Inducing Daytime Circadian Phase Shifts Using Chemogenetic and Spectral Approaches

Summary

This study introduces chemogenetic and violet light–based strategies to enable reliable daytime circadian phase shifting.

Abstract

In mammals, circadian phase shifting during the daytime is limited by reduced photic responsiveness of the suprachiasmatic nucleus (SCN), restricting the ability to experimentally manipulate the circadian clock during this phase. Reliable methods to induce daytime phase shifts are therefore essential for investigating mechanisms of circadian plasticity and photic entrainment. Complementary genetic and spectral strategies are described to enable robust, temporally precise manipulation of the circadian clock during the day. The first approach, currently limited to mice, employs a chemogenetic strategy involving intravitreal delivery of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to selectively activate intrinsically photosensitive retinal ganglion cells (ipRGCs). This approach permits controlled activation of the retinohypothalamic pathway and induces reproducible daytime phase shifts independent of ambient lighting conditions. The second approach utilizes wavelength-specific optical stimulation as a non-invasive alternative. Exposure to violet light exploits the spectral sensitivity of ipRGCs to reduce depolarization block and promote sustained activation, enabling reliable phase resetting during the subjective day. This method is broadly applicable across mammalian systems and does not require genetic manipulation or pharmacological intervention. Detailed protocols are provided for experimental preparation, stimulation timing, validation of phase shifts using locomotor activity, and assessment of neuronal activation via c-Fos immunohistochemistry. Key considerations, including circadian timing, stimulus parameters, and experimental controls, are outlined to facilitate reproducibility. Together, these approaches provide versatile and experimentally tractable tools for inducing daytime circadian phase shifts and enable direct investigation of mechanisms underlying daytime circadian responsiveness.

Introduction

The suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinates essential daily rhythms in physiology and behavior, including sleep–wake cycles and feeding patterns. To maintain alignment with the external environment, the SCN receives direct photic input from intrinsically photosensitive retinal ganglion cells (ipRGCs), which transmit retinal light information to synchronize internal circadian rhythms with the solar day^1,2.

A defining feature of circadian systems across organisms is phase-dependent responsiveness: the same stimulus delivered at different circadian times can produce markedly different effects. In mammals, nocturnal light reliably induces phase shifts of the SCN clock, whereas exposure during the subjective day produces little or no resetting^3,4. This apparent daytime insensitivity has been consistently observed in both in vivo and in vitro preparations, where attempts to mimic photic input fail to elicit significant phase shifts^{5,6,7,8,9,10,11,12,13,14,15,16}.

Daytime insensitivity has been attributed, at least in part, to insufficient retinal drive to engage phase-resetting mechanisms, alongside circadian gating mechanisms within the SCN. Consistent with this idea, ipRGCs are not uniformly responsive under photopic conditions; a subset enters depolarization block, limiting sustained action potential firing^17,18,19. This physiological constraint may reduce the effectiveness of conventional photic stimulation during the day. Overcoming this limitation would enable direct interrogation of daytime clock plasticity, a phase traditionally considered refractory to photic resetting.

Two complementary methodologies are described to enable robust phase shifting of the circadian clock during the subjective day. The first approach employs a chemogenetic strategy to selectively activate ipRGCs independent of ambient lighting conditions. Specifically, Opn4^Cre/+ mice receive intravitreal injections of an adeno-associated virus (AAV) encoding the excitatory Designer Receptor Exclusively Activated by Designer Drugs (DREADD), hM3Dq, in a Cre-dependent manner. Administration of clozapine-N-oxide (CNO) at circadian time 4 (CT4) induces burst-like firing of ipRGCs, providing sustained activation of the retinohypothalamic pathway¹⁹. Phase shifts are quantified using locomotor activity recordings, and molecular activation within the SCN is assessed by c-Fos immunohistochemistry.

The second approach uses wavelength-specific optical stimulation as a non-invasive strategy for broader applicability. Wild-type mice are exposed to violet light (385 nm) at circadian time 6 (CT6), during the middle of the subjective day. Violet light produces reduced depolarization block and supports sustained ipRGC activation compared to blue or white light¹⁹. Phase shifts are quantified as described above.

Both approaches aim to enhance ipRGC-driven signaling to the SCN, thereby overcoming the reduced photic responsiveness characteristic of the subjective day. Together, these methodologies provide robust, experimentally tractable strategies for manipulating the circadian clock during a phase traditionally resistant to photic resetting.

