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Paradoxical sleep deprivation impairs spatial learning and affects membrane excitability and mitochondrial protein in the hippocampus
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
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Research Report
Paradoxical sleep deprivation impairs spatial learning and affects membrane excitability and mitochondrial protein in the hippocampus Rui-Hua Yang a,⁎, San-Jue Hu b , Yuan Wang a , Wen-Bin Zhang a , Wen-Jing Luo a , Jing-Yuan Chen a,⁎ a b
Department of Occupational and Environmental Health, the Fourth Military Medical University, Xi'an 710032, PR China Institute of Neuroscience, the Fourth Military Medical University, Xi'an 710032, PR China
A R T I C LE I N FO
AB S T R A C T
Article history:
Previous research has demonstrated that paradoxical sleep has a key role in learning and
Accepted 9 July 2008
memory, and sleep deprivation interferes with learning and memory. However, the
Available online 17 July 2008
mechanism of memory impairment induced by sleep deprivation is poorly understood. The present study investigated the effect of paradoxical sleep deprivation (PSD) on spatial
Keywords:
learning and memory using the Morris Water Maze. Effects of PSD on CA1 pyramidal
paradoxical sleep deprivation
neurons in hippocampus were also examined. PSD impaired spatial learning of rats. PSD
Morris Water Maze
induced translocation of Bax to mitochondria and cytochrome c release into the cytoplasm,
spatial learning
and decreased the membrane excitability of CA1 pyramidal neurons, effects which may
membrane excitability
contribute to the deficits in learning behavior. These results may partially explain the
mitochondria
mechanism of the effect of PSD on learning. Modulating the excitability of hippocampal
hippocampus
neurons and protecting mitochondrial function are possible targets for preventing the effects of paradoxical sleep deprivation. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
There is growing evidence that sleep and learning are interconnected. Both human and animal studies have shown that sleep has a key role in learning and memory. Rapid eye movement (REM) sleep is increased following learning sessions (Smith and Rose, 1997; Mandai et al., 1989), and sleep deprivation interferes with learning and memory (Silvestri, 2005; Guan et al., 2004). A number of animal studies have gone on to explore the potential cellular and molecular underpinnings of sleep deprivation-induced learning deficits, and
many of these studies have focused on changes in transmitters (Banks et al., 2002; Hipolide et al., 2005), the impairment of formation of long-term potentiation (Campbell et al., 2002; Davis et al., 2003), and effects on extracellular signal-regulated kinases (Kelleher et al., 2004; Guan et al., 2004). Less attention has been given to the effects of sleep deprivation on the intrinsic properties of neurons. Limited animal studies indicated that the firing patterns of neurons in the hippocampus and cortex involved in the learning experience were replayed during subsequent sleep (Louie and Wilson, 2001; Skaggs and McNaughton, 1996; Sutherl and McNaughton,
⁎ Corresponding authors. Fax: +86 29 84774863. E-mail addresses: [email protected] (R.-H. Yang), [email protected] (J.-Y. Chen). Abbreviations: Rapid eye movement sleep, REM sleep; Paradoxical sleep deprivation, PSD; Large platform, LP; Action potential, AP; Cytochrome c, Cyt-c; Mitochondria permeability transition pore, MTP 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.07.033
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2000), and REM sleep deprivation reduced the basic excitability of hippocampal neurons (McDermott et al., 2003). However, the molecular mechanism of changes of neuron properties induced by sleep deprivation remains poorly understood. Given the well recognized cognitive effects and the prevalence of sleep deprivation in society today, investigating the effects of sleep deprivation on cellular physiology and related mechanisms is of interest. The present study investigated the effect of paradoxical sleep deprivation (PSD) on spatial learning and memory with the Morris Water Maze, which is commonly used to examine hippocampal function (Walker and Stickgold, 2006; Smith and Rose, 1996). In addition we examined possible mechanisms by investigating the effects of PSD on membrane excitability and mitochondria of hippocampal CA1 pyramidal neurons. Using the Morris Water Maze spatial learning set protocol, we have found that PSD rats are slower to learn the location of the platform than control groups, but are capable of successfully performing the task with repeated exposure to the test. During the probe trial, the PSD rats tended to spend slightly less time in the target quadrant compared with the control groups, but this effect did not quite reach significance. The data from the behavior tests indicated that PSD impairs the ability of spatial learning of rats. There were indications of minor effects on working memory and maintenance of established memory as well, but most of these did not quite reach significance. We further have found that PSD induces translocation of Bax to mitochondria and cytochrome c release into cytoplasm, and decreases the membrane excitability of CA1 pyramidal neurons, which may contribute the deficits in performing the spatial learning task.
2.
Results
2.1.
PSD impairs spatial learning
To test for the impact of PSD on memory formation, a group of rats was REM sleep-deprived for 5 days, and a second group of rats (LP) was placed on large platforms as a control group to simulate the PSD condition while allowing sleep to occur, and the third group of rats was maintained in their home cages as another control group. Daily, the rats were tested between 150:0 h and 18:00 h, in a place-learning set paradigm using the water maze as described in Experimental procedures. Initial body weights and weight change of the rats are given in Table 1. PSD rats lost weight, LP rats maintained weight, and control rats gained weight during the 5-day experiment. Body weight changes in PSD, LP and control groups were significantly different from one another (F = 55.009, p b 0.001). The latency and distance traveled to find the platform in either Trial 1 or Trial 2 of Experiment 1 were shown in Fig. 1. The ANOVA for the three groups over the 5 training days indicated that all groups exhibited significant, substantial reductions in their times and distance to find the platform over the training sessions. However, the rate of learning in Trial 1 was different between groups (F = 5.038, p b 0.008). Post hoc Newman–Keuls comparisons revealed that, compared to the other two groups, PSD rats took significantly longer to find the platform, implying a significant impairment of reference
memory, and this impairment occurred from training day 3 onward (p b 0.01). These results are shown in Figs. 1A and C. Swimming speed was calculated from distance and latency. The swimming speeds of PSD rats in Trial 2 tended to be higher than in controls, but this was not significant (p N 0.128; Figs. 1E and F). There were no statistically significant differences of latency in Trial 2 between the three groups (Fig. 1B), but the distance traveled to the platform during Trial 2 on day 5 was significantly increased (Fig. 1D). Experiment 2 was designed to observe the effect of PSD on spatial memory maintenance. In the probe trial, the platform was removed and the animal was placed into quadrant 2, which is opposite to the target quadrant (quadrant 4). The time that an animal spent in the target quadrant and the number of times that the same animal crossed the former platform area were recorded and compared with the baseline obtained after the 5 days of training. During the 120 s of the probe trial, the sleep-deprived rats spent a similar percentage of time in the target quadrant, as the LP and control group, showing only a small decrease which did not quite reach significance (34.76 ± 1.19% vs. 37.26 ± 1.58% and 37.32 ± 1.53%, respectively, p = 0.054) (Fig. 2). The times spent in the target quadrant of all of three groups were numerically lower than baseline value (40.45 ± 1.18%), but this difference was not significant. There was no significant difference between crossing times of PSD and LP or control group (4.4 ± 0.84, 3.6 ± 0.96 and 3.5 ± 1.27, respectively, p = 0.079).
