Neurobiology of Learning and Memory 123 (2015) 140–148

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Effect of castration on the susceptibility of male rats to the sleep deprivation-induced impairment of behavioral and synaptic plasticity Vahid Hajali a,⇑, Vahid Sheibani b, Hamed Ghazvini b, Tahereh Ghadiri c, Toktam Valizadeh d, Hakimeh Saadati e, Mohammad Shabani b a

Quchan Higher Health Education Center, Mashhad University of Medical Sciences, Mashhad, Iran Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran Iranian National Center of Addiction Studies, Tehran University of Medical Sciences, Tehran, Iran d Department of Statistics, School of Mathematics and Computer Sciences, Amirkabir University of Technology, Tehran, Iran e Faculty of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran b c

a r t i c l e

i n f o

Article history: Received 10 February 2015 Revised 26 May 2015 Accepted 26 May 2015 Available online 12 June 2015 Keywords: Sleep deprivation Spatial memory Long-term potentiation Brain derived neurotropic factor Gonadectomy

a b s t r a c t In both human and animal studies, the effect of sleep deficiency on cognitive performances has mostly been studied during adulthood in males, but very little data exist concerning the effects of poor sleep in gonadal hormones-depleted status, such as aging or gonadectomized (GDX) male animal models. The present study investigated the potential modulatory effects of the endogenous male sex hormones on the 48 h REM sleep deprivation (SD)-induced cognitive and synaptic impairments by comparing the gonadally intact with castrated male rats, a rodent model of androgen-deprived male animals. The multiple platform method was used for inducing REM-SD and spatial performances were evaluated using Morris water maze (MWM) task. Early long-term potentiation (E-LTP) was measured in area CA1 of the hippocampus and PCR and western blotting assays were employed to assess brain derived neurotrophic factor (BDNF) gene and protein expression in the hippocampus. To reveal any influence of sleep loss on stress level, we also evaluated the plasma corticosterone levels of animals. Regardless of reproductive status, REM-SD significantly disrupted short-term memory and LTP, as well as hippocampal BDNF expression. The corticosterone levels were not significantly changed following REM-SD neither in intact nor in GDX male rats. These findings suggest that depletion of male sex steroid hormones by castration does not lead to any heightened sensitivity of male animals to the deleterious effects of 48 h REM-SD on cognitive and synaptic performances. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction The average amount of sleep per 24 h has declined by 1.5 h over the past century, a figure that seems to continue to increase (Rajaratnam & Arendt, 2001). A great body of evidence from both human and rodent studies suggests that sleep has a remarkable role in certain types of learning and memory (Diekelmann & Born, 2010). It was primarily believed that sleep after a learning task (post-training sleep) contributes to the consolidation of new information into long-term memory (Stickgold & Walker, 2007). Abbreviations: REM, rapid eye movement; GDX, gonadectomized; SD, sleep deprivation; MWM, Morris water maze; WP, wide platform; LTP, long-term potentiation; BDNF, brain derived neurotrophic factor; fEPSPs, field excitatory post synaptic potentials; PPF, paired-pulse facilitation. ⇑ Corresponding author. Fax: +98 51462217118. E-mail addresses: [email protected], [email protected] (V. Hajali). http://dx.doi.org/10.1016/j.nlm.2015.05.008 1074-7427/Ó 2015 Elsevier Inc. All rights reserved.

Accordingly, sleep deprivation (SD) following a learning task causes subsequent memory deficits in both humans (Stickgold, James, et al., 2000) and rodents (Smith, Conway, et al., 1998). However, as post-training sleep, sleep prior to learning (pre-training sleep) can also improve memory consolidation by promoting the capability of related neuronal systems to process new information and encode new memories (Stickgold & Walker, 2007). Human studies show that total SD for a single night disrupts different kinds of subsequent memory (Van Der Werf, Altena, et al., 2009; Yoo, Hu, et al., 2007). Numerous animal studies also show that one to five days of SD before training task can lead to impairment in subsequent behavioral efficiency (Alvarenga, Patti, et al., 2008; Hagewoud, Havekes, et al., 2010; McDermott, LaHoste, et al., 2003; Ruskin & Lahoste, 2008; Ruskin, Liu, et al., 2004; Silva, Chehin, et al., 2004; Tiba, Oliveira, et al., 2008). In parallel with these alterations in cognitive performances, a lot of cellular

