ΔFosB immunoreactivity in the rat brain in response to chronic sleep restriction

Behavioural Brain Research 322 (2017) 9–17

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Region-specific increases in FosB/FosB immunoreactivity in the rat brain in response to chronic sleep restriction Shannon Hall a , Samüel Deurveilher a , Kristin Robin Ko b , Joan Burns a , Kazue Semba a,c,d,∗ a

Department of Medical Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada School of Biomedical Engineering, Dalhousie University, Halifax, Nova Scotia, Canada Department of Psychology & Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada d Department of Psychiatry, Dalhousie University, Halifax, Nova Scotia, Canada b c

h i g h l i g h t s • • • • •

Rats were partially sleep-deprived for 99 h using motorized wheels (3 h on/1 h off). FosB/FosB immunohistochemistry was used as a marker of chronic neuronal activation. FosB/FosB was induced in several sleep/wake, autonomic, and limbic brain regions. After 6 days of recovery from sleep restriction, FosB/FosB was at control levels. FosB/FosB may play a role in the allostatic responses to chronic sleep restriction.

a r t i c l e

i n f o

Article history: Received 27 September 2016 Received in revised form 5 January 2017 Accepted 11 January 2017 Available online 12 January 2017 Keywords: Sleep deprivation Allostasis Immunohistochemistry Fos family proteins Limbic-hypothalamic areas

a b s t r a c t Using a rat model of chronic sleep restriction (CSR) featuring periodic sleep deprivation with slowly rotating wheels (3 h on/1 h off), we previously observed that 99 h of this protocol induced both homeostatic and allostatic (adaptive) changes in physiological and behavioural measures. Notably, the initial changes in sleep intensity and attention performance gradually adapted during CSR despite accumulating sleep loss. To identify brain regions involved in these responses, we used FosB/FosB immunohistochemistry as a marker of chronic neuronal activation. Adult male rats were housed in motorized activity wheels and underwent the 3/1 CSR protocol for 99 h, or 99 h followed by 6 or 12 days of recovery. Control rats were housed in home cages, locked activity wheels, or unlocked activity wheels that the animals could turn freely. Immunohistochemistry was conducted using an antibody that recognized both FosB and FosB, and 24 brain regions involved in sleep/wake, autonomic, and limbic functions were examined. The number of darkly-stained FosB/FosB-immunoreactive cells was increased immediately following 99 h of CSR in 8/24 brain regions, including the medial preoptic and perifornical lateral hypothalamic areas, dorsomedial and paraventricular hypothalamic nuclei, and paraventricular thalamic nucleus. FosB/FosB labeling was at control levels in all 8 brain areas following 6 or 12 recovery days, suggesting that most of the immunoreactivity immediately after CSR reflected FosB, the more transient marker of chronic neuronal activation. This region-specific induction of FosB/FosB following CSR may be involved in the mechanisms underlying the allostatic changes in behavioural and physiological responses to CSR. © 2017 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: ABC, avidin-biotin-horseradish peroxidase complex; ANOVA, analysis of variance; CSR, chronic sleep restriction; EC, exercise control; GABA, ␥aminobutyric acid; HC, home cage; ir, immunoreactive; LW, locked wheel; MCH, melanin-concentrating hormone; NREM, non-rapid eye movement; REC, recovery; REM, rapid eye movement; SEM, standard error of the mean; SR, sleep restricted. ∗ Corresponding author at: Department of Medical Neuroscience, Dalhousie University, 5850 College Street, PO Box 15000, Halifax, Nova Scotia B3H 4R2, Canada. E-mail address: [email protected] (K. Semba). http://dx.doi.org/10.1016/j.bbr.2017.01.024 0166-4328/© 2017 Elsevier B.V. All rights reserved.

Although sleep is a vital physiological process, chronic sleep restriction (CSR) is common in modern society due to long or irregular work hours, social and familial obligations, increased use of technology, and sleep disorders [1,2]. CSR not only impairs cognitive functions, such as attention, learning, and memory [3,4], but also has serious health consequences including increased risk of heart disease, diabetes, obesity, and immune system impairment [5,6].

