Novel object recognition memory in REM sleep-deprived rats: Role of the cannabinoid CB1 receptor

Journal Pre-proof Novel object recognition memory in REM sleep-deprived rats: role of the cannabinoid CB1 receptor Kaveh Shahveisi, Mehdi Khodamoradi

PII:

S0166-4328(19)30499-1

DOI:

https://doi.org/10.1016/j.bbr.2019.112311

Reference:

BBR 112311

To appear in:

Behavioural Brain Research

Received Date:

28 March 2019

Revised Date:

12 September 2019

Accepted Date:

15 October 2019

Please cite this article as: Shahveisi K, Khodamoradi M, Novel object recognition memory in REM sleep-deprived rats: role of the cannabinoid CB1 receptor, Behavioural Brain Research (2019), doi: https://doi.org/10.1016/j.bbr.2019.112311

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Novel object recognition memory in REM sleep-deprived rats: role of the cannabinoid CB1 receptor

Kaveh Shahveisi , Mehdi Khodamoradi

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Kermanshah, Iran , 6719851451 , [email protected]

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Substance Abuse Prevention Research Center, Kermanshah University of Medical Sciences,

Highlights

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RSD impaired consolidation, retrieval, and reconsolidation of NOR memory. Both RSD and rimonabant did not affect the acquisition of NOR memory. Rimonabant improved the RSD-induced impairment of the consolidation and retrieval of NOR memory. It seems that the CB1R can be targeted to, at least partially, modulate the adverse effects of RSD on memory.

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ABSTRACT

A survey of the literature indicates that both rapid eye movement sleep deprivation (RSD) and activation of cannabinoid CB1 receptor (CB1R) may impair novel object recognition (NOR)

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memory in rodents. To our knowledge, so far, no previous study has investigated the probable effects of RSD on the different phases of NOR memory. Moreover, far too little attention has been paid to the potential role of the CB1R in the effects of RSD on object memory. Therefore, the major objective of this study was to investigate the probable role of the CB1R in the acquisition, consolidation, retrieval, and reconsolidation of NOR memory in the RSD rats. A 12h paradigm of RSD using the multiple platform method did not affect acquisition, but it impaired the consolidation, retrieval, and reconsolidation of NOR memory. Administration of the CB1R 1

antagonist rimonabant (1 or 3 mg/kg, i.p.) did not have significant effects on the acquisition and reconsolidation, but it improved RSD-induced impairment of the consolidation and retrieval of object memory, especially at the dose of 3 mg/kg. In addition, the RSD paradigm did not affect the levels of plasma corticosterone as an important marker of stress in rat. The results revealed that RSD may have different effects on the different phases of NOR memory which may not be attributable to the effects of stress. Our findings would seem to suggest that the CB1R can be targeted to, at least partially, modulate the adverse effects of RSD on the process of NOR

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memory.

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Keywords: REM sleep deprivation; novel object recognition memory; cannabinoid CB1

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receptor; rimonabant.

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1. Introduction

It is well documented that sleep plays a crucial role in various cognitive functions,

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particularly in memory processing [1,2]. Sleep has been shown to be necessary for memory consolidation [1,3] as sleep after learning facilitates memory retrieval [4]. Thus, sleep deficiency, including total and rapid eye movement (REM) sleep deprivation (RSD), may lead to memory

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deficits, including spatial [5,6] and novel object recognition (NOR) [7,8] memory impairment. Because of the fact that sleep deficiency is increasingly recognized as a high prevalence, worldwide public health concern [9], especially in individuals who work irregular or rotating shifts [10], cognitive dysfunction should be considered in individuals with sleep problems.

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A literature survey indicates that the cannabinoid system may play a modulatory role in sleep

and memory processing [11–17]. In the brain, cannabinoids bind to a transmembrane G-proteincoupled receptor known as central type 1 receptor (CB1R) to modulate various processes, particularly sleep and memory [13,14,17]. The endogenous cannabinoids, such as anandamide and 2-arachidonylglycerol, and activation of the CB1R may promote sleep; however, blocking the CB1R may enhance wakefulness [13]. It has been shown that delta-9-tetrahydrocannabinol (THC; the main psychoactive ingredient in marijuana), as a potent agonist of the CB1R, impairs 2

spatial memory which can be prevented by administration of the CB1R antagonist rimonabant [18]. Furthermore, it has also been reported that the CB1R agonist WIN-55,212-2 deteriorates NOR memory in rat [19,20]. Moreover, acute administration of the CB1R agonist JWH-081 has been reported to impair hippocampal synaptic plasticity and NOR memory in CB1 receptor wild type, but not in knockout mice [21]. On the other hand, rimonabant has been reported to antagonize the adverse effects of THC or anandamide on memory [22]. Previous research has shown that blocking the CB1R using SLV330 ameliorates cognitive impairment in several

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preclinical models, including scopolamine-induced NOR memory deficits [23]. It has also been shown that CB1 receptor knock-out mice have an enhanced NOR memory in comparison with the wild-type controls [24]. Thus, it seems that the CB1R may play a critical role in the process

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of object memory and, therefore, it can be targeted to modulate NOR memory.

