Occurrence of epilepsy at different zeitgeber times alters sleep homeostasis differently in rats
Pei-Lu Yi, MSc1,2, Ying-Ju Chen1, MSc, Chung-Tien Lin, D.V.M., Ph.D.1*, Fang-Chia Chang, Ph.D.1,3,4 * 1 Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University,
Taipei, Taiwan
2 Department of Sports, Health & Leisure, College of Sports Knowledge, Aletheia University, Matou
Campus, Taiwan
3 Graduate Institute of Acupuncture Science, College of Chinese Medicine, China Medical University,
Taichung, Taiwan
4 Graduate Institute of Brain and Mind Sciences, National Taiwan University, Taipei, Taiwan
Abbreviated title: Epilepsy alters sleep homeostasis
Corresponding author: Fang-Chia Chang, Ph.D. & Chung-Tien Lin, D.V.M., Ph.D., Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4., Roosevelt Road, Taipei 106, Taiwan
Tel: +886-2-3366-3883; Fax: +886-2-2366-1475, Email: fchang@ ntu.edu.tw
All of authors contributed equally to this work.
Conflict of Interest: This is not an industry supported study. Authors have indicated no financial conflicts of interest.
A
cknowledgments
Authors thank Mr. Brian Chang for his help with English revision and Mr. Yi-Fong Tsai’s technical assistance in this project. This work was supported by National Science Council grant NSC99-2320-B-002-026-MY3.
ABSTRACT
Study Objectives: Controversial sleep disruptions (e.g. poor nighttime sleep and daytime somnolence) are common in epilepsy patients. Sleep is known to be regulated by homeostatic factors, which mediate sleep propensity, and circadian oscillator, a clocklike mechanism. However, how epilepsy occurred at different zeitgeber times (ZTs) alters sleep regulation remains unknown. Current study was designed to elucidate the sleep disruptions and their underlying mechanisms by delivering kindled epilepsy at different ZTs－ZT0, ZT6 and ZT13.
Design: Kindled epilepsy was induced at three different ZTs, and sleep-wake activities were analyzed before and after full-blown seizure. Ribonuclease protection assay, radioimmunoassay and
immunohistochemistry were respectively employed to determine the levels of interleukin-1 mRNA, corticosterone and PER1 protein.
Setting: The experiments were performed at Neurophysiology Laboratory at National Taiwan University. Participant and Interventions: Male Sprague-Dawley rats were implanted with electroencephalogram (EEG) electrodes, a bipolar stimulating electrode and a guide cannula. Kindling stimuli delivered via a bipolar electrode placing in the right central nucleus of amygdala.
Measurement and Results: Kindled epilepsy occurring at ZT0 and ZT13 predominantly affected the homeostatic factors, whereas ZT6-kindling stimuli altered the circadian oscillator. ZT0-kindling decreased rapid eye movement (REM) and non-REM (NREM) sleep, which was mediated by
corticotrophin-releasing hormone, but did not alter the rhythm of sleep fluctuation. On the other hand, ZT13-kindling enhanced interleukin-1 and consequently increased NREM sleep, without altering the sleep-wake fluctuation. Nevertheless, the expression of PER1 protein in suprachiasmatic nucleus of the
hypothalamus and the circadian rhythm of sleep fluctuation were respectively shifted 6 and 2 hours in advance when kindling stimulation was delivered at ZT6. Shifts of sleep circadian rhythm and PER1 oscillation induced by ZT6-kindling were blocked by administration of hypocretin receptor antagonist, SB334867, into the SCN, indicating the involvement of hypocretin.
Conclusion: These observations suggest that the occurrence of epilepsy at different ZTs alters sleep processes differently.
INTRODUCTION
According to the two-process model, sleep is regulated by two oscillatory processes: the sleep
homeostasis (process S) and the circadian pacemaker (process C).1,2 The homeostatic process S mediates
the rise of sleep propensity during waking and dissipates during sleep. Circadian process C is a clocklike mechanism which is independent of prior sleep-wake activities and determines the alteration of periods with high and low sleep propensity.2 Slow wave activity (SWA) during slow wave sleep (SWS) and other
circadian fluctuated factors (e.g. interleukin (IL)-1, tumor necrosis factor (TNF)-,3,4
corticotrophin-releasing hormone (CRH)5,6 and growth hormone releasing hormone (GHRH)7 may represent the
homeostatic variable S. On the other hand, the circadian pacemaker, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, coordinates daily oscillations in the transcription and translation of clock-component genes, the periods (Pers), cryptochromes (Crys), Clock and Bmal1 genes.8 The
circadian clock system is entrained by time cues and photic signals.8
Clinical and experimental observations demonstrate that sleep and epilepsy reciprocally affect and influence each other. Much attention has been focused on the influence of sleep (e.g. rapid eye movement (REM) and non-REM (NREM) sleep) on epileptiform discharges and seizure occurrences.9,10
Nonetheless, disturbances in the sleep-wake continuum in patients with epilepsy are common but often overlooked. Many patients with different types of epilepsy controversially experience either daytime somnolence or insufficient nocturnal sleep.11-14 These contradictory sleep disturbances caused by epilepsy
sleep,15 and another study observed increases in deep SWS and decreases of light SWS and wakefulness
after full-blown seizures.16 These conflicting results may be due to the lack of control for seizures or
medication in humans, or because of the manipulation of kindling at different zeitgeber times (ZTs) in animal studies. Epilepsy-induced sleep disruptions may be attributed to alterations in homeostatic factors or the circadian process, or both. However, little evidence has been found in literature to support this. The present study was therefore designed to systematically explore the nature and underlying mechanisms of amygdaloid kindling, which resembles the temporal lobe epilepsy (TLE),-induced alterations in sleep at three different ZTs: ZT0, ZT6 and ZT13. Melatonin or temperature rhythms was not altered in TLE patients,17 while one animal study demonstrated that the phase of temperature rhythm was shifted after
seizure in free-running rats.18 These suggest that the zeitgeber may mask the effect of epilepsy on the
circadian rhythm. In order to simulate clinical situation, rats in current study were well accommodated to the 12:12h light:dark (L:D) cycle, as opposed to being in an environment lacking of time cues (e.g., constant light or constant dark). Our previous results suggest that epilepsy enhances both CRH and IL-1 concentrations in the CNS and the alteration of sleep-wake activity is determined by the out-of-phase fluctuation between CRH and IL-1.19 Based upon the nature of CRH and IL-1 in the sleep-wake
regulations,3-6 we further investigated the role of CRH and IL-1 in the ZT0 and ZT13 kindling-induced
sleep alteration, respectively. Furthermore, the topographic organization of projections from central nucleus of amygdala (CeA) to lateral hypothalamic area (LHA) has been confirmed,20 and hypoccretin
receptors type 1 (HcrtR1) are dominantly distributed in the SCN.21 A circadian phase shift could be
shift circadian rhythm and the effect is mediated by hypocretin in the SCN.
