Nicotine-prevented learning and memory impairment in REM sleep-deprived rat is modulated by DREAM protein in the hippocampus

2.1 Animal preparation

Seventy-two adult male Sprague Dawley (230–280 g) rats were obtained from the Animal Research and Service Centre (ARASC), Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia, were placed in a room with 12-hr light/dark (lights on at 7:00 am and turn off at 7.00 pm) cycle at 28OC, with free access to food and water. There were six groups of rats in this study: control (C, n = 12), control treated with nicotine (C + N, n = 12), wide platform (W, n = 12), wide platform treated with nicotine (W + N, n = 12), REM sleep deprivation (REMsd, n = 12), and REM sleep deprivation treated with nicotine (REMsd + N, n = 12). The nicotine groups (C + N, W + N, and REMsd  +  N) were treated with 1 mg/kg nicotine (Sigma, St. Louis, MO, USA) subcutaneously twice a day, for 72 hr. The nontreatment groups were treated with subcutaneous saline injection. The nicotine dose in this study was based on previous study which was known to produce nicotine blood levels similar to those of chronic smokers (Le Houezec, Jacob, & Benowitz, 1993). All experiments were approved by the Animal Care and Use Committee of Universiti Sains Malaysia [USM/Animal Ethics Approval/2012/ (81) (408)].

2.2 Induction of REM sleep deprivation

The inverted flowerpot method was modified based on our previously study (Siran, Ahmad, Abdul Aziz, & Ismail, 2014) and used to selectively induce REM sleep deprivation for 72 hr. Before the start of the experiment, all rats except from control group were isolated and adapted individually in a dry tank model for 72 hr before being exposed to the REM sleep deprivation model. The control group rats were adapted in their normal dry cage for 72 hr. The purpose of the adaptation was to expose and adapt the rat to the glass tank environment before being exposed to the REM sleep deprivation model. For REM sleep deprivation procedure, two small platforms of 6.5 cm diameter, 8.5 cm height, and 8 cm length between both platforms were placed in a glass tank measuring 50 cm in height, 50 cm in width, and 100 cm in length. The rats were deprived of REM sleep by placing one rat at a time on top of one of the two platforms (6.5 cm in diameter) placed in the middle of a glass tank filled with water (platform was 1 cm above water) for 72 hr. REM sleep was prevented by the muscular atonia due to the rats awakening when the body came into contact with water. For wide platform group, each rat was placed in the same experimental condition as REM sleep deprivation model except that the diameter of the platform was larger (14 cm diameter, 8.5 cm height, and 8 cm length between both platforms) which allowed REM sleep to occur. The purpose of this W group was to expose them to the same aquatic environment as the REM sleep deprivation rat but allowed them to experience both NREM and REM sleeps ad libitum (May et al., 2005). This platform technique has been validated by other studies using electroencephalography and has been shown to deprive a small degree of non-REM sleep (Maloney, Mainville, & Jones, 1999). The temperature of the water in the tub was 30°C. Food and water were available ad libitum throughout the time the rats were on the platforms.

2.3 Morris Water Maze test

The water maze test is a widely accepted method for learning and memory test, and this test was performed as described by (Vorhees & Williams, 2006). Maze testing was performed by the SMART-CS (Panlab, Barcelona, Spain) program and equipment. A circular plastic pool (110 cm in diameter and 20 cm in depth) was filled to a depth of 14 cm with milky water kept at 22–25°C. An escape platform was submerged 0.5–1.0 cm below the surface of the water in position. On training trials, the rats were placed in a pool of water, and allowed to remain on the platform for 10 s and were then returned to the home cage during the second-trial interval. The rats that do not find the platform within 60 s were placed on the platform for 10 s at the end of trial. They were allowed to swim until they sought the escape platform. These trials were performed in single platform and in three rotational starting positions. Escape latency, swimming distance, and swimming speed of each rat were monitored by a camera above the center of the pool connected to a SMART-CS program (Panlab).

A probe trial in order to assess memory consolidation was performed 24 hr after the 5-day acquisition tests. In this trial, the platform was removed from the tank, and the rats were allowed to swim freely. For these tests, the percentage time in the target quadrant and target site crossings within 60 s was recorded. The time spent in the target quadrant was recorded to indicate the degree of memory consolidation that has taken place after learning. The time spent in the target quadrant was used as a measure of spatial memory. Swimming pattern of each rat was monitored by a camera above the center of the pool connected to a SMART-CS program described above.

2.4 Immunohistochemistry analysis

Rats were sacrificed by an overdose intraperitoneal injection of sodium pentobarbitone. This method was used to avoid damage to the spinal cord (Hao, Takahata, Mamiya, & Iwasaki, 2002). Thoracotomy was done to expose the heart. An 18G branula was then inserted into the left ventricle, and a snip was made to the right atrium for outlet. Perfusion was performed using gravity method with phosphate-buffered saline (PBS) followed by 500 ml of cold 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB) (pH 7.4) (Hayati et al., 2008). The brain was dissected out from the rats. Following overnight cryoprotection in 20% sucrose in 0.1 mol/L PB, the brain was cut using cryostat and the hippocampus region was collected as free-floating section in PBS. Sections then were rinsed with Tris-buffered saline (TBS) and incubated overnight with rabbit polyclonal primary antibodies (dilution 1:500) for DREAM protein (Pierce, USA). Then, the sections were incubated with biotinylated secondary antibody anti-rabbit (dilution 1:200) for 1 hr. After 1 hr, sections were reacted with avidin–biotin complex (ABC) and stained with diaminobenzidine and hydrogen peroxide until brown coloration was seen. Sections were mounted on slides, air-dried, dehydrated, and covered with a cover slip.

