Chronic sleep restriction promotes brain inflammation and synapse loss, and potentiates memory impairment induced by amyloid-β oligomers in mice

Accepted Manuscript Chronic sleep restriction promotes brain inflammation and synapse loss, and potentiates memory impairment induced by amyloid-β oligomers in mice Grasielle C. Kincheski, Isabela S. Valentim, Julia R. Clarke, Danielle Cozachenco, Morgana T.L. Castelo-Branco, Angela M. Ramos-Lobo, Vivian M.B.D. Rumjanek, José Donato Jr. , Fernanda G. De Felice, Sergio T. Ferreira PII: DOI: Reference:

S0889-1591(17)30109-5 http://dx.doi.org/10.1016/j.bbi.2017.04.007 YBRBI 3120

To appear in:

Brain, Behavior, and Immunity

Received Date: Revised Date: Accepted Date:

21 December 2016 2 April 2017 10 April 2017

Please cite this article as: Kincheski, G.C., Valentim, I.S., Clarke, J.R., Cozachenco, D., Castelo-Branco, M.T.L., Ramos-Lobo, A.M., Rumjanek, V.M.B., Donato, J. Jr., De Felice, F.G., Ferreira, S.T., Chronic sleep restriction promotes brain inflammation and synapse loss, and potentiates memory impairment induced by amyloid-β oligomers in mice, Brain, Behavior, and Immunity (2017), doi: http://dx.doi.org/10.1016/j.bbi.2017.04.007

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Chronic sleep restriction promotes brain inflammation and synapse loss, and potentiates memory impairment induced by amyloid-β oligomers in mice

Grasielle C. Kincheski1, Isabela S. Valentim1*, Julia R. Clarke2*, Danielle Cozachenco1, Morgana T. L. Castelo-Branco3, Angela M. Ramos-Lobo5; Vivian M. B. D. Rumjanek1, José Donato Jr.5, Fernanda G. De Felice1,6, Sergio T. Ferreira1,4

1

Institute of Medical Biochemistry Leopoldo de Meis, 2School of Pharmacy,

3

Institute of Biomedical Sciences, 4Institute of Biophysics Carlos Chagas Filho,

Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil 5

Department of Physiology and Biophysics, Institute of Biomedical Sciences,

University of São Paulo, São Paulo, Brazil 6

Centre for Neuroscience Studies, Department of Biomedical and Molecular

Sciences, Queen’s University, Kingston, ON, Canada

*Both authors contributed equally to this work. Correspondence to: Sergio T. Ferreira, Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Email: [email protected]

1

Abstract It is increasingly recognized that sleep disturbances and Alzheimer’s disease (AD) share a bidirectional relationship. AD patients exhibit sleep problems and alterations in the regulation of circadian rhythms; conversely, poor quality of sleep increases the risk of development of AD. The aim of the current study was to determine whether chronic sleep restriction potentiates the brain impact of amyloid-β oligomers (AβOs), toxins that build up in AD brains and are thought to underlie synapse damage and memory impairment. We further investigated whether alterations in levels of pro-inflammatory mediators could play a role in memory impairment in sleep-restricted mice. We found that a single intracerebroventricular (i.c.v.) infusion of AβOs disturbed sleep pattern in mice. Conversely, chronically sleep-restricted mice exhibited higher brain expression of pro-inflammatory mediators, reductions in levels of pre- and postsynaptic marker proteins, and exhibited increased susceptibility to the impact of i.c.v. infusion of a sub-toxic dose of AβOs (1 pmol) on performance in the novel object recognition memory task. Sleep-restricted mice further exhibited an increase in brain TNF-α levels in response to AβOs. Interestingly, memory impairment in sleep-restricted AβO-infused mice was prevented by treatment with

the

TNF-α

neutralizing

monoclonal

antibody,

infliximab.

Results

substantiate the notion of a dual relationship between sleep and AD, whereby AβOs disrupt sleep/wake patterns and chronic sleep restriction increases brain vulnerability to AβOs, and point to a key role of brain inflammation in increased susceptibility to AβOs in sleep-restricted mice.

2

Keywords: Sleep, chronic sleep restriction, Alzheimer’s disease, amyloid-β oligomers, cytokines, TNF-α, IL-1β, IL-6, hippocampus, synaptic proteins, synaptophysin, PSD-95, inflammation

3

1. Introduction Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, affecting over 24 million people worldwide (Reitz and Mayeux, 2014). Patients initially present with relatively mild cognitive impairment whereas at later stages even vital functions are compromised (Thies et al., 2013). Brain accumulation of soluble oligomers of the amyloid-β peptide (AβOs) is a central feature of AD pathogenesis. Mounting evidence implicates AβOs in multiple pathological aspects of AD, including synapse loss, cognitive impairment, generation of reactive oxygen species and tau hyperphosphorylation (for reviews, see Ferreira et al., 2015; Ferreira and Klein, 2011; Mucke and Selkoe, 2012; Selkoe and Hardy, 2016). Importantly, presence of AβOs or larger Aβ aggregates such as amyloid plaques leads to neuroinflammation and activation of microglial cells, contributing to neurodegeneration (Czirr and Wyss-Coray, 2012; De Felice and Ferreira, 2014; Ferreira et al., 2014; Ledo et al., 2013; Ledo et al., 2016; Perry et al., 2010; Swardfager et al., 2010). Although aging is still considered the main risk factor for AD, increasing evidence supports a cumulative hypothesis for AD (De Felice, 2013). According to this hypothesis, poor lifestyle habits and certain conditions such as obesity, type 2 diabetes, smoking and sleep disturbances increase the risk of AD development later in life (Benedict et al., 2015; Mayeux and Stern, 2012; Ownby et al., 2006; Wilson et al., 2007). Chronic or sub-clinical inflammation is a common denominator for all these disorders (Ferreira et al., 2014). Among the conditions that could favor development of AD, the link between sleep

