PNP-08771; No of Pages 7 Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
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PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation
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Department of Pharmacology, Peking University, School of Basic Medical Science, Beijing 100191, China
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Article history: Received 6 February 2015 Received in revised form 28 April 2015 Accepted 6 May 2015 Available online xxxx
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Keywords: Dorsal raphe nucleus Protein kinase C Serotonin Sleep Slow wave sleep
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Studies suggest a tight relationship between protein kinase C (PKC) and circadian clock. However, the role of PKC in sleep–wake regulation remains unclear. The present study was conducted to investigate the role of PKC signaling in sleep–wake regulation in the rat. Our results showed that the phosphorylation level of PKC in dorsal raphe nucleus (DRN) was decreased after 6 h sleep deprivation, while no alterations were found in ventrolateral preoptic nucleus (VLPO) or locus coeruleus (LC). Microinjection of a pan-PKC inhibitor, chelerythrine chloride (CHEL, 5 or 10 nmol), into DRN of freely moving rats promoted non-rapid eye movement sleep (NREMS) without influences on rapid-eye-movement sleep (REMS). Especially, CHEL application at 5 nmol increased light sleep (LS) time while CHEL application at 10 nmol increased slow wave sleep (SWS) time and percentage. On the other hand, microinjection of CaCl2 into DRN not only increased the phosphorylation level of PKC, but also reduced NREMS time, especially SWS time and percentage. While CHEL abolished the inhibitory effect of CaCl2 on NREMS and SWS. These data provide the first direct evidence that inhibition of intracellular PKC signaling in DRN could increase NREMS time including SWS time and percentage, while activation of PKC could suppress NREMS and reduce SWS time and percentage. These novel findings further our understanding of the basic cellular and molecular mechanisms of sleep–wake regulation. © 2015 Published by Elsevier Inc.
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1. Introduction
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Protein kinase C (PKC) is highly expressed within the brain, where it plays important roles in regulating neurotransmission, coordinating intracellular signaling in response to external stimuli, and modulating changes in gene expression and neuronal plasticity (Calabrese and Halpain, 2005; Ramakers et al., 1997; Zarate and Manji, 2009). Intensive studies on circadian clock have provided an insight into the role of PKC in it. Conventional Ca2+-sensitive PKC signaling pathway is not limited to relaying external stimuli but is rhythmically activated by internal processes, forming an integral part of the circadian feedback loop (Robles et al., 2010). The alternative between sleep and arousal is one of the most typical behaviors that have circadian rhythm. The tight relativity between PKC and circadian clock implies the potential role of PKC signaling in sleep–wake regulation. The PKC family is composed of twelve isozymes that are classified as either conventional, novel, or atypical according to the nature of their regulatory domains. The conventional PKCs (cPKCs) possess a Ca2+regulated domain that binds Ca2+ to be activated. cPKCs are designed
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Sheng-Jie Li 1, Su-Ying Cui 1, Xue-Qiong Zhang, Bin Yu, Zhao-Fu Sheng, Yuan-Li Huang, Qing Cao, Ya-Ping Xu, Zhi-Ge Lin, Guang Yang, Xiang-Yu Cui, Yong-He Zhang ⁎
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⁎ Corresponding author. Tel./fax.: +86 10 82801112. E-mail address: [email protected] (Y.-H. Zhang). 1 Sheng-Jie Li and Su-Ying Cui contributed equally to the present study.
to exactly decode two second messengers, Ca2+ and signaling lipids. cPKCs reside in the cytosol during resting periods waiting to be triggered by rises in intracellular Ca2 + concentration (Lipp and Reither, 2011). Circadian oscillations of free Ca2 + have been widely observed (Harrisingh et al., 2007; Imaizumi et al., 2007), which may be involved in the rhythmical activation of PKC. Several reports have revealed that Ca2 + oscillations drive oscillations of the activity of cPKCs (Bartlett et al., 2005; Violin et al., 2003). In addition, our recent study has demonstrated that the elevation of calcium function by BAY-K-8644, the L-type calcium channel agonist, and CaCl2 reduces NREMS and REMS, whereas down-regulation of Ca2 + function by diltiazem, the L-type calcium channel antagonist, and EGTA, the Ca2 + chelating agent in dorsal raphe nucleus (DRN) may promote NREMS especially the SWS percentage in pentobarbital treated rats (Cui et al., 2011a). And pharmacological evidences strongly suggest a correlation between L-type Ca2 + channel and serotonergic (5-HT) system in signaling transduction involved in sleep regulation (Cui et al., 2011b). Based on the important role of PKC on the circadian regulation, the pharmacological actions of calcium in DRN, and the fact that cPKCs can be activated by elevation of intracellular calcium concentration, we hypothesized that the intracellular PKC signaling in the DRN may be involved in the regulation of physiological sleep. In the present study, we investigated the role of PKC in DRN on sleep– wake regulation.
http://dx.doi.org/10.1016/j.pnpbp.2015.05.005 0278-5846/© 2015 Published by Elsevier Inc.
Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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2. Materials and methods
2.4. EEG and EMG recordings and analysis
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Male Sprague–Dawley rats (220–240 g, Grade I, purchased from the Animal Center of Peking University, Beijing) were used. All experiments were conducted in accordance with the European Community guidelines for the use of experimental animals and approved by the Peking University Committee on Animal Care and Use. The rats were housed in acrylfiber cages individually and had ad libitum access to food and water. They were exposed to a 12:12 h light/dark schedule with lights on at 9:00 A.M. The ambient temperature averaged 23 ± 1 °C and the relative humidity was 50 ± 10%.
Seven days after implantation the animals were adapted to the recording and injection procedures. For CHEL experiment, recording was started at 9 P.M. and lasted for 6 h during the night phase. For CHEL and CaCl2 combination experiment, recording was started at 9 A.M. and lasted for 6 h during the light phase. Sleep–wake states were manually classified as wakefulness, light sleep (LS), slow wave sleep (SWS) and rapid eye movement sleep (REMS). Non-rapid eye movement sleep (NREMS) time equals LS time plus SWS time. Total sleep (LS) time equals NREMS time plus REMS time. The mean number and duration of TS, NREMS, LS, SWS, and REMS were also quantified, as well as LS, SWS and REMS percentage in TS. Details of the EEG and EMG recordings and analysis were described previously (Wang et al., 2014).