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and molecular correlates of membrane excitability and synaptic plasticity within the hippocampus have been shown to be affected by SD (Guzman-Marin, Ying, et al., 2006; McDermott et al., 2003; Ravassard, Pachoud, et al., 2009). The effects of sleep deficiency on cognitive functions have frequently been investigated during adulthood in both human and animal studies but very few reports exist concerning the effects of poor sleep in gonadal hormones-depleted status (eg, androgen deprivation therapy, aging, gonadectomized animal models, or postmenopausal women). The effect of sleep loss on physical (Goldman, Stone, et al., 2007) and cognitive performances (Blackwell, Yaffe, et al., 2006) of postmenopausal women has been explored, but to our knowledge, to date no study has examined the effects of poor sleep on cognitive functions of androgen-depleted status. A few available studies regarding the effects of SD in elderly subjects suggest that the aged men may be more susceptible to the negative effects of sleep loss on cognitive performances (Webb, 1985; Webb & Levy, 1982). Robust evidence suggests the potential neurotrophic and neuroprotective effects of sex steroid hormones in the nervous system (Pike, Carroll, et al., 2009; Pike, Nguyen, et al., 2008). Testosterone levels are decreased in aged men (Nankin & Calkins, 1987) and it has been shown to have protective potentials against a variety of neuronal insults, brain injuries in experimental models, as well as cognitive and neurodegenerative disorders (Bialek, Zaremba, et al., 2004; Cherrier, Matsumoto, et al., 2005a, 2005b; Roglio, Bianchi, et al., 2007). Accordingly, it is plausible that the stronger effects of SD at older age might be the consequence of lower levels of testosterone. Therefore, in light of the fact that chronic SD is a common problem in industrialized societies (National Sleep Fundation, 2005) and that age-related testosterone depletion in men may result in the increased susceptibility to the negative effects of sleep loss, it is important to fully understand the responsivity of androgen-depleted subjects to the impairing effects of SD on cognitive performances. In the present study, we compared the gonadally intact male with gonadectomized (GDX) rats to examine any potential protective effects of endogenous male sex steroid hormones against the 48 h REM-sleep deprivation-induced cognitive and synaptic modulations. Brain-derived neurotropic factor (BDNF), as the second member of neurotrophin family, has been shown to strongly affect synaptic plasticity, as well as learning and memory processes (Cunha, Brambilla, et al., 2010). Several lines of evidence suggest that BDNF is a potential mediator for the central effects of gonadal steroid hormones (Rasika, Alvarez-Buylla, et al., 1999; Scharfman & MacLusky, 2006). There has been, therefore, a long history of studies showing the extensive similarities between the actions of sex steroids and BDNF in the CNS (Scharfman & Maclusky, 2005; Yang & Arnold, 2000). Along these lines of evidence, in this study, we first aimed to compare the extent of the effects of 48 h REM-SD on cognitive functions between intact and GDX male rats in Morris water maze (MWM) and then to determine the possible cellular and molecular mechanisms responsible for REM-sleep deprivation-induced modulations of memory performances, we investigated the synaptic efficacy by measuring long-term potentiation (LTP) and BDNF expression in the hippocampus of the experimental groups.

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in groups of four in the Plexiglas cages with free access to food and water and housed in a climate-controlled room (23 °C ± 1 °C) on a 24-h light-dark cycle (lights-on 07:00–19:00 h). In this study, three sets of intact male and GDX animals were submitted to SD (narrow platform), wide platform (WP; sham groups), or maintained in home cages as controls for behavioral, electrophysiological, and molecular experiments. All experimental protocols and animal handling procedures were in accordance with the Animal Ethics Committee of Kerman Neuroscience Research Center (EC/KNRC/89/46). 2.2. Gonadectomy Bilateral gonadectomy was carried out under general anesthesia (60 mg/kg ketamine and 10 mg/kg xylazine). All the gonadectomized rats were at the same ages as the gonadally intact animals and entered in the experiments four weeks after the surgery to insure that all gonadally synthesized steroids had been cleared from circulation. 2.3. REM sleep deprivation

2. Experimental protocols

REM sleep deprivation was induced as described previously (Hajali, Sheibani, et al., 2012, 2015; Joukar, Ghorbani-Shahrbabaki, et al., 2013). In this study, animals were sleep deprived for 48 h using the multiple platforms model. It started and ended somewhere in the beginning of the light phase and the room was maintained under controlled temperature (23 ± 1 °C) and a light–dark cycle (lights on 07:00–19:00 h). The procedure involves placing 4 rats from a same group in a water tank (90 cm  50 cm  50 cm) containing 10 round platforms (7 cm diameter, 10 cm height, rising 2 cm above the water level) arranged in two lines and spaced 10 cm away from each other (edge to edge), in which the animals can move around freely by leaping from one platform to the another. Loss of the muscle tone at the beginning of each REM (paradoxical) sleep episode causes rats to touch the water, thus being awakened. To test the possible effects of the chamber atmosphere on the stress level of sleep deprived animals, we also utilized a wide platform (WP, sham group) (15 cm in diameter) version, which allowed the rats to sleep comfortably. During the SD period, the animals had free access to water bottles and chow pellets attaching from a grill located on the top of the chamber. The water in the tank was refreshed daily throughout this period. This protocol involves repeated awakenings, which predominantly, but not exclusively, affects the REM stage of sleep which has been confirmed by the electroencephalogram (EEG) recording in the previous studies (Machado, Hipolide, et al., 2004; Ravassard et al., 2009). Machado et al have shown that animals in the small platforms display 100% and 31% reduction of REM and non-REM sleep respectively, compared to the home cage control group (Machado et al., 2004). Ravassard et al reported that compared to WP group, REM sleep was suppressed by 60%, but non-REM sleep was increased by 10% in the small platforms group (Ravassard et al., 2009). As the animals can move freely within such multiplatform chambers, it has been reported that they will experience less immobilization stress compared to the single version of platform technique. Moreover, this paradigm allows rats from the same cages and groups to be deprived of sleep at the same time thus, remaining socially connected and some probable separation stress associated with the single flowerpot model is avoided. (Machado et al., 2004; Suchecki & Tufik, 2000).

