Neuroscience Letters 580 (2014) 114–118
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Sleep deprivation impairs spontaneous object-place but not novel-object recognition in rats Hiroko Ishikawa, Kazuo Yamada ∗ , Constantine Pavlides, Yukio Ichitani Institute of Psychology and Behavioral Neuroscience, University of Tsukuba, Tsukuba 305-8577, Ibaraki, Japan
h i g h l i g h t s • Effects of sleep deprivation on one-trial recognition memory were examined. • Sleep deprivation immediately after learning disrupted place recognition but not object recognition. • Sleep deprivation 4–8 h after learning had no effects on both recognitions.
a r t i c l e
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Article history: Received 9 May 2014 Received in revised form 31 July 2014 Accepted 2 August 2014 Available online 12 August 2014 Keywords: Sleep deprivation Spontaneous novel-object recognition Object-place Recognition Rat Hippocampus
a b s t r a c t Effects of sleep deprivation (SD) on one-trial recognition memory were investigated in rats using either a spontaneous novel-object or object-place recognition test. Rats were allowed to explore a field in which two identical objects were presented. After a delay period, they were placed again in the same field in which either: (1) one of the two objects was replaced by another object (novel-object recognition); or (2) one of the sample objects was moved to a different place (object-place recognition), and their exploration behavior to these objects was analyzed. Four hours SD immediately after the sample phase (early SD group) disrupted object-place recognition but not novel-object recognition, while SD 4–8 h after the sample phase (delayed SD group) did not affect either paradigm. The results suggest that sleep selectively promotes the consolidation of hippocampal dependent memory, and that this effect is limited to within 4 h after learning. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Numerous studies have demonstrated that sleep contributes to memory consolidation. In humans, both short- and long-term total sleep deprivation (SD) and partial SD (less than 5 h SD per day) impaired cognitive task performance [15,24]. Especially, sleep is thought to promote the consolidation of declarative memory, which is dependent on the hippocampus [10]. Rodent models have previously been used to investigate the role of sleep in learning and memory. SD before or after the training of hippocampal-dependent tasks, such as, contextual fear conditioning [12,13,19,25,31], radial-arm maze [28], radial-arm water maze [1,2] and Morris water maze [27,29], impaired performance, however, SD before or after non-hippocampal dependent tasks did not produce any effects [5,33]. Furthermore, a number of studies have focused on the role of REM sleep on learning and memory. In rats, long-term potentiation (LTP), regarded as a neural basis of
∗ Corresponding author. Tel.: +81 29 853 2961; fax: +81 29 853 2961. E-mail address: [email protected] (K. Yamada). http://dx.doi.org/10.1016/j.neulet.2014.08.004 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
learning and memory, was impaired by REM SD prior to the application of high frequency stimulation (HFS) [8]. Also, neural replay of hippocampal awake activity during sleep was observed in the CA1 field during sleep [16,23]. These results suggest that REM sleep is strongly involved in learning and memory, and it especially promotes the consolidation of hippocampal-dependent memory. In addition, 4 h REM SD immediately after training, but not 4 h or 8 h after training, impaired the performance in Morris water maze [29] and radial-arm maze [28]. These findings suggest that REM sleep is essential within a specific time window following the training in order to facilitate learning and memory, specifically for hippocampally dependent tasks. In the present study, we investigated effects of 4 h SD on spontaneous novel-object and object-place recognition. The novel-object recognition and object-place recognition tests typically consist of a “sample phase” and a “test phase”, with a delay inserted between them. In the sample phase of novel-object recognition, two identical objects are placed in an open field and the subjects can freely explore, following which they are returned to their home cage. After a delay interval, they are returned to the open field for the test phase. In the test phase, two objects are placed in the same place
H. Ishikawa et al. / Neuroscience Letters 580 (2014) 114–118
as the sample phase, with one object being the same as the sample phase, while the other is a novel object. As the rats innately prefer novel objects, they spend more time exploring the novel object. In the object-place recognition, the sample and delay phases are the same as the novel-object recognition. In the test phase, two objects which are the same as the sample phase are presented, but one object is placed in a novel location. The subjects explore the object in the novel place more because they have the spontaneous innate tendency for novelty. The role of sleep in learning and memory is complex and appears to depend on the nature of the task. Most of the tasks that have been used to examine the effects of sleep on learning and memory, thus far, have certain disadvantages. For example, the radial arm maze task requires food restriction and repetitive training. The Morris water maze task and fear conditioning are based on negative reinforcement and are stressful. These factors may, in and of themselves, influence learning and memory. On the other hand, spontaneous object and object-place recognition tests offer a number of advantages in that they require: (1) one-trial learning; (2) do not involve stressful procedures or previous treatments like food deprivation; and (3) are based on a rodent’s innate tendency to explore novel stimuli longer than familiar stimuli [7]. Since animals should be able to retrieve the object or location information in a complex spatial scene when tested 24 h later, it has been suggested that memories of objects in rodents could be compared to human episodic memory [9]. Another advantage of these tasks is that they allow a comparison in performance between object recognition and object-place recognition in a within subject design, by using different objects. According to previous studies the hippocampus is involved in object-place recognition but not object recognition [3,20]. Thus, SD would be expected to impair only object-place recognition but not object memory. The present study is, to our knowledge, the first attempt to examine whether there is a specific timing of sleep that may be more important for memory consolidation of object or object-place in rats.
