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\begin{document}

\title{Circasemidian, circadian, and longer-period activity rhythms in
caffeine-treated molecular clock deficient cryptochrome (Cry) 1 and Cry
2 double knockout mice}



\author[1]{Satoru Masubuchi}%
\author[1]{Takako Yano}%
\author[1]{Kouji Komatsu}%
\author[1]{Keisuke Ikegami}%
\author[2]{Takeshi Todo}%
\author[3]{Wataru Nakamura}%
\affil[1]{Aichi Medical University Graduate School of Medicine School of Medicine}%
\affil[2]{Osaka University School of Medicine Graduate School of Medicine}%
\affil[3]{Nagasaki University Graduate School of Biomedical Sciences}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Mammalian circadian rhythms are driven by the
transcriptional-translational feedback loop of clock genes in the
hypothalamic suprachiasmatic nucleus. However, chronic methamphetamine
treatment induces circadian activity rhythms in arrhythmic animals with
suprachiasmatic nucleus lesions or clock gene deletions. Activation of
dopaminergic neurotransmission by methamphetamine is considered to
induce activity rhythms. Adenosine antagonizes the actions of dopamine
at heteromers of dopamine and adenosine receptors (dopamine D1 and
adenosine A1 receptors, dopamine D2 and adenosine A2A receptors). In
this study, we considered that adenosine inhibition acts similarly to
methamphetamine, and administered an antagonist of adenosine A1 and A2A
receptors, caffeine, in drinking water. Chronic caffeine treatment
extended the circadian activity period of wild-type mice under constant
darkness. The circadian period extension continued for three weeks after
the replacement of caffeine with water. Chronic caffeine treatment
induced circasemidian (\textasciitilde{}12 h), circadian, and
longer-period activity rhythms in clock gene deficient, cryptochrome
(Cry) 1 and Cry 2 double knockout mice under constant darkness. These
activity rhythms changed periods spontaneously over time and became
arrhythmic upon caffeine withdrawal. In humans, rhythms with shorter or
longer than 24 h periods are hypothesized to cause internal
desynchronization of the sleep-wake rhythm from the \textasciitilde{} 24
h body temperature rhythm under temporal isolation. Circasemidian
rhythms are hypothesized to cause afternoon sleepiness and nap.
Caffeine-induced rhythms may help in understanding rhythms with not
around 24 h periods in humans.%
\end{abstract}%



\sloppy


The total word count: 3487

\emph{SHORT COMMUNICATION}

\textbf{Circasemidian, circadian, and longer-period activity rhythms in
caffeine-treated molecular clock deficient \emph{cryptochrome (Cry) 1}
and \emph{Cry 2} double knockout mice}

running title: caffeine induced activity rhythms

Satoru Masubuchi\textsuperscript{1}, Takako Yano\textsuperscript{1},
Kouji Komatsu\textsuperscript{1}, Keisuke Ikegami\textsuperscript{1},
Takeshi Todo \textsuperscript{2}, Wataru Nakamura \textsuperscript{3}

\textsuperscript{1} Department of Physiology, Aichi Medical University
School of Medicine, Nagakute, Aichi 480-1195, Japan

\textsuperscript{2} Department of Radiation Biology and Medical
Genetics, Graduate School of Medicine, Osaka University, Suita, Osaka
565-0871, Japan

\textsuperscript{3} Department of Oral-Chrono Physiology, Graduate
School of Biomedical Sciences, Nagasaki University, Nagasaki, Nagasaki
852-8588, Japan

*Corresponding author

Satoru Masubuchi\emph{masubuchi.satoru.488@mail.aichi-med-u.ac.jp}

ACKNOWLEDGEMENT

We thank Dr. Natsuki Matsushita (Division of Laboratory Animal Research,
Aichi Medical University School of Medicine) for the management of
animal care.