Protocol

All animal procedures were approved by and performed in accordance with the guidelines of the National Institute of Mental Health (NIMH) Animal Care and Use Committee (ASP-SLCR-01). All efforts were made to minimize pain and reduce the number of animals used. The research tools used are listed in the Table of Materials.

1. Chemogenetic strategy

1. Intravitreal injection of adeno-associated virus in Opn4^Cre/+ mice
   1. Clean and sterilize the stereotaxic stage and all surgical instruments using appropriate sterilization procedures. Set up the injection apparatus and attach a glass capillary injection needle.
   2. Anesthetize the mouse using isoflurane. Set the oxygen flow rate to approximately 1.5 L/min. Adjust isoflurane to 3%–5% for induction and reduce to 1–3% for maintenance throughout the procedure.
   3. Verify adequate anesthesia using a toe pinch. Consider the animal fully anesthetized when no withdrawal or flinching reflex is observed.
   4. Place the mouse on a heating pad to maintain body temperature throughout the procedure.
   5. Pull glass capillary needles. Needles were marked at ~1.5 mm from the tip using a fine marker under a stereomicroscope to ensure consistent penetration depth delivery.
   6. Gently apply pressure to the inferior eyelid with blunt-tipped forceps to slightly protrude the eyeball and expose the dorsal limbus. Using a dorsal approach, insert the needle tangentially to the surface of the eye and penetrate the sclera 0.5–1 mm posterior to the limbus while avoiding vascularized regions limbus.
   7. Insert the needle into the vitreous chamber to the predetermined depth. Withdraw slightly to relieve intraocular pressure and prevent blockage of the needle tip by tissue.
   8. Inject a total volume of 1.5 µL AAV solution (at 2.4 x 10¹³ GC/ml), delivered as five sequential injections of 300 nL each (at the rate of 50nl/s). Pause for 5 s between injections to allow diffusion and minimize reflux. After the final injection, wait 30 s before slowly withdrawing the needle. Repeat the same procedure in the contralateral eye using identical parameters, taking care to minimize trauma and reflux.
   9. Administer ketoprofen (10 mg/kg, subcutaneously) immediately following surgery.
   10. Allow mice to recover for at least 2 weeks to ensure adequate viral expression before transferring them to the behavioral facility.
2. Wheel-running activity and CNO administration (Figure 1A)
   1. House mice individually in cages equipped with running wheels. Record wheel-running activity using an automated data acquisition system.
   2. Entrained mice to a 12:12 h light–dark cycle for 4–7 days. Confirm stable entrainment and then transition animals to constant darkness (DD).
   3. Determine activity onset using analysis software. Define activity onset as CT12 in nocturnal rodents. Calculate CT4 accordingly.
   4. On day 8 in DD, mice receive intraperitoneal injections of CNO (1 mg/kg) or saline at CT4 under dim red light.
   5. Return animals to their home cages and maintain them under constant darkness. Record locomotor activity continuously for 8 days.
   6. Determine activity onsets and perform regression analysis on pre- and post-injection periods. Calculate phase shifts as the difference between projected and observed activity onset.
3. SCN c-Fos immunohistochemistry following chemogenetic activation
   1. Entrained mice to a 12:12 h light–dark cycle and release into constant darkness. On the second day in DD, administer CNO at CT6. Perfuse animals 30 min after injection.
      Note: CT4 was used in behavioral experiments to minimize the possibility of CNO effects extending into the subjective night, as CNO can remain active for several hours. In contrast, CT6 was used for immunostaining experiments to assess the acute effects of CNO on the circadian clock, with animals perfused 30 min after injection.
   2. Dissect the brain and post-fix in 4% paraformaldehyde overnight at 4 °C. Cryoprotect in 30% sucrose until the tissue sinks. Section coronally at 40 µm.
   3. Incubate sections in blocking solution, followed by primary antibody incubation overnight at 4 °C. Wash and incubate with secondary antibodies for 1 h. Counterstain with DAPI and mount sections.
   4. Acquire images using consistent microscope settings. Process images using analysis software. Identify AAV-infected regions by mCherry-positive cells.

