Secretin receptor-deficient mice exhibit altered circadian rhythm in wheel-running activity

Journal Pre-proof Secretin receptor-deficient mice exhibit altered circadian rhythm in wheel-running activity Mizuki Sugiyama (Methodology) (Validation) (Formal analysis) (Investigation) (Writing - original draft) (Visualization), Ichiko Nishijima (Conceptualization) (Methodology) (Resources) (Writing review and editing) (Funding acquisition), Shota Miyazaki (Validation) (Formal analysis) (Investigation), Takahiro J. Nakamura (Conceptualization) (Methodology) (Investigation) (Resources) (Writing - review and editing) (Visualization) (Supervision) (Project administration) (Funding acquisition)

PII:

S0304-3940(20)30084-7

DOI:

https://doi.org/10.1016/j.neulet.2020.134814

Reference:

NSL 134814

To appear in:

Neuroscience Letters

Received Date:

21 October 2019

Revised Date:

26 January 2020

Accepted Date:

1 February 2020

Please cite this article as: Sugiyama M, Nishijima I, Miyazaki S, Nakamura TJ, Secretin receptor-deficient mice exhibit altered circadian rhythm in wheel-running activity, Neuroscience Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.134814

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Secretin receptor-deficient mice exhibit altered circadian rhythm in wheel-running activity

Mizuki Sugiyamaa, Ichiko Nishijimab*, Shota Miyazakia, Takahiro J. Nakamuraa*

aLaboratory

of Animal Physiology, School of Agriculture, Meiji University, Kawasaki,

bTohoku

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Kanagawa 214-8571, Japan Medical Megabank Organization, Tohoku University, Sendai, Miyagi 980-8575 Japan

to: Takahiro J. Nakamura, Ph.D. and Ichiko Nishijima, Ph.D.

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*Correspondence

Laboratory of Animal Physiology, School of Agriculture, Meiji University

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1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 Japan

E-mail: [email protected]

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Tel & Fax: +81-44-934-7823

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Abstract (250 words limit) In mammals, the timing of behavior and physiological activity is controlled by the suprachiasmatic nucleus (SCN) in the hypothalamus. Incidentally, secretin is a peptide hormone that promotes digestive activities and regulates water reabsorption. In recent studies, exogenous administration of secretin has been reported to induce secretion of oxytocin in the supraoptic nucleus of the hypothalamus and modulate social behavior. These results indicate that secretin is involved in the neural network that controls social behavior

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and plays important roles in the central nervous system. In the present study, we

investigated the effects of secretin on circadian rhythms, by assessing circadian rhythms during wheel-running behavior in secretin receptor-deficient (Sctr-/-) mice. Male adult

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wild-type (WT) and Sctr-/- mice were housed in separate cages containing a wheel. Every

minute of the wheel-running activity was monitored during the normal light-dark (LD) cycle

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(12:12 h) and in constant darkness (DD). Significant differences were observed in the free-running period between the WT and Sctr-/- mice. However, no significant differences

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were observed in the daily wheel-running revolutions between WT and Sctr-/- mice, in the LD and DD conditions. Moreover, the ratio of the daily activity phase to the rest phase (α/ρ) was

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significantly smaller in Sctr-/- than that in WT mice in the DD condition. Secretin receptors were expressed in the SCN cells. These findings suggest that secretin receptors are involved

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in the central circadian clock in the SCN and the circadian system in general.

Abbreviations: α/ρ, activity phase to rest phase ratio; AVP, arginine vasopressin; DD

constant darkness; LD, light-dark; Pac1, pituitary adenylate cyclase-activating peptide receptor 1; Sctr, secretin receptor; Sctr-/-, secretin receptor-deficient; SCN, suprachiasmatic nucleus; V1a, AVP receptor; VIP, vasoactive intestinal peptide; Vpac, VIP receptor; WT, wild-type

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Keywords: Circadian rhythm; Secretin; Wheel running; Free-running period

1. Introduction Most organisms have evolved circadian clocks that anticipate environmental

changes and establish endogenous 24-h rhythms, to obtain the correct physiology and behavior according to the appropriate time window each day [1]. In mammals, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the central pacemaker that

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coordinates daily rhythms in behavior and physiology, including sleep, hormone secretion,

etc. [1]. The SCN contains about 20,000 neurons, most of which have the ability to generate autonomous circadian oscillations by transcriptional/translational feedback loops [2].