4

disturbance, neurodegeneration and memory defects is the less extensively understood. Sleep is an essential behavior for the survival and integrity of organisms, and plays a major role in body homeostasis (Dang-Vu et al., 2006; Siegel, 1995; Zepelin et al., 1994). Even though many studies have tried to correlate the many functions of sleep (Breder et al., 1993; Brown et al., 2012; Inqué et al., 1995; McGinty and Szymusiak, 1990; Mignot, 2008; Millers, 2010; Stickgold and Walker, 2007; Tononi and Cirelli, 2006), its importance is easily appreciated when an individual is deprived of sleep (Chen, Wynne and Kushida, 2005). Lack of sleep causes deleterious effects on health, including cognitive impairment, mood disorders, deficits in attention and sensorial perception (Goel et al., 2009; McCoy and Strecker, 2011; Stickgold, 2006; Walker, 2009, 2008; Zhang et al., 2014). Recent findings have shown that sleep is essential for clearance of Aβ that accumulates in the brain as a result of normal metabolism during wakefulness (Iliff et al., 2012; Xie et al., 2013), and sleep deprivation leads to increased levels of Aβ in the cerebrospinal fluid of healthy subjects (Benedict et al., 2015; Lucey et al., 2016; Ooms et al., 2014) and of APP/PS1 transgenic mice (Kang Jae-Eun, 2009). Sleep disturbances could contribute to neurodegeneration by promoting higher neuronal excitability due to oxidative stress, which could alter the physiology of neurons, impairing synaptic plasticity and promoting cell death (Benedict et al., 2014; Buzsáki, 1998; Cedernaes et al., 2015; D’Almeida et al., 1998; Graves et al., 2001; Musiek and Holtzman, 2016; Novati et al., 2012; Ramanathan et al., 2002). Moreover, increased 5

plasma and brain levels of pro-inflammatory cytokines, notably tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), were reported after sleep deprivation in humans and animal models (Krueger et al., 1984; Shearer et al., 2001). Besides the evidence that sleep disturbance could increase the risk of development of AD (Osorio et al., 2011), sleep disorders and disruption of circadian rhythms accompany memory loss in patients, and have been referred to as early and non-cognitive symptoms of AD (Ju et al., 2014; Sperling et al., 2011). Clinical/epidemiological data have shown that AD patients show sleep fragmentation, excessive daytime napping and decreased slow-wave sleep (McCurry et al., 1999; Moran et al., 2005). Post-mortem analysis of AD brains has shown a decreased number of suprachiasmatic cells and a reduction in orexin-positive neurons compared to control individuals (Fronczek et al., 2012; Swaab et al., 1985). It is not clear, however, whether these effects are an early consequence of Aβ toxicity or a result of extensive cell death seen in late stages of the disease. Here,

we

used a

well-established mouse model consisting in

intracerebroventricular (i.c.v.) infusion of AβOs (Figueiredo et al., 2013; Ledo et al., 2016, 2013; Lourenco et al., 2013) to investigate whether these toxins affect normal sleep/wake behavior. We further assessed whether chronic sleep restriction influences the susceptibility to AβO-induced memory impairment, and if alterations in brain levels of pro-inflammatory mediators could account for this behavioral impact. We found that a single i.c.v. infusion of AβOs disrupted sleep pattern in mice. Moreover, sleep-restricted mice were more susceptible to the 6

impact of a sub-toxic dose of AβOs on memory, and had an exacerbated proinflammatory profile. Finally, cognitive impairment in sleep-restricted, AβOinjected mice was prevented by the TNF-α neutralizing monoclonal antibody, infliximab. 2. Methods 2.1. Animals Experiments were performed in 8-10 week-old male Swiss mice. Animals were housed in groups of five per cage with free access to food and water, under a 12h light/dark cycle with controlled room temperature (21 ± 2 ºC). Mice were randomly assigned into one of three groups: control (Ctrl), paradoxical sleep deprivation for 72 h (PSD 72h), or chronic sleep restriction group (CSR). The Institutional Animal Care and Use Committee of the Federal University of Rio de Janeiro (protocol IBqM#071-05/16)

approved

all

experimental

procedures. 2.2. Paradoxical sleep deprivation and chronic sleep restriction protocols Paradoxical sleep deprivation (PSD) was performed using the multiple platforms method (Van Hulzen and Coenen, 1980). Briefly, the procedure consisted of placing eight cylindrical platforms (3.3 cm diameter x 5.0 cm high) inside a polypropylene box (45 cm x 35 cm x 17 cm) filled with water up to 1 cm below the platform surface. This arrangement allows mice to move between platforms and maintain social contact during the duration of the experiment (72 hours).

7

Chronic sleep restriction (CSR) was performed using the gentle touch method. Sleep restriction sessions lasted 3h (from 1 to 4 pm) and were performed 5 days a week (on weekdays) for 30 days. During these sessions, mice were gently disturbed with a brush every time they showed signs of sleepiness (Frank et al., 1998; Yang et al., 2014). During the entire period, animals assigned to the control group remained in the animal facility, without any stimulus, and were allowed to sleep at will. For sleep recovery (when indicated in “Results"), animals were kept in their home cages and allowed to sleep normally. Mice were then tested again in behavioral paradigms seven days later. 2.3. Assessment of sleep and wakefulness Assessment of sleep and wakefulness was performed by recording periods of activity and inactivity measured using the CLAMS system (Comprehensive Laboratory Animal Monitoring System; Columbus Instruments, Columbus, OH). Animals were placed in a Plexiglas chamber (11×31×12 cm) that has light beams positioned 0.5 inch apart on the horizontal plane, providing a high-resolution grid covering the XY-plane. Dedicated software provides counts of beam brakes by the mouse, binned in 10s epochs. Sleep and wakefulness indices were estimated as described by Pack and colleagues (2007). Mice were considered inactive if there were no beam-brakes for 40s. Two days after i.c.v. injection of AβOs, animals were placed in the CLAMS chambers for habituation, where they remained for the following 72h. Recording was performed between the 5th and 8th day after i.c.v. injections, a time frame in which we have previously shown that AβOs induce cognitive impairment and 8

hippocampal synapse loss, amongst other neuropathological features of AD (Clarke et al., 2015; Figueiredo et al., 2013; Ledo et al., 2016, 2013; Lourenco et al., 2013). 2.4. Preparation of AβOs Aβ oligomers were prepared weekly from synthetic Aβ1–42 (American Peptide, Sunnyvale, CA) (Lambert et al., 1998) and were routinely characterized by size-exclusion chromatography. Occasionally, preparations were also characterized by western immunoblots, as previously described (De Felice et al., 2008, 2007; Jürgensen et al., 2011; Sebollela et al., 2012). Oligomers were kept at 4 °C and were used within 48 h of preparation. 2.5. Intracerebroventricular (i.c.v.) injections For i.c.v. injections, animals were anesthetized for 7 min with 2.5% isoflurane (Cristália, Itapira, Brazil) using a vaporizer system, and were gently restrained only during the injection procedure itself, as described (Figueiredo et al., 2013; Ledo et al., 2013). A 2.5 mm long needle was unilaterally inserted 1 mm to the right of the midline point equidistant from each eye and 1 mm posterior to a line drawn through the anterior base of the eye. AβOs (1, 10 or 100 pmol, as indicated in “Results”) or Vehicle (Veh) were injected in a final volume of 3 µl, and the needle was kept in place for 30 s to avoid backflow. At the end of experiments, injection of blue dye in the same injection site used for AβOs or vehicle was employed to verify the accuracy of injection into the lateral ventricle. Mice showing any signs of misplaced injections or brain hemorrhage were excluded from further analysis. 2.6. Treatment with Infliximab 9

