2 mL of 8.1% sodium lauryl sulfate, 1.5 mL of 20% acetic acid (pH 3.5) and 1.5 mL of 0.8% aqueous solution of thiobarbituric acid. The mixture was made up to 4 mL with distilled water and heated at 95 °C for 60 min. After cooling with tap water, 5 mL of n-butanol and pyridine (15:1, v/v) and 1 mL of distilled water was added and centrifuged at 4000 rpm for 10 min. The colored complex was extracted into n-butanol, and the absorption at 532 nm was measured and MDA content was expressed as nmol/mg protein.