Huang et al. BMC Neurosci (2016) 17:74 DOI 10.1186/s12868-016-0309-0 BMC Neuroscience RESEARCH ARTICLE Open Access Interleukin‑1 receptor (IL‑1R) mediates epilepsy‑induced sleep disruption Tzu‑Rung Huang1†, Shuo‑Bin Jou2†, Yu‑Ju Chou1, Pei‑Lu Yi3*, Chun‑Jen Chen4 and Fang‑Chia Chang1,5,6* Abstract Background: Sleep disruptions are common in epilepsy patients. Our previous study demonstrates that homeo‑ static factors and circadian rhythm may mediate epilepsy-induced sleep disturbances when epilepsy occurs at different zeitgeber hours. The proinflammatory cytokine, interleukin-1 (IL-1), is a somnogenic cytokine and may also be involved in epileptogenesis; however, few studies emphasize the effect of IL-1 in epilepsy-induced sleep disrup‑ tion. We herein hypothesized that IL-1 receptor type 1 (IL-1R1) mediates the pathogenesis of epilepsy and epilepsy- induced sleep disturbances. We determined the role of IL-1R1 by using IL-1R1 knockout (IL-1R1 / KO) mice. − − Results: Our results elucidated the decrease of non-rapid eye movement (NREM) sleep during the light period in IL-1R / mice and confirmed the somnogenic role of IL-1R1. Rapid electrical amygdala kindling was performed to induce− epilepsy− at the particular zeitgeber time (ZT) point, ZT13. Our results demonstrated that seizure thresholds induced by kindling stimuli, such as the after-discharge threshold and successful kindling rates, were not altered in IL-1R / mice when compared to those obtained from the wildtype mice (IL-1R / mice). This result suggests that IL−-1R1− is not involved in kindling-induced epileptogenesis. During sleep, ZT13+ kindling+ stimulation significantly enhanced NREM sleep during the subsequent 6 h (ZT13-18) in wildtype mice, and sleep returned to the baseline the following day. However, the kindling-induced sleep alteration was absent in the IL-1R / KO mice. − − Conclusions: These results indicate that the IL-1 signal mediates epilepsy-induced sleep disturbance, but dose not participate in kindling-induced epileptogenesis. Keywords: Amygdala, Epilepsy, IL-1 receptor, Kindling, Sleep Background acting at the basal forebrain [11] and the ventrolateral Epilepsy results from the imbalance of excitability and preoptic area (VLPO) [12]. Two types of IL-1 receptors inhibition of neuronal networks [1, 2]. Patients with dif- (IL-1Rs) have been identified: the type 1 receptor (IL- ferent types of epilepsy may experience either daytime 1R1) and the type 2 receptor (IL-1R2). IL-1R1 domi- sleepiness or nighttime sleep disturbance [3–6]. The nantly distributes throughout the brain and carries out prevalence of sleep disorders, such as excessive daytime the main function of NREM sleep enhancement [13]. sleepiness [7, 8], insomnia [9, 10] and obstructive sleep IL-1R1 KO mice decrease sleep during the dark period apnea [8, 10] is higher among patients with epilepsy. of the light:dark cycle when compared with the wildtype Interleukin-1β (IL-1β), one of the somnogenic factors, mice [13]. IL-1 is also identified as one of the proin- enhances non-rapid eye movement (NREM) sleep by flammatory cytokines, which leads to the pathogenesis of epilepsy during inflammation. The concentrations of IL-1β increase and the number of IL-1 receptors are *Correspondence: [email protected]; [email protected] †Tzu-Rung Huang and Shuo-Bin Jou contributed equally to this work and up-regulated during seizures [14, 15]. Existing evidence should be considered cofirst authors indicates that the increased concentrations of IL-1β may 1 Department of Veterinary Medicine, School of Veterinary Medicine, affect the seizure threshold, but the role of IL-1β in the National Taiwan University, No. 1, Sec. 4., Roosevelt Road, Taipei 106, Taiwan 3 Department of Sport Management, College of Tourism, Leisure epileptogenesis is still controversial, as both a proconvul- and Sports, Aletheia University, New Taipei City, Taiwan sant and an anticonvulsant have been suggested. Grow- Full list of author information is available at the end of the article ing evidence supports IL-1 as a proconvulsant substance. © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. BMC Neurosci (2016) 17:74 Page 2 of 8 Seizure activities are inhibited after the injection of the experiments and to confirm any genetic drift during breed- IL-1 receptor antagonist (IL-1ra) and seizures are also ing (the detail genotyping methods as described later). All suppressed in mice with over-expressed IL-1ra [14, 16]. experiments and animal care were performed following On the other hand, the anticonvulsant effects of IL-1 are the principles outlined in the Institutional Animal