Mice received daily intraperitoneal injections of infliximab (20 mg/day) or vehicle, starting 5 days before the end of the chronic sleep restriction protocol and continuing until mice were euthanized (Lourenco et al., 2013). 2.7. Behavioral Tests 2.7.1. Open Field Mice were placed at the center of the open field apparatus and their activity was recorded during 5 min. The open field apparatus consisted of a 30 cm × 30 cm × 45cm chamber with the floor divided by lines into nine equal rectangles. The total distance travelled and number of lines crossed were automatically quantified using Any-maze® video-tracking system (Stoelting Inc., Kiel, WI, USA). The arena was cleaned with 20% ethanol between trials to eliminate olfactory cues. All open field sessions were performed during the light phase of the cycle (8 am – 5 pm). 2.7.2. Novel object recognition test (NOR) The novel object recognition test was performed in an open field arena where objects were fixed to the box using tape, as described (Figueiredo et al., 2013). During training and test sessions, animals were placed at the center of the arena and exploratory behavior towards both objects was recorded for 5 min. The arena was cleaned with 20% ethanol between trials to eliminate olfactory cues. The training session was performed in the presence of two identical objects. For the test session, carried out one and a half hour after training, one of the two objects used in the training session was replaced by a novel object. Sniffing and touching the object were considered exploratory behavior, and the amount of time spent exploring each object was recorded by 10

a trained researcher. Sessions were always performed during the light phase of the cycle (8 am – 5 pm). Open field tests were performed before training, and no significant difference in total number of crossings and total distance travelled during session were observed between groups. These results indicate that pharmacological treatments or sleep-disturbance protocols did not affect locomotor/exploratory behaviors. Moreover, animals did not show any preference for objects used during training sessions and total exploration times were comparable between groups in every experiment (Suppl. Figs. 1, 2 and 3). 2.7.3. Contextual Fear Conditioning (CFC) The conditioning chamber (25 x 25 x 25 cm) was made of aluminum walls, a methacrylate door and a grid floor with stainless steel bars connected to a shock generator (Panlab®, Harvard Apparatus, Cornellà, Spain). During training sessions, mice were allowed to freely explore the conditioning box for 3 min, and then received 2 footshocks (0.35 mA shock for 2 s, with 30s interval). Animals remained in the chamber for 30 s after the last shock, and were placed back in their home cages. After 24h, animals were again placed in the conditioning chamber for a 5 min-long test session. Freezing behavior was automatically quantified using the Freezing® software version 1.3.04 (Panlab®, Harvard Apparatus). Sessions were always performed during the light phase of the cycle (8 am – 5 pm). 2.8. Tissue collection and preparation Mice were intraperitoneally anaesthetized with 1.5 ml/kg of a solution containing 10% ketamine and 2% xylazine immediately after chronic sleep 11

restriction or after behavioral tests. Bilateral hippocampi and cortex of control and CSR animals were collected, immediately frozen in liquid nitrogen and stored at -80 ºC until use in ELISA or Western blotting assays. Blood samples were collected from the abdominal aorta with a heparinized syringe connected to a 26 G needle. Samples were spun immediately (15,000 rpm for 10 min at 4 °C) and plasma was stored at -80 °C until use. Tissues used for analyses of gene expression were immediately frozen in liquid nitrogen and stored at -80 ºC until the qPCR experiment. 2.9. Measurement of hippocampal cytokine levels Hippocampi were homogenized in ice-cold PBS with protease and phosphatase inhibitors and centrifuged for 10 min at 15,000 rpm (4 ºC). Supernatants were collected and assayed in duplicate by ELISA for each of the following cytokines: IL-1β (Thermo Scientific, Rockford, IL), TNF-α (Biolegend, San Diego, CA) and interleukin-6 (IL-6; R&D Systems, Minneapolis, MN), following manufacturer’s instructions. 2.10. Corticosterone measurements Plasma levels of corticosterone were measured using an ELISA kit following manufacturer’s instructions (Corticosterone (Human, Rat, Mouse) ELISA; IBL International, Hamburg, Germany). 2.11. Western immunoblotting Samples were thawed and homogenized in 25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP‐40 (Invitrogen), 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1% Triton X‐100 and phosphatase and protease inhibitor cocktail 12

(Pierce–Thermo

Scientific,

Rockford,

IL).

Protein

concentrations

were

determined using the BCA kit (Pierce–Thermo Scientific, Rockford, IL), and samples containing 30 µg of protein were resolved in 4–20% polyacrylamide Tris-glycine gels (Novex; Invitrogen, Grand Island, NY) and electrotransferred to nitrocellulose membranes at 300 mA for 1 h. Blots were incubated with Odyssey® blocking buffers (Li-Cor; Lincoln, Nebraska) at room temperature for 1 h and incubated with primary antibody diluted in blocking buffer at 4 °C overnight. Primary antibodies used were anti-PSD-95 (1:1,000; Santa Cruz Biotechnology, Inc., Dallas, Texas), anti-synaptophysin (1:1,000; Sigma, St Louis, MO), anti-β-tubulin (1:10,000; Abcam, Cambridge, MA) and anticyclophilin (1:10,000; Abcam, Cambridge, MA). Membranes were then incubated with IRDye secondary antibodies (1:10,000; Li-Cor) at room temperature for 1 h, imaged on an Odyssey Imaging System (Li-Cor) and analyzed using NIH Image J. 2.12. Hippocampal gene expression analysis Hippocampal RNA extraction was performed using an RNA isolation kit (Promega, Fitchburg, WI) following manufacturer’s instructions. Purity and integrity of RNA were determined by the 260/280 and 260/230 nm absorbance ratios and by agarose gel electrophoresis. Only preparations with ratios >1.8 and no signs of RNA degradation were used. One µg total RNA was used for cDNA synthesis using the SuperStrand III Reverse Transcriptase kit (Invitrogen; Carlsbad, CA). Expression of genes of interest was analyzed by qPCR on an Applied Biosystems 7500 RT–PCR system using the Power SYBR kit (Applied Biosystems; Foster City, CA). Actin was used as an endogenous control. Cycle 13

threshold (Ct) values were used to calculate fold changes in gene expression (Livak and Schmittgen, 2001). 2.13. Statistical analysis All datasets were submitted to the Shapiro-Wilk normality test. Specific statistical analyses employed are mentioned in Figure Legends. Briefly, datasets showing normal distribution were analyzed by Student’s t-test or ANOVA followed by Dunnet's post-test, as appropriate. Non-parametric data were analyzed using the Kruskal-Wallis test. Effect sizes were calculated using g of Hedges (Hedges, 1981). Data from the novel object recognition task were analyzed by one-sample t-test, compared against the fixed value of 50% (as previously described; Figueiredo et al., 2013). All analyses were performed using GraphPad Prism 6 (GraphPad Software; La Jolla, CA). Values are expressed as means ± SEM. t- and F-values, as well as confidence levels and effect sizes are described throughout Results. Criteria for animal exclusion were the following: mice that did not explore objects used in the NOR test; animals showing any signs of sickness or stress; misplaced i.c.v. injection site or brain hemorrhage; RNA samples showing any signs of degradations or 260/280 nm absorbance ratios < 1.8.