Materials and Methods Substances
Stock solutions of CRH receptor antagonist astressin (Bachem, Torrance, CA, USA) and human recombinant IL-1 receptor antagonist (IL-1 ra) (Bachem) were dissolved in pyrogen-free saline (PFS). Hypocretin receptor type 1 (HcrtR1) antagonist, N-(2-methyl-6-benzoxazolyl)-N’-1,5-naphthyridin-4-yl urea (SB334867) (Tocris, Bristol, UK) was dissolved in 50 % dimethyl sulfoxide (DMSO, Sigma, St. Louis, USA). The doses of substances used in these experiments were as follows: 0.5, 2.5 and 12.5 g for astressin; 0.01, 0.1 and 0.25 g for human rec IL-1 ra; 1.5, 3.0 and 6.0 g for SB334867. Both astressin and IL-1ra were administered via intracerebroventricular (ICV) route, whereas SB334867 was directly microinjected into the SCN (AP, -1.4 mm from bregma; ML, 0.3 mm; DV, 9.3 mm).23 The total volume
for ICV and SCN administration was 3 and 1 l, respectively. ICV and SCN administration were given 20 minutes prior to ZT0, ZT6 or ZT13.
Animals
Male Sprague-Dawley rats (250 - 300 g; National Laboratory Animal Breeding and Research Center, Taiwan) were anesthetized by ketamine/xylazine (87/13 mg/kg), with an analgesic (morphine) and an antibiotic (penicillin G benzathine) to reduce pain and avoid infection. Rats were implanted with three electroencephalogram (EEG) electrodes and a thermistor (model: Pt100; Enercrop, Toronto, Canada) as previously described.24 Either an ICV guide cannulae (AP: -1.0 mm from bregma; ML: 1.6 mm; DV: 3.5
mm) or a SCN microinjection guide cannulae (AP, -1.4 mm from bregma; ML, 0.3 mm; DV, 9.3 mm) was implanted.23 The insulated leads from the EEG electrodes were routed to a Teflon pedestal (Plastics
One, Roanoke, VA, USA). Additional implantation of a bipolar electrode (model # MS303/9, Plastics One, Roanoke, VA) was placed in the right CeA; the coordinates were as follows: AP, -2.0 mm from bregma; ML, 4.0 mm; DV, 8.0 mm.23 The Teflon pedestal and bipolar electrode were then cemented to
the skull with dental acrylic (Tempron, GC Co., Tokyo, Japan). The incision was treated topically with polysporin (polymixin B sulfate – bacitracin zinc), and the animals were allowed to recover for seven days prior to the initiation of experiments. The rats were housed separately in individual recording cages in an isolated room, in which the temperature was maintained at 23 ± 1 °C and the L:D rhythm was controlled in a 12:12 h cycle (40 Watt x 4 tubes illumination). Food and water were available ad libitum. All procedures performed in this study were approved by the Institutional Animal Care and Use
Committee of National Taiwan University.
On the second postsurgical day, rats were connected to the recording apparatus via a flexible tether. Three days after surgery, the patency and free drainage of the ICV cannulae for rats implanted with ICV guide cannulae was assessed by administering 200 - 400 ng of angiotensin II (Tocris); angiotensin elicits a drinking response mediated by structures in the preoptic area.25 Rats implanted with ICV guide cannulae
were again injected with angiotensin at the end of each experimental protocol; only data from those rats that elicited a positive drinking response were included in the subsequent analyses. The location of the SCN microinjection cannulae was confirmed by injecting 0.5 % trypan blue dye at the end of experiment.
After recovery from surgery, the digitized EEG waveform and integrated values for body movement and brain temperature were recorded and stored as binary computer files pending for subsequent analyses. Postacquisition determination of the vigilance state was done by the visual scoring of 12-s epochs using custom software (ICELUS, M. R. Opp) written in LabView for Windows (National Instruments). The animal’s behavior was classified as either NREM sleep, REM sleep or waking based on previously defined criteria.24
Kindling manipulation
Seven days after surgery, rats were subjected to a kindling procedure. A stimulator-isolator unit (A360 Stimulus Isolator, World Precision Instruments, Sarasota, FL, USA) triggered by a main stimulator (Accupulser A310, World Precision Instruments) was used to deliver kindling stimuli. Kindling stimulus was performed in two different protocols, the traditional kindling stimuli26,27 and the rapid kindling
stimuli28. For the traditional kindling protocol, each kindling stimulus was simultaneously applied once a
day to the right CeA. The stimulus was a train of biphasic pulses (1 milliseconds duration each) of 50 Hz for 1 second. The intensity was ranged from 50 to 200 A. The kindling stimulation was performed once a day either at ZT0 or ZT13. Criteria for establishing the presence of a kindled seizure required
behavioral seizures that attained stage 5 by Racine’s criteria29 as well as the appearance of continuous
epileptiform EEGs as shown in Figure 1A. For rats underwent traditional kindling protocol, three to four weeks were required until Racine’s stage 5 (full-blown) seizure developed. In addition to the requirement
(Figure 1B). The epileptiform EEG discharges were simultaneously monitored and analyzed with the sleep-wake EEGs during the whole experimental protocol. Spontaneous interictal epileptiform EEGs after ZT0 kindling or ZT13 kindling stimuli were respectively demonstrated in Figure 1C and 1D. In order to shorten the manipulation time and to reduce spontaneous epileptiform EEGs, a rapid kindling protocol was employed to precisely induce kindled epilepsy at ZT6. The stimulus for rapid kindling protocol was a train of biphasic pulses of 20 Hz for 10 second, the standard rate for train presentation was once every 5 minutes, the total of 60 stimulation trains was given with 5 hours in a day, and this protocol was
continued for 5 hours in the next day until full-blown seizure appears. The intensity was ranged from 100 to 300 A. As long as the full-blown seizure appears, 1 second of a stimulation train successfully induced continuous epileptiform EEGs (Figure 1E). Secondary spontaneous epileptiform EEGs were seldom observed when the stimuli were absent (Figure 1F). Both traditional and rapid kindling stimuli successfully developed stage-5 full-blown seizures in 60-80% of rats which received the kindling protocol. Rats which exhibited full-blown seizures in two consecutive days after kindling protocol were recruited in current study.
