Somatostatin regulates the clock sensitivity to evening light

Yamaguchi, Yoshiaki; Masubuchi, Satoru; Kiyokage, Emi; Toida, Kazunori; Mizoro, Yasutaka; Suzuki, Toru; Sato, Miho; Fustin, Jean-Michel; Tominaga, Keiko; Okamura, Hitoshi

doi:10.1038/s41598-025-26904-2

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Published: 28 November 2025

Somatostatin regulates the clock sensitivity to evening light

Yoshiaki Yamaguchi^1,2,
Satoru Masubuchi^3,4,
Emi Kiyokage^5,6,
Kazunori Toida⁵,
Yasutaka Mizoro¹,
Toru Suzuki¹,
Miho Sato^1,3,
Jean-Michel Fustin^1,7,
Keiko Tominaga⁸ &
…
Hitoshi Okamura^1,3,9

Scientific Reports volume 15, Article number: 42664 (2025) Cite this article

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Abstract

Entrainment of the internal clock to the light–dark cycle is a fundamental feature of biological rhythms. Here, we show that animals genetically deficient for somatostatin (SST) are unable to respond normally to long photoperiodic conditions, showing a significantly delayed phase of activity. The phase delay is also reproduced in the cognate SSTR1 receptor knockout mice and in mice in which SSTR1 is pharmacologically inhibited. We also provide histological evidence that SST inhibits light-induced activation of SSTR1 cells. Furthermore, chronic administration of CH-275, an SSTR1 agonist, normalizes the phase delay of the circadian clock in SST-deficient mice under long photoperiod. Together, these results provide insights into the inhibitory effect of the SST-SSTR1 system in the regulation of light sensitivity of the central clock in the SCN at dusk.

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Introduction

Dynamic resetting of the internal clock by light is an important property of the mammalian circadian clock. It has been shown experimentally that a single short light stimulus (~ 30 min) given early or late in the nighttime delays or advances the locomotor activity rhythm, respectively^1,2. This phase-dependent resetting of the internal clock is considered the basis of daily entrainment to environmental 24 h light–dark (LD) cycles.

Mammalian circadian rhythms in physiology and behavior are driven by the master pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus^3,4. At the molecular level, a variety of clock proteins constitute a transcriptional-(post)translational negative feedback loop that generates daily rhythms throughout the body⁵. The SCN is conventionally divided into two compartments, the ventrolateral (VL) and the dorsomedial (DM) regions⁶. The VL SCN receives direct neural input from the retina through the retinohypothalamic tract via melanopsin-containing neurons⁷. Light exposure in early subjective night induces gene expression of immediate early genes (e.g., cFos and Zif268) and clock genes (e.g., Per1 and Per2) in the VL SCN, which plays a role in the phase delay of the circadian clock¹.

Several types of neurons form neural circuits within the SCN⁶. Somatostatin (SST) neurons are discretely located in the DM SCN and project their axon to the VL SCN^8,9, where SST receptor subtype 1 (SSTR1) is abundantly expressed^10,11. This unique anatomical property provides an attractive hypothesis that SST signal from the light-irresponsive DM SCN regulates cell activities in the light-responsive VL SCN.

Seasonal changes in the duration of days and nights, i.e. the photoperiod, can lead to changes in psychology (e.g., mood disorders¹²) and metabolism (e.g., body weight and brown adipose tissue activity^13,14). In many ways, changes in photoperiod are considered the strongest natural stress on the mammalian clock system. Electrophysiological and clock gene expression analyses have clarified how changing the photoperiodic light schedule affects the SCN^15,16,17,18. However, the mechanisms that maintain the correct phase of locomotor activity rhythms in the photoperiodic environment are still unclear.

Here, by releasing mice acclimated to different photoperiods to constant darkness, we show that mutant mice lacking SST expression (SST^m/m mice)¹¹ entrain to a long photoperiodic condition (summer) with a delayed phase relationship to the lighting cycle compared to wild-type (WT) mice, with abnormal Per1 expression in the VL SCN at dusk. By advancing the start of the continuous dark period by 4 h, thereby excluding evening lights on the last day under LD cycles, the delay in the circadian rhythms of locomotor activity and the abnormal induction of Per1 expression at dusk were normalized in the mutant mice, suggesting their hypersensitivity to evening lights. We further demonstrate that SSTR1, an SST receptor in the SCN, also regulates light sensitivity in the evening based on behavioral and histochemical experiments performed under long photoperiod using SSTR1 knockout (SSTR1^−/−) mice or WT mice in which SSTR1 had been pharmacologically inhibited by administering SRA880, an SSTR1 antagonist. Finally, we succeeded in normalizing the phase delay in circadian rhythms of SST^m/m mice caused by long photoperiod when CH-275, an SSTR1 agonist, was chronically administered to the SCN. Together with a recent report by Joye and colleagues¹⁹ demonstrating that SST expression in the SCN is modulated by photoperiod, with longer daylight exposure increasing SST levels, our present in vivo physiological experiments support the hypothesis that SST contributes to photoperiodic entrainment by regulating the light responsiveness of cells in the VL SCN, especially under long photoperiod.

