Aerobic exercise attenuates inhibitory avoidance memory deficit induced by paradoxical sleep deprivation in rats

(1)

www.elsevier.com/locate/brainres

Available online at www.sciencedirect.com

Research Report

Aerobic exercise attenuates inhibitory avoidance

memory de

ﬁ

cit induced by paradoxical sleep

deprivation in rats

Jansen Fernandes

a

, Luiz Guilherme Zaccaro Baliego

a

,

Luiz Fernando Peixinho-Pena

a

, Alexandre Aparecido de Almeida

a

,

Daniel Paulino Venancio

c

, Fulvio Alexandre Scorza

b

, Marco Tulio de Mello

c

,

Ricardo Mario Arida

a,n

a_{Department of Physiology, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil}

b_{Department of Neurology and Neurosurgery, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil}

c_{Department of Psychobiology, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil}

a r t i c l e i n f o

Article history: Accepted 11 July 2013 Available online 26 July 2013

Keywords: Sleep deprivation Treadmill exercise Memory

Synaptic proteins Hippocampus Neuroplasticity

a b s t r a c t

The deleterious effects of paradoxical sleep deprivation (SD) on memory processes are well documented. Physical exercise improves many aspects of brain functions and induces neuroprotection. In the present study, we investigated the influence of 4 weeks of treadmill aerobic exercise on both long-term memory and the expression of synaptic proteins (GAP-43, synapsin I, synaptophysin, and PSD-95) in normal and sleep-deprived rats. Adult Wistar rats were subjected to 4 weeks of treadmill exercise training for 35 min,five times per week. Twenty-four hours after the last exercise session, the rats were sleep-deprived for 96 h using the modified multiple platform method. To assess memory after SD, all animals underwent training for the inhibitory avoidance task and were tested 24 h later. The aerobic exercise attenuated the long-term memory deficit induced by 96 h of paradoxical SD. Western blot analysis of the hippocampus revealed increased levels of GAP-43 in exercised rats. However, the expression of synapsin I, synaptophysin, and PSD-95 was not modified by either exercise or SD. Our results suggest that an aerobic exercise program can attenuate the deleterious effects of SD on long-term memory and that this effect is not directly related to changes in the expression of the pre- and post-synaptic proteins analyzed in the study.

1. Introduction

Although the precise function of sleep is not known, it is widely accepted that sleep affects a variety of physiological functions, including those involved in learning and memory

(Blissitt, 2001;Diekelmann and Born, 2010). Memory is classi-cally deﬁned as the ability to retain and manipulate pre-viously acquired information by means of neuronal plasticity (Thompson et al., 2002). Indeed, sleep plays a critical role in fostering connections among neuronal networks for memory

n

Correspondence to: Departamento de Fisiologia, Universidade Federal de São Paulo, Rua Botucatu 862, Ed. Ciências Biomédicas, 51andar. Vila Clementino, 04023-900 São Paulo, SP, Brasil. Fax: 55 11 55739304.

(2)

consolidation in the hippocampus, a critical structure for learning and memory processes (Blissitt, 2001; Diekelmann and Born, 2010; Kim et al., 2005; McDermott et al., 2006). Animal studies have demonstrated that theﬁring patterns of hippocampal neurons during a learning experience are replayed during the subsequent paradoxical sleep period (Louie and Wilson, 2001; Skaggs and McNaughton, 1996). Moreover, there is compelling evidence indicating that mem-ory is impaired by SD. For example, in rodents, the memmem-ory in different hippocampus-dependent tasks, such as contextual fear conditioning, the Morris water maze (MWM), and the inhibitory avoidance task (IA), are disrupted by SD (Bueno et al., 1994;Graves et al., 2003;Smith and Rose, 1996).

The neural processing of new memories requires alterations in the protein synthesis, gene expression and structural prop-erties of neurons and synapses (Alberini, 2009;Sultan and Day, 2011). Interestingly, some reports have provided evidence that the sleep/wake cycle may modulate the expression of certain genes implicated in synaptic plasticity and memory, such as brain derived neurotrophic factor (BDNF), synapsin I, calcium– calmodulin-dependent protein kinase II (CAMKII) and the cAMP response element binding protein (CREB) (Cirelli and Tononi, 1998,2000;Sei et al., 2000;Taishi et al., 2001). Accord-ingly, in a previous study,Guzman-Marin et al. (2006)observed that the hippocampal expression of BDNF, synapsin I, CAMKII and CREB were reduced after 48 h of paradoxical SD.

Several studies have shown the ability of physical exer-cise, unlike SD, to ameliorate many aspects of brain function (Cotman et al., 2007;Hamer and Chida, 2009;van Praag, 2008). We recently reported that 8 weeks of endurance or resistance exercise improved the acquisition and retention in the MWM task (Cassilhas et al., 2012a). This ﬁnding corroborates pre-vious studies conducted in aging and young rodents that showed physical exercise-induced improvements in various hippocampus-dependent memory tasks (O’Callaghan et al., 2007; Radak et al., 2006; Schweitzer et al., 2006; Vaynman et al., 2004). The mechanism underlying exercise-induced synaptic plasticity requires the involvement of a myriad of molecules implicated in the maintenance and regulation of brain function, including neurotrophic factors, signal trans-duction proteins, transcription factors and synaptic proteins (Cassilhas et al., 2012a;Cotman et al., 2007;Ding et al., 2004; Lista and Sorrentino, 2010).