2.5. Drugs and drug administration
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The following drugs were used in this study: chelerythrine chloride (CHEL), CaCl2 (both from Sigma-Aldrich, St. Louis, USA). CHEL inhibits PKC activation by blocking the site of DAG/phorbol-ester binding and inhibiting PKC translocation to the membrane for activation (Brennan et al., 2009). CHEL was dissolved in physiological saline and was microinjected into DRN either alone at 9 P.M. (5 or 10 nmol) or 20 min prior to CaCl2 administration at 5 nmol. CaCl2 was dissolved in saline and the pH of the solution was adjusted to 7.3 with NaOH. CaCl2 was microinjected into the DRN at 9 A.M. (25 or 50 nmol). Each agent was microinjected in a volume of 0.2 μl. The control groups in each experiment were microinjected with saline.
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2.6. Tissue sample preparation
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The procedure was based on our previous study (Wang et al., 2015). For sleep deprivation experiment, immediately after 6 h sleep deprivation (3 P.M.), rats were decapitated, and the brains were quickly removed to a pre-chilled brain matrix. Ventrolateral preoptic nucleus (VLPO), dorsal raphe nucleus (DRN), and locus coeruleus (LC, with some of their surrounding tissue) were punched (1 mm diameter for VLPO, between 0 mm and 1 mm posterior to the bregma, bilaterally; 2 mm diameter for DRN, between 7 mm and 9 mm posterior to the bregma; 2 mm for LC, between 9 mm and 11 mm posterior to the bregma, bilaterally) using a hypodermic needle (12 guage) guided by the rat brain atlas of Paxinos and Watson. For CaCl2 administration experiment, DRN was dissected 3 h after CaCl2 administration (12 A.M.). All the
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2.2. Sleep deprivation
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Rats were handled by procedures as reported (Estabrooke et al., 2001). Sleep deprivation was performed by gentle handling while animals were kept in their home cages respectively. The rats were monitored continuously during sleep deprivation. They were aroused by tapping lightly on their cages or touching whenever they appeared to go to sleep. And the rats were never lifted out of the cages. When light tapping did not awaken the animals, crumpled paper was inserted into the cages to stimulate the rats. If needed, louder tapping of the cage was repeated. The deprivation started at light onset (9 A.M.) and lasted for 6 h and the rats were sacrificed at 3 P.M. Control undisturbed rats were sacrificed at the corresponding time point (3 P.M.).
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2.3. Surgery
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Details of the surgical procedure were described previously (Wang et al., 2014). The animals were implanted chronically with stainless steel screws over the frontal–parietal cortex and a pair of wire electrodes through the nuchal muscles for recording of electroencephalogram (EEG) and electromyogram (EMG), respectively. Additionally, a guide cannula (26 gauge) was implanted 1 mm above the DRN at coordinates, AP = − 8.0; L = 0.0 and DV = − 5.8 (Paxinos and Watson, 1998). Drug or vehicle was injected into the DRN with an injection cannula (29 gauge), which extended 1 mm beyond the guide, in a 0.2 μl volume over a 2 min period. Histological verification of cannula/ injection sites was carried out at the end of the experiments. All the data presented in the sleep recording experiments are derived from animals whose injection site was within the limits of DRN. The location of cannula/injection is shown in Fig. 1B.
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Fig. 1. Photomicrographs of representative cannula placements in DRN. Sections were according to Paxinos and Watson (1998).
Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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2.8. Statistical analysis
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The data were analyzed using SPSS software 17.0 and are expressed as mean ± S.E.M. The biochemical data for comparisons between the control and sleep-deprivation groups were analyzed using unpaired Student's t-test. For multiple comparisons, data were analyzed by oneway analysis of variance (ANOVA) followed by Student–Newman– Keuls post hoc test. In all of the tests, p b 0.05 was considered statistically significant.
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3. Results
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3.1. Effects of sleep deprivation on pPKC and PKC expression level
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A western blot analysis was performed to probe the pPKC and PKC expression level within different brain regions under physiological and 6 h sleep-deprivation conditions. The protein level of pPKC in DRN of the sleep-deprived (SD) group was markedly reduced (by 46.97%, t = 4.33, df = 6, p b 0.01, Fig. 2A) compared to that of the control group. This effect was regionally specific, as an analysis of pPKC expression within the VLPO and LC revealed no difference between control and sleep-deprived groups. Total PKC level revealed no difference between control and sleep-deprived groups in all the three brain regions.
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3.2. Effects of CHEL microinjection into DRN on sleep parameters
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Microinjections of CHEL (5 or 10 nmol) into DRN augmented TS (by 73.40% and 55.14%, F2, 32 = 6.70, p b 0.01, respectively), with a
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3.3. Effects of CaCl2 microinjection into DRN on pPKC and PKC expression level
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Application of CaCl2 into DRN significantly reduced NREMS time. To determine whether PKC mediated the effect of CaCl2 on sleep–wake regulation, we used western blot to observe the changes of PKC phosphorylation level and total protein level in the DRN 3 h after CaCl2 administration. The results showed that the expression levels of pPKC were significantly increased (by 64.01% and 94.90%, F2, 9 = 14.56, p b 0.01, respectively) after microinjections of CaCl2 (25 or 50 nmol). While total PKC expression levels revealed no difference between saline and CaCl2 groups. These results indicated that the microinjection of CaCl2 into DRN increased the active form of PKC in DRN (Fig. 4).
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The protein concentrations of all samples were determined using the BCA assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin as the standard. Loading buffer (5 × SDS-PAGE Sample/Loading Buffer B1012, Applygen) was added to each sample before boiling for 5 min. Equal amounts of protein (25 μg) were separated by 10% SDSpolyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membrane (Millipore, MA, USA). The membranes were blocked overnight at 4 °C in TBST buffer (Tris-buffered saline + 0.1% Tween-20) supplemented with 5% bovine serum albumin (BSA), and incubated with primary antibodies, including anti-GAPDH (1:1000; sc25778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pPCK (1:1000; #9379, Cell Signaling Technology, MA, USA), and anti-PKC (1:1000; ab23511, Abcam, Cambridge, MA, USA), in TBS-T buffer containing 5% BSA at 4 °C overnight. After 3 × 10 min TBS-T washes, the blots were incubated with horseradish peroxidaseconjugated secondary antibodies (1:1000; Cell Signaling Technology, MA, USA) in TBS-T containing 5% nonfat dry milk for 2 h at 37 °C and then washed with TBS-T buffer for 3 × 10 min. The blots were then treated with an enhanced chemiluminescence detection kit (CoWin Bioscience, Beijing, China). Western blot bands were scanned with a GelDoc XR System (Bio-Rad, Hercules, CA, USA) and subsequently analyzed densitometrically with Image Lab software. The results were normalized to the protein expression level of GAPDH in each sample.