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Secretin is a 27-amino acid peptide belonging to the secretin-glucagon peptide hormone family. It promotes digestive activities and regulates water reabsorption by

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inducing the secretion of pancreatic juice in the duodenum [3, 4]. It was recently reported

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that secretin is also produced in the cerebellum [5] and hippocampus [6]. The receptor for secretin is a member of the type II G-protein-coupled receptor family, which is also widely expressed in the pancreas [7], stomach [8], kidney [9], and brain [cerebellum, hippocampus,

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and central amygdala] [6, 10].

An increasing amount of evidence indicates a neuroactive role of secretin in the

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central nervous system, and its functions have been attracting attention. Recent studies

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have reported that both peripheral and intracranial administration of secretin activate oxytocin and vasopressin expressing neurons in the supraoptic nucleus of the hypothalamus [12, 13] and improve social recognition behavior. Moreover, exogenous administration of secretin alleviates the symptoms of some patients with autism [11] and the negative symptoms of patients with schizophrenia [14]. Furthermore, it was reported that the administration of secretin alters dopamine metabolism in the central nervous system in autism [15], and that autism-specific single-nucleotide polymorphisms exist in the 3

transcriptional regulatory region of the secretin gene [16]. These results indicate that secretin is involved in the neural network that controls social behavior, and that it plays important roles in the central nervous system. In the present study, we evaluated the expression of secretin receptor in the SCN and the effects of secretin in circadian rhythms, using secretin receptor-deficient (Sctr-/-) mice. We also assessed the wheel-running activity of Sctr-/- mice and alterations in circadian

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rhythms, including the duration of the activity phase.

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2. Materials and methods 2.1. Animals and housing Sctr-/- mice were generated in the 129/SvEv background. The Sctr-/- mice were created by replacing exon 1 of the secretin receptor with lacZ reporter and PGKneobpA selection marker, which are in frame with the endogenous start codon of the Sctr gene, as described previously [17]. Heterozygous mutants were back crossed ten generations onto C57BL/6J mice to obtain homozygous (Sctr-/-) and wild-type (WT) mice. Genotyping was

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performed with PCR, using sets of primers that amplified the WT or the disrupted gene for each Sctr allele. We only used male mice to accurately examine the influence of knocking

out secretin receptors on mice, because female mice have regular 4 or 5-day estrous cycles

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that are accompanied by fluctuating activity levels, depending on the stage of the cycle. The animals were maintained in a 12:12-h light-dark (LD) cycle under controlled air conditions

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(room temperature, 23 ± 1° C; humidity, 50 ± 10%) with food and water available ad libitum.

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All animal housing conditions, and experimental procedures were in accordance with the guidelines of the Japanese Physiological Society and were approved by the Institutional Animal Care and Use Committee at School of Agriculture Meiji University (permission

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number: IACUC16-0012).

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2.2. Measuring of wheel-running activity

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Each mouse was housed in a separate cage (183 × 340 × 148 mm; CL-0135, CLEA Japan, Tokyo Japan) that contained a running wheel (12-cm diameter, SANKO, Osaka, Japan). The cages were placed in light-tight, ventilated boxes, in which the light intensity at the bottom of the cage was 200–300 lx. The number of wheel revolutions was counted by a magnet-sensor-activated signal between a button magnet on the running wheel and a magnet relay (59070-010, Littelfuse, Inc., Chicago, IL, USA) fixed on a side wall of the cage and was fed into a computer every minute. A chronobiology kit (Stanford Software Systems, 5

Naalehu, HI, USA) and ClockLab software (Version 2.72, Actimetrics, Wilmette, IL, USA) were used to collect and display the activity data.