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platforms were located 2 cm above the water surface, 8–9 cm apart from each other. 2.3. Spontaneous recognition test All rats were tested on two spontaneous recognition tests in the following order at 24–48 h intervals: (1) object recognition test, (2) object-place recognition test. Handling and habituation to the apparatus preceded the recognition test. Rats received 5 min handling and 10 min habituation to the OF for 2 days. During the habituation, rats individually explored the OF freely. In addition, animals in the SD (early and delayed) groups were habituated to the apparatus for SD. Two or three rats that were raised in the same home cage were simultaneously placed on one of the platforms in the pool for 30 min. The spontaneous recognition test began between 08:30 and 10:00. The object recognition test consisted of a sample phase (20 min) and a test phase (5 min). In the sample phase, two identical objects were placed diagonally in the OF, and the rat explored the field freely. Twenty-four hours later the rat was introduced into the field again (test phase). In this phase, two objects were placed at the same position as in the sample phase, and one object was identical to that in the sample phase (familiar object) while the other was a novel object. The positions of objects in the test phase were counterbalanced between animals. In the object-place recognition test, the procedure was the same as the object recognition except on the test phase. In the test phase, two objects that were identical to the ones in the sample phase were placed in the OF, but one was in the same location as the sample phase (familiar object-place) and the other was moved to a different location (novel object-place). After each phase the floor of the OF was wiped with a damp cloth, and the objects were wiped with 70% ethanol. Time spent exploring each object during the test phase was scored by the experimenter viewing the rats’ behavior on a monitor screen. Exploration was defined as the rat’s nose being directed toward the object within a distance of 2 cm.
2. Materials and methods
2.4. Sleep deprivation
2.1. Animals
SD was accomplished by a modified multiple platform method [30]. Briefly, 2 or 3 rats that were raised in the same home cage were simultaneously placed on one of the platforms in the pool for 4 h and were able to travel over the platforms freely. If rats slept and relaxed their muscles, they fell into the water and awoke. Rats were subjected to this procedure immediately after the sample phase (early SD group) or starting 4 h after the sample phase (delayed SD group). Control animals were returned to their home cage after the sample phase, and left undisturbed in the same room as the SD groups for 8 h. Sleep was assessed based on video recorded behavior by standard visual procedures validated in previous studies [32]. Basically, sleep was scored when the animal displayed a typical sleep posture and kept immobile for at least 5 s. Scores of the control animals in the early and delayed SD periods indicated an average of 1.96 ± 0.47 and 3.10 ± 0.53 h of sleep, respectively. On the other hand, no sleep was observed during the SD periods in the experimental animals.
Twenty-five male Wistar-Imamichi rats (8–10 weeks old) were used as subjects. These were divided into three groups: Control (n = 9); early SD (n = 8); and delayed SD (n = 8). They were housed in groups (2–3/cage) under constant temperature and humidity conditions on a 12 h light/12 h dark cycle (lights on 08:00–20:00) with food and water available ad libitum. All experiments were approved by the University of Tsukuba Committee on Animal Research.
2.2. Apparatus For the spontaneous recognition test, an open field (OF) arena (90 cm × 90 cm × 45 cm) made of polyvinylchloride was used. Its walls were colored black, and the floor was gray. The objects used were black and white triangular cast materials, black and white cylinders of cast metal, yellow and brown soda cans, duck toys colored in yellow and orange, pink plastic cups, blue plastic soap cases, and silver and black bottles. All objects were sufficiently heavy that rats could not move them. A video camera was suspended above the arena, and the image was projected to a monitor to allow the experimenter to observe the animal’s behavior. For SD, a circular swimming pool measuring 150 cm in diameter and 45 cm in height was used. The pool was filled to a depth of 10 cm with 24 ± 2 ◦ C water. The platform was made of transparent glass, and its top was a circular plate measuring 7 cm in diameter. Eight
2.5. Statistical analysis One rat of early SD group, that did not explore both objects in the test phase, was excluded from further analysis. The exploration time to each object in the test phase was analyzed by a two-way ANOVA with repeated measure. Individual comparisons were evaluated using a simple main effect test and a post-hoc Bonferroni test. A discrimination ratio (DR), which was calculated by dividing the amount of exploration of the novel object by the total amount
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Fig. 1. Effects of 4 h SD on spontaneous novel-object recognition. (A) Schematic drawing of novel-object recognition test. (B) Time spent exploring each object in the first 2 min of test phase. (C) Discrimination ratio (DR) in the test phase, calculated by dividing the amount of exploration of the novel object by the total amount of object exploration. DRs of all groups were above chance (50%) level. In both panels means ± SEM are shown; * p < 0.05, ** p < 0.01.