3. Results 3.1. AβOs disrupt sleep pattern in mice We recently demonstrated that intracerebroventricular (i.c.v.) infusion of AβOs in mice induces memory impairment, depressive-like behavior and 14

neuropathological features of AD (Lourenco et al., 2013; Figueiredo et al., 2013; Ledo et al., 2013; Clarke et al., 2015; Ledo et al., 2016). To evaluate the impact of AβOs on circadian rhythms, we measured sleep and wakefulness patterns in i.c.v.-infused mice over a three-day recording period in the CLAMS system starting on the 5th day after oligomer administration (Fig. 1A). The rationale for recording activity between 5 and 8 days post-injection was based on previous work from our group (Figueiredo et al., 2013; Lourenço et al., 2013; Clarke et al., 2015; Ledo et al., 2013) which showed that several behavioral and biochemical effects of AβOs are detected in this time window after i.c.v. injection. Results showed that all experimental groups exhibited bimodal activity profiles, with sleep behavior prevailing during the light phase of the dark/light cycle (Fig. 1B). Compared to vehicle-infused mice, no differences in sleep pattern were found in mice infused 10 pmol AβOs (Fig. 1B, D). However, mice infused 100 pmol AβOs spent less time asleep during the three-day recording period (Fig. 1B, D; F(2,29) = 7.212; p = 0.0029; g = 1.15). The decrease in sleep time induced by AβOs was significant during the light phase of the cycle (Fig. 1E; F(2,29) = 7.587; p = 0.0022; g = 1.26), whereas a trend of decrease was noted in the dark phase

(Fig. 1F; F(2,29) = 4.500; ANOVA p-value=

0.0199; Dunnet-adjusted p-value = 0.0622, g = 0.87). Total locomotion during the three-day recording period did not change between experimental groups (Fig. 1C, G; K(2,29) = 4.599; p = 0.1003).

3.2. Chronic sleep restriction induces memory impairment, hippocampal synapse damage and inflammation in mice 15

It is increasingly thought that poor lifestyle habits cumulatively contribute to brain susceptibility to the onset and progression of AD (De Felice, 2013). To determine the impact of sleep restriction on memory, we initially subjected mice to a 72-h paradoxical sleep deprivation (PSD) protocol (as described in “Methods”). Control measurements showed that the 72-h PSD protocol had no effect on motor/exploratory behavior in mice (Suppl. Fig. 1A, B; crossings: t(1,15) = 0.2900, p =0.7758; distance travelled: t(1,15) = 1.014, p = 0.3266). Memory performance was then assessed in both the contextual fear conditioning

paradigm

(CFC;

a

test

that

is

dependent

on

hippocampus/amygdala circuits) and the novel object recognition paradigm (NOR; a test that is dependent on cortex/hippocampus circuits) (Fig. 2A). Compared to control animals, mice subjected to 72-h PSD showed reduced freezing behavior when tested in the CFC task immediately after sleep deprivation (Fig. 2B; t(1,13) = 3.204; p = 0.0069; g = 1.56). Moreover, while control animals exhibited a clear preference for the novel object in the NOR test session [t(1,9) = 4.443; p = 0.0016], PSD mice failed to recognize the familiar object as such and spent comparable amounts of time exploring familiar and novel objects (Fig. 2C; t(1,6) = 0.4683; p = 0.6561). Furthermore, the PSD protocol induced a significant increase in plasma levels of corticosterone (Suppl. Fig. 1C; t(1,14) = 3.471; p = 0.0037; g = 1.64), an indicator of stress. We next subjected a separate group of mice to a chronic sleep restriction (CSR) protocol for 1 month (see “Methods”). The CSR protocol had no effect on motor/exploratory behavior (Suppl. Fig. 1D and E; crossings: t(1,15) = 0.1761, p = 0.8626; distance traveled: t(1,15) = 0.4947, p = 0.6280). Further, the CSR protocol caused no changes in plasma levels of corticosterone (Suppl. Fig. 1F; 16

t(1,14) = 0.9676; p = 0.3497), indicating lack of a significant stress response. Similar to the result obtained using the PSD protocol, CSR caused a decrease in freezing behavior of mice in the CFC test (Fig. 2D; t(1,13) = 2.890; p = 0.0127; g = 1.41). However, performance in the NOR test was normal in animals subjected to the CSR protocol (t(1,9) = 5.429; p= 0.0004 for CSRsubjected mice; t(1,6) = 2.829; p = 0.0300 for control mice) (Fig. 2E). Collectively, these results demonstrate that the PSD protocol per se had a negative impact on memory measured by both CFC and NOR tests. Moreover, the PSD protocol caused a significant increase in plasma levels of corticosterone, a marker of stress, which could interfere with subsequent behavioral analyses. Thus, using the PSD protocol would introduce a potential confound in the examination of the combined effects of i.c.v. injection of AβOs and sleep deprivation in mice. In contrast, the CSR protocol caused no increase in circulating corticosterone levels, and only affected performance in the CFC test, but not in the NOR memory test. Therefore, in subsequent experiments we used the CSR protocol followed by the NOR test to address the impact of AβOs on memory in the absence of potential confounds related to the sleep restriction protocol. Use of the CSR protocol further ruled out or greatly diminished any potential contribution of stress in memory assessments. To determine biochemical/signaling alterations that could underlie memory impairment induced by CSR, we analyzed hippocampal and frontal cortex levels of pre- and post-synaptic markers, as well as inflammatory mediators in the hippocampus of mice subjected to sleep restriction. For biochemical analyses, animals were euthanized immediately after the last CSR 17

session, without being subjected to any behavioral assessment that could modulate cytokine levels. Levels of synaptophysin (Syp) and PSD-95 (pre- and post-synaptic

markers,

respectively)