Results

SST ^m/m mice are hypersensitive to evening light and exhibit phase-delay and altered Per1 expression profile in the SCN

We studied the effects of SST on circadian rhythms using SST mutant (SST^m/m) mice, which lack SST mRNA and protein expression¹¹. First, we measured conventional circadian properties in SST^m/m and WT (SST^+/+) mice, but found no significant differences in the length of the free-running period (tau) and the magnitudes of the phase-shifts to brief light exposure measured at CT2, CT6, CT10, CT14, CT18, and CT22 (30 min, 200 lx) (Supplementary Fig. 1). Moreover, the amounts of phase-delay in the locomotor activity rhythm induced by long-term light exposure (12 h, 200 lx) did not change in SST^m/m mice. Taken together, from the perspective of classical and basic circadian rhythm analysis, clock oscillation and light-induced resetting appeared normal in SST^m/m mice. Quantifying their activity profile however led to an interesting observation: while locomotor activity was mainly observed during the dark period in both SST^+/+ and SST^m/m mice, SST^m/m mice showed significantly higher activity levels at dawn compared to SST^+/+ under 12 h light:12 h dark (LD 12 h:12 h) cycles (P < 0.0001, two-way ANOVA), (Fig. 1A upper panel). Moreover, under a long photoperiod with 18 h of light and 6 h of darkness (LD 18 h:6 h), SST^m/m mice showed a prolonged active phase, with significantly higher levels of activity than SST^+/+ mice from ZT19 to ZT21 (ZT12 represents the time of light off, P < 0.005, two-way ANOVA) (Fig. 1A lower panel). We next evaluated the actual phase difference between the endogenous clock and the LD cycles by calculating the timing of activity onsets after the transfer to constant darkness (DD)²⁰ (Fig. 1B, C). When SST^m/m and SST^+/+ mice were entrained to LD 12 h:12 h and released to DD, the actual active phase of both mice almost coincided with ZT12, and no significant differences were observed between the two genotypes (Fig. 1B, Fig. 1C). However, when mice were entrained to LD 18 h:6 h and released into DD, the actual activity onsets were significantly different between SST^+/+ and SST^m/m mice: the activity onset advanced for approximately 1 h only in SST^+/+ but not in SST^m/m mice (Fig. 1B, C).

To examine whether there was any difference at the molecular level in the SCN, we examined the expression patterns of Per1, a canonical clock gene involved in the resetting of the clock by light, at six different time points (ZT0, 4, 8, 12, 16, and 20) using in situ hybridization with digoxigenin-labelled cRNA probes. Under LD 12 h:12 h condition, the expression patterns of Per1 mRNA were quite similar between SST^+/+ and SST^m/m mice (Fig. 1D). Under LD 18 h:6 h, however, Per1 expression was noticeably higher in the VL SCN of SST^m/m mice at ZT12 (Fig. 1D, arrows in lower panels), although no significant difference was observed at any other time points between SST^+/+ and SST^m/m mice. Per1 is a core clock gene that generates circadian oscillations, but is also an immediate early gene that is locally induced in the VL SCN, a retinorecipient area to which light signals are directly transmitted²¹. Since the abnormal expression of Per1 in SST^m/m mice at ZT12 under long photoperiod was restricted to the VL SCN, we speculated that SST^m/m mice are more sensitive to evening light than SST^+/+ mice, resulting in enhanced induction of Per1 expression. We tested this hypothesis by measuring the induction of the immediate early genes cFos and Zif268^22,23 in the SCN of mice exposed to a light pulse (30 min, 200 lx) on the second day after transferring mice to DD. In the SCN of SST^m/m mice, the expression of cFos and Zif268 was significantly increased only at CT12 compared to SST^+/+ mice; no significant differences were observed at other time points (CT0, CT4, CT8, CT16, and CT20) (Fig. 1E). These results further suggest that SST^m/m mice are more sensitive to evening light.

If the phase delay in mutant mice is due to an abnormal sensitivity to evening light, then eliminating evening light should prevent the phase delay in SST^m/m under long photoperiod. To test this, we advanced the lights-off time under LD 18 h:6 h conditions by 4 h, so that the lights were turned off at ZT8 instead of ZT12 (evening light restriction, ELR), and released the mice to DD conditions (Fig. 1F). Surprisingly, this ELR was sufficient to restore the abnormal delayed locomotor activity rhythms and the ectopic expression of Per1 in the VL SCN in SST^m/m mice (Fig. 1C, F). These results suggest that the abnormal behavior and clock gene expression in SST^m/m mice under long photoperiod are not due an impaired circadian clock system, but rather to an excessive sensitivity to evening light.