Experimental p rotocols
Six distinct groups of rats were used. A diagram was delineated in Figure 2. Rats in groups 1, 3 and 5 (n = 8 for each group) were respectively used to determine the sleep-wake alterations induced by CeA kindling occurring at ZT0, ZT6 and ZT13, and to demonstrate the involvement of CRH, hypocretin and IL-1. Rats in groups 2 (n = 24), 4 (n = 72) and 6 (n = 12) were employed to elucidate the concentrations of
plasma corticosterone, SCN PER1 protein and IL-1 mRNA, respectively, after ZT0-, ZT6- and ZT13-kindling stimuli. Rats in group 1 received ICV administration of PFS prior to ZT0, and a 23-h continuous EEG was recorded as a vehicle control. Then, the traditional kindling manipulation was performed. Until stage 5 seizures appeared, rats were given PFS injection combined with ZT0-kindling stimuli on the 1st
recording day. ICV administration of three doses of astressin, 0.5, 2.5 and 12.5 g, prior to the kindling stimulation was randomly initiated on the subsequent 2nd, 3rd and 4th recording days, respectively. All
recordings began at ZT0 and continued for 23 h. Group 1 was used to determine the sleep alteration after ZT0 kindling stimuli and the effect of CRH receptor antagonist astressin in the ZT0 kindling-induced sleep alteration. Rats in group 2 were employed to determine the corticosterone concentrations and were sub-divided into three groups. Rats in sub-group 1 received PFS ICV administration without kindling stimuli. Others in sub-groups 2 and 3 underwent traditional kindling protocol and respectively received ICV injections with PFS and 12.5 g astressin at ZT0 on the experimental day. Rats were decapitated at ZT1 after manipulations. Trunk blood was collected for corticosterone radioimmunoassay as previously described.19 Group 3 was used to determine the sleep alteration after ZT6 kindling stimuli and the
involvement of hypocretin in the ZT6 kindling-induced sleep alteration. Rats in group 3 were
microinjected with PFS and DMSO at ZT6, and sleep recordings began at ZT6 and lasted for 42 hours. After full-blown seizure developed, SCN administration of DMSO and one-second single train
stimulation was given at ZT6. Thereafter, three similar manipulations and recordings were employed, except that DMSO was substituted with three different doses (1.5, 3.0 and 6.0 g) of SB334867. Three doses of SB334867 were randomly given at three injection times as shown in Figure 2. Rats in group 3
received total of 5 times of 50 % DMSO administrations (1 l for each injection) and the interval between administrations was at least 42 hours. Rats in group 4 were used to determine the expression of PER1 protein in the SCN and were divided into three sub-groups. In sub-group 1, rats were gently handled at ZT6 and decapitated at ZT18, ZT’0, 3, 6, 9 and 12. Brains were removed and tissue blocks containing SCN of hypothalamus were dissected and fixed in 10 % neutral buffered formalin for at least 36-48 hours. These tissue blocks were embedded in paraffin wax. Sections of 6 m brain slice were prepared and used for PER1 immunohistochemistry (IHC). Rats in sub-groups 2 and 3 received the similar protocol as those in sub-group 1, except that these rats were rapidly kindled and developed full-blown seizure before experiments. Rats in sub-group 3 randomly received SB334867. Group 5 was employed to determine the sleep alteration after ZT13 kindling stimuli and the effect of IL-1 receptor antagonist in the ZT13
kindling-induced sleep alteration. Rats in group 5 received all injections and traditional kindling manipulation at ZT13, in which rats were ICV administered IL-1ra in doses of 0.01, 0.1 and 0.25 g. Three doses of IL-1ra were randomly given at three injection times as shown in Figure 2. Rats in group 6 were used to determine the effects of kindling on cytokine mRNA expression in brain. They were
maintained under the same conditions as those of group 5. Traditional kindling protocol was performed in sub-group 2, but not in the sub-group 1. Rats in sub-group 1 were decapitated at ZT15. Rats in sub-group 2 were injected with PFS with kindling stimulation at ZT13 and decapitated at ZT15. Hippocampus and a section of the parietal cortex were then dissected out and the content of IL-1 mRNA was then determined. We used a ribonuclease protection assay (RPA), as previously described,19 to detect and quantify specific
PER1 immunohistochemistry (IHC)
The coronal section of brain slices containing SCN region was sliced at 6 m thickness as previously described. Six slices obtained from the middle of the SCN block in one rat were used for the PER1 IHC. The Super SensitiveTM Non-biotin Polymerized Horseradish-peroxidase (Polymer-HRP) Detection
System (BioGenex Laboratories, San Ramon, CA, USA) was employed for PER1 protein detection. The primary antibody was anti-PER1 polyclonal antibody (PA1-524, Thermo Scientific, Waltham, MA, USA). Tissue sections were dewaxed in xylene and rehydrated in a graded alcohol series (100 %, 95 %, 90 %, 80 % and 60 %). Antigen unmasking was performed by immersion of sections in ethylene
diaminetetraacetic acid (EDTA) and boiling for 15 minutes at 121 C in an autoclave. Sections were then rinsed in Tris-buffered saline (TBS). Endogenous peroxidase activity was quenched with 3 % hydrogen peroxide in methanol (Merck, Darmstadt, Germany) for 10 minutes at room temperature. This is followed by rinsing in TBS and incubating in 5 % normal horse serum in phosphate buffered saline (PBS) with 0.3 % Trition X-100 for 1 hour at room temperature, which is capable of reducing the nonspecific
background staining. The primary antibody, anti-PER1 polyclonal antibody, was diluted by 5 % normal horse serum in 1/500 ratio and was then applied to the brain slices for 24 hours at 4 C. The sections were then rinsed in TBS, incubated with Super EnhancerTM (BioGenex) for 60 minutes at room temperature,
rinsed in TBS, and incubated in Polymer-HRP reagent (BioGenex) for 60 minutes at room temperature. The subsequent procedures consisted of rinsing in TBS once more, incubating with AEC (3-amino, 9 ethyl-carbazole) chromogen solution (BioGenex) for 3-5 minutes at room temperature, counterstaining
with GM hematoxylin stain solution (H3136, Sigma-Aldrich, St. Louis, MO, USA), and mounting with Super Mount® (BioGenex). The sections were then examined under microscope (Olympus BX 501,
Tokyo, Japan) and the images were photographed by DP2-BSW (Olympus). The number of PER1-positive cells in one rat was counted by Image-Pro Plus 5.1 (Media Cybernetics, Silver Spring, USA), and was determined by averaging the cell numbers of the six slices. The cell counting was based upon the assumption-based technique. While using stereological technique for cell counting may reduce the bias, assumption-based counting techniques have been used in most studies in estimating neurons and synapse number.30
Statistical a nalyses
All values are presented as mean ± SEM. One-way analyses of variance (ANOVA) for the duration of sleep states were performed across each 4-h, 6-h and 12-h time blocks. If statistically significant differences were detected, post hoc (Scheffe) multiple comparisons were made to determine which condition(s) contributed to the effect. Circulating corticosterone concentrations are presented as mean ± SEM in unit of ng/ml. PER1 protein expression is elucidated by the number of PER1-positive cells. An level of p 0.05 was taken as indicating a statistically significant difference between the manipulations.