Funding information

MEXT \textbar{} Japan Society for the Promotion of Science (JSPS),
Grant/Award Number: 22K08246, 16K08532; Akiyama Life Science Foundation;
Suzuken Memorial Foundation; Takeda Medical Research Foundation;

Present address

Keisuke Ikegami, Laboratory of Regulation in Metabolism and Behavior,
Faculty of Agriculture, Kyushu University, Fukuoka, Japan

\textbf{Abstract}

Mammalian circadian rhythms are driven by the
transcriptional-translational feedback loop of clock genes in the
hypothalamic suprachiasmatic nucleus. However, chronic methamphetamine
treatment induces circadian activity rhythms in arrhythmic animals with
suprachiasmatic nucleus lesions or clock gene deletions. Activation of
dopaminergic neurotransmission by methamphetamine is considered to
induce activity rhythms. Adenosine antagonizes the actions of dopamine
at heteromers of dopamine and adenosine receptors (dopamine D1 and
adenosine A1 receptors, dopamine D2 and adenosine A2A receptors). In
this study, we considered that adenosine inhibition acts similarly to
methamphetamine, and administered an antagonist of adenosine A1 and A2A
receptors, caffeine, in drinking water. Chronic caffeine treatment
extended the circadian activity period of wild-type mice under constant
darkness. The circadian period extension continued for three weeks after
the replacement of caffeine with water. Chronic caffeine treatment
induced circasemidian (\textasciitilde{}12 h), circadian, and
longer-period activity rhythms in clock gene
deficient,\emph{cryptochrome (Cry) 1} and \emph{Cry 2} double knockout
mice under constant darkness. These activity rhythms changed periods
spontaneously over time and became arrhythmic upon caffeine withdrawal.
In humans, rhythms with shorter or longer than 24 h periods are
hypothesized to cause internal desynchronization of the sleep-wake
rhythm from the \textasciitilde{} 24 h body temperature rhythm under
temporal isolation. Circasemidian rhythms are hypothesized to cause
afternoon sleepiness and nap. Caffeine-induced rhythms may help in
understanding rhythms with not around 24 h periods in humans.

KEYWORDS

caffeine, adenosine receptors, circasemidian rhythm, circadian rhythm,
cryptochrome

\textbf{1 \textbar{} INTRODUCTION}

Circadian physiological and behavioral rhythms in mammals are driven by
the molecular clock in the hypothalamic suprachiasmatic nucleus (SCN).
Molecular clock is a transcriptional-translational feedback loop of
clock genes (Takahashi, 2017). CLOCK/BMAL1 heterodimer transactivates
the expression of \emph{Period} (\emph{Per} ) and\emph{Cryptochrome}
(\emph{Cry} ) via E-box. Periodic repression of\emph{Per} and \emph{Cry}
expression by their products, PER/CRY complex, generates circadian
rhythm. Clock gene deficient mice (Lowrey \& Takahashi, 2011), such as
\emph{Cry1} and \emph{Cry2} double knockout mice (CryDKO) (van der Horst
et al., 1999; Vitaterna et al., 1999), show arrhythmic behavior under
constant darkness (DD) and an increase in nighttime activity under light
dark cycle.

Chronic methamphetamine (MAP) treatment in drinking water causes the
rhythm of locomotor activity to desynchronize from the light dark cycle
and free run (Honma et al., 1986). Because MAP generates locomotor
activity rhythms even in SCN-lesioned animals (Honma et al., 1987;
Tataroglu et al., 2006), we examined gene expression rhythms in the
brain. In parallel with locomotor activity rhythm desynchronization from
the light dark cycle by MAP, clock gene and clock controlled gene
expression rhythms in the striatum and parietal cortex desynchronized
from the SCN which was entrained to the light dark cycle (Masubuchi et
al., 2000, 2007). Because these results suggest that clock genes outside
the SCN drive the MAP-induced activity rhythm, we administered MAP to
arrhythmic\emph{Clock} mutant mice. Contrary to expectations, locomotor
activity rhythms appeared after MAP treatment (Masubuchi et al., 2001).
MAP has also been found to induce activity rhythms in arrhythmic clock
gene deficient mice including CryDKO (Honma et al., 2008; Mohawk et al.,
2009). These results indicate that the MAP-induced locomotor rhythm is
not driven by the transcriptional-translational feedback loop of clock
genes.

