CIRCADIAN CLOCK GENE CSNK1E REGULATES REM SLEEP AMOUNT http://dx.doi.org/10.5665/sleep.3590

The Circadian Clock Gene Csnk1e Regulates Rapid Eye Movement Sleep Amount, and Nonrapid Eye Movement Sleep Architecture in Mice Lili Zhou, PhD1,2; Camron D. Bryant, PhD3; Andrew Loudon, PhD4; Abraham A. Palmer, PhD5,6; Martha Hotz Vitaterna, PhD1,2; Fred W. Turek, PhD1,2

Center for Sleep and Circadian Biology, Northwestern University, Evanston, IL; 2Department of Neurobiology, Northwestern University, Evanston, IL; Departments of Pharmacology and Experimental Therapeutics and Psychiatry, Boston University School of Medicine, Boston, MA; 4Faculty of Life Sciences, University of Manchester, Manchester, UK; 5Department of Human Genetics, University of Chicago, Chicago, IL; 6Department of Psychiatry and Behavioral Neuroscience, University of Chicago, Chicago, IL

1 3

Study Objectives: Efforts to identify the genetic basis of mammalian sleep have included quantitative trait locus (QTL) mapping and gene targeting of known core circadian clock genes. We combined three different genetic approaches to identify and test a positional candidate sleep gene — the circadian gene casein kinase 1 epsilon (Csnk1e), which is located in a QTL we identified for rapid eye movement (REM) sleep on chromosome 15. Measurements and Results: Using electroencephalographic (EEG) and electromyographic (EMG) recordings, baseline sleep was examined in a 12-h light:12-h dark (LD 12:12) cycle in mice of seven genotypes, including Csnk1etau/tau and Csnk1e-/- mutant mice, Csnk1e B6.D2 and Csnk1e D2.B6 congenic mice, and their respective wild-type littermate control mice. Additionally, Csnk1etau/tau and wild-type mice were examined in constant darkness (DD). Csnk1etau/tau mutant mice and both Csnk1e B6.D2 and Csnk1e D2.B6 congenic mice showed significantly higher proportion of sleep time spent in REM sleep during the dark period than wild-type controls — the original phenotype for which the QTL on chromosome 15 was identified. This phenotype persisted in Csnk1etau/tau mice while under free-running DD conditions. Other sleep phenotypes observed in Csnk1etau/tau mice and congenics included a decreased number of bouts of nonrapid eye movement (NREM) sleep and an increased average NREM sleep bout duration. Conclusions: These results demonstrate a role for Csnk1e in regulating not only the timing of sleep, but also the REM sleep amount and NREM sleep architecture, and support Csnk1e as a causal gene in the sleep QTL on chromosome 15. Keywords: Csnk1e, circadian rhythms, QTL, REM, sleep Citation: Zhou L; Bryant CD; Loudon A; Palmer AA; Vitaterna MH; Turek FW. The circadian clock gene Csnk1e regulates rapid eye movement sleep amount, and nonrapid eye movement sleep architecture in mice. SLEEP 2014;37(4):785-793.

INTRODUCTION Great progress has been made in the identification of mammalian circadian clock genes and their interactions involving transcriptional and translational feedback loops over the past two decades,1,2 leading to studies of the role of these genes in sleep regulation. A surprising result from these studies is that genetic manipulation of core clock genes can alter not only the timing of sleep, but also the quantity and quality of sleep. For example, Clock mutant mice are awake more and sleep less than wildtype mice.3 Mice deficient in NPAS2, a paralog of CLOCK, showed a reduction in both nonrapid eye movement (NREM) and rapid eye movement (REM) sleep during the dark period,4 and a deficit in homeostasis of NREM sleep.5 Although it was argued that mouse Per genes were not required for homeostatic regulation of the amount of sleep,6,7 Per gene mutant mice exhibited altered daily distribution of sleep.6 In Cry1 and Cry2 double-null mutant mice, slow wave sleep, but not REM sleep, is higher under both entrained and free-running conditions, and the electroencephalographic (EEG) delta power during NREM sleep is also higher.8 Mice deficient in BMAL1 have an increase in total sleep time and sleep fragmentation, and they also lack the diurnal pattern of sleep drive as evidenced by the flat distribution of NREM EEG delta power under baseline conditions.9

Submitted for publication June, 2013 Submitted in final revised form November, 2013 Accepted for publication November, 2013 Address correspondence to: Fred W. Turek, PhD, Center for Sleep and Circadian Biology, Northwestern University, 2205 Tech Drive, Evanston, IL USA, 60208; Tel: (847) 467-6512; Fax: (847) 467-4065 ; E-mail: fturek@ northwestern.edu SLEEP, Vol. 37, No. 4, 2014 785 Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

Transgenic mice carrying a point mutation in Dec2 show decreased NREM sleep and REM sleep during the light period, as well as altered structure of NREM sleep under baseline conditions.10 The combined results indicate that clock genes can affect more than just the timing of sleep. A separate experimental approach to identify sleep-regulatory genes is the analysis of quantitative trait loci (QTLs) linked to sleep phenotypes.11-17 In a previous study of a genetically segregating population of mice, we uncovered 52 QTLs regulating multiple aspects of baseline sleep.18 Considering the close relationship between circadian rhythms and sleep, one QTL was of particular interest because it was in a region containing a canonical circadian clock gene, casein kinase 1epsilon (Csnk1e). This QTL was located on chromosome 15 (peak logarithm of odds [LOD] = 4.04), and was associated with the percentage of total sleep time spent in REM sleep (%REM/ TS) in the dark period. Although REM sleep comprises only a small proportion of adult mammalian sleep, and its function and underlying mechanisms are still unclear,19 it is important to study REM sleep because abnormal REM sleep has been reported to be associated with different diseases and disorders such as neurodegenerative disease,20 posttraumatic stress disorder,21 and affective disorders.22,23 The tau mutant hamster was first identified as a circadian mutant in mammals in 1988, but the mutated gene was not identified as Csnk1e until several years later.24,25 The tau point mutation shortens the free-running period by approximately 4 h in homozygous mutants by hyperphosphorylating PER proteins, whereas a small but significant lengthening of the free-running period was observed in homozygous Csnk1e null mutant (Csnk1e-/-) mice.26 The paralogous gene, CSNK1E in humans, is associated with familial advanced sleep-phase syndrome Csnk1e Alters Sleep Amount and Architecture—Zhou et al