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According to the above-mentioned studies, RSD impairs and antagonism of the CB1R modulates NOR memory. Nonetheless, what is not yet clear is whether RSD affects the different

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phases of NOR memory, including the acquisition, consolidation, retrieval, and reconsolidation of object memory. Furthermore, so far, no attempt has been made to examine the possible role of the CB1R in sleep deprivation-induced NOR memory impairment. Indeed, we hypothesized that

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blocking the CB1R may affect RSD-induced object memory impairment. Therefore, the purpose of this study was to assess the extent to which rimonabant administration affects the acquisition,

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consolidation, retrieval, and reconsolidation of NOR memory in REM sleep-deprived rats. 2. Materials and methods 2.1. Subjects

A total of 329 male Wistar rats (~ 3-month-old, 200–250 g; n = 8–10 in each group) were

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used as subjects throughout this study. They were maintained in Plexiglas cages (four per cage) under constant temperature (23 ± 1C) and humidity (50 ± 5%) and a 12L:12D cycle (light phase between 07:00 h and 19:00 h). Behavioral procedures were carried out during the active/dark phase of the cycle. All experiments and measurements were carried out by experimenters blind to the groups and treatments. The protocols were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and confirmed by the

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Regional Ethics Committee of Kermanshah University of Medical Sciences, Kermanshah, Iran (Grant No. KUMS.REC.1396.291). 2.2. Apparatus and testing room All protocols were carried out in an experimental room dimly lit by a 15 W overhead lamp. The behavioral experiments were conducted using an open field box made of polyvinyl chloride (50 × 50 × 50 cm). The performance of the animals was videotaped using a camera installed

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above the apparatus. The animals were moved to the experimental room 45 min prior to each experiment. Different objects made of glass, plastic, or porcelain differing in color, shape, surface texture, and size (between 7 × 7 × 7 cm and 12 × 12 × 12 cm), with no biological

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relevance, were used as the stimuli (objects). The objects were too heavy to be displaced by the

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animals. Between the trials, the objects and floor of the arena were cleaned with ethanol (70%). 2.3. Novel object recognition memory

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The NOR task consisted of one or two sample phase(s), a delay of 24 h, and a test phase as described in previous studies [8,25–28]. A period of 24 h for the delay phase was chosen to

on the performance of animals.

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evaluate long-term memory retention and also to prevent the possible effects of circadian rhythm

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The animals were handled for 5 min each day over a period of one week. They were then habituated to the empty arena (without any objects/stimuli) for 10–15 min each day during three consecutive days. Twenty-four hours after the last habituation day, during the sample phase, the animals were subjected to two identical objects (A1 and A2) at two adjacent corners of the arena, each 10 cm away from the sidewalls (Figs. 1A, 2A, 3A, and 4A). The animals were placed in the

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middle of the box facing away from the objects and allowed to explore the objects for a period of 5 min. After a delay of 24 h, the test phase was conducted for a further period of 5 min. During the test phase (Figs. 1A, 2A, 3A, and 4A), the animals were introduced to two objects at the same places; one of the objects (A3) was the same copy of the A1 and A2, but another object was a novel one (B). For reconsolidation experiment, the animals underwent two sample phases with a delay of 24 h [25]. During the second sample phase, the animals were introduced to two copies of the A1 and A2 (A3 and A4) (Fig. 4A). The test phase of the reconsolidation task was the same as that described for other object memory phases; the animals were introduced to a 4

familiar (A5) and a novel (B) object, 24 h after the second sample phase (Fig. 4A). It is worth mentioning that the role (as familiar or novel) and positions of the objects were counterbalanced to decrease potential biases due to preferences for particular objects or positions. We expected that the animals spend more time exploring the novel objects (B) than the familiar ones (A3 or A5). Object exploration was determined as touching the objects or exploring them with the nose at a distance ≤ 2 cm; however, turning around or sitting on the objects were not defined as exploratory behavior. Those animals (about 2% of the rats) which explored the objects less than

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a total period of 15 s in the sample phase(s) or less than 10 s in the test phase were not considered for the subsequent analyses [26].

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2.4. REM sleep deprivation

The multiple platform method was used to induce RSD as described previously [6,29]. We

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used two apparatus; the RSD apparatus for sleep-deprived animals and the wide platform (WP) apparatus for control/WP animals. The RSD apparatus (100 × 60 × 40 cm) comprised of 15

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platforms; 10 cm in height and 6 cm in diameter. The WP apparatus had the same shape and dimensions as the RSD apparatus, except that the platforms were 14 cm in diameter. Therefore,

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wide platforms in the WP apparatus allowed the animals to sleep without the risk of falling (into the water). Furthermore, the animals were able to jump from one platform to another in both apparatus. Prior to place the animals in the WP or RSD apparatus (for the WP or RSD groups,

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respectively), both the apparatus were filled with water to approximately 2 cm below the surface of the platforms. During the 12 h period of RSD, the cage-mate animals were placed in the same (WP or RSD) apparatus at the same time. Pellet baskets and water bottles were provided above the WP and RSD apparatus (attached to the wire-mesh lids). The WP and RSD protocols were carried out in a room under standard conditions, such as a temperature of 23 ± 1 C and a

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12L:12D cycle. To induce RSD, the animals were placed in the RSD (or in the WP apparatus for controls) for a period of 12 h. 2.5. Effects of rimonabant on NOR memory in REM sleep-deprived rats The sample phase, a delay of 24 h, and a test phase of the NOR task was carried out as described before. A 12-h period of RSD was conducted at different time points; either just before the sample trial (for acquisition; Fig. 1A), immediately after the sample trial (for consolidation; 5

Fig. 2A), or just before the test phase (for retrieval; Fig. 3A). For reconsolidation, the 12-h RSD paradigm was conducted immediately after the second sample phase (Fig. 4A). For control (WP) groups, the animals were placed in the WP apparatus at the (four) corresponding time points, as described for the RSD groups. In the treatment groups, the animals received intraperitoneal administration of the CB1R antagonist rimonabant (SR141716A; Cayman, USA) or vehicle at two doses; 1 or 3 mg/kg. Since we assessed the effects of rimonabant in sleep-deprived rats, these two doses were administered

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as arousal enhancing doses in male rats [30]. Rimonabant was dissolved in dimethyl sulfoxide (DMSO) before further dilution in distilled water (maximum final DMSO concentration, 1%v/v).