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significant increase in the number of TS episodes (by 63.36% and 68.95%, F2, 32 = 8.95, p b 0.001, respectively) and mean duration remaining unchanged. The results revealed no difference in REMS time or REMS percentage. NREMS time was significantly increased (by 76.55% and 58.48%, F2, 32 = 7.21, p b 0.01, respectively) after microinjections of CHEL (5 or 10 nmol) compared with vehicle control microinjections, which was attributed to the increase in number of NREMS episodes (by 64.43% and 58.03%, F2, 32 = 8.95, p b 0.001, respectively). Microinjection of 5 nmol concentration of CHEL prolonged LS time (by 74.18%, F2, 32 = 6.74, p b 0.01), due to an increased number of LS episodes (by 74.12%, F2, 32 = 4.79, p b 0.05). Microinjection of 10 nmol concentration of CHEL prolonged SWS time (by 405.09%, F2, 32 = 3.70, p b 0.05), increased number (by 243.75%, F2, 32 = 3.30, p b 0.05) and mean duration (by 51.62%, F2, 32 = 6.27, p b 0.01) of SWS episodes, as well as the percentage of SWS in TS (by 183.78%, F2, 32 = 3.85, p b 0.05) (Fig. 3).
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dissected tissues were stored separately in pre-chilled microcentrifuge tubes at −80 °C until assayed. Brain tissues were pooled and homogenized in ice-cold lysis buffer containing 50 mM Tris buffer (pH 7.2), 6 mM MgCl2, 1 mM EDTA, 10% (wt/vol) sucrose, 1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, 5 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, 5 μg/ml bestatin, 2 μg/ml E-64 and protein phosphatase inhibitor cocktail (All-in-One, 100 ×, P1260, Applygen). The homogenate was then sonicated (5 s × 3, 5 s interval) and ultracentrifuged (12,000 g) for 10 min at 4 °C. The supernatant was used as whole-cell extract.
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3.4. Effects of CaCl2 microinjection into DRN on sleep parameters in rats 240 pretreated with CHEL 241 To further verify the role of PKC in calcium induced sleep reduction, rats received PKC inhibitor before CaCl2 administration and then were subjected to recording session for 6 h (between 9:00 A.M. and 3:00 P.M.), as mentioned earlier. Significant decreases in TS time (by 25.31%, F3, 29 = 5.10, p b 0.01), NREMS time (by 25.29%, F3, 29 = 5.83, p b 0.01), SWS time (by 82.78%, F3, 29 = 4.37, p b 0.05) and SWS percentage (by 73.96%, F3, 29 = 4.82, p b 0.01) were observed after CaCl2(25 nmol) administration, which could be reversed by CHEL (5 nmol). Microinjection of CHEL (5 nmol) alone into DRN did not interfere with sleep structure of rats in the light period. These results indicated that pretreatment of CHEL in DRN, which blocked the function of PKC, could at least partially antagonize the sleep disturbance caused by CaCl2 administration (Fig. 5).
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The present study showed that 6 h sleep deprivation caused a lower phosphorylation level of PKC in DRN and the inhibition of PKC by the pan-PKC inhibitor CHEL in DRN significantly increased NREMS time, but did not influence REMS. Especially, CHEL application at 5 nmol increased LS time while CHEL application at 10 nmol increased SWS time and percentage. On the other hand, microinjection of CaCl2 into DRN not only increased pPKC expression, but also reduced NREMS time especially SWS time and percentage. While CHEL abolished the inhibitory effect of CaCl2 on NREMS and SWS. These results, for the first time, provide evidence that intracellular CHEL-sensitive PKC in DRN may play an important role in sleep–wake regulation. The sleep–wake cycle is a kind of homeostatic process which involves mutually inhibitory interactions between sleep and arousalpromoting systems, and is represented by the build-up of sleep pressure during wake and its decay in the course of sleep (Rachalski et al., 2014). It has been demonstrated that the activity of DRN 5-HT neurons was
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Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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Fig. 2. Acute sleep deprivation decreased pPKC level in DRN in a regional-specific way. Representative of western blots and quantification of pPKC and PKC in DRN (A), VLPO (B) and LC (C). GAPDH was shown as quantitative loading control. Data are represented as mean ± S.E.M. (n = 6 pooled samples and repeated for 3–5 times, **p b 0.01 vs Control). SD, sleep-deprived group; DRN, dorsal raphe nucleus; VLPO, ventrolateral preoptic nucleus; LC, locus coeruleus.
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elevated initially and lowered during the later phase of sleep deprivation in cat (Gardner et al., 1997). The decline may be due to diminished arousal and/or increase in sleep pressure. It has been reported that 6 h sleep deprivation in the beginning of the 12-h light period prolonged NREMS and markedly enhanced EEG power density in the lowfrequency range (0.75–6.0 Hz) (Tobler and Borbely, 1990). The present study shows that 6 h sleep deprivation in rats significantly reduced phosphorylation level of PKC in DRN. The lower phosphorylation level of PKC in DRN may be related to the weaker activity of arousal promoting system and the reinforcement of sleep initiating system after sleep
deprivation. Consequently, the lower phosphorylation level of PKC in DRN after sleep deprivation may facilitate the body to initiate the subsequent recovery sleep and probably correspond to the rebound of NREMS especially SWS after 6 h sleep deprivation. Supporting evidence illustrates that DRN 5-HT and non-5-HT neurons are involved in sleep–wake regulation (Monti, 2010a). It is currently accepted that DRN 5-HT neurons function to promote wakefulness and to inhibit REMS (Monti, 2010b). As interneurons, most of DRN non-5-HT neurons modulate the activity of 5-HT neurons by expressing a variety of substances including GABA and glutamate (Monti, 2010a;
Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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Soiza-Reilly and Commons, 2014). Previous studies provide persuasive evidence which indicated that the effect of non-5-HT neurons in DRN on sleep–wake regulation was eventually mediated by 5-HT neurons (Monti, 2010a). Previous reports also showed that activation of PKC signaling pathway could potentiate the function of serotonergic system. PKC activation induces phosphorylation of serotonin transporter (SERT), which inhibits SERT activity (Jayanthi et al., 2005). Besides, PKC induces phosphorylation and desensitization of 5-HT1A receptor (Raymond, 1991). The activation of PKC could disinhibit 5-HT1A receptor's inhibitory effect on serotonergic neurons. Studies indicated that SERT and 5-HT1A are closely related to sleep regulation (Alexandre et al., 2006; Wisor et al., 2003), which implies the involvement of PKC signaling pathway. The present study shows that intraDRN application of the PKC signaling activation inhibitor, CHEL,
increased NREMS time, especially SWS time and SWS percentage. Considering the important role of DRN 5-HT neurons on sleep–wake regulation and the function of PKC on serotonergic system, we hypothesized that the hypnotic effect of CHEL was related to down regulation of PKC signaling on serotonergic system through downstream substrates of PKC. Previous studies reported that microinjection of certain neurotransmitters/neuromodulators or their receptor agonists (including glutamate, serotonin, melanin-concentrating hormone, dopamine, orexin), which could modulate PKC, into DRN caused alterations in sleep variables (Gao et al., 2002; Kohlmeier et al., 2004; Lagos et al., 2009; Monti, 2011; Tao et al., 2006). For example, microinjection of glutamate into DRN induced enhancement of wakefulness, and reduced TS time, especially SWS time (Gao et al., 2002). Hcrt/Orx produced a dose-
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Fig. 3. Microinjection of CHEL into DRN altered sleep parameters. (A) Total sleep (TS), non-rapid eye movement sleep (NREMS), light sleep (LS), slow wave sleep (SWS) and rapid eye movement sleep (REMS), LS percentage (LS%), SWS percentage (SWS%) and REMS percentage (REMS%). (B) Number and mean duration of TS, NREMS, LS, SWS episodes (n = 11–15/group). Data are represented as mean ± S.E.M. (*p b 0.05 and **p b 0.01 vs Saline, #p b 0.05 vs CHEL 5 nmol). CHEL, chelerythrine chloride.