2.2. Effect of PSD on the membrane excitability of hippocampus pyramidal neurons Whole-cell patch-clamp recordings were obtained from 41 normal and 36 PSD CA1 pyramidal neurons. The passive and action potential properties of the neurons are summarized in Table 2. PSD had no effect on resting potential. The amplitude, peak voltage and duration of the action potential (AP) of hippocampal pyramidal neurons were not significantly changed by PSD, except the single spike threshold was higher after 72 h of PSD (p b 0.05). The injection current required to evoke an AP was significantly increased in the PSD group. Increases in rheobase are illustrated in the typical recordings shown in Fig. 2. The top records are a series of 8 voltage responses
Table 1 – Effect of PSD on body weight and weight change of rats (mean ± SE, n = 10)
Starting weight (g) Day 1 (g) Day 2 (g) Day 3 (g) Day 4 (g) Day 5 (g) Weight change (g)
Control
PSD
LP
Significance
172.6 ± 2.9
172.1 ± 3.1
173.3 ± 2.6
NS
178.1 ± 3.3 185.9 ± 4.2 193.4 ± 4.4 199.1 ± 5.0 208.3 ± 5.4 34.4 ± 3.1
168.2 ± 3.4 163.9 ± 4.1 162.7 ± 4.2 163.9 ± 3.8 164.4 ± 3.8 −16.8 ± 2.5
175.8 ± 2.8 177.9 ± 3.2 178.1 ± 3.1 180.8 ± 4.2 183.8 ± 4.2 10.8 ± 4.3
NS F = 8.4, p b 0.001 F = 14.7, p b 0.001 F = 16.4, p b 0.001 F = 21.8, p b 0.001 F = 55.0, p b 0.001
Statistically significant differences were determined by one-way analysis of variance and calculation of least significant difference (p b 0.05). Body weight changes in the 3 groups were significantly different from one another.
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Fig. 1 – Effect of sleep deprivation on spatial learning and memory. (A) The mean escape latencies in Trial 1 were significantly higher in the PSD group compared with control groups from the third day onward. There was no significant difference between LP and control group. (B) There were no significant differences of latency in Trial 2 between the three groups. (C, D) The distance traveled during Trial 1 and Trial 2. (E, F) The swimming speeds of PSD rats were similar with that of LP and control group in Trial 1 and Trial 2. Data are means ± SE. Statistical comparisons between treatments were made by 1-way analysis of variance on each day. *p b 0.05.
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Fig. 2 – The time that the animal spent in each quadrant during the probe trial. Animals showed a preference for the platform quadrant but without significant differences between PSD group and LP or control group. The times spent in the target quadrant of all of three groups were numerically lower than baseline value, but this difference was not significant.
induced by the 8-incrementing current commands shown in the bottom record. In the control cell (Fig. 3A), only the first command current failed to generate an AP, the other seven current pulses stimulated the generation of one or multiple APs, whereas in the PSD cell (Fig. 3B), the first five current commands produced only passive responses, and only the three most intense currents elicited APs. Spike accommodation was quantified by measuring difference in the latency between the first two APs (first interspike interval) and the last two APs evoked by a 1200 ms injection of a depolarizing pulse using 1.5× the rheobase current determined with the 100 ms pulse protocol (see Fig. 4A). The latency of the interspike interval was increased significantly in PSD neurons (Table 1, Fig. 4B), suggesting that spike frequency accommodation was enhanced by PSD. Repetitive firing was evoked by 1200 ms current steps of increasing amplitude in control and PSD hippocampus pyramidal neurons. In current injections ranging from 10
Table 2 – Effect of 72 h PSD on electrophysiological properties in the hippocampus pyramidal neurons (mean ± SE)
RMP (mV) AP amplitude (mV) AP–Peak voltage (mV) AP width (ms) Rheobase (pA) Single spike threshold (mV) Accommodation (ms)
Control
PSD
p
− 66.4 ± 5.4 79.9 ± 5.7 33.9 ± 7.7 1.2 ± 0.2 37.7 ± 14.7 − 46.5 ± 3.9 92.3 ± 34.5
− 63.8 ± 2.9 76.6 ± 6.4 31.2 ± 7.1 1.3 ± 0.2 55.4 ± 11.5 − 42.5 ± 2.9 132.1 ± 44.6
0.5296 0.0570 0.2565 0.9305 0.0051⁎ 0.0002⁎ 0.0341⁎
RMP = resting membrane potential, AP = action potential. ⁎t-test, p b 0.05.
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Fig. 3 – Recordings to illustrate increase of rheobase seen in PSD neurons. Top traces are voltage recordings of subthreshold phenomena and APs. Bottom recording illustrates amplitude and duration of current pulse injected. (A) A recording from a control hippocampus pyramidal neuron. (B) A recording from a PSD hippocampus pyramidal neuron. Note increased number of APs seen in control cell compared with PSD cell for a given current amplitude.
to 180 pA, the firing frequency of PSD neurons was dramatically and significantly reduced for all current amplitudes above 10 pA.
2.3. PSD induced Bax translocation and cytochrome c release Levels of Bax and Cyt-c in the mitochondria and cytoplasm of the hippocampus were detected by Western blot and quantitatively analyzed (Fig. 5). To confirm the purity of the subcellular fractions, we used antibodies against the mitochondria-specific form of cytochrome c oxidase. As shown in Fig. 5A-d, the mitochondria fraction was pure. β-Actin staining (Fig. 5A-c), used as a gel loading control, showed similar protein loading in all the wells. In controls, Bax was distributed with a higher intensity in the cytoplasmic than in the mitochondrial fractions. In the PSD animals, Bax immunoreactivity was detectable in both fractions, but with higher intensity in the mitochondria (Fig. 5A-a). The opposite result was observed with cytochrome c: immunoreactivity was primarily found in the mitochondrial fractions in control animals, but in the cytoplasmic fraction in the PSD group (Fig. 5A-b). The results of the densitometric analysis of cytoplasmic and mitochondrial cytochrome c and Bax in both groups are shown in Fig. 5B.
3.
Discussion
Sleep is composed of two prominent types: rapid eye movement (REM) sleep and non-REM (NREM) sleep, and both of them differently modulate the consolidation of declarative and nondeclarative memories, respectively (Peigneux et al.,
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2004). Rapid eye movement (REM) sleep is increased following learning sessions (Smith and Rose, 1997; Mandai et al., 1989), previously learned information and new associations can be formed during REM, and information processed during REM can be transferred to the awake state and be expressed in behavior (Hennevin et al., 1995). Sleep deprivation interferes with learning and memory in both humans (Plihal and Born, 1999; Stickgold et al., 2000) and animals (Silvestri, 2005; Guan et al., 2004). In the present study, with the Morris Water Maze spatial learning set protocol, the escape latencies of PSD rats in Trial 1 were significantly more delayed than in the control groups, which indicated that PSD decreased spatial learning ability. There were no statistically significant differences of latency in Trial 2, indicating that PSD may not affect working memory of the rats. These results agree with those of Youngblood et al. (1997). However, the PSD rats traveled a longer distance to find the platform during Trial 2 on day 5 than the control groups, suggesting that there was some PSD impairment on working memory task. Some impairment of
working memory might have been masked by the fact that the PSD rats, who lost weight during the experiment, tended to swim faster (which should have reduced latency below controls), although this effect on swimming speed did not reach significance. The results from a probe trial indicated that there were no significant differences between PSD and control groups, however, the p value (0.054) indicated a trend for PSD animals to spend less time in the platform quadrant, so we cannot rule out a small effect of PSD on the maintenance of established memory. The importance of the hippocampal formation for memory is well established on the basis of neuroanatomical and electrophysiological studies (Squire and Zola, 1996; Eichenbaum, 1999). In particular, spatial memory is understood to be strongly dependent on hippocampal activity in rats (Henninger et al., 2007; Eichenbaum, 2000). The firing patterns of neurons in the hippocampus involved in the spatial learning experience are replayed during the subsequent sleep (Louie and Wilson, 2001; Skaggs and McNaughton, 1996). To study the
Fig. 4 – Neuron excitability was reduced after sleep deprivation. (A) Spike frequency accommodation was enhanced in PSD neurons. Representative traces showing membrane potential from a control (top) and a PSD neuron (bottom) in response to 1.5× rheobase current injection to each cell. (B) The spike accommodation (“Last” minus “First”) was increased significantly in PSD neurons. (C) Membrane potential responses to current injection ranging from 10 to 180 pA. The number of the action potentials was reduced in PSD neurons. (D) Summary frequency–current plot showed that action potential frequency was significantly reduced in PSD group compared with controls. Differences were significant for all current values above 10 pA.