Abnormal locomotor behavior in SSTR1 ^−/− mice under long photoperiod

Among 5 SST receptors (SSTR1-SSTR5), SSTR1 is the only SST receptor subtype expressed in the SCN¹¹, and its expression is localized in the ventral SCN¹⁰. We examined the circadian changes in the expression profile of SSTR1 mRNA in the SCN under LD and DD conditions. We found that VL-localized expression of SSTR1 exhibited a mild circadian rhythm under both LD (one-way ANOVA; P < 0.0005) and DD (one-way ANOVA; P < 0.001) conditions, peaking at (subjective) dusk (ZT12 and CT12), respectively (Fig. 2A, B).

To investigate whether SSTR1 mediates the effects of SST we have revealed above, we monitored locomotor activity rhythms of SSTR1 knockout (SSTR1^−/−) mice under long photoperiod. When SSTR1^−/− mice were entrained to LD 18 h:6 h and then released to DD, the advancement of activity onset in SSTR1^−/− mice was significantly reduced compared to SSTR1^+/+ mice (unpaired t-test, P < 0.001, Fig. 2C). Moreover, 4 h dark ELR rescued the abnormal delayed onset of locomotor activity rhythm in a manner similar to that seen in SST^m/m mice (Fig. 2D). These results strongly suggest that SST-SSTR1 signaling exerts an inhibitory effect to prevent the VL region from receiving excessive light stimulation at dusk.

Pharmacological blocking of SSTR1 also shows abnormal phase-delay under long photoperiod

To investigate whether pharmacological local inhibition of SSTR1 in the SCN of SSTR1^+/+ mice causes the phase delay observed in SST^m/m or SSTR1^−/− mice under long photoperiod, SRA880, an SSTR1 antagonist, was continuously infused into the SCN using a cannula connected to an osmotic minipump implanted directly over the SCN. Similar to SST^m/m or SSTR1^−/− mice, SRA880-infused mice also showed a significant decrease in the advancement of the activity onset compared to vehicle-infused mice under LD 18 h:6 h condition (unpaired t-test, P < 0.001, Fig. 2E). In addition, abnormally high expression of Per1 mRNA was observed in the VL SCN of SRA880-infused mice at ZT12 under long photoperiod (LD 18 h:6 h condition) (Fig. 2F), similar to that observed in SST^m/m mice (see Fig. 1D). Thus, it is clear that SST-SSTR1 signal in the SCN plays an important role in light sensitivity under long photoperiodic conditions.

SST-SSTR1 signal disruption causes abnormally high sensitivity to a light pulse at dusk: Inhibitory SSTergic projections to retinorecipient neurons

From the above results, inhibition of SST-SSTR1 signaling would specifically increase light sensitivity at dusk. To examine this more directly, we measured the magnitude of phase-shifts in locomotor activity rhythms when a light pulse was given at CT12 under DD conditions. When mice were exposed to 200 lx light for 30 min (CT12-12.5), SST^m/m mice showed a significantly greater amounts of phase delay than SST^+/+ mice (Fig. 3A). We also examined the effects of dim light pulses (~ 2 lx) on the locomotor activity rhythm in SST^+/+ and SST^m/m mice (Fig. 3B). At this intensity, there was almost no phase shift in SST^+/+ mice. However, dim light pulses at CT12 induced a significant phase delay in SST^m/m compared to SST^+/+ mice. We further examined the effects of dim light pulses on SCN neurons by concurrent expression of immediate early genes and the activation of extracellular signal-regulated kinase (ERK)^24,25. Dim light pulse at CT12 induced abundant cFos mRNA expression and ERK phosphorylation in the VL SCN of SST^m/m mice, but not in the SCN of SST^+/+ mice (Fig. 3C). These inductions were also observed in the VL SCN of SRA-infused mice (Fig. 3C). Considering that SSTR1 expression fluctuates in a circadian manner, peaking at ZT12 or CT12 in the VL SCN (Fig. 2B), the abundant expression of SSTR1 at this time point in the VL SCN may generate the time-specific inhibitory effect of SST-SSTR1 signaling in the SCN.

We next investigated how SST-SSTR1 affects retinorecipient neurons in the SCN. To examine whether SST neurons terminate onto the retinorecipient neurons, we injected cholera toxin subunit B (CTB) conjugated with Alexa Fluor 488 into the mouse eyeball. CTB is taken up by ganglion cells and transported to their axon terminals located in the SCN through axoplasmic flow. Imaging of triple-labeled sections (CTB, SST and nuclei) by confocal fluorescence microscopy revealed that abundant VL SCN cells were innervated by both CTB- and SST-positive fibers in SST^+/+ mice, although only CTB-positive retinal terminals were detected in the VL SCN of SST^m/m mice (Fig. 3D). We also investigated whether SST-SSTR1 is involved in the light-induced activation of retinorecipient neurons. When dim light pulses were applied at CT12, cFOS protein was induced in many of the SSTR1-expressing cells in the VL SCN of SST^m/m mice, whereas cFOS protein expression was barely induced in SSTR1-expressing cells in SST^+/+ mice (Fig. 3E). The ratio of cFOS-protein-positive cells among SSTR1-expressing cells was significantly higher in SST^m/m mice than in SST^+/+ mice (unpaired t-test, P = 0.00142, Fig. 3E). This result strongly suggests that SST acts on SSTR1-expressing cells in the SCN and exerts an inhibitory effect on their activation by light stimulation. We further performed immunoelectron microscopy to confirm that SST neurons project to the VL SCN. Indeed, we found that SST-immunoreactive dendrite received asymmetrical synapse from SST-negative axon (Fig. 3F: left). On the other hand, SST-immunoreactive axon formed symmetrical synapse to SST-negative cell body in the VL SCN (Fig. 3F right), suggesting that SST axons provide more effective inputs. These results indicate that SST neurons form inhibitory innervation onto retinorecipient neurons in the VL SCN and regulate the input of retinal light signals.