were

significantly

lower

in

the

hippocampus (Fig. 2F,G; Syp: t(1,14) = 2.743, p = 0.0159; PSD-95: t(1,14) = 2.652, p = 0.0190), but not in the frontal cortex (Fig. 2H,I; Syp: t(1,14) = 0.1033, p = 0.9192; PSD-95: t(1,14) = 0.7719, p = 0.4530) of chronically sleep-restricted mice. Results thus showed that the CSR protocol caused a reduction in hippocampal levels of synaptophysin and PSD-95, suggesting that CSR impacts synapse structure and density in the hippocampus. Although this damage per se did not impair performance in the NOR test, we hypothesized that it could suffice to poise the system and make it more vulnerable to the impact of AβOs, as described below. Hippocampal expression (mRNA levels) of the pro-inflammatory cytokines, IL-1β [t(1,18) = 2.463; p = 0.0120; g = 1.06] and IL-6 [t(1,16) = 2.560; p = 0.0105; g = 1.15], but not of TNF-α [t(1,17) = 0.4734; p = 0.3210], were markedly increased in sleep-restricted mice (Fig. 2J-L). However, when hippocampal protein levels were measured by ELISA, no relevant changes in IL-1β [t(1,18) = 3.672; p = 0.0017; g = 1.46], IL-6 [t(1,18) = 0.2644; p= 0.795] or TNF-α [t(1,18) = 1.933; p = 0.0691] were detected in CSR mice compared to control animals (Figure 2 M-O).

3.3. Chronic sleep restriction potentiates the impact of AβOs on memory

18

We next asked whether sleep-restricted mice would be differentially susceptible to the impact of AβOs on memory. To this end, male Swiss mice subjected to CSR for 30 days received a single i.c.v. infusion of either 1 or 10 pmol AβOs. Control measurements showed that neither dose of AβOs had any effect on locomotor/exploratory behavior in mice (Suppl. Fig. 2A,B; crossings: F(5,51) = 0.4500, p = 0.8113; distance travelled: F(5,51) = 0.4802, p = 0.7894). The two doses of AβOs used were chosen based on our previous report (Figueiredo et al., 2013) that i.c.v. infusion of 1 pmol AβOs does not cause memory impairment in mice (hence, a dose we refer to as “sub-toxic”), whereas infusion of 10 pmol AβOs causes rapid and persistent memory impairment, as confirmed here (Fig. 3B, white bars; Ctrl-Veh: t(1,6) = 2.829, p = 0.0300; CtrlAβOs1pmol: t(1,9) = 6.512, p = 0.0001; Ctrl-AβOs10pmol: t(1,9) = 2.545, p = 0.0315). Interestingly,

sleep-restricted

mice

were

susceptible

to

memory

impairment by a low dose (1 pmol) of AβOs (Fig. 3B, grey bars; CSR-Veh: t(1,9) = 5.429, p = 0.0004; CSR-AβOs1pmol: t(1,9) = 1.306, p = 0.2238; CSRAβOs10pmol: t(1,9) = 0.44361, p = 0.6731). NOR memory impairment induced by 1 pmol AβOs in CSR mice persisted even when mice were allowed to recover sleep for seven days at the end of the restriction protocol (Fig. 3C, grey bars; Ctrl-Veh: t(1,6) = 6.397, p = 0.0007; Ctrl-AβOs1pmol: t(1,9) = 7.967, p = 0.0001; Ctrl-AβOs10pmol: t(1,9) = 0.3359, p = 0.7446; CSR-Veh: t(1,8) = 5.167, p = 0.0009; CSR-AβOs1pmol: t(1,9) = 1.083, p = 0.3071; CSR-AβOs10pmol: t(1,8) = 1.392, p = 0.2013).

19

We then asked whether this persistent memory impairment was accompanied by a hippocampal inflammatory response to low doses of AβOs (1pmol) in CSR mice. While levels of IL-1β were comparable amongst all experimental groups (Fig. 3D; F(5, 49) = 1.087; p = 0.3796), hippocampal IL6 levels were significantly decreased (Fig. 3E; [F (5, 49) = 9.573; p = 0.0001; g Ctrl-Veh X CSR-AβOs1pmol = 2.32; g Ctrl-Veh X CSR-AβOs10pmol = 2.64), while TNF-α levels were increased (Fig. 3F; [F (5, 49) = 5.737; p = 0.0003; g Ctrl-Veh X CSR-AβOs1pmol = 1.37; g Ctrl-Veh X CSR-AβOs10pmol = 1.99]) in AβO-infused mice that had been previously submitted to chronic sleep restriction.

3.4. Infliximab prevented memory impairment induced by AβOs in sleeprestricted mice Finally,

we

examined

whether

infliximab,

a

TNF-α-neutralizing

monoclonal antibody, would prevent memory impairment induced by 1 pmol AβOs in sleep-restricted mice. Control measurements showed that infliximab had no effect on locomotor activity in mice (Suppl. Fig. 3A,B; crossings: F(7, 122) = 1.295, p = 0.2585; distance travelled: F(7, 122) = 0.7745, p = 0.6098). Interestingly, mice previously subjected to CSR for 30 days and given daily injections of infliximab for 5 days (20 mg/kg, i.p. during the last 5 days of CSR) exhibited normal performance in the NOR memory test when infused 1 pmol AβOs (Fig. 4B), in sharp contrast with CSR mice infused with 1 pmol AβOs but not treated with infliximab [Ctrl-Veh: t(1,18) = 2.448, p = 0.0248; CtrlAβOs1pmol: t(1,16) = 4.411, p=0.0004; Ctrl-AβOs10pmol: t(1,19) = 0.3377, p = 20

0.7393; CSR-Veh: t(1,17) = 3.933, p = 0.0011; CSR-AβOs1pmol: t(1,16) = 0.9070, p = 0.3798; CSR-AβOs10pmol: t(1,19) = 0.1602, p = 0.8744; CtrlAβOs1pmol-Inflix: t(1,9) = 4.407, p = 0.0017; CSR-AβOs1pmol-Inflix t(1,8) = 7.784, p = 0.0001]. We then asked whether the prevention of memory impairment by infliximab in CSR-AβO-injected mice was accompanied by hippocampal or plasma changes in the levels of TNF-α. Somewhat surprisingly, hippocampal and plasma levels of TNFα were not reduced to control levels in infliximabtreated groups (Fig. 4C,D; hippocampus [F (7, 64) = 7.341, p = 0.0001, g CtrlVeh X CSR-AβOs1pmol = 2.52; g Ctrl-Veh X CSR-AβOs10pmol = 1.41; g = Ctrl-Veh x Ctrl-AβOs1pmol-Inflix = 2.11; g Ctrl-Veh X CSR-AβOs1pmol-Inflix = 2.77]; plasma [F (7, 68) = 22.52; p = 0.0001, g Ctrl-Veh X CSR-AβOs1pmol = 2.93; g = Ctrl-Veh x Ctrl-AβOs1pmol-Inflix = 3.52; g Ctrl-Veh X CSRAβOs1pmol-Inflix = 2.69]).