2.1. Animals

2.4. Spatial learning and memory in Morris water maze (MWM)

Adult male Sprague-Dawley rats, (200–250 g, 10–12 weeks of age) were purchased from the colony maintained by Kerman Neuroscience Research Center Animal Facility. Animals were kept

The MWM was a black circular metal tank (160 cm diameter and 80 cm height) filled with 40 cm of water maintained at room temperature (21 °C ± 1 °C). The pool was geographically divided

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into four quadrants of equal size and release points were designated in each quadrant as, northeast, northwest, southeast and southwest. A square platform (10 cm diameter) was located just below (1.5 cm) the water surface in the center of the northeast quadrant. The experiments were performed in a dimly lit room in which a variety of fixed extra maze geometric images (e.g., squares, circles or triangles) were attached at different points on the walls around the maze. Animal performance was recorded by a smart video tracing system (NoldusEthovisionÒ system, version 5, USA) and it could be traced on the screen of a computer. Behavioral experiments in MWM task were performed as described earlier (Hajali et al., 2012). In a single day training protocol of MWM task, each rat accomplished three training blocks separated by 30 min interval. Each block comprised of four successive trials of 60 s duration and three 60 s inter-trial intervals. In this protocol, rats become fully trained in approximately 1.5 h which is sufficient for evaluating the effects of long-lasting treatments, such as 48 h sleep deprivation. In fact, the classic version of MWM task (5 days training protocol) is not applicable for 48 h pre-training sleep deprivation. On each trial, animal was randomly released into the water from one of the four quadrants of the maze with its face toward the wall of the quadrant where it was released. Each rat had 4 different starting points. During acquisition trials, the position of the platform was constant and rats were allowed to swim to the hidden escape platform in 60 s (maximum time). When the animal found the platform, it was allowed to remain on it for 20–30 s and was then transferred to its home cage to wait 20–30 s before the start of the next trial. Rats that failed to find the platform within 60 s were manually guided to the platform. The time and distance to find the escape platform were collected and analyzed later. A single probe trial was carried out 2 h after the last training trial to test the spatial short-term memory in the water maze. In this trial, the platform was removed, and then the rat was allowed to swim freely for 60 s. The time and distance spent in the target quadrant (northeast quadrant) were analyzed and considered as the spatial memory parameters. Following the probe trial, rats accomplished a visible platform test to reveal any possibility of sleep deprivation interference with sensory and motor coordination or motivation. In this test, the animal’s ability to escape to an apparent platform (platform was raised 2 cm above the water surface and became visible with aluminum foil) was evaluated. The animals were placed back on the SD platforms during the 2 h period between training and probe trails. The MWM tasks were conducted during the lights-on phase and were started about 15 min after the end of SD (at 8:15 am) and lasted until 11:45 am. 2.5. Electrophysiological experiments In vivo electrophysiological recording of field excitatory post synaptic potentials (fEPSPs) from CA1 region was performed as described previously (Hajali et al., 2015; Saadati, Sheibani, et al., 2014a, 2014b). A new set of six groups of intact and GDX male rats (control, WP, and SD groups in each set) was used. Animals were weighed and anesthetized with urethane injected intraperitoneally (1.2–1.4 g/kg) 10 min after the end of exposure to the narrow and wide platforms in SD and WP groups, respectively. The skull was fixed in a stereotaxic frame (nose bar at 3.3) and exposed enough to allow electrode placement. After two small holes were drilled in the skull, a concentric bipolar stimulating electrode was inserted into the ipsilateral Shaffer collateral pathway (AP: 3.00 mm; ML: 3.5 mm; DV: 2.8 mm from the skull) and a recording electrode into the stratum radiatum of area CA1 of left hippocampus (AP: 4.1 mm; ML: mm; 3.00 DV: mm 2.5 from the skull) to record the evoked fEPSPs. Both electrodes consisted of a pair of twisted stainless steel wires (0.008 in. in diameter) insulated except at the tip (A-M