2. Spectral strategy

1. Transfer wild-type mice to the behavioral facility and house them individually in cages equipped with running wheels, as described above. Maintain animals under constant darkness (DD) to monitor free-running circadian activity.
2. On day 8 in DD, at circadian time 6 (CT6), remove the cage from the light-tight cabinet and expose the mouse to a 15 min light pulse.
3. Expose mice to either a blue light pulse (λmax = 470 nm; 1.8 × 10¹⁴–2.2 × 10¹⁴ photons/cm²/s) or a violet light pulse (λmax = 385 nm; 1.45 × 10¹⁴–3.3 × 10¹⁴ photons/cm²/s), with intensity measured at the center and all four corners of the cage at animal eye level.
4. For both wavelength conditions, keep mice in their home cages with the cage tops removed. Position cages directly beneath a fixed light source such that the center of the light beam is aligned with the center of the cage to ensure uniform illumination.
5. Immediately return cages to their original position under constant darkness following light exposure. Continue wheel-running activity recording uninterrupted for phase-shift analysis.

Results

Opn4^Cre/+ mice expressing AAV-DREADD that received saline at CT4 did not exhibit a significant phase shift in wheel-running activity (n = 5; Figure 1B). In contrast, CNO administration at CT4 produced a significant phase delay in activity onset (mean ± SEM: 5.03 ± 0.48 h; n = 5; Figure 1C, D), demonstrating that DREADD activation at this circadian time shifts locomotor activity rhythms. A schematic of the experimental design is shown in Figure 2A. In the same mice, CNO administration increased c-Fos expression in the SCN 30 min after injection (Figure 2B) compared to saline controls (Figure 2C, D), indicating acute activation of SCN neurons during the subjective day.

Wavelength-specific light stimulation (Figure 3A) was next examined to determine whether it could induce daytime phase shifts in wild-type mice. Blue light stimulation (λmax = 470 nm) did not induce a measurable phase shift (−0.05 ± 0.04 h; n = 5 mice; Figure 3B). In contrast, a 15 min pulse of violet light (λmax = 385 nm) delivered at CT6 produced a significant phase delay (0.67 ± 0.04 h; n = 5 mice; Figure 3C, D).

Figure 1: Chemogenetic activation of ipRGCs phase-shifts behavioral rhythms during the daytime. (A) Schematic of the experimental design. Opn4^CRE/+ mice received bilateral intravitreal AAV-DREADD injections. After 2 weeks, mice were entrained to a 12:12 h light-dark (LD) cycle and then released into constant darkness (DD). On day 8 in DD, CNO (or saline) was administered intraperitoneally at CT4, and wheel-running activity was monitored to assess phase shifts. (B) Double-plotted actogram of a Opn4^CRE/+ mouse expressing AAV-DREADD. Wheel-running activity is shown as black bars. On day 8 in DD, the mouse received a saline injection at CT4. Activity onsets are indicated by orange lines, and the dashed purple line highlights the absence of a phase shift. (C) Same as in B, but following a CNO injection at CT4 on day 8 in DD. Activity onsets are indicated by orange lines, and the dashed purple line highlights the phase delay induced by the CNO injection. (D) Quantification of phase shifts following saline and CNO injections. Each dot represents an individual mouse; bars indicate mean ± S.E.M. Statistical significance was determined using an unpaired two-tailed t-test (****P < 0.0001). Please click here to view a larger version of this figure.

Figure 2: Chemogenetic activation of ipRGCs induces c-Fos expression in the SCN during the daytime. (A) Schematic of the experimental design. Opn4^CRE/+ mice received bilateral intravitreal AAV-DREADD injections. After 2 weeks, mice were entrained to 12:12 h light-dark (LD) cycle and released into DD. On day 2 in DD, mice received IP CNO (or saline) at CT6 and were then perfused with PBS and PFA for c-Fos analysis. (B) Immunohistochemistry showing c-Fos induction in the SCN following saline injection at CT6. The SCN (dashed outline) is visualized with DAPI. ipRGC projections are labeled by mCherry expression. Left: merged image (DAPI, c-Fos, mCherry); middle: c-Fos; right: c-Fos and mCherry. (C) Same as in B, but following CNO injection at CT6. Increased c-Fos expression is observed in the SCN. Scale bars, 100 µm. Please click here to view a larger version of this figure.

Figure 3: Violet, but not blue, light induces phase shifts in circadian behavioral rhythms during the daytime. (A) Schematic of the experimental design. Wildtype mice were entrained to a 12:12 h light-dark (LD) cycle and then released into constant darkness (DD). On DD day 8, mice received a 15 min light pulse (blue or violet) at CT6, and wheel-running activity was subsequently recorded in DD to assess phase shifts. (B) Double-plotted actogram of a wildtype mouse exposed to a blue light pulse at CT6 on DD day 8. Wheel-running activity is shown as black bars. Activity onsets are indicated by orange lines, and the dashed purple line highlights the minimal phase shift. (C) Same as in B, but following a violet light pulse at CT6 on day 8 in DD. Activity onsets are indicated by orange lines, and the dashed purple line highlights the phase delay induced by violet light. (D) Quantification of phase shifts following blue and violet light exposure. Each dot represents an individual mouse; bars indicate mean ± S.E.M. Statistical significance was determined using an unpaired two-tailed t-test (****P < <0.0001). Please click here to view a larger version of this figure.