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Animal models provide a useful approach to studying the physiological and behavioural effects of CSR [7–11]. To better understand the cognitive and physiological consequences of CSR and their underlying mechanisms, we developed a rat model of CSR featuring continuous cycles of sleep deprivation and sleep opportunity using programmed (3 h on, 1 h off) slow rotation of activity wheels for 4 days [12–14]. This ‘3/1’ protocol reduced total daily sleep amounts to approximately 40% of baseline levels across the 4 days, and initiated both homeostatic (increased sleep duration and intensity during sleep opportunities) and allostatic or adaptive sleep responses (decline in compensatory increases in sleep intensity during sleep opportunities and muted or even negative compensatory sleep responses during a 2-day recovery period) [12]. The 3/1 CSR protocol also resulted in changes in body temperature and heart rate, which lasted for 3–4 days following CSR [15]. The ability to perform a psychomotor vigilance task involving sustained attention was impaired initially but improved gradually [13], while forebrain levels of brain-derived neurotrophic factor were reversibly increased, during the same 4-day CSR protocol [14]. These results indicate different degrees of adaptation in the response to CSR depending on physiological and behavioural measures. The neurobiological mechanisms underlying the physiological and behavioural consequences of CSR are not well understood. In a recent study, following 5 days of CSR in rats, the density of adenosine A1 receptor in the basal forebrain, thalamus, and hippocampus was increased, while the density of ␤-adrenergic receptors in the basal forebrain was decreased; these changes persisted after 3 days of recovery following CSR [8]. Another recent study using mice showed that two measures of sleep propensity, namely, non-rapid eye movement (NREM) sleep slow wave activity and adenosine tone assayed in hippocampal slices, increased initially, but then declined over 3 days of CSR; when challenged with acute (6 h) sleep deprivation after 2 weeks following the initial CSR protocol, compensatory increases in both the sleep propensity measures remained diminished [16]. While these studies have revealed some of the molecular mechanisms underlying the long-term impairments associated with CSR, little is known about the neural network that responds to chronic sleep loss which may account for lasting alterations in sleep and other physiological processes. One way to address this question is to investigate regional patterns of chronic neuronal activation associated with CSR. The Fos family of proteins, including c-Fos and FosB, are encoded by a group of immediate early genes whose expression is rapidly induced in response to various stimuli such as sensory stimulation, drug administration, exercise, stress, and sleep deprivation [17]. While c-Fos and FosB proteins have a half-life of one to several hours, FosB, a truncated splice variant of the full length FosB protein, has a considerably longer half-life of about 8.5 days, and accumulates in response to repeated stimulation, persisting for weeks to even months following the cessation of stimulation [18]. FosB acts as a transcription factor to alter gene expression, and its particular stability and accumulation over time have led to its use as a molecular marker for chronic neuronal activation. For example, FosB levels increased in the dorsal striatum and nucleus accumbens following 2 weeks of repeated (5 days on, 2 days off) cocaine administration in rats [19]. FosB levels also increased in the frontal cortex, nucleus accumbens, and basolateral amygdala 24 h after 5 or 10 days of chronic restraint stress in rats [20]. Additionally, a brief period of social defeat stress daily for 5 days resulted in increased levels of FosB/FosB immediately following the last stress session that persisted up to 21 days in the frontal cortex and medial amygdala, and up to 14 days in the nucleus accumbens [21]. These unique temporal properties of FosB could therefore be useful for studying patterns of long-term neuronal activation of sleep-wake and other regulatory systems in response to CSR.

Towards this goal, in the current study we used an antibody selective for the N-terminus region shared by FosB and FosB, and assessed the pattern of FosB/FosB immunoreactivity in select brain regions with sleep/wake, autonomic, and limbic functions following 99 h of the 3/1 CSR protocol in rats. FosB/FosB immunoreactivity was also assessed after 6 or 12 days of recovery following CSR. We hypothesized that FosB/FosB would be expressed in brain regions involved in sleep/wake, limbic, and autonomic functions that show adaptive responses to 99 h of CSR. In light of the temporal stability of FosB relative to FosB, we predicted that FosB/FosB immunoreactivity observed immediately after 99 h of CSR would represent both FosB and FosB, and that virtually all of the immunostaining after 6 or 12 days of recovery following CSR would represent FosB. To the best of our knowledge, this is the first study to report patterns of FosB/FosB induction following CSR. 2. Materials and methods 2.1. Animals A total of 50 adult male Wistar rats (350–400 g at experiment onset; Charles River Canada, St. Constant, Quebec, Canada) were used. Animals were initially pair-housed in standard housing cages in a colony room under a 12 h:12 h light:dark cycle (lights on at 07:00 AM). Rats were given ad libitum access to food and water throughout acclimation and the experiment. All animal handling protocols followed the guidelines of the Canadian Council on Animal Care and were approved by the Dalhousie University Committee on Laboratory Animals. As this study used multiple control groups and analyzed a large number of brain regions, only male rats were used to keep it manageable. 2.2. The 3/1 CSR protocol Rats were housed individually and sleep-restricted under the 3/1 protocol using programmable, motorized activity wheels (11 cm in width, 36 cm in diameter; Model 80860, Lafayette Instrument, Lafayette, IN, USA). The activity wheels were placed inside individual melamine-coated wooden boxes, each equipped with a light and fan, under a 12 h:12 h light:dark cycle with lights on at 07:00AM as in the animal colony room. Sleep was restricted through continuous cycles of 3 h of slow (∼2.5 m/min) wheel rotation, which sleep deprived the animal housed within the wheel, followed by 1 h of no wheel rotation, which provided the animal with opportunities to sleep [12]. This 3/1 cycle was repeated for a total of 99 h to model CSR. Throughout this protocol, rats had ad libitum access to food and water via a water bottle and food hopper attached to the unmoving side wall of the wheel. 2.3. Experimental design Rats were assigned to 8 treatment groups (n = 3–9/group): a sleep restricted (SR) group underwent the 3/1 CSR protocol, starting at lights on (i.e., 7:00 AM), for a duration of 99 h; two recovery groups underwent the 3/1 CSR protocol for 99 h followed by recovery (unrestricted sleep) in locked wheels for either 6 (REC6) or 12 (REC12) days; three locked wheel control (LW, LW6, and LW12) groups were housed in locked wheels for a duration matched to the SR, REC6, and REC12 groups, respectively; an exercise control (EC) group was kept in unlocked wheels that rats could rotate freely for voluntary exercise for 99 h; and a home cage control (HC) group was housed in pairs in standard home cages in the animal colony room for 1–3.5 months before sacrifice. Prior to the start of these experimental protocols, all groups, except the HC group, underwent 5 days of habituation in activ-