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For acquisition, rimonabant was administered at the two doses (in different groups) 1 h before the sample phase (the first sample phase in the reconsolidation task). Thus, the animals received

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rimonabant after the 12-h WP or RSD paradigm and 1 h before the sample phase (Fig. 1A). Rimonabant was also injected immediately after the sample phase and prior to the WP/RSD

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paradigm for the consolidation (Fig. 2A) or administered 1 h before the test phase (after the 12-h WP/RSD episode) for the retrieval (Fig. 3A) of NOR memory. For reconsolidation, the animals received rimonabant immediately after the second sample phase and just before the WP/RSD

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paradigm (Fig. 4A). In all experiments, rimonabant was administered at 1 (in RSD-R 1 mg/kg groups) or 3 mg/kg (in RSD-R 3 mg/kg groups) and their corresponding control groups received

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vehicle (in RSD-Veh groups). Furthermore, all the above-mentioned protocols were also carried out for the control WP groups. To do so, for the acquisition, consolidation, retrieval, and reconsolidation of NOR memory, the animals were placed in the WP apparatus (instead of the RSD apparatus) and received vehicle or rimonabant at 1 or 3 mg/kg for the WP-Veh, WP-R 1 mg/kg, and WP-R 3 mg/kg groups, respectively. Therefore, each object recognition (acquisition,

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consolidation, retrieval or reconsolidation) task included eight groups; the WP, RSD, WP-Veh, WP-R 1 mg/kg, WP-R 3 mg/kg, RSD-Veh, RSD-R 1 mg/kg, and RSD-R 3 mg/kg groups. 2.6. Plasma corticosterone We also examined the levels of plasma corticosterone in the animals to find out whether the effects of RSD and/or rimonabant on NOR memory may have affected by stress. Two groups of animals underwent either a (12-h period of) RSD or WP paradigm. Animals of the third group were maintained in their home cages. After the WP or RSD paradigm, the animals were 6

immediately anesthetized with CO2 and the blood specimens were collected in plastic polyethylene tubes on ice containing Na2–EDTA. The samples were then centrifuged at 2600 rpm for a period of 20 min at 4 °C. The specimens were maintained into microtubes at a temperature of −80 °C to examine the levels of corticosterone. Finally, the plasma specimens were evaluated using an ELISA kit specific for rats and mice (DRG International Inc., USA) [31]. 2.7. Data analysis

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A discrimination ratio (DR) of 0.5 was defined as an equal exploration of the familiar and novel objects, indicating exploration by chance (DR = 0.5 was defined as chance level or

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theoretical mean); however, a DR > 0.5 was determined as a significant preference for the novel object. For the test phases, the DR was determined as the time spent exploring the novel object

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(T B) divided by the time spent exploring the familiar object (T A3 or TA5) plus the time spent exploring the novel object (T B); T B/(T A3 + T B) (Figs. 1A, 2A, and 3A) or T B/(T A5 + T B)

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(Fig. 4A). For the sample phases, the DR was calculated as the time spent exploring the object that was replaced by the novel object (in the test phase) divided by the total exploration time; T

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A2/(T A1 + T A2) (Figs. 1A, 2A, and 3A) or T A4/(T A3 + T A4) (Fig. 4A) [8,32]. Student’s one-sample t-test was used to compare the DR of all groups with the chance level/theoretical mean (DR = 0.5). The between-group comparisons for the sample and test phases were

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performed using the independent samples t-test or one-way analysis of variance (ANOVA) followed by Tukey's test. 3. Results

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3.1. Effects of rimonabant on NOR memory in REM sleep-deprived rats The sample phases in all groups in the four (acquisition, consolidation, retrieval, and

reconsolidation) memory experiments were not affected by the RSD paradigm or rimonabant administration as none of the groups performed significantly above or below the chance level/theoretical mean (DR = 0.5; p > 0.05). Moreover, for the sample phases, the between-group comparisons did not show significant differences between the groups (p > 0.05; Figs. and Tables 1, 2, 3, and 4). The 12-h period of RSD and rimonabant administration, however, induced different effects on the test phases in the four memory experiments (as described below). 7

3.1.1. The acquisition of NOR memory In the acquisition experiment, we first conducted and analyzed the WP and RSD groups. Analysis revealed that the DR of the test phases of both the WP (p < 0.001) and RSD (p = 0.039) groups were above the chance level; however, no significant differences were found between the two (WP and RSD) groups (p > 0.05; Fig. 1B and Table 1). This indicates that the RSD episode did not affect the acquisition of object memory. The DR of the test phases of all other groups, including the WP-Veh (p < 0.001), WP-R 1 mg/kg (p < 0.001), WP-R 3 mg/kg (p < 0.001),

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RSD-Veh (p = 0.001), RSD-R 1 mg/kg (p = 0.018), and RSD-R 3 mg/kg (p = 0.037) groups, were above the chance level. Nonetheless, the between-group comparisons of the test phases

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revealed no significant differences between the groups (F7,70 = 1.94, p > 0.05; Fig. 1C and Table 1). Therefore, rimonabant administration did not affect the acquisition of NOR memory in the

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control (WP) and sleep-deprived (RSD) animals.