In addition to MAP, methylphenidate induces an activity rhythm in
SCN-lesioned rats (Honma \& Honma, 1992). Both MAP and methylphenidate
activate dopaminergic neurotransmission by acting as reuptake inhibitors
of the dopamine released from dopaminergic neurons (Dela Pe\selectlanguage{ngerman}ña et al.,
2015), which suggesting that the activation of dopaminergic
neurotransmission plays a role in locomotor rhythm generation (Honma \&
Honma, 2009). Blum et al. (2014) demonstrated the generation of
locomotor rhythms by chemogenetic activation of dopaminergic neurons.

Adenosine is a physiological sleep factor (Porkka-Heiskanen \&
Kalinchuk, 2011). Adenosine A1 and dopamine D1 receptors (A1AR and D1R),
and adenosine A2A and dopamine D2 receptors (A2AAR and D2R) form
heteromers (A1AR/D1R and A2AAR/D2R). Adenosine antagonizes dopamine
activity at the A1AR/D1R and A2AAR/D2R sites. These heteromers are
involved in the pathophysiology of various diseases, including
Parkinson's disease, schizophrenia, drug addiction, and restless legs
syndrome (Fuxe et al., 2010; Nazario et al., 2017; Cortés et al., 2019).
Caffeine is a psychostimulant that acts as an A1AR and A2AAR antagonist
(Ferré, 2016). We expected that caffeine would act similarly to the
dopamine stimulant MAP and generate activity rhythm. Here, we
demonstrate that chronic caffeine administration in drinking water,
similar to MAP (Masubuchi et al., 2001) generates activity rhythms in
CryDKO.

\textbf{2 \textbar{} METHODS}

2.1. Animals

Specific pathogen-free (SPF) male C57BL/6J mice (WT) were purchased from
Japan SLC. \emph{Cry1} \selectlanguage{english}-/\selectlanguage{english}- and \emph{Cry2} \selectlanguage{english}-/\selectlanguage{english}- mice of C57BL/6J
background (CryDKO) were generated by Takasu et al. (2015). Animals were
maintained in the animal facility of Aichi Medical University. The
experimental protocols used in this study were approved by the Committee
for Animal Research at Aichi Medical University.

2.2. Behavioral rhythm monitoring

Mice aged 2-12 months were housed individually, and their behaviors were
monitored under a 12 h light (fluorescent light, \textasciitilde{}100
lux) - 12 h dark cycle (LD) or constant darkness (DD). The light-off
time was defined as Zeitgeber Time (ZT) 12. The locomotor activity of
the mice was monitored using passive (pyroelectric) infrared sensors
(PS-3241; EK-Japan, Japan). Data were collected and analyzed as
previously described (Masubuchi et al., 2005 Ikegami et al., 2020) using
The Chronobiology kit (Stanford Software Systems, Stanford, CA, USA).
Mice were entrained to the LD for more than two weeks and used for
experiments. Circasemidian/circadian periods of activity were identified
using a chi-square periodogram (Masubuchi et al., 2001). Differences in
periods between WT with and without caffeine administration were
evaluated using Student's t-test. Period values of Balb/c mice before
and after MAP reported by Masubuchi et al. (2001) were re-evaluated by
Student's t-test. Period changes caused by caffeine administration and
withdrawal in WT were evaluated using one-way ANOVA and Tukey HSD. All
statistical analyses were performed using EZR (Saitama Medical Center,
Jichi Medical University, Saitama, Japan), which is a graphical user
interface for R (The R Foundation for Statistical Computing, Vienna,
Austria).

2.3. Caffeine administration

For chronic caffeine treatment, caffeine (FUJIFILM Wako 031-06792) was
dissolved in distilled water (0.05\%) (Nagasawa et al., 2001) and
administered via drinking water.