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Fig. 5 – Western blot analyses of cytochrome c and Bax in cytoplasmic (Cyt) and mitochondrial (Mit) fractions from the hippocampus obtained from control and PSD rats. (A) Representative blots of Bax and cytochrome c proteins. (a) Bax immunoreactivity presented a higher intensity in the mitochondria in PSD rats than control. (b) Significant increase in the amount of cytochrome c was found in the cytoplasmic fraction from PSD rats. (c) β-Actin showed similar protein loading in the mitochondrial and cytoplasmic fractions. (d) Cox IV, used as a marker for mitochondrial contamination, was only present in the mitochondrial fractions. (B) Densitometric analyses for protein levels. The changes were expressed as the percentage of β-actin. Data represent mean ± SE (n = 9). *p b 0.05 compared to controls.
effects of PSD on hippocampal neurons, changes in membrane excitability after PSD were observed by electrophysiological method. We found that, after 72 h of PSD, the overall membrane excitability of CA1 pyramidal neurons was significantly reduced. McDermott et al. (2003) have reported that sleep deprivation reduced the membrane excitability of CA1 neurons by enhancing spike frequency adaptation. We further observed that the rheobase for generation of an action potential and the threshold were increased significantly, while spike accommodation was enhanced. It was reported that changes in membrane excitability are critical for the
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formation of memory (Bliss and Collingridge, 1993), and the neuronal properties could be altered during sleep and by the lack of sleep (Buzsaki, 1998; Graves et al., 2001). Our data have led us to conclude that the decrease in membrane excitability affected the neuron's activity, and by extension contributed to the deficits of performing certain learning and memory tasks after PSD. Mitochondria are complex in both structure and function and are the energy metabolism sites of cells including neurons. Mitochondria play a very significant role in maintaining the physiological functions of the brain. Besides their role in supplying energy, mitochondria are also important in the regulation of neuron excitability (Berghe et al., 2002; Bergmann and Keller, 2003; Nowicky and Duchen, 1998). Nowicky and Duchen (1998) proved that inhibiting mitochondrial metabolism made hyperpolarized the membrane potential and silenced spontaneous activity. To investigate whether the decrease of excitability induced by PSD is related to an effect on mitochondrial function, we examined subcellular localization of cytochrome c, which is located in mitochondrial intermembranous space and released into the cytoplasm during mitochondrial changes following various stimuli. Release of cytochrome c from mitochondria to the cytoplasm can occur either following opening of a mitochondria permeability transition pore (MTP) or subsequent to the translocation of Bax into mitochondria (Jurgensmeier et al., 1998). Bax forms a channel which is permeable to cytochrome c. In the present study, we demonstrated both PSD-induced cytochrome c release into the cytoplasm and Bax translocation into the mitochondria. However, whether the cytochrome c release induced by PSD resulted from opening of the MTP or from increased Bax translocation into mitochondria remains to be determined. It has been reported that sleep deprivation-induced apoptosis of hippocampal neurons (Eilan et al., 1995; Cheng et al., 2004). PSD might decrease membrane excitability by affecting mitochondrial function, and initiate apoptosis of neurons by increasing Bax levels in mitochondria and promoting cytochrome c release. Further studies are needed to explore the mechanisms of these excitability changes. In summary, paradoxical sleep deprivation impaired spatial learning ability of rats, while effects on repeated working memory as well as maintenance of established memory were minor or did not quite reach significance. PSD induces Bax translocation to mitochondria and cytochrome c release into cytoplasm and decreases the membrane excitability of CA1 pyramidal neurons, which may contribute to the deficits in learning behavior. These results partially explain the mechanism of the effect of PSD on learning ability. Further studies are needed to understand how to modulate the excitability of neurons and protect mitochondrial function for preventing the effect of sleep deprivation.
4.
Experimental procedures
4.1.
Subjects
A total of 77 adult (180–200 g) male Sprague–Dawley rats were used in the experiments. Animal care was in accord with the “Principles of Medical Laboratory Animal Care” issued by the
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National Ministry of Health. All experiments conformed to the guidelines of the “National Ordinances on Experimental Animals” for the ethical use of animals. All animals were kept at 24 °C (room temperature) with light from 07:00 to 19:00 h.
4.2.
Paradoxical sleep deprivation
Ten small platforms (8.5 cm in height and 6.0 cm in diameter) were placed (8–10 cm apart) inside a water tank made of sheet iron. The bottom of the tank was filled with 24 °C water which reached up to ~2 cm below the surface of the platforms. The platforms were of small diameter permitting the rat to sit, but not lie down, on the platform. Furthermore the rats could easily move between the platforms but could not stretch across any two platforms to sleep, thus the animals were awoken when they experienced paradoxical sleep-induced atonia by touching the water. The water in the tank was changed daily. All rats had free access to food and water and were weighed daily.
the rat to find the platform and time (latency) to locate the platform were recorded by an MT-200 Morris image motion system (Chengdu Technology, Market Corp., China). Swimming speed was calculated from distance and latency. Experiment 2 was designed to observe the effect of PSD on spatial memory maintenance. A total of 27 animals were divided into the PSD group (n = 9), the LP group (n = 9) and the control group (n = 9) after they finished 5 days of training in the Morris Water Maze. A probe trial was given at the end of the 5day training as a baseline value. After a 3-day PSD, a probe trial was given again to detect the effect of PSD on maintenance of memory. In the probe trial, the hidden platform was removed from the Water Maze. The animal was placed in quadrant 2, which was opposite to the target quadrant (quadrant 4), and was allowed to swim for 120 s in the Water Maze. The time that an animal spent in the target quadrant and the number of times that the same animal crossed the former platform area were used to measure the spatial memory maintenance.
4.4. 4.3.