RESULTS
Sleep disruptions induced by ZT0 kindling and the involvement of CRH
Our results demonstrated that kindled epilepsy occurring at different ZTs altered homeostatic variables and circadian fluctuation differently. CeA kindling stimuli delivered at ZT0 significantly decreased
NREM sleep during the first 8 hours (ZT1－8) and pronouncedly reduced REM sleep during the 12-h light period of the L:D cycle (ZT1－ZT12) (Figure 3A and 3B). The percentage of time spent in NREM sleep significantly decreased from 61.0 ± 4.0 % and 58.8 ± 3.3 % obtained from control to 41.9 ± 3.8 % (n = 8, p < 0.05) and 41.3 ± 2.5 % (p < 0.05) after ZT0 kindling stimuli during ZT1－4 and ZT5－8, respectively (Figure 3A). ZT0 kindling stimuli also suppressed REM sleep from 7.0 ± 1.1 %, 11.2 ± 0.9 and 9.4 ± 1.1 % after control to 3.0 ± 0.9 % (n = 8, p < 0.05), 5.4 ± 1.0 % (p < 0.05) and 3.3 ± 0.9 % (p < 0.05) during ZT1－4, ZT5－8 and ZT9－12, respectively (Figure 3B). Neither NREM sleep nor REM sleep was altered in the subsequent dark period (ZT13－24; Figure 3A and 3B). Analyzing the cortical temperature revealed that the ZT0 kindling stimulation enhanced cortical temperature during ZT1－8 (Figure 8A). Furthermore, the zenith-nadir fluctuations of circadian sleep and cortical temperature were prominently observed during the transition from the light period to the dark period (Figure 3A & 8A), indicating that the sleep circadian rhythm and the temperature oscillation were not altered when kindled epilepsy occurred at ZT0. ICV administration of the CRH receptor antagonist astressin (12.5 g) 20 minutes prior to ZT0 blocked ZT0 kindling-induced NREM sleep reduction, but had limited effect on the REM sleep suppression (Figure 3A and 3B). However, REM sleep was reduced during ZT17－20 after receiving 12.5 g astressin (Figure 3B). The percentage of time spent in NREM sleep was restored to 58.9 ± 3.9 % (n = 8, p < 0.05; when comparing to the values obtained after ZT0 kindling stimuli) and 55.3 ± 3.1 % (p < 0.05; when comparing to the values obtained after ZT0 kindling stimuli) during ZT1－4 and ZT5－8 after administration of astressin (Figure 3A). The effect of astressin on blocking ZT0 kindling-induced NREM alteration exhibited a dose-dependent manner as demonstrated in Figure 3C. Astressin
(12.5 g) also blocked the ZT0 kindling-induced increase of cortical temperature (Figure 8A). In
addition, plasma concentrations of corticosterone at ZT1 were significantly enhanced from 199.0 ± 32.3 ng/ml (n = 6) obtained from control to 563.3 ± 42.5 ng/ml after ZT0 kindling stimuli (n = 6, p < 0.05), and the concentrations were restored down to 220.0 ± 63.3 ng/ml by 12.5 g astressin (n = 6; p < 0.05; when compared to the values obtained after ZT0 kindling stimuli; Figure 3E). Our results also
demonstrated that ZT0 kindling significantly increased SWAs of NREM sleep during the 12-h light period (ZT1-12; Figure 3D). Nevertheless, astressin (12.5 g) was not capable of blocking ZT0 kindling-induced increases of SWA after the suppression of NREM sleep was reversed. Parts of the
aforementioned results were adopted from our previous observations.19
Alterations of sleep circadian rhythm and SCN PER1 expression after ZT6 kindling and the role of hypocretin
When CeA kindling stimuli was delivered at ZT6, no significant alteration in both REM sleep and NREM sleep was found during ZT6－22, ZT’1－10 and ZT’13－24 of the following L:D cycle (ZT’ represents the zeitgeber time of the second L:D cycle). However, ZT6 kindling stimuli enhanced the amount of NREM sleep during ZT23－24 (the last two zeitgeber hours of the first L:D cycle) from 9.6 ± 3.4 % obtained after control to 27.4 ± 4.7 % (n = 8, p < 0.05; Figure 4A and 4B). ZT6 kindling stimuli suppressed both NREM sleep and REM sleep during ZT’11－12 of the following L:D cycle; the percentage spent in NREM sleep decreased from 42.8 ± 4.2 % to 19.0 ± 3.2 % (n = 8, p < 0.05) and the amount of REM sleep was reduced from 12.1 ± 1.7 % to 4.9 ± 1.4 % (n = 8, p < 0.05; Figure 4A & 4B).