of object exploration in the test phase, was analyzed by one-way ANOVA, followed by post hoc comparisons using the Bonferroni test. For statistical analysis of exploration time and DR, only the exploration in the first 2 min of the test phase was used, because novelty of object decreases with time and novelty preference could no longer be reliably assessed [11,21]. DR data were also compared to chance level (50%) using a one-sample t-test. 3. Results Exploration time of each object in the first 2 min of the test phase in the object recognition task is shown in Fig. 1B. There was a significant main effect of object (F(1,21) = 42.35, p < 0.01). Exploration time of the novel object was significantly longer than that of the familiar object for the three groups. Fig. 1C shows the DRs in the first 2 min of the test phase. One-way ANOVA showed no significant main effect (F(2,21) = 0.69, n.s.). DR scores in all groups were significantly higher than chance level (control group, t(8) = 8.33, p < 0.01; early SD group, t(6) = 2.89, p < 0.01; delayed SD group, t(7) = 4.14, p < 0.01). Exploration time of each object in the first 2 min of the test phase in object-place recognition task is shown in Fig. 2B. There was a significant main effect of object (F(1,21) = 11.67, p < 0.01), as well as a significant interaction between group and object (F(2,21) = 6.40, p < 0.01). An analysis of simple effects indicated that the object effect was significant in both control and delayed SD groups, and the group effect was significant for the familiar object. Exploration of the novel object was significantly longer than that of the familiar object in the control group (F(1,21) = 27.72, p < 0.01) and delayed SD group (F(1,21) = 5.98, p < 0.05), but not in the early SD group. A post hoc comparison revealed that exploration of the familiar object in the early SD group was significantly longer than that of the delayed SD group (p < 0.05) and control group (p < 0.01). Fig. 2C shows DRs for each group in the test phase. There was a significant
main effect of group (F(2,21) = 12.03, p < 0.01), and the post hoc test revealed that the DR of the early SD group was lower than that of the delayed SD group (p < 0.01) and control group (p < 0.01). There was no significant difference between the delayed SD group and control group. Furthermore, DRs of the delayed SD group and control group were higher than chance level (control group, t(8) = 6.24, p < 0.01; delayed SD group, t(7) = 6.03, p < 0.01). Table 1 shows the total exploration time in the entire duration of the sample and test phase. In the sample phase, a two-way ANOVA (Group (3) × Task (2)) revealed significant main effects of group (F(2,21) = 6.17, p < 0.01) and task (F(1,21) = 9.00, p < 0.01). Exploration time of the control group was longer than that of the delayed SD group (p < 0.05). In addition, exploration time in the object recognition task was longer than that in the object-place recognition task (p < 0.01). In the test phase, there was a significant main effect of task (F(1,21) = 9.38, p < 0.01). As in the sample phase, total exploration time in the object recognition task was longer than that in the object-place recognition task (p < 0.01).
4. Discussion The present results show that 4 h SD immediately after the sample phase (early SD group) disrupted object-place recognition but not novel-object recognition memory. The procedure for SD used was in accordance with the modified multiple platform method by Sushecki and Tufik [30]. Machado et al. [17] reported that using this method a total deprivation of REM and a decrement of 31% of non-REM sleep were achieved. On the other hand, all rats in both SD groups (early SD and delayed SD) in the present study appeared to be deprived of total sleep according to our observation of rat’s behavior. This discrepancy may be accounted by the difference in the length of SD (4 d vs. 4 h) between Machado et al. [17] and the present study. Rats could be on the narrow platform without sleep
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Fig. 2. Effects of 4 h SD on spontaneous object-place recognition. (A) Schematic drawing of object-place recognition test. (B) Time spent exploring each object in the first 2 min of test phase. (C) Discrimination ratio (DR) in the test phase, calculated by dividing the amount of exploration of the novel object by the total amount of object exploration. DR of early SD group was significantly lower than those of Control and delayed SD groups. In both panels means ± SEM are shown; * p < 0.05, ** p < 0.01.