In vitro electrophysiological recording
Test of spatial learning and memory
In Experiment 1, 30 animals were divided into three groups of ten. The PSD animals were deprived of paradoxical sleep by the multiple platform technique described above. To simulate the circumstances of the PSD condition, a large platform (LP) group was used as a control group. Ten large platforms (8.5 cm in height and 15 cm in diameter) were placed inside another water tank, which allowed rats to lie down without touching the water. Another control group of animals was housed in their home cage and permitted to sleep during the same period. Daily, each rat was tested between 15:00 h and 18:00 h, in the Morris Water Maze. The maze, 80 cm deep and 150 cm in diameter, was divided into four quadrants of equal size on the monitor screen of a computer, filled to a depth of 24 cm with water. The water in the tank was fresh each day and was maintained at 23–24 °C. A white, 10 cm diameter platform was placed in the center of quadrant 4 and submerged 2 cm below the water surface. Rats with red tags attached to their backs, were trained to find the hidden platform according to the spatial cues in the experimental room. Each rat was released facing the wall of the Water Maze in the four quadrants respectively. The order of quadrants was changed each day such that subjects were never exposed to a sequence of trials that they had had before. Each rat was tested in 8 trials (2 consecutive trials from each starting position) per day. Reference memory was assessed by the reduction in distance and latency required to find the platform during the first trial from successive starting positions. Working memory was indicated by the reduction in distance and latency required to find the platform during the second trial from each starting location, compared with the first trial from that same location. Each animal was allowed to swim for a maximum duration of 120 s in each trial to find the platform. After the animal found and got onto the platform, it was allowed to stay on the platform for 20 s. If the rat did not find the platform, the rat was guided to it and left there for 20 s. After training, the animal was dried with a fabric towel and returned to its home cage, large platform or PSD tank cage. The distance swum by
In Experiment 3, the experimental rats were deeply anesthetized with pentobarbital sodium (40 mg/kg) and decapitated after 3 days of PSD (n = 11). The control animals (n = 9) were allowed to sleep during the same period. Hippocampal slices (300 μm in thickness) were prepared with a vibratome (Vibroslice 752M, Campden Instruments, Loughborough, UK) and incubated with artificial cerebrospinal fluid (ACSF) containing (in millimolars): 124 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.0 MgCl2, 2.0 CaCl2, 25 NaHCO3, 10 Glucose. Slices were maintained in ACSF at 28 °C for at least 1 h before being moved into the recording chamber. During the recordings, the slices were kept submerged in a chamber perfused with ACSF. In the experiments, the ACSF was saturated with 95% O2/5% CO2 and the temperature was kept at 26 °C. Individual neurons were visualized with a 40× water-immersion objective under a microscope (BX51WI; Olympus, Tokyo, Japan) equipped with infrared differential interference contrast optics. Whole-cell recordings were obtained from pyramidal cells using recording pipettes with a resistance of 4–7 MΩ. Patch pipettes were filled with a solution containing (in millimolars): 140 potassium gluconate, 10 HEPES, 10 phosphocreatine sodium salt, 2 ATP sodium salt, 0.4 GPT sodium salt and 2 MgCl2. For patchclamp recordings, a Multiclamp 700B amplifier (Molecular Devices Corporation, Sunnyvale, CA, USA) was used. The series resistance was 10–20 MΩ. All potentials were corrected online for the junction potential by adjusting the offset of the pipette using the Multiclamp 700B commander software. Neurons were selected for further study if they had a resting membrane potential that was more negative than −50 mV and if they exhibited overshooting action potentials. Each cell was injected with a sequence of hyperpolarizing and depolarizing square wave current pulses to measure input resistance and to assess intrinsic electrophysiological characteristics. Rheobase was defined as the minimal amount of depolarizing current (100 ms) required to evoke a spike, and the spike threshold was defined as the voltage at which the value of dVm/dt preceding a spike first became 1/30 of dVm/dt(max) (Azouz and Gray, 1999). Spike width was measured at 50% maximum amplitude.
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4.5. Isolation of mitochondrial fraction and Western blot analysis In Experiment 4, we examined changes of proteins in 3-day PSD rats (n = 9) and control rats (n = 9) in the subcellular fractions where they are reported to be localized and/or translocated. Cytochrome c is present in mitochondria and translocates into cytoplasm, and Bax can translocate from the cytoplasm to the mitochondria. Protein from enriched fractions of mitochondria and cytosol were extracted as described by Ghribi et al. (2003) with slight modifications. Tissue from the entire hippocampus was gently homogenized using a glass–glass homogenizer in nine volumes of ice-cold homogenizing buffer (10 mM Tris–HCl pH 7.4, 250 mM sucrose, 1 mM KCl) and centrifuged at 600 ×g at 4 °C for 15 min. The supernatants were re-centrifuged at 18,000 ×g at 4 °C for 15 min. The pellets represented the mitochondrial fraction while the supernatants were the cytosolic fraction (Lee et al., 2004). The protein content of each sample was determined using a BCA™ Protein Assay Kit (Pierce, Rockford, IL, USA). Equal quantities of total protein (30 μg per lane) from mitochondrial and cytosolic fractions were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by transfer to nitrocellulose membranes. The membranes were incubated in 5% milk at room temperature for 2 h. Blots were then incubated with primary antibodies including mouse monoclonal antibody for cytochrome c (diluted 1:2000), or with rabbit polyclonal antibody against Bax (diluted 1:2000), cytochrome oxidase subunit IV (Cox IV) (diluted 1:2000), or β-actin (1:2000). After washing with buffer, the blots were incubated in anti-mouse (Zhong Shan, Beijing, China, 1:1000) and antirabbit (Zhong Shan, Beijing, China, 1:1000) secondary antibody conjugated with horseradish peroxide in TBS-T with 5% milk at 4 °C overnight. The blots were visualized using a West Pico Chemiluminescent Kit (Pierce, Rockford, IL, USA), and the density of protein bands was quantified by transmittance densitometry using volume integration with LumiAnalyst Image Analysis software.
4.6.
Data acquisition and statistics
Data values are presented as means ± SE Student's t-tests (SigmaStat version 2.03) were used to determine the statistical significance of differences between means obtained from two different groups of neurons. One-way ANOVAs followed by post hoc pairwise comparisons (Student–Newman–Keuls method) (SigmaStat 2.03) were used to determine the statistical significance of differences between means obtained from three experimental groups. Chi-square tests were used to assess differences in the percentages of time the rats swam in each quadrant of the Morris Water Maze. In all experiments, a probability of 0.05 or less was considered statistically significant.
Acknowledgments We thank Dr Judith Strong for her helpful comments on this manuscript. This work was supported by an NSFC grant
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(30530260) from China and a grant from the Fourth Military Medical University (XJ200502).
REFERENCES
Azouz, R., Gray, C.M., 1999. Cellular mechanisms contributing to response variability of cortical neurons in vivo. J. Neurosci. 19, 2209–2223. Banks, M.I., Hardie, J.B., Pearce, R.A., 2002. Development of GABAA receptor-mediated inhibitory postsynaptic currents in hippocampus. J. Neurophysiol. 88, 3097–3107. Berghe, P.V., Kenyon, J.L., Smith, T.K., 2002. Mitochondrial Ca2+ uptake regulates the excitability of myenteric neurons J. Neurosci. 22, 6962–6971. Bergmann, F., Keller, B.U., 2003. Impact of mitochondrial inhibition on excitability and cytosolic Ca2+ levels in brainstem motoneurones from mouse. J. Physiol. 555, 45–59. Bliss, T.V.P., Collingridge, G.L., 1993. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39. Buzsaki, G., 1998. Memory consolidation during sleep: a neurophysiological perspective. J. Sleep Res. 7, 17–23. Campbell, I.G., Guinan, M.J., Horowitz, J.M., 2002. Sleep deprivation impairs long-term potentiation in rat hippocampal slices J. Neurophysiol. 88, 1073–1076. Cheng, J.T., Zhang, Q., Qiao, P., 2004. Apoptosis of hippocampus neurons and upexpression of related gene induced by sleep deprivation in rats. Basic Medical Sciences and Clinics 24, 205–208. Davis, C.J., Harding, J.W., Wright, J.W., 2003. REM sleep deprivation-induced deficits in the latency-to-peak induction and maintenance of long-term potentiation within the CA1 region of the hippocampus. Brain Res. 973, 293–297. Eichenbaum, H., 1999. The hippocampus and mechanisms of declarative memory. Behav. Brain Res. 103, 123–133. Eichenbaum, H., 2000. A cortical–hippocampal system for declarative memory. Nat. Rev. Neurosci. 1, 41–50. Eilan, M.M., Gilliland, M., Bergnmnn, B.M., 1995. Neuronal degeneration in sleep deprivation rats. Sleep 2, 21–24. Ghribi, O., Herman, M.M., Savory, J., 2003. Lithium inhibits Abeta-induced stress in endoplasmic reticulum of rabbit hippocampus but does not prevent oxidative damage and tau phosphorylation. J. Neurosci. Res. 71, 853–862. Graves, L., Pack, A., Abel, T., 2001. Sleep and memory: a molecular perspective. Trends Neurosci. 24, 237–243. Guan, Z., Peng, X., Fang, J., 2004. Sleep deprivation impairs spatial memory and decreases extracellular signal-regulated kinase phosphorylation in the hippocampus. Brain Res. 1018, 38–47. Hennevin, E., Hars, B., Maho, C., Bloch, V., 1995. Processing of learned information in paradoxical sleep: relevance for memory. Behav. Brain Res. 69, 125–135. Henninger, N., Feldmann Jr., R.E., Futterer, C.D., Schrempp, C., Maurer, M.H., Waschke, K.F., Kuschinsky, W., Schwab, S., 2007. Spatial learning induces predominant downregulation of cytosolic proteins in the rat hippocampus. Genes Brain and Behavior 6, 128–140. Hipolide, D.C., Moreira, K.M., Barlow, K.B.L., Wilson, A.A., Nobrega, J.N., Tufik, S., 2005. Distinct effects of sleep deprivation on binding to norepinephrine and serotonin transporters in rat brain. Progress in Neuro-Psychopharmacology & Biological Psychiatry 29, 297–303. Jurgensmeier, J., Xie, Z., Deveraux, Q., Ellerby, L., Bredsen, D., Reed, J.C., 1998. Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. Acad. Sci. U. S. A. 95, 4997–5002.