System, Inc.). Stereotaxic coordinates are based on the atlas of Paxinos & Watson, 2007 (Paxinos & Watson, 2007). The depth of the electrodes was adjusted to maximize the amplitude of the evoked field potentials by infrequent stimulation (square wave pulses, 0.2 ms duration and 0.4–0.8 mA intensity). The evoked field potentials were amplified (1000), transformed by an analog/digital interface (mentioned below) and stored on a computer. After assurance of stabilization of the response for about 30 min, an input–output (I/O) curve was measured by gradually increasing the stimulus intensity (input) and recording the generated fEPSPs (output). A baseline recording was obtained by giving a test stimulus, adopted to evoke 40–50% of the maximal response, every 10 s for a duration of 20 min. Early-LTP (E-LTP) was induced by high frequency stimulation (HFS, 400 Hz, a train of 10 pulses applied every 7 s for a period of 70 s) of Shafer collateral pathway at the same stimulus intensity used for baseline recordings. Thereafter, a test stimulus at the same intensity was delivered every 10 s and continued for 120 min. Paired-pulse facilitation (PPF) with an inter-stimulus interval of 20, 50, and 100 ms was also examined before inducing LTP. All recorded responses were averaged from 10 consecutive signals at each time point and normalized to the mean value of the baseline. Computer-based stimulation and recording was achieved using Neurotrace software version 9 and Electromodule 12 (ScienceBeam Institute, Tehran, Iran). For analyzing the responses, the software Potentalise from the same institute was used. 2.6. Molecular experiments 2.6.1. Tissue dissection and preparation for Western blot and semi quantitative PCR analysis For molecular experiments, the third set of intact and GDX male rats was used and divided into control, WP and SD subgroups. Animals were decapitated under CO2 anesthesia and both whole hippocampi were rapidly dissected out on ice and frozen in liquid nitrogen. The dissected hippocampi from each rat were randomly distributed for further western blot and PCR assays and stored at 80 until homogenization. All rats were decapitated between 8.00 and 8.30 am. 2.6.2. BDNF immunoblot analysis Sample preparation for immunoblot analysis was performed as reported (Saadati et al., 2014a, 2014b). The dissected hippocampal tissues were homogenized in ice-cold buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1% SDS, 0.1% Na-deoxycholate, 1% NP-40 with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2.5 lg/ml of leupeptin, 10 lg/ml of aprotinin) and 1 mM sodium orthovanadate, using a tissue homogenizer (Silent Crusher S Homogenizer, Germany) at a medium speed for 5 s, repeated 3 to 5 times. The homogenate was centrifuged at 14,000 rpm for 15 min at 4 °C. Protein concentrations were determined using the Bradford protein assay (Bio-Rad). After adding an equal volume of 2 SDS sample buffer, the fractions were incubated at 100 °C for 5 min, and then the protein samples (10 ll per well) were separated by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto PVDF (polyvinylidene fluoride) membranes. During electrophoresis, the molecular weight was monitored with a pre-stained protein ladder (Fermentas, Life Science). Blots were blocked with 5% nonfat powdered milk in Tris-buffered saline-Tween 20 (TBS-T) (0.1% Tween 20 in 150 mM Tris-HCl, pH 7.5) for 1.5 h at room temperature and thereafter, the membranes were incubated overnight with a primary rabbit polyclonal antibody for BDNF (1:1000, sc-20981; Santa Cruz Biotechnology, Santa Cruz, USA,) at 4 °C. After washing in TBS-T buffer (three times for 5 min each, at room temperature) the blots were incubated for 2 h at room temperature with an anti-rabbit IgG secondary antibody conjugated with horseradish

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peroxidase (1:15,000; GE Healthcare Bio-Sciences). Both primary and secondary antibodies were diluted in blocking buffer. The antibody-antigen complexes were visualized using the ECL system (Amersham Biosciences) and then exposed to Lumi-Film chemiluminescent detection film (Roch, Germany). Lab Work analyzing software (UVP, UK) was used to analyze the intensity of the expression. To control for loading, the membranes were stripped and reprobed using an antibody for b-actin (Cell Signaling Technology Inc., Beverly, MA, USA; 1:1000). 2.6.3. Semi-quantitative PCR Hippocampal tissues were homogenized in 1 ml RNA extraction buffer according to the manufacturer’s guideline. Total cellular RNAs were isolated by a modified guanine isothiocyanate-phe nol-chloroform procedure using RNX+ reagent. (Ausubel, Brent, et al., 2002) A semi-quantitative PCR method was used (Hajializadeh, Esmaeili-Mahani, et al., 2010). Briefly, to produce a PCR template, single-strand cDNA was synthesized from 5 ll of purified total RNA using M-Mulv reverse transcriptase and oligo-dTprimer. PCR was accomplished in a final volume of 50 ll containing 2 ll tissue cDNA, 1 ll of each BDNF sense and antisense primers, 2 ll dNTP mixture, 1.5 ll MgCl2, 5 ll 10  PCR buffer, 0.3 ll Taq DNA polymerase, and 37.2 ll distillate water. Three separate PCR reactions were performed to study gene expression in the samples obtained from each rat. Each PCR reaction was performed using selective forward and reverse primers for BDNF and b-actin (as an internal standard) genes. The sequences of the primers used were as follows: BDNF-forward: 5’-GAC GAC GAC GTC CCT GGC TGA-30 , BDNF-reverse: 50 -ACG ACT GGG TAG TTC GGC ACT GG-30 ; and b-actin-forward: 50 -CCC AGA GCA AGA GAG GCA TC-30 , b-actin-reverse: 50 -CTC AGG AGG AGC AAT GAT CT-30 . All sequences of primers were synthesized by Metabion International AG (Martinsried, Germany). Taq DNA polymerase (Roche, Germany) used for DNA amplification and reactions were setup according to the manufacturer’s protocol. After denaturation at 95 °C for 5 min, PCR was performed by 25 cycles (45 s at 94 °C, 45 s at 60 °C and 45 s at 72 °C), followed by final extension for 3 min at 72 °C. The PCR products were subsequently analyzed on 1.5% agarose LMMP (Roche, Germany) gel and the obtained bands were quantified by a densitometric analysis (Lab Works analyzing software, UVP, UK). 2.7. Plasma corticosterone assay Immediately after decapitation, the trunk blood was collected into ice-cold EDTA-coated tubes from the same animals killed for the molecular experiments. Samples were then centrifuged at 4 °C for 15 min at 2600g. The plasma fraction was isolated and stored at 80 °C until the assay time. All samples were collected during a same time, between 8:00 am and 8:30 am (10 min after the end of exposure to the narrow and wide platforms in SD and WP groups, respectively). Samples were analyzed blindly using an ELISA kit specific for rat and mice (DRG International Inc., USA) (Hajali et al., 2012). The sensitivity of assay was 0.25 ng/ml and the antibody cross-reacted 100% with corticosterone, 0.7% with progesterone, 0.3% with cortisol, 0.2% with aldosterone and non-detectable with other steroids.