for 4 h, while rats had to sleep on the platform for 4 d in Machado et al. [17]. The present findings showing that 4 h SD impaired only objectplace recognition are consistent with some previous studies, but at adds with others. For example, Inostroza et al. [14] showed that 80 min SD immediately after the sample phase impaired objectplace recognition but not novel-object recognition. Furthermore, 2 h SD immediately after the sample phase was also reported to impair object-place recognition [4]. In contrast, a different study reported that 6 h SD immediately after the sample phase disturbed memory consolidation in a novel-object recognition test [6,22]. This discrepancy may be accounted for by differences in the number of objects used in the sample phase. While the above studies used two or three different objects, we used two identical objects in the sample phase. Additional contextual cues or object-object associations due to using multiple different objects in the sample phase may complicate the memory load sufficiently to also require the hippocampus for novel-object recognition memory [3]. Lesion studies demonstrated that the hippocampus is involved in object-place recognition, while novel-object recognition is dependent on the prefrontal and entorhinal cortex [3,20]. In other memory tasks, performance that depends on the hippocampus has been shown to be impaired by SD. For example, REM SD impaired contextual fear conditioning (hippocampus dependent) but not cued conditioning (amygdala dependent) [19]. Four hours
SD immediately after training impaired performance in the Morris water maze [29] and 8-arm radial maze task [28]. In addition, Romcy-Pereira and Pavlides [26] reported that 4 h REM SD immediately after HFS impaired the maintenance of LTP 48 h after HFS in the dentate gyrus, but it enhanced LTP in the medial prefrontal cortex. Taken together, these findings strongly suggest that hippocampal dependent learning and memory is selectively promoted by sleep. On the other hand, SD initiated 4 h after the sample phase (delayed SD group) did not affect either object or object-place recognition. These results are consistent with previous reports showing that 4 h REM SD immediately after training, but not 4 h or 8 h after training, impaired performance in the Morris water maze [29] and radial-arm maze [28]. Thus it is suggested that only the early intervention by motor activity, sensory input or additional learning occurring during waking might be interfered with the process of memory consolidation necessary for optimal performance in the recognition test, while 4 h of undisturbed sleep immediately after the sample phase was sufficient for memory consolidation. A possible alternative explanation for the disruption of objectplace recognition memory by SD could be the lack of motivation in the SD animals. However, this could be discounted since there was no difference in the total amount of object exploration between the early SD group and control group (Table 1). The differences in the total exploration time between the tasks may be due to an order
Table 1 The total exploration time (s) in the sample phase (20 min) and the test phase (5 min) in spontaneous object recognition task and object-place recognition task. Means (±SEM) are shown. Sample phase (20 nub)
Object recognition Object-place recognition
Test phase (5 min)
Control
Early SD
Delayed SD
Control
Early SD
Delayed SD
142.52 (±17.32) 109.95 (±11.31)
111.61 (±13.77) 73.56 (±5.73)
95.34 (±12.39) 74.70 (±9.27)
50.01 (±2.90) 37.14 (±3.89)
45.89 (±7.28) 35.91 (±3.91)
37.26 (±4.80) 29.10 (±5.04)
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effect, because the object recognition task was followed by the object-place recognition task for all animals. In addition, although the different amount of exploration during the sample phase could have affected the performance in object-place recognition, this possibility could be also discounted since there was no difference in the total amount of object exploration during the sample phase between the early SD and delayed SD groups. A second possibility is that stress during the SD could have affected memory consolidation or the performance of the recognition test. In general, SD has been considered to be stressful for animals, especially using the platform method. Since stress itself may cause memory impairment, stressful factors should be excluded by all possible means. The multiple platform method used in the present study is thought to avoid stress from immobilization. In addition, Sushecki and Tufik [30] reported that the method in which multiple animals, raised in the same home cage, are simultaneously placed in the SD apparatus, attenuated stress from social instability during the SD, even though plasma ACTH levels of SD group were higher than the control group that was not sleep deprived. In the present study, although the possibility exists that the stress during SD affected the performance at the test phase, this is unlikely since 20 h recovery from SD, in the early SD group, allowed sufficient time to recover from the SD. In addition, no deficits in the object-place recognition was observed in the delayed SD group suggesting that this was not the case. Nonetheless, to further eliminate the possibility that stress immediately after training or learning could affect encoding and/or consolidation processes of object-place memory [18], further studies using stress control animals that, for example, are subjected to SD during early dark phase at which rats rarely sleep, are needed. 5. Conclusions A period of 4 h SD impaired object-place recognition but not novel-object recognition. In addition, SD immediately after, but not 4 h after the sample phase, impaired object-place recognition. It is suggested that sleep selectively promotes the consolidation of hippocampal-dependent memory, and that this effect is limited to within 4 h after learning. Acknowledgments This study was supported by grants from Japan Society for the Promotion of Science to K.Y. (24530909) and Y.I. (24653209). References [1] A.M. Aleisa, K.H. Alzoubi, K.A. Alkadhi, Post-learning REM sleep deprivation impairs long-term memory: reversal by acute nicotine treatment, Neurosci. Lett. 499 (2011) 28–31. [2] I.M. Alhaider, A.M. Aleisa, T.T. Tran, K.H. Alzoubi, K.A. Alkadhi, Chronic caffeine treatment prevents sleep deprivation-induced impairment of cognitive function and synaptic plasticity, Sleep 33 (2010) 437–444. [3] G.R.I. Barker, E.C. Warburton, When is the hippocampus involved in recognition memory? J. Neurosci. 31 (2011) 10721–10731. [4] S. Binder, P.C. Baier,.M. Mölle, M. Inostroza, J. Born, L. Marshall, Sleep enhances memory consolidation in the hippocampus-dependent object-place recognition task in rats, Neurobiol. Learn. Mem. 97 (2012) 213–219. [5] E.T. Bjorness, B.T. Riley, M.K. Tysor, G.R. Poe, REM restriction persistently alters strategy used to solve a spatial task, Learn. Mem. 12 (2005) 352–359. [6] L. Chen, S. Tian, J. Ke, Rapid eye movement sleep deprivation disrupts consolidation but not reconsolidation of novel object recognition memory in rats, Neurosci. Lett. 563 (2014) 12–16.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Sleep deprivation impairs spontaneous object-place but not novel-object recognition in rats Hiroko Ishikawa, Kazuo Yamada ∗ , Constantine Pavlides, Yukio Ichitani Institute of Psychology and Behavioral Neuroscience, University of Tsukuba, Tsukuba 305-8577, Ibaraki, Japan
h i g h l i g h t s • Effects of sleep deprivation on one-trial recognition memory were examined. • Sleep deprivation immediately after learning disrupted place recognition but not object recognition. • Sleep deprivation 4–8 h after learning had no effects on both recognitions.