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3.1.2. The consolidation of NOR memory

We first evaluated the WP and RSD groups. The one-sample t-test showed that the DR of the test phase of the WP (p < 0.001), but not the RSD group (p > 0.05), showed a significant increase

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compared with the chance level. Furthermore, the RSD group significantly decreased in comparison with the WP group (p = 0.001; Fig. 2B and Table 2). Thus, RSD impaired the consolidation of NOR memory. Further analysis revealed that, except the RSD-Veh group (p >

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0.05), the test phases of other groups, including the WP-Veh (p < 0.001), WP-R 1 mg/kg (p < 0.001), WP-R 3 mg/kg (p < 0.001), RSD-R 1 mg/kg (p = 0.026), and RSD-R 3 mg/kg (p < 0.001) groups, significantly increased from chance level (Fig. 2C and Table 2). For the betweengroup comparisons of the test phases (F7,67 = 6.68, p < 0.001), the WP-R 1 mg/kg and WP-R 3

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mg/kg groups did not show significant differences in comparison with the WP-Veh group (p > 0.05). In addition, the RSD-R 3 mg/kg group (p = 0.012), but not the RSD-R 1 mg/kg group (p > 0.05), showed a significant increase compared with the RSD-Veh group (Fig. 2C and Table 2). Thus, rimonabant did not affect object memory in the WP groups; however, it increased novel object exploration in the RSD animals compared with the RSD-Veh group. 3.1.3. The retrieval of NOR memory

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Like other memory stages, we first examined the WP and RSD groups. Analysis indicated that animals of the WP (p < 0.001), but not the RSD group (p > 0.05), performed above chance. Moreover, the independent samples t-test showed that the RSD group showed a significant decrease in novel object exploration in comparison with the WP group (p < 0.001; Fig. 3B and Table 3). Therefore, the RSD paradigm impaired the retrieval of object memory. Analysis also revealed that, similar to the consolidation experiment, except the RSD-Veh group (p > 0.05), other groups, including the WP-Veh (p < 0.001), WP-R 1 mg/kg (p = 0.002), WP-R 3 mg/kg (p <

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0.001), RSD-R 1 mg/kg (p < 0.001), and RSD-R 3 mg/kg (p < 0.001) groups, showed significant increases in novel object exploration as their DRs (in the test phases) were significantly above the chance level (Fig. 3C and Table 3). The between-group analysis (F7,66 = 6.94, p < 0.001)

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showed that the test phases of the WP-R 1 mg/kg and WP-R 3 mg/kg groups did not show

significant differences compared with the WP-Veh group (p > 0.05). Nonetheless, the RSD-R 3

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mg/kg (p = 0.035), but not the RSD-R 1 mg/kg group (p > 0.05), showed a significant increase in novel object exploration with respect to the RSD-Veh group (Fig. 3C and Table 3). Thus,

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rimonabant did not have significant effects on NOR memory in the WP groups, whereas it enhanced the time spent exploring the novel object in the sleep-deprived groups in comparison

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with the RSD-Veh group. 3.1.4. The reconsolidation of NOR memory

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In the reconsolidation experiment, analysis using the one-sample t-test demonstrated that the test phase of the WP (p < 0.001), but not the RSD group (p > 0.05), was significantly above the chance level. Analysis using the independent samples t-test indicated that novel object exploration in the RSD group significantly decreased in comparison with the WP group (p < 0.001; Fig. 4B and Table 4). Therefore, the RSD paradigm impaired the reconsolidation of object

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recognition memory. For other groups, exploratory behavior (for the novel object) in the test phases of the WP-Veh, WP-R 1 mg/kg, WP-R 3 mg/kg, and RSD-R 3 mg/kg groups significantly increased (p < 0.001 for the four groups), but in the test phases of the RSD-Veh and RSD-R 1 mg/kg (p > 0.05) groups did not show significant differences compared with the chance level (Fig. 4C and Table 4). For the between-group analysis of the test phases (F7,67 = 6.3, p < 0.001), the DRs in the WP-R 1 mg/kg and WP-R 3 mg/kg groups did not have significant differences in comparison with the WP-Veh group (p > 0.05). Moreover, the RSD-R 1 mg/kg and RSD-R 3 9

mg/kg groups did not show significant differences when compared with the RSD-Veh group (p > 0.05; Fig. 4C and Table 4). Therefore, rimonabant did not also affect the reconsolidation of NOR memory in the WP and RSD groups. 3.2. Plasma corticosterone levels The one-way ANOVA demonstrated that no significant differences were found in the levels of plasma corticosterone between the home cage, WP, and RSD groups (F2,24= 0.85, p > 0.05;

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Table 5). 4. Discussion

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To the best of our knowledge, this study is the first to demonstrate that the CB1R may play an important role in RSD-induced NOR memory impairment. Our findings revealed that a 12-h

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period of RSD impaired the consolidation, retrieval, and reconsolidation, but it did not affect the acquisition of NOR memory. Furthermore, rimonabant administration did not have a significant

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effect on the acquisition and reconsolidation, whereas it improved the RSD-induced impairment of the consolidation and retrieval of object memory, especially at the dose of 3 mg/kg. Our

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findings provide new insights into the role of sleep and cannabinoid system in NOR memory. The empirical findings of this study are in agreement with previous reports [8,33] and provide a new understanding of the role of sleep in the process of object recognition memory. It

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is well documented that sleep plays a critical role in memory consolidation as hippocampal replay, a reactivation of the hippocampal circuits related to a recent experience, during sleep may enhance memory consolidation [3]. Sleep also regulates the essential genes for synaptic plasticity and memory processing [34,35]. Thus, it is not surprising that RSD may result in memory

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impairment [6, 8,31]. In the current study, the acute RSD paradigm produced different effects on the various stages of NOR memory. The RSD episode did not affect the acquisition and reconsolidation, but it impaired the consolidation and retrieval of NOR memory. The adverse effects of RSD on the consolidation, retrieval, and reconsolidation of NOR memory, in this study, could possibly be explained by the fact that the RSD protocol was induced just before or immediately after memory consolidation or reactivation. It should be mentioned that memory reactivation/retrieval (and the subsequent reconsolidation) may involve de novo protein synthesis which may convert the consolidated memory to an unstable state, thereby causes the memory 10

become susceptible to interference [36,37]. Regarding the reconsolidation experiment, it is worth mentioning that the second sample phase reactivates the consolidated/established memory and, therefore, returns the memory to an unstable state [25]. This, however, may not happen for encoding or acquisition of a new memory. It is, therefore, reasonable to assume that, in this study, the RSD paradigm may have impaired the consolidation, retrieval, and reconsolidation, but not the acquisition, of NOR memory because of the adverse effects of RSD on the process of memory storage and reactivation. In addition, neither the RSD protocol nor the rimonabant

that only specific cognitive processes are susceptible to interference [8].