(FASPS).27,28 A mutation in the CSNK1E binding region of the human PER2 gene was associated with early sleep onset and early wake-up times compared to normal individuals.29 However, to date, no functions related to noncircadian aspects of sleep have been ascribed to Csnk1e. In the current study, we made use of multiple genetic tools to assess the role of Csnk1e in baseline sleep regulation: Csnk1etau/tau knock-in mutant mice,26 Csnk1e-/- null mutant mice,26 reciprocal congenic mice (Csnk1eB6.D2 and Csnk1eD2.B6), which differ in only one restricted Cskn1e-containing region by repeatedly crossing a C57BL/6J (B6) strain onto a DBA/2J (D2) strain and vice versa,30 and their respective wild-type control mice. We first studied the baseline sleep in Csnk1etau/tau and Csnk1e-/- mutant mice and their littermate controls under 12-hr light: 12-hr dark cycle (LD 12:12) entrained conditions. We then examined sleep in constant darkness (DD) in Csnk1e+/+ and Csnk1etau/tau mice to ensure that the sleep phenotypes associated with this circadian gene mutation were not the result of a masking effect of light, or a consequence of the tau mutants’ abnormal entrainment to LD 12:12 cycles.25,26 Finally, we tested baseline sleep in a set of reciprocal congenic mice to determine if the observed sleep phenotypes in mutant mice would also be found as a result of a naturally occurring genetic variation. Our results derived from multiple genetic approaches support the same conclusion: Csnk1e plays an important role in regulating the amount of REM sleep and the architecture of NREM sleep. METHODS Reevaluation of Previous Sleep QTL Data In a previous sleep QTL study by Winrow et al.,18 two inbred strains that had different sleep-wake characteristics, the C57BL/6J (B6) strain and the BALB/cByJ (BALB) strain, were selected to create a genetically segregating population of 269 [B6×(BALBxB6)F1]N2 (N2) male progeny. Thus, the 269 N2 mice had a 50:50 probability of being either homozygous B6/ B6 or heterozygous BALB/B6 at any genomic region. QTL mapping in this N2 population identified a large number of sleep related genomic regions. To further refine specific candidate genes for sleep regulation from those QTL regions, data from this sleep QTL study were reevaluated. Csnk1e Mutant Mice All mutant animals used in this experiment were coisogenic B6 male mice between 3 and 6 mo of age. Recordings were performed in mice homozygous for either a point mutation that duplicates the spontaneous tau mutation of the golden hamster (Csnk1etau/tau; n = 13), or a null mutation in the Csnk1e gene (Csnk1e-/-; n = 12) as well as wild-type littermate controls (Csnk1e+/+; n = 15; eight from the null breeding and seven from the tau breeding). The production and characterization of circadian rhythms in these mutant mice have been described previously.26 The mice were housed individually with food and water available ad libitum under 22-24°C ambient temperature. All genotype groups were born and maintained on a 12-h light:12-h dark cycle (LD 12:12; lights on 06:00, lights off 18:00). Baseline sleep was carried out while mice were housed under these conditions. In addition, after their entrained baseline sleep, both Csnk1e+/+ and Csnk1etau/tau mice were maintained in constant SLEEP, Vol. 37, No. 4, 2014 786 Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

darkness (DD) recordings for 5 days. All procedures were approved in advance by the Institutional Animal Care and Use Committee of Northwestern University. Csnk1e Congenic Mice The creation of the reciprocal congenic mice was as previously described.31 Briefly, Csnk1eB6.D2 mice, with the DBA/2J (D2) chromosomal region introgressed onto a B6 genetic background, were derived from a larger congenic line that had been backcrossed to B6 for more than 10 generations.30 Csnk1eD2.B6 mice, with the B6 chromosomal region introgressed onto a D2 genetic background, were derived from a larger congenic line produced by Valerie Bolivar at the Wadsworth Center (Albany, NY) that had been backcrossed to the D2 strain for more than 10 generations. We further backcrossed both congenic lines to their respective background strains until we obtained smaller congenic regions capturing the Csnk1e locus, and then we intercrossed these new lines to yield congenics (Csnk1eB6.D2 and Csnk1eD2.B6) and littermate controls (Csnk1eB6.B6 and Csnk1eD2.D2). Surgical Procedures and Protocol Mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (9 mg/kg) and then surgically prepared for electroencephalography (EEG) and electromyographic (EMG) recordings as described previously.9 Following 7 days of singly-housed postsurgical recovery, mice were placed in sound-attenuated sleep recording chambers and connected to a lightweight rotating tether that permitted free movement throughout the cage. Mice were allowed a 5-day acclimation period to adjust to the tether. A 24-h baseline recording, starting at lights on, was collected. For sleep recording in DD, mice were released from the LD 12:12 entrained condition into DD by permitting lights off at the usual time and to remain off for 5 days. Beam break activity was recorded simultaneously with sleep recording in the same sleep chamber, and one circadian day in DD was determined by two adjacent onsets of the infrared monitored activity. The beginning of the subjective night was defined by the onset of beam break activity, and the beginning of the subjective day was defined by the offset of the beam break activity. Sleep recording occurred on all 5 days in DD; only the data of one circadian day with the clearest onset of activity was used for further sleep scoring and analysis, which was usually the second or the third circadian day in DD. The mice remained tethered to the cables throughout the experiment. Total activity was monitored using infrared motion sensors (A1 Securing and Electrical Ltd., Huyton Merseyside, UK) located on the side of the bottom of each cage. Activity patterns were recorded and analyzed with ClockLab software (Actimetrics, Wilmette, IL, USA). Infrared activity monitoring was used to ensure entrainment to the LD cycle and provide information about onset and offset of activity to determine sleep patterns for individual mice in DD. Sleep Recording and Analyses Sleep recording and analyses was as previously described.18 Briefly, using SleepReport software (Actimetrics, Wilmette, IL, USA), EEG and EMG recordings were divided into 10-sec epochs and scored via visual inspection, according to the amplitude and frequency of EEG and the amplitude of EMG, as Csnk1e Alters Sleep Amount and Architecture—Zhou et al

wakefulness (low-amplitude, high-frequency EEG, and highamplitude EMG), NREM sleep (high-amplitude or occasionally low-amplitude, low-frequency EEG, and low-amplitude EMG), or REM sleep (low-amplitude EEG constituted mainly by theta wave activity and EMG atonia). Examples of each stage were shown in Figure S4 (supplemental material). Sleep architecture was examined by determining the number of sleep and wake bouts and the average bout duration for each state. For quantitative analysis of the EEG signal, fast Fourier transformation, which included a range of 1-25 Hz with a frequency resolution of 0.5 Hz, was used. For all epochs of NREM sleep, the EEG power in the delta (1-4 Hz), theta (4-8 Hz), and sigma (11-15 Hz) frequency ranges were calculated. Activity Monitoring in Running Wheel Cages Mice of different genotypes (10 Csnk1e+/+, 13 Csnk1etau/tau, 9 Csnk1e-/-, 10 Csnk1eB6.B6, 14 Csnk1eB6.D2, 9 Csnk1eD2.B6, and 10 Csnk1eD2.D2), were individually housed in cages equipped with running wheels within ventilated, light-tight chambers (interior dimensions: 56 × 44 × 180 cm3) with timer-controlled lighting. Cages were 32 × 15 × 13 cm3 and fitted with a 12.5-cm diameter stainless steel running wheel. Lighting within the chamber was provided by a 40 W fluorescent light, with intensity measured at 300 to 600 lux inside the cage. Animals were maintained on a LD 12:12 cycle (lights on 06:00, lights off 18:00) with food and water available ad libitum. After 1 w of acclimation to the recording environment and running wheels, activity was recorded as the number of wheel revolutions per 5 min using ClockLab software (Actimetrics, Wilmette, IL, USA). Activity in each individual mouse was averaged over the 7 days of recording to obtain an average activity distribution. Statistical Analysis All statistical analyses and figure plotting were performed using R software (http://www.r-project.org/).32 Comparisons between mutant genotypes were assessed via one-way analysis of variance (ANOVA), followed by the indicated TukeyKramer post hoc tests. Comparisons between congenic mice were assessed via two-way ANOVA, followed by TukeyKramer post hoc tests, for the significance of genotype, genetic background, and their interactions. Comparisons between the time course data of different genotypes were accessed via two-way ANOVA, followed by Tukey-Kramer post hoc tests. Significant differences were defined as P ≤ 0.05. Group values were reported as mean ± standard error of the mean. RESULTS A QTL on Chromosome 15 in the Region of Csnk1e was Associated With the Proportion of the Total Sleep Time Spent in REM Sleep During the Dark Period Reevaluation of data presented by Winrow et al.18 in 2009 indicated that a QTL region on chromosome 15 had a LOD score of 4.04 (significance threshold = 2.7, confidence interval = 20-30 cM, corresponding to a physical interval of 77.2 Mb-89.6 Mb), with a peak at 26.3 cM (corresponding to a physical location at 82.4 Mb in this study; Figure 1A). Mice with the BALB allele at the QTL peak exhibited significantly higher proportion of the total sleep time spent in REM sleep SLEEP, Vol. 37, No. 4, 2014 787 Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