a r t i c l e
i n f o
Article history: Received 9 May 2014 Received in revised form 31 July 2014 Accepted 2 August 2014 Available online 12 August 2014 Keywords: Sleep deprivation Spontaneous novel-object recognition Object-place Recognition Rat Hippocampus
a b s t r a c t Effects of sleep deprivation (SD) on one-trial recognition memory were investigated in rats using either a spontaneous novel-object or object-place recognition test. Rats were allowed to explore a field in which two identical objects were presented. After a delay period, they were placed again in the same field in which either: (1) one of the two objects was replaced by another object (novel-object recognition); or (2) one of the sample objects was moved to a different place (object-place recognition), and their exploration behavior to these objects was analyzed. Four hours SD immediately after the sample phase (early SD group) disrupted object-place recognition but not novel-object recognition, while SD 4–8 h after the sample phase (delayed SD group) did not affect either paradigm. The results suggest that sleep selectively promotes the consolidation of hippocampal dependent memory, and that this effect is limited to within 4 h after learning. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Numerous studies have demonstrated that sleep contributes to memory consolidation. In humans, both short- and long-term total sleep deprivation (SD) and partial SD (less than 5 h SD per day) impaired cognitive task performance [15,24]. Especially, sleep is thought to promote the consolidation of declarative memory, which is dependent on the hippocampus [10]. Rodent models have previously been used to investigate the role of sleep in learning and memory. SD before or after the training of hippocampal-dependent tasks, such as, contextual fear conditioning [12,13,19,25,31], radial-arm maze [28], radial-arm water maze [1,2] and Morris water maze [27,29], impaired performance, however, SD before or after non-hippocampal dependent tasks did not produce any effects [5,33]. Furthermore, a number of studies have focused on the role of REM sleep on learning and memory. In rats, long-term potentiation (LTP), regarded as a neural basis of
∗ Corresponding author. Tel.: +81 29 853 2961; fax: +81 29 853 2961. E-mail address: [email protected] (K. Yamada). http://dx.doi.org/10.1016/j.neulet.2014.08.004 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
learning and memory, was impaired by REM SD prior to the application of high frequency stimulation (HFS) [8]. Also, neural replay of hippocampal awake activity during sleep was observed in the CA1 field during sleep [16,23]. These results suggest that REM sleep is strongly involved in learning and memory, and it especially promotes the consolidation of hippocampal-dependent memory. In addition, 4 h REM SD immediately after training, but not 4 h or 8 h after training, impaired the performance in Morris water maze [29] and radial-arm maze [28]. These findings suggest that REM sleep is essential within a specific time window following the training in order to facilitate learning and memory, specifically for hippocampally dependent tasks. In the present study, we investigated effects of 4 h SD on spontaneous novel-object and object-place recognition. The novel-object recognition and object-place recognition tests typically consist of a “sample phase” and a “test phase”, with a delay inserted between them. In the sample phase of novel-object recognition, two identical objects are placed in an open field and the subjects can freely explore, following which they are returned to their home cage. After a delay interval, they are returned to the open field for the test phase. In the test phase, two objects are placed in the same place
H. Ishikawa et al. / Neuroscience Letters 580 (2014) 114–118
as the sample phase, with one object being the same as the sample phase, while the other is a novel object. As the rats innately prefer novel objects, they spend more time exploring the novel object. In the object-place recognition, the sample and delay phases are the same as the novel-object recognition. In the test phase, two objects which are the same as the sample phase are presented, but one object is placed in a novel location. The subjects explore the object in the novel place more because they have the spontaneous innate tendency for novelty. The role of sleep in learning and memory is complex and appears to depend on the nature of the task. Most of the tasks that have been used to examine the effects of sleep on learning and memory, thus far, have certain disadvantages. For example, the radial arm maze task requires food restriction and repetitive training. The Morris water maze task and fear conditioning are based on negative reinforcement and are stressful. These factors may, in and of themselves, influence learning and memory. On the other hand, spontaneous object and object-place recognition tests offer a number of advantages in that they require: (1) one-trial learning; (2) do not involve stressful procedures or previous treatments like food deprivation; and (3) are based on a rodent’s innate tendency to explore novel stimuli longer than familiar stimuli [7]. Since animals should be able to retrieve the object or location information in a complex spatial scene when tested 24 h later, it has been suggested that memories of objects in rodents could be compared to human episodic memory [9]. Another advantage of these tasks is that they allow a comparison in performance between object recognition and object-place recognition in a within subject design, by using different objects. According to previous studies the hippocampus is involved in object-place recognition but not object recognition [3,20]. Thus, SD would be expected to impair only object-place recognition but not object memory. The present study is, to our knowledge, the first attempt to examine whether there is a specific timing of sleep that may be more important for memory consolidation of object or object-place in rats.