Fig. 4. Microinjections of CaCl2 (25 or 50 nmol) into DRN increased pPKC, but not PKC protein levels. Representative of western blots and quantification of pPKC and PKC in the DRN were shown. GAPDH was shown as quantitative loading control (n = 6 pooled samples and repeated for 4 times). Data are expressed as mean ± S.E.M. (**p b 0.01 vs saline).
Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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In summary, the molecular, pharmacological, and behavioral data of this study, for the first time, demonstrate a novel wake-promoting and SWS-suppressing role for PKC signaling within DRN neurons. These findings are critical for our complete understanding of the basic cellular
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and molecular mechanisms of sleep–wake regulation. However, the heterogeneity of PKC isoforms raises the possibility that the supposed PKC activity imbalance may actually consist in a more complex schema, where the various isozymes would be differentially involved in the regulation of sleep–wake cycle. Future studies are therefore needed to clarify the respective contribution of specific isozymes to sleep–wake regulation and to mood disorders.
This study was funded by grants from the National Natural Science 375 Foundation of China (Nos. 81173031, 81202511 and 81302746). 376 References
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dependent elevation of calcium concentration and serotonin in significant numbers of neurons in DRN (Kohlmeier et al., 2004; Tao et al., 2006). Microinjection of PKC inhibitor, CHEL, into DRN could block the effects of endogenous neurotransmitters/neuromodulators, which may be possible mechanisms underlying the hypnotic effect of CHEL. However, CHEL induces apoptosis by causing rapid release of cytochrome c from mitochondria (Wan et al., 2008). Although the apoptosis was detected 16 h after CHEL application, the rapid release of cytochrome c from mitochondria could possibly lead to reductions of ATP synthase and ATP availability in the neurons and further make neurons cease firing. Although Nissl staining revealed no obvious changes in cell morphology and the number of neurons in the DRN between saline and CHEL (5 or 10 nmol) groups (not show), we could not exclude the possibility that the hypnotic effect of CHEL limited in 6 h was induced by its toxic effect on serotonergic neurons. The PKC family is classified as either conventional, novel, or atypical as mentioned earlier. Only cPKCs possess a Ca2+-regulated domain that binds Ca2+ to be activated. Other types are all activated in a calcium independent manner. Intracellular Ca2+ plays an important role in the activation of Ca2 +-dependent PKC. Our previous study proved that Ca2 + in DRN plays an important role in sleep–wake regulation. And the present study indicates that the arousal effect of Ca2+ in DRN was associated with PKC signaling pathway. Administration of CaCl2 into DRN significantly increased the expression level of pPKC, and decreased NREMS time, especially SWS time and SWS percentage. The intra-DRN application of PKC inhibitor CHEL reversed the arousal effect of CaCl2 by recovery of NREMS and SWS time. These results indicate that the activation of PKC signaling in the DRN suppresses NREMS and could decrease sleep intensity. Thus, there is a clear rationale to suggest that the inhibition of PKC signaling in the DRN may promote NREMS, especially SWS and SWS percentage at higher dose (10 nmol), but activation of PKC may suppress NREMS and reduce SWS time and percentage. Through phosphorylation of a large variety of substrates, PKC is able to modulate a multiplicity of neuronal functions, such as neurotransmitter synthesis and release, gene expression, ion channel and receptor regulation. A growing body of studies have identified the involvement of PKC in bipolar disorder (DiazGranados and Zarate, 2008). In addition, PKC is a cellular target of most current mood stabilizing and antimanic agents (Abrial et al., 2013; Jensen and Mork, 1997; Sabioni et al., 2008). The data of the present study raise the question whether PKC modulation may also be effective on the sleep disorders or the mood disorders associated with sleep disorders.
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Fig. 5. Microinjection of CHEL into DRN eliminated the inhibitory effect of CaCl2 on non-rapid eye movement sleep (NREMS) and slow wave sleep (SWS). Sleep parameters including total sleep (TS) time, NREMS time, light sleep (LS) time, SWS time, rapid eye movement sleep (REMS) time, LS percentage (LS%), SWS percentage (SWS%) and REM sleep percentage (REMS%) were assessed (n = 7 ~ 10/group). Data are expressed as mean ± S.E.M. [*p b 0.05 and **p b 0.01 vs Saline + Saline; #p b 0.05 vs Saline + CaCl2 (25 nmol)]. CHEL, chelerythrine chloride.
Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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Sabioni P, Baretta IP, Ninomiya EM, Gustafson L, Rodrigues AL, Andreatini R. The antimanic-like effect of tamoxifen: behavioural comparison with other PKCinhibiting and antiestrogenic drugs. Prog Neuropsychopharmacol Biol Psychiatry 2008;32:1927–31. Soiza-Reilly M, Commons KG. Unraveling the architecture of the dorsal raphe synaptic neuropil using high-resolution neuroanatomy. Front Neural Circ 2014;8:105. Tao R, Ma Z, McKenna JT, Thakkar MM, Winston S, Strecker RE, et al. Differential effect of orexins (hypocretins) on serotonin release in the dorsal and median raphe nuclei of freely behaving rats. Neuroscience 2006;141:1101–5. Tobler I, Borbely AA. The effect of 3-h and 6-h sleep deprivation on sleep and EEG spectra of the rat. Behav Brain Res 1990;36:73–8. Violin JD, Zhang J, Tsien RY, Newton AC. A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol 2003;161:899–909. Wan KF, Chan SL, Sukumaran SK, Lee MC, Yu VC. Chelerythrine induces apoptosis through a Bax/Bak-independent mitochondrial mechanism. J Biol Chem 2008;283:8423–33. Wang ZJ, Yu B, Zhang XQ, Sheng ZF, Li SJ, Huang YL, et al. Correlations between depression behaviors and sleep parameters after repeated corticosterone injections in rats. Acta Pharmacol Sin 2014;35:879–88. Wang ZJ, Zhang XQ, Cui XY, Cui SY, Yu B, Sheng ZF, et al. Glucocorticoid receptors in the locus coeruleus mediate sleep disorders caused by repeated corticosterone treatment. Sci Rep 2015;5:9442. Wisor JP, Wurts SW, Hall FS, Lesch KP, Murphy DL, Uhl GR, et al. Altered rapid eye movement sleep timing in serotonin transporter knockout mice. Neuroreport 2003; 14:233–8. Zarate CA, Manji HK. Protein kinase C inhibitors: rationale for use and potential in the treatment of bipolar disorder. CNS Drugs 2009;23:569–82.
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Contents lists available at ScienceDirect
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PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation
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Department of Pharmacology, Peking University, School of Basic Medical Science, Beijing 100191, China
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Article history: Received 6 February 2015 Received in revised form 28 April 2015 Accepted 6 May 2015 Available online xxxx
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Keywords: Dorsal raphe nucleus Protein kinase C Serotonin Sleep Slow wave sleep
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Studies suggest a tight relationship between protein kinase C (PKC) and circadian clock. However, the role of PKC in sleep–wake regulation remains unclear. The present study was conducted to investigate the role of PKC signaling in sleep–wake regulation in the rat. Our results showed that the phosphorylation level of PKC in dorsal raphe nucleus (DRN) was decreased after 6 h sleep deprivation, while no alterations were found in ventrolateral preoptic nucleus (VLPO) or locus coeruleus (LC). Microinjection of a pan-PKC inhibitor, chelerythrine chloride (CHEL, 5 or 10 nmol), into DRN of freely moving rats promoted non-rapid eye movement sleep (NREMS) without influences on rapid-eye-movement sleep (REMS). Especially, CHEL application at 5 nmol increased light sleep (LS) time while CHEL application at 10 nmol increased slow wave sleep (SWS) time and percentage. On the other hand, microinjection of CaCl2 into DRN not only increased the phosphorylation level of PKC, but also reduced NREMS time, especially SWS time and percentage. While CHEL abolished the inhibitory effect of CaCl2 on NREMS and SWS. These data provide the first direct evidence that inhibition of intracellular PKC signaling in DRN could increase NREMS time including SWS time and percentage, while activation of PKC could suppress NREMS and reduce SWS time and percentage. These novel findings further our understanding of the basic cellular and molecular mechanisms of sleep–wake regulation. © 2015 Published by Elsevier Inc.
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1. Introduction
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Protein kinase C (PKC) is highly expressed within the brain, where it plays important roles in regulating neurotransmission, coordinating intracellular signaling in response to external stimuli, and modulating changes in gene expression and neuronal plasticity (Calabrese and Halpain, 2005; Ramakers et al., 1997; Zarate and Manji, 2009). Intensive studies on circadian clock have provided an insight into the role of PKC in it. Conventional Ca2+-sensitive PKC signaling pathway is not limited to relaying external stimuli but is rhythmically activated by internal processes, forming an integral part of the circadian feedback loop (Robles et al., 2010). The alternative between sleep and arousal is one of the most typical behaviors that have circadian rhythm. The tight relativity between PKC and circadian clock implies the potential role of PKC signaling in sleep–wake regulation. The PKC family is composed of twelve isozymes that are classified as either conventional, novel, or atypical according to the nature of their regulatory domains. The conventional PKCs (cPKCs) possess a Ca2+regulated domain that binds Ca2+ to be activated. cPKCs are designed
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Sheng-Jie Li 1, Su-Ying Cui 1, Xue-Qiong Zhang, Bin Yu, Zhao-Fu Sheng, Yuan-Li Huang, Qing Cao, Ya-Ping Xu, Zhi-Ge Lin, Guang Yang, Xiang-Yu Cui, Yong-He Zhang ⁎
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⁎ Corresponding author. Tel./fax.: +86 10 82801112. E-mail address: [email protected] (Y.-H. Zhang). 1 Sheng-Jie Li and Su-Ying Cui contributed equally to the present study.
to exactly decode two second messengers, Ca2+ and signaling lipids. cPKCs reside in the cytosol during resting periods waiting to be triggered by rises in intracellular Ca2 + concentration (Lipp and Reither, 2011). Circadian oscillations of free Ca2 + have been widely observed (Harrisingh et al., 2007; Imaizumi et al., 2007), which may be involved in the rhythmical activation of PKC. Several reports have revealed that Ca2 + oscillations drive oscillations of the activity of cPKCs (Bartlett et al., 2005; Violin et al., 2003). In addition, our recent study has demonstrated that the elevation of calcium function by BAY-K-8644, the L-type calcium channel agonist, and CaCl2 reduces NREMS and REMS, whereas down-regulation of Ca2 + function by diltiazem, the L-type calcium channel antagonist, and EGTA, the Ca2 + chelating agent in dorsal raphe nucleus (DRN) may promote NREMS especially the SWS percentage in pentobarbital treated rats (Cui et al., 2011a). And pharmacological evidences strongly suggest a correlation between L-type Ca2 + channel and serotonergic (5-HT) system in signaling transduction involved in sleep regulation (Cui et al., 2011b). Based on the important role of PKC on the circadian regulation, the pharmacological actions of calcium in DRN, and the fact that cPKCs can be activated by elevation of intracellular calcium concentration, we hypothesized that the intracellular PKC signaling in the DRN may be involved in the regulation of physiological sleep. In the present study, we investigated the role of PKC in DRN on sleep– wake regulation.
http://dx.doi.org/10.1016/j.pnpbp.2015.05.005 0278-5846/© 2015 Published by Elsevier Inc.
Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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2. Materials and methods
2.4. EEG and EMG recordings and analysis
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Male Sprague–Dawley rats (220–240 g, Grade I, purchased from the Animal Center of Peking University, Beijing) were used. All experiments were conducted in accordance with the European Community guidelines for the use of experimental animals and approved by the Peking University Committee on Animal Care and Use. The rats were housed in acrylfiber cages individually and had ad libitum access to food and water. They were exposed to a 12:12 h light/dark schedule with lights on at 9:00 A.M. The ambient temperature averaged 23 ± 1 °C and the relative humidity was 50 ± 10%.