Previous studies have extensively demonstrated the dele-terious effects of SD on memory and the contrasting beneﬁcial effects of physical exercise on this behavior. However, only one study was conducted to investigate the interaction of the two at the molecular level. Zagaar et al. (2012) observed that 4 weeks of aerobic exercise was able to attenuate the short-term memory loss induced by 24 h of paradoxical SD in rats. Additionally, physical exercise abrogated the detrimental effects of SD on early phase of long-term potentiation (LTP) and rescued hippocampal levels of BDNF and CAMKII. How-ever, given the positive effects of exercise on neurobiology, the molecular mechanisms through which exercise prevents the SD-induced cognitive decline still remain to be explored. Considering that no study, to date, has investigated the effects of physical exercise on long-term memory after prolonged SD periods, the present study aimed to verify the inﬂuence of 4 weeks of aerobic treadmill exercise on long-term memory in

normal and sleep-deprived rats subjected to an inhibitory avoidance task and to analyze the expression of synaptic proteins (GAP-43, synapsin I, synaptophysin, and PSD-95) with critical roles for synaptic plasticity and memory process.

2. Results

2.1. Inhibitory avoidance

No signiﬁcant differences were observed in the latency to cross the aversive compartment between groups [F(3, 31)¼ 1.653; p¼0.200] during the training session for the IA task (Fig. 1). However, the latency in the test session was reduced in the SSD group compared with that of the SC group (221.5740.69 s vs. 514726.00 s; po0.001, respectively) and between the ExSD and Ex groups (405.5748.24 s vs. 540.07 0.00 s; p¼0.044, respectively). Additionally, the ExSD group showed a higher latency to cross the aversive compartment than shown by the SSD group (405.5748.24 s vs. 221.57 40.69 s;p¼0.004, respectively). No signiﬁcant differences were observed in the SC group relative to the Ex and ExSD groups.

2.2. Synaptic protein expression

To verify the underlying mechanisms of the beneficial effects of aerobic exercise on the memory deficits induced by 96 h of paradoxical SD, we conducted western blot analysis of pre-and post-synaptic proteins. Significant differences were observed in the hippocampal levels of GAP-43 [Fig. 2a;F(3, 19) ¼4.789;p¼0.014]. These increases were observed in the Ex (167715%; p¼0.015) and ExSD (156715%; p¼0.047) groups relative to the SC group. In contrast, no significant differences were found in the hippocampal expression of the other analyzed proteins: synapsin I (Fig. 2b;F(3, 19)¼0.55;p¼0.65); synaptophysin (Fig. 2c;F(3, 19)¼1.241;p¼0.328) and PSD-95 (Fig. 2d;F(3, 19)¼2.754;p¼0.077).

Fig. 1–Effects of aerobic exercise and paradoxical sleep deprivation on the mean latency to cross to black compartment during training and test session of the inhibitory avoidance task. Sedentary control group (SC, n¼8), sedentary sleep-deprived group (SSC,n¼8), exercise group (Ex,n¼8) and exercise sleep-deprived group (ExSD, n¼8). Values are expressed as mean7SEM. *Indicates signiﬁcant difference from SC group and#_indicates

(3)

3. Discussion

Memory impairment is one of the classic behavioral effects of SD (Bueno et al., 1994; Graves et al., 2003; Smith and Rose, 1996). This study demonstrated that 4 weeks of aerobic exercise attenuated the long-term memory loss induced by 96 h of paradoxical SD in rats. However, this behavior was not directly correlated with changes in pre- and post-synaptic protein expression.

Previous studies have shown that SD negatively affects memory in rodents subjected to various hippocampus-dependent tasks, such as MWM, IA, contextual fear condi-tioning and the radial water maze (Bueno et al., 1994;Graves et al., 2003; Smith and Rose, 1996; Zagaar et al., 2012). Consistent with thesefindings, our results demonstrate that rats that were sleep deprived for 96 h (SSD and ExSD) had impaired IA performance. However, this deficit was mitigated by physical exercise, given that the latency to cross the aversive compartment did not differ between the ExSD and SC groups. To date, only one study investigated the effects of exercise on the memory impairment triggered by SD in animals (Zagaar et al., 2012). The authors found that aerobic exercise performed for 4 weeks prevented the short-term memory deficit induced by 24 h of paradoxical SD. Never-theless, the effects of physical exercise on long-term memory after prolonged SD periods (96 h) had not yet been investi-gated. Therefore, this study is the first to report that an exercise program can reduce the long-term memory deficit induced by long periods of sleep disruption in rats.