A mirror effect on wakefulness was also observed. Further analysis of the circadian fluctuation of sleep-wake activities and architectures, we found that the sleep-sleep-wake rhythm was forwardly shifted 2-h in the following L:D cycle after the ZT6-kindled epilepsy (Figure 4C, left panel), and the sleep-wake
architectures of ZT23－24 (block b) and ZT’11－12 (block e) after ZT6 kindling stimuli were
respectively similar to those of ZT’1－2 (block a) and ZT’13－14 (block d) obtained from vehicle control (Figure 4C, right panel). Furthermore, analyzing the cortical temperature demonstrated that the
temperature oscillation was shifted forward 2-h for both light period and dark period in the following zeitgeber cycle when rats received ZT6 kindling stimuli (Figure 8B). Whether the SCN oscillation is altered by ZT6 kindling stimuli was investigated by determining the expression of PER1 protein in the SCN. This is because the circadian oscillation of Per1 expression is essential for the regulation of circadian clocks, and the behavioral rhythms and oscillation of Per1 were disrupted in Per1 mutant mice.31,32 The number of PER1-immunoreactive cells peaked around ZT’12 of the second L:D cycle in the
sham control rats (Figure 6A & 6D), which is consistent with previous reports.33 However, ZT6 kindling
stimuli forwarded the peak of PER1 protein expression to ZT’6 (Figure 6B & 6D), which may be responsible for the 2-h shift in the sleep-wake fluctuation during the second L:D cycle. The LHA of the hypothalamus containing hypocretin as its neurotransmitter possesses first-order afferents to the SCN.34
The topographic organization of projections from CeA to LHA has been further confirmed,20 and HcrtR1
are dominantly distributed in the SCN.21 Therefore, we hypothesized that the multisynaptic afferents of
CeA-LHA-SCN and the hypocretin in the SCN are involved in ZT6 kindling-induced shifts in both PER1 oscillations and the sleep-wake rhythm. Our results demonstrated that when the HcrtR1 antagonist
SB334867 (6 g/l) was administered directly into the SCN 20 minutes prior to the ZT6 kindling stimuli the acrpohase of PER1 protein oscillations was delayed to peak at ZT’9 (Figure 6C and 6D),
demonstrating that the 6-h forward shifting of the PER1 protein oscillation induced by ZT6 kindling stimuli was mediated by the hypocretin. SB334867 also dose-dependently blocked ZT6 kindling-induced NREM alterations during ZT23－24 (Figure 4A & 4B upper panel) and ZT’11－12 (Figure 4A & 4B lower panel) when it was microinjected 20 minutes prior to the ZT6 kindling stimuli. The percentage of time spent in NREM sleep during ZT23－24 was restored down to 12.4 ± 4.3 % (n = 8, p < 0.05; when compared with the values obtained after ZT6 kindling), 13.3 ± 3.4 % (p < 0.05; when compared with the values obtained after ZT6 kindling) and 10.3 ± 3.1 (p < 0.05; when compared with the values obtained after ZT6 kindling; Figure 4B) after administrations of 1.5, 3.0 and 6 g SB334867, respectively. The amount of NREM sleep during ZT’11－12 was increased to 27.5 ± 3.6 % (n = 8, p < 0.05; when
compared with the values obtained after ZT6 kindling), 33.6 ± 5.5 % (p < 0.05; when compared with the values obtained after ZT6 kindling) and 31.6 ± 3.2 (p < 0.05; when compared with the values obtained after ZT6 kindling; Figure 4B) after administrations of 1.5, 3.0 and 6 g SB334867, respectively. The 2-h shifting of the sleep-wake fluctuation had also been reversed by 6 g/l SB334867 (Figure 4C).
Furthermore, SB334867 (6 g/l) also reverted ZT6 kindling-induced shift of temperature oscillation (Figure 8B). Our previous results also demonstrated that microinjection of 6 g SB334867 directly into the SCN at ZT6 in non-kindled rats exhibited no alteration in any aspects of sleep-wake activity and sleep circadian rhythm (Figure 5), suggesting the role of SCN hypocretin in ZT6 kindling-induced shift of sleep-wake rhythm is specific. We also found that the SWAs during NREM sleep were altered neither by
ZT6 kindling stimuli nor by SB334867 (data not shown), suggesting no change in the process S.
One concern in this experiment is the effect of 50 % DMSO per se, since SB334867 was dissolved in 50 % DMSO. Our results demonstrated that administration of 1 l of 50% DMSO directly into the SCN did not alter sleep-wake activity when comparing to the results obtained from PFS administration (Figure 5), suggesting that the effect of SB334867 is not contaminated by DMSO.
Sleep disruptions induced by ZT13 kindling and the involvement of IL-1
Kindled epilepsy occurring at ZT13 significantly enhanced NREM sleep during ZT13－16 and suppressed REM sleep during ZT’1－12 of the subsequent light period of L:D cycle. The amount of NREM sleep during ZT13－16 was enhanced from 19.5 ± 3.4 % obtained from control to 37.1 ± 1.9 % after ZT13 kindling stimuli (n = 8, p < 0.05; Figure 7A & 7B). The amounts of REM sleep during ZT’1－ 4, ZT’5－8 and ZT’9－12 were reduced from 3.1 ± 0.8 %, 5.7 ± 1.0 % and 6.9 ± 1.5 % obtained from control to 1.2 ± 0.5 % (n = 8, p < 0.05), 2.8 ± 0.7 % (p < 0.05) and 3.7 ± 0.8 % (p < 0.05) after ZT13 kindling stimuli (Figure 7B). Analyzing the cortical temperature depicted that the ZT13 kindling
stimulation decreased cortical temperature during ZT13－16 (Figure 8C). The zenith-nadir fluctuations of circadian sleep and cortical temperature were prominently observed during the transition from the dark period to the light period (Figure 7A & 8C), indicating that homeostatic variables, rather than the process C, are dominant in ZT13 kindling-induced NREM sleep enhancement. IL-1, acting as a somnogenic factor, is one of the targeted homeostatic factors to be further elucidated, due to the fact that it exerts circadian fluctuation lowest during the dark period3,4 and expresses augmentation after epilepsy in both
humans and animals.35,36 The increases of IL-1 expressions at ZT15 were further confirmed in the cortex
and hippocampus of ZT13-kindled rats by ribonuclease protection assay (Figure 7E). The expression of IL-1 was significantly increased from 2.01 ± 0.21 (the optic density ratio) to 7.97 ± 0.24 (n = 6; p < 0.05) in the cortex and was enhanced from 2.84 ± 0.82 to 4.55 ± 0.84 in the hippocampus, although it did not reach statistical significance.19 ICV administration of IL-1 receptor antagonist (IL-1ra; 0.25 g), given
20 minutes prior to the ZT13 kindling stimuli, blocked ZT13 kindling-induced NREM sleep
enhancement; the amount of NREM sleep was restored back to 24.8 ± 2.8 % (n = 8, p < 0.05; when compared with the values acquired after ZT13 kindling stimuli; Figure 7A). The effect of IL-1ra on blocking ZT13 kindling-induced NREM enhancement exhibited a dose-dependent manner as demonstrated in Figure 7C. IL-1ra (0.25 g) partially blocked ZT13 kindling-induced REM sleep suppression during ZT’1－4 in the subsequent L:D cycle (Figure 7B). The amount of REM sleep increased to 3.1 ± 0.8 % after administration of IL-1ra (n = 8, p < 0.05; when compared to the values obtained after ZT13 kindling stimuli; Figure 7B), indicating IL-1 partially participates in this event. Nevertheless, REM sleep was reduced during ZT13－24 when IL-1ra was given 20 minutes prior to the ZT13 kindling stimuli (Figure 7B), though REM sleep was not altered by the kindling itself. SWAs during NREM sleep were enhanced during both ZT13－24 and ZT’1－12 of the subsequent light period (Figure 7D), and the increases of SWA were not altered by IL-1ra, which excluded the effect of IL-1 on SWA alterations. As for the temperature oscillation, IL-1ra (0.25 g) reverted the ZT13 kindling-induced decrease of cortical temperature (Figure 8C). Parts of the aforementioned results were adopted from our previous observations.19