Figure 1—Map of QTL on chromosome 15 and congenic regions. (A) The quantitative trait locus (QTL) on chromosome 15 was associated with the proportion of the total sleep in rapid eye movement sleep (%REM/TS) during the dark period. The logarithm of the odds (LOD) for linkage of this trait along chromosome 15 is shown. The QTL on chromosome15 has a LOD score of 4.04 (significance threshold = 2.7, confidence interval = 2030 cM) and a peak at 26.3 cM (corresponding to a physical location at 82.4 Mb in this study). This figure was derived from previous data (Winrow et al., 2009). (B) Individuals with the BALB/B6 genotype at the QTL peak exhibit higher %REM/TS during the dark period than those with the B6/ B6 genotype (P < 0.01). Group values were reported as mean ± standard error of the mean. (C) The B6.D2 congenic line contained an introgressed region (gray) on chromosome 15 spanning 77.1 to 86.8 Mb (Build 37.2). The D2.B6 congenic line contained a smaller introgressed region (black) spanning 78.7 to 81.6 Mb. The rest of genome is greater than 99% homozygous for the respective background strains (B6 = black, D2 = gray) at genomic loci outside of the congenic regions. The physical position of Csnk1e is located at 79.42-79.44 Mb (white vertical line; Build 37.2). The diagonally striped region was not genotyped. (D) SNP comparison between different strains. The SNP histogram was binned at a 0.1 Mb resolution. Gray bars in the background represent the total number of SNP locations where allele data for both strains is available. Black bars represent the number of SNP locations where the two strains are polymorphic. The physical position of Csnk1e is represented by a black arrow on the x-axis at the bottom.

(%REM/TS) during the dark period than mice homozygous for the B6 allele (P < 0.01; Figure 1B). Genomewide epistasis screening33 revealed a significant interaction between the genotypes at chromosome 13 and chromosome 15 for this sleep Csnk1e Alters Sleep Amount and Architecture—Zhou et al

Csnk1e+/+ = 6.16 ± 0.41%, Csnk1etau/tau = 8.73 ± 0.47%, P < 0.001). The time course data showed that Csnk1etau/tau mice exhibited significantly higher %REM/TS at Zeitgeber Time (ZT; with ZT0 being when the lights turn on) 14, ZT18 and ZT24 (Figure 2B). Csnk1e-/- mice showed a similar level of %REM/ TS in the dark period to wild-type control mice. (Figure 2A; Csnk1e+/+ = 6.16 ± 0.41%, Csnk1e-/- = 4.87 ± 0.62%, P = 0.12). The time course data revealed significantly lower %REM/TS in Csnk1e-/- mice than wild-type mice only at ZT16 (Figure 2B).

Figure 2—Increased percent of total sleep spent in REM sleep (%REM/ TS) during the dark period and active phase in Csnk1e mutant mice. (A) Csnk1etau/tau mice show significantly higher %REM/TS in the dark period than wild-type controls (P ≤ 0.001; n = 12-15). (B) Time of day distribution of %REM/TS in Csnk1e mutant mice while exposed to a 12-hr light: 12-hr dark cycle (LD 12:12 cycle; asterisk, P ≤ 0.05; n = 12-15). ZT, Zeitgeber Time. (C) Csnk1etau/tau mice show significantly higher %REM/TS in the active phase in constant darkness (DD) than wild-type mice (P ≤ 0.001; n = 7-8). (D) Circadian phase distribution of %REM/TS in Csnk1e+/+ and Csnk1etau/tau mice in DD. Comparisons between mutants and controls at different time points are not available, because data were binned at a 2-h resolution so that Csnk1e+/+ mice had 12 time points but Csnk1etau/tau mice had only 10 time points. CT, Circadian Time. Group values were reported as mean ± standard error of the mean.

phenotype (Figure S1A and S1B, supplemental material). Individuals with the B6/B6 genotype on chromosome 13 but the BALB/B6 genotype on chromosome 15 exhibited the greatest REM sleep in the dark period (Figure S1C, supplemental material). However, when the genotype on chromosome 13 was BALB/B6, regardless of their genotypes on chromosome 15, mice exhibited a similar level of REM sleep in the dark period (Figure S1C, supplemental material). Hence, the effect of the QTL of chromosome 15 on REM sleep was strongly influenced by the genetic background from chromosome 13. Csnk1etau/tau Mutant Mice Showed Higher %REM/TS During the Dark Period Under Entrained Conditions We recorded sleep under LD 12:12 entrained conditions in two types of mutants, Csnk1etau/tau and Csnk1e-/- mutant mice, and compared them to their wild-type littermates. Because there were no significant differences between wildtype littermates produced by null mutant matings and tau mutant matings, all wild-type mice were combined as a single group of Csnk1e+/+ for data analysis and presentation. The sleep trait showing the greatest difference between mutants and wild-types was the QTL-associated trait, %REM/TS in the dark period. Csnk1etau/tau mice had significantly higher %REM/TS in the dark period than Csnk1e+/+ mice (Figure 2A; SLEEP, Vol. 37, No. 4, 2014 788 Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

Csnk1etau/tau Mice Showed Higher Proportion of the %REM/TS During Active Phase Under DD Conditions Csnk1etau/tau mice had a much shorter free-running period than 24 h (20 h in homozygous mutants), and their entrainment to the LD cycle was abnormal—the onset of activity was several hours earlier than the time of lights off (Csnk1e+/+ = ZT 11.6 ± 0.2 h, Csnk1etau/tau = ZT 8.8 ± 0.5 h, P < 0.001; Figure S2A, supplemental material). Such an advanced phase angle of entrainment might affect the distribution of sleep in entrained conditions and influence the interpretation of sleep phenotypes observed in Csnk1etau/tau mice. To eliminate possible environmental effects on sleep, such as light, we recorded sleep in both Csnk1e+/+ and Csnk1etau/tau mice under DD conditions. Locomotor activity showed that each rest and active phase spanned approximately half of a circadian period, and the activity patterns of Csnk1e+/+ and Csnk1etau/tau mice under DD overlapped (Figure S2B, supplemental material). Indeed, the advanced phase angle of entrainment of Csnk1etau/tau mice did cause a change in the distribution of wakefulness and NREM sleep in the light versus dark periods. Under LD 12:12 entrained conditions, in the dark period, the percentage of time spent in wakefulness (Wake%) was significantly decreased (Figure S3A, supplemental material), and the percentage of time spent in NREM sleep (NREM%) was increased in Csnk1etau/tau mice (Figures S3A and S3C). However, these phenotypes disappeared when mice were exposed to DD (Figures S3B and S3D). In contrast, %REM/TS in Csnk1etau/tau mice during the active phase, i.e. subjective night, was still significantly higher than that of Csnk1e+/+ mice under DD conditions (Figures 2C and 2D; Csnk1e+/+ = 6.07 ± 0.57%, Csnk1etau/tau = 9.41 ± 0.52%, P < 0.001), which was consistent with the observation in Csnk1etau/tau mice under LD 12:12 entrained conditions, indicating that this phenotype was not simply a result of the advanced phase angle in Csnk1etau/tau mice. Other Sleep Phenotypes in Csnk1e Mutant Mice In addition to the primary QTL-associated %REM/TS trait, there were other sleep changes observed in Csnk1e mutants, especially the tau mutants. A higher percentage of time spent in REM sleep (REM%) in the dark period was observed in tau mutants (Figures 3A and 3B; Csnk1e+/+ = 2.08 ± 0.19%, Csnk1etau/tau = 4.07 ± 0.28%, P < 0.001). The phenotypic differences in REM% in the active phase were also consistently observed in Csnk1etau/tau mice under DD conditions (Figure 3C; Csnk1e+/+ = 2.38 ± 0.39%, Csnk1etau/tau = 3.87 ± 0.24%, P < 0.01). Noticeably, the change in REM sleep amount in Csnk1etau/tau mice was the result of an increased number of bouts of REM sleep (nbREM) in the dark period (Figures 3D and 3E; Csnk1e+/+ = 11.95 ± 0.93, Csnk1etau/tau = 21.71 ± 1.48, P < 0.001), Csnk1e Alters Sleep Amount and Architecture—Zhou et al