Seven days after implantation the animals were adapted to the recording and injection procedures. For CHEL experiment, recording was started at 9 P.M. and lasted for 6 h during the night phase. For CHEL and CaCl2 combination experiment, recording was started at 9 A.M. and lasted for 6 h during the light phase. Sleep–wake states were manually classified as wakefulness, light sleep (LS), slow wave sleep (SWS) and rapid eye movement sleep (REMS). Non-rapid eye movement sleep (NREMS) time equals LS time plus SWS time. Total sleep (LS) time equals NREMS time plus REMS time. The mean number and duration of TS, NREMS, LS, SWS, and REMS were also quantified, as well as LS, SWS and REMS percentage in TS. Details of the EEG and EMG recordings and analysis were described previously (Wang et al., 2014).
2.5. Drugs and drug administration
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The following drugs were used in this study: chelerythrine chloride (CHEL), CaCl2 (both from Sigma-Aldrich, St. Louis, USA). CHEL inhibits PKC activation by blocking the site of DAG/phorbol-ester binding and inhibiting PKC translocation to the membrane for activation (Brennan et al., 2009). CHEL was dissolved in physiological saline and was microinjected into DRN either alone at 9 P.M. (5 or 10 nmol) or 20 min prior to CaCl2 administration at 5 nmol. CaCl2 was dissolved in saline and the pH of the solution was adjusted to 7.3 with NaOH. CaCl2 was microinjected into the DRN at 9 A.M. (25 or 50 nmol). Each agent was microinjected in a volume of 0.2 μl. The control groups in each experiment were microinjected with saline.
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The procedure was based on our previous study (Wang et al., 2015). For sleep deprivation experiment, immediately after 6 h sleep deprivation (3 P.M.), rats were decapitated, and the brains were quickly removed to a pre-chilled brain matrix. Ventrolateral preoptic nucleus (VLPO), dorsal raphe nucleus (DRN), and locus coeruleus (LC, with some of their surrounding tissue) were punched (1 mm diameter for VLPO, between 0 mm and 1 mm posterior to the bregma, bilaterally; 2 mm diameter for DRN, between 7 mm and 9 mm posterior to the bregma; 2 mm for LC, between 9 mm and 11 mm posterior to the bregma, bilaterally) using a hypodermic needle (12 guage) guided by the rat brain atlas of Paxinos and Watson. For CaCl2 administration experiment, DRN was dissected 3 h after CaCl2 administration (12 A.M.). All the
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Rats were handled by procedures as reported (Estabrooke et al., 2001). Sleep deprivation was performed by gentle handling while animals were kept in their home cages respectively. The rats were monitored continuously during sleep deprivation. They were aroused by tapping lightly on their cages or touching whenever they appeared to go to sleep. And the rats were never lifted out of the cages. When light tapping did not awaken the animals, crumpled paper was inserted into the cages to stimulate the rats. If needed, louder tapping of the cage was repeated. The deprivation started at light onset (9 A.M.) and lasted for 6 h and the rats were sacrificed at 3 P.M. Control undisturbed rats were sacrificed at the corresponding time point (3 P.M.).
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Details of the surgical procedure were described previously (Wang et al., 2014). The animals were implanted chronically with stainless steel screws over the frontal–parietal cortex and a pair of wire electrodes through the nuchal muscles for recording of electroencephalogram (EEG) and electromyogram (EMG), respectively. Additionally, a guide cannula (26 gauge) was implanted 1 mm above the DRN at coordinates, AP = − 8.0; L = 0.0 and DV = − 5.8 (Paxinos and Watson, 1998). Drug or vehicle was injected into the DRN with an injection cannula (29 gauge), which extended 1 mm beyond the guide, in a 0.2 μl volume over a 2 min period. Histological verification of cannula/ injection sites was carried out at the end of the experiments. All the data presented in the sleep recording experiments are derived from animals whose injection site was within the limits of DRN. The location of cannula/injection is shown in Fig. 1B.
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Fig. 1. Photomicrographs of representative cannula placements in DRN. Sections were according to Paxinos and Watson (1998).
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2.8. Statistical analysis
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The data were analyzed using SPSS software 17.0 and are expressed as mean ± S.E.M. The biochemical data for comparisons between the control and sleep-deprivation groups were analyzed using unpaired Student's t-test. For multiple comparisons, data were analyzed by oneway analysis of variance (ANOVA) followed by Student–Newman– Keuls post hoc test. In all of the tests, p b 0.05 was considered statistically significant.
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3.1. Effects of sleep deprivation on pPKC and PKC expression level
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A western blot analysis was performed to probe the pPKC and PKC expression level within different brain regions under physiological and 6 h sleep-deprivation conditions. The protein level of pPKC in DRN of the sleep-deprived (SD) group was markedly reduced (by 46.97%, t = 4.33, df = 6, p b 0.01, Fig. 2A) compared to that of the control group. This effect was regionally specific, as an analysis of pPKC expression within the VLPO and LC revealed no difference between control and sleep-deprived groups. Total PKC level revealed no difference between control and sleep-deprived groups in all the three brain regions.
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Microinjections of CHEL (5 or 10 nmol) into DRN augmented TS (by 73.40% and 55.14%, F2, 32 = 6.70, p b 0.01, respectively), with a
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Application of CaCl2 into DRN significantly reduced NREMS time. To determine whether PKC mediated the effect of CaCl2 on sleep–wake regulation, we used western blot to observe the changes of PKC phosphorylation level and total protein level in the DRN 3 h after CaCl2 administration. The results showed that the expression levels of pPKC were significantly increased (by 64.01% and 94.90%, F2, 9 = 14.56, p b 0.01, respectively) after microinjections of CaCl2 (25 or 50 nmol). While total PKC expression levels revealed no difference between saline and CaCl2 groups. These results indicated that the microinjection of CaCl2 into DRN increased the active form of PKC in DRN (Fig. 4).