Care also suggested in other studies. IL-1 augments the effect and Use Committee (IACUC) of National Taiwan Univer- of gamma-aminobutyric acid (GABA)-A receptors and sity. Mice were anesthetized with zoletil (20 mg/kg, i.p.) increases the pentylenetetrazol (PTZ)-induced seizure and xylazine (12 mg/kg, i.p.), treated with antibiotics (pen- threshold in mice [17]. Intracerebroventricular (ICV) icillin G benzathine) to prevent infection, and surgically injections of IL-1β suppress the amygdaloid kindling- implanted with two electroencephalographic (EEG) elec- induced seizures in rats [18]. Furthermore, IL-1 exhibits a trodes (wire-wrapping-wire 30 AWG) and a bipolar stimu- temporal and dose-dependent influence on sleep regula- lating electrode. The placements of EEG electrodes were tion. Low doses of IL-1 increase NREM sleep during the at the left frontal lobe and right parietal lobe. The bare night and slow wave activity during the day; in contrast, ends of the insulated leads from the EEG electrodes were high doses of IL-1 suppress NREM sleep during the day connected to Dupont female terminals and a 2.54 mm 2P and slow wave activity at night [19]. The opposite actions Dupont connector. Bipolar insulated electrodes (model of high- and low-doses of IL-1 may explain the divergent # M148340, California fine wire company, Grover beach, effects. Because of the conflicts between those observa- CA) were placed in the left basolateral nucleus of the tions, the role of IL-1 in epileptogenesis needs to be fur- amygdala (BLA) as the target of kindling stimuli. The coor- ther determined. We herein hypothesized that IL-1R1 dinates of the BLA were 1.9 mm caudal to bregma, 2.8 mm mediates the pathogenesis of epilepsy and the epilepsy- lateral to bregma, and 4.6 mm ventral to the dura [22]. The induced sleep disturbances. In this study, we examined Dupont connector and bipolar electrodes were cemented IL-1R1 in the kindling-induced epileptogenesis and sleep to the skull with dental acrylic (Tempron, GC Co., Tokyo, disruptions by employing IL-1R1 −/− mice. Japan). Ibuprofen was added to their drinking water for Our previous results have demonstrated that epi- 5 days after the surgery to reduce pain. Five days after the lepsy occurring at different zeitgeber time (ZT) points EEG implantation, the Dupont connector was connected alters sleep differently [20, 21]. Kindled epilepsy at ZT0 to the amplifier system for habituation. All animals were decreases NREM sleep during the light period; in con- housed separately in a recording cage and were housed in trast, kindling stimulation at ZT13 increases NREM a 12:12 h light:dark (L:D) cycle in an isolated room where sleep during the dark period [20]. At ZT0, corticotropin- the temperature was maintained at 23 ± 1 °C. Food and releasing hormone (CRH) mediates kindling-induced water were provided ad libitum. NREM sleep reduction, and at ZT13, the increases in IL-1 are attributed to kindling-induced NREM sleep PCR genotyping enhancement [20]. Furthermore, we further found that For the PCR genotyping analysis in each mouse, 0.5 cm at ZT6, kindling epilepsy shifts the fluctuation of period sections of the tail tips were dissolved in 0.2 ml of Direct- circadian protein homolog 1 protein (PER1) in the supra- PCR Lysis Reagent (Viagen Biotech) and 0.5 mg/ml of chiasmatic nucleus (SCN) of the hypothalamus and alters proteinase K (Roche) solutions under the following con- sleep circadian rhythm [20]. These results suggest that ditions: 55 °C for 6–7 h, 85 °C for 45 min, 25 °C for 5 min epilepsy may alter either homeostatic factors or circadian (1 cycle), and then precipitated by centrifuging for 10 s. rhythms to cause sleep disturbances. In this study, we One μl of lysate was used for 50 μl PCR reactions with further determined our hypothesis that at ZT13, IL-1R1 MyTaq HS Mix (Bioline, Taunton, MA). We used the mediates kindling-induced sleep disruption by using primers recommended by Jackson Laboratory for geno- IL-1R1 +/+ and IL-1R1 −/− mice. typing (Table 1). The expected PCR band for IL1R1+ /+ is 310 bps and the band for IL-1R1 / is 150 bps. The Methods − − heterozygous (IL-1R+/−) mice revealed both bands of Animals 310 and 150 bps. PCR was performed by a C1000 ther- Male C57BL/6 (IL-1R1 +/+ and IL-1R1 −/−) mice (4- mocycler (BioRad, Hercules, CA). The parameters for to 6-weeks old) were used in present study. The original reaction temperature cycles were 94 °C for 2 min, and IL-1R1 −/− KO mice were obtained from Jackson Labo- then 10 cycles of 94 °C for 20 s, 65 °C for 15 s (−0.5 °C ratory (strain: B6.129S7-I/1r1tm1Imx/J) and bred in house.