PNAS Nexus, 2024, 3, 1–8
https://doi.org/10.1093/pnasnexus/pgad481
Advance access publication 29 December 2023

Research Report

Circadian control of sleep-related neuronal
activity in lizards
a,1, Koki T. Kotakea, Zhiwen Zhoua

, Kazuhiko Kumeb

, Sam Reiterca

Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan
Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japanc
Computational Neuroethology Unit, Okinawa Institute of Science and Technology (OIST) Graduate University, Okinawa 904-0495, Japan*To whom correspondence should be addressed: Email: norimoto@pop.med.hokudai.ac.jp; (H.N.); Email: sho.yamaguchi723@gmail.com (S.T.Y.)
1
S.T.Y. and S.H. contributed equally to this work.
Edited By: Andrey AbramovbAbstract
Although diurnal animals displaying monophasic sleep patterns exhibit periodic cycles of alternating slow-wave sleep (SWS) and rapid
eye movement sleep (REMS), the regulatory mechanisms underlying these regular sleep cycles remain unclear. Here, we report that in theAustralian dragon Pogona vitticeps exposed to constant darkness (DD), sleep behavior and sleep-related neuronal activity emerged over a
24-h cycle. However, the regularity of the REMS/SWS alternation was disrupted under these conditions. Notably, when the lizards werethen exposed to 12 h of light after DD, the regularity of the sleep stages was restored. These results suggest that sleep-related neuronal
activity in lizards is regulated by circadian rhythms and that the regularity of REMS and SWS cycling is influenced by daytime light
exposure.
Keywords: circadian rhythms, REMS/SWS alternation, Pogona vitticeps

Significance Statement
The influence of circadian rhythms on the regular REMS/SWS alternation in sleep remains unclear. By recording sleep-related neur­
onal activity in the diurnal lizard Pogona vitticeps under constant darkness (DD) conditions, we revealed the presence of circadian regu­
lation of the sleep/wake cycle. Furthermore, we found that REMS/SWS alternation was disrupted under DD conditions and restored by
12 h of light exposure, thus demonstrating circadian regulation and sensory modulation of sleep patterns in P. vitticeps and offeringvaluable insights into the regulatory mechanisms of REMS/SWS alternation.

Introduction
Diurnal animals with monophasic sleep patterns exhibit
species-specific cycles of slow-wave sleep (SWS) alternating with
rapid eye movement sleep (REMS) (1–6). Humans typically haveinter-REMS intervals averaging approximately 90 min, with fourto six cycles occurring throughout an 8-h sleep (1). In contrast,
marmosets have shorter sleep-stage cycles lasting approximately
50 min, with a range of 10 to 17 cycles during the night (2). A recent
study revealed that in the diurnal reptile Pogona vitticeps, SWS and
REMS regularly alternate with approximately 2-min cycles
throughout the night, generating more than 300 SWS/REMS cycles(3). Although the cycle varies significantly across species, com­
mon neural mechanisms may be involved. However, the precise
mechanisms responsible for maintaining these regular cycles
remain elusive.
Sleep-wake patterns are predominantly regulated by the circa­
dian clock, which maintains an ∼24-h sleep/wake rhythm even in
the absence of external cues (7–9). Long-term activity recordings

under constant dark (DD) conditions are commonly used to study
circadian behavior. While these methods allow for the assess­ment of free-running rhythms in spontaneous activity, accurately
tracking sleep-wake stages such as SWS, REMS, and wakefulness
poses significant challenges. Several studies addressing this issue
report that each vigilance stage, notably wakefulness and REMS,
relies on circadian rhythms (10, 11). Nevertheless, to date, few
studies have explored circadian effects on the dynamics of
REMS/SWS alternation in diurnal animals. In this study, we fo­
cused on the impact of DD conditions on the regular alternationof REMS and SWS in P. vitticeps.