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ity wheels. During this period, rats in the SR, REC6, and REC12 groups were habituated to the rotation of the activity wheels for 5–20 min per day at a speed of ∼2.5 m/min; the wheels were otherwise locked. During the same habituation period, rats in the EC group were housed in unlocked activity wheels which they could freely rotate. Body weights of rats in all groups, except the HC group, were recorded on the last day of habituation prior to the start of each experimental protocol and again immediately following completion of each respective protocol. Body weights of rats in the HC group were recorded at the time of sacrifice only.

2.4. Perfusion and tissue collection Rats were sacrificed immediately after their respective experimental protocols, at 10:00 AM (lights on at 7:00 AM). At this time point, the SR group just completed a 3 h sleep deprivation period, while the HC, LW, LW6, LW12, REC6, and REC12 groups, which had been left undisturbed, likely had spent most of their time asleep as typical of nocturnal rodents during early light phase. Rats were deeply anaesthetized (208 mg/kg ketamine, 9.6 mg/kg xylazine, and 1.8 mg/kg acepromazine; intraperitoneal injection). Once unresponsive to a hind-limb toe pinch, animals were intracardially perfused with 100 ml of phosphate-buffered saline (pH 7.4), followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. Immediately following perfusion, brains were removed and post-fixed in the same fixative solution for approximately 5 h, followed by cryoprotection in a solution of 30% sucrose in 0.1 M phosphate buffer at 4 ◦ C for 2 days. Once brains had sunk in the sucrose solution, they were cut into 40 ␮m thick coronal sections on a freezing microtome, and sections were collected in 0.05 M Tris buffered saline (pH 7.2–7.4).

2.5. Immunohistochemistry

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2.6. Microscopy and image acquisition FosB/FosB immunoreactivity was assessed in 24 brain regions with sleep/wake, autonomic, and limbic functions (see Supplementary Table S1 for respective location relative to bregma, number of sections examined, and the size and placement of analysis boxes/ovals used for each region). An experimenter (S.H.) who was blind to the experimental conditions of the brain sections conducted image acquisition and cell counting. Sections selected for cell counting for each brain region were viewed using an Axioplan MOT II microscope (Zeiss) at 100 or 200X magnification, and photographed digitally (1300 × 1030 pixels) with an AxioCam camera directly mounted on the microscope. All images were taken using the same microscope and camera settings. 2.7. Cell counts Captured images were cropped to delineate the region of interest (i.e., box or oval, Supplementary Table S1) using Photoshop (Version 12.0; Adobe System Inc., San Jose, CA). To count only ‘darkly stained’ cells [19], a threshold gray level for ‘positive’ FosB/FosB labeling was set for each image using ImageJ software (Version 1.48; NIH, Bethesda, MD) as follows: for each image, the pixel distribution was first examined to determine the peak background intensity (mode gray value), and a threshold for positive staining was set at 50% of this peak intensity. Thus, this threshold highlighted immunopositive cells that were judged visually to be dark, and took into account differences in background staining intensity between animals and sections. Stereological cell counting was not used as we were interested in relative differences between treatment conditions, and the mean cross-sectional area of FosB/FosB-immunoreactive (ir) cell nuclei (range: 20–65 ␮m2 ) did not significantly differ between groups (P = 0.062–0.80). For each region, unilateral or, for 5 regions, bilateral (see Supplementary Table S1) counts were obtained from 2 or 3 sections spaced 200 ␮m apart and averaged for each rat. 2.8. Statistics