In contrast to the results from previous studies (Cassilhas et al., 2012b; Liu et al., 2009; Radak et al., 2006), the IA performance was not enhanced by physical exercise in the present study.Cassilhas et al. (2012b)found a memory improve-ment in rats subjected to 8 weeks of resistance exercise when compared with the memory of their sedentary counterparts. Moreover, the performance in this task appears to be dependent on the type of exercise employed. For example,Liu et al. (2009) demonstrated that moderate treadmill exercise (forced) and voluntary wheel running affected the IA performance differently; animals subjected to the former had an improvement in long-term memory, but the latency of the voluntary group did not differ from that of the sedentary controls. The lack of differences in IA performance between the Ex and SC groups can be explained, at least in part, by the use of a very intense protocol during the training for the behavioral task. Thus, studies that veriﬁed a memory improvement as a result of physical exercise used a lower negative reinforcer (1 footshock of 0.2–0.5 mA) during IA training (Cassilhas et al., 2012b;Liu et al., 2009;Radak et al., 2006) than that used in this work (5 footshocks of 0.8 mA). The higher number and intensity of footshocks during the IA training may have led to the occurrence of a ceiling effect on the IA performance. For example, Cruz-Morales et al. (1992)found that the amnesic effect triggered by systemic administration of scopolamine, a cholinergic antagonist, was not present when the intensity of footshocks was increased during IA training. There-fore, it is possible that the observed absence of differences between the Ex and SC groups in the present study was due to the occurrence of a ceiling effect.

(4)

Previous studies have extensively documented that the formation of long-term memories requires changes in pro-teins synthesis, gene expression and the structural properties of neurons and synapses (Costa-Mattioli et al., 2009; Sultan and Day, 2011). Furthermore, one of the mechanisms under-lying learning and memory requires the involvement of several synaptic proteins needed for the proper synaptic transmission, such as synapsin I, synaptophysin, GAP-43 and PSD-95 (Clare et al., 2010;Powell, 2006;Silva et al., 1996; Xu, 2011).

The growth-associated protein GAP-43 is a neuron-specific protein found in high concentrations in growth cones and pre-synaptic terminals and is closely associated with neuritogen-esis, synaptic plasticity and regenerative processes (Aigner et al., 1995; Oehrlein et al., 1996; Oestreicher et al., 1997). Moreover, GAP-43 plays a central role in learning and memory. For instance,Rekart et al. (2005)demonstrated that mice with decreased expression of GAP-43 exhibited memory deficits in contextual fear conditioning, whereas genetic overexpression of this protein dramatically enhanced learning and LTP in transgenic mice (Routtenberg et al., 2000). In our current experiment, there were no differences in the hippocampal levels of GAP-43 between the SC and SSD rats. These data are consistent with those found by Gao et al. (2010), where the GAP-43 expression did not change after daily total SD (12 h for 3 days). In contrast, we observed an increase in the hippo-campal levels of GAP-43 in exercised rats even after 5 days of exercise cessation. Although we did notfind differences in IA performance between the Ex and SC groups, the increased expression of GAP-43 may have mediated, at least in part, the prevention of memory loss in the ExSD group.

Synapsin I is a nerve terminal-speciﬁc synaptic vesicle associated phosphoprotein that is involved in both the synap-togenesis and the plasticity of mature synapses by controlling synaptic vesicle trafﬁcking at pre- and post-docking levels (Evergren et al., 2007). Previous studies showed an increase in synapsin I immunoreactivity during LTP (Sato et al., 2000) and revealed that 6 days of spatial learning in the MWM increased synapsin I mRNA and protein expression (Gomez-Pinilla et al., 2001). The hippocampal levels of synapsin I did not change in any of the experimental conditions in the present study. Guzman-Marin et al. (2006)observed a reduction in synapsin I mRNA expression in the hippocampus after 8 and 48 h of SD. In contrast, a recent study demonstrated that 96 h of paradoxical SD increased the levels of total synapsin I and its phosphory-lated form in the synaptosomes from the whole brain of rats (Singh et al., 2012). These discrepancies may be due the different periods and methods of SD used in the two studies as well as in the method for analyzing synapsin I.

The expression of synapsin I is modulated differently depending on the type and volume of exercise (Cassilhas et al., 2012a; Ferreira et al., 2011; Vaynman et al., 2004). A recent study conducted in our laboratory demonstrated that, independent of the type of exercise (aerobic or resistance), 8 weeks of exercise was able to increase the levels of synapsin I in the rat hippocampus (Cassilhas et al., 2012a). Moreover, studies have shown an increase in hippocampal levels of this protein after just 3 (Vaynman et al., 2004) or 7 (Ferreira et al., 2011) days of aerobic exercise. In the present study, the absence of changes in synapsin I expression after 20 days of exercise is in accordance

with previous studies (Ferreira et al., 2011;Molteni et al., 2002) where no signiﬁcant differences were found after longer periods (28 and 15 days) of exercise.