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administration affected object familiarity processing (in the sample phases) which may indicate

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The results also indicated that the cannabinoid system may play a modulatory role in the

process of NOR memory in sleep-deprived rats. In line with our findings, it is stated that CB1R

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knock-out mice are able to retain object memory for a longer time when compared with wildtype controls [24]. Clarke et al. [20] reported that the CB1R agonist WIN-55,212-2 and the

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endocannabinoid membrane transporter inhibitor VDM-11 blocked the retention of NOR memory when they, immediately after the sample phase, infused in the CA1 region of the hippocampus. Mouro et al. [38] found that a prolonged, intermittent (30 days) administration of

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WIN 55,212‐2 (1 mg/kg) disrupts NOR memory. They also showed that chronic exposure to WIN 55,212‐2 alters brain functional connectivity and produces hypometabolism in the brain

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regions involved in memory, including the hippocampus and mediodorsal nucleus of thalamus [38]. Since the hippocampus is involved in memory formation [39,40], it would seem to suggest that, based on our findings and also according to the above-mentioned literature, targeting the hippocampal CB1R via agonists or antagonists may be a potential strategy to modulate NOR memory. Our results may, therefore, suggest that the CB1 receptor can also be targeted to

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modulate object memory deficits induced by sleep disorders. According to the literature discussed above, the CB1R may be a target for various drugs and pathological conditions; nonetheless, it is also involved in various functions in the normal brain, such as memory and sleep [13,41]. The CB1R is one of the most abundant neuromodulatory receptors in various regions of the brain, including the prefrontal cortex, anterior cingulate cortex, and hippocampus which are involved in cognitive processes, especially memory [42–44]. Further evidence indicates that activation of the CB1 receptor modulates neurotransmitter release due to inhibition of calcium (Ca2+) or activation of potassium (K+) channels [42,43]. Using retrograde signaling, 11

the endocannabinoids can control synaptic plasticity; they activate presynaptic CB1 receptors, thereby leading to inhibition of presynaptic Ca2+ influx and neurotransmitter release [45]. Therefore, due to a decrease in presynaptic glutamate release [46], long-term potentiation (LTP), as a synaptic model of memory in the hippocampus [47], has been shown to be suppressed by activation of the CB1 receptor [48]. As also noted by Sullivan [41], activation of the CB1R decreases neurotransmitter release below the levels required to produce persistent modifications in synaptic plasticity in the hippocampus. Furthermore, LTP in the hippocampus has been found

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to be enhanced in CB1R knock-out mice [49]. In further agreement with the above-mentioned studies and mechanisms, the probable effects of rimonabant on neurotransmitter release and

synaptic plasticity, in the hippocampus and other brain regions involved in memory, may, at least

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partially, account for the effects of rimonabant on NOR memory in the present study.

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Regarding the effects of cannabinoids on sleep, Santucci et al. [30] reported that blocking the CB1R by systemic administration of rimonabant at 0.1, 0.3, 1, 3, and 10 mg/kg doses increased

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the time spent in wakefulness, whereas it reduced the duration of REM and non-rapid eye movement (NREM) stages of sleep in a dose-dependent manner. They also indicated that rimonabant increased REM sleep latency; however, it did not affect motor behavior [30]. They,

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finally, concluded that the arousal-enhancing effect of rimonabant may be mediated by blocking the CB1 receptor [30]. Furthermore, it has been claimed that the expression of the CB1R can be

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modulated by circadian rhythm. The maximum expression of the protein and mRNA of the CB1 receptor in the brainstem has been reported to be at 13:00 h and 21:00 h, respectively [50]. In addition, for the protein and mRNA of the CB1R, the minimum expression has been found to be at 01:00 h and 09:00 h, respectively [50]. Considering these effects of the circadian clock on the expression of the CB1 receptor, as mentioned before, a period of 24 h was chosen for the delay

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phase (in the present study) to minimize the probable effects of the circadian rhythm on the results. Moreover, it has been reported that a 2-h period of sleep rebound subsequent to a 24-h period of RSD increases the expression of the CB1 protein in the pons, which is involved in the modulation of sleep, whereas the 24-h period of RSD has no effect by itself (when performed without sleep rebound) on the expression of the CB1 protein and mRNA [50,51]. Thus, the effects of rimonabant on NOR memory in the present study may not be attributable to changes in the expression of the CB1 receptor in the brain.

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In addition to the mechanisms discussed above, another possible mechanism may be related to the interaction between the CB1 receptor and hyperpolarization activated cyclic nucleotidegated (HCN) channels. Due to impairment of dendritic integration of excitatory inputs, activation of the CB1-HCN pathway impairs LTP and memory formation [52]. Furthermore, in this study, it is somewhat surprising that antagonism of the CB1 receptor affected object recognition memory in the RDS animals; nonetheless, it did not affect NOR in the control WP animals. It should, however, be mentioned that similar results have been reported previously [53]. It has, for

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example, been reported that that administration of rimonabant prevents memory deficits induced by the endocannabinoids, exogenous cannabinoids, and WIN-55,212-2; nonetheless, when

administered alone in control animals, rimonabant did not have any effects on memory [54,55].

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Therefore, it would seem reasonable to assume that the CB1R can be targeted to modulate NOR

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memory deficits following pathological conditions.

Finally, and interestingly, evaluation of the levels of plasma corticosterone following the

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RSD paradigm also revealed that the observed effects of RSD (and/or rimonabant) on NOR memory in the present study may not be related to stress. In general, the above-mentioned mechanisms and explanations may be involved in the effects of rimonabant on RSD-induced

5. Conclusions

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NOR memory impairment; nonetheless, the precise mechanisms remain to be elucidated.