Figure 3—Changes in percent time spent in REM sleep (REM%) and number of bouts of REM sleep (nbREM) during the dark period in Csnk1e mutant mice. (A) Csnk1etau/tau mice show significantly higher REM% in the dark period than wild-type mice under entrained conditions (asterisk, P ≤ 0.001; n = 12-15). (B) Time-of-day distribution of REM% in Csnk1etau/tau mice under entrained conditions (asterisk, P ≤ 0.05; n = 1215). ZT, Zeitgeber Time. (C) Csnk1etau/tau mice show significantly higher REM% than wild-type mice during the active phase (CT12-24) in DD (asterisk, P ≤ 0.05; n = 12-15). CT, Circadian Time. (D) Csnk1etau/tau mice show significantly higher nbREM in the dark period than wild-type mice under entrained conditions (asterisk, P ≤ 0.001; n = 12-15). (E) Time-ofday distribution of nbREM in Csnk1etau/tau mice under entrained conditions (asterisk, P ≤ 0.05; n = 12-15). ZT, Zeitgeber Time. (F) Csnk1etau/tau mice show significantly higher nbREM than wild-type mice in the active phase (CT 12-24) in DD (asterisk, P ≤ 0.05; n = 12-15). CT, Circadian Time. Group values were reported as mean ± standard error of the mean.

rather than the duration of bouts of REM sleep (Table S1, supplemental material). Csnk1etau/tau mice still exhibited higher nbREM than wild-type mice in the active phase (Figure 3F; Csnk1e+/+ = 15.29 ± 3.07, Csnk1etau/tau = 22.80 ± 1.97, P < 0.05) under DD conditions. Changes were also found in fragmentation parameters of NREM sleep, such as the number of bouts of NREM sleep (nbNREM) and the duration of bouts of NREM sleep (dbNREM). Csnk1etau/tau mice had a significant decrease in nbNREM compared with Csnk1e+/+ mice during the light period under LD 12:12 conditions (Figures 4A and 4B; Csnk1e+/+ = 231.42 ± 9.21, Csnk1etau/tau = 180.79 ± 11.24, P < 0.001), and this phenotype persisted during the rest phase in DD, corresponding to the light period under entrained conditions (Figure 4C; Csnk1e+/+ = 170.86 ± 12.47, Csnk1etau/tau = 138.75 ± 11.49, P < 0.05). SLEEP, Vol. 37, No. 4, 2014 789 Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

Figure 4—Changes in the number of bouts (nb) and duration of bouts (db) of nonrapid eye movement (NREM) sleep in Csnk1e mutant mice. (A) Csnk1etau/tau mice show significantly lower nbNREM in the light period than wild-type mice under entrained conditions (asterisk, P ≤ 0.001; n = 12-15). (B) Time-of-day distribution of nbNREM in Csnk1etau/tau mice under entrained conditions (asterisk, P ≤ 0.05; n = 12-15). ZT, Zeitgeber Time. (C) Csnk1etau/tau mice show significantly higher nbNREM than wildtype mice in the rest phase (CT 0-12) in constant darkness (DD; asterisk, P ≤ 0.05; n = 12-15). CT, Circadian Time. (D) Csnk1etau/tau mice show significantly higher dbNREM than wild-type mice in all phases under entrained conditions (asterisk, P ≤ 0.05; n = 12-15). (E) Time-of-day distribution of dbNREM of Csnk1etau/tau mice under entrained conditions (asterisk, P ≤ 0.05; n = 12-15). ZT, Zeitgeber Time. (F) Csnk1etau/tau mice show significantly higher dbNREM in all phases than wild-type mice in DD (asterisk, P ≤ 0.05; n = 12-15). Group values were reported as mean ± standard error of the mean.

The dbNREM in Csnk1etau/tau mice was higher than that in Csnk1e+/+ mice in all phases under entrained conditions (Figures 4D and 4E; Light: Csnk1e+/+ = 1.78 ± 0.08 min, Csnk1etau/tau = 2.13 ± 0.16 min, P = 0.05; Dark: Csnk1e+/+ = 1.69 ± 0.08 min, Csnk1etau/tau = 2.28 ± 0.17 min, P < 0.001; 24 h: Csnk1e+/+ = 1.74 ± 0.07 min, Csnk1etau/tau = 2.19 ± 0.16 min, P < 0.01), and these changes in dbNREM in Csnk1etau/tau mice persisted when mice were exposed to DD (Figure 4F; Rest phase: Csnk1e+/+ = 2.66 ± 0.15 min, Csnk1etau/tau = 3.17 ± 0.20 min, P < 0.05; Active phase: Csnk1e+/+ = 2.15 ± 0.10 min, Csnk1etau/tau = 2.65 ± 0.23 min, P < 0.05; Total: Csnk1e+/+ = 2.44 ± 0.12 min, Csnk1etau/tau = 2.89 ± 0.15 min, P < 0.05). We did not find significant differences in EEG power among mice with different genotypes. We only observed a few significant changes at certain time points in Csnk1e-/- mice, e.g., decreased %REM/TS and nbREM in Csnk1e-/- mice at ZT16 (Figure 2B) and ZT20 (Figure 3E), respectively. It is worth mentioning that all Csnk1e Alters Sleep Amount and Architecture—Zhou et al

Figure 5—Increased percent total sleep spent in rapid eye movement sleep (%REM/TS) during the dark period in congenic mice. (A) Against the B6 genetic background, congenic mice with the D2 allele (B6.D2) show higher %REM/TS in the dark period than congenic mice with the B6 allele (B6.B6) (asterisk, P ≤ 0.05; n = 5-6). (B) Time-of-day distribution of %REM/TS in congenic mice with the B6 background (asterisk, P ≤ 0.05; n = 5-6). ZT, Zeitgeber Time. (C) Against the D2 genetic background, congenic mice with the B6 allele (D2.B6) show higher %REM/TS in the dark period than congenic mice with the D2 allele (D2.D2) (asterisk, P ≤ 0.05; n = 6-9). (D) Time-of-day distribution of %REM/TS in congenic mice with the D2 background (asterisk, P ≤ 0.05; n = 6-9). ZT, Zeitgeber Time. Group values were reported as mean ± standard error of the mean.

significant sleep phenotypes shown in homozygous tau mutants were also found in heterozygous tau mutants, indicating that the tau allele was dominant in sleep regulation (data not shown). Csnk1e B6.D2 Congenic Mice Showed Higher %REM/TS During the Dark Period To take advantage of the existing genetic tools, we used a set of reciprocal congenic lines derived from B6 and D2 inbred stains for chromosome 15. The Csnk1eB6.D2 line had a congenic region of 9.71 Mb (77.09-86.80 Mb; 4.63 cM; Figure 1C) from the D2 strain on a B6 background, and the Csnk1eD2.B6 line possessed a congenic region of 2.89 Mb (78.73-81.62 Mb; 0.56 cM; Figure 1C) from the B6 strain on a D2 background. The genetic background was greater than 99% homozygous in each congenic line.31 Our reciprocal congenic lines had narrow congenic regions that were close to the QTL peak on chromosome 15. Csnk1e was one of the 35 candidate genes, which have polymorphic single nucleotide polymorphisms (SNPs) between BALB and B6 strains, as well as polymorphic SNPs between DBA2 and B6 strains, within the narrowest congenic region of 78.73-81.62 Mb (Table S2, supplemental material). The physical position of Csnk1e was 79.42-79.44 Mb (NCBI Build 37.2), which was contained within both congenic regions. We determined that the polymorphisms between B6 and D2 strains in the congenic regions were similar in number compared with those between B6 and BALB strains, and a large portion of these SLEEP, Vol. 37, No. 4, 2014 790 Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