21

4. Discussion Even though memory loss is a hallmark of AD, patients exhibit a broader group of symptoms ranging from anxiety and depression to sleep and circadian rhythm disturbances. Robust clinical/epidemiological data show that sleep disorders, including fragmented sleep, decreased slow-wave sleep, shorter latency for the first REM sleep episode and increased daytime sleepiness are present in one third of AD patients (Bliwise et al., 1989; McCurry et al., 1999; Moran et al., 2005). Here, we show that soluble oligomers of the amyloid-β peptide, toxins that accumulate in AD brains and are thought to instigate synapse failure and memory loss (Ferreira et al., 2015; Selkoe and Hardy, 2016), disrupt the sleep/wakefulness pattern when injected into the lateral brain ventricle of mice. We have previously shown that a single i.c.v. infusion of 10 pmol AβOs in mice causes pathological and behavioral outcomes that recapitulate AD, including synapse loss, memory impairment and depressive-like behavior (Figueiredo et al., 2013; Ledo et al., 2016, 2013). Current results show that sleep disturbances were only seen upon i.c.v. injection of a dose of AβOs 10 times higher (100 pmol) than the dose we have previously shown to be sufficient to cause cognitive impairment (10 pmol). Previous work from our group has shown that i.c.v.-infused

AβOs

preferentially

target

memory-related

regions

(e.g.,

hippocampus and cortex) over brain regions implicated in control of sleep behavior, such as the hypothalamus (Forny-Germano et al., 2014). It is possible, therefore, that the differential targeting of distinct brain structures by

22

AβOs accounts for the discrepancy in oligomer doses that affect memory and sleep in mice. Sleep disturbances and impaired circadian rhythm have been reported in several transgenic mouse models of AD (Ambrée et al., 2006; Bedrosian et al., 2011; Duncan et al., 2012; Kaushal et al., 2012; Platt et al., 2011; Roh et al., 2012; Sterniczuk et al., 2010). Current findings suggest that Aβ oligomer accumulation could underlie sleep disturbances in those transgenic models. However, we note that a limitation of our study concerns the fact that we did not perform EEG measurements, and so we are not able to make direct claims concerning the effects of AβOs on duration or frequency of sleep stages, or the most affected sleep wave frequency. This question remains to be addressed in future studies. We further investigated whether sleep restriction in mice could increase susceptibility to AβO-induced memory impairment. We initially established a protocol for paradoxical sleep deprivation (PSD) in mice and found that sleepdeprived mice showed impaired memory formation in two different paradigms. Those effects were accompanied by increased plasma levels of corticosterone, suggesting that this sleep deprivation protocol was accompanied by development of a stress response that could interfere with subsequent behavioral assessments. We then established a chronic sleep restriction protocol (CSR), whereby mice were kept awake for 3 hours daily (during weekdays) during the light phase cycle. Using this chronic protocol, we found that mice failed in a fearrelated memory task, but had normal performance in the object recognition 23

memory test. Moreover, this experimental protocol did not appear to induce significant stress, as demonstrated by the fact that sleep-restricted mice had plasma corticosterone levels comparable to those seen in control mice. We therefore chose to further investigate the interplay between sleep restriction using the CSR protocol and AβO-induced memory impairment using the NOR task. The task-selective memory impairment in CSR mice may likely be explained by the fact that fear and object recognition memories involve distinct brain structures (LeDoux, 2000). Whereas fear memory is amygdala- and hippocampus-dependent (Anagnostaras et al., 2001; Ehrlich et al., 2009; Radulovic and Tronson, 2010), the hippocampus and adjacent cortical areas, including entorhinal, perirhinal, and parahippocampal cortices, are crucial for recognition memory (Baxter, 2010). To determine whether CSR differentially affected distinct brain areas, we examined levels of synaptic proteins in the hippocampus and frontal cortex of sleep-restricted and control mice. Levels of synaptophysin and PSD-95, considered useful readouts of synapse density and loss (Kornau et al., 1995; Sze et al., 1997), were significantly decreased in the hippocampus, but not in the frontal cortex, of CSR mice. Therefore, the fear memory impairment seen in chronically sleep-restricted mice could be explained by reduced synaptic density in the hippocampus. On the other hand, the lack of CSR-induced synapse loss in the frontal cortex likely accounts for the lack of impact on object recognition memory in these mice. Our findings are in agreement with previous studies showing a decrease in density of synaptic proteins in the hippocampi of rats subjected to PSD for 48h (Wadhwa et al.,

24

2015) and in young rats submitted to sleep deprivation for 4 h during 3 consecutive days (Lopez et al., 2008). Besides alterations in synaptic proteins, previous studies

have

demonstrated that sleep deprivation disturbs physiological neuronal function via additional mechanisms (Buzsáki, 1998; Cedernaes et al., 2015; McDermott et al., 2003; Musiek and Holtzman, 2016). Particularly in the context of AD, production of inflammatory cytokines could be a link explaining the bidirectional relationship between dementia and sleep disturbances (Ju et al., 2014). Previous studies have shown that sleep deprivation increases IL-1β in the mouse brain, whereas TNF-α levels varied depending upon the study (Weil et al., 2009; Zielinski et al., 2014). Although most studies agree on the involvement of IL-6, IL-1β and TNF-α on sleep deprivation-mediated brain inflammation, there is substantial variation in findings depending on sleep disruption protocols used (Mullington et al., 2010). We hypothesized that memory impairment and synapse damage associated with chronic sleep restriction might be accompanied by changes in brain levels of pro-inflammatory cytokines, particularly TNF-α, IL1-β and IL-6. We then evaluated hippocampal levels of these mediators immediately after CSR. IL1-β and IL-6 expression (mRNA levels) were indeed increased in the hippocampi of CSR mice, with no relevant changes in protein levels seen for any of these cytokines. Previous studies have found similar discrepancies between mRNA and protein levels of IL-1β and other cytokines (Gruol, 2016; Gruol et al., 2014). Several conditions associated with elevated inflammatory state have been shown to increase susceptibility to AD (De Felice, 2013; Ferreira et al., 25

2014). We thus investigated the possibility that an increased pro-inflammatory status induced by chronic sleep restriction might increase susceptibility to memory impairment induced by 1 pmol AβOs, a “sub-toxic” dose we have previously shown not cause memory impairment per se (Figueiredo et al., 2013). Surprisingly, we found that 1 pmol AβOs induced memory impairment and increased hippocampal levels of TNF-α in CSR mice, but not in mice allowed to sleep undisturbed. In contrast, hippocampal levels of IL-6 were decreased following AβO infusion in CSR mice. More studies will be necessary to fully understand the implications of this biological effect, which could be a result of the complex kinetics and interactions between pro-inflammatory cytokines (Krueger, 2008; Medzhitov, 2010).