\textbf{3 \textbar{} RESULTS}

\textbf{3.1 Chronic caffeine treatment extended circadian activity
period in WT.}

We administered 0.05\% caffeine to WT (n=7) under LD. After 7 days, the
caffeine-treated and control (n=6) mice were moved to DD. For 21 days in
DD, the activity period of caffeine-treated mice (23.85+-0.022 h, Figure
1a) was longer than that of controls (23.72+-0.032 h, Figure 1b). After
replacing caffeine by water, the period did not shorten for 26 days, and
caffeine re-administration for 21 days extended this period (Figure 1a,
1c upper, one-way ANOVA P=0.008). In the control mice, caffeine
administration was started at day 21 in DD. Caffeine treatment for 26
days extended the period, as previously reported (Oike et al., 2011).
After replacing caffeine by water, the period did not shorten for the
following 21 days (Figure 1b, 1c lower, one-way ANOVA, P=0.003).

\textbf{3.2 Chronic caffeine treatment induced circasemidian
(\textasciitilde{}12h), circadian, and longer activity rhythms in
CryDKO.}

We administered 0.05\% caffeine to CryDKO (n=16; Table 1) under LD.
After 7 days, the mice were moved to DD. In DD, CryDKO exhibited
behavioral rhythms that changed over time (Table 1). Of the 16 CryDKO,
15 exhibited circasemidian (8.75-17.16 h period) activity rhythms. 7
animals demonstrated circadian (20.75-26.66 h period) activity rhythms.
For example, CryDKO\#13 displayed an altered activity period from 13.75
h (7d) to 23.83 h (3d) and 15.16 h (6d) (Figure 2a). CryDKO\#09
displayed an altered activity period from 17.08 h (7d) to 14.33 h (4d)
and 12.00 h (5d) (Figure 2b). CryDKO\#14 displayed an altered activity
period from 15.16 h (6d) to 20.75 h (6d) and 13.08 h (3d) (Figure 2c). 7
animals demonstrated much longer activity periods (28.16-39.41 h). For
example, CryDKO\#07 displayed an altered activity period from 29.25 h
(5d) to 13.33 h (2d) and 30.00 h (10d) (Figure 3a). CryDKO\#10 displayed
an altered activity period from 28.33 h (5d) to 17.16 h (3d) and 30.91 h
(5d) (Figure 3b). One animal did not demonstrate circasemidian or
circadian rhythms for 37 days. The average amount of spontaneous
caffeine intake was 2.8+-0.19 mg/day/animal. After caffeine withdrawal,
all the CryDKO became arrhythmic.

\textbf{4 \textbar{} DISCUSSION}

The extension of the activity period in WT by chronic caffeine treatment
continued for at least 3 weeks after caffeine removal. This means that
temporal caffeine exposure brought about extension of period of the
biological clock. Chronic MAP treatment extended the circadian activity
period and induced activity rhythms with approximately 30 h period in
wild-type Balb/c mice (Masubuchi et al., 2001). Data obtained from this
study show that after MAP withdrawal, the activity period was
significantly longer than that before MAP (23.8+-0.0 h vs 23.4+-0.071 h,
n=4, P=0.001). Period extensions after chronic caffeine or MAP
administration suggest their similar effects on the circadian period.
After-effect is long lasting circadian period change after exposure to
various environmental lights (Pittendrigh \& Daan, 1976). We showed that
PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1) is
involved in after-effect of activity rhythm by light induced phase shift
(Masubuchi et al., 2010). Although the mechanism of after-effect is not
well known, it is possible that a common mechanism drives after-effect
by light and after-effect by non-photic intervention, that is, the
period extension after temporal caffeine treatment.