DISCUSSION
Epilepsy patients with different types or different recurrent times (i.e. nocturnal or diurnal) during the zeitgeber time controversially experience either daytime somnolence or insomnia,11-14 however the
underlying mechanisms remain unknown. This current study was designed to elucidate the nature of sleep alterations induced by amygdala kindling stimulation at different ZTs. Our results demonstrated that ZT0 kindling stimuli decreased both NREM sleep and REM sleep during ZT1－12 (the light period of the light:dark cycle). Reductions of NREM sleep and REM sleep are consistent with the clinical observations of profound decreases in REM sleep and in sleep efficiency during the night when TLE occurred early in the night.37 The zenith-nadir fluctuation of circadian sleep was prominently observed during the transition
from the light period to the dark period after ZT0 kindling stimulation, suggesting that sleep circadian rhythm was not altered by ZT0-kindled epilepsy in rats. In contrast, the homeostatic variables may be dominant in the ZT0 kindling-induced sleep alteration. CRH, acting as a waking promoter,24 is one of the
targeted homeostatic factors to be further investigated, since it exhibits circadian fluctuation lowest during the light period5,6 and its expression increases after epilepsy in both humans and animals.38-40 Our
results demonstrated that ZT0 kindling stimuli enhanced corticosterone concentrations in plasma, and ICV administration of CRH receptor antagonist astressin blocked ZT0 kindling-induced NREM sleep suppression, but not the REM sleep reduction. These results elucidate the contribution of CRH to the ZT0 kindling-induced alteration in NREM sleep, whereas mechanisms other than CRH may be involved in the REM sleep alteration. As for the REM sleep reduction observed during ZT17－20 after astressin
administration, it might be simply due to the effect of astressin per se on the physiological REM sleep, which was consistent with our previous study.24 However, it needs to be further confirmed in future study.
EEG SWAs during the NREM sleep are thought to reflect both sleep debt and intensity,2 and is a
characteristic of the homeostatic response to sleep deprivation. Therefore, the increases in SWA during NREM sleep could conceivably be a response to sleep loss per se after ZT0 kindling. Nevertheless, astressin was not capable of blocking ZT0 kindling-induced increases of SWA after the suppression of NREM sleep was reversed, depicting that this SWA enhancement is not attributed to CRH action and was not simply due to the consequence of sleep loss. Achermann and Borbély (1990) have postulated that a periodically activated REM sleep triggers a signal that rapidly declines SWA, and the inactivation of this REM sleep signal permits the buildup of SWA in the SWS.41 Based upon Achermann and Borbély’s
postulation, the enhancement of SWA during NREM sleep after ZT0 kindling stimuli may result from the suppression of REM sleep. Collectively, kindled epilepsy occurring at ZT0 decreased sleep and increased SWAs, but did not alter the sleep circadian rhythm. The homeostatic factor CRH is involved in ZT0 kindling-induced sleep alteration. A hypothetical diagram for this summary was depicted in Figure 3F. As for the effect of ZT0 kindling-induced enhancement of CRH in the epileptogenesis, our previous study had demonstrated that ZT0 kindling stimulation induced secondary epileptiform EEGs, which was blocked by astressin, indicating the epileptogenic property of CRH.19 Based upon these observations, it is
hard to differentiate that the revert of ZT0 kindling-induced sleep alteration by astressin is due to a reduction in epileptic seizures, a direct effect on sleep activity or both. Nevertheless, our results suggest that blockade of CRH effect is beneficial to both of the anti-epileptogenesis and seizure-induced sleep
alteration.
Sleep-wake activities were not immediately altered after the ZT6 kindling stimuli, and the sleep-wake alteration continued to be observed in the dark period (ZT23－24) and the following light period (ZT11 －12) of the L:D cycle, suggesting the involvement of process C rather than that of homeostatic
variables. Analysis of sleep circadian fluctuation and the sleep architecture indicated that the sleep rhythm was advanced 2 hours after ZT6 kindling stimuli. PER1 protein expression in the SCN was forwardly shifted 6 hours after kindled epilepsy occurring at ZT6. These observations indicate that the change of circadian rhythm in the SCN causes the sleep alterations induced by ZT6 kindling. However, NREM sleep during ZT23－24 after the ZT6 kindling stimuli did not reach the same amount as that obtained during ZT’1－2 when rats were not kindled, suggesting that － if the process C is involved in the consequence of ZT6 kindling stimuli － entrainment factors (e.g. time cue or photic signal) are still dominant in the sleep-wake rhythm and somehow confound or mask the alterations in the sleep circadian rhythm as reported by Bazil et al,17 since experimented rats were well accommodated to the 12:12h L:D
cycle. The SCN receives photic-related pathways from the retina and intergeniculate nucleus;42
nervertheless, a variety of other CNS structures also innervate the SCN, though little knowledge is known of their functions.34 The LHA is one of the hypothalamic regions, containing hypocretin as its
neurotransmitter and possessing first-order afferents to the SCN.34 Using a retrograde transneuronal
tracer, the pseudorabies virus, to identify high order afferents of the SCN demonstrates that CeA multisynaptically innervates the SCN.34 The topographic organization of projections from CeA to LHA
has been further confirmed,20 and HcrtR1 are dominantly distributed in the SCN.21 Furthermore, Marston
et al. (2008) have demonstrated that the SCN circadian clock can be reset by hypocretin, while the activation of hypocretin neurons is controlled by the SCN circadian rhythm,43 suggesting the existence of
a reciprocal relationship between the SCN circadian rhythm and LHA neuronal activity. Therefore, we hypothesized that the multisynaptic afferents of CeA-LHA-SCN and the hypocretin in the SCN are involved in ZT6 kindling-induced shifts in both PER1 oscillations and the sleep-wake rhythm. Our results have demonstrated that microinjection of hypocretin receptor antagonist SB334867 into the SCN partially reversed the shifting of PER1 protein expression and blocked alteration of sleep circadian rhythm induced by ZT6 kindling stimuli. Conclusively, kindled epilepsy occurring at ZT6 shifted both the expression of PER1 protein in the SCN with 6-h advance and the sleep-wake rhythm with 2-h advance, implicating the involvement of process C. Hypocretin receptors contributed to the adjustment of the circadian rhythm after ZT6 kindling stimuli. A hypothetical diagram of this conclusion was demonstrated in Figure 6F. The role of hypocretin in epileptogenesis is controversial. A study demonstrated that hypocretin inhibits epileptiform discharges induced by bicuculline in CA1 hippocampal slice.44 In contrast, an in vivo study
indicates that hypocretin potentiates the cortical epileptic activity induced by focal application of
penicillin-G.45 In our study, blockade of HcrtR by SB334867 did not induce the appearance of secondary
epileptic activity in ZT6 kindled rats, suggesting that hypocretin in SCN is not anti-epileptogenic. Rapid kindling protocol did not induce secondary epileptogenesis as aforementioned; therefore, the role of SCN hypocretin in epileptogenesis needs to be further confirmed in future.