Current findings suggest that

chronic sleep restriction leads to a pro-inflammatory brain profile, and increased susceptibility to memory impairment induced by Aβ oligomers. Lastly, we hypothesized that the increase in hippocampal TNF-α levels in AβO-infused CSR mice could account for impaired recognition memory in mice given a sub-toxic dose of AβOs. To test this possibility, we used infliximab, a TNF-α neutralizing monoclonal antibody, as a pharmacological tool to determine whether increased brain levels of TNF-α were linked to AβO-induced cognitive impairment. We found that TNF-α blockade prevented memory loss induced by a low dose (1 pmol) of AβOs in chronically sleep-restricted mice. Results further showed that the biological activity of infliximab in preventing AβO-induced cognitive impairment in sleep-restricted mice was not accompanied by a reduction in TNF-α levels, either in the hippocampus or in plasma. While this could, at first glance, appear counter-intuitive, we note that previous studies have shown that the clinical actions of infliximab are not always accompanied 26

by reduced TNF-α levels (Chen et al., 2013; Kim et al., 2016). In fact, in some cases an increase in plasma levels of TNF-α has been reported following longterm treatment in humans (Takeshita et al., 2015). Collectively, those studies show that clinical improvement was seen independently of TNF-α reduction, and suggest that the beneficial actions of infliximab may be related to blockade of the interaction of TNF-α with its receptors. In addition, previous reports have shown that binding by infliximab could further stabilize TNF-α, increasing its circulating half-life (Scallon et al., 2002), an effect that could also explain the lack of reduction in TNF-α levels we now report. Finally, it is also possible that infliximab and the anti-TNF-α antibody employed in the commercial ELISA kit target distinct epitopes or binding sites in the TNF-α molecule. This could explain why TNF-α detection continues even when it is bound to infliximab, and therefore unable to exert its biological effect through binding to its receptor. In interpreting our results, some limitations should be kept in mind. First, while our study was performed only with male mice, sex appears to interact with age to alter the risk of dementia (Pike, 2017; Podcasy and Epperson, 2016). Second, sleep disturbances become more frequent with aging (Nowakowski et al., 2015; Ohayon et al., 2004) and this could reduce the brain’s ability to remove toxins, such as Aβ, and to maintain neuroprotective balance. Therefore, further experiments performed in aged mice, and especially in aged transgenic AD mice, could potentially contribute to further our understanding of sleep disturbances in AD patients. On the other hand, aging itself is accompanied by sleep disturbances and by increased levels of pro-inflammatory markers 27

(Brüünsgaard and Pedersen, 2003; Dinarello, 1998; Maggio et al., 2006), which could represent potential confounding factors in the assessment of the impact of Aβ oligomers in sleep-deprived animals.

5. Conclusions Our results reinforce a dual relationship between sleep and AD, demonstrating that (1) AβOs disrupt sleep/wake pattern in a dose-dependent manner, and (2) chronic sleep restriction increases vulnerability of mice to memory impairment caused by a sub-toxic dose of AβOs. We further show that TNF-α plays an important role in oligomer-induced memory impairment following chronic sleep restriction. Our model provides a useful approach to investigate the contribution of sleep disruption to the pathogenesis of AD, as well as the role of the immune response in these conditions.

28

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Figure Legends Figure 1. Intracerebroventricular infusion of AβOs alters sleep pattern in mice. Male Swiss mice were submitted to sequential three days of habituation and three days of recording in the Comprehensive Laboratory Animal Monitoring

System

(CLAMS),

starting

two

days

after

a

single

intracerebroventricular (i.c.v.) injection of vehicle or AβOs (10 pmol or 100 pmol) (A). (B) Two-hour total sleep time and (C) total locomotor activity measured for vehicle- or AβO-injected groups during the three days of recording. The dark phase of the cycle is indicated by shaded rectangles. (D) Area under the curve of total sleep time for data shown in B. (E, F) Percentages of total time spent asleep during the light (E) and dark (F) phases of the cycle for vehicle- or AβOinjected groups, obtained from data shown in B. (G) Area under the curve of locomotor activity for vehicle- or AβO-injected mice, obtained from data shown in C. Data are expressed as means ± S.E.M. (N = 16 vehicle-injected mice, 8 mice injected with 10 pmol AβOs, 8 mice injected with 100 pmol AβOs). *p < 0.05, one-way ANOVA followed by Dunnet’s post-hoc test. Figure 2. Chronic sleep restriction induces cognitive impairment, hippocampal synapse loss and inflammation in mice. A: Male Swiss mice subjected to the paradoxical sleep deprivation protocol for 3 days (PSD) and control animals (Ctrl) were trained either in the Contextual Fear Conditioning (CFC) or in the Novel Object Recognition (NOR) paradigms one day after sleep restriction (left panel). Mice subjected to the chronic sleep restriction protocol for 30 days (CSR; see Methods) and control animals (Ctrl) were trained in CFC or NOR one day after the last sleep restriction session (right panel). B, C: PSD mice showed impaired performance in the CFC (B; N = 7 Ctrl mice, 8 PSD 43

mice) and NOR (C; N = 10 Ctrl mice, 7 PSD mice) tasks compared to control animals. D, E: CSR mice showed impaired performance in the CFC (D; N = 7 Ctrl mice, 8 CSR mice) but not in the NOR (E; N = 7 Ctrl mice, 10 CSR mice) compared to control animals. F-I: Levels of pre- and post-synaptic proteins synaptophysin and PSD-95 were measured by Western blotting in the hippocampi (F, G; N = 9 Ctrl mice, 7 CSR mice) and frontal cortex (H, I; N = 8 Ctrl mice, 8 CSR mice) and were normalized by β-tubulin (β-tub) or cyclophilin (Cyclo), used as loading controls. J-O: IL-1β (J; N = 10 Ctrl mice, 10 CSR mice), IL-6 (K; N = 9 Ctrl mice, 9 CSR mice) and TNF-α mRNA (L; N = 10 Ctrl mice, 9 CSR mice) and protein (M-O; N = 10 Ctrl, 10 CSR) levels were measured by qPCR and ELISA, respectively, in hippocampi of control (Ctrl) or CSR mice. Data are expressed as means ± S.E.M. In C and E: *p < 0.05, onesample Student’s t-test compared to the fixed value of 50%. F = familiar object; N = novel object. Other panels: *p < 0.05, Student’s t-test. Figure 3. Chronic sleep restriction increases susceptibility to memory impairment induced by a sub-toxic dose of AβOs in mice. Male Swiss mice subjected to chronic sleep restriction (CSR, grey bars) for 30 days or control animals (Ctrl, white bars) received a single i.c.v. injection of vehicle, 1 or 10 pmol AβOs on the day of the last sleep restriction session, and were trained in in the novel object recognition (NOR) test 24hs and seven days thereafter (A). CSR-mice injected with 1 pmol AβOs fail to acquire NOR memory 24hs (B; N = 7 Ctrl-Veh mice; 10 Ctrl-AβOs 1pmol mice; 10 Ctrl-AβOs 10pmol mice; 10 CSRVeh mice; 10 CSR-AβOs 1pmol mice; 10 CSR-AβOs 10pmol mice) and seven days after sleep restriction (C; N = 7 Ctrl-Veh mice; 10 Ctrl-AβOs 1pmol mice; 10 Ctrl-AβOs 10pmol mice; 9 CSR-Veh mice; 10 CSR-AβOs 1pmol mice; 9 44