Activity rhythm generation by chronic caffeine administration in CryDKO
supports the idea that caffeine acts as an inhibitor of adenosine at
A1AR/D1R and A2AAR/D2R heteromers and acts similarly to the dopamine
increase in the synaptic cleft by MAP. D1R, D2R, and A2AAR are strongly
expressed in the striatum, whereas A1AR is widely expressed in the brain
(Fuxe et al., 2010; Mishra et al., 2018). If MAP- and caffeine-induced
rhythms are driven by a common system that acts through A1AR/D1R and/or
A2AAR/D2R, it may also involve the striatum.

Caffeine induced activity rhythms with various periods, including
circasemidian and circadian rhythms, and longer periods in CryDKO. In
addition to circadian rhythm, rhythms with a period longer or shorter
than 24 h have been hypothesized in human physiology. Internal
desynchronization of biological rhythms is known under isolated constant
condition. The period of the sleep-wake rhythm becomes either much
longer or shorter than 24 h, whereas the period of the body temperature
rhythm remains close to 24 h (Aschoff et al., 1967; Wever, 1979).
Dissociation of two oscillators, a circadian oscillator and an
oscillator with a period longer or shorter than 24 h, is considered to
cause internal desynchronization. Analyzing caffeine-induced rhythms
with longer and shorter periods than 24 h may be important for
clarifying the mechanism of internal desynchronization.

The caffeine-induced rhythms spontaneously changed over time.
Interestingly, the long period in CryDKO\#07 (29.25 h, Figure 3a) and
\#10 (28.33 h, Figure 3b) quickly became approximately half the
(circasemidian) period (13.33 h, 17.16 h) and returned to a long period
(30.00 h, 30.91 h). In humans, sleep propensity increases not only at
night, but also in the afternoon. 2 per day sleep/wake regulation is
thought to cause afternoon sleepiness and nap. The harmonics of
circadian oscillators, circadian oscillator splitting into 2 antiphase
circadian oscillators, model, and the independent circasemidian
oscillator model are hypothesized to cause 2 per day regulation. The
former does not account for the fact that naps are often skipped
(Broughton \& Mullington, 1992). The short-term appearance and
disappearance of the circasemidian rhythm, as observed in CryDKO\#07 and
\#10, may account for the skipping of naps and variable afternoon
sleepiness.

Caffeine is the most consumed psychoactive drug in the world (Ferre,
2016). Caffeine at bedtime has significant effects on sleep disturbance
(Drake et al., 2013). However, rhythm induction by caffeine provides a
possibility that caffeine is useful not only to increase wakefulness but
also to control the sleep-wake rhythm.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

SM designed research. SM, TY, and KK performed experiments. KI, TT, and
WN contributed new reagents/analytic tools. SM wrote the manuscript. TY,
KK, KI, TT, and WN reviewed the manuscript.

CONSENT FOR PUBLICATION

The authors consent to the publication of this manuscript.

DATA AVAILABILITY STATEMENT

All data and materials are available for review

ORCID

Satoru Masubuchi\emph{https://orcid.org/0000-0003-0377-088X}

REFERENCES

Aschoff, J., Gerecke, U., \& Wever, R. (1967). Desynchronization of
human circadian rhythms. \emph{Japanese Journal of Physiology,
17,}450-457.\emph{https://doi.org/10.2170/jjphysiol.17.450}.

\par\null

Blum, I. D., Zhu, L., Moquin, L., Kokoeva, M. V., Gratton, A., Giros,
B., \& Storch, K. F. (2014). A highly tunable dopaminergic oscillator
generates ultradian rhythms of behavioral arousal.\emph{Elife, 3,}
e05105.\emph{https://doi.org/10.7554/eLife.05105} .

Broughton, R., \& Mullington, J. (1992). Circasemidian sleep propensity
and the phase-maintenance model of human sleep/wake regulation.
\emph{Journal of Sleep Research,
1,}93-98.\emph{https://doi.org/10.1111/j.1365-2869.1992.tb00017.x}.