Results
Behavioral and pigmentation rhythms under
DD conditions
In the DD experiments, the lizards were exposed to 12:12-h lightdark (LD) conditions (days 1 and 2), followed by 5 days of constantCompeting Interest: The authors declare no competing interest.
Received: September 18, 2023. Accepted: December 18, 2023
© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences. This is an Open Access article
distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits
unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Sho T. Yamaguchi a,*,1, Sena Hatoriand Hiroaki Norimoto a,*2 | PNAS Nexus, 2024, Vol. 3, No. 1

darkness (DD), followed by the LD condition for 1 day (Figs. 1A and S1).
Lizards in the control group were maintained under LD conditionsfor 6–8 consecutive days (referred to as “LD experiments,” Fig. S1).
The temperature was maintained at 30 °C. Videos and local fieldpotentials (LFP) were recorded continuously during the experi­ments. Food and water were withheld during the experiments.
The effect of food and water deprivation for 8 days on body weight
was remarkably subtle, and these differences were not significant
between the LD and DD experiments (Fig. S1, −3.14 ± 0.50% for the
LD group and −3.56 ± 0.53% for the DD group, mean ± SEM).
To quantify the locomotor activity of the lizards, we used aframe subtraction method on video data to detect body move­ments. These movements were converted into locomotor counts(Fig. S2; see the Material and methods section). Under LD condi­
tions, the lizards exhibited a diurnal behavioral pattern, being
most active during the light phase and displaying minimal move­
ment during the dark phase (Fig. S3). Despite the lack of food andwater, total daily locomotor activity remained constant through­out the LD experiments (Fig. S3). Under DD conditions, the lizards
showed reduced locomotor activity compared with LD conditions(Fig. 1B). Total daily locomotion during the DD condition (days 3–7)
was significantly lower than that during the LD condition (day 2)(Fig. 1C). Nevertheless, lizard behavior under DD conditions
exhibited a diurnal rhythm, with a significant difference in total
activity between the inferred subjective day (SD) and subjective
night (SN, Fig. 1D). Repeated-measures analysis of variance(RM-ANOVA) and the Lomb–Scargle periodogram demonstrated
the presence of a behavioral rhythm under DD conditions with a∼24-h cycle in all individuals (Fig. 1E). The brightness of the
body surface also exhibited circadian fluctuation in four of seven
lizards under DD conditions (Fig. S4), consistent with previous re­search that showed that about 50% of P. vitticeps individuals exhib­
ited a circadian rhythm in body brightness (12). These results
indicate that lizard behavior is regulated by endogenous circadianrhythms.

The circadian sleep/wake cycle under the
DD condition
To examine the effects of DD conditions on neuronal activity dur­ing sleep, we recorded LFPs from the claustrum or the adjacentdorsal ventricular ridge (DVR), the dominant noncortical pallial
domain of sauropsid brains (13–16), because sleep/wake stages
in lizards are distinguished by neuronal activity in these regions(3, 16, 17). We used the ratio of delta (0.1–3 Hz) power to beta
(10–30 Hz) power, referred to as the delta/beta (δ/β) ratio, as thebasis of the classification of sleep stages (3). Under DD conditions,δ/β oscillated on a 24-h cycle, with the maximum at SN (Fig. 2A
and B). RM-ANOVA and Lomb–Scargle periodogram analyses con­firmed the circadian rhythmicity of the δ/β ratio (Fig. 2C). Thesefindings indicate the presence of a circadian rhythm in the elec­
trophysiological sleep-wake cycle of lizards.

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Fig. 1. Behavioral rhythmicity under the DD condition. A) Experimental image. The lizards were maintained under a 12:12-h LD cycle, followed byconstant DD for 5 days, followed by LD for 1 day. The ambient temperature was maintained at 30 °C. B) Shown is the 30-min interval locomotor activity
during days 1–7. Blue lines are individual data; black lines are the average over all individuals (n = 7 animals). An enlarged view of the section enclosed by
the gray dashed line is shown in the center. C) Total daily locomotor activity for each day. Results of one-way ANOVA followed by the Tukey–Kramer
posthoc test vs. day 2 are as follows: day 3, P = 7.87 × 10−4, Q 7, 7 = 6.43; day 4, P = 0.001, Q 7, 7 = 6.31; day 5, P = 5.62 × 10−4, Q 7, 7 = 6.59; day 6, P = 0.001, Q 7, 7
= 6.16; day 7, P = 0.001, Q 7, 7 = 6.18. D) Activity profile under constant DD conditions. The 30-min interval locomotor activity of each individual under DD
conditions is binned into 24-h intervals, and the average locomotion at each phase (horizontal axis) is calculated from all data (n = 35 recordings fromseven animals for 5 days). The black line is the mean; blue shading is the SEM. The mean total activity in the SD and SN for each animal is shown in the top
right (n = 7 animals). The activity during SD and SN were summed for each day and the mean of the total activity was calculated for each animal. ***P <1.0 × 10−7 two-sided, by a two-sample hypothesis test using bootstrapping. E) Lomb–Scargle periodograms of locomotor activity under DD conditions for 5
days. The red circle on each periodogram indicates the peak of the estimated power. The period lengths of the peaks are 1,349, 1,450, 1,517, 1,423, 1,430,
1,510, and 1,443 min, reading from top to bottom.