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treatment, or interactions between the two factors. The values are expressed as Means ± SEM, and P < 0.05 was considered statistically significant. The partial eta squared was used for the effect size estimation of ANOVA. 3. Results 3.1. Spatial learning and short-term memory During learning phase, animals in individual groups learned to find the location of the hidden platform as displayed by the decrease in escape latency across subsequent blocks of training. Two-way repeated measures ANOVA revealed no significant difference in escape latency blocks between the SD and control groups. There was also no effect of reproductive status on the mean values of learning blocks (Fig. 1A). These results indicate that regardless of reproductive status, spatial learning in MWM is not altered by sleep loss. The results of the spatial memory in probe trial are shown in Fig. 1B. The percentage of time and distance spent in the target quadrant (Q4) in a single probe trial, which was accomplished 2 h after acquisition, have been measured as the memory consolidation criteria in MWM task. Two-way ANOVA revealed significant effect for treatment (Ctl, WP or SD) [time: F(2,44) = 4.81, P = 0.01; effect size = 0.17 and distance: F(2,44) = 5.36, P = 0.001; effect size = 0.19 ], but not for reproductive status (gonadally intact or gonadectomized) [time: F(1,44) = 2.58, P = 0.11; effect size = 0.05 and distance: F(1,44) = 2.19, P = 0.14; effect size = 0.04] or for the interaction of treatment  reproductive status [time: F(2,42) = 0.04, P = 0.95; effect size = 0.00 and distance: F(2,42) = 0.07, P = 0.92; effect size = 0.00]. These results show that 48 h REM-SD impairs spatial memory regardless of reproductive status. There was no effect of reproductive status or treatment on the results of swimming speed and visible platform test in MWM, demonstrating that these parameters were not influenced by reproductive status or sleep deprivation (Table 1). 3.1. LTP and PPF measurements The increase in the slope of the fEPSPs after HFS and its maintenance over 2 h were considered as E-LTP. The results of the electrophysiological experiments have been presented in Fig. 2A and B. As indicated in Fig 2A, two-way ANOVA revealed that there is a significant effect of treatment [F(24,492) = 1.92, P = 0.006; effect size = 0.08], but not of reproductive status (gonadally intact or gonadectomized) [F(12,492) = 0.56, P = 0.86; effect size = 0.01] or of the interaction of treatment  reproductive status [F(24,492) = 0.47, P = 0.98; effect size = 0.02]. Post hoc Tukey analysis revealed that LTP was impaired in sleep deprived animals compared with both control and WP groups, (P < 0.001). We were also interested in assessing whether presynaptic mechanisms contributed in 48 h REM sleep deprivation-induced impairment of LTP by measuring paired-pulse facilitation (PPF) ratios. PPF was tested at inter-stimulus intervals of 20, 50, and 100 ms and represented as the ratio of the slope of the second fEPSP to that of the first (fEPSP 2/fEPSP 1, PPF ratio). Two-way ANOVA revealed no appreciable effect of reproductive status or treatment on PPF ratios, suggesting that these factors have no impact on presynaptic mechanisms (Fig. 2B).