Cortes, A., Casado-Anguera, V., Moreno, E., \& Casado, V. (2019). The
heterotetrameric structure of the adenosine A1-dopamine D1 receptor
complex: Pharmacological implication for restless legs
syndrome.\emph{Advances in Pharmacology, 84,}
37-78.\emph{https://doi.org/10.1016/bs.apha.2019.01.001}

\par\null

Dela Pena, I., Gevorkiana, R., \& Shi, W. X. (2015). Psychostimulants
affect dopamine transmission through both dopamine transporter-dependent
and independent mechanisms.\emph{European Journal of Pharmacology, 764,}
562-570.\emph{https://doi.org/10.1016/j.ejphar.2015.07.044}.

Drake, C., Roehrs, T., Shambroom, J., \& Roth, T. (2013). Caffeine
effects on sleep taken 0, 3, or 6 hours before going to
bed.\emph{Journal of Clinical Sleep Medicine, 9,}
1195-1200.\emph{https://doi.org/10.5664/jcsm.3170}.

Ferre, S. (2016). Mechanisms of the psychostimulant effects of caffeine:
implications for substance use disorders.\emph{Psychopharmacology
(Berl),233,} 1963-1979.\emph{https://doi.org/10.1007/s00213-016-4212-2}.

Fuxe, K., Marcellino, D., Borroto-Escuela, D. O., Guescini, M.,
Fernandez-Duenas, V., Tanganelli, S., Rivera, A., Ciruela, F., \&
Agnati, L. F. (2010). Adenosine-dopamine interactions in the
pathophysiology and treatment of CNS disorders. \emph{CNS Neuroscience
\& Therapeutics, 16,}
e18-42.\emph{https://doi.org/10.1111/j.1755-5949.2009.00126.x} .

Honma, K., Honma, S., \& Hiroshige, T. (1986). Disorganization of the
rat activity rhythm by chronic treatment with
methamphetamine.\emph{Physiology \& Behavior, 38,}
687--695.\emph{https://doi.org/10.1016/0031-9384(86)90265-9}

Honma, K., Honma, S., \& Hiroshige, T. (1987). Activity rhythms in the
circadian domain appear in suprachiasmatic nuclei lesioned rats given
methamphetamine.\emph{Physiology \& Behavior, 40,}
767--774.\emph{https://doi.org/10.1016/0031-9384(87)90281-2}

Honma, K., \& Honma, S. (2009). The SCN-independent clocks,
methamphetamine and food restriction. \emph{European Journal of
Neuroscience, 30,}
1707-1717.\emph{https://doi.org/10.1111/j.1460-9568.2009.06976.x}.

Honma, S., \& Honma, K. (1992). Locomotor rhythms induced by
methylphenidate in suprachiasmatic nuclei-lesioned
rats.\emph{Neuroscience Letters, 137,}
24-28.\emph{https://doi.org/10.1016/0304-3940(92)90289-j}.

Honma, S., Yasuda, T., Yasui, A., van der Horst, G. T. J., \& Honma, K.
(2008). Circadian behavioral rhythms in Cry1 \selectlanguage{english}/ Cry2 double-deficient
mice induced by methamphetamine.\emph{Journal of Biological Rhythms,
23,} 91--94.\emph{https://doi.org/10.1177/0748730407311124}

Ikegami, K., Shigeyoshi, Y., \& Masubuchi, S. (2020). Circadian
Regulation of IOP Rhythm by Dual Pathways of Glucocorticoids and the
Sympathetic Nervous System. \emph{Investigative Ophthalmology \& Visual
Science, 61,} 26.\emph{https://doi.org/10.1167/iovs.61.3.26}.