Yamaguchi et al. |3

The regularity of the REMS/SWS alternation isdiminished under DD conditions

Restoration of the REMS/SWS alternations after
12-h light exposure

Next, we examined the regularity of the REMS and SWS alterna­
tions under different conditions. Under the LD condition, on day
2, the δ/β ratio oscillated regularly with a fixed period (Fig. 3A).
However, under the DD condition, the oscillation in δ/β became
irregular and arrhythmic by day 7. The auto-correlogram of theδ/β ratio revealed that under DD conditions, the patterns were
irregular and their amplitudes were dampened in comparison
with those recorded under LD conditions (Fig. 3B). The sliding
auto-correlogram revealed that the δ/β oscillation occurred pre­dominantly during the night, irrespective of LD or DD conditions(Fig. 3C), but the peaks of the auto-correlogram were less dis­tinct under DD conditions. These data suggest that REMS/SWS
alternations became unstable under DD conditions. This infer­ence is further supported by the cumulative distribution of
REMS/SWS bouts, the interval between a given SWS bout andthe subsequent SWS onset. Under the DD condition, theREMS/SWS bouts were shifted to longer durations, and theslopes of the cumulative plots were moderate compared withthose observed in the LD condition (Fig. 3D), indicating that
the REMS/SWS bouts are variable and relatively long under
the DD condition. Notably, we did not observe any prolongation
or instability of the REMS/SWS under the LD condition (Fig. 3E),
indicating that the changes were specifically associated with
the DD condition. Despite the changes in sleep architecture,
the proportions of SWS and REMS did not differ between the
LD and DD conditions (Fig. 3F), suggesting that the long-term
visual deprivation and/or decreased locomotion associatedwith the DD condition disrupts the regularity of the REMS/SWS cycles.

Finally, we examined whether the lengthened and unstable
REMS/SWS alternations observed under DD conditions were re­stored after returning to LD conditions, during which time the liz­ards were more active and provided with visual information. The
LFP on day 8 showed more regular REMS/SWS alternations com­pared with those of the DD condition on day 7 (Fig. 4A). The cumu­
lative distribution of REMS/SWS bouts on day 8 resembled that on
day 2 (LD condition) rather than that on day 7 (DD condition)(Fig. 4B). The increased mean and coefficient of variation (CV) of
the REMS/SWS bouts observed under the DD condition decreased
significantly on day 8 (Fig. 4C). Locomotor activity also significant­ly increased on day 8 (Fig. 4D). These results indicate that a 12-h
light exposure restores the longer and more irregular REMS/SWS
alternations that emerge under the DD condition.
We also examined whether locomotion influenced REMS/SWS
alternation by computing the correlation coefficients between
the total daily locomotion and the properties of the REMS/SWS
bouts. Neither the mean nor the CV of the REMS/SWS bouts wascorrelated with locomotion (Fig. 4E). These results suggest thatchanges in REMS/SWS alternations under the DD condition wereprimarily driven by visual deprivation rather than by a reduction
in locomotor activity.