Synaptophysin, a major integral glycoprotein attached to the membrane of synaptic vesicles, was not affected by SD or by exercise (Tarsa and Goda, 2002). Synaptophysin acts as an important protein in the biogenesis of synaptic vesicles (Thiele et al., 2000) and appears to be involved in the formation of the fusion pore between the vesicle and the plasma membrane (Fykse et al., 1993) as well as in the endocytosis and recycling of synaptic vesicles (Evans and Cousin, 2005). Recently, Zunzunegui et al. (2011) observed that 12 h of SD during the light phase of the sleep-wake cycle for 3 days did not significantly alter the synaptophysin levels in rat brains; this result is in accordance with our findings. Furthermore, 4 weeks of aerobic exercise did not induce significant changes in synaptophysin expression compared with that in all other groups. Ourfinding is in agreement with previous studies that demonstrated that hippocampal levels of this protein were not altered after 3, 7, 15 (Ferreira et al., 2011) and 20 (Hescham et al., 2009) days of forced and voluntary exercise. Conversely, other reports have demon-strated increased expression of synaptophysin in the hippo-campus (Cassilhas et al., 2012a;Vaynman et al., 2004), striatum and substantia nigra (Ferreira et al., 2010) after different exercise regimens.

We also investigated the effects of exercise and SD on the expression of PSD-95, a synaptic scaffolding protein com-posed of modular domains for protein interactions, along with studying their effects on presynaptic proteins. PSD-95 is enriched in the postsynaptic density (PSD), an electron-dense specialization of the postsynaptic membrane that contains macromolecular protein complexes (Cho et al., 1992;Kistner et al., 1993). This postsynaptic protein is an important regulator of synaptic strength and plasticity. For example, PSD-95 overexpression increases synaptic AMPA receptor clustering, enhances the frequency of miniature excitatory postsynaptic currents, occludes LTP and enhances long-term depression (Han and Kim, 2008;Xu et al., 2008). In a previous study,Lopez et al. (2008)showed that 4 h of paradoxical SD for 3 days did not alter the PSD-95 expression in young and adolescent rats. Although the PSD-95 expression levels increased with short- (Dietrich et al., 2005) and long-term (Hu et al., 2009) voluntary exercise, we did notﬁnd signiﬁcant changes induced by exercise or by SD.

(5)

on alternating days. Therefore, it is possible that detraining changes the expression pattern of the proteins analyzed in this study. Although it is feasible that these and other exercise-regulated molecules in the hippocampus undergo distinct temporal patterns of decay after exercise ends, as occurs in others tissues (Esposito et al., 2011;Léger et al., 2006), little is known about the effects of detraining in proteins other than BDNF.

In conclusion, ourﬁndings demonstrated that 4 weeks of aerobic exercise can attenuate the long-term memory impair-ment induced by 96 h of paradoxical SD. The lack of change in proteins other than GAP-43 after the exercise program can be related to the distinct temporal patterns of decay of these proteins after exercise ends. Further investigation is required to elucidate the mechanisms underlying the ability of exer-cise to prevent the long-term memory deﬁcit induced by paradoxical SD.

4. Experimental procedures

4.1. Animals

Fifty-two male Wistar rats, 60-days-old, were provided by the Center for Development of Experimental Models for Medicine and Biology (CEDEME/UNIFESP). The animals were housed in groups of ﬁve in standard polypropylene cages. The room temperature was maintained at 22711C, and the relative humidity was 5573%. The animals were kept on a 12 h light/ dark schedule (with the lights on at 7:00 AM) and had free access to food and water. All experimental protocols were approved by the ethics committee of the Universidade Fed-eral de São Paulo (#0607/09), and all efforts were made to minimize animal suffering, in accordance with the proposals of the International Ethical Guideline for Biomedical Research (CIOMS, 1985).

4.2. Exercise training

At the beginning of the study, all animals were subjected to a recruitment process, which consisted of three days of running familiarization sessions on a motorized treadmill (Columbus Instruments). During this period, the rats ran for 10 min/day at a speed of 8 m/min at 01incline. Electric shocks were used sparingly to motivate the rats to run. To provide a measure of their trainability, we rated each animal’s tread-mill performance on a scale of 1–5, according to the following classiﬁcations [1, refused to run; 2, below average runner (sporadic, stop and go, wrong direction); 3, average runner; 4, above average runner (consistent runner occasionally fell back on the treadmill); and 5, good runner (consistently stayed at the front of the treadmill)] (Arida et al., 2011; Dishman et al., 1988). Animals with a mean rating of 3 or higher were randomly distributed into four groups of 13 animals: sedentary control (SC), exercise (Ex), sedentary sleep-deprived (SSD) and exercise sleep-deprived (ExSD). The animals that did not meet this criterion were excluded from the experiment. This proce-dure was used to exclude the possibility of different levels of stress between the animals. Afterward, the rats of the Ex and ExSD groups were subjected to daily running (5 days/week) for

4 weeks, while the SC and SSD groups were handled in exactly the same manner as the exercised groups and maintained on the turned-off treadmill for the same times. Each training session started with a 5-min warm-up at 8 m/min, followed by a bout of 25 min of running at a speed of 12 m/min (ﬁrst week), 14 m/min (second week), 16 m/min (third week) and 18 m/min (fourth week).