ZT13 kindling stimulation enhanced NREM sleep during ZT13－16, which was opposite to the result of ZT0 kindling. ZT13 kindling stimuli also increased SWA during NREM sleep, demonstrating that both the amount and the intensity of NREM sleep were increased by ZT13 kindling stimuli. We further elicited that ZT13 kindling enhanced somnogenic IL-1 mRNA expression in cortex and hippocampus, and ICV administration of IL-1ra blocked ZT13 kindling-induced NREM sleep enhancement, but had no effect on the SWA. Although daytime somnolence is often blamed upon the antiepileptic medications, poor seizure control, or sleep disruptions after nocturnal seizure, our results implicated that IL-1 may play an
important role in epilepsy-induced sleepiness. In contrast, REM sleep during ZT’1－12 was suppressed after ZT13 kindling stimuli, which is consistent with the previous observations of a significant decrease in REM sleep during the following night after the daytime TLE in patients.37 Furthermore, blockade of REM
sleep suppression during ZT’1－4 by IL-1ra indicated that IL-1 partially involved in ZT13 kindling induced REM sleep alteration. Unexpectedly, IL-1ra reduced REM sleep during ZT13－24 after ZT13 kindling stimulation, although REM sleep within the ZT period was not altered by kindling itself. Our previous results had shown that IL-1ra 0.25 g administered ICV at the beginning of the dark period did not alter sleep-wake activity during the dark period in the normal control rat.19 This observation suggests
that other mechanisms regulated by IL-1 in the CNS may become predominant in REM sleep generation after ZT13 kindling stimulation. For example, IL-1 stimulates growth hormone (GH) secretion through a hypothalamic effector, growth hormone releasing hormone (GHRH). Both GH and GHRH exhibit REM sleep promotion.46 If the GH/GHRH system induced by IL-1 were to become predominant in REM sleep
subsequently reduce GH/GHRH-mediated REM sleep. However, this hypothesis needs to be determined in future studies. In summary, kindled epilepsy occurring at ZT13 increased NREM sleep and SWAs, but did not alter sleep circadian rhythm. Homeostatic factor IL-1 is involved in ZT13 kindling-induced sleep alteration. A hypothetical diagram of this conclusion was demonstrated in Figure 7F. As for the effect of ZT13 kindling-induced enhancement of IL-1 in the epileptogenesis, our previous study had demonstrated that ZT13 kindling stimulation induced secondary epileptiform EEGs, which was aggravate by IL-1ra, suggesting the anti-epileptogenic property of IL-1.19
Nevertheless, clarification as to why kindling stimuli delivered at different ZTs (ZT0 and ZT13) enhanced CRH and IL-1 differently and caused opposite alterations in NREM sleep is still necessary. Our previous results have demonstrated that ZT0 kindling stimuli only increases limited IL-1, while IL-1 concentration is highest during the light period, and that this minimal increase in IL-1 may not produce additional somnogenic action in behavior.19 In contrast, ZT13 kindling stimuli increases limited corticosterone
concentration during the dark period while its concentration is high.19 The out-of-phase characteristics in
the circadian fluctuations of CRH and IL-1 cause distinct alterations of NREN sleep when kindled epilepsy occurs at different ZTs.
In conclusion, this current study is the first paper to demonstrate that kindled epilepsy occurring at distinct ZTs alters sleep processes differently. Homeostatic variables are involved in ZT0 and ZT13 kindling-induced sleep-wake alterations; in contrast, shifting of the circadian rhythm contributes to ZT6 kindling-induced sleep alterations.
FIGURE LEGENDS
Figure 1. The kindled epileptiform EEGs (panel A) and secondary spontaneous epileptic polyspikes (panel B) induced by traditional kindling stimuli. The percentages of spontaneous polyspikes during the subsequent 24-h ZTs after ZT0 and ZT13 kindling stimuli were respectively demonstrated in panels C and D. Each point represents the percentage of time when the kindling-induced polyspike activity was appeared in the individual rat. Closed bar represents the dark period (ZT13-24) and open bar depicts the light period (ZT1-12). The kindled epileptiform EEGs (panel E), but not the secondary spontaneous polyspikes (panel F), were induced by rapid kindling stimuli.
Figure 2. A diagram of experimental protocol. Total of 132 rats was used in this study and was divided into 6 groups. Open bar depicts the light period (ZT1-12) and closed bar represents the dark period (ZT13-24). “//” indicates the traditional kindling protocol and arrow depicts the manipulation.
Abbreviations: K, 1-s kindling stimuli; A, astressin injection; A1, astressin dose 1; A2, astressin dose 2; A3, astressin dose 3; S, sacrifice; R, sleep recording; IL-1ra, IL-1 receptor antagonist.