Discussion
We here describe the circadian and ultradian sleep-related
rhythms in lizards under DD conditions. Under DD conditions,P. vitticeps exhibits circadian behavioral rhythms. Furthermore,
SWS and REMS occurred during the SN, even in the absence of

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Fig. 2. Circadian rhythmicity in the power of the LFP under the DD condition. A) Top: a representative trace of the δ/β ratio during days 1–7. Middle: arepresentative image showing the normalized δ/β ratio during days 1–7. Each vertical column represents a 30-min segment, progressing from bottom totop, and successive 30-min segments run continuously from left to right. Bin size, 1 s. Bottom: the mean δ/β ratio at 30-min intervals during days 1–7. The
data from each lizard was aligned and then standardized using z-scores. Blue lines represent individual data; black line represents the average of all
individuals (n = 5 animals). White, gray, and black horizontal bars indicate light, SD, and dark (or SN) phases, respectively. B) The profile of the z-scored δ/βratio under DD conditions. The 30-min interval mean δ/β ratios (z-scored) of each individual under DD conditions are binned into 24-h intervals, and themean δ/β ratio at each phase (horizontal axis) is calculated from all data (n = 25 sessions of five recordings for 5 days from five animals). Blue shading, SEM.
C) Lomb–Scargle periodogram of the δ/β ratio under DD conditions for 5 days. The red circles indicate the peaks of the estimated power. The period
lengths of the peaks are 1,456, 1,488, 1,442, 1,440, and 1,520 min, reading from top to bottom.

4 | PNAS Nexus, 2024, Vol. 3, No. 1

external cues (e.g. light, temperature, and feeding). These findings
indicate that lizard locomotion and sleep/wake cycles are regu­
lated by an endogenous circadian rhythm.
Intriguingly, under the DD condition, the REMS/SWS alterna­
tions became longer and more variable than under the LD condi­
tion. The disrupted rhythms were restored when the lizards werereturned to the LD condition. The decreased locomotor activity
observed under DD conditions was also reinstated by exposure
to light; however, the amount of locomotor activity did not correl­
ate with the duration or variation of the REMS/SWS alternations.
These results suggest that the REMS/SWS cycle observed at night
may be maintained by light exposure during the day, which isconsistent with previous studies showing disrupted sleep-stagetransitions in sensory-deprived ponies, which is known to be

diurnal (18). In addition, several studies using nocturnal or cre­puscular animals have investigated the effects of DD conditions
on each sleep stage. These studies imply that the effect of DD con­ditions on the REMS/SWS alternation may be widely observed
across species (19–22, but see Ref. 23). However, it is important
to note that the animals used in these studies are nocturnal or cre­puscular and their sleep is largely fragmented, with frequent oc­
currences of wakefulness, which may also contribute to themodifications of each sleep stage.
A possible mechanism for changes in sleep architecture causedby visual deprivation is the impact of visual deprivation on the cir­cadian clock system (24). In squirrel monkeys, rhythmic REMS/
SWS alternations with a period of 60 min have been observed.
However, damage to the suprachiasmatic nucleus, a dominant

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Fig. 3. Modification of REMS/SWS alternations under DD conditions. A) Top: short segments of LFP (top) and δ/β ratio (bottom), displaying the REMS/SWS
alternations (low/high δ/β ratio), on days 2 (left), and 7 (right). The δ/β ratio data are shown after median filtering (20-s window). B) Auto-correlogram of theδ/β ratio shown in (A). C) Representative heatmap for the auto-correlogram of the filtered δ/β ratio at 30-min intervals. Each vertical column represents
the auto-correlogram of the δ/β ratio for 30 min from the time point indicated on the horizontal axis. The auto-correlograms of successive 30-min
segments of the δ/β ratio run continuously from left to right. The white, gray, and black horizontal bars indicate light, SD, and dark (or SN) phases,
respectively. D) Cumulative fractions of REMS/SWS bout length for each day from day 1 to day 7. Data from each individual are aggregated by day, and thecumulative fraction is calculated (n = 5 animals for each day). E) Mean (left) and CV (right) of REMS/SWS bout lengths for each day (n = 5–7 recordings eachfrom four animals for LD experiments, and n = 5 recordings each from five animals for DD experiments). The data from each individual are normalized
by division by the mean of the corresponding data recorded on days 1 and 2. The results of the two-way ANOVA, LD vs. DD, are as follows: F1, 68 = 36.35;***P < 1.0 × 10−7 for the mean REMS/SWS bout (left); F1, 68 = 13.35; ***P = 5.05 × 10−4 for the CV of the REMS/SWS bout lengths (right). Blue shading, SEM.
F) SWS ratio during sleep (n = 5–7 recordings each from four animals for LD experiments, and n = 5 recordings each from five animals for DD experiments).
n.s., not significant by two-way ANOVA, LD vs. DD; F1, 68 = 2.70, P = 0.105. Blue shading, SEM.