Immunohistochemistry was performed on every fifth section, as previously described [22]. Briefly, sections were incubated with a goat anti-FosB(N) antibody (1:400; catalog #: sc-48 g; lot #: F1011; Santa Cruz Biotechnology, Dallas, TX: see below for specificity and characterization) on a shaker at room temperature overnight or at 4 ◦ C for two days. Sections were then rinsed and incubated with a biotinylated donkey anti-goat IgG secondary antibody (1:1000; Jackson Laboratories, West Grove, PA) for 1 h. Sections were rinsed and incubated in avidin-biotin-horseradish peroxidase complex (1:200; ABC Elite PK-6100; Vector Laboratories, Burlingame, CA) for 1 h. After rinsing, sections were placed in a 0.02% diaminobenzidine solution with 0.6% nickel ammonium sulphate, followed by the addition of 0.006% hydrogen peroxide to produce a black/purple staining. Sections were then mounted on gelatin-coated slides, dehydrated, and coverslipped. The anti-FosB(N) antibody used in this study was raised against a recombinant protein corresponding to amino acids 102–117 within an internal region of mouse FosB protein that is common to both FosB and FosB [23]. Western blot analysis using homogenates of rat frontal cortex confirmed that this antibody recognized the full-length FosB protein (46–48 kDa), FosB isoforms (35–37 kDa), and 2FosB (24 kDa) (Supplementary Fig. S1). In addition, the anti-FosB(N) antibody was incubated with 5× and 20× (by weight) excess of blocking peptide (sc-38 g P; Santa Cruz), and the preadsorption of the antibody resulted in no immunopositive staining (data not shown). As the antibody recognizes both FosB and FosB, we describe FosB(N) immunostaining as FosB/FosB immunoreactivity.

GraphPad Prism 6 (GraphPad Software, Inc.; La Jolla, CA) was used for statistical analyses. For each brain region, one-way analysis of variance (ANOVA) was used to compare average cell counts between groups, followed by Newman-Keuls post-hoc test, when applicable. For brain regions in which the data showed heterogeneity of variance, statistics were performed on log (X + 1) transformed data. Body weights were also analysed using one-way ANOVA. Probability values of less than 0.05 were considered statistically significant. Values are expressed as mean ± standard error of the mean (SEM). 3. Results The SR group (n = 8) underwent 99 h of the 3/1 CSR protocol. As non-sleep deprived controls, the HC group (n = 9) was housed in standard home cages without activity wheels, the LW group (n = 9) was housed in locked wheels, whereas the EC group (n = 9) was housed in unlocked wheels that they could rotate freely. The EC rats typically rotated the wheels during the dark (active) phase; their average daily number of wheel rotations (980 ± 431 rotations) was lower than that imposed upon the SR group (2454 rotations). The REC6 (n = 4) and REC12 (n = 3) groups underwent 99 h of CSR followed by 6 and 12 days of undisturbed recovery sleep, respectively, while the time-matched LW6 (n = 4) and LW12 (n = 5) control groups were housed in locked wheels. The number of dark-stained FosB/FosB-ir cells did not significantly differ between the LW6 and LW12 groups for any brain regions examined (all P > 0.05), or

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between the REC6 and REC12 groups (all P > 0.05); therefore, these groups were combined into LWR (n = 9) and REC (n = 7) groups, respectively. Thus, a total of 6 groups were included in the final statistical analyses for cell counting.

3.1. Region-specific increases in FosB/FosB immunoreactivity following CSR The number of darkly-stained FosB/FosB-ir cells was counted in 24 brain regions with sleep/wake, autonomic, and limbic functions. Fig. 1 shows examples of FosB/FosB-ir cells under different treatment conditions, as well as placement of counting boxes/ovals, in the paraventricular thalamic nucleus (left) and the perifornical lateral hypothalamic area (right). In 8 out of the 24 brain regions examined, the number of FosB/FosB-ir cells was significantly increased immediately after CSR, compared to the non-sleep deprived control (HC, LW, and EC) groups. These regions include the medial preoptic area, perifornical lateral hypothalamic area, dorsomedial hypothalamic nucleus, lateral periaqueductal gray, paraventricular hypothalamic and thalamic nuclei, and dorsomedial striatum (Fig. 2). Unexpectedly, in the posterior hypothalamic nucleus, the highest number of FosB/FosB-ir cells were observed in the EC group (Fig. 3). In the other 15 brain regions examined, no significant treatment group effect was found (Supplementary Table S2). Of the 8 ‘CSR-responsive’ regions, two were known to be directly involved in sleep/wake regulation. In the medial preoptic area (Fig. 2A), which contains many sleep- and wake-active neurons [24], the number of FosB/FosB-ir cells was significantly increased in the SR group compared to the non-deprived control groups (+88–309%; F5,44 = 6.98, P < 0.0001; P < 0.05 vs. HC, LW, and EC). In the perifornical lateral hypothalamic area (Fig. 2B), which is abundantly populated by wake-active orexin/hypocretin and rapid eye movement (REM) sleep-active melanin-concentrating hormone (MCH) neurons [25,26], there was a significant increase (+343%) in the SR group vs. the HC group (F5,44 = 5.44, P = 0.0006; P < 0.05 vs. HC). The other nuclei of the sleep-wake regulatory system examined, including the median and ventrolateral preoptic nuclei, ventral tuberomammillary nucleus, dorsal raphe nucleus, lateral parabrachial nucleus, and locus coeruleus [27], did not show any significant treatment group differences in FosB/FosB labeling (Supplementary Table S2). CSR also had an effect in two regions involved in central autonomic control as well as sleep/wake and other functions. In the dorsomedial hypothalamic nucleus (Fig. 2C), the number of FosB/FosB-ir cells was significantly increased in the SR group compared to the HC (+116%) and LW (+128%) groups (F5,45 = 6.57, P = 0.0001; P < 0.05 vs. HC and LW). Likewise, for the lateral periaqueductal gray (Fig. 2D), an increase was observed in the SR group compared to the control groups (+55–199%; F5,45 = 3.76, P = 0.0062; P < 0.05 vs. HC, LW, and EC). FosB/FosB labeling was also elevated in the neuroendocrine and limbic forebrain regions in SR rats. In the paraventricular hypothalamic nucleus (Fig. 2E), a brain area critical for stress response [28], the number of FosB/FosB-ir cells was markedly higher in the SR group than the control groups (+346–510%; F5,45 = 6.21, P = 0.0002; P < 0.05 vs. HC, LW, and EC). Similarly, for the paraventicular thalamic nucleus, which is involved in habituation to chronic stress [29], there was a significant increase in FosB/FosB immunoreactivity in the SR group in both the anterior (+84–191%; F5,45 = 7.46, P < 0.0001; P < 0.05 vs. HC, LW, and EC; Fig. 2F) and posterior (+73–295%; F5,45 = 7.90, P < 0.0001; P < 0.05 vs. HC, LW, and EC; Fig. 2G) divisions. Other nuclei of the limbic system examined, such as the nucleus accumbens, anterior cingulate cortex, bed nucleus of the stria terminalis, amygdala, and dentate gyrus