2.8. Statistical analysis 3.2. BDNF mRNA and protein expression A two-way ANOVA with reproductive status and treatment as between factors with repeated measures on training blocks or LTP time points was employed for group comparisons in MWM training and LTP scales. All comparisons of data collected in probe trials, swimming speed, corticosterone levels, BDNF expression, and paired-pulse facilitation ratios were analyzed with two-way ANOVA followed by Tukey’s test for effects of reproductive status,

Next, we sought to examine if there is a probable molecular mechanism that underlies the memory and LTP impairments, and especially the possible differential sensitivity of two reproductive conditions to 48 h REM sleep deprivation. Regarding both mRNA and protein expression in Fig 3A and B, two-way ANOVA revealed significant effect for treatment (Ctl, WP or SD) [mRNA:

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Fig. 1. Effects of 48 h sleep deprivation (SD) on spatial learning (A) and memory (B) in the Morris water maze (MWM) test in gonadally intact and gonadectomized (GDX) male rats (Ctl: Control; WP: Wide platform and SD: Sleep deprivation). Each block in (A) represents the mean latency of four consecutive trials to find the hidden platform. Animals in all groups learned to find the location of the hidden platform as shown by the decrease in escape latency across subsequent blocks of training. There is no effect of reproductive status or treatment on the average of training block values. Spatial memory in MWM test (B) was defined as the time and distance spent in the target quadrant. Regardless of reproductive status, SD impaired the spatial memory parameters. ⁄ P < 0.05 and ⁄⁄ P < 0.01 indicate significant difference from respective Ctl group. There was no main effect of reproductive status or the interaction of treatment  reproductive status on memory parameters. Data are shown as mean ± S.E.M. (n = 8 per each group). (Two-way ANOVA with repeated measures on training blocks and Two-way ANOVA followed by Tukey test).

Table 1 Swimming speed, latency to the visible platform, and plasma corticosterone levels. Groups

Swim speed (cm/s)

Escape latency to the visible platform (s)

Corticosterone (ng/ml)

Male Ctl WP SD

26.8 ± 0.52 28.85 ± 0.69 26.55 ± 1.02

16.06 ± 3.38 13.53 ± 2.51 17.55 ± 2.13

245.12 ± 45.85 298.43 ± 22.51 283.12 ± 62.32

GDX Ctl WP SD

24.28 ± 0.89 26.35 ± 1.6 24.08 ± 1.53

18.31 ± 3.27 14.78 ± 3.3 17.05 ± 3.69

296.16 ± 58.35 348.66 ± 67.23 303.00 ± 56.09

Ctl: Control; WP: Wide platform; SD: Sleep deprivation; GDX: Gonadectomized. Two-way analysis of variance (ANOVA) was used for main effects of treatment, reproductive status or interaction between two factors. Regardless of reproductive status, plasma corticosterone levels were not affected by sleep deprivation. Data are means ± S.E.M. for groups of 8 rats each.

F(2,28) = 8.54, P = 0.001; effect size = 0.379 and protein: F(2,68) = 12.72, P = 0.000; effect size = 0.272], but not for reproductive status (gonadally intact or gonadectomized) [mRNA: F(1,28) = 0.895, P = 0.352; effect size = 0.031 and protein: F(1,68) = 0.011, P = 0.917; effect size = 0.000] or for the interaction of treatment  reproductive status [mRNA: F(2,26) = 0.967, P = 0.394; effect size = 0.069 and protein: F(2,66) = 0.434, P = 0.65; effect size = 0.013]. Post hoc Tukey analysis revealed that BDNF levels were decreased in sleep deprived animals compared with both control (mRNA: P < 0.001 and protein: P < 0.0001) and WP (mRNA and protein: P < 0.001 groups. These results show that REM-SD inhibits BDNF expression regardless of reproductive status. 3.3. Plasma corticosterone levels The results of plasma corticosterone levels are shown in Table 1. Two-way ANOVA revealed no significant effect of reproductive status or treatment on plasma corticosterone levels, suggesting that regardless of reproductive status, corticosterone levels were not affected by 48 h REM sleep deprivation. 4. Discussion A large body of human and animal studies has documented the impairing effects of sleep loss on learning and memory processes