2.3. Experimental schedule When animals were 8 weeks old, they were housed in a normal 12:12 h LD cycle for 2 weeks and transferred into DD for 2 weeks. When the animals were transferred and entrained to LD cycle again, they were sacrificed under isoflurane anesthesia, to harvest the

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brain at zeitgeber time (ZT) [8-10]. Zeitgeber time is a 24-h normalized notation of the phase in a circadian cycle entrained to the LD condition, with lights on from ZT0 to ZT12. On the other hand, circadian time (CT) is a normalized 24-h notation of the phase in a circadian

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cycle, which represents an estimation of the animal’s subjective time, and CT12 indicates the beginning of subjective night (activity onset). Activity onset was manually determined using

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an activity profile in ClockLab, as the time at which the amount of activity near the baseline

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started to exceed the average amount of activity.

2.4. X-gal staining

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To assess Sctr expression in the SCN, we performed X-gal staining. Both WT and Sctr-/- brains were placed in 4% paraformaldehyde fixative at 4 °C for 24 h, then transferred

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to a sucrose gradient, and stored in 30% sucrose at 4 °C until sinking of the brains. Frozen

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coronal sections of 30 μm that included the SCN region (bregma, -0.22 to -0.82 nm) were cut using a Cryostat (Cryostar NX50, Thermo Fisher Scientific, Tokyo, Japan) and stained with Beta-Galactosidase Staining Kit (Clontech Laboratories, Inc. A Takara Bio Company, Shiga, Japan), according to the manufacturer’s instructions. Sections were placed onto slide glasses, and then counterstained with neutral red (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to label the cell nucleus.

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2.5. Data analysis and statistics Daily wheel-running revolutions and the duration of the active phase, as well as the free-running period, were calculated by ClockLab software (Actimetrics). The free-running period of wheel-running activity for 14 days in the DD condition were calculated using chi-squared periodograms. Daily activities for 7 days in the LD and 14 days in the DD conditions were quantified using the activity profile function in ClockLab. Since the study included fewer Sctr-/- mice than WT mice from the beginning, we performed each

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experiment with the maximum number of samples and analyzed the collected data for

securing the number of samples. On the other hand, the corresponding data was excluded from evaluation and statistical analysis when continuous activities exceeding the standard

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day were not recorded. The number of analyzed samples for each experiment is shown in the results. Differences between the WT and Sctr-/- mice were examined using unpaired

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Student’s t-test. Statistical analysis was performed using IBM SPSS Statistics software (SPSS

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Inc., Chicago, IL, USA). All results are presented as the mean ± standard error of mean, and p-values < 0.05 were considered significant.

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3. Results

3.1. Sctr-/- mice exhibit shorter circadian period

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Both the WT and Sctr-/- mice showed diurnal and circadian wheel-running rhythms

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in both the LD (Fig. 1A) and DD (Fig. 1B) conditions. We calculated the free-running period and daily wheel-running revolutions. The circadian period during the wheel-running activity was significantly shorter in Sctr-/- mice than that in the WT mice (WT: n = 10, 23.697 ± 0.028 h; Sctr-/-: n = 7, 23.527 ± 0.055; p < 0.01; Fig. 1C). However, no significant differences were observed in daily wheel-running revolutions between WT and Sctr-/- mice in both the LD (WT: n = 12, 22,882 ± 1,813 rev/day; Sctr-/-: n = 9, 18,239 ± 3,040 rev/day; Fig. 1D) and DD (WT: n = 12, 18,828 ± 2,127 rev/cycle; Sctr-/-: n = 7, 22,575 ± 2,423 rev/cycle; Fig. 1E) 7

condition.

3.2. Sctr-/- mice exhibit shorter activity phase We calculated the ratio of the activity (α) to rest (ρ) phase in both the LD and DD condition. No significant differences in α/ρ ratio between WT and Sctr-/- mice were observed in the LD condition (WT: n =12, 0.808 ± 0.053; Sctr -/-: n = 9, 0.641 ± 0.074; Fig. 1F). In contrast, the α/ρ ratio was significantly smaller in Sctr-/- than in WT mice in the DD condition