of the hippocampus, did not show any significant group differences in the number of FosB/FosB-ir cells (Supplementary Table S2). Lastly, a significant effect of CSR on FosB/FosB immunoreactivity was found in the dorsomedial striatum, a brain region primarily involved in motor coordination [30]. In this region, FosB/FosB-ir cell counts were significantly higher in the SR group than in the control groups (+73–142%; F5,45 = 3.68, P = 0.0071; P < 0.05 vs. HC, LW, and EC; Fig. 2H). This increase was specific to the dorsomedial subregion; no significant changes in FosB/FosB labeling after CSR were seen in the combined dorsolateral and ventral subregions (Supplementary Table S2). After 6 or 12 days following CSR, the number of FosB/FosB-ir cells was significantly decreased, compared to immediately after CSR (P < 0.05, REC vs. SR), in all 8 CSR-responsive brain regions, excluding the dorsomedial striatum. In the latter region, a trend of a decrease was observed but it was not statistically significant (P > 0.05, REC vs. SR). The number of FosB/FosB-ir cells in the REC group was not significantly different from the values of the timematched controls in any of the 8 CSR-responsive regions (all P > 0.05 vs. LWR). Although the posterior hypothalamic nucleus (Fig. 3), a brain region involved in locomotor and autonomic functions [31] as well as wake behaviour [27], was not responsive to CSR, a significant increase in FosB/FosB immunoreactivity occurred in the EC group compared to the HC (+247%, P < 0.05) and LW (+299%, P < 0.05) groups (F3,31 = 5.76, P = 0.0030). However, no significant correlation was found between the total number of wheel rotations and the number of FosB/FosB-ir cells in the EC group for this brain region (r = 0.34, P = 0.37; n = 9).

3.2. Body weights The average percent change in body weight relative to pre-protocol values differed between the 8 treatment groups (F6,35 = 21.05, P < 0.001; Supplementary Table S3). While both the LW and EC groups gained weight over the 99 h of their respective experimental protocols (on average +7.2% and +6.3%, respectively), the SR group lost weight (on average −7.0%) after 99 h of CSR. The REC6 and REC12 groups tended to gain less weight over the 11 and 17 days of their respective protocols (+10% and +18%, respectively) than the time-matched LW controls (+21.5% and +24.4%, for the LW6 and LW12 groups, respectively), with a significant difference between the LW6 and REC6 groups (P < 0.05). There were no significant correlations between body weight change after 99 h of CSR and FosB/FosB labeling in any of the 8 CSR-responsive brain regions (r = 0.045–0.34; P = 0.41–0.92; n = 8/brain region), with the exception of the paraventricular hypothalamic nucleus. For the paraventricular hypothalamic nucleus, the greater the body weight loss after CSR, the greater the number of FosB/FosB-ir cells (r = 0.73; P = 0.041; n = 8) in the SR group.

4. Discussion We found that 99 h of the 3/1 CSR protocol resulted in increased numbers of FosB/FosB-ir cells in 8/24 brain regions examined that are known to be involved in sleep/wake, autonomic, neuroendocrine and limbic functions. When examined after 6 and 12 days of recovery following CSR, however, the number of FosB/FosB-ir cells was back to control levels in all 8 CSR-responsive regions. In the posterior hypothalamic nucleus, FosB/FosB immunoreactivity was increased following 9 days (including 5 days of habituation) of voluntary wheel running.