Is the expression level of SSTR1 altered in the SCN of SST^m/m mice? Quantitative PCR analysis using SCN samples isolated by laser microdissection revealed that the expression level of SSTR1 mRNA in SST^m/m SCN was approximately twice that of SST^+/+ SCN (Fig. 3G). This result suggests that SSTR1 mRNA expression is upregulated in a compensatory manner. This finding led us to investigate whether SST agonist administration would restore the abnormal phase delay of the behavioral rhythm in SST^m/m mice under long photoperiod. When CH-275, an SSTR1 agonist, was continuously infused around the SCN of SST^m/m mice using a cannula and an osmotic pump, the onset of locomotor activity rhythm was significantly advanced in CH-275-infused mice compared to vehicle-infused mice (unpaired t-test, P < 0.05, Fig. 3H). This result indicates that the pharmacological increase in SSTR1 activity can mitigate the evening light hypersensitivity due to the lack of SST in SST^m/m mice.

Discussion

In this study, we analyzed SST^m/m mice, which lack SST expression, and found a marked increase in the sensitivity of the circadian clock to evening light under long photoperiodic conditions. We hypothesize that SST neurons located in the DM part of the SCN project to SSTR1 neurons in the VL part (Supplementary Fig. 2), forming a gate to regulate environmental light signaling from the retina at dusk. In SST^m/m mice, which lack this gating system, the endogenous circadian clock exhibited a delayed phase under long photoperiodic conditions. Presumably, this is caused by excessive light input at dusk, since Per1 mRNA was abnormally high in retinorecipient VL cells, which is consistent with the observation that elimination of evening light rescued both delayed onset and Per1 expression in SST^m/m mice entrained to a long photoperiod.

Moreover, a time-specific effect of SST-SSTR1 signaling was demonstrated by short light pulse-induced phase shifts of locomotor activity rhythms: a light pulse at CT12 induced significantly larger amounts of phase delay in SST^m/m mice, but there was no difference in the amounts of phase shifts induced by a light pulse at the other time points, such as CT2, 6, 10, 14, 18, and 22, under constant dark conditions. These results indicate that the increased light sensitivity observed in SST^m/m mice at the onset of the dark phase is not solely due to exposure to a long photoperiod. Thus, by taking advantage of an inhibitory gating system and reducing the light response in VL cells, SST-SSTR1 signaling plays a fundamental role, mitigating the phase-delaying effects of light at the subjective early night under long photoperiod, keeping animals synchronized with their environment. This is ecologically important, since the internal clock is entrained to the natural photoperiodic environment by dusk and dawn as time cues².

SRA880 is highly specific to the SSTR1 receptor, exhibiting extremely low affinity for the SSTR2–5 receptors²⁶. However, it has been reported that SRA880 may also act on other receptors (e.g., dopamine D4 receptor)²⁶, and we cannot rule out the possibility that its effects may differ from those of SSTR1 inhibition.

How does SST decrease the light sensitivity of neurons in the VL SCN? Except for the hypothalamic periventricular neurosecretory cells projecting to the median eminence, SST neurons are inhibitory interneurons modulating proximal neurons in various brain areas²⁷. In these proximal neurons, SST causes membrane hyperpolarization by increasing K⁺ outward current through SSTR1, resulting in a decrease in cell activity^28,29. Thus, SST-SSTR1 signaling at dusk would inhibit the light-induced activation of retinorecipient cells, whose activation is considered to be critical for the phase shift^1,30. We acknowledge that this study lacks a direct causal manipulation required to support the proposed SST-SSTR1 model (Supplementary Fig. 2). Still, our results strongly suggest that SST neurons are indeed functional interneurons in the SCN.

Immunohistochemically, several SST cell bodies are observed in the DM SCN along the border with the VL SCN^31,32. SST fibers originating from these cells in the DM SCN extend to the VL SCN and may form synapses onto VIP cells, while some SST fibers innervate AVP neurons in the DM SCN^33,34. Importantly, SST in the SCN shows an overt rhythm under both LD and DD conditions^35,36,37. The circadian patterns of gene expression of SST in the SCN are similar to that of AVP: SST in the SCN is at its highest level in the early subjective day and at its lowest level during the subjective night^{19,32,38,39,40}.