Yamaguchi et al. |5circadian pacemaker in the mammalian brain, disrupted the
REMS/SWS switching process (6). In P. vitticeps, decreased light in­
put to the homologous master clock may have led to irregular
REMS/SWS alternations. Further experiments, such as lesioning
the master clock in lizards (25–29) or simultaneously recording
in lizards the LFPs in the DVR and the neuronal activity in the mas­ter clock, would allow a deeper understanding of how thecircadian clock system influences the regulation of REMS/SWS
alternation.
In conclusion, we demonstrated that the sleep of P. vitticeps isregulated by the internal circadian clock but that the clock alone,
without the presence of light, is not sufficient to maintain the re­gularity of the REMS/SWS alternation. Our findings reveal add­
itional similarities in sleep between lizards and other amniotes,
emphasizing the utility of lizards in sleep research. Further

investigations using reptiles could enable us to access in detailthe fundamental mechanisms of REMS/SWS alternation that areconserved across species.

Limitations of the study
We only examined P. vitticeps in our study; however, it is necessary
to investigate additional animal species to determine whether our
findings can be generally applied to other animals.

Materials and methods
Animals
Ten lizards (Pogona vitticeps; seven males, two females, and one
unknown), weighing 150–300 g, were obtained from external

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Fig. 4. Restoration of the REMS/SWS alternation upon return to LD conditions. A) Short segments of LFP (top) and δ/β ratio (bottom), displaying thealternations in REMS/SWS (low/high δ/β ratio), on day 7 (left) and 8 (right). B) Cumulative fraction of REMS/SWS bout length for days 2, 7, and 8. Data fromeach individual are aggregated by day, and the cumulative fraction is calculated. C) Mean (left) and CV (right) of REMS/SWS bout lengths for days 7 and 8(n = 5 animals). The data from each individual are normalized by division by the mean of the corresponding data recorded between days 1 and 2.
**P = 0.008; *P = 0.036, by a two-sample hypothesis test using bootstrapping (one-sided). D) Total daily locomotion on days 7 and 8 (n = 7). ***P < 1.00 × 10−7
by a two-sample hypothesis test using bootstrapping (one-sided). E) The mean (left) and CV (right) of REMS/SWS bout length are plotted against the amountof daily locomotion (n = 52 sessions, comprising six recordings of LD experiments from four animals and three recordings of DD experiments from three
animals). Red dots represent data from days with the LD condition (days 2 and 8 of DD experiments and from day 2 to day 8 of LD experiments); blue dots
represent data from days with the DD condition (from days 3 to 7 of DD experiments). R, Pearson’s correlation coefficient; P, confidence level to reject the null
hypothesis.

6 | PNAS Nexus, 2024, Vol. 3, No. 1

Surgery
The day before surgery, the lizards were administered meloxicam(Boehringer Ingelheim Animal Health Japan; 0.2 mg/kg s.c.). On
the day of the surgery, the animal was initially placed in a box
filled with isoflurane (Mylan Inc., USA) and, slightly later, topicalanesthesia (xylocaine; Sandoz Pharma, Japan) was administeredaround the trachea. After induction of deep anesthesia, the ani­
mal was intubated via the trachea and placed in a stereotactic
apparatus. During surgery, anesthesia was maintained with
isoflurane (0.5–3 vol%), and body temperature was maintainedat around 32 °C using a heating pad. The heart rate was monitored
using an ultrasonic blood flow sensor.
A craniotomy was performed around the parietal eye, and theoverlying skin was disinfected using a 7.5% povidone–iodine solu­
tion (Meiji, Japan) before removal with a scalpel. The dural andarachnoid layers covering the forebrain were removed using fineforceps, and the pia was gently removed over the area targeted
for electrode insertion. The exposed skull was covered with ultra­
violet (UV)-curing glue, and the bare ends of two insulated
stainless-steel wires (0.005 in, A-M Systems, USA) were placed
subdurally to serve as the electrical reference and ground, re­spectively, and fixed with UV-curing glue.
To insert the electrodes, a stainless-steel wire (A-M Systems)
was mounted on an OpenEphys shuttleDrive (30) or a 3D-printed
custom microdrive based on the shuttleDrive and secured toa stereotactic adaptor. The electrodes were lowered about
1–1.5 mm from the brain surface, targeting the DVR. The brain sur­face was covered with Dura-gel (Neurotech, UK). After connecting
the reference to the ground, the skull, craniotomy, and microdrive
were tightly secured using UV-curing glue and dental cement. After
surgery, the lizards were injected s.c. with meloxicam (0.2 mg/kg),
released from the stereotactic apparatus, and maintained on aheating pad until fully recovered from anesthesia.