polymorphic regions were identical by descent (IBD) between BALB and D2 strains (Figure 1D). Importantly, there were no polymorphic SNPs between the BALB and the D2 strains in the D2.B6 congenic region and the Csnk1e containing region, which allowed us to substitute the D2 allele for the BALB allele in the Csnk1e containing region for validating the QTL. To verify that naturally occurring allelic differences between strains in Csnk1e can produce phenotypic alterations in the proportion of REM sleep during the dark period, we tested sleep in these reciprocal congenic lines. Sleep of Csnk1e B6.D2 and Csnk1e D2.B6 congenic mice and their littermate controls, Csnk1e B6.B6 and Csnk1e D2.D2, respectively, were examined under LD 12:12 entrained conditions using the same protocol as the mutant mice. Because there was a strong genetic background effect and our interest was the QTL region on chromosome 15, comparisons between different congenic groups were focused on the evaluation within each background. On a B6 genetic background, the QTL-associated trait, %REM/ TS during the dark period, was significantly higher in mice with the D2 allele than in mice with the B6 allele (Figure 5A; Csnk1e B6.B6 = 3.70 ± 0.76%, Csnk1e B6.D2 = 6.87 ± 0.41%, P < 0.001). In contrast, on a D2 background, %REM/TS during the dark period was significantly higher in mice with the B6 allele than mice with the D2 allele (Figure 5C; Csnk1e D2.D2 = 6.00 ± 0.63%, Csnk1e D2.B6 = 8.95 ± 0.69%, P < 0.01), indicating that the B6 and D2 alleles containing Csnk1e interact with other loci on each different genetic background. Remarkably, on both genetic backgrounds, the differences were significant only during the dark period, but not the light period. The time course data showed that %REM/TS was significantly higher at ZT14, ZT18, and ZT20 on each background (Figures 5B and 5D). To determine whether the sleep phenotypic differences in congenic mice were associated with alterations of circadian rhythms, we examined circadian rhythms of wheel-running locomotor activity in these congenic mice. The circadian properties we studied included the free-running period in constant darkness and constant light, phase shifts in response to a 6-h pulse of light beginning at Circadian Time 16 (CT16; the onset of activity of nocturnal organisms defines CT12) or CT23, and the phase angle of entrainment under LD 12:12 conditions. Congenic mice were stably entrained by the LD cycle (Figure S2C, supplemental material). We did not observe any circadian phenotypic differences between B6 and D2 genotypes on each genetic background, although there were significant differences between backgrounds, primarily in activity level (data not shown). Other Sleep Phenotypes in Congenic Mice With a D2 background, we did not find any other significant sleep differences between the B6 and the D2 alleles except the QTL-associated trait %REM/TS in the dark period. All other significant sleep alterations were observed exclusively with the B6 strain background. Therefore, results of other sleep phenotypes from mice with only the B6 strain background are reported here. Results from the congenic mice were remarkably consistent with those from mutant mice. As expected, REM% during the dark period in Csnk1eB6.D2 mice was significantly higher than that in Csnk1eB6.B6 mice (Figure 6A, B; Csnk1eB6.B6 = 1.53 ± Csnk1e Alters Sleep Amount and Architecture—Zhou et al

0.39%, Csnk1eB6.D2 = 3.09 ± 0.40%, P < 0.01). These REM sleep phenotypes were also due to the increased nbREM in the dark period (Figure 6C, D; Csnk1eB6.B6 = 8.50 ± 1.84, Csnk1eB6.D2 = 20.80 ± 2.31, P < 0.05) rather than the dbREM (Table S1, supplemental material). Furthermore, we observed increased dbNREM in Csnk1eB6.D2 mice in all phases across a LD 12:12 cycle (Figures 6E and 6F; Light: Csnk1eB6.B6 = 2.08 ± 0.14 min, Csnk1eB6.D2 = 2.94 ± 0.30 min, P < 0.05; Dark: Csnk1eB6.B6 = 1.68 ± 0.12 min, Csnk1eB6.D2 = 2.41 ± 0.24 min, P < 0.05; 24 h: Csnk1eB6.B6 = 1.90 ± 0.11 min, Csnk1eB6.D2 = 2.71 ± 0.27 min, P < 0.05), and decreased nbNREM in Csnk1eB6.D2 mice in the light period (Csnk1eB6.D2 = 158.4 ± 11.5, Csnk1eB6.B6 = 221.0 ± 12.5, P < 0.01). In summary, results from congenic mice indicated that the effects of the Csnk1e D2 allele with a B6 genetic background on sleep were highly similar to those of the Csnk1e tau allele, and consistent with the effect of the BALB allele in the previous QTL analysis of the N2 mice.18 DISCUSSION Sleep studies of specific circadian genes have been reported for most core circadian genes, but for most of them only a limited number of targeted mutant alleles have been available. The observed phenotypes could be biased by compensation effects from other genes, by genetic background effects from the flanking regions, and/or by atypical functions of mutant alleles not reflective of naturally occurring polymorphisms. A unique strength of the current study is that we integrated multiple genetic tools to examine sleep with respect to the LD 12:12 entrained conditions or the circadian free-running conditions in different genetic variants of the circadian clock gene, Csnk1e. The major phenotypic differences in sleep that were associated with Csnk1e variants included the amount of REM sleep and the architecture of NREM sleep, such as nbNREM and dbNREM. To our knowledge, there have been no mammalian sleep studies using such comprehensive genetic approaches and achieving such consistent results. The results show that Csnk1e regulates not only the timing of sleep, but also the amount and architecture of sleep, and the effects of Csnk1e on sleep were strongly influenced by other genetic loci on the background. We found that REM sleep during the dark period exhibited the greatest and most consistent difference between mice with different genotypes, and the effects of Csnk1e on REM sleep appeared to change the number of REM sleep episodes without altering the average duration of the episodes. In addition to the QTL-associated REM sleep trait, we found that Csnk1e was also associated with the architecture of NREM sleep, as observed in nbNREM and dbNREM in both Csnk1etau/tau and Csnk1e B6.D2 mice. The changes of nbNREM and dbNREM were in opposite directions, offsetting one another and resulting in an unchanged total amount of NREM%. However, these NREM sleep traits were not significantly associated with the QTL in our mapping studies from the B6 × BALB cross. This may be because the effects of Csnk1e were masked by interactions between Csnk1e and other genes in mice with a mixed B6/BALB genetic background, but could be detected with a uniform genetic background, such as in mutants or congenic mice. It is also possible that the effects on nbNREM and dbNREM were too small to be identified by a population with the limited sample size in our N2 mice. SLEEP, Vol. 37, No. 4, 2014 791 Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

Figure 6—Increased percent time spent in rapid eye movement sleep (REM%), number of bouts of REM sleep (nbREM), and number of bouts of nonrapid eye movement sleep (dbNREM) in congenic mice. (A) Against the B6 genetic background, congenic mice with the D2 allele (B6. D2) show higher REM% in the dark period than congenic mice with the B6 allele (B6.B6) (asterisk, P ≤ 0.05; n = 5-6). (B) Time-of-day distribution of REM% in congenic mice with the B6 background (asterisk, P ≤ 0.05; n = 5-6). ZT, Zeitgeber Time. (C) Against the B6 genetic background, congenic mice with the D2 allele (B6.D2) show higher nbREM in the dark period than congenic mice with the B6 allele (B6.B6) (asterisk, P ≤ 0.05; n = 5-6). (D) Time-of-day distribution of nbREM in congenic mice with the B6 background (asterisk, P ≤ 0.05; n = 5-6). ZT, Zeitgeber Time. (E) Against the B6 genetic background, congenic mice with the D2 allele (B6.D2) show higher dbNREM than congenic mice with the B6 allele (B6.B6) in all phases (asterisk, P ≤ 0.05; n = 5-6). (F) Time-ofday distribution of dbNREM in congenic mice with the B6 background (asterisk, P ≤ 0.05; n = 5-6). ZT, Zeitgeber Time. Group values were reported as mean ± standard error of the mean.