4.3. Paradoxical sleep deprivation

Twenty-four hours after the last exercise session, the animals from the SSD and ExSD groups were sleep-deprived for 96 h using the modiﬁed multiple platform method (Suchecki and Tuﬁk, 2000), while the other groups remained in their home cages in the same room where the SD procedures took place. The rats were placed in a water tank (123 cm44 cm44 cm) containing circular platforms (6.5 cm diameter). The water level remained at approximately 2 cm below the surface of the platforms, and the number of platforms always exceeded the number of animals, allowing the rats to move around freely inside the tank by jumping from one platform to another. During the paradoxical SD period, the rats had free access to water bottles and food pellet baskets located on a grid on top of the tank. This method relies on the muscle atonia that accompanies paradoxical sleep. Therefore, when the animals on the platforms reached this sleep stage, they lost muscle tone, touched or fell into the surrounding water, and then awakened. Before the onset of the SD period, the animals were habituated to the method for two days (1 h/day) to avoid unnecessary drops in the water. This method completely abolishes paradoxical sleep and also decreases slow wave sleep by approximately 35% (Machado et al., 2004).

4.4. Inhibitory avoidance task

Immediately after the paradoxical sleep deprivation period, the animals (n¼8 for each group) were gently dried and subjected to the IA. The IA apparatus consisted of two acrylic boxes, each measuring (212627.5 cm3), connected by a sliding door. A box with white acrylic walls was designated as the safe compartment, whereas the other black acrylic box was the aversive compartment. The ﬂoor of the apparatus was made of parallel metallic rods (0.4 cm diameter), which were separated by a distance of 1.2 cm, and connected to an electric shock generator. During the task training, each animal was placed in the safe compartment with the sliding door closed. Ten seconds later, the door was opened. As soon as the animal crossed to the aversive compartment with its four paws, the door was closed, the latency to enter was recorded, and the animal receivedﬁve footshocks (0.8 mA/1 s) separated by 15 s (Esumi et al., 2011). After the shocks, the animal was removed from the apparatus and returned to the home cage.

(6)

4.5. Western blot analysis

Immediately after the 96 h of SD, the rats (n¼5 for each group) were euthanized by decapitation, and the hippocampi were dissected and immediately frozen in liquid nitrogen. Tissues and serum were stored at 801C until use. Thereafter, the hippocampi were homogenized in lysis buffer (1% Triton X-100; 0.5% sodium deoxycholate; 100 mM Tris–HCl, pH 8.3; 150 mM NaCl; 10 mM EDTA; 0.1% SDS; 10% glycerol; 1% NP-40; and protease inhibitor cocktails), and the total protein con-centration was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA) (Bradford, 1976). The samples were loaded on 10% (PSD-95, 20mg/lane; synapsin 1, synaptophysin and GAP-43, 30mg/lane) SDS-polyacrylamide gels, separated using electrophoresis and then transferred to nitrocellulose mem-branes (Amersham GE, Little Chalfont, UK). Immunodetection was performed at room temperature. The membranes were blocked with 2% non-fat milk for 1 h and then incubated with primary antibodies for 1 h at the indicated dilutions: anti-PSD-95 (1:20.000); GAP-43 (1:5.000); synapsin 1 (1:1000); synaptophy-sin (Abcam, Cambridge, MA, USA; 1:10.000); anti-β-actin (1:10.000);β-tubulin (Sigma, St. Louis, MO, USA; 1:50.000). After 3 5 min washes, the membranes were incubated for 45 min with Alexa-680-conjugated anti-rabbit IgG (1:10.000, Invitro-gen, Carlsbad, CA, USA). After 5 5-min washes, digital images of the membranes were acquired and quantiﬁed using the Odyssey Infrared Image System (LICOR, Baltimore, MD, USA). The band intensity of the protein of interest was normalized to the band intensity ofβ-actin orβ-tubulin. The relative protein expression in the SSD, Ex and ExSD groups was expressed as the percentage of the SC mean.

4.6. Statistical analysis

Data were analyzed using SPSS (version 17.0), and in all analyses,po0.05 was considered statistically signiﬁcant. After conﬁrmation of the normality of variables using the Shapiro– Wilk test, the values were compared using one-way analysis of variance (one-way ANOVA) followed by the Tukey post hoc test for both the western blotting and the behavioral task data. Data were presented as the mean7standard error.

Acknowledgments

Supported by CAPES, CNPq, CEPE, CEMSA, FAPESP, CEPID/ SONO/FAPESP and INNT (Brazil).

r e f e r e n c e s

Aigner, L., Arber, S., Kapfhammer, J.P., Laux, T., Schneider, C., Botteri, F., Brenner, H.R., Caroni, P., 1995. Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice. Cell 83, 269–278.

Alberini, C.M., 2009. Transcription factors in long-term memory and synaptic plasticity. Physiol. Rev. 89, 121–145.

Arida, R.M., Scorza, F.A., Gomes da Silva, S., Cysneiros, R.M., Cavalheiro, E.A., 2011. Exercise paradigms to study brain

injury recovery in rodents. Am. J. Phys. Med. Rehabil. 90, 452–465.

Berchtold, N.C., Chinn, G., Chou, M., Kesslak, J.P., Cotman, C.W., 2005. Exercise primes a molecular memory for brain-derived neurotrophic factor protein induction in the rat hippocampus. Neuroscience 133, 853–861.

Berchtold, N.C., Castello, N., Cotman, C.W., 2010. Exercise and time-dependent beneﬁts to learning and memory. Neuroscience 167, 588–597.