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platforms were located 2 cm above the water surface, 8–9 cm apart from each other. 2.3. Spontaneous recognition test All rats were tested on two spontaneous recognition tests in the following order at 24–48 h intervals: (1) object recognition test, (2) object-place recognition test. Handling and habituation to the apparatus preceded the recognition test. Rats received 5 min handling and 10 min habituation to the OF for 2 days. During the habituation, rats individually explored the OF freely. In addition, animals in the SD (early and delayed) groups were habituated to the apparatus for SD. Two or three rats that were raised in the same home cage were simultaneously placed on one of the platforms in the pool for 30 min. The spontaneous recognition test began between 08:30 and 10:00. The object recognition test consisted of a sample phase (20 min) and a test phase (5 min). In the sample phase, two identical objects were placed diagonally in the OF, and the rat explored the field freely. Twenty-four hours later the rat was introduced into the field again (test phase). In this phase, two objects were placed at the same position as in the sample phase, and one object was identical to that in the sample phase (familiar object) while the other was a novel object. The positions of objects in the test phase were counterbalanced between animals. In the object-place recognition test, the procedure was the same as the object recognition except on the test phase. In the test phase, two objects that were identical to the ones in the sample phase were placed in the OF, but one was in the same location as the sample phase (familiar object-place) and the other was moved to a different location (novel object-place). After each phase the floor of the OF was wiped with a damp cloth, and the objects were wiped with 70% ethanol. Time spent exploring each object during the test phase was scored by the experimenter viewing the rats’ behavior on a monitor screen. Exploration was defined as the rat’s nose being directed toward the object within a distance of 2 cm.
2. Materials and methods
2.4. Sleep deprivation
2.1. Animals
SD was accomplished by a modified multiple platform method [30]. Briefly, 2 or 3 rats that were raised in the same home cage were simultaneously placed on one of the platforms in the pool for 4 h and were able to travel over the platforms freely. If rats slept and relaxed their muscles, they fell into the water and awoke. Rats were subjected to this procedure immediately after the sample phase (early SD group) or starting 4 h after the sample phase (delayed SD group). Control animals were returned to their home cage after the sample phase, and left undisturbed in the same room as the SD groups for 8 h. Sleep was assessed based on video recorded behavior by standard visual procedures validated in previous studies [32]. Basically, sleep was scored when the animal displayed a typical sleep posture and kept immobile for at least 5 s. Scores of the control animals in the early and delayed SD periods indicated an average of 1.96 ± 0.47 and 3.10 ± 0.53 h of sleep, respectively. On the other hand, no sleep was observed during the SD periods in the experimental animals.
Twenty-five male Wistar-Imamichi rats (8–10 weeks old) were used as subjects. These were divided into three groups: Control (n = 9); early SD (n = 8); and delayed SD (n = 8). They were housed in groups (2–3/cage) under constant temperature and humidity conditions on a 12 h light/12 h dark cycle (lights on 08:00–20:00) with food and water available ad libitum. All experiments were approved by the University of Tsukuba Committee on Animal Research.
2.2. Apparatus For the spontaneous recognition test, an open field (OF) arena (90 cm × 90 cm × 45 cm) made of polyvinylchloride was used. Its walls were colored black, and the floor was gray. The objects used were black and white triangular cast materials, black and white cylinders of cast metal, yellow and brown soda cans, duck toys colored in yellow and orange, pink plastic cups, blue plastic soap cases, and silver and black bottles. All objects were sufficiently heavy that rats could not move them. A video camera was suspended above the arena, and the image was projected to a monitor to allow the experimenter to observe the animal’s behavior. For SD, a circular swimming pool measuring 150 cm in diameter and 45 cm in height was used. The pool was filled to a depth of 10 cm with 24 ± 2 ◦ C water. The platform was made of transparent glass, and its top was a circular plate measuring 7 cm in diameter. Eight
2.5. Statistical analysis One rat of early SD group, that did not explore both objects in the test phase, was excluded from further analysis. The exploration time to each object in the test phase was analyzed by a two-way ANOVA with repeated measure. Individual comparisons were evaluated using a simple main effect test and a post-hoc Bonferroni test. A discrimination ratio (DR), which was calculated by dividing the amount of exploration of the novel object by the total amount
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Fig. 1. Effects of 4 h SD on spontaneous novel-object recognition. (A) Schematic drawing of novel-object recognition test. (B) Time spent exploring each object in the first 2 min of test phase. (C) Discrimination ratio (DR) in the test phase, calculated by dividing the amount of exploration of the novel object by the total amount of object exploration. DRs of all groups were above chance (50%) level. In both panels means ± SEM are shown; * p < 0.05, ** p < 0.01.