Figure 3. The effects of ZT0 kindling stimuli. A: ZT0 kindling stimuli decreased NREM sleep during ZT1－4 and ZT5－8, which was blocked by 12.5 g astressin. B: ZT0 kindling stimuli profoundly decreased REM sleep during ZT1－12; however, 12.5 g astressin exhibited little effect on it. Open circles represent the values obtained after PFS injection (before kindling protocol), closed circles indicate those values acquired after PFS+ZT0 kindling stimuli, and closed triangles demonstrate the results when
12.5 g astressin administered prior to the ZT0 kindling stimuli. C: The dose dependent response of astressin. D: SWAs during NREM sleep was significantly enhanced after ZT0 kindling stimuli. Astressin (12.5 g) has no effect on ZT0 kindling-induced enhancement of SWAs. Blue, red and black colors represent the SWA values obtained after PFS injection (before receiving kindling protocol), PFS+ZT0 kindling stimuli, and astressin+ZT0 kindling stimuli, respectively. E: Plasma corticosterone
concentrations were elevated at ZT2 after ZT0 kindling stimuli, and this elevation was blocked by 12.5 g astressin. F: Summary depicting the alterations in CRH fluctuation, sleep fluctuation, SWA and circadian rhythm after receiving ZT0 kindling stimuli. Dashed lines indicate the alterations after ZT0 kindling stimuli. * represents statistically significant difference from the PFS-injected control and # depicts statistically significant difference from the PFS+ZT0 kindling stimuli. The sleep-wake activity was recorded after ZT0 kindling stimuli and lasted for 24 hours.
Figure 4. The effects of ZT6 kindling stimuli. A: ZT6 kindling stimuli enhanced NREM sleep during ZT23－24 and decreased NREM sleep during ZT’11－12 of the subsequent L:D cycle. REM sleep was suppressed during ZT’11－12 of the subsequent L:D cycle. Microinjection of HcrtR1 antagonist,
SB334867 (6 g), blocked ZT6 kindling-induced sleep alterations. Blue shaded areas represent the values obtained after DMSO injection (before kindling protocol), red circles indicate those values acquired after DMSO+ZT6 kindling stimuli, and black triangles demonstrate the results when 6 g/l SB334867 administered prior to the ZT6 kindling stimuli. B: Summary of alteration in NREM sleep during ZT23－ 24 and ZT’11－12. In panels A and B, * represents statistically significant difference between the
DMSO-injected control and DMSO+ZT6 kindling stimuli, and # depicts statistically significant difference between the DMSO+ZT6 kindling stimuli and SB334867+ ZT6 kindling stimuli. C: ZT6 kindling stimuli advanced sleep circadian oscillation by 2 hours, and SB334867 blocked ZT6 kindling-induced circadian shift (data obtained from a representative rat). Blue lines indicate wakefulness and yellow lines depict sleep, including both REM sleep and NREM sleep. Blocks a－f indicate the sleep architectures extracted from particular 2-h time period of the zeitgeber time. The sleep-wake activity was recorded after ZT6 kindling stimuli and lasted for 42 hours.
Figure 5. Effects of DMSO and SB334867 on spontaneous sleep. Open circles represent the values obtained after administration of PFS into the SCN at ZT6, shade areas demonstrate the results acquired after receiving DMSO microinjection, and closed circles depict the values obtained after 6 g SB334867 administration. Dashed line indicates the time point for receiving SCN microinjection. The sleep-wake activity was recorded beginning from ZT6 and lasted for 42 hours.
Figure 6. The expression of PER1 protein in the SCN. Blue staining represents the cell nucleus and red staining depicts the immunoreactive signal for PER1 protein. Panels A, B and C represent the PER 1-immunoreactive staining at different ZTs obtained from sham control rats, rats received ZT6 kindling stimuli, and those administered with SB334867 prior to the ZT6 kindling stimuli, respectively. D: Summary of PER1 expression obtained from sham control group, ZT6-kindling group and
SB334867+ZT6-kindling group. Both * and # represent the values statistically differ from the sham control group. E: The summary depicting the alterations in sleep fluctuation, SWA and the oscillation of
PER1 expression after receiving ZT6 kindling stimuli. Dashed lines indicate the alterations after ZT6 kindling stimuli. The dark and open portions of bars represent the dark and light periods of the 12:12-h L:D cycle. The red dashed line indicates the timing for receiving kindling stimuli.
Figure 7. The effects of ZT13 kindling stimuli. A: ZT13 kindling stimuli increased NREM sleep during ZT13－16, which was blocked by 0.25 g IL-1ra. B: ZT13 kindling stimuli profoundly decreased REM sleep during ZT’1－12 of subsequent L:D cycle. However, 0.25 g IL-1ra further suppressed REM sleep during ZT13－24. Open circles represent the values obtained after PFS injection (before kindling
protocol), closed circles indicate those values acquired after PFS+ZT13 kindling stimuli, and closed triangles demonstrate the results when 0.25 g IL-1ra administered prior to the ZT13 kindling stimuli. C: The dose dependent response of IL-1ra. D: SWAs during NREM sleep was significantly enhanced after ZT13 kindling stimuli. IL-1ra (0.25 g) has no effect on ZT13 kindling-induced enhancement of SWAs. Blue, red and black colors represent the SWA values obtained after PFS injection (before kindling protocol), PFS+ZT13 kindling stimuli, and 0.25 g IL-1ra+ZT13 kindling stimuli, respectively. E: Expression of IL-1 mRNA was elevated in the cortex and hippocampus at ZT15 after ZT13 kindling stimuli in one representative rat. F: Summary depicting the alterations in IL-1 fluctuation, sleep fluctuation, SWA and circadian rhythm after receiving ZT13 kindling stimuli. Dashed lines indicate the alterations after ZT13 kindling stimuli. * represents statistically significant difference from the PFS-injected control and # depicts statistically significant difference from the PFS+ZT13 kindling stimuli. The sleep-wake activity was recorded after ZT13 kindling stimuli and lasted for 24 hours.
Figure 8. Effects of temperature alterations after kindling epilepsy. A: ZT0 kindling enhanced cortical temperature during ZT1－8, and this effect was blocked by 12.5 g astressin. B: ZT6 kindling decreased cortical temperature during ZT23－24 and enhanced temperature during ZT’11－12. The temperature shift was reverted by SB334867. C: ZT13 kindling decreased temperature during ZT13－16, and this effect was blocked by 0.25 g IL-1ra. Open circles represent the results of vehicle control. Closed circles demonstrate the values obtained after ZT0, ZT6 and ZT13 kindling in panels A, B and C, respectively. Closed triangles depict the results acquired after administration of 12.5 g astressin, 6 g SB334867 and 0.25 g IL-1ra in panels A, B and C, respectively. * represents the statistical significance from the vehicle control, and # demonstrates the statistical significance from the kindling stimuli.
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