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The protein concentrations of all samples were determined using the BCA assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin as the standard. Loading buffer (5 × SDS-PAGE Sample/Loading Buffer B1012, Applygen) was added to each sample before boiling for 5 min. Equal amounts of protein (25 μg) were separated by 10% SDSpolyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membrane (Millipore, MA, USA). The membranes were blocked overnight at 4 °C in TBST buffer (Tris-buffered saline + 0.1% Tween-20) supplemented with 5% bovine serum albumin (BSA), and incubated with primary antibodies, including anti-GAPDH (1:1000; sc25778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pPCK (1:1000; #9379, Cell Signaling Technology, MA, USA), and anti-PKC (1:1000; ab23511, Abcam, Cambridge, MA, USA), in TBS-T buffer containing 5% BSA at 4 °C overnight. After 3 × 10 min TBS-T washes, the blots were incubated with horseradish peroxidaseconjugated secondary antibodies (1:1000; Cell Signaling Technology, MA, USA) in TBS-T containing 5% nonfat dry milk for 2 h at 37 °C and then washed with TBS-T buffer for 3 × 10 min. The blots were then treated with an enhanced chemiluminescence detection kit (CoWin Bioscience, Beijing, China). Western blot bands were scanned with a GelDoc XR System (Bio-Rad, Hercules, CA, USA) and subsequently analyzed densitometrically with Image Lab software. The results were normalized to the protein expression level of GAPDH in each sample.
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significant increase in the number of TS episodes (by 63.36% and 68.95%, F2, 32 = 8.95, p b 0.001, respectively) and mean duration remaining unchanged. The results revealed no difference in REMS time or REMS percentage. NREMS time was significantly increased (by 76.55% and 58.48%, F2, 32 = 7.21, p b 0.01, respectively) after microinjections of CHEL (5 or 10 nmol) compared with vehicle control microinjections, which was attributed to the increase in number of NREMS episodes (by 64.43% and 58.03%, F2, 32 = 8.95, p b 0.001, respectively). Microinjection of 5 nmol concentration of CHEL prolonged LS time (by 74.18%, F2, 32 = 6.74, p b 0.01), due to an increased number of LS episodes (by 74.12%, F2, 32 = 4.79, p b 0.05). Microinjection of 10 nmol concentration of CHEL prolonged SWS time (by 405.09%, F2, 32 = 3.70, p b 0.05), increased number (by 243.75%, F2, 32 = 3.30, p b 0.05) and mean duration (by 51.62%, F2, 32 = 6.27, p b 0.01) of SWS episodes, as well as the percentage of SWS in TS (by 183.78%, F2, 32 = 3.85, p b 0.05) (Fig. 3).
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dissected tissues were stored separately in pre-chilled microcentrifuge tubes at −80 °C until assayed. Brain tissues were pooled and homogenized in ice-cold lysis buffer containing 50 mM Tris buffer (pH 7.2), 6 mM MgCl2, 1 mM EDTA, 10% (wt/vol) sucrose, 1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, 5 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, 5 μg/ml bestatin, 2 μg/ml E-64 and protein phosphatase inhibitor cocktail (All-in-One, 100 ×, P1260, Applygen). The homogenate was then sonicated (5 s × 3, 5 s interval) and ultracentrifuged (12,000 g) for 10 min at 4 °C. The supernatant was used as whole-cell extract.
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3.4. Effects of CaCl2 microinjection into DRN on sleep parameters in rats 240 pretreated with CHEL 241 To further verify the role of PKC in calcium induced sleep reduction, rats received PKC inhibitor before CaCl2 administration and then were subjected to recording session for 6 h (between 9:00 A.M. and 3:00 P.M.), as mentioned earlier. Significant decreases in TS time (by 25.31%, F3, 29 = 5.10, p b 0.01), NREMS time (by 25.29%, F3, 29 = 5.83, p b 0.01), SWS time (by 82.78%, F3, 29 = 4.37, p b 0.05) and SWS percentage (by 73.96%, F3, 29 = 4.82, p b 0.01) were observed after CaCl2(25 nmol) administration, which could be reversed by CHEL (5 nmol). Microinjection of CHEL (5 nmol) alone into DRN did not interfere with sleep structure of rats in the light period. These results indicated that pretreatment of CHEL in DRN, which blocked the function of PKC, could at least partially antagonize the sleep disturbance caused by CaCl2 administration (Fig. 5).
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The present study showed that 6 h sleep deprivation caused a lower phosphorylation level of PKC in DRN and the inhibition of PKC by the pan-PKC inhibitor CHEL in DRN significantly increased NREMS time, but did not influence REMS. Especially, CHEL application at 5 nmol increased LS time while CHEL application at 10 nmol increased SWS time and percentage. On the other hand, microinjection of CaCl2 into DRN not only increased pPKC expression, but also reduced NREMS time especially SWS time and percentage. While CHEL abolished the inhibitory effect of CaCl2 on NREMS and SWS. These results, for the first time, provide evidence that intracellular CHEL-sensitive PKC in DRN may play an important role in sleep–wake regulation. The sleep–wake cycle is a kind of homeostatic process which involves mutually inhibitory interactions between sleep and arousalpromoting systems, and is represented by the build-up of sleep pressure during wake and its decay in the course of sleep (Rachalski et al., 2014). It has been demonstrated that the activity of DRN 5-HT neurons was
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Fig. 2. Acute sleep deprivation decreased pPKC level in DRN in a regional-specific way. Representative of western blots and quantification of pPKC and PKC in DRN (A), VLPO (B) and LC (C). GAPDH was shown as quantitative loading control. Data are represented as mean ± S.E.M. (n = 6 pooled samples and repeated for 3–5 times, **p b 0.01 vs Control). SD, sleep-deprived group; DRN, dorsal raphe nucleus; VLPO, ventrolateral preoptic nucleus; LC, locus coeruleus.