Recording
Before recording, the lizards were habituated to an acrylic cage
(30 × 30 × 30 cm) and placed in an incubator for at least one night.
On the initial day of recording, the lizard was fed crickets in add­
ition to normal feeding. One to two hours before lights went off,the lizard was moved into an acrylic cage inside an incubator, inwhich the ambient temperature was maintained at 30 °C, andthe recording for day 1 was then started. For DD experiments,
the lizards were exposed to 12:12-h LD conditions (day 2), followedby 5 days of DD conditions (days 3–7), then returned to LD condi­
tions for 1 day (day 8). For the LD experiments, the lizards weremaintained under the LD condition for 6–8 consecutive days, in­cluding day 1. The lizards in both groups were not provided withfood or water during the experiments.
The body weight of the lizards was measured before and after
the experiment. To calculate the changes in body weight, thebody weights after the experiments were subtracted from thoseat baseline (before or a week after the experiments), followed bydivision by the body weights at baseline, and expressed as percen­
tages (%). Body weight data from LD experiments lasting 6 or 7days were excluded from the analysis.
Video recordings were conducted using a Universal Serial Bus
camera (BFS-U3-16S2M-CS; Teledyne FLIR, USA) positioned above
the acrylic cage, and illumination was provided by an infraredtransmitting plate. The video data were acquired using
SpinView software (Teledyne FLIR, Wilsonville, Oregon, USA) at
a rate of 10 frames per second.
The LFP recordings were performed using the OpenEphys sys­
tem and 32-channel head stages (Intan Technologies, Los
Angeles, CA, USA). The recordings were referenced to one of thewires. Signals were sampled at 1 kHz and band-pass filtered at
0.1–100 Hz.

Video analysis
To detect locomotion, frames were converted to grayscale, andeach of the two consecutive frames was subtracted. The resulting
subtraction images were binarized, and connected components
with fewer than 10 pixels in each frame were excluded. The num­
ber of pixels that exhibited changes between two successiveframes (referred to as the pseudo-velocity) was then calculated.
The pseudo-velocity was thresholded to remove lights-on/off
events. The difference between the pseudo-velocities at two con­secutive time points was computed as the pseudo-acceleration.
Locomotor activities were counted as events when thepseudo-acceleration exceeded 2 × standard deviations.
For the extraction of body brightness, the videos were down­
sampled to 1 Hz. The body parts of the lizards were tracked using
DeepLabCut (31). Before extracting the pixel intensity, the videos
were converted to grayscale. The sum of the pixel intensities ob­
tained from a specific area on the back of the lizard was normal­
ized to the intensity obtained from the background. After
detrending, data points that deviated by more than 3 × local
standard deviation from the local mean over a 30-min interval
or data points with a pseudo-velocity exceeding 2 × standard devi­
ation were excluded. The remaining data points were linearly in­terpolated following standardization to z-scores.

LFP analysis and sleep staging
After binning the data in 10-s windows and 1-s steps, the power
spectrum for each bin was calculated using the fast Fourier trans­
form algorithm. The δ/β ratio was calculated by dividing the mean
spectrum over frequencies lower than 3 Hz by the mean spectrum
over frequencies between 10 and 30 Hz in each bin. The medianfiltered (20-s window) δ/β ratio was used for the calculation of
auto-correlograms. Electrostatic noise was removed by setting avisually guided threshold on the LFP plot.
Sleep-stage analysis was performed using the data obtained
during a 4-h period centered around the peak of the median fil­tered (6-h window) δ/β ratio. The onsets/offsets of the SWS/REMS alternations were calculated by upward/downward thresh­
old crossing of the median filtered (20-s window) δ/β ratio. The
threshold was set as the average between the 100 smallest and
100 largest points of the median filtered (1,000-s window) δ/β ratio.
The interval between two consecutive SWS onsets was computed
as the duration of a REMS/SWS alternation. REMS/SWS bouts thatdeviated by more or less than 2 × standard deviations from themean were excluded from further analysis. The SWS ratio was