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Kelleher 3rd, R.J., Govindarajan, A., Tonegawa, S., 2004. Translational regulatory mechanisms in persistent forms of synaptic plasticity. Neuron 44, 59–73. Lee, J.H., Jung, K.J., Kim, J.W., Kim, H.J., Yu, B.P., Chung, H.Y., 2004. Suppression of apoptosis by calorie restriction in aged kidney. Exp. Gerontol. 39, 1361–1368. Louie, K., Wilson, M.A., 2001. Temporally structured replay of awake hippocampal ensemble activity during rapid eye movement sleep. Neuron 29, 145–156. Mandai, O., Guerrien, A., Sockeel, P., Dujardin, K., Leconte, P., 1989. REM sleep modifications following a Morse code learning session in humans. Physiol. Behav. 46, 639–642. McDermott, C.M., LaHoste, G.J., Chen, C., Musto, A., Bazan, N.G., Magee, J.C., 2003. Sleep deprivation causes behavioral, synaptic, and membrane excitability alterations in hippocampal neurons. J. Neurosci. 23, 9687–9695. Nowicky, A.V., Duchen, M.R., 1998. Changes in (Ca2+)i and membrane currents impaired mitochondrial metabolism in dissociated rat hippocampal neurons. J. Physiol. 507, 131–145. Peigneux, P., Laureys, S., Fuchs, S., Collette, F., Perrin, F., Reggers, J., 2004. Are spatial memories strengthened in the human hippocampus during slow wave sleep. Neuron 44, 535–545. Plihal, W., Born, J., 1999. Effects of early and late nocturnal sleep on priming and spatial memory. Psychophysiology 36, 571–582.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Paradoxical sleep deprivation impairs spatial learning and affects membrane excitability and mitochondrial protein in the hippocampus Rui-Hua Yang a,⁎, San-Jue Hu b , Yuan Wang a , Wen-Bin Zhang a , Wen-Jing Luo a , Jing-Yuan Chen a,⁎ a b
Department of Occupational and Environmental Health, the Fourth Military Medical University, Xi'an 710032, PR China Institute of Neuroscience, the Fourth Military Medical University, Xi'an 710032, PR China
A R T I C LE I N FO
AB S T R A C T
Article history:
Previous research has demonstrated that paradoxical sleep has a key role in learning and
Accepted 9 July 2008
memory, and sleep deprivation interferes with learning and memory. However, the
Available online 17 July 2008
mechanism of memory impairment induced by sleep deprivation is poorly understood. The present study investigated the effect of paradoxical sleep deprivation (PSD) on spatial
Keywords:
learning and memory using the Morris Water Maze. Effects of PSD on CA1 pyramidal
paradoxical sleep deprivation
neurons in hippocampus were also examined. PSD impaired spatial learning of rats. PSD
Morris Water Maze
induced translocation of Bax to mitochondria and cytochrome c release into the cytoplasm,
spatial learning
and decreased the membrane excitability of CA1 pyramidal neurons, effects which may
membrane excitability
contribute to the deficits in learning behavior. These results may partially explain the
mitochondria
mechanism of the effect of PSD on learning. Modulating the excitability of hippocampal
hippocampus
neurons and protecting mitochondrial function are possible targets for preventing the effects of paradoxical sleep deprivation. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
There is growing evidence that sleep and learning are interconnected. Both human and animal studies have shown that sleep has a key role in learning and memory. Rapid eye movement (REM) sleep is increased following learning sessions (Smith and Rose, 1997; Mandai et al., 1989), and sleep deprivation interferes with learning and memory (Silvestri, 2005; Guan et al., 2004). A number of animal studies have gone on to explore the potential cellular and molecular underpinnings of sleep deprivation-induced learning deficits, and
many of these studies have focused on changes in transmitters (Banks et al., 2002; Hipolide et al., 2005), the impairment of formation of long-term potentiation (Campbell et al., 2002; Davis et al., 2003), and effects on extracellular signal-regulated kinases (Kelleher et al., 2004; Guan et al., 2004). Less attention has been given to the effects of sleep deprivation on the intrinsic properties of neurons. Limited animal studies indicated that the firing patterns of neurons in the hippocampus and cortex involved in the learning experience were replayed during subsequent sleep (Louie and Wilson, 2001; Skaggs and McNaughton, 1996; Sutherl and McNaughton,
⁎ Corresponding authors. Fax: +86 29 84774863. E-mail addresses: [email protected] (R.-H. Yang), [email protected] (J.-Y. Chen). Abbreviations: Rapid eye movement sleep, REM sleep; Paradoxical sleep deprivation, PSD; Large platform, LP; Action potential, AP; Cytochrome c, Cyt-c; Mitochondria permeability transition pore, MTP 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.07.033
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2000), and REM sleep deprivation reduced the basic excitability of hippocampal neurons (McDermott et al., 2003). However, the molecular mechanism of changes of neuron properties induced by sleep deprivation remains poorly understood. Given the well recognized cognitive effects and the prevalence of sleep deprivation in society today, investigating the effects of sleep deprivation on cellular physiology and related mechanisms is of interest. The present study investigated the effect of paradoxical sleep deprivation (PSD) on spatial learning and memory with the Morris Water Maze, which is commonly used to examine hippocampal function (Walker and Stickgold, 2006; Smith and Rose, 1996). In addition we examined possible mechanisms by investigating the effects of PSD on membrane excitability and mitochondria of hippocampal CA1 pyramidal neurons. Using the Morris Water Maze spatial learning set protocol, we have found that PSD rats are slower to learn the location of the platform than control groups, but are capable of successfully performing the task with repeated exposure to the test. During the probe trial, the PSD rats tended to spend slightly less time in the target quadrant compared with the control groups, but this effect did not quite reach significance. The data from the behavior tests indicated that PSD impairs the ability of spatial learning of rats. There were indications of minor effects on working memory and maintenance of established memory as well, but most of these did not quite reach significance. We further have found that PSD induces translocation of Bax to mitochondria and cytochrome c release into cytoplasm, and decreases the membrane excitability of CA1 pyramidal neurons, which may contribute the deficits in performing the spatial learning task.
2.
Results
2.1.