Because of a very short endogenous period in tau mutants, the activity rhythm of Csnk1etau/tau mice under LD 12:12 entrained condition was entrained with an advanced phase angle to the onset of darkness, or it failed to be stably entrained in some cases.25,26 This abnormal entrainment appeared to be responsible for some of the alterations in sleep phenotypes in Csnk1etau/tau mice. For example, the phase-advanced NREM sleep in Csnk1etau/tau mice on a LD 12:12 cycle was similar to the FASPS in individuals who had an earlier onset of waking and sleep due to a mutation affecting the phosphorylation site of the human PER2 gene.28 A serine to a glycine mutation within the CSNK1E binding region of the PER2 gene was Csnk1e Alters Sleep Amount and Architecture—Zhou et al

also associated with FASPS in humans, and individuals with this syndrome woke up and went to sleep earlier compared with controls.27 A missense mutation in casein kinase 1 delta (CSNK1D) led to the same FASPS phenotypes.27 In these FASPS cases, however, the quantity and quality of sleep in FASPS affected individuals appeared to be normal,34 which was different from the altered REM sleep amount observed in Csnk1etau/tau mice in the current study. Because only the total amount of sleep/wake stages in a few subjects under the entrained conditions were reported in the study by Jones and colleagues,34 it is still unknown whether the REM sleep distribution and/or NREM sleep architecture is altered in a particular LD phase in the FASPS subjects. Csnk1e and Csnk1d are the two closest members of casein kinase family,35 and genetic studies suggest that Csnk1d plays a more dominant role in regulating circadian period length than Csnk1e.36,37 This conclusion is also supported by pharmacological studies in vitro, in which CSNK1E specific inhibitor (PF-4800567) minimally altered the circadian period, while an inhibitor of both CSNK1E and CSNK1D (PF-670462) largely increased the circadian period.38 Therefore, it is possible that Csnk1d may also play a more prominent role in sleep regulation relative to Csnk1e, and this hypothesis could be tested pharmacologically in the future to avoid the potential compensation effects in Csnk1e-/- mice. With respect to the molecular mechanism underlying REM sleep regulation by Csnk1e, it is worth emphasizing that our results showed that Csnk1e influenced the level of REM sleep, specifically during the dark period but not the light period. This dark-period specificity was remarkably consistent throughout all the current studies, indicating that this was not a chance observation. It has been known that despite the fact that Csnk1e is constitutively expressed in both SCN and peripheral tissues,39,40 the tau mutation is a gain-of-function mutation that hyperphosphorylates the cyclically expressed PER2 protein after the peak expression of PER2 at approximately ZT12 or CT1226,41; as a result, the effects of these molecular changes on circadian rhythms in tau mutants occur primarily during the dark period or active phase. This might be one explanation for the dark period-specific sleep phenotype observed in the current study. However, some sleep parameters, such as dbNREM, exhibited constant changes across the entire LD cycle or circadian cycle in tau mutant mice, regardless of the phase. In addition, we did not observe any significant opposing effects on sleep between the null mutation and the tau mutation, as we observed in their circadian periods. Therefore, it is possible that other noncircadian proteins may also be involved in the regulation of sleep in Csnk1e mutant mice. The strong interaction between genotypes and genetic backgrounds in both QTL mapping and congenic mice was another interesting observation. It demonstrates the complexity of sleep regulation and implies that additional molecular components outside the Csnk1e containing region are important for sleep regulation. In fact, the strong effect of strain background has been observed in other circadian gene mutants, such as Clock. Mice carrying ClockΔ19 mutation, but on different strain backgrounds, exhibited different circadian and metabolic phenotypes.42-45 All of these findings stress the importance of considering the effect of genetic background when studying the effects of a single gene on complex traits such as sleep. The identification of the upstream regulators or downstream SLEEP, Vol. 37, No. 4, 2014 792 Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

substrates of Csnk1e in the future will be very important for understanding how REM sleep is regulated by Csnk1e with its interaction with the other molecular components. In summary, after identifying a QTL that was associated with REM sleep, and finding that a circadian clock gene, Csnk1e, was located within the QTL, we studied sleep in Csnk1etau/tau, Csnk1e-/- mutant mice, and a set of reciprocal congenic mice. We found that both Csnk1etau/tau mice and Csnk1eB6.D2 mice showed significantly higher %REM/TS in the dark period than controls when they were entrained to a LD 12:12 cycle. Such a sleep alteration was also consistently observed in Csnk1etau/tau mutants during the active period under free-running conditions. Although it is not proved by the current data that the Csnk1e causes the QTL, consistent sleep alterations in different genetic variants suggest that Csnk1e is likely to be one of the genes in the QTL linked to %REM/TS during the dark period. The involvement of Csnk1e in REM sleep amount and NREM sleep fragmentation opens up new research areas to determine the underlying molecular mechanisms by which Csnk1e regulates sleep. ABBREVIATIONS QTL, quantitative trait locus Csnk1e, casein kinase 1 epsilon B6, C57BL/6J mouse inbred strain D2, DBA/2J mouse inbred strain EEG, electroencephalography EMG, electromyography LD 12:12, 12-hr light: 12-hr dark cycle DD, constant darkness REM, rapid eye movement NREM, nonrapid eye movement SNP, single nucleotide polymorphism LOD, logarithm of odds %REM/TS, percentage of total sleep time spent in REM sleep FASPS, familial advanced sleep-phase syndrome BALB, BALB/cByJ mouse inbred strain N2, backcross generation 2 ANOVA, analysis of variance cM, centimorgan, a unit for measuring genetic linkage Mb, megabase Wake%, percentage of time spent in wakefulness NREM%, percentage of time spent in NREM sleep REM%, percentage of time spent in REM sleep nbREM, number of bouts of REM sleep nbNREM, number of bouts of NREM sleep dbNREM, duration of bouts of NREM sleep IBD, identical by descent Csnk1d, casein kinase 1 delta ACKNOWLEDGMENTS The authors thank Joseph Takahashi and Caroline Ko for kindly providing Csnk1e mutant mice; Christopher Olker for animal care, breeding and comments on the manuscript; Peng Jiang for comments on the manuscript; and Kazuhiro Shimomura for discussions and suggestions. DISCLOSURE STATEMENT This was not an industry supported study. This work was supported by R01DA021336 (to Dr. Palmer), K99DA029635 (to Csnk1e Alters Sleep Amount and Architecture—Zhou et al

Dr. Bryant), F32DA026697 (to Dr. Bryant), and P01 AG11412 (to Dr. Turek). This work was supported by, or in part by, the Defense Advanced Research Projects Agency (DARPA) and the United States Army Research Laboratory and the U. S. Army Research Office under contract/grant number DAAD 19-02-10038. The work was performed at Northwestern University. Dr. Loudon’s laboratory is supported in part by funding from GlaxoSmithKline (UK), but none of the data or material presented in the present study is or has been supported by GSK. Dr. Vitaterna has participated in research supported in part by Merck & Co., Inc. and in a study funded by Institute de Recherches Inernationale de Servier. Dr. Turek has received consultant fees from Vanda Pharmaceuticals and Ingram Barge companies. The other authors have indicated no financial conflicts of interest. REFERENCES