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Taken together, our findings may suggest that a 12-h period of RSD impairs NOR memory. It seems that acute RSD might affect the different phases of NOR memory differently, depending on whether memory consolidation or reactivation occurs (or not) during the process of NOR memory. In other words, when NOR memory is stored (consolidated) or reactivated (restored), it

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can be modulated by RSD. Therefore, and returning to the question raised at the beginning of this study, it is now possible to assume that the CB1R may be involved in the adverse effects of RSD on NOR memory. Furthermore, according to our results and previous findings, it seems that the observed effects of rimonabant on object recognition memory in the sleep-deprived animals (in this study) may not be attributable to the effects of stress and circadian rhythm. Therefore, our findings would seem to suggest that the cannabinoid CB1 receptor can be targeted to, at least partially, modulate the adverse effects of RSD on memory.

13

Conflict of interest The authors declare no conflict of interest.

Acknowledgements

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The authors gratefully acknowledge the Research Council of Kermanshah University of Medical Sciences, Kermanshah, Iran for the financial support (Grant No.

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KUMS.REC.1396.291). The authors would also like to acknowledge Dr. Mohammad Ghazvini

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for his critical proofreading of the manuscript.

14

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Fig. 1. Effects of rimonabant on the acquisition of object recognition memory in REM sleep-deprived rats. (A) The experimental timeline used for the acquisition experiment. The discrimination ratios of sample phases in all groups were not significantly different from the chance level/theoretical mean (the dashed line; discrimination ratio = 0.5) and also between the groups. Further analysis revealed that the test phases of the WP and RSD groups (B) were above the chance level; however, no significant differences were found between the two (WP and RSD) groups. The test phases of other groups (C) also showed similar results; all groups showed significant increases (in discrimination ratio) compared with the chance level, whereas there were no significant differences between the groups. Data are expressed as mean ± S.E.M. (n = 9-10 rats in each group). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the chance level. A1-3, familiar objects; B, novel object; R, rimonabant administration, WP/RSD, wide

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platform or REM sleep deprivation paradigm; WP, wide platform group; RSD, REM sleep-deprived group; WPVeh, wide platform-vehicle group; WP-R 1 mg/kg, wide platform-rimonabant 1 mg/kg group; WP-R 3 mg/kg, wide platform-rimonabant 3 mg/kg group; RSD-Veh, REM sleep deprivation-vehicle group; RSD-R 1 mg/kg, REM sleep

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deprivation-rimonabant 1 mg/kg group; RSD-R 3 mg/kg, REM sleep deprivation-rimonabant 3 mg/kg group.

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Fig. 2. Effects of rimonabant on the consolidation of object memory in REM sleep-deprived animals. (A) Experimental timeline. The (discrimination ratios of) sample phases in all groups did not show significant differences compared with the chance level/theoretical mean (the dashed line; discrimination ratio = 0.5) and also between the groups. Comparison of test phases between the control WP and RSD groups (B) showed that the WP group (p < 0.001), but not the RSD group (p > 0.05), performed significantly above the chance level. Furthermore, the discrimination ratio in the RSD group significantly decreased compared with the WP group (p < 0.01). Regarding the test phases of other groups (C), except the RSD-Veh group (p > 0.05), all other groups were above the chance level. In addition, the WP-R 1 mg/kg and WP-R 3 mg/kg groups did not show significant differences compared with the WP-Veh groups; however, the RSD-R 3 mg/kg group (p < 0.05), but not the RSD-R 1 mg/kg

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group, showed a significant increase compared with the RSD-Veh group. Data are expressed as mean ± S.E.M. (n = 8-10 rats in each group). * p < 0.05 and *** p < 0.001 vs. the chance level; ## p < 0.01 vs. the test phase of the WP group; + p < 0.05 vs. the test phase of the RSD-Veh group. A1-3, familiar objects; B, novel object; R, rimonabant

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administration, WP/RSD, wide platform or REM sleep deprivation paradigm; WP, wide platform group; RSD, REM sleep-deprived group; WP-Veh, wide platform-vehicle group; WP-R 1 mg/kg, wide platform-rimonabant 1 mg/kg

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group; WP-R 3 mg/kg, wide platform-rimonabant 3 mg/kg group; RSD-Veh, REM sleep deprivation-vehicle group; RSD-R 1 mg/kg, REM sleep deprivation-rimonabant 1 mg/kg group; RSD-R 3 mg/kg, REM sleep deprivation-

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rimonabant 3 mg/kg group.

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of ro -p re lP ur na Jo Fig. 3. Effects of rimonabant on the retrieval of object recognition memory in REM sleep-deprived rats. (A) The experimental timeline used for the retrieval of object memory. The sample phases in all groups did not show

21

significant differences compared with the chance level/theoretical mean (the dashed line; discrimination ratio = 0.5) and also between the groups. Comparisons of test phases between the control WP and RSD groups (B) revealed that discrimination ratio of the WP group (p < 0.001), but not the RSD group (p > 0.05), was above the chance level. Furthermore, the RSD group significantly decreased compared with the WP group (p < 0.001). Regarding the test phases of other groups (C), except the RSD-Veh group (p > 0.05), all other groups performed above the chance level. In addition, the between-group analysis demonstrated that the WP-R 1 mg/kg and WP-R 3 mg/kg groups did not show significant differences in comparison with the WP-Veh group; however, the RSD-R 3 mg/kg group (p < 0.05), but not the RSD-R 1 mg/kg group, showed a significant increase compared with the RSD-Veh group. Data are expressed as mean ± S.E.M. (n = 8-10 rats in each group). ** p < 0.05 and *** p < 0.001 vs. the chance level; ### p

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< 0.001 vs. the test phase of the WP group; + p < 0.05 vs. the test phase of the RSD-Veh group. A1-3, familiar objects; B, novel object; R, rimonabant administration, WP/RSD, wide platform or REM sleep deprivation

paradigm; WP, wide platform group; RSD, REM sleep-deprived group; WP-Veh, wide platform-vehicle group; WP-

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R 1 mg/kg, wide platform-rimonabant 1 mg/kg group; WP-R 3 mg/kg, wide platform-rimonabant 3 mg/kg group; RSD-Veh, REM sleep deprivation-vehicle group; RSD-R 1 mg/kg, REM sleep deprivation-rimonabant 1 mg/kg

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group; RSD-R 3 mg/kg, REM sleep deprivation-rimonabant 3 mg/kg group.