Since SSTR1 is highly expressed in the VL SCN in a circadian manner, and SST nerves specifically innervate cells in the VL SCN^11,19,33, it is highly likely that SST-SSTR1 neurotransmission regulates the circadian function of cells in the VL SCN. Joye and colleagues found a significant phase difference between the VL and the DM SCN in SST^−/− SCN¹⁹. They also showed that the number of VIP/GRP-immunoreactive cells was increased in the VL SCN of SST^−/− mice, and that SSTR1 expression in VIP cells in the VL SCN was increased under long photoperiodic conditions¹⁹. These findings strongly support our hypothesis that SST is a key factor in controlling the circadian function of the VL SCN.

It is also well known that AVP-expressing neurons, which co-express GABA, also express V1a receptors in the DM SCN^41,42. This suggests a reciprocal interaction between AVP and V1a, forming a local circuit in the SCN⁴³. Transcripts of AVP and V1a show robust antiphasic rhythms⁴², suggesting that this communication exhibits a robust circadian rhythm⁴⁴, resulting in the circadian oscillation of AVP released into the cerebrospinal fluid^45,46. SST cells in the DM SCN that co-express GABA also show an oscillatory profile similar to AVP/V1a/GABA cells^8,39, and their axons project to VIP/GABA cells in the VL SCN^9,33. CRF/AVP neurons in the paraventricular nucleus (PVN) of the hypothalamus, modulated by VIP/GABA neurons in the VL SCN⁴⁷, project to the median eminence where they secrete CRF/AVP into the portal vessels. They then act on V1b cells in the anterior pituitary and finally connect to SST neurons in the SCN¹¹. Therefore, SST could be a critical factor in both the input and output of the SCN in a SCN-PVN-pituitary loop coordinating the circadian clock.

Over the past decades, it has become increasingly clear that information about seasonal rhythms is encoded in the neural networks of the SCN^15,16,17,18. While both the DM SCN and the VL SCN are capable of generating circadian rhythms, the DM SCN, which receives light information from the VL SCN, plays a dominant role in the autonomous synchrony of the circadian network⁴⁸. The ability of the SCN to encode photoperiodic information requires the existence of a functional neural network, and it appears that the SST neurons in the SCN play a key role by controlling the function of the cells in the VL SCN (e.g., VIP cells), which are light-receptive clock cells^19,49. By encoding photoperiodic information, the SCN creates an internal representation of day length that is transmitted to the PVN. In the PVN, it has been demonstrated that photoperiod-driven neurotransmitter switching occurs between SST and dopamine phenotype⁵⁰. Further studies showed that this switch is mediated by circadian inputs from SCN neurons⁵¹. The role of the SST signal within the SCN in the control of seasonal rhythms needs to be investigated in future studies.

When it comes to the effects of daylight hours on human health, there is a tendency to focus on the impact of short daylight hours (i.e., a short photoperiod), linked to seasonal affective disorder (SAD). Long daylight hours (i.e., a long photoperiod) can, however, also cause stress and physical and mental problems. In clinical medicine, the incidence of manic episodes is known to be high in early summer, when days are longer^52,53. Moreover, long photoperiods increase the incidence of insomnia/sleep disorders, which might be related to delayed or reduced melatonin secretion. For some people, long photoperiod may increase a risk of developing circadian rhythm sleep disorder (Delayed Sleep Phase Syndrome⁵⁴) due to abnormal phase-shifts of the circadian clock. Finally, sleep deprivation increases stress and emotional instability associated with overactivity of the sympathetic nervous system.

The SSTergic system in the SCN may help stabilize the SCN neural network under long photoperiods. People in modern society live in an environment with an artificial long photoperiod, with a day of natural light and a dusk and night of strong artificial lights. Actually, we are constantly exposed to light except when we sleep. Under such an artificial long photoperiodic condition, not only bright light in the morning advances the clock⁵⁵, but also artificial light at night (bed light; ~ 40 lx) delays the clock⁵⁶. Therefore, both morning and night light determine the internal clock phase in this socially long day environment. Humans who are more sensitive to light at dusk, maybe with low SSTergic signaling like SST^m/m mice, may have a delayed internal clock and difficulty waking up early in the morning.

Methods

Animals

All animal experiments were approved by the Animal Care and Experimentation Committee of Kyoto University and the Committee for Animal Research of Kobe University and conducted in accordance with the relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Mutant mice for the gene encoding SST were generated by gene targeting with mouse embryonic stem (ES) cells as described previously⁵⁷. We confirmed SST^m/m mice were deficient in SST mRNA and its protein expression¹¹. Resultant heterozygous mutant mice (SST^+/m) were backcrossed to the C57BL/6 J background for more than 9 generations and used to obtain homozygous mutant mice (SST^m/m). SST^m/m mice develop normally without somatic gigantism, as reported in a line of SST^–/– mice⁵⁸. SSTR1^–/– mice were generated as described previously⁵⁹, and backcrossed to the C57BL/6 background for more than 8 generations. Male SST^m/m, SSTR1^–/–, and their respective WT littermates SST^+/+ and SSTR1^+/+ mice (2 to 5 mo old, age-matched between genotypes) were used for behavioral rhythm, immunohistochemistry, and gene expression analyses. Mice were euthanized with high-flow CO₂ if their health was significantly impaired and recovery was unlikely.