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21. Mellman TA, Pigeon WR, Nowell PD, Nolan B. Relationships between REM sleep findings and PTSD symptoms during the early aftermath of trauma. J Trauma Stress 2007;20:893-901. 22. Kyung Lee E, Douglass AB. Sleep in psychiatric disorders: where are we now? Can J Psychiatrye 2010;55:403-12. 23. van der Helm E, Yao J, Dutt S, Rao V, Saletin JM, Walker MP. REM sleep depotentiates amygdala activity to previous emotional experiences. Curr Biol 2011;21:2029-32. 24. Lowrey PL, Shimomura K, Antoch MP, et al. Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science 2000;288:483-91. 25. Ralph MR, Menaker M. A mutation of the circadian system in golden hamsters. Science 1988;241:1225-7. 26. Meng Q-J, Logunova L, Maywood ES, et al. Setting Clock Speed in Mammals: The CK1tau Mutation in Mice Accelerates Circadian Pacemakers by Selectively Destabilizing PERIOD Proteins. Neuron 2008;58:78-88. 27. Xu Y, Padiath QS, Shapiro RE, et al. Functional consequences of a CKI[delta] mutation causing familial advanced sleep phase syndrome. Nature 2005;434:640-4. 28. Toh KL, Jones CR, He Y, et al. An hPer2 Phosphorylation site mutation in familial advanced sleep phase syndrome. Science 2001;291:1040-3. 29. Xu Y, Toh KL, Jones CR, Shin JY, Fu YH, Ptáček LJ. Modeling of a human circadian mutation yields insights into clock regulation by PER2. Cell 2007;128:59-70. 30. Iakoubova OA, Olsson CL, Dains KM, et al. Genome-tagged mice (GTM): two sets of genome-wide congenic strains. Genomics 2001;74:89-104. 31. Bryant CD, Parker CC, Zhou L, et al. Csnk1e is a genetic regulator of sensitivity to psychostimulants and opioids. Neuropsychopharmacology 2012;37:1026-35. 32. Team RDC. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing, 2008. 33. Shimomura K, Low-Zeddies SS, King DP, et al. Genome-wide epistatic interaction analysis reveals complex genetic determinants of circadian behavior in mice. Genome Res 2001;11:959-80. 34. Jones CR, Campbell SS, Zone SE, et al. Familial advanced sleep-phase syndrome: A short-period circadian rhythm variant in humans. Nat Med 1999;5:1062-5. 35. Fish KJ, Cegielska A, Getman ME, Landes GM, Virshup DM. Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family. J Biol Chem 1995;270:14875-83. 36. Etchegaray JP, Yu EA, Indic P, Dallmann R, Weaver DR. Casein kinase 1 delta (CK1delta) regulates period length of the mouse suprachiasmatic circadian clock in vitro. PLoS One 2010;5:e10303. 37. Etchegaray JP, Machida KK, Noton E, et al. Casein kinase 1 delta regulates the pace of the mammalian circadian clock. Mol Cell Biol 2009;29:3853-66. 38. Walton KM, Fisher K, Rubitski D, et al. Selective inhibition of casein kinase 1 epsilon minimally alters circadian clock period. J Pharmacol Exp Ther 2009;330:430-9. 39. Ishida Y, Yagita K, Fukuyama T, et al. Constitutive expression and delayed light response of casein kinase Iepsilon and Idelta mRNAs in the mouse suprachiasmatic nucleus. J Neurosci Res 2001;64:612-6. 40. Lowrey PL, Takahashi JS. Mammalian circadian biology: elucidating genome-wide levels of temporal organization. Annu Rev Genomics Hum Genet 2004;5:407-41. 41. Yoo SH, Yamazaki S, Lowrey PL, et al. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U S A 2004;101:5339-46. 42. Turek FW, Joshu C, Kohsaka A, et al. Obesity and metabolic syndrome in circadian clock mutant mice. Science 2005;308:1043-5. 43. Kennaway DJ, Voultsios A, Varcoe TJ, Moyer RW. Melatonin and activity rhythm responses to light pulses in mice with the Clock mutation. Am J Physiol Regul Integr Comp Physiol 2003;284:R1231-40. 44. Kennaway DJ, Owens JA, Voultsios A, Boden MJ, Varcoe TJ. Metabolic homeostasis in mice with disrupted Clock gene expression in peripheral tissues. Am J Physiol Regul Integr Comp Physiol 2007;293:R1528-37. 45. Shimomura K, Kumar V, Koike N, et al. Usf1, a suppressor of the circadian Clock mutant, reveals the nature of the DNA-binding of the CLOCK:BMAL1 complex in mice. Elife 2013;2:e00426.

Csnk1e Alters Sleep Amount and Architecture—Zhou et al

SUPPLEMENTAL MATERIAL Table S1—Sleep traits in all genotype groups (mean ± standard error of the mean). Trait