CSR-AβOs 10pmol mice). Hippocampal IL-1β (D), IL-6 (E) and TNF-α (F) levels (7 Ctrl-Veh mice; 10 Ctrl-AβOs 1pmol mice; 10 Ctrl-AβOs 10pmol mice; 9 CSRVeh mice; 10 CSR- AβOs 1pmol mice; 9 CSR-AβOs 10pmol mice for all cytokines) were measured by ELISA one day after the last behavioral session. Data are expressed as means ± S.E.M. In B and C: *p < 0.05, one-sample Student’s t-test compared to the fixed value of 50%. F = familiar object; N = novel object. In E and F: *p < 0.05, one-way ANOVA followed by Dunnet’s posthoc test. Figure 4. Infliximab prevents memory impairment caused by a low dose of AβOs in chronically sleep-restricted mice. Male Swiss mice subjected to chronic sleep restriction (CSR, grey bars) for 30 days or control mice (Ctrl, white bars) received a single i.c.v. injection of vehicle, 1 pmol AβOs or 10 pmol AβOs on the day of the last sleep restriction session, and were trained in in the novel object recognition (NOR) test 24hs thereafter. When indicated, treatment with infliximab was performed during the last five days of sleep restriction (20 mg/kg, i.p., daily) until the end of the experiment (A). Infliximab rescued the performance of CSR-mice injected with 1 pmol AβOs in the NOR memory task (B; N = 19 Ctrl-Veh mice; 17 Ctrl-AβOs 1pmol mice; 20 Ctrl-AβOs 10pmol mice; 18 CSR-Veh mice; 17 CSR- AβOs 1pmol mice; 20 CSR-AβOs 10pmol mice; 10 Ctrl-AβOs 1pmol-infliximab mice; 9 CSR- AβOs 1pmol-infliximab mice). Hippocampal (C; N = 8 Ctrl-Veh mice; 10 Ctrl-AβOs 1pmol mice; 8 Ctrl-AβOs 10pmol mice; 10 CSR-Veh mice; 9 CSR- AβOs 1pmol mice; 9 CSR-AβOs 10pmol mice; 9 Ctrl-AβOs 1pmol-infliximab mice; 9 CSR-AβOs 1pmol-infliximab mice) and plasma TNF-α levels (E; N = 10 Ctrl-Veh mice; 10 Ctrl-AβOs 1pmol mice; 9 Ctrl-AβOs 10pmol mice; 10 CSR-Veh mice; 10 CSR- AβOs 1pmol mice; 45

9 CSR-AβOs 10pmol mice; 9 Ctrl-AβOs 1pmol-infliximab mice; 9 CSR- AβOs 1pmol-infliximab mice) were measured by ELISA eight days after last behavioral session. Data are expressed as means ± S.E.M. In B: *p < 0.05, one-sample Student’s t-test compared to the fixed value of 50%. In C and D: *p < 0.05, oneway ANOVA followed by Dunnet’s post-hoc test.

Supplementary Figure Legends Suppl. Fig. S1. Sleep restriction does not affect exploratory or locomotor activities in the open field test. Male Swiss mice were subjected to paradoxical sleep deprivation for 72 hours (PSD; A-C; N = 10 Ctrl mice, 7 PSD mice) or to chronic sleep restriction (CSR; D-F; N = 7 Ctrl mice, 10 CSR mice) for 30 days, and were tested in the open field one day after the last sleep restriction session. (A, D) Number of lines crossed during the open field test. (B, E) Total distance travelled during the open field test. Plasma corticosterone levels were measured immediately after the last sleep restriction session in PSD (C; N = 8 Ctrl mice; 8 PSD mice) or CSR (D; N = 8 Ctrl mice; 8 CSR mice) mice and were compared to control (Ctrl) animals. Data are expressed as means ± S.E.M. *p < 0.05, Student’s t test. Suppl. Fig. S2. I.c.v. infusion of AβOs after chronic sleep restriction does not affect exploratory or locomotor activities in the open field test. Male Swiss mice were subjected to chronic sleep restriction (CSR) for 30 days and received an i.c.v. injection of vehicle, 1 or 10 pmol AβOs 24 hours before the open field test. (A) Number of line crossings in the open field test. (B) Distance travelled in the open field test. Data are expressed as means ± S.E.M. (N = 7

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Ctrl-Veh mice; 10 Ctrl-AβOs 1pmol mice; 10 Ctrl-AβOs 10pmol mice; 10 CSRVeh mice; 10 CSR-AβOs 1pmol mice; 10 CSR-AβOs 10pmol mice). Suppl. Fig. S3. Infliximab treatment does not affect exploratory or locomotor activities in the open field test. Male Swiss mice were subjected to chronic sleep restriction (CSR) for 30 days and received a single i.c.v. injection of vehicle, 1 pmol or 10 pmol AβOs 24 hours before the novel object recognition test. Mice were pre-treated for five days with infliximab (20 mg/kg, i.p.) before i.c.v. AβO injection until the end of the experiment. (A) Number of line crossings in the open field test. (B) Distance travelled in the open field test. Data are expressed as means ± S.E.M. (N = 19 Ctrl-Veh mice; 17 Ctrl-AβOs 1pmol mice; 20 Ctrl-AβOs 10pmol mice; 18 CSR-Veh mice; 17 CSR-AβOs 1pmol mice; 20 CSR-AβOs 10pmol mice; 10 Ctrl-AβOs 1pmol-infliximab mice; 9 CSR-AβOs 1pmol-infliximab mice).

Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank Ana Claudia Rangel, Maíra Oliveira and Mariângela Viana for administrative and technical support. This work was supported by grants from National Institute for Translational Neuroscience (to STF), Conselho Nacional de Desenvolvimento Científico e Tecnológico (to STF, FGF, JRC), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (to STF, FGF and JRC), and Fundação de Amparo à Pesquisa do Estado de São Paulo (to JD, # 2014/50140-6).

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A

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Figure 1 Kincheski et al.

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Figure 2 Kincheski et al.

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Figure 3 Kincheski et al.

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Figure 4 Kincheski et al.

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