Lowrey, P. L., \& Takahashi, J. S., (2011). Genetics of circadian
rhythms in Mammalian model organisms.\emph{Advances in Genetics, 74,}
175-230.\emph{https://doi.org/10.1016/B978-0-12-387690-4.00006-4}

Masubuchi, S., Honma, S., Abe, H., Ishizaki, K., Namihira, M., Ikeda,
M., \& Honma, K. (2000). Clock genes outside the suprachiasmatic nucleus
involved in manifestation of locomotor activity rhythm in rats.
\emph{European Journal of Neuroscience, 12,}
4206-4214.\emph{https://doi.org/10.1111/j.1460-9568.2000.01313.x}

\par\null

Masubuchi, S., Honma, S., Abe, H., Nakamura, W., \& Honma, K. (2001).
Circadian activity rhythm in methamphetamine-treated Clock mutant
mice.\emph{European Journal of Neuroscience,14,}
1177-1180.\emph{https://doi.org/10.1046/j.0953-816x.2001.01749.x}

Masubuchi, S., Kataoka, N., Sassone-Corsi, P., \& Okamura, H. (2005).
Mouse Period1 (mPER1) acts as a circadian adaptor to entrain the
oscillator to environmental light/dark cycles by regulating mPER2
protein. \emph{Journal of Neuroscience, 25,}
4719-4724.\emph{https://doi.org/10.1523/JNEUROSCI.4761-04.2005}

Masubuchi, S., Honma, S., Abe, H., Namihira, M., \& Honma, K. (2007).
Methamphetamine induces circadian oscillation in the brain outside the
suprachiasmatic nucleus in rats. \emph{Sleep and Biological Rhythms,
5,}132--140.\emph{https://doi.org/10.1111/j.1479-8425.2007.00263.x}

Masubuchi, S., Gao, T., O'Neill, A., Eckel-Mahan, K., Newton, A. C., \&
Sassone-Corsi, P. (2010). Protein phosphatase PHLPP1 controls the
light-induced resetting of the circadian clock. \emph{Proceedings of the
National Academy of Sciences of the United States of America,
107,}1642-1647.\emph{https://doi.org/10.1073/pnas.0910292107}

Mishra, A., Singh, S., \& Shukla, S. (2018). Physiological and
Functional Basis of Dopamine Receptors and Their Role in Neurogenesis:
Possible Implication for Parkinson's disease. \emph{Journal of
Experimental Neuroscience, 12}
,1179069518779829.\emph{https://doi.org/10.1177/1179069518779829}.

Mohawk, J. A., Baer, L. B. \& Menaker, M. (2009). The methamphetamine
sensitive circadian oscillator does not employ canonical clock
genes.\emph{Proceedings of the National Academy of Sciences of the
United States of America, 106,}
3519--3524.\emph{https://doi.org/10.1073/pnas.0813366106}

Nazario, L. R., da Silva, R. S., \& Bonan, C. D. (2017). Targeting
Adenosine Signaling in Parkinson's Disease: From Pharmacological to
Non-pharmacological Approaches. \emph{Frontiers in Neuroscience,
11,}658.\emph{https://doi.org/10.3389/fnins.2017.00658}

Nagasawa, H., Nakajima, T., Yamamura, M., Kaku, S., \& Hongou, N.
(2001). Effects of chronic treatment with caffeine on behaviour and
related parameters in male and female mice. \emph{In Vivo. 15,} 495-501.

Oike, H., Kobori, M., Suzuki, T., \& Ishida, N. (2011). Caffeine
lengthens circadian rhythms in mice. \emph{Biochemical and Biophysical
Research Communications,} \emph{410,}
654-658.\emph{https://doi.org/10.1016/j.bbrc.2011.06.049}.

Pittendrigh, C. S., \& Daan, S. (1976). A functional analysis of
circadian pacemakers in nocturnal rodents: I. The stability and lability
of spontaneous frequency. \emph{Journal of Comparative Physiology, 106,}
223--252.