Phase

+/+

–/–

tau/tau

B6.B6

B6.D2

D2.B6

D2.D2

WT in DD

Tau in DD

W%

Total Light Dark

51.73 ± 2.62 37.91 ± 1.37 65.57 ± 2.76

49.66 ± 2.09 34.49 ± 1.82 64.85 ± 2.67

48.44 ± 1.85 43.48 ± 2.92 53.42 ± 1.97

45.04 ± 1.81 30.21 ± 1.50 59.88 ± 2.90

43.11 ± 2.36 31.12 ± 1.10 55.11 ± 4.40

44.72 ± 1.60 31.78 ± 2.19 57.67 ± 2.89

43.15 ± 1.30 28.10 ± 1.95 58.20 ± 3.16

46.96 ± 2.40 34.75 ± 3.18 62.18 ± 3.60

48.71 ± 1.60 38.81 ± 2.77 58.63 ± 2.45

NREM%

Total Light Dark

44.30 ± 1.54 56.24 ± 1.22 32.37 ± 2.63

46.72 ± 1.98 60.00 ± 1.77 33.46 ± 2.54

46.94 ± 1.75 51.35 ± 2.67 42.54 ± 1.81

51.05 ± 1.98 63.49 ± 2.21 38.62 ± 2.66

51.88 ± 2.52 61.96 ± 1.47 41.83 ± 4.06

50.94 ± 1.48 63.37 ± 1.20 38.52 ± 2.57

53.16 ± 1.48 66.94 ± 0.83 39.40 ± 3.17

48.49 ± 2.04 58.51 ± 2.33 35.47 ± 3.26

46.02 ± 1.47 54.52 ± 2.60 37.53 ± 2.30

REM%

Total Light Dark

3.98 ± 0.19 5.88 ± 0.30 2.08 ± 0.19

3.63 ± 0.30 5.54 ± 0.43 1.72 ± 0.24

4.63 ± 0.16 5.19 ± 0.30 4.07 ± 0.28

3.92 ± 0.58 6.32 ± 0.96 1.53 ± 0.39

5.02 ± 0.25 6.95 ± 0.76 3.09 ± 0.40

4.36 ± 0.24 4.88 ± 0.30 3.83 ± 0.44

3.70 ± 0.23 4.99 ± 0.33 2.42 ± 0.25

4.57 ± 0.39 6.76 ± 0.50 2.38 ± 0.39

5.29 ± 0.24 6.70 ± 0.32 3.87 ± 0.24

%REM/TS

Total Light Dark

8.29 ± 0.38 9.43 ± 0.42 6.16 ± 0.41

7.22 ± 0.59 8.47 ± 0.67 4.87 ± 0.62

9.03 ± 0.28 9.19 ± 0.32 8.73 ± 0.47

7.21 ± 1.13 9.17 ± 1.49 3.70 ± 0.76

8.93 ± 0.78 10.11 ± 1.17 6.87 ± 0.41

7.88 ± 0.37 7.17 ± 0.45 8.95 ± 0.69

6.60 ± 0.49 6.93 ± 0.41 6.00 ± 0.63

8.53 ± 0.40 9.84 ± 0.53 6.07 ± 0.57

10.31 ± 0.41 10.95 ± 0.44 9.41 ± 0.52

nbNREM

Total Light Dark

370.47 ± 14.65 231.42 ± 9.21 139.32 ± 10.93

385.08 ± 14.02 239.08 ± 9.14 146.62 ± 8.65

319.93 ± 16.68 180.79 ± 11.24 139.43 ± 7.45

386.50 ± 11.58 221.00 ± 8.22 165.83 ± 20.59

288.60 ± 36.54 158.40 ± 19.14 130.80 ± 19.10

451.88 ± 25.87 248.75 ± 11.91 203.38 ± 18.83

431.80 ± 36.56 240.60 ± 19.17 191.60 ± 19.74

292.71 ± 25.42 170.86 ± 12.47 122.14 ± 16.19

254.40 ± 19.41 138.75 ± 11.49 115.80 ± 10.59

dbNREM

Total Light Dark

1.74 ± 0.07 1.78 ± 0.08 1.69 ± 0.08

1.75 ± 0.08 1.83 ± 0.09 1.63 ± 0.07

2.19 ± 0.16 2.13 ± 0.16 2.28 ± 0.17

1.90 ± 0.11 2.08 ± 0.14 1.68 ± 0.12

2.71 ± 0.27 2.94 ± 0.30 2.41 ± 0.24

1.63 ± 0.07 1.85 ± 0.09 1.38 ± 0.08

1.84 ± 0.12 2.10 ± 0.16 1.51 ± 0.08

2.44 ± 0.12 2.66 ± 0.15 2.15 ± 0.10

2.89 ± 0.15 3.17 ± 0.20 2.65 ± 0.23

nbREM

Total Light Dark

45.37 ± 2.38 33.42 ± 2.08 11.95 ± 0.93

40.15 ± 2.64 31.15 ± 2.35 9.00 ± 0.91

51.68 ± 1.83 30.14 ± 1.82 21.71 ± 1.48

44.83 ± 6.26 36.33 ± 5.06 8.50 ± 1.84

59.80 ± 3.51 39.00 ± 3.73 20.80 ± 2.31

52.50 ± 3.09 29.25 ± 1.70 23.25 ± 2.54

50.60 ± 2.94 31.60 ± 1.94 19.00 ± 1.30

53.57 ± 4.47 38.29 ± 2.08 15.29 ± 3.07

57.30 ± 3.30 34.50 ± 1.87 22.80 ± 1.97

dbREM

Total Light Dark

1.26 ± 0.04 1.28 ± 0.04 1.25 ± 0.06

1.29 ± 0.07 1.28 ± 0.08 1.33 ± 0.09

1.29 ± 0.05 1.24 ± 0.05 1.35 ± 0.05

1.24 ± 0.06 1.22 ± 0.05 1.32 ± 0.19

1.21 ± 0.10 1.29 ± 0.11 1.06 ± 0.08

1.19 ± 0.09 1.20 ± 0.07 1.18 ± 0.09

1.04 ± 0.08 1.12 ± 0.05 0.99 ± 0.09

1.20 ± 0.06 1.24 ± 0.07 1.22 ± 0.06

1.33 ± 0.07 1.30 ± 0.07 1.26 ± 0.10

Table S2—Genes with polymorphic single nucleotide polymorphisms (SNPs) between BALB and B6 strains, as well as polymorphic SNPs between DBA2 and B6 strains within region 78.7-81.6 Mb on chromosome 15. Apobec3 Card10 Cbx6 Cbx7 Cby1 Csnk1e Ddx17 Dmc1 Eif3l

Fam227a Gtpbp1 Kcnj4 Kdelr3 Lgals2 Mfng MicaIl1 Mir1943 Nol12

Genelist Npcd Pdgfb Pick1 Pla2g6 Polr2f Sox10 Sun2 Tmem184b Tomm22

Triobp 1700088E04Rik 4930500E03Rik D730005E14Rik Gm10856 Gm10863 Gm16576 Gm20420

SLEEP, Vol. 37, No. 4, 2014 793A Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

Csnk1e Alters Sleep Amount and Architecture—Zhou et al

Figure S1—Epistatic interaction between loci on chromosomes 13 and 15 affecting the REM sleep spent during the dark period. (A) The lower triangle displays joint logarithm of odds (LOD) scores for the full model fitting each pair of loci and their interaction. The upper triangle indicates model fitting for the interaction alone. LODfull = 11.47, LODconditional-interactive = 5.55, LODinteractive = 2.65, LODadditive = 8.82. The color scale at the right indicates separate scales for the joint LOD scores (right) and the epistasis LOD scores (left). (B) A magnified view of the intersection between chromosome 13 and chromosome 15. (C) Quantitative trait locus effect size of pair-wise interaction for the markers of rs3688207 on chromosome 13 (45.45 Mb) and rs13459189 on chromosome 15 (79.56 Mb). Because all mice were on a B6 background, the genotype labels are simplified as B6 = B6/B6 and BALB = BALB/B6. Error bars represent standard error of the mean. SLEEP, Vol. 37, No. 4, 2014 793B Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

Figure S2—Wheel-running activity patterns in mutant and congenic mice. (A) Wheel running activity patterns in mutant mice under a 12-h light: 12-h dark cycle (LD 12:12) entrained conditions (n = 9-14). The mean values of the time of activity onset for each genotype were calculated. Comparisons between mutant genotypes were assessed via one-way analysis of variance, followed by the Tukey post hoc tests. Csnk1e+/+ = 11.6 ± 0.2 h; Csnk1e-/- = 12.0 ± 0.1 h, P = 0.654; Csnk1etau/tau = 8.8 ± 0.5 h, P < 0.001. Group values were reported as mean ± standard error of the mean. ZT, Zeitgeber Time. (B) Wheel-running activity patterns in mutant mice in constant darkness (DD) conditions (n = 9-13). Data were normalized by circadian time (CT). (C) Wheel-running activity patterns in congenic mice under LD 12:12 entrained conditions (n = 9-13). B6.B6, congenic mice with the B6 allele on the B6 genetic background. B6.D2, congenic mice with the D2 allele against the B6 genetic background. D2.B6, congenic mice with the B6 allele against the D2 genetic background. D2.D2, congenic mice with the D2 allele on the D2 genetic background. ZT, Zeitgeber Time. Csnk1e Alters Sleep Amount and Architecture—Zhou et al

Figure S3—The advanced phase angle of entrainment of Csnk1etau/tau mice causes the phenotypic alterations of percentage of time spent in wakefulness (Wake%) and percentage of time spent in nonrapid eye movement sleep (NREM%) under entrained conditions, but these sleep differences are eliminated when exposed to DD. (A) Csnk1etau/tau mice show decreased Wake% in dark period under LD 12:12 entrained conditions (asterisk, P < 0.001, n = 12-15). (B) While exposed to constant darkness (DD), Csnk1etau/tau mice show same level of Wake% in the active phase compared to Csnk1e+/+ mice (n = 7-8). (C) Csnk1etau/tau mice show increased NREM% in the dark period in LD 12:12 entrained conditions (asterisk, P < 0.001, n = 12-15). (D) While exposed to DD, Csnk1etau/tau mice show the same level of NREM% in the active phase compared to Csnk1e+/+ mice (n = 7-8), indicating that the phenotype under entrained condition is an artifact of phase advance. Group values were reported as mean ± standard error of the mean. NREM, nonrapid eye movement.

SLEEP, Vol. 37, No. 4, 2014 793C Downloaded from https://academic.oup.com/sleep/article-abstract/37/4/785/2416973 by University of New England user on 01 February 2018

Figure S4—Examples of each sleep/wake stage. (A) NREM sleep with high-amplitude, low-frequency electroencephalography (EEG), and lowamplitude electromyography (EMG). (B) Nonrapid eye movement sleep with high-amplitude, low-frequency EEG, and low-amplitude EMG. (C) Rapid eye movement sleep with low amplitude EEG constituted mainly by theta wave activity and EMG atonia. (D) Wakefulness with low-amplitude, high-frequency EEG, and high-amplitude EMG.

Csnk1e Alters Sleep Amount and Architecture—Zhou et al