Blissitt, P.A., 2001. Sleep, memory, and learning. J. Neurosci. Nurs. 33, 208–215.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Bueno, O.F., Lobo, L.L., Oliveira, M.G., Gugliano, E.B., Pomarico, A.C.,

Tuﬁk, S., 1994. Dissociated paradoxical sleep deprivation effects on inhibitory avoidance and conditioned fear. Physiol. Behav. 56, 775–779.

Cassilhas, R.C., Lee, K.S., Fernandes, J., Oliveira, M.G., Tuﬁk, S., Meeusen, R., de Mello, M.T., 2012a. Spatial memory is improved by aerobic and resistance exercise through divergent molecular mechanisms. Neuroscience 202, 309–317. Cassilhas, R.C., Lee, K.S., Venâncio, D.P., Oliveira, M.G., Tuﬁk, S.,

Mello, M.T., 2012b. Resistance exercise improves

hippocampus-dependent memory. Brazilian J. Med. Biol. Res. 45, 1215–1220.

Cho, K.O., Hunt, C.A., Kennedy, M.B., 1992. The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9, 929–942.

CIOMS, 1985. Council for international organizations of medical sciences. International Guiding Principles for Biomedical Research Involving Animals. Geneva, Switzerland. Cirelli, C., Tononi, G., 1998. Differences in gene expression

between sleep and waking as revealed by mRNA differential display. Brain Res. Mol. Brain Res. 56, 293–305.

Cirelli, C., Tononi, G., 2000. Differential expression of plasticity-related genes in waking and sleep and their regulation by the noradrenergic system. J. Neurosci. 20, 9187–9194.

Clare, R., King, V.G., Wirenfeldt, M., Vinters, H.V., 2010. Synapse loss in dementias. J. Neurosci. Res. 88, 2083–2090.

Costa-Mattioli, M., Sossin, W.S., Klann, E., Sonenberg, N., 2009. Translational control of long-lasting synaptic plasticity and memory. Neuron 61, 10–26.

Cotman, C.W., Berchtold, N.C., Christie, L.A., 2007. Exercise builds brain health: key roles of growth factor cascades and inﬂammation. Trends Neurosci. 30, 464–472.

Cruz-Morales, S.E., Duran-Arevalo, M., Diaz Del Guante, M.A., Quirarte, G., Prado-Alcala, R.A., 1992. A threshold for the protective effect of over-reinforced passive avoidance against scopolamine-induced amnesia. Behav. Neural Biol. 57, 256–259.

Diekelmann, S., Born, J., 2010. The memory function of sleep. Nat. Rev. Neurosci. 11, 114–126.

Dietrich, M.O., Mantese, C.E., Porciuncula, L.O., Ghisleni, G., Vinade, L., Souza, D.O., Portela, L.V., 2005. Exercise affects glutamate receptors in postsynaptic densities from cortical mice brain. Brain Res. 1065, 20–25.

Ding, Y.H., Luan, X.D., Li, J., Rafols, J.A., Guthinkonda, M., Diaz, F.G., Ding, Y., 2004. Exercise-induced overexpression of angiogenic factors and reduction of ischemia/reperfusion injury in stroke. Curr. Neurovasc. Res. 1, 411–420.

Dishman, R.K., Armstrong, R.B., Delp, M.D., Graham, R.E., Dunn, A.L., 1988. Open-ﬁeld behavior is not related to treadmill

performance in exercising rats. Physiol. Behav. 43, 541–546. Esposito, F., Ronchi, R., Milano, G., Margonato, V., Di Tullio, S.,

(7)

Esumi, L.A., Palma, B.D., Gomes, V.L., Tufik, S., Hipólide, D.C., 2011. Inflammatory markers are associated with inhibitory avoidance memory deficit induced by sleep deprivation in rats. Behav. Brain Res. 221, 7–12.

Evans, G.J., Cousin, M.A., 2005. Tyrosine phosphorylation of synaptophysin in synaptic vesicle recycling. Biochem. Soc. Trans. 33, 1350–1353.

Evergren, E., Benfenati, F., Shupliakov, O., 2007. The synapsin cycle: a view from the synaptic endocytic zone. J. Neurosci. Res. 85, 2648–2656.

Ferreira, A.F., Real, C.C., Rodrigues, A.C., Alves, A.S., Britto, L.R., 2010. Moderate exercise changes synaptic and cytoskeletal proteins in motor regions of the rat brain. Brain Res. 1361, 31–42.

Ferreira, A.F., Real, C.C., Rodrigues, A.C., Alves, A.S., Britto, L.R., 2011. Short-term, moderate exercise is capable of inducing structural, BDNF-independent hippocampal plasticity. Brain Res. 1425, 111–122.

Fykse, E.M., Takei, K., Walch-Solimena, C., Geppert, M., Jahn, R., De Camilli, P., Südhof, T.C., 1993. Relative properties and localizations of synaptic vesicle protein isoforms: the case of the synaptophysins. J. Neurosci. 13, 4997–5007.

Gao, B., Cam, E., Jaeger, H., Zunzunegui, C., Sarnthein, J., Bassetti, C.L., 2010. Sleep disruption aggravates focal cerebral ischemia in the rat. Sleep 33, 879–887.