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Fig. 1. Examples of FosB/FosB-immunoreactive cells in the posterior paraventricular thalamic nucleus (PVP; A, C, E) and perifornical lateral hypothalamic area (PeF/LH; B, D, F), two brain regions that show increases in FosB/FosB immunoreactivity under LW (A), EC (B), SR (C, D) and REC (E, F) conditions (see Section 2.3 in Materials and Methods for further details). The box or oval used for cell counts and their placement over the PVP (black oval) and PeF/LH (box encompasses the whole image) are shown. Inset in C shows dark-stained FosB/FosB-immunoreactive neuronal nuclei (white arrows). See Supplementary Table S1 for sizes and placements of analysis box/oval for the other brain regions studied. Other abbreviations: 3 V, third ventricle; fx, fornix. Scale bars: 300 ␮m (A) and 50 ␮m (C).

4.1. Methodological considerations As the antibody we used recognized both FosB and FosB as confirmed by our Western blot analysis, the identity of the immunolabel should be discussed relative to the experimental paradigm. FosB induction peaks about 6 h after an acute stimulus, and can persist for up to 24 h [32,33]. When stimulation is repeated, it desensitizes; for example, FosB expression returned to control levels in all brain regions following 10 or 14 days of repeated restraint stress [20,34]. In contrast, FosB, due to its long half-life (∼8.5 days [18]), accumulates over many days in response to repeated stimulation and can be detected for several weeks after cessation of the stimulus [20,35]. In our study, FosB/FosB

immunostaining was no longer elevated after 6 or 12 days following CSR in any brain region examined. This absence of persistent expression suggests that most of the immunostaining observed immediately after CSR represented the full-length FosB isoform rather than FosB. It remains possible that FosB might be induced after longer periods of CSR. The use of rotating wheels as a means of sleep deprivation required the inclusion of several control groups. First, the wheel housing condition was controlled for by using the HC and LW, LW6, and LW12 groups, which were housed without sleep restriction in either standard home cages in the animal colony room for 1–3.5 months, or in locked activity wheels in the experimental room for 9–21 days. The lack of significant difference between these two

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Fig. 3. Number (means ± SEM) of FosB/FosB immunopositive cells in the posterior hypothalamus (PH) showing a significant increase in FosB/FosB immunoreactivity in the EC group. Cell counts in the HC (white, n = 9), LW (light gray, n = 9), EC (dark gray, n = 9), and SR (black, n = 8) groups are expressed per section per side of the brain. *P < 0.05, Newman-Keuls post-hoc tests.

Fig. 2. Numbers (means ± SEM) of FosB/FosB-immunoreactive cells in 8 brain regions showing significant increases in FosB/FosB immunoreactivity following chronic sleep restriction. These regions include: medial preoptic area (MPA; A), perifornical lateral hypothalamic area (PeF/LH; B), dorsomedial hypothalamic nucleus (DMH; C), lateral periaqueductal gray (LPAG; D), paraventricular hypothalamic nucleus (PVH; E), anterior paraventricular thalamic nucleus (PVA; F), posterior paraventricular thalamic nucleus (PVP; G), and dorsomedial striatum (DMS; H). No significant difference was found between REC rats that were allowed 6 or 12 days of undisturbed recovery sleep following CSR, and thus these rats were combined into a single REC group. Likewise, there was no significant difference between timematched LWR rats, so they were combined into a single LWR group. Cell counts in the HC (white, n = 9), LW (light gray, n = 9), EC (dark gray, n = 9), SR group (black, n = 8), LWR (vertical lines, n = 9), and REC (horizontal lines, n = 7) groups are expressed per section per side of the brain. *P < 0.05, Newman-Keuls post-hoc tests after one-way ANOVA.

groups in FosB/FosB immunoreactivity in any brain region examined suggests that wheel housing did not contribute to the elevated immunoreactivity in the SR group. Second, we attempted to control for the physical activity associated with wheel rotations by including the EC group which was housed in unlocked activity wheels that animals could turn freely. Unlike in our previous studies [13,14], in the present study EC rats accumulated less than half the number of wheel rotations over the 99 h period compared to those imposed on the SR group. We therefore cannot exclude the possibility that increased FosB/FosB immunostaining in the SR group may be, at least in part, due to increased physical activity. Unlike the control groups, which gained weight, the SR group lost weight (−7.0% on average) over 99 h of CSR. This result is consistent with previous studies using the 3/1 and other CSR protocols [11–14]. The weight loss in the SR animals is unlikely to be attributable to the physical activity associated with wheel rotation, as the EC group gained weight during the same period although the EC group was generally less active than the SR group in terms of wheel rotations. As reported previously using a repeated CSR protocol [36], the weight loss in the SR animals may be due to increased energy expenditure compared to energy intake, although food and water consumption was not monitored in this study. 4.2. Region-specific patterns of FosB/FosB immunoreactivity immediately following CSR Several brain regions showed an increase in the number of FosB/FosB-ir cells following 99 h of the 3/1 CSR protocol, suggesting that neurons in these brain areas had been persistently active over a prolonged period (at least for several hours [18] and most likely longer) preceding the end of the CSR protocol. Previous studies have shown region-specific increases in immediate early gene expression in the rodent brain in response to acute sleep deprivation. Increases in the number of c-Fos-ir cells was observed following either 3 or 6 h of sleep deprivation in several regions of the rat brain including those examined in the present