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(WT: n = 12, 1.072 ± 0.077; Sctr-/-: n = 7, 0.612 ± 0.032, p < 0.001; Fig. 1G). We also plotted

hourly activity profiles in Fig. 1H and I. Significant differences in the hourly activity at ZT21

and ZT22 were observed between WT and Sctr-/- mice in the LD condition (ZT21: WT: n = 11,

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4.107 ± 0.417; Sctr-/-: n =7, 2.034 ± 0.462, p < 0.01; ZT22: WT: 3.966 ± 0.353; Sctr-/-: 2.296 ± 0.332, p < 0.01; Fig. 1H). Moreover, in the DD condition, significant differences were also

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observed in the normalized hourly activity at CT19 and CT21 between the WT and Sctr-/mice (CT19—WT: n = 12, 8.609 ± 0.495; Sctr-/-: n = 7, 10.939 ± 0.187; p < 0.05; CT21—WT:

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5.8196 ± 0.438; Sctr-/- 7.965 ± 0.166; p < 0.01; Fig. 1I).

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3.3. Secretin receptors are expressed in the SCN

Exon 1 of the secretin receptor was replaced with the lacZ reporter and a

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PGKneobpA selection marker in Sctr-/- mice [17]. In the present study, we assessed the

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expression of the lacZ reporter in the brains of WT and Sctr-/- mice using X-gal and counterstained the cell nuclei with neutral red to analyze the expression of secretin receptors in the SCN. LacZ expressing cells were observed in the entire SCN of Sctr-/- but not that of WT mice (Fig. 2).

4. Discussion In the present study, we assessed circadian rhythms of wheel-running behavior in 8

Sctr-/- mice aiming to investigate the effects of secretin depletion on circadian rhythms. No significant difference was observed between the daily-activity levels of WT and Sctr-/- mice in the LD and DD conditions. However, the free-running period of Sctr-/- mice was significantly shorter than that of WT mice. Sctr-/- mice also showed a shorter activity phase in the DD condition. Moreover, the expression of Sctr was observed in the SCN. These results suggest that secretin receptor signaling modulates circadian behavioral rhythms via secretin receptors in the SCN.

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Previous studies reported that the α/ρ is positively correlated with the circadian

period [18, 19]. Thus, the shorter activity phase of Sctr-/- mice observed in the DD condition authenticates the observation of a shorter free-running period in Sctr-/- mice. Decreased

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hourly activity was observed in the second half of the activity phase the LD condition,

phase and enhances the result of the α/ρ.

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although it was not observed in the DD condition. This result indicates the shorter activity

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Sctr is a member of the type II G-protein-coupled receptor family, which is mainly expressed in the pancreas [7], stomach [8], and kidneys [9]. It was also reported that it is expressed in several regions of the brain, including the cerebellum, hippocampus, central

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amygdala, median frontal gyrus, entorhinal cortex, hypothalamic paraventricular nucleus, perifornical region, lateral hypothalamic area, head of the caudate nucleus, and spinal

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trigeminal nucleus [6, 10, 20]. However, it has not been ascertained whether secretin

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receptors are expressed in the SCN. In the present study, we observed a few scattered Sctr expressing cells in the SCN, which represents the central circadian clock and defines the circadian behavioral period [21, 22]. Therefore, our findings suggest that the secretin receptor is involved in the central circadian clock and regulates the circadian period of behavioral rhythms. The SCN is divided into dorsal (shell) and ventral (core) subdivisions and contains approximately 20,000 neurons that have heterogeneous properties, expressing several 9

neurotransmitters, which are located within specific regions. For example, arginine vasopressin (AVP)-expressing neurons are mainly located in the SCN shell, while vasoactive intestinal peptide (VIP)-expressing neurons are mainly located in the SCN core region [23]. It was reported that VIP receptors (Vpac2) are essential for circadian function in mouse SCN cells [24], and that AVP receptor (V1a) regulates circadian rhythms of locomotor activity and the expression of clock-controlled genes in the SCN [25]. Moreover, the family of type II G-protein-coupled receptors includes the VIP receptors Vpac1 and Vpac2, as well as pituitary