Gomez-Pinilla, F., So, V., Kesslak, J.P., 2001. Spatial learning induces neurotrophin receptor and synapsin I in the hippocampus. Brain Res. 904, 13–19.

Graves, L.A., Heller, E.A., Pack, A.I., Abel, T., 2003. Sleep deprivation selectively impairs memory consolidation for contextual fear conditioning. Learn. Mem. 10, 168–176. Guzman-Marin, R., Ying, Z., Suntsova, N., Methippara, M., Bashir, T.,

Szymusiak, R., Gomez-Pinilla, F., McGinty, D., 2006. Suppression of hippocampal plasticity-related gene expression by sleep deprivation in rats. J. Physiol. 575, 807–819.

Hamer, M., Chida, Y., 2009. Physical activity and risk of

neurodegenerative disease: a systematic review of prospective evidence. Psychol. Med. 39, 3–11.

Han, K., Kim, E., 2008. Synaptic adhesion molecules and PSD-95. Prog. Neurobiol. 84, 263–283.

Hescham, S., Grace, L., Kellaway, L.A., Bugarith, K., Russell, V.A., 2009. Effect of exercise on synaptophysin and calcium/ calmodulin-dependent protein kinase levels in prefrontal cortex and hippocampus of a rat model of developmental stress. Metab. Brain Dis. 24, 701–709.

Hu, S., Ying, Z., Gomez-Pinilla, F., Frautschy, S.A., 2009. Exercise can increase small heat shock proteins (sHSP) and pre- and post-synaptic proteins in the hippocampus. Brain Res. 1249, 191–201.

Kim, E.Y., Mahmoud,G.S., Grover, L.M., REM sleep deprivation inhibits LTP in vivo in area CA1 of rat hippocampus. Neuroscience Letters. 388. pp. 163–167.

Kistner, U., Wenzel, B.M., Veh, R.W., Cases-Langhoff, C., Garner, A.M., Appeltauer, U., Voss, B., Gundelﬁnger, E.D., Garner, C.C., 1993. SAP90, a rat presynaptic protein related to the product of the Drosophila tumor suppressor gene dlg-A. J. Biol. Chem. 268, 4580–4583.

Langfort, J., Barańczuk, E., Pawlak, D., Chalimoniuk, M., Lukacova, N., Marsala, J., Górski, J., 2006. The effect of endurance training on regional serotonin metabolism in the brain during early stage of detraining period in the female rat. Cell Mol. Neurobiol. 26, 1327–1342.

Léger, B., Cartoni, R., Praz, M., Lamon, S., Dériaz, O., Crettenand, A., Gobelet, C., Rohmer, P., Konzelmann, M., Luthi, F., Russell, A.P., 2006. Akt signalling through GSK-3beta, mTOR and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy. J. Physiol. 576, 923–933.

Lista, I., Sorrentino, G., 2010. Biological mechanisms of physical activity in preventing cognitive decline. Cell Mol. Neurobiol. 30, 493–503.

Liu, Y.F., Chen, H.I., Wu, C.L., Kuo, Y.M., Yu, L., Huang, A.M., Wu, F.S., Chuang., J.I., Jen, C.J., 2009. Differential effects of treadmill running and wheel running on spatial or aversive learning and memory: roles of amygdalar brain-derived neurotrophic factor and synaptotagmin I. J. Physiol. 587, 3221–3231.

Lopez, J., Roffwarg, H.P., Dreher, A., Bissette, G., Karolewicz, B., Shaffery, J.P., 2008. Rapid eye movement sleep deprivation decreases long-term potentiation stability and affects some glutamatergic signaling proteins during hippocampal development. Neuroscience 153, 44–53.

Louie, K., Wilson, M.A., 2001. Temporally structured replay of awake hippocampal ensemble activity during rapid eye movement sleep. Neuron 29, 145–156.

Machado, R.B., Hipólide, D.C., Benedito-Silva, A.A., Tufik, S., 2004. Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery. Brain Res. 1004, 45–51.

McDermott, C.M., Hardy, M.N., Bazan, N.G., Magee, J.C., 2006. Sleep deprivation-induced alterations in excitatory synaptic transmission in the CA1 region of the rat hippocampus. J. Physiol. 570, 553–565.

Molteni, R., Ying, Z., Gómez-Pinilla, F., 2002. Differential effects of acute and chronic exercise on plasticity-related genes in the rat hippocampus revealed by microarray. Eur. J. Neurosci. 16, 1107–1116.

Moreira, K.M., Hipólide, D.C., Nobrega, J.N., Bueno, O.F., Tuﬁk, S., Oliveira, M.G., 2003. Deﬁcits in avoidance responding after paradoxical sleep deprivation are not associated with altered [3H]pirenzepine binding to M1 muscarinic receptors in rat brain. Brain Res. 977, 31–37.

Nelson, A.J., Iwamoto, G.A., 2006. Reversibility of exercise-induced dendritic attenuation in brain cardiorespiratory and locomotor areas following exercise detraining. J. Appl. Physiol. 101, 1243–1251.

O’Callaghan, R.M., Ohle, R., Kelly, A.M., 2007. The effects of forced exercise on hippocampal plasticity in the rat: a comparison of LTP, spatial- and non-spatial learning. Behav. Brain Res. 176, 362–366.