and their neural mechanisms in adult males. However, to our knowledge, so far, very little attention has been paid to the magnitude of the negative consequences of poor sleep in gonadal hormones-depleted status (eg, aging, postmenopausal women or gonadectomized animal models). The main objective of the present study was to test the probable protective effects of endogenous male gonadal hormones against the 48 h REM sleep deprivation-induced cognitive and synaptic impairments by comparing the intact and GDX male rats. The data showed that both gonadally intact and GDX male animals showed memory deficit to the same extent after REM sleep loss. Likewise, in electrophysiological and molecular experiments, we found that REM-SD caused significant impairment in hippocampal E-LTP and BDNF expression in both intact and GDX male rats, but there was still no difference in the response of two reproductive statuses. The present data showed no significant change in cognitive and synaptic functions of the rats held on the wide platforms for 48 h with respect to the home cage control animals. Consistently, in comparison to the control and WP groups, plasma corticosterone levels were not significantly changed in REM-SD groups, confirming the previous studies that used the same paradigm for REM-SD induction (McDermott et al., 2003; Ravassard et al., 2009; Saadati, Esmaeili-Mahani, et al., 2015). Moreover, administration of metyrapone, a glucocorticoid synthesis inhibitor, to the rats immediately after 96 h REM-SD induced by the same model, could not reverse the memory deficiencies (Tiba et al., 2008). These findings diminish the probability of any stress contribution for cognitive and synaptic impairments observed in animals deprived of REM sleep on the small platforms. Corticosterone levels in nocturnal animals like rats exhibit a distinct circadian variation with peak values in the evening followed by a nadir in the morning (D’Agostino et al., 1982). Unexpectedly, our data showed that the corticosterone concentrations independent of treatment or reproductive status were generally higher than the normal levels in undisturbed male rats. Since both control and SD groups displayed elevated corticosterone levels, it is difficult to conclude with certainty about this finding, but it can be due to the altered reactivity of the hypothalamic-pituitary-adrenal axis (HPA) system during the handling or transferring the animals to the lab. However, as hormone levels, as well as behavioral and synaptic functions were not differently altered in two reproductive statuses after REM-SD, it could

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Fig. 2. Effects of 48 h sleep deprivation (SD) on the induction and maintenance of long term potentiation (LTP) over a period of 120-min (early phase) (A) in the hippocampal CA1 area of gonadally intact (n = 9 in Ctl; control and n = 8 in WP; wide platform and SD groups) and gonadectomized (GDX) (n = 8 in Ctl, n = 7 in WP and SD groups) male rats. The magnitude of LTP was expressed as a relative change (%) in the slope of fEPSPs after HFS with respect to the average of responses during a 20-min period of baseline recording. The fEPSP slopes have independently of reproductive status been decreased significantly following SD. There was no main effect of reproductive status or the interaction of treatment  reproductive status on LTP scales. The presented traces are the typical average examples of fEPSPs recorded from individual rats at different time points. All recorded responses were averaged from 10 consecutive signals at each time point and normalized to the mean value of the baseline. There was no effect of reproductive status or treatment on PPF ratios at any interpulse interval (male and GDX: n = 5 in Ctl and WP and n = 7 in SD groups). (B). Data are shown as mean ± S.E.M. (Two-way ANOVA or Two-way ANOVA with repeated measures on time point).

not be interpreted as a causative role of probable differences in sensitivity to 48 h REM-SD. Several studies have demonstrated the impairing effects of SD on cognitive performances examined using a variety of learning and memory tasks (Alhaider, Aleisa, et al., 2010; Guan, Peng, et al., 2004; McDermott et al., 2003). Since no significant difference was observed in the visible platform test and swimming speed data in MWM between control and SD groups, it can be claimed that the observed behavioral and synaptic impairments were not likely due to the poor motivation, fatigue, reduced arousal, or altered sensorimotor coordination of animals which might be raised from insufficient sleep. Rather, it replicates the assumptions that SD-induced memory impairment is primarily attributed to an alteration of vigilance state quantities. Accordingly, the sequential hypothesis (SH) claims that SD-induced memory deficits are based on altered proportion of different phases of sleep (Giuditta, 2014; Ota, Moreira, et al., 2013). In addition to these well-known effects on cognitive functions, several neurotransmitters, cellular and molecular factors involved in synaptic plasticity within the hippocampus of rodents have also been shown to be influenced by SD (Guzman-Marin et al., 2006; Longordo, Kopp, et al., 2009; Ravassard et al., 2009).

LTP is considered as a long-lasting amplification of synaptic efficacy which is commonly accepted as a cellular correlate for storing new information within neuronal networks (Bliss & Collingridge, 1993). Late-LTP (L-LTP) is assumed to be related to long-term memory, while early-LTP (E-LTP) corresponds to short-term memory consolidation (Jones, Errington, et al., 2001). The deleterious effects of REM-SD induced by single (Davis, Harding, et al., 2003) and multiple platform technique (Alhaider et al., 2010; McDermott, Hardy, et al., 2006; Saadati et al., 2014a, 2014b) on E-LTP have previously been established in both in vivo and in vitro studies. LTP deficits have also been reported after total SD and fragmented sleep (Campbell, Guinan, et al., 2002;Tartar, Ward, et al., 2006). LTP induction seems to display a graded sensitivity to SD. Extended periods of SD for 24–72 h appear to inhibit or reduce LTP induction (Campbell et al., 2002; McDermott et al., 2003). However, short periods of SD only appear to lead to disruption of signaling underlying LTP maintenance while induction remains intact (Florian, Vecsey, et al., 2011; Vecsey, Baillie, et al., 2009). Thus, in the second experiment, we recorded hippocampal E-LTP and PPF to assess whether the observed similarity in