PSD impairs spatial learning
To test for the impact of PSD on memory formation, a group of rats was REM sleep-deprived for 5 days, and a second group of rats (LP) was placed on large platforms as a control group to simulate the PSD condition while allowing sleep to occur, and the third group of rats was maintained in their home cages as another control group. Daily, the rats were tested between 150:0 h and 18:00 h, in a place-learning set paradigm using the water maze as described in Experimental procedures. Initial body weights and weight change of the rats are given in Table 1. PSD rats lost weight, LP rats maintained weight, and control rats gained weight during the 5-day experiment. Body weight changes in PSD, LP and control groups were significantly different from one another (F = 55.009, p b 0.001). The latency and distance traveled to find the platform in either Trial 1 or Trial 2 of Experiment 1 were shown in Fig. 1. The ANOVA for the three groups over the 5 training days indicated that all groups exhibited significant, substantial reductions in their times and distance to find the platform over the training sessions. However, the rate of learning in Trial 1 was different between groups (F = 5.038, p b 0.008). Post hoc Newman–Keuls comparisons revealed that, compared to the other two groups, PSD rats took significantly longer to find the platform, implying a significant impairment of reference
memory, and this impairment occurred from training day 3 onward (p b 0.01). These results are shown in Figs. 1A and C. Swimming speed was calculated from distance and latency. The swimming speeds of PSD rats in Trial 2 tended to be higher than in controls, but this was not significant (p N 0.128; Figs. 1E and F). There were no statistically significant differences of latency in Trial 2 between the three groups (Fig. 1B), but the distance traveled to the platform during Trial 2 on day 5 was significantly increased (Fig. 1D). Experiment 2 was designed to observe the effect of PSD on spatial memory maintenance. In the probe trial, the platform was removed and the animal was placed into quadrant 2, which is opposite to the target quadrant (quadrant 4). The time that an animal spent in the target quadrant and the number of times that the same animal crossed the former platform area were recorded and compared with the baseline obtained after the 5 days of training. During the 120 s of the probe trial, the sleep-deprived rats spent a similar percentage of time in the target quadrant, as the LP and control group, showing only a small decrease which did not quite reach significance (34.76 ± 1.19% vs. 37.26 ± 1.58% and 37.32 ± 1.53%, respectively, p = 0.054) (Fig. 2). The times spent in the target quadrant of all of three groups were numerically lower than baseline value (40.45 ± 1.18%), but this difference was not significant. There was no significant difference between crossing times of PSD and LP or control group (4.4 ± 0.84, 3.6 ± 0.96 and 3.5 ± 1.27, respectively, p = 0.079).
2.2. Effect of PSD on the membrane excitability of hippocampus pyramidal neurons Whole-cell patch-clamp recordings were obtained from 41 normal and 36 PSD CA1 pyramidal neurons. The passive and action potential properties of the neurons are summarized in Table 2. PSD had no effect on resting potential. The amplitude, peak voltage and duration of the action potential (AP) of hippocampal pyramidal neurons were not significantly changed by PSD, except the single spike threshold was higher after 72 h of PSD (p b 0.05). The injection current required to evoke an AP was significantly increased in the PSD group. Increases in rheobase are illustrated in the typical recordings shown in Fig. 2. The top records are a series of 8 voltage responses
Table 1 – Effect of PSD on body weight and weight change of rats (mean ± SE, n = 10)
Starting weight (g) Day 1 (g) Day 2 (g) Day 3 (g) Day 4 (g) Day 5 (g) Weight change (g)
Control
PSD
LP
Significance
172.6 ± 2.9
172.1 ± 3.1
173.3 ± 2.6
NS
178.1 ± 3.3 185.9 ± 4.2 193.4 ± 4.4 199.1 ± 5.0 208.3 ± 5.4 34.4 ± 3.1
168.2 ± 3.4 163.9 ± 4.1 162.7 ± 4.2 163.9 ± 3.8 164.4 ± 3.8 −16.8 ± 2.5
175.8 ± 2.8 177.9 ± 3.2 178.1 ± 3.1 180.8 ± 4.2 183.8 ± 4.2 10.8 ± 4.3
NS F = 8.4, p b 0.001 F = 14.7, p b 0.001 F = 16.4, p b 0.001 F = 21.8, p b 0.001 F = 55.0, p b 0.001
Statistically significant differences were determined by one-way analysis of variance and calculation of least significant difference (p b 0.05). Body weight changes in the 3 groups were significantly different from one another.
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Fig. 1 – Effect of sleep deprivation on spatial learning and memory. (A) The mean escape latencies in Trial 1 were significantly higher in the PSD group compared with control groups from the third day onward. There was no significant difference between LP and control group. (B) There were no significant differences of latency in Trial 2 between the three groups. (C, D) The distance traveled during Trial 1 and Trial 2. (E, F) The swimming speeds of PSD rats were similar with that of LP and control group in Trial 1 and Trial 2. Data are means ± SE. Statistical comparisons between treatments were made by 1-way analysis of variance on each day. *p b 0.05.
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Fig. 2 – The time that the animal spent in each quadrant during the probe trial. Animals showed a preference for the platform quadrant but without significant differences between PSD group and LP or control group. The times spent in the target quadrant of all of three groups were numerically lower than baseline value, but this difference was not significant.
induced by the 8-incrementing current commands shown in the bottom record. In the control cell (Fig. 3A), only the first command current failed to generate an AP, the other seven current pulses stimulated the generation of one or multiple APs, whereas in the PSD cell (Fig. 3B), the first five current commands produced only passive responses, and only the three most intense currents elicited APs. Spike accommodation was quantified by measuring difference in the latency between the first two APs (first interspike interval) and the last two APs evoked by a 1200 ms injection of a depolarizing pulse using 1.5× the rheobase current determined with the 100 ms pulse protocol (see Fig. 4A). The latency of the interspike interval was increased significantly in PSD neurons (Table 1, Fig. 4B), suggesting that spike frequency accommodation was enhanced by PSD. Repetitive firing was evoked by 1200 ms current steps of increasing amplitude in control and PSD hippocampus pyramidal neurons. In current injections ranging from 10
Table 2 – Effect of 72 h PSD on electrophysiological properties in the hippocampus pyramidal neurons (mean ± SE)
RMP (mV) AP amplitude (mV) AP–Peak voltage (mV) AP width (ms) Rheobase (pA) Single spike threshold (mV) Accommodation (ms)
Control
PSD
p
− 66.4 ± 5.4 79.9 ± 5.7 33.9 ± 7.7 1.2 ± 0.2 37.7 ± 14.7 − 46.5 ± 3.9 92.3 ± 34.5
− 63.8 ± 2.9 76.6 ± 6.4 31.2 ± 7.1 1.3 ± 0.2 55.4 ± 11.5 − 42.5 ± 2.9 132.1 ± 44.6
0.5296 0.0570 0.2565 0.9305 0.0051⁎ 0.0002⁎ 0.0341⁎
RMP = resting membrane potential, AP = action potential. ⁎t-test, p b 0.05.
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Fig. 3 – Recordings to illustrate increase of rheobase seen in PSD neurons. Top traces are voltage recordings of subthreshold phenomena and APs. Bottom recording illustrates amplitude and duration of current pulse injected. (A) A recording from a control hippocampus pyramidal neuron. (B) A recording from a PSD hippocampus pyramidal neuron. Note increased number of APs seen in control cell compared with PSD cell for a given current amplitude.
to 180 pA, the firing frequency of PSD neurons was dramatically and significantly reduced for all current amplitudes above 10 pA.
2.3. PSD induced Bax translocation and cytochrome c release Levels of Bax and Cyt-c in the mitochondria and cytoplasm of the hippocampus were detected by Western blot and quantitatively analyzed (Fig. 5). To confirm the purity of the subcellular fractions, we used antibodies against the mitochondria-specific form of cytochrome c oxidase. As shown in Fig. 5A-d, the mitochondria fraction was pure. β-Actin staining (Fig. 5A-c), used as a gel loading control, showed similar protein loading in all the wells. In controls, Bax was distributed with a higher intensity in the cytoplasmic than in the mitochondrial fractions. In the PSD animals, Bax immunoreactivity was detectable in both fractions, but with higher intensity in the mitochondria (Fig. 5A-a). The opposite result was observed with cytochrome c: immunoreactivity was primarily found in the mitochondrial fractions in control animals, but in the cytoplasmic fraction in the PSD group (Fig. 5A-b). The results of the densitometric analysis of cytoplasmic and mitochondrial cytochrome c and Bax in both groups are shown in Fig. 5B.