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Fig. 4. Effects of rimonabant on the reconsolidation of object memory in REM sleep-deprived rats. (A) Experimental timeline. The first and second sample phases in all groups were not significantly different with respect to the chance level/theoretical mean (the dashed line; discrimination ratio = 0.5) and also between the groups. Comparison of test phases between the control WP and RSD groups (B) revealed that the WP group (p < 0.001), but not the RSD group (p > 0.05), showed a significant increase in discrimination ratio compared with the chance level. Furthermore, the RSD group significantly decreased in comparison with the WP group (p < 0.001). Regarding novel object exploration in the test phases of other groups (C), except the RSD-Veh and RSD-R 1 mg/kg groups (p > 0.05), other groups performed significantly above the chance level. The between-group comparisons demonstrated that the WP-R 1 mg/kg and WP-R 3 mg/kg groups were not significantly different compared with the WP-Veh

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group (p < 0.05). Moreover, the RSD-R 1 mg/kg and RSD-R 3 mg/kg groups did not show significant differences in comparison with the RSD-Veh group (p < 0.05). Data are expressed as mean ± S.E.M. (n = 8-10 rats in each group). ***p < 0.001 vs. the chance level; ### p < 0.001 vs. the test phase of the WP group. A1-5, familiar objects; B, novel

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object; R, rimonabant administration, WP/RSD, wide platform or REM sleep deprivation paradigm; WP, wide

platform group; RSD, REM sleep-deprived group; WP-Veh, wide platform-vehicle group; WP-R 1 mg/kg, wide

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platform-rimonabant 1 mg/kg group; WP-R 3 mg/kg, wide platform-rimonabant 3 mg/kg group; RSD-Veh, REM sleep deprivation-vehicle group; RSD-R 1 mg/kg, REM sleep deprivation-rimonabant 1 mg/kg group; RSD-R 3

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mg/kg, REM sleep deprivation-rimonabant 3 mg/kg group.

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Table 1 The mean time spent exploring objects during the sample and test phases and also the mean time spent exploring the familiar and novel objects during the test phase in the acquisition experiment of the object recognition task. Time spent exploring novel object during the test phase (s)

Time spent exploring Time spent exploring objects during objects during the sample phase (s) the test phase (s)

WP

26.51 ± 2.54

22.03 ± 3.07

11.54 ± 2.00

32.53 ± 3.42

RSD

19.34 ± 1.83

17.37 ± 2.25

13.08 ± 2.39

21.67 ± 3.29

WP-Veh

26.29 ± 2.84

22.01 ± 3.43

10.96 ± 1.74

33.06 ± 4.13

WP-R 1 mg/kg

26.27 ± 2.8

21.84 ± 2.97

12.24 ± 2.12

31.45 ± 3.21

WP-R 3 mg/kg

25.29 ± 2.5

21.05 ± 2.48

11.86 ± 1.62

30.23 ± 2.26

RSD-Veh

20.72 ± 2.84

16.00 ± 1.97

11.64 ± 2.10

20.36 ± 2.72

RSD-R 1 mg/kg

21.06 ± 2.07

18.20 ± 2.13

13.33 ± 2.05

23.07 ± 3.05

RSD-R 3 mg/kg

19.45 ± 2.17

18.57 ± 2.27

12.99 ± 1.75

24.14 ± 3.37

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Groups

Time spent exploring familiar object during the test phase (s)

Data are presented as mean ± S.E.M. WP, wide platform group; RSD, REM sleep-deprived group; WP-Veh, wide

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platform-vehicle group; WP-R 1 mg/kg, wide platform-rimonabant 1 mg/kg group; WP-R 3 mg/kg, wide platformrimonabant 3 mg/kg group; RSD-Veh, REM sleep deprivation-vehicle group; RSD-R 1 mg/kg, REM sleep deprivation-

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rimonabant 1 mg/kg group; RSD-R 3 mg/kg, REM sleep deprivation-rimonabant 3 mg/kg group.

26

Table 2 The mean time spent exploring objects during the sample and test phases and also the mean time spent exploring the familiar and novel objects during the test phase in the consolidation experiment of the object recognition task. Time spent exploring familiar object during the test phase (s)

Time spent exploring novel object during the test phase (s) 35.99 ± 3.20

WP

27.82 ± 2.65

23.93 ± 3.29

11.86 ± 1.97

RSD

27.8 ± 2.93

18.02 ± 2.34

17.64 ± 3.44

18.40 ± 3.18

WP-Veh

27.48 ± 2.89

22.2 ± 3.4

11.59 ± 1.82

32.8 ± 3.86

WP-R 1 mg/kg

27.66 ± 2.81

19.97 ± 2.68

11.07 ± 1.56

28.88 ± 2.94

WP-R 3 mg/kg

26.24 ± 2.77

20.99 ± 2.4

12.7 ± 1.66

29.28 ± 2.58

RSD-Veh

27.64 ± 3.09

18.56 ± 2.17

18.47 ± 3.06

18.64 ± 3.09

RSD-R 1 mg/kg

26.25 ± 2.81

22.19 ± 2.96

17.48 ± 3.72

26.91 ± 4.11

27.60 ± 2.30

24.25 ± 2.82

15.05 ± 2.82

33.45 ± 2.63

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RSD-R 3 mg/kg

Time spent exploring objects during the test phase (s)