Animals and behavioral rhythm monitoring and analysis

SST^m/m and SST^+/+ mice were housed in an environment with a LD 12 h: 12 h cycle (fluorescent light, 200 lx) for more than two weeks with food and water ad libitum and subsequently moved to each experimental condition (LD 12 h:12 h, 18 h:6 h, and constant darkness). Locomotor activity was detected by passive (pyroelectric) infrared sensors (FA-05 F5B; Omron, Kyoto, Japan). Data were monitored and analyzed by Chronobiology kit (Stanford Software Systems, Stanford, CA) as described previously²⁰. Circadian period and phase shift of the activity rhythms were analyzed by Clocklab software (Actimetrics). The phase was determined by fitting a regression line across several days in DD and extrapolating it back to the first day, rather than using only the raw Day 1 onset data. The spectral distribution of the 2-lx light (15 mW/m²) was measured using a MK350 UPRtek spectrometer and is shown in Supplementary Table 1.

SRA880 and CH-275 infusion over the SCN

For pharmacological inhibition of SSTR1, SRA880 (Novartis Pharmaceuticals), dissolved at 40 mM in a mixture of 1-methyl-2-pyrrolidone and ethanol (1:1) supplemented with 20 mg/ml ascorbic acid, was continuously delivered to the SCN using alzet micro-osmotic pump (model 1002) and brain infusion kit 2. For pharmacological activation of SSTR1, CH-275 (Tocris Bioscience) was dissolved at 200 μM in artificial cerebrospinal fluid (147 mM NaCl, 4 mM KCl, and 1.2 mM CaCl₂, pH 7.0) and delivered to the SCN in the same way. At the end of the experiment, brain sections were prepared and examined under a microscope to confirm that the cannula was positioned directly above the SCN.

Electron microscopy

Mice were perfused through the left cardiac ventricle with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brain was removed and immersed in the same fixative for 2 h. The removed brains were cut into 50-μm-thick transverse slices using a MicroSlicer (DTK-3000; DOSAKA, Kyoto, Japan). For preservation of ultrastructural details, we performed immunocytochemistry with the complete omission of Triton X-100 from all solutions. Instead, to enhance antibody penetration, sections were incubated in 30% sucrose overnight at 4 °C and freeze–thawed sequentially three times in liquid nitrogen. The sections were incubated with anti-SST serum (1:1000)⁶⁰ in 0.1 M PBS and then avidin–biotin peroxidase complex-3–3’-diaminobenzidine-4HCl immunocytochemistry was processed as described above. After a coloring reaction, sections were fixed in chilled 1% OsO₄ in 0.1 M PB for 1 h, dehydrated through a graded series of ethanol (the 70% ethanol contained 1% uranyl acetate; 1 h) and flat embedded in a Quetol 812 (Nisshin EM, Tokyo, Japan) between silicon-coated slides and coverslips. The reacted neurons and fibers were selected under a light microscope. Ultrathin sections were cut on a Reichert Ultracut E ultramicrotome, collected on nickel grids and examined under a JEM-1220 electron microscope (JEOL, Tokyo, Japan).

Cholera toxin subunit B (CTB) injection and SST immunostaining

Under isofluran anesthesia, CTB conjugated with Alexa Fluor 488 (C34775; Thermo Fisher Scientific) was injected into the eyeball using a Hamilton syringe. After 2–3 days of injection, the animals were sacrificed and fixed with a fixative consisting of 4% paraformaldehyde. The isolated brains were post-fixed with the same fixative for 24 h at 4 °C and then transferred to 30% sucrose in 0.1 M PB for cryoprotection. To minimize technical variations in immunostaining, sections from different genotypes were immunolabelled simultaneously. Coronal brain cryosections (30 μm thick) were processed for free-floating immunohistochemistry with anti-SST serum (1:1000)⁶⁰. Then, the sections were incubated with donkey anti-rabbit IgG antibody Alexa594 (A21207; Thermo Fisher Scientific; 1:500).