of object exploration in the test phase, was analyzed by one-way ANOVA, followed by post hoc comparisons using the Bonferroni test. For statistical analysis of exploration time and DR, only the exploration in the first 2 min of the test phase was used, because novelty of object decreases with time and novelty preference could no longer be reliably assessed [11,21]. DR data were also compared to chance level (50%) using a one-sample t-test. 3. Results Exploration time of each object in the first 2 min of the test phase in the object recognition task is shown in Fig. 1B. There was a significant main effect of object (F(1,21) = 42.35, p < 0.01). Exploration time of the novel object was significantly longer than that of the familiar object for the three groups. Fig. 1C shows the DRs in the first 2 min of the test phase. One-way ANOVA showed no significant main effect (F(2,21) = 0.69, n.s.). DR scores in all groups were significantly higher than chance level (control group, t(8) = 8.33, p < 0.01; early SD group, t(6) = 2.89, p < 0.01; delayed SD group, t(7) = 4.14, p < 0.01). Exploration time of each object in the first 2 min of the test phase in object-place recognition task is shown in Fig. 2B. There was a significant main effect of object (F(1,21) = 11.67, p < 0.01), as well as a significant interaction between group and object (F(2,21) = 6.40, p < 0.01). An analysis of simple effects indicated that the object effect was significant in both control and delayed SD groups, and the group effect was significant for the familiar object. Exploration of the novel object was significantly longer than that of the familiar object in the control group (F(1,21) = 27.72, p < 0.01) and delayed SD group (F(1,21) = 5.98, p < 0.05), but not in the early SD group. A post hoc comparison revealed that exploration of the familiar object in the early SD group was significantly longer than that of the delayed SD group (p < 0.05) and control group (p < 0.01). Fig. 2C shows DRs for each group in the test phase. There was a significant
main effect of group (F(2,21) = 12.03, p < 0.01), and the post hoc test revealed that the DR of the early SD group was lower than that of the delayed SD group (p < 0.01) and control group (p < 0.01). There was no significant difference between the delayed SD group and control group. Furthermore, DRs of the delayed SD group and control group were higher than chance level (control group, t(8) = 6.24, p < 0.01; delayed SD group, t(7) = 6.03, p < 0.01). Table 1 shows the total exploration time in the entire duration of the sample and test phase. In the sample phase, a two-way ANOVA (Group (3) × Task (2)) revealed significant main effects of group (F(2,21) = 6.17, p < 0.01) and task (F(1,21) = 9.00, p < 0.01). Exploration time of the control group was longer than that of the delayed SD group (p < 0.05). In addition, exploration time in the object recognition task was longer than that in the object-place recognition task (p < 0.01). In the test phase, there was a significant main effect of task (F(1,21) = 9.38, p < 0.01). As in the sample phase, total exploration time in the object recognition task was longer than that in the object-place recognition task (p < 0.01).
4. Discussion The present results show that 4 h SD immediately after the sample phase (early SD group) disrupted object-place recognition but not novel-object recognition memory. The procedure for SD used was in accordance with the modified multiple platform method by Sushecki and Tufik [30]. Machado et al. [17] reported that using this method a total deprivation of REM and a decrement of 31% of non-REM sleep were achieved. On the other hand, all rats in both SD groups (early SD and delayed SD) in the present study appeared to be deprived of total sleep according to our observation of rat’s behavior. This discrepancy may be accounted by the difference in the length of SD (4 d vs. 4 h) between Machado et al. [17] and the present study. Rats could be on the narrow platform without sleep
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Fig. 2. Effects of 4 h SD on spontaneous object-place recognition. (A) Schematic drawing of object-place recognition test. (B) Time spent exploring each object in the first 2 min of test phase. (C) Discrimination ratio (DR) in the test phase, calculated by dividing the amount of exploration of the novel object by the total amount of object exploration. DR of early SD group was significantly lower than those of Control and delayed SD groups. In both panels means ± SEM are shown; * p < 0.05, ** p < 0.01.