Porkka-Heiskanen, T., \& Kalinchuk A. V. (2011). Adenosine, energy
metabolism and sleep homeostasis.\emph{Sleep Medicine Reviews, 15,}
123-135. \emph{https://doi.org/10.1016/j.smrv.2010.06.005.}

Takahashi, J. S., (2017). Transcriptional architecture of the mammalian
circadian clock. \emph{Nature Reviews Genetics, 18,}
164-179.\emph{https://doi.org/10.1038/nrg.2016.150}

\par\null

Takasu, N. N., Nakamura, T. J., Tokuda, I. T., Todo, T., Block, G. D.,
\& Nakamura, W. (2015). Recovery from Age-Related Infertility under
Environmental Light-Dark Cycles Adjusted to the Intrinsic Circadian
Period. \emph{Cell Reports, 12,}
1407-1413.\emph{https://doi.org/10.1016/j.celrep.2015.07.049}

Tataroglu, O., Davidson, A. J., Benvenuto, L. J., \& Menaker, M. (2006).
The methamphetamine-sensitive circadian oscillator (MASCO) in
mice.\emph{Journal of Biological Rhythms,} 21,
185-94.\emph{https://doi.org/10.1177/0748730406287529} .

van der Horst, G. T., Muijtjens, M., Kobayashi, K., Takano, R., Kanno,
S., Takao, M., de Wit, J., Verkerk, A., Eker, A. P., van Leenen, D.,
Buijs, R., Bootsma, D., Hoeijmakers, J. H., \& Yasui, A., (1999).
Mammalian Cry1 and Cry2 are essential for maintenance of circadian
rhythms. \emph{Nature, 398,}
627-630.\emph{https://doi.org/10.1038/19323}.

Vitaterna, M. H., Selby, C. P., Todo, T., Niwa, H., Thompson, C.,
Fruechte, E. M., Hitomi, K., Thresher, R. J., Ishikawa, T., Miyazaki,
J., Takahashi, J. S., \& Sancar, A., (1999). Differential regulation of
mammalian period genes and circadian rhythmicity by cryptochromes 1 and
2. \emph{Proceedings of the National Academy of Sciences of the United
States of America} , 96,
12114-12119.\emph{https://doi.org/10.1073/pnas.96.21.12114}.

Wever, R. (1979). The Circadian System of Man. Springer-Verlag, New York

\textbf{FIGURE 1 Circadian period extension in WT by chronic caffeine
treatment.}

(a) and (b) are representative double-plotted actograms of WT, which are
plotted on a 2 X 24-h time base. The black and white bars above the
recordings represent LD cycles. Black triangles indicate the day of
movement from LD to DD. The durations of the caffeine treatment are
presented on the right side of the actograms. (c) Changes in the period
value caused by caffeine administration and withdrawal. (c) Upper and
(c) lower panels correspond to (a) and (b), respectively.

\textbf{FIGURE 2 Circasemidian and circadian rhythms of CryDKOby chronic
caffeine treatment.}

Representative double plotted actograms of CryDKO. (a), (b), and (c) are
plotted on a 2 X 24-h time base. Parts of (a) are replotted on 2 X
13.75-h (7d), 2 X 23.83-h (3d), and 2 X 15.16-h (6d) time bases. Parts
of (b) are replotted on 2 X 17.08-h (7d), 2 X 14.33-h (4d), and 2 X
12.00-h (5d). Parts of (c) are replotted on 2 X 15.16-h (6d), 2 X
20.75-h (6d), and 2 X 13.08-h (3d) time bases. The black and white bars
above the recordings represent the LD cycles. Black triangles indicate
the day of moving from LD to DD.

\textbf{FIGURE 3 Apploximately 30-h rhythms of CryDKO by chronic
caffeine treatment.}

Representative double plotted actograms of CryDKO. (a) and (b) are
plotted on a 2 X 24-h time base. Parts of (a) are replotted on 2 X
29.25-h (5d), 2 X 13.33-h (2d), and 2 X 30.00-h (10d) time bases. Parts
of (b) are replotted on 2 X 28.33-h (5d), 2 X 17.16-h (3d), and 2 X
30.91-h (5d) time base. The black and white bars above the recordings
represent the LD cycles. Black triangles indicate the day of moving from
LD to DD.

\textbf{Hosted file}

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