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study, such as the medial preoptic area, cortex, paraventricular thalamic nucleus, amygdala, and striatum [37,38]. Another study found widespread increases in c-fos and fosB mRNA expression in the mouse brain after 6 h of sleep deprivation, including the cortex, basal forebrain, thalamus, and cerebellum, which were at baseline levels following 4 h of recovery sleep [39]. These results indicate diversely distributed expression of c-Fos and c-fos/fosB mRNA following acute sleep deprivation, suggesting that neurons in this wide range of brain regions were acutely activated following a short period of sleep loss. In contrast, FosB/FosB immunoreactivity after CSR in the present study was regionally more restricted, likely reflecting the requirements of persistent neuronal activity. This elevated neuronal activity could reflect the cumulative effects of repeated sleep deprivation, repeated sleep opportunity, or both. Additionally, the last 3 h episode of sleep deprivation directly before euthanasia could have had a proximity effect. Two brain regions implicated in sleep/wake regulation, namely, the perifornical lateral hypothalamic area and medial preoptic area, showed increased FosB/FosB immunoreactivity after CSR. The perifornical lateral hypothalamic area houses orexin/hypocretin neurons, which play a key role in the arousal network [25], MCH neurons, which are REM sleep-active and promote sleep [40,41], and ␥-aminobutyric acid (GABA)-ergic neurons, at least some of which are either sleep-active [26] or wake-promoting [42]. The medial preoptic area contains sleep- as well as wake-active neurons [43,44]. It is possible, therefore, that the increases in FosB/FosB levels in the lateral hypothalamic and medial preoptic areas are associated with the activation of wake-active neurons in these regions that are involved in the maintenance of sustained wakefulness during sleep deprivation periods, as well as the activation of sleep-active neurons in these areas that are involved in the increase in sleep propensity, or increases in REM and NREM sleep amounts, during sleep opportunity periods [12]. The lateral hypothalamus is also involved in cardiovascular regulation, feeding, and energy metabolism [45,46], while the medial preoptic area has a wellestablished role in thermoregulation, which is linked to sleep/wake regulation [47]. We previously observed that the 3/1 CSR protocol causes elevation in core body temperature [15]. Therefore, it is possible that some of the FosB/FosB labeling in these regions could reflect changes in the regulation of these additional processes as a result of CSR. The dorsomedial hypothalamic nucleus also showed increased FosB/FosB labeling following CSR. This nucleus plays an important role in mediating the circadian signal to multiple sleep/wake regulatory nuclei to promote wakefulness and integrate behavioural and endocrine processes [48]. It is also involved in cardiovascular functions [49] including the cardiovascular response to stress [50]. In addition, an increase in the number of FosB/FosB-ir cells was observed after CSR in the lateral periaqueductal gray, a region mediating the somatic and autonomic components of active emotional coping to a stressor [51]. The lateral periaqueductal gray is also an important “relay station” for the descending projections from the cardiovascular-responsive dorsomedial hypothalamic nucleus [52]. We previously observed increased heart rate in rats following 4 days of the 3/1 CSR protocol [15]. Thus, the activation of neurons in the dorsomedial hypothalamic nucleus may be part of the mechanisms underlying altered sleep/wake patterns and cardiovascular responses to CSR, while the lateral periaqueductal gray may be involved in mediating emotional behaviours associated with cardiovascular responses to CSR, such as tachycardia and hypertension [15,53]. Two areas involved in the stress response, the paraventricular hypothalamic and thalamic nuclei, showed increased FosB/FosB levels after CSR. The paraventricular hypothalamic nucleus contains neurosecretory neurons involved in initiating glucocorticoid secretion through the hypothalamic-pituitary-adrenal axis [28].