Oehrlein, S.A., Parker, P.J., Herget, T., 1996. Phosphorylation of GAP-43 (growth-associated protein of 43 kDa) by conventional, novel and atypical isotypes of the protein kinase C gene family: differences between oligopeptide and polypeptide phosphorylation. Biochem. J. 317, 219–224.

Oestreicher, A.B., De Graan, P.N., Gispen, W.H., Verhaagen, J., Schrama, L.H., 1997. B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system. Prog. Neurobiol. 53, 627–686.

Powell, C.M., 2006. Gene targeting of presynaptic proteins in synaptic plasticity and memory: across the great divide. Neurobiol. Learn. Mem. 85, 2–15.

Radak, Z., Toldy, A., Szabo, Z., Siamilis, S., Nyakas, C., Silye, G., Jakus, J., Goto, S., 2006. The effects of training and detraining on memory, neurotrophins and oxidative stress markers in rat brain. Neurochem. Int. 49, 387–392.

Rekart, J.L., Meiri, K., Routtenberg, A., 2005. Hippocampal-dependent memory is impaired in heterozygous GAP-43 knockout mice. Hippocampus 15, 1–7.

Routtenberg, A., Cantallops, I., Zaffuto, S., Serrano, P., Namgung, U., 2000. Enhanced learning after genetic overexpression of a brain growth protein. Proc. Natl. Acad. Sci. USA 97, 7657–7662. Sato, K., Morimoto, K., Suemaru, S., Sato, T., Yamada, N., 2000.

(8)

Schweitzer, N.B., Alessio, H.M., Berry, S.D., Roeske, K., Hagerman, A.E., 2006. Exercise-induced changes in cardiac gene expression and its relation to spatial maze performance. Neurochem. Int. 48, 9–16. Sei, H., Saitoh, D., Yamamoto, K., Morita, K., Morita, Y., 2000.

Differential effect of short-term REM sleep deprivation on NGF and BDNF protein levels in the rat brain. Brain Res. 877, 387–390.

Silva, A.J., Rosahl, T.W., Chapman, P.F., Marowitz, Z., Friedman, E., Frankland, P.W., Cestari, V., Ciofﬁ, D., Südhof., T.C.,

Bourtchuladze, R., 1996. Impaired learning in mice with abnormal short-lived plasticity. Curr. Biol. 6, 1509–1518. Singh, S., Amar, M., Mallick, B.N., 2012. Rapid eye movement sleep

deprivation modulates synapsinI expression in rat brain. Neurosci. Lett. 520, 62–66.

Skaggs, W.E., McNaughton, B.L., 1996. Replay of neuronalﬁring sequences in rat hippocampus during sleep following spatial experience. Science 271, 1870–1873.

Smith, C., Rose, G.M., 1996. Evidence for a paradoxical sleep window for place learning in the Morris water maze. Physiol. Behav. 59, 93–97.

Suchecki, D., Tuﬁk, S., 2000. Social stability attenuates the stress in the modiﬁed multiple platform method for paradoxical sleep deprivation in the rat. Physiol. Behav. 68, 309–316. Sultan, F.A., Day, J.J., 2011. Epigenetic mechanisms in memory

and synaptic function. Epigenomics 3, 157–181.

Taishi, P., Sanchez, C., Wang, Y., Fang, J., Harding, J.W., Krueger, J.M., 2001. Conditions that affect sleep alter the expression of molecules associated with synaptic plasticity. Am. J. Physiol.-Regul. Integrative Comp. Physiol. 281, R839–R845.

Tarsa, L., Goda, Y., 2002. Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons. Proc. Natl. Acad. Sci. USA 99, 1012–1016. Thiele, C., Hannah, M.J., Fahrenholz, F., Huttner, W.B., 2000.

Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles. Nat. Cell Biol. 2, 42–49. Thompson, A.M., Gosnell, B.A., Wagner, J.J., 2002. Enhancement of

long-term potentiation in the rat hippocampus following cocaine exposure. Neuropharmacology 42, 1039–1042. van Praag, H., 2008. Neurogenesis and exercise: past and future

directions. NeuroMolecular Med. 10, 128–140.

Vaynman, S., Ying, Z., Gomez-Pinilla, F., 2004. Hippocampal BDNF mediates the efﬁcacy of exercise on synaptic plasticity and cognition. Eur. J. Neurosci. 20, 2580–2590.

Xu, W., Schlüter, O.M., Steiner, P., Czervionke, B.L., Sabatini, B., Malenka, R.C., 2008. Molecular dissociation of the role of PSD-95 in regulating synaptic strength and LTD. Neuron 57, 248–262.

Xu, W., 2011. PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs) and synaptic plasticity. Curr. Opinion Neurobiol. 21, 306–312.

Zagaar, M., Alhaider, I., Dao, A., Levine, A., Alkarawi, A., Alzubaidy, M., Alkadhi, K., 2012. The beneﬁcial effects of regular exercise on cognition in REM sleep deprivation: behavioral, electrophysiological and molecular evidence. Neurobiol. Dis. 45, 1153–1162.