3.
Discussion
Sleep is composed of two prominent types: rapid eye movement (REM) sleep and non-REM (NREM) sleep, and both of them differently modulate the consolidation of declarative and nondeclarative memories, respectively (Peigneux et al.,
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2004). Rapid eye movement (REM) sleep is increased following learning sessions (Smith and Rose, 1997; Mandai et al., 1989), previously learned information and new associations can be formed during REM, and information processed during REM can be transferred to the awake state and be expressed in behavior (Hennevin et al., 1995). Sleep deprivation interferes with learning and memory in both humans (Plihal and Born, 1999; Stickgold et al., 2000) and animals (Silvestri, 2005; Guan et al., 2004). In the present study, with the Morris Water Maze spatial learning set protocol, the escape latencies of PSD rats in Trial 1 were significantly more delayed than in the control groups, which indicated that PSD decreased spatial learning ability. There were no statistically significant differences of latency in Trial 2, indicating that PSD may not affect working memory of the rats. These results agree with those of Youngblood et al. (1997). However, the PSD rats traveled a longer distance to find the platform during Trial 2 on day 5 than the control groups, suggesting that there was some PSD impairment on working memory task. Some impairment of
working memory might have been masked by the fact that the PSD rats, who lost weight during the experiment, tended to swim faster (which should have reduced latency below controls), although this effect on swimming speed did not reach significance. The results from a probe trial indicated that there were no significant differences between PSD and control groups, however, the p value (0.054) indicated a trend for PSD animals to spend less time in the platform quadrant, so we cannot rule out a small effect of PSD on the maintenance of established memory. The importance of the hippocampal formation for memory is well established on the basis of neuroanatomical and electrophysiological studies (Squire and Zola, 1996; Eichenbaum, 1999). In particular, spatial memory is understood to be strongly dependent on hippocampal activity in rats (Henninger et al., 2007; Eichenbaum, 2000). The firing patterns of neurons in the hippocampus involved in the spatial learning experience are replayed during the subsequent sleep (Louie and Wilson, 2001; Skaggs and McNaughton, 1996). To study the
Fig. 4 – Neuron excitability was reduced after sleep deprivation. (A) Spike frequency accommodation was enhanced in PSD neurons. Representative traces showing membrane potential from a control (top) and a PSD neuron (bottom) in response to 1.5× rheobase current injection to each cell. (B) The spike accommodation (“Last” minus “First”) was increased significantly in PSD neurons. (C) Membrane potential responses to current injection ranging from 10 to 180 pA. The number of the action potentials was reduced in PSD neurons. (D) Summary frequency–current plot showed that action potential frequency was significantly reduced in PSD group compared with controls. Differences were significant for all current values above 10 pA.
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Fig. 5 – Western blot analyses of cytochrome c and Bax in cytoplasmic (Cyt) and mitochondrial (Mit) fractions from the hippocampus obtained from control and PSD rats. (A) Representative blots of Bax and cytochrome c proteins. (a) Bax immunoreactivity presented a higher intensity in the mitochondria in PSD rats than control. (b) Significant increase in the amount of cytochrome c was found in the cytoplasmic fraction from PSD rats. (c) β-Actin showed similar protein loading in the mitochondrial and cytoplasmic fractions. (d) Cox IV, used as a marker for mitochondrial contamination, was only present in the mitochondrial fractions. (B) Densitometric analyses for protein levels. The changes were expressed as the percentage of β-actin. Data represent mean ± SE (n = 9). *p b 0.05 compared to controls.
effects of PSD on hippocampal neurons, changes in membrane excitability after PSD were observed by electrophysiological method. We found that, after 72 h of PSD, the overall membrane excitability of CA1 pyramidal neurons was significantly reduced. McDermott et al. (2003) have reported that sleep deprivation reduced the membrane excitability of CA1 neurons by enhancing spike frequency adaptation. We further observed that the rheobase for generation of an action potential and the threshold were increased significantly, while spike accommodation was enhanced. It was reported that changes in membrane excitability are critical for the
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formation of memory (Bliss and Collingridge, 1993), and the neuronal properties could be altered during sleep and by the lack of sleep (Buzsaki, 1998; Graves et al., 2001). Our data have led us to conclude that the decrease in membrane excitability affected the neuron's activity, and by extension contributed to the deficits of performing certain learning and memory tasks after PSD. Mitochondria are complex in both structure and function and are the energy metabolism sites of cells including neurons. Mitochondria play a very significant role in maintaining the physiological functions of the brain. Besides their role in supplying energy, mitochondria are also important in the regulation of neuron excitability (Berghe et al., 2002; Bergmann and Keller, 2003; Nowicky and Duchen, 1998). Nowicky and Duchen (1998) proved that inhibiting mitochondrial metabolism made hyperpolarized the membrane potential and silenced spontaneous activity. To investigate whether the decrease of excitability induced by PSD is related to an effect on mitochondrial function, we examined subcellular localization of cytochrome c, which is located in mitochondrial intermembranous space and released into the cytoplasm during mitochondrial changes following various stimuli. Release of cytochrome c from mitochondria to the cytoplasm can occur either following opening of a mitochondria permeability transition pore (MTP) or subsequent to the translocation of Bax into mitochondria (Jurgensmeier et al., 1998). Bax forms a channel which is permeable to cytochrome c. In the present study, we demonstrated both PSD-induced cytochrome c release into the cytoplasm and Bax translocation into the mitochondria. However, whether the cytochrome c release induced by PSD resulted from opening of the MTP or from increased Bax translocation into mitochondria remains to be determined. It has been reported that sleep deprivation-induced apoptosis of hippocampal neurons (Eilan et al., 1995; Cheng et al., 2004). PSD might decrease membrane excitability by affecting mitochondrial function, and initiate apoptosis of neurons by increasing Bax levels in mitochondria and promoting cytochrome c release. Further studies are needed to explore the mechanisms of these excitability changes. In summary, paradoxical sleep deprivation impaired spatial learning ability of rats, while effects on repeated working memory as well as maintenance of established memory were minor or did not quite reach significance. PSD induces Bax translocation to mitochondria and cytochrome c release into cytoplasm and decreases the membrane excitability of CA1 pyramidal neurons, which may contribute to the deficits in learning behavior. These results partially explain the mechanism of the effect of PSD on learning ability. Further studies are needed to understand how to modulate the excitability of neurons and protect mitochondrial function for preventing the effect of sleep deprivation.
4.
Experimental procedures
4.1.
Subjects
A total of 77 adult (180–200 g) male Sprague–Dawley rats were used in the experiments. Animal care was in accord with the “Principles of Medical Laboratory Animal Care” issued by the
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National Ministry of Health. All experiments conformed to the guidelines of the “National Ordinances on Experimental Animals” for the ethical use of animals. All animals were kept at 24 °C (room temperature) with light from 07:00 to 19:00 h.
4.2.