Quantitative in situ hybridization using radiolabelled probes

In situ hybridization using radiolabelled probes was performed as described in detail previously²¹. The SSTR1, cFos and Zif268 cDNA fragment-containing vectors were linearized with restriction enzymes and then used as templates for sense or antisense cRNA probes. Radiolabelled probes for SSTR1, cFos and Zif268 were made using [³³P]UTP (New England Nuclear, Boston, MA, U.S.A.) with a standard protocol for cRNA synthesis. Each DNA fragment was PCR amplified using the following oligonucleotides: 5′-GAACTGGCAAGCAGGAAAGGAGCT-3′ and 5′-GTAGATGAAAGAGATGAGAATGGC-3′ for SSTR1, 5′-CGTCTTCCTTTGTCTTCACCTACC-3′ and 5′-AACACGCTATTGCCAGGAACACAG-3′ for cFos, 5′-GAGATGCAATTGATGTCTCCGCTG-3′ and 5′-AAAGACCAGTTGAGGTGCTGAAGG-3′ for Zif268. These fragments were then cloned into the pCR-BluntII-TOPO (Invitrogen). We used X-ray film (Kodak BioMax) for macroscopic analysis to quantify isotope signals. The radioactivity of each SCN section on BioMax film was analyzed using a microcomputer interfaced to an image analyzing system (MCID, Imaging Research Inc., Canada) after conversion into the relative optical densities produced by the ¹⁴C-acrylic standards (Amersham). The data were normalized with respect to the difference in signal intensity between equal areas of the SCN and the corpus callosum. The optical density intensities of sections ranging from the most rostral to the most caudal of the SCN (10 sections per mouse) were then summed, and this sum was considered a measure of mRNA expression.

In situ hybridization using digoxigenin-labelled probes

In situ hybridization using digoxigenin-labelled probes was performed as described in detail previously²¹. Antisense Per1, cFOS, and SSTR1 cRNA probes labelled using digoxigenin-UTP (Boehringer Mannheim, Mannheim, Germany) were made following standard protocols for cRNA synthesis supplied by Boehringer Mannheim. In the experiment shown in Fig. 1F, we first prepared a complete series of sections from the rostral to caudal regions of the SCN using a free-floating method. From these, we selected three central SCN sections where the distinction between the DM and VL regions was most evident. We then counted the number of Per1-positive cells in reference to SSTR1 expression area.

Immunohistochemistry for pERK and cFOS

For immunohistochemical labeling of pERK and cFOS, animals were anesthetized and systemically perfused with 25 mL of cold fixative containing 4% paraformaldehyde in 0.1 M PB²⁵. Isolated brains were transferred to the same fixative for 12 h at 4 °C, and then immersed in 20% sucrose in 0.1 M PB for cryoprotection. Coronal brain cryosections (20 μm-thick) were processed for free-floating immunohistochemistry. We used a rabbit polyclonal antibody specific for the phosphorylated forms of ERK 1 and 2 (Cell Signaling; catalog code 9101) and a rabbit antibody for cFOS protein identification (Abcam; catalog code 7963). Free-floating sections were pretreated with hydrogen peroxide (1.5% in 0.1 M PB, for 20 min at 4 °C) and blocked with 5% horse serum (in 0.1 M PB) for 1 h at room temperature. The sections were then incubated with pERK antibody [1:500 dilution, in 0.1 M PB containing 0.3% Triton X-100 (PBX)], or with cFOS antibody [1:8000] for 12 h at room temperature. After washing with PBX, the sections were incubated with a biotinylated anti-rabbit IgG secondary antibody (1:500 dilution in PBX) (Vector Laboratories) for 1 h at room temperature. The tissues were then subjected to standard avidin–biotin-immunoperoxidase staining (Vectorstain Elite ABC kit, Vector Laboratories). Immunoreactivity was visualized with 3,3’-diaminobenzidine (DAB). Immunostained sections were then washed with 50 mM Tris–HCl buffer (pH 7.5), dehydrated in ethanol, and coverslipped with Entellan mounting medium.

Double-labeling histochemistry for SSTR1 mRNA and cFOS protein

To examine whether a dim light illumination induces cFOS protein expression in SSTR1-expressing cells in the VL SCN, we performed double-labeling immunocytochemistry and in situ hybridization. Free-floating sections were incubated with the cFOS antibody (1:8000 dilution), followed by treatment with the goat biotinylated anti-rabbit IgG and avidin–biotin-complex. Following the visualization of cFOS by DAB, sections were processed for subsequent in situ hybridization with digoxigenin-labeled antisense SSTR1 cRNA probe. Pre-hybridization, hybridization, and post-hybridization washes were performed as described above. DAB-stained reactions were observed in the nucleus for cFOS. Digoxigenin-labeled SSTR1 mRNA signals were stained blue with a nucleic acid detection kit (Roche Diagnostics) in the cytoplasm. Photomicrographs were captured using a Zeiss microscope and imaging software. As described above, we selected three central SCN sections and counted the number of SSTR1-positive cells. We then counted the number of cFOS-positive cells within these cells.