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elevated initially and lowered during the later phase of sleep deprivation in cat (Gardner et al., 1997). The decline may be due to diminished arousal and/or increase in sleep pressure. It has been reported that 6 h sleep deprivation in the beginning of the 12-h light period prolonged NREMS and markedly enhanced EEG power density in the lowfrequency range (0.75–6.0 Hz) (Tobler and Borbely, 1990). The present study shows that 6 h sleep deprivation in rats significantly reduced phosphorylation level of PKC in DRN. The lower phosphorylation level of PKC in DRN may be related to the weaker activity of arousal promoting system and the reinforcement of sleep initiating system after sleep
deprivation. Consequently, the lower phosphorylation level of PKC in DRN after sleep deprivation may facilitate the body to initiate the subsequent recovery sleep and probably correspond to the rebound of NREMS especially SWS after 6 h sleep deprivation. Supporting evidence illustrates that DRN 5-HT and non-5-HT neurons are involved in sleep–wake regulation (Monti, 2010a). It is currently accepted that DRN 5-HT neurons function to promote wakefulness and to inhibit REMS (Monti, 2010b). As interneurons, most of DRN non-5-HT neurons modulate the activity of 5-HT neurons by expressing a variety of substances including GABA and glutamate (Monti, 2010a;
Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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Soiza-Reilly and Commons, 2014). Previous studies provide persuasive evidence which indicated that the effect of non-5-HT neurons in DRN on sleep–wake regulation was eventually mediated by 5-HT neurons (Monti, 2010a). Previous reports also showed that activation of PKC signaling pathway could potentiate the function of serotonergic system. PKC activation induces phosphorylation of serotonin transporter (SERT), which inhibits SERT activity (Jayanthi et al., 2005). Besides, PKC induces phosphorylation and desensitization of 5-HT1A receptor (Raymond, 1991). The activation of PKC could disinhibit 5-HT1A receptor's inhibitory effect on serotonergic neurons. Studies indicated that SERT and 5-HT1A are closely related to sleep regulation (Alexandre et al., 2006; Wisor et al., 2003), which implies the involvement of PKC signaling pathway. The present study shows that intraDRN application of the PKC signaling activation inhibitor, CHEL,
increased NREMS time, especially SWS time and SWS percentage. Considering the important role of DRN 5-HT neurons on sleep–wake regulation and the function of PKC on serotonergic system, we hypothesized that the hypnotic effect of CHEL was related to down regulation of PKC signaling on serotonergic system through downstream substrates of PKC. Previous studies reported that microinjection of certain neurotransmitters/neuromodulators or their receptor agonists (including glutamate, serotonin, melanin-concentrating hormone, dopamine, orexin), which could modulate PKC, into DRN caused alterations in sleep variables (Gao et al., 2002; Kohlmeier et al., 2004; Lagos et al., 2009; Monti, 2011; Tao et al., 2006). For example, microinjection of glutamate into DRN induced enhancement of wakefulness, and reduced TS time, especially SWS time (Gao et al., 2002). Hcrt/Orx produced a dose-
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Fig. 3. Microinjection of CHEL into DRN altered sleep parameters. (A) Total sleep (TS), non-rapid eye movement sleep (NREMS), light sleep (LS), slow wave sleep (SWS) and rapid eye movement sleep (REMS), LS percentage (LS%), SWS percentage (SWS%) and REMS percentage (REMS%). (B) Number and mean duration of TS, NREMS, LS, SWS episodes (n = 11–15/group). Data are represented as mean ± S.E.M. (*p b 0.05 and **p b 0.01 vs Saline, #p b 0.05 vs CHEL 5 nmol). CHEL, chelerythrine chloride.
Fig. 4. Microinjections of CaCl2 (25 or 50 nmol) into DRN increased pPKC, but not PKC protein levels. Representative of western blots and quantification of pPKC and PKC in the DRN were shown. GAPDH was shown as quantitative loading control (n = 6 pooled samples and repeated for 4 times). Data are expressed as mean ± S.E.M. (**p b 0.01 vs saline).
Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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In summary, the molecular, pharmacological, and behavioral data of this study, for the first time, demonstrate a novel wake-promoting and SWS-suppressing role for PKC signaling within DRN neurons. These findings are critical for our complete understanding of the basic cellular
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and molecular mechanisms of sleep–wake regulation. However, the heterogeneity of PKC isoforms raises the possibility that the supposed PKC activity imbalance may actually consist in a more complex schema, where the various isozymes would be differentially involved in the regulation of sleep–wake cycle. Future studies are therefore needed to clarify the respective contribution of specific isozymes to sleep–wake regulation and to mood disorders.
This study was funded by grants from the National Natural Science 375 Foundation of China (Nos. 81173031, 81202511 and 81302746). 376 References
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dependent elevation of calcium concentration and serotonin in significant numbers of neurons in DRN (Kohlmeier et al., 2004; Tao et al., 2006). Microinjection of PKC inhibitor, CHEL, into DRN could block the effects of endogenous neurotransmitters/neuromodulators, which may be possible mechanisms underlying the hypnotic effect of CHEL. However, CHEL induces apoptosis by causing rapid release of cytochrome c from mitochondria (Wan et al., 2008). Although the apoptosis was detected 16 h after CHEL application, the rapid release of cytochrome c from mitochondria could possibly lead to reductions of ATP synthase and ATP availability in the neurons and further make neurons cease firing. Although Nissl staining revealed no obvious changes in cell morphology and the number of neurons in the DRN between saline and CHEL (5 or 10 nmol) groups (not show), we could not exclude the possibility that the hypnotic effect of CHEL limited in 6 h was induced by its toxic effect on serotonergic neurons. The PKC family is classified as either conventional, novel, or atypical as mentioned earlier. Only cPKCs possess a Ca2+-regulated domain that binds Ca2+ to be activated. Other types are all activated in a calcium independent manner. Intracellular Ca2+ plays an important role in the activation of Ca2 +-dependent PKC. Our previous study proved that Ca2 + in DRN plays an important role in sleep–wake regulation. And the present study indicates that the arousal effect of Ca2+ in DRN was associated with PKC signaling pathway. Administration of CaCl2 into DRN significantly increased the expression level of pPKC, and decreased NREMS time, especially SWS time and SWS percentage. The intra-DRN application of PKC inhibitor CHEL reversed the arousal effect of CaCl2 by recovery of NREMS and SWS time. These results indicate that the activation of PKC signaling in the DRN suppresses NREMS and could decrease sleep intensity. Thus, there is a clear rationale to suggest that the inhibition of PKC signaling in the DRN may promote NREMS, especially SWS and SWS percentage at higher dose (10 nmol), but activation of PKC may suppress NREMS and reduce SWS time and percentage. Through phosphorylation of a large variety of substrates, PKC is able to modulate a multiplicity of neuronal functions, such as neurotransmitter synthesis and release, gene expression, ion channel and receptor regulation. A growing body of studies have identified the involvement of PKC in bipolar disorder (DiazGranados and Zarate, 2008). In addition, PKC is a cellular target of most current mood stabilizing and antimanic agents (Abrial et al., 2013; Jensen and Mork, 1997; Sabioni et al., 2008). The data of the present study raise the question whether PKC modulation may also be effective on the sleep disorders or the mood disorders associated with sleep disorders.
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Fig. 5. Microinjection of CHEL into DRN eliminated the inhibitory effect of CaCl2 on non-rapid eye movement sleep (NREMS) and slow wave sleep (SWS). Sleep parameters including total sleep (TS) time, NREMS time, light sleep (LS) time, SWS time, rapid eye movement sleep (REMS) time, LS percentage (LS%), SWS percentage (SWS%) and REM sleep percentage (REMS%) were assessed (n = 7 ~ 10/group). Data are expressed as mean ± S.E.M. [*p b 0.05 and **p b 0.01 vs Saline + Saline; #p b 0.05 vs Saline + CaCl2 (25 nmol)]. CHEL, chelerythrine chloride.
Please cite this article as: Li S-J, et al, PKC in rat dorsal raphe nucleus plays a key role in sleep–wake regulation, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.005
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