for 4 h, while rats had to sleep on the platform for 4 d in Machado et al. [17]. The present findings showing that 4 h SD impaired only objectplace recognition are consistent with some previous studies, but at adds with others. For example, Inostroza et al. [14] showed that 80 min SD immediately after the sample phase impaired objectplace recognition but not novel-object recognition. Furthermore, 2 h SD immediately after the sample phase was also reported to impair object-place recognition [4]. In contrast, a different study reported that 6 h SD immediately after the sample phase disturbed memory consolidation in a novel-object recognition test [6,22]. This discrepancy may be accounted for by differences in the number of objects used in the sample phase. While the above studies used two or three different objects, we used two identical objects in the sample phase. Additional contextual cues or object-object associations due to using multiple different objects in the sample phase may complicate the memory load sufficiently to also require the hippocampus for novel-object recognition memory [3]. Lesion studies demonstrated that the hippocampus is involved in object-place recognition, while novel-object recognition is dependent on the prefrontal and entorhinal cortex [3,20]. In other memory tasks, performance that depends on the hippocampus has been shown to be impaired by SD. For example, REM SD impaired contextual fear conditioning (hippocampus dependent) but not cued conditioning (amygdala dependent) [19]. Four hours
SD immediately after training impaired performance in the Morris water maze [29] and 8-arm radial maze task [28]. In addition, Romcy-Pereira and Pavlides [26] reported that 4 h REM SD immediately after HFS impaired the maintenance of LTP 48 h after HFS in the dentate gyrus, but it enhanced LTP in the medial prefrontal cortex. Taken together, these findings strongly suggest that hippocampal dependent learning and memory is selectively promoted by sleep. On the other hand, SD initiated 4 h after the sample phase (delayed SD group) did not affect either object or object-place recognition. These results are consistent with previous reports showing that 4 h REM SD immediately after training, but not 4 h or 8 h after training, impaired performance in the Morris water maze [29] and radial-arm maze [28]. Thus it is suggested that only the early intervention by motor activity, sensory input or additional learning occurring during waking might be interfered with the process of memory consolidation necessary for optimal performance in the recognition test, while 4 h of undisturbed sleep immediately after the sample phase was sufficient for memory consolidation. A possible alternative explanation for the disruption of objectplace recognition memory by SD could be the lack of motivation in the SD animals. However, this could be discounted since there was no difference in the total amount of object exploration between the early SD group and control group (Table 1). The differences in the total exploration time between the tasks may be due to an order
Table 1 The total exploration time (s) in the sample phase (20 min) and the test phase (5 min) in spontaneous object recognition task and object-place recognition task. Means (±SEM) are shown. Sample phase (20 nub)
Object recognition Object-place recognition
Test phase (5 min)
Control
Early SD
Delayed SD
Control
Early SD
Delayed SD
142.52 (±17.32) 109.95 (±11.31)
111.61 (±13.77) 73.56 (±5.73)
95.34 (±12.39) 74.70 (±9.27)
50.01 (±2.90) 37.14 (±3.89)
45.89 (±7.28) 35.91 (±3.91)
37.26 (±4.80) 29.10 (±5.04)
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effect, because the object recognition task was followed by the object-place recognition task for all animals. In addition, although the different amount of exploration during the sample phase could have affected the performance in object-place recognition, this possibility could be also discounted since there was no difference in the total amount of object exploration during the sample phase between the early SD and delayed SD groups. A second possibility is that stress during the SD could have affected memory consolidation or the performance of the recognition test. In general, SD has been considered to be stressful for animals, especially using the platform method. Since stress itself may cause memory impairment, stressful factors should be excluded by all possible means. The multiple platform method used in the present study is thought to avoid stress from immobilization. In addition, Sushecki and Tufik [30] reported that the method in which multiple animals, raised in the same home cage, are simultaneously placed in the SD apparatus, attenuated stress from social instability during the SD, even though plasma ACTH levels of SD group were higher than the control group that was not sleep deprived. In the present study, although the possibility exists that the stress during SD affected the performance at the test phase, this is unlikely since 20 h recovery from SD, in the early SD group, allowed sufficient time to recover from the SD. In addition, no deficits in the object-place recognition was observed in the delayed SD group suggesting that this was not the case. Nonetheless, to further eliminate the possibility that stress immediately after training or learning could affect encoding and/or consolidation processes of object-place memory [18], further studies using stress control animals that, for example, are subjected to SD during early dark phase at which rats rarely sleep, are needed. 5. Conclusions A period of 4 h SD impaired object-place recognition but not novel-object recognition. In addition, SD immediately after, but not 4 h after the sample phase, impaired object-place recognition. It is suggested that sleep selectively promotes the consolidation of hippocampal-dependent memory, and that this effect is limited to within 4 h after learning. Acknowledgments This study was supported by grants from Japan Society for the Promotion of Science to K.Y. (24530909) and Y.I. (24653209). References [1] A.M. Aleisa, K.H. Alzoubi, K.A. Alkadhi, Post-learning REM sleep deprivation impairs long-term memory: reversal by acute nicotine treatment, Neurosci. Lett. 499 (2011) 28–31. [2] I.M. Alhaider, A.M. Aleisa, T.T. Tran, K.H. Alzoubi, K.A. Alkadhi, Chronic caffeine treatment prevents sleep deprivation-induced impairment of cognitive function and synaptic plasticity, Sleep 33 (2010) 437–444. [3] G.R.I. Barker, E.C. Warburton, When is the hippocampus involved in recognition memory? J. Neurosci. 31 (2011) 10721–10731. [4] S. Binder, P.C. Baier,.M. Mölle, M. Inostroza, J. Born, L. Marshall, Sleep enhances memory consolidation in the hippocampus-dependent object-place recognition task in rats, Neurobiol. Learn. Mem. 97 (2012) 213–219. [5] E.T. Bjorness, B.T. Riley, M.K. Tysor, G.R. Poe, REM restriction persistently alters strategy used to solve a spatial task, Learn. Mem. 12 (2005) 352–359. [6] L. Chen, S. Tian, J. Ke, Rapid eye movement sleep deprivation disrupts consolidation but not reconsolidation of novel object recognition memory in rats, Neurosci. Lett. 563 (2014) 12–16.
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