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adenylate cyclase-activating peptide receptor 1 (Pac1), and the mice deficient in these

receptors show altered circadian rhythms. It was also reported that the significantly lower

overall activity was reported in Vpac2-deficient mice than in WT mice, while Vpac2-deficient

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mice were incapable of sustaining normal rhythms of rest/activity behavior [24]. Dissociation between light-induced phase shift of the circadian rhythm and clock gene expression in mice

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lacking Pac1 was also reported [26]. Despite having low affinity, secretin was shown to bind

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to these receptors [27]. Moreover, VIP elicits a biological response at the rat secretin receptor through a low-affinity binding site [28]. After considering these studies, although we do not have a direct evidence, we speculated that secretin signaling influences circadian

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rhythms via Vpac1, Vpac2, or Pac1, or that VIP signaling acts in the same manner, via secretin receptors, indicating that secretin and secretin receptors might also be a part of the

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circadian regulatory network.

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A previous study reported that Sctr-/- mice displayed autism-like social interactions [17]. It has been reported that children with autism exhibit greater variability in the circadian rhythm [29]. These reports support the findings of the present study, suggesting a possible relationship between circadian rhythm, secretin receptor signaling, and autism. In conclusion, a shorter circadian period and activity phase were observed in Sctr-/mice. Moreover, a few scattered Sctr-expressing cells were observed in the SCN. Hence, our findings indicate that secretin receptor signaling modulates the circadian period of the 10

behavioral rhythm via secretin receptors. However, we were unable to ascertain whether secretin directly modulates the circadian rhythm, which will require further investigations at the cellular or tissue level.

Credit author statement Mizuki Sugiyama: Methodology, Validation, Formal analysis, Investigation, Writing - Original Draft, Visualization.

acquisition. Shota Miyazaki: Validation, Formal analysis, Investigation.

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Ichiko Nishijima: Conceptualization, Methodology, Resources, Writing - Review & Editing, Funding

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Takahiro J. Nakamura: Conceptualization, Methodology, Investigation, Resources, Writing - Review &

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Declarations of interest: None.

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Editing, Visualization, Supervision, Project administration, Funding acquisition.

Acknowledgements: This work was supported by JSPS KAKENHI grant numbers 17K08564

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(to I.N.), 17H04022, and 19K06360 (to T.J.N).

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Figure captions

Fig. 1. Effects of secretin receptor depletion on the circadian rhythm of wheel-running activity in mice. (A, B) Representative double-plotted actograms showing the wheel-running activity of (A) WT and (B) Sctr-/- mice under light/dark (LD) and constant darkness (DD) conditions. The light schedule is indicated by black and white bars above the graphs (C) The free-running

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period of wheel-running activity in DD is shown. (D, E) Daily amounts of wheel-running

activity in the LD (D) and DD (E) conditions. (F, G) The ratio of the activity phase (α) to the

rest phase (ρ) in the LD (F) and DD (G) conditions. (H, I) Normalized hourly activity of WT and

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Sctr-/- mice during the day in both LD and DD conditions. Significant differences were

observed at ZT21 and ZT22 in the LD condition (H) and also at CT19 and CT21 in the DD

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condition (I). Data are presented as mean ± standard error of the mean, n = 7-9 per group,

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*p < 0.05, **p < 0.01, ***p < 0.001 for WT vs SCTR-/- (Student’s t-test).

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CT:circadian time, ZT:zeitgeber time

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Fig. 2. Secretin receptor expression in the adult mouse brain revealed by staining of the lacZ reporter. (A) Expression of beta-galactosidase in the suprachiasmatic nucleus (SCN) of wild-type (WT) mice (negative control). (B) Enlargement of the box area in (A). (C) Expression of beta-galactosidase in the SCN of secretin receptor-deficient (Sctr-/-) mice. (D) Enlargement of the box area in (C). (E) Expression of beta-galactosidase in the SCN of WT mice counterstained with neutral red (negative control). (F) Enlargement of the box area in (E). (G) Expression of beta-galactosidase in the SCN of Sctr-/- mice counterstained with neutral red.

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(H) Enlargement of the box area in (G). Scale bars, 50 µm. 3V, third ventricle. OC, optic

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chiasm.

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