Six tissue sections were randomly taken from one rat each group; therefore, the total tissue sections that were taken from one group were 36 tissue sections (n = 6). The tissue sections were scrutinized at hippocampal CA1, CA2, and CA3 and dentate gyrus regions. The DREAM positive neuron in every unit area (360,000 μm²) was then identified and captured at a 40× magnification. On average, four area units, that is, three in the hippocampus and one in the dentate gyrus, could be obtained from each section. The mean number of DREAM positive neuron per unit area in the hippocampus and dentate gyrus of each animal group was then calculated.

2.5 Western Blot analysis

In order to verify the DREAM protein quantification results, we performed Western blot analysis on the hippocampal protein extract. The brain tissue was removed directly from the rats without a fixation process being performed and separated into hippocampus region. The hippocampus region was immediately deep freezer by liquid nitrogen and kept at −80°C until further analysis. Protein was extracted from the hippocampus region using the NE-PER extraction reagents (Pierce). Before being used, NE-PER extraction reagents were mixed with concentrated Halt^™ Protease Inhibitor cocktail kit, EDTA-free (Pierce) in a volume of 10 μl/ml per reagent. The protein concentration of extracted samples was measured using the bicinchoninic acid (BCA) protein assay kit. Sample protein containing 40–50 μg total protein (after optimization) was denatured and subjected to SDS-PAGE using 12% resolving gel. The protein from polyacrylamide gels was transferred to nitrocellulose membrane (Bio-Rad, USA). The nitrocellulose membrane was washed with deionized water before incubated in blocking solution (5% BSA in PBS) for 1 hr at room temperature. Following that, the nitrocellulose membrane was washed three times for 10 min in Tris buffer saline-Tween 20 (TBS-T20). The nitrocellulose was then incubated with rabbit polyclonal DREAM antibody (dilution 1: 500 in TBST) or mouse monoclonal ß actin antibody (dilution 1:2000 in TBST,) overnight at 4°C. The nitrocellulose membrane was then incubated with HRP-conjugated goat anti-rabbit antibody (dilution 1:5000 in TBST) or mouse secondary antibody (dilution 1:5000 in TBST) for 1 hr at room temperature. In between incubations, the nitrocellulose membrane was washed three times in TBS-T20 for 10 min each. Finally, the blot was examined using Immobilon Western Chemiluminescent HRP substrate and an image was taken using an image analyzer. The integrated density values (IDV) of DREAM and ß-actin protein were measured using Spot Denso AlphaView^™ software programmed in the image analyzer. The mean relative intensity or fold change was determined by the following formula:

2.6 Statistical analysis

Statistical analyses were performed using Statistical Package of Social Sciences (SPSS) software version 21. Escape latency, swimming distance, and speed were analyzed using repeated measurements, and differences between specific groups were analyzed by analysis of variance (ANOVA) and Bonferroni post hoc test. The differences between specific groups for time spent in target quadrant, DREAM positive neuron expression and mean relative of DREAM protein level were measured using one-way ANOVA, Bonferroni, and Dunnet's post hoc test. All data are reported as mean ± standard error mean (SEM), and the level of significance was set at p < .05.

3.1 Escape latency and swim distance

By using the Morris Water Maze test, we found that within training trial days, rats in all groups showed significant improvement in escape latency time to finding the submerged platform (p < .001) and swim in short distance (p < .001). However, when compared between groups, the REM sleep-deprived group (REMsd) took the longest time to locate the hidden platform compared to the other groups (p < .001) in all training trial days. Similar pattern could be seen in swim distance, in which the REMsd group took the longest route before reaching the target platform compared to the other groups which swim in shorter distance and to escape on the platform (p < .001) (Figure 1). When comparison makes between nicotine-treated and nontreated groups, all nicotine-treated groups took least time and swim in shorter distance to find the submerged platform with the (C + N) group showed the shortest time and swim in short distance to find the target platform, while wide platform (W) and the control (C) groups took similar time and swim distance to get to submerged platform (Figure 1).

3.2 Probe test and Swim speed

During probe test, experimental rats will try to remember the hidden platform location, and swim around the quadrant in which the platform was located previously. In general, all rats were swimming mostly in quadrant 1 as the hidden platform was located in this quadrant. We found that rats in REMsd group spent least time in quadrant 1 when compared to other groups during probe test (p < .05) (Figure 2A). However, there was no significant difference in swim speed within groups or comparison between all groups in all training trial days (Figure 2B).

3.3 DREAM protein expression in hippocampus

DREAM protein expression was significantly increased in the REMsd group when compared to the other groups in CA1 (p < .001) (Figure 3C), CA2 (p < .001) (Figure 3D), CA3 (p < .001) (Figure 3E), and dentate gyrus hippocampus (p < .001) regions (Figure 3F). Treatment with nicotine significantly attenuated this effect and decreased DREAM protein expression in CA1, CA2, CA3 and dentate gyrus hippocampus regions as showed in REMsd + N group when compared to REMsd group (p < .001) (Figure 3C–F).

3.4 Mean relative of DREAM protein level in hippocampus

Mean relative of DREAM protein level in hippocampus was significantly increased in the REMsd group when compared to the C + N (p < .01), W (p < .01), and W + N groups (p < .01) (Figure 4A,B). Treatment with nicotine (REMsd + N) slightly reduced this effect but not significantly decreased mean relative of DREAM protein level in hippocampus when compared to REMsd group. In addition, mean relative of DREAM protein level in hippocampus of the REMsd + N group was still higher if compared to the C + N (p < .05), W (p < .05), and W + N groups (p < .05) (Figure 4A,B).