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Fig. 3. Effects of 48 h sleep deprivation (SD) on BDNF gene expression in the hippocampus of gonadally intact (n = 5 in Ctl; control, WP; wide platform and SD groups) and gonadectomizaed (GDX) (n = 5 in Ctl and WP groups and n = 7 in SD group) male rats (A). (B) Shows the effects of 48 h SD on BDNF protein expression in the hippocampus of above mentioned animals (n = 12 in each group). Regardless of reproductive status, SD suppressed the both gene and protein expression of hippocampal BDNF. ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 indicate significant difference from respective Ctl group and  P < 0.01 indicate significant difference from respective WP group. There was no main effect of reproductive status or the interaction of treatment  reproductive status on BDNF gene and protein levels. Each value in the graphs represents the mean ± S.E.M. band density ratio for each group. b Actin was used as an internal control. (Two-way ANOVA followed by Tukey test).

vulnerability of two reproductive statuses to the negative effects of REM-SD on memory performances are also emerged at the cellular level. In agreement with the behavioral data described above, LTP was also significantly, but equally disrupted in both intact and GDX male animals following REM-SD. Paired pulse facilitation (PPF) which is believed to be related to short-term synaptic plasticity depends on presynaptic mechanisms, including increased neurotransmitter release probability and residual calcium level in presynaptic terminals (Citri & Malenka, 2008). Consistent with Tartar et al (Tartar, Ward, et al., 2009) and Saadati et al. (2014a, 2014b), the PPF ratios were not influenced by REM-SD, proposing that firstly, presynaptic factors were probably not involved in the LTP impairment following REM-SD and secondly, male gonadal hormones exerted no presynaptic involvement in LTP deficit after sleep loss. However, a lack of change in the PPF ratio may not exclude all potential presynaptic effects of SD or gonadal hormones. It has been demonstrated that L-LTP involves an elevation in endogenous BDNF for its long-lasting maintenance, however, evidence suggests that endogenous BDNF is also essential for the

induction and maintenance of early phase of LTP (Lu, Pang, et al., 2005). In addition to the synaptic plasticity, BDNF has also been suggested to have neuroprotective potentials against a variety of neurological insults and neurodegenerative disorders (Nagahara, Merrill, et al., 2009). The decrease in BDNFlevels and its downstream targets in the hippocampus of male rats after 8 h and 48 h total sleep deprivation (TSD) have been previously reported by Guzman-Marin et al (Guzman-Marin et al., 2006). Consistently, the present results showed that regardless of reproductive status, REM-SD suppressed the hippocampal BDNF gene and protein expression, suggesting that disrupted BDNF signaling could be involved in the observed impairing effects of sleep loss on cognitive and synaptic plasticity. Androgens have been demonstrated to affect cognitive functions and to modulate structural, biochemical, and electrical properties of the hippocampus (Schulz & Korz, 2010; Skucas, Duffy, et al., 2013). Male sex steroid hormones have also potential neuroprotective capabilities against neural damages, experimental brain injuries, and neurodegenerative disease (Bialek et al., 2004; Pike et al., 2008, 2009). Accordingly, some studies have reported the

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beneficial effects of testosterone replacement on cognitive deficiencies in elderly men and also in men with Alzheimer disease (Cherrier et al., 2005a, 2005b; Hogervorst, Bandelow, et al., 2005; Moffat, 2005). Nevertheless, in this study we could not establish any significant effect of reproductive status or interaction of treatment and reproductive status on cognitive functions or their neural correlates. Indeed, the present results suggest that male sex steroid hormones do not exert any protective role against the deleterious effects of 48 h REM-SD on behavioral and synaptic plasticity. There may be an evolutionary reason for the lack of a protective effect of male gonadal hormones in this context, however, using different cognitive tasks or protocols may clear this up. It is also worthy to mention that although depletion of male sex gonadal hormones had no significant effect on our measures after REM sleep deprivation, it may have an impact in animals that underwent total sleep deprivation. In addition to the gonadal-derived sex steroids, there has been increasing evidence that some brain regions, especially the hippocampus also have their own steroids (neurosteroids) which might be synthesized either de novo or from endogenous precursors by enzymes present in these regions. In fact, it has been shown that sex steroids remain in the hippocampal neurons long after gonadectomy and adrenalectomy and that these brain-derived steroids have neuroprotective potentials and are essential for synaptic plasticity. (Mukai, Tsurugizawa, et al., 2006; Rune & Frotscher, 2005; Wojtal, Trojnar, et al., 2006). Thus, it cannot be ruled out that elimination of neurosteroids may even result in the altered sensitivity of the gonadectomized animals to the impairing effects of sleep loss. In conclusion, this study benefits the behavioral, cellular, and molecular approaches to explore the possibilities of differences in susceptibility of gonadally intact and GDX male animals to the deleterious effects of 48 h REM-SD on cognitive and synaptic plasticity. Although these findings failed to establish any significant difference in cognitive vulnerability of two reproductive conditions to sleep loss, however, it does not reduce the need to consider sex, age, or reproductive status when exploring the consequences of sleep disturbances on health.

Acknowledgment This work was supported by funds from the Kerman Neuroscience Research Center (KNRC/89/46), Kerman, Iran. The authors have no financials conflict of interest.

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