Paradoxical sleep deprivation
Ten small platforms (8.5 cm in height and 6.0 cm in diameter) were placed (8–10 cm apart) inside a water tank made of sheet iron. The bottom of the tank was filled with 24 °C water which reached up to ~2 cm below the surface of the platforms. The platforms were of small diameter permitting the rat to sit, but not lie down, on the platform. Furthermore the rats could easily move between the platforms but could not stretch across any two platforms to sleep, thus the animals were awoken when they experienced paradoxical sleep-induced atonia by touching the water. The water in the tank was changed daily. All rats had free access to food and water and were weighed daily.
the rat to find the platform and time (latency) to locate the platform were recorded by an MT-200 Morris image motion system (Chengdu Technology, Market Corp., China). Swimming speed was calculated from distance and latency. Experiment 2 was designed to observe the effect of PSD on spatial memory maintenance. A total of 27 animals were divided into the PSD group (n = 9), the LP group (n = 9) and the control group (n = 9) after they finished 5 days of training in the Morris Water Maze. A probe trial was given at the end of the 5day training as a baseline value. After a 3-day PSD, a probe trial was given again to detect the effect of PSD on maintenance of memory. In the probe trial, the hidden platform was removed from the Water Maze. The animal was placed in quadrant 2, which was opposite to the target quadrant (quadrant 4), and was allowed to swim for 120 s in the Water Maze. The time that an animal spent in the target quadrant and the number of times that the same animal crossed the former platform area were used to measure the spatial memory maintenance.
4.4. 4.3.
In vitro electrophysiological recording
Test of spatial learning and memory
In Experiment 1, 30 animals were divided into three groups of ten. The PSD animals were deprived of paradoxical sleep by the multiple platform technique described above. To simulate the circumstances of the PSD condition, a large platform (LP) group was used as a control group. Ten large platforms (8.5 cm in height and 15 cm in diameter) were placed inside another water tank, which allowed rats to lie down without touching the water. Another control group of animals was housed in their home cage and permitted to sleep during the same period. Daily, each rat was tested between 15:00 h and 18:00 h, in the Morris Water Maze. The maze, 80 cm deep and 150 cm in diameter, was divided into four quadrants of equal size on the monitor screen of a computer, filled to a depth of 24 cm with water. The water in the tank was fresh each day and was maintained at 23–24 °C. A white, 10 cm diameter platform was placed in the center of quadrant 4 and submerged 2 cm below the water surface. Rats with red tags attached to their backs, were trained to find the hidden platform according to the spatial cues in the experimental room. Each rat was released facing the wall of the Water Maze in the four quadrants respectively. The order of quadrants was changed each day such that subjects were never exposed to a sequence of trials that they had had before. Each rat was tested in 8 trials (2 consecutive trials from each starting position) per day. Reference memory was assessed by the reduction in distance and latency required to find the platform during the first trial from successive starting positions. Working memory was indicated by the reduction in distance and latency required to find the platform during the second trial from each starting location, compared with the first trial from that same location. Each animal was allowed to swim for a maximum duration of 120 s in each trial to find the platform. After the animal found and got onto the platform, it was allowed to stay on the platform for 20 s. If the rat did not find the platform, the rat was guided to it and left there for 20 s. After training, the animal was dried with a fabric towel and returned to its home cage, large platform or PSD tank cage. The distance swum by
In Experiment 3, the experimental rats were deeply anesthetized with pentobarbital sodium (40 mg/kg) and decapitated after 3 days of PSD (n = 11). The control animals (n = 9) were allowed to sleep during the same period. Hippocampal slices (300 μm in thickness) were prepared with a vibratome (Vibroslice 752M, Campden Instruments, Loughborough, UK) and incubated with artificial cerebrospinal fluid (ACSF) containing (in millimolars): 124 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.0 MgCl2, 2.0 CaCl2, 25 NaHCO3, 10 Glucose. Slices were maintained in ACSF at 28 °C for at least 1 h before being moved into the recording chamber. During the recordings, the slices were kept submerged in a chamber perfused with ACSF. In the experiments, the ACSF was saturated with 95% O2/5% CO2 and the temperature was kept at 26 °C. Individual neurons were visualized with a 40× water-immersion objective under a microscope (BX51WI; Olympus, Tokyo, Japan) equipped with infrared differential interference contrast optics. Whole-cell recordings were obtained from pyramidal cells using recording pipettes with a resistance of 4–7 MΩ. Patch pipettes were filled with a solution containing (in millimolars): 140 potassium gluconate, 10 HEPES, 10 phosphocreatine sodium salt, 2 ATP sodium salt, 0.4 GPT sodium salt and 2 MgCl2. For patchclamp recordings, a Multiclamp 700B amplifier (Molecular Devices Corporation, Sunnyvale, CA, USA) was used. The series resistance was 10–20 MΩ. All potentials were corrected online for the junction potential by adjusting the offset of the pipette using the Multiclamp 700B commander software. Neurons were selected for further study if they had a resting membrane potential that was more negative than −50 mV and if they exhibited overshooting action potentials. Each cell was injected with a sequence of hyperpolarizing and depolarizing square wave current pulses to measure input resistance and to assess intrinsic electrophysiological characteristics. Rheobase was defined as the minimal amount of depolarizing current (100 ms) required to evoke a spike, and the spike threshold was defined as the voltage at which the value of dVm/dt preceding a spike first became 1/30 of dVm/dt(max) (Azouz and Gray, 1999). Spike width was measured at 50% maximum amplitude.
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4.5. Isolation of mitochondrial fraction and Western blot analysis In Experiment 4, we examined changes of proteins in 3-day PSD rats (n = 9) and control rats (n = 9) in the subcellular fractions where they are reported to be localized and/or translocated. Cytochrome c is present in mitochondria and translocates into cytoplasm, and Bax can translocate from the cytoplasm to the mitochondria. Protein from enriched fractions of mitochondria and cytosol were extracted as described by Ghribi et al. (2003) with slight modifications. Tissue from the entire hippocampus was gently homogenized using a glass–glass homogenizer in nine volumes of ice-cold homogenizing buffer (10 mM Tris–HCl pH 7.4, 250 mM sucrose, 1 mM KCl) and centrifuged at 600 ×g at 4 °C for 15 min. The supernatants were re-centrifuged at 18,000 ×g at 4 °C for 15 min. The pellets represented the mitochondrial fraction while the supernatants were the cytosolic fraction (Lee et al., 2004). The protein content of each sample was determined using a BCA™ Protein Assay Kit (Pierce, Rockford, IL, USA). Equal quantities of total protein (30 μg per lane) from mitochondrial and cytosolic fractions were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by transfer to nitrocellulose membranes. The membranes were incubated in 5% milk at room temperature for 2 h. Blots were then incubated with primary antibodies including mouse monoclonal antibody for cytochrome c (diluted 1:2000), or with rabbit polyclonal antibody against Bax (diluted 1:2000), cytochrome oxidase subunit IV (Cox IV) (diluted 1:2000), or β-actin (1:2000). After washing with buffer, the blots were incubated in anti-mouse (Zhong Shan, Beijing, China, 1:1000) and antirabbit (Zhong Shan, Beijing, China, 1:1000) secondary antibody conjugated with horseradish peroxide in TBS-T with 5% milk at 4 °C overnight. The blots were visualized using a West Pico Chemiluminescent Kit (Pierce, Rockford, IL, USA), and the density of protein bands was quantified by transmittance densitometry using volume integration with LumiAnalyst Image Analysis software.
4.6.
Data acquisition and statistics
Data values are presented as means ± SE Student's t-tests (SigmaStat version 2.03) were used to determine the statistical significance of differences between means obtained from two different groups of neurons. One-way ANOVAs followed by post hoc pairwise comparisons (Student–Newman–Keuls method) (SigmaStat 2.03) were used to determine the statistical significance of differences between means obtained from three experimental groups. Chi-square tests were used to assess differences in the percentages of time the rats swam in each quadrant of the Morris Water Maze. In all experiments, a probability of 0.05 or less was considered statistically significant.
Acknowledgments We thank Dr Judith Strong for her helpful comments on this manuscript. This work was supported by an NSFC grant
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(30530260) from China and a grant from the Fourth Military Medical University (XJ200502).
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