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Groups

Time spent exploring objects during the sample phase (s)

Data are presented as mean ± S.E.M. WP, wide platform group; RSD, REM sleep-deprived group; WP-Veh, wide

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platform-vehicle group; WP-R 1 mg/kg, wide platform-rimonabant 1 mg/kg group; WP-R 3 mg/kg, wide platformrimonabant 3 mg/kg group; RSD-Veh, REM sleep deprivation-vehicle group; RSD-R 1 mg/kg, REM sleep

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deprivation-rimonabant 1 mg/kg group; RSD-R 3 mg/kg, REM sleep deprivation-rimonabant 3 mg/kg group.

27

Table 3 The mean time spent exploring objects during the sample and test phases and also the mean time spent exploring the familiar and novel objects during the test phase in the retrieval experiment of the object recognition task. Time spent exploring familiar object during the test phase (s)

Time spent exploring novel object during the test phase (s) 32.50 ± 3.20

WP

27.27 ± 3.16

21.84 ± 3.17

11.17 ± 1.28

RSD

27.95 ± 2.44

19.98 ± 2.20

18.03 ± 2.32

21.94 ± 3.62

WP-Veh

25.65 ± 2.66

20.97 ± 3.12

10.52 ± 1.68

31.42 ± 3.47

WP-R 1 mg/kg

27.95 ± 2.42

19.46 ± 2.64

11.74 ± 2.32

27.19 ± 3.05

WP-R 3 mg/kg

26.68 ± 2.47

22.67 ± 3.06

12.53 ± 1.8

32.8 ± 3.35

RSD-Veh

25.96 ± 2.59

19.33 ± 2.04

17.68 ± 2.94

20.99 ± 2.73

RSD-R 1 mg/kg

28.36 ± 2.41

22.76 ± 2.51

16.09 ± 2.41

29.42 ± 3.26

26.62 ± 2.49

23.13 ± 2.53

14.91 ± 2.25

31.35 ± 2.66

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RSD-R 3 mg/kg

Time spent exploring objects during the test phase (s)

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Groups

Time spent exploring objects during the sample phase (s)

Data are presented as mean ± S.E.M. WP, wide platform group; RSD, REM sleep-deprived group; WP-Veh, wide

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platform-vehicle group; WP-R 1 mg/kg, wide platform-rimonabant 1 mg/kg group; WP-R 3 mg/kg, wide platformrimonabant 3 mg/kg group; RSD-Veh, REM sleep deprivation-vehicle group; RSD-R 1 mg/kg, REM sleep

Jo

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deprivation-rimonabant 1 mg/kg group; RSD-R 3 mg/kg, REM sleep deprivation-rimonabant 3 mg/kg group.

28

Table 4 The mean time spent exploring objects during the sample and test phases and also the mean time spent exploring the familiar and novel objects during the test phase in the reconsolidation experiment of the object recognition task. Time spent exploring novel object during the test phase (s)

11.21 ± 1.24

35.36 ± 2.07

28.59 ± 2.18

23.70 ± 1.97

23.28 ± 2.95

RSD

27.77 ± 2.24

23.24 ± 2.41

20.08 ± 2.41

WP-Veh

27.98 ± 2.67

22.32 ± 2.2

24.37 ± 3.58

WP-R 1 mg/kg

29.61 ± 2.37

23.86 ± 1.98

21.43 ± 2.89

WP-R 3 mg/kg

29.57 ± 2.6

24.25 ± 2

RSD-Veh

26.81 ± 2.46

RSD-R 1 mg/kg

22.44 ± 3.50

12.27 ± 1.39

36.48 ± 3.57

10.64 ± 1.15

32.21 ± 2.97

19.84 ± 3.14

9.59 ± 2

30.09 ± 3.48

24.37 ± 2.48

20.19 ± 2.26

16.92 ± 2.73

23.47 ± 3.27

29.18 ± 2.48

23.68 ± 2.54

20.45 ± 2.23

17.36 ± 2.74

23.54 ± 3.24

27.83 ± 2.57

23.48 ± 2.73

23.72 ± 3.18

14.27 ± 3.15

33.17 ± 3.57

re

ro

17.73 ± 3.12

-p

WP

of

Time spent exploring familiar object during the test phase (s)

Groups

RSD-R 3 mg/kg

Time spent exploring objects during the 2nd sample phase (s)

Time spent exploring objects during the test phase (s)

Time spent exploring objects during the 1st sample phase (s)

Data are presented as mean ± S.E.M. WP, wide platform group; RSD, REM sleep-deprived group; WP-Veh, wide platform-vehicle

lP

group; WP-R 1 mg/kg, wide platform-rimonabant 1 mg/kg group; WP-R 3 mg/kg, wide platform-rimonabant 3 mg/kg group; RSDVeh, REM sleep deprivation-vehicle group; RSD-R 1 mg/kg, REM sleep deprivation-rimonabant 1 mg/kg group; RSD-R 3 mg/kg,

Jo

ur na

REM sleep deprivation-rimonabant 3 mg/kg group.

29

Table 5 Plasma corticosterone levels in the control and REM sleep-deprived animals. Plasma corticosterone levels (pg/ml)

Groups Home cage

41.86 ± 2.58

WP

44.18 ± 3.07

RSD

47.63 ± 3.2

Jo

ur na

lP

re

-p

ro

of

Comparison of plasma corticosterone levels between the groups. There were not significant differences between the groups. (n = 9 in each group). Data are presented as mean ± S.E.M. WP, wide platform group; RSD, REM sleep-deprived group.

30