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breeders and housed individually in reptile terraria (45 × 45 ×
30 cm) maintained on a 12; 12-h light/dark cycle. The lights-on
and lights-off times were referred to as 0 and 12 h, respectively.
A halogen spotlight for basking was placed above the terraria
and turned on between 3 and 9 h each day. The lizards had accessto water ad libitum and were fed either vegetables (bok choy, car­rots, and lettuce) or crickets on alternate days. Calcium and vita­
min D3 supplementation (GEX Corporation, Japan) were provided
in the vegetables.
All procedures involving animals were approved by the Animal
Care and Use Committee of Hokkaido University (21-0092).

Yamaguchi et al. |7computed as the intervals between the SWS onsets and the subse­
quent REMS onsets summed over 4 h, and then dividing by 4 h.

foundation, the Naito foundation, and the Toray Science andTechnology Grant to H.N.

Circadian rhythm analysis

Author Contributions

Calculation of correlation coefficients
The mean and CV of the REMS/SWS bout durations from each in­dividual were divided by the mean of the corresponding data re­corded between days 1 and 2. The locomotor activity of eachindividual was scaled by the corresponding data recorded on
day 2. Pearson’s correlation coefficient was calculated after out­
liers had been deleted, and outliers were detected using the
Grubbs’ test.

Statistical analysis
The presented data are the mean ± SEM or individual data with/without means. Data were analyzed using MATLAB version
9.12.0 (R2022a; The MathWorks Inc., Natick, MA, USA) (33) andPython (Python 3 Reference Manual, 2009; CreateSpace; Scotts
Valley, CA, USA) (34), as described above and in the figure legends.

Acknowledgments
We thank Dr. Lorenz A. Fenk for the discussions; and all the mem­bers of the Norimoto Laboratory for their daily discussions andadvice.

Supplementary Material
Supplementary material is available at PNAS Nexus online.

Funding
This work was supported by JSPS KAKENHI (22K15369) and grant
form Hirose Foundation to Z.Z., KAKENHI (23K14225 and
22K20675), the Akiyama Life Science Foundation and the
Sasakawa Scientific Research from the Japan Science Society
Grant to S.T.Y., and JST PRESTO (JPMJPR2048), AMED
(22wm0525003s0202), KAKENHI (23K18251), the Murata Science
Foundation, Takeda Science Foundation, the Uehara Memorial
Foundation, the Mochida Memorial Foundation for Medical andPharmaceutical Research, a Grant for Basic Science Research
Projects from the Sumitomo Foundation, the Astellas
Foundation for Research on Metabolic Disorders, the Nakajima

S.T.Y., K.K., and H.N. designed the study. S.T.Y. and S.H. per­formed the experiments. S.T.Y. and K.T.K. analyzed the dataand prepared the figures. R.S. and Z.Z. assisted with the analysis.
All authors discussed and interpreted the results. S.T.Y. and H.N.
wrote the manuscript with input from all authors.

Data Availability
The data are openly available in Mendeley Data at https://data.
mendeley.com/datasets/v7kkwgnyn5/1.

References1

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The data were summed (for locomotion) or averaged (for body col­or and δ/β ratio) over 30-min intervals, then binned into 24-h inter­vals. The phase profiles of each parameter were calculated byaveraging all the data at each time point. RM-ANOVA was per­formed using individual profile data to estimate whether the indi­vidual was rhythmic (P < 0.05) or not (32). In addition, the data
were smoothed by calculating the 1-min interval sum (for locomo­tion) or average (for body color and δ/β ratio). The Lomb–Scargle
periodogram was constructed using 1-min interval data, and apeak in the periodogram ranging from 1,200 to 1,680 min was
detected. A peak in the Lomb–Scargle periodogram was defined
as significant if it had a power greater than that of the minimumof the peaks from individuals estimated as rhythmic by
RM-ANOVA. Any individual parameter exhibiting a significant
peak in the Lomb–Scargle was classified as “rhythmic.” The data
were z-scored before the calculation of profiles for body color,the δ/β ratio, and the Lomb–Scargle periodograms for body color.

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