Laser microdissection of the SCN

Mice were killed by cervical dislocation, and the eyes were removed under a safety red light. The brain was then isolated from the skull under room light and was frozen immediately on dry ice. Coronal brain Sects. (30 μm thick) were prepared using a cryostat microtome (CM3050S, Leica) and mounted on POL-membrane slides (Leica). Sections were fixed for 3 min in an ice-cold mixture of ethanol and acetic acid (19:1), rinsed briefly in ice-cold water, stained for 30 s in ice-cold water containing 0.05% toluidine blue, followed by two brief washes in ice-cold water (all the solutions were RNase-free). After wiping off excess water, slides were quickly air dried at room temperature. As soon as moisture in the sections decreased enough for laser-cutting, cells in the SCN were microdissected using a LMD7000 device (Leica; 10 × magnification) and lysed in TRIzol reagent (Thermo Fisher Scientific), and total RNA was purified using the RNeasy micro kit (Qiagen).

Quantitative RT-PCR

Total RNA in the SCN dissected by a laser microdissection technique was converted to cDNA using SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative PCR analysis of the individual cDNAs was performed using the Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific) with StepOnePlus real-time PCR monitoring system (Applied Biosystems). Results were normalized to 36b4 mRNA levels. The primer sets used for this study were as follows: SSTR1, Fw: 5′-GGAAAGGAGCTGCTGACGCGA-3′, Rv: 5′-ACCCAGCGGCAGCTTGCTTG-3′; 36b4, Fw: 5′-CTCACTGAGATTCGGGATATG-3′, Rv: 5′-CTCCCACCTTGTCTCCAGTC-3′.

Data analysis

Statistical analyses were conducted using GraphPad Prism 10. All numerical results are presented as mean ± SEM. Significant differences were determined using an unpaired t-test or analysis of variance (ANOVA) followed by a multiple comparison post hoc test recommended by Prism. P values < 0.05 were considered statistically significant, with P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 represented by *, **, ***, and †, respectively.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Dr. Paola Bagnoli and Dr. Kazuto Kobayashi for providing SSTR1^–/– mice and SST^m/m mice, respectively. We also thank Dr. Masahiko Yamakawa and Takayuki Yamasaki for their help with the data analysis and behavioral study, respectively.

Funding

This work was supported in part by the Ministry of Education, Culture, Sports, Science, and Technology of Japan: Grant-in-aid for Challenging Research (Pioneering) (22K18384, H.O.), Basic Research B (23H02796, H.O.), Young Scientists A (15H05642, Y.Y.), Scientific Research C (22K06594, Y.Y.), Exploratory Challenging Research (20K20864, Ke.T.), Specially Promoted Research (H.O.), grants from Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (CREST/JPMJCR14W3, H.O.), Kobayashi International Scholarship Foundation and SRF (H.O.), Young Scientists B (18790169, S.M.) and from Hyogo Science and Technology Association (18S012, S.M.).

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Authors and Affiliations

Department of Systems Biology, Kyoto University Graduate School of Pharmaceutical Sciences, 46-29 Yoshida-Shimo-Adachi-Cho, Sakyo-Ku, Kyoto, 606-8501, Japan
Yoshiaki Yamaguchi, Yasutaka Mizoro, Toru Suzuki, Miho Sato, Jean-Michel Fustin & Hitoshi Okamura
Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka, 564-8680, Japan
Yoshiaki Yamaguchi
Division of Molecular Brain Science, Kobe University Graduate School of Medicine, Chuo-Ku, Kobe, 650-0017, Japan
Satoru Masubuchi, Miho Sato & Hitoshi Okamura
Department of Physiology, School of Medicine, Aichi Medical University, Nagakute, Aichi , 480-1195, Japan
Satoru Masubuchi
Department of Anatomy, Kawasaki Medical School, Kurashiki, Okayama, 701-0192, Japan
Emi Kiyokage & Kazunori Toida
Department of Medical Technology, Faculty of Health Science and Technology, Kawasaki University of Medical Welfare, Kurashiki, Okayama, 701-0193, Japan
Emi Kiyokage
Centre for Biological Timing, The University of Manchester, Manchester, M13 9PL, UK
Jean-Michel Fustin
Graduate School of Frontier Biosciences, The University of Osaka, Suita, Osaka, 565-0871, Japan
Keiko Tominaga
Department of Neuroscience, Graduate School of Medicine, Kyoto University, 46-29 Yoshida-Shimo-Adachi-Cho, Sakyo-Ku, Kyoto, 606-8501, Japan
Hitoshi Okamura

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Yoshiaki Yamaguchi
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Contributions

S.M. and H.O. designed research; Y.Y, S.M., E.K., Ka.T., Y.M., T.S., M.S., J.-M.F., and Ke.T. performed research; and Y.Y. and H.O. wrote the paper.

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Yamaguchi, Y., Masubuchi, S., Kiyokage, E. et al. Somatostatin regulates the clock sensitivity to evening light. Sci Rep 15, 42664 (2025). https://doi.org/10.1038/s41598-025-26904-2

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Received: 21 July 2025
Accepted: 30 October 2025
Published: 28 November 2025
Version of record: 28 November 2025
DOI: https://doi.org/10.1038/s41598-025-26904-2