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Sleep restriction for 7 days resulted in sustained increases in plasma adrenocorticotropic hormone and corticosterone levels when measured directly after sleep deprivation periods [54]. Although corticosterone levels were not measured in the present study, the increased FosB/FosB immunoreactivity in the paraventricular hypothalamic nucleus after CSR most likely reflects a level of neuroendocrine activation during the CSR protocol. Increased FosB/FosB expression was also observed in the paraventricular thalamic nucleus, which has been suggested to play a role in regulating adaptation to various types of chronic stress [29]. The FosB/FosB labeling in this nucleus after CSR may reflect processes that attempt to reduce the stress response and autonomic activation to CSR. An increase in the number of FosB/FosB-ir cells following CSR was found in the dorsomedial striatum, a region recognized as the “associative” striatum with roles in locomotion, reactivity to novelty, and early motor learning [30,55]. It is possible that neurons in this region were activated as rats in the SR group were most likely forced to learn to walk and balance inside the rotating wheels. In the posterior hypothalamic nucleus, FosB/FosB immunoreactivity was increased in the EC but not in the SR group. The posterior hypothalamic nucleus acts as a control centre for the integration of both autonomic and locomotor functions [31,56], and an increase in FosB/FosB labeling in this region in the EC group is consistent with this role. Voluntary activity appears to be necessary for increased FosB/FosB in the posterior hypothalamic nucleus, as the SR group did not show such increases despite their higher levels of physical activity compared to the EC group (see section 4.1). Consistent with this interpretation, voluntary and forced exercises are known to have different effects on cerebral metabolism [57], neurotransmitter levels [58], and neurogenesis [59]. In addition, the temporal pattern of activity cannot be excluded as a contributing factor for the differential results; the EC group was typically engaged in short bouts of wheel running with more activity during the dark phase, whereas the SR group experienced cycles of sustained physical activity around the clock. Although 99 h of CSR resulted in increased FosB/FosB immunoreactivity in several brain areas as discussed above, many regions showed no changes. These included regions involved in sleep/wake regulation (median and ventrolateral preoptic nuclei, locus coeruleus, ventral tuberomammillary nucleus, dorsal raphe and lateral parabrachial nuclei), behavioural, autonomic, and neuroendocrine responses to stress (bed nucleus of the stria terminalis and amygdala) [60,61], and reward circuitry (anterior cingulate cortex and nucleus accumbens) [62]. These results are somewhat surprising as most of these regions have been reported to express increased levels of c-Fos following acute sleep deprivation [37,39,63]. The reason for this discrepancy is unclear. One possibility is the lack of persistent neuronal activity despite initial activation. Alternatively and not exclusive to the first possibility, certain differences in function and physiology might result in some brain regions being more responsive, or vulnerable, to CSR than others. Given their role as transcription factors [18], it is likely that increased levels of FosB/FosB following CSR initiate and promote changes in the expression of other genes. The absence of fosB reduced the amount of time spent in REM sleep in mice, suggesting that FosB may be involved in the regulation of sleep, in particular REM sleep [64]. In addition to the FosB/FosB pathways, other transcription factors might be involved in the long-term effects of CSR, particularly in those brain regions without changes in FosB/FosB levels. For example, elevations in the immediate early gene NGFI-A mRNA have been observed following forced (6 h sleep deprivation) and spontaneous wakefulness in the cerebral cortex, basal forebrain, and basal ganglia [39,65]. Elevated expression of mRNA for ARC, a transcription factor implicated in activity-dependent synap-

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tic plasticity, has also been reported in the cerebral cortex following 7 days of sleep deprivation [66]. The main goal of this study was to understand the neural network that could be activated by CSR. Based on the observed pattern of FosB/FosB immunoreactivity, at the core of this network might be an antagonistic activation of sleep-promoting and wake-promoting neurons in the medial preoptic and perifornical lateral hypothalamic areas, along with projections in particular of the wake-promoting lateral hypothalamic neurons to the dorsomedial hypothalamic nucleus (as evidenced for orexin neurons [67]), then to the lateral periaqueductal gray. This network may serve to keep animals awake during sleep deprivation while integrating homeostatic responses to sleep loss with other physiological and motivational/emotional functions. The paraventricular hypothalamic nucleus, which receives projections from the lateral hypothalamus [68], most likely provides a stress-neuroendocrine component of the response, while the paraventricular thalamic nucleus, which receives lateral hypothalamic orexin projections [67], may promote physiological adaptation to CSR. The activation of the dorsomedial striatum appears to be specific to the use of rotating wheels for sleep deprivation and is likely not central to the general neural response to CSR. This model is consistent with our hypothesis that CSR would result in the activation of a neural network involved in sleep/wake, limbic, and autonomic functions that show adaptation to CSR, and suggests that CSR may initiate a systematic response in a neural network to alter its functions to maximize adaptation. 5. Conclusions In the present study, an increase in FosB/FosB immunoreactivity was observed in select sleep/wake, autonomic, and limbic brain regions following 99 h of the 3/1 CSR protocol. FosB/FosB levels were at baseline levels in these brain areas after 6 and 12 recovery days following CSR, indicating that most of the observed immunoreactivity reflects full-length FosB. Additional work involving the identification of the transmitter phenotype(s) of FosB/FosB-ir cells and their selective inactivation during CSR would help determine the specific roles of these cell populations within a network that organizes the allostatic changes in sleep regulation and other behavioural/physiological responses to CSR. Funding sources This work was supported by the Canadian Institutes of Health Research (MOP-259183) and the Dalhousie Medical Research Foundation.

[4] [5] [6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

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Acknowledgements

[23]

We thank Ms. Kay Murphy for technical assistance and Dr. William Currie for his support in our Western blot analysis.

[24]

Appendix A. Supplementary data

[26]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2017.01.024.

[27]

[25]

[28]

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