Discussion

The results demonstrate that activation of ipRGCs during the subjective day can induce circadian phase shifts in mice, a time typically characterized by reduced sensitivity to photic stimulation. A chemogenetic approach enabled selective activation of ipRGCs independent of external light and produced robust phase delays in wheel-running behavior. This activation also induced acute c-Fos expression in the SCN, confirming activation of the central circadian pacemaker. These findings indicate that strong ipRGC-driven stimulation of the retinohypothalamic pathway is sufficient to overcome reduced daytime responsiveness of the circadian system.

Wavelength-specific optical stimulation provides a complementary, non-invasive strategy to achieve daytime phase resetting. A brief pulse of violet light induced significant phase delays, whereas a comparable-intensity, comparable-duration pulse of blue light did not. These findings suggest that spectral properties of light critically influence the ability to modulate circadian timing during the subjective day. Unlike chemogenetic manipulation, wavelength-specific stimulation does not require viral expression or pharmacological intervention, increasing accessibility and potential for broader application.

In humans, light exposure during the daytime generally produces minimal phase shifts, although under certain experimental conditions the circadian system can respond to bright white or blue light^20,21. Therapeutic interventions for circadian rhythm disorders remain limited. Although nighttime light exposure can shift the circadian clock, it may also disrupt sleep–wake patterns, restricting clinical utility.

Based on the known spectral sensitivities of retinal photoreceptors in primates and humans, melanopsin-expressing ipRGCs peak around 480 nm and exhibit reduced sensitivity at shorter wavelengths near 420 nm. Short-wavelength stimulation may reduce depolarization block in ipRGCs and permit more effective daytime signaling to the SCN, consistent with observations in rodents. Targeted short-wavelength light exposure during the daytime may represent a potential strategy for addressing circadian misalignment in conditions such as shift work, jet lag, and related disorders.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AAV2-hSyn-DIO-hM3D(Gq)-mCherry	Addgene	44361	Cre-dependent viral vector encoding excitatory DREADD (hM3Dq) fused to mCherry for selective ipRGC activation
Anti-c-Fos antibody (rabbit monoclonal)	Cell Signaling Technology	2250S	Primary antibody used to detect c-Fos as a marker of neuronal activation
Anti-tdTomato antibody (goat)	LSBio	LS-C340696	Primary antibody used to detect mCherry/tdTomato reporter expression
Blue LED driver	Thorlabs	LEDD1B	Power supply for controlling intensity and output of blue LED
Blue LED light source	Thorlabs	M470L5	LED source emitting blue light (λmax ≈ 470 nm) for photic stimulation
ClockLab software	Actimetrics	Version 6.0.53	Software for acquisition and analysis of circadian locomotor activity data
Clozapine-N-oxide (CNO)	Sigma-Aldrich	C0832-5MG	Ligand used to activate hM3Dq DREADD receptors in vivo
DAPI	Invitrogen	62248	Fluorescent nuclear stain for labeling cell nuclei
Donkey anti-goat Alexa Fluor 555	Invitrogen	A21432	Fluorescent secondary antibody for detection of goat primary antibodies
Donkey anti-rabbit Alexa Fluor 488	Invitrogen	A21206	Fluorescent secondary antibody for detection of rabbit primary antibodies
Fluoromount-G	Invitrogen	00-4958-02	Mounting medium used to preserve fluorescence in tissue sections
ImageJ (Fiji) software	NIH	Version 2.16.0/1.54g	Open-source software for image processing and quantitative analysis
Opn4cre Mice	The Jackson Laboratry	RRID:IMSR_JAX:035925	Mice used for chemogenetic activation
Violet LED driver	Mightex	SLA-1000-2	Power supply for controlling violet LED output
Violet LED light source	Mightex	BLS-LCS-0385-04-22	LED source emitting violet light (λmax ≈ 385 nm) for wavelength-specific stimulation
Wild-type mice	The Jackson Laboratry	Jackson strain #101043	Mice used for violet light experiment