Distinct ipRGC subpopulations mediate light’s acute and circadian effects on body temperature and sleep

The light environment greatly impacts human alertness, mood, and cognition by both acute regulation of physiology and indirect alignment of circadian rhythms. These processes require the melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), but the relevant downstream brain areas involved remain elusive. ipRGCs project widely in the brain, including to the central circadian pacemaker, the suprachiasmatic nucleus (SCN). Here we show that body temperature and sleep responses to acute light exposure are absent after genetic ablation of all ipRGCs except a subpopulation that projects to the SCN. Furthermore, by chemogenetic activation of the ipRGCs that avoid the SCN, we show that these cells are sufficient for acute changes in body temperature. Our results challenge the idea that the SCN is a major relay for the acute effects of light on non-image forming behaviors and identify the sensory cells that initiate light’s profound effects on body temperature and sleep.


Introduction
Many essential functions are influenced by light both indirectly through alignment of circadian rhythms (photoentrainment) and acutely by a direct mechanism (sometimes referred to as 'masking') Altimus et al., 2008;Lupi et al., 2008;Tsai et al., 2009;LeGates et al., 2012). Dysregulation of the circadian system by abnormal lighting conditions has many negative consequences, which has motivated decades of work to identify the mechanisms of circadian photoentrainment (Golombek and Rosenstein, 2010). In contrast, it has only recently become apparent that light exposure can also acutely influence human alertness, cognition, and physiology (Chellappa et al., 2011). As a result, there is a developing awareness of light quality in everyday life (Lucas et al., 2014). It is therefore essential to human health and society to elucidate the circuitry and coding mechanisms underlying light's acute effects.
Intriguingly, a single population of retinal projection neurons-intrinsically photosensitive retinal ganglion cells (ipRGCs)-have been implicated in the circadian and acute effects of light on many functions, including activity, sleep, and mood (Gö z et al., 2008;Güler et al., 2008;Hatori et al., 2008;LeGates et al., 2012;Fernandez et al., 2018). ipRGCs integrate light information from rods, cones, and their endogenous melanopsin phototransduction cascade (Schmidt et al., 2011), and relay that light information to over a dozen central targets (Hattar et al., 2006;Ecker et al., 2010). However, the circuit mechanisms mediating ipRGC-dependent functions are largely unknown.
One notable exception is the control of circadian photoentrainment. It is accepted that ipRGCs mediate photoentrainment by direct innervation of the master circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus (Gö z et al., 2008;Güler et al., 2008;Hatori et al., 2008;Jones et al., 2015). This is supported by studies demonstrating that genetic ablation of ipRGCs results in mice with normal circadian rhythms that 'free-run' with their endogenous rhythm, independent of the light/dark cycle (Gö z et al., 2008;Güler et al., 2008;Hatori et al., 2008). Further, mice with genetic ablation of all ipRGCs except those that project to the SCN and intergeniculate leaflet (IGL) display normal circadian photoentrainment , suggesting that ipRGC projections to the SCN/IGL are sufficient for photoentrainment.
In comparison, the mechanisms by which ipRGCs mediate acute light responses remain largely a mystery. Genetic ablation of ipRGCs or their melanopsin phototransduction cascade blocks or attenuates the acute effects of light on sleep Lupi et al., 2008;Tsai et al., 2009), wheel-running activity (Mrosovsky and Hattar, 2003;Güler et al., 2008), and mood (LeGates et al., 2012;Fernandez et al., 2018). This dual role of ipRGCs in circadian and acute light responses suggests they may share a common circuit mechanism. However, whether the circuit basis for ipRGCs in the acute effects of light and circadian functions is through common or divergent pathways has yet to be determined. ipRGCs project broadly in the brain beyond the SCN (Hattar et al., 2002;Hattar et al., 2006;Gooley et al., 2003;Baver et al., 2008). Additionally, ipRGCs are comprised of multiple subpopulations with distinct genetic, morphological, and electrophysiological signatures (Baver et al., 2008;Schmidt and Kofuji, 2009;Ecker et al., 2010;Schmidt et al., 2011) and distinct functions Schmidt et al., 2014). Though there are rare exceptions Schmidt et al., 2014), the unique roles played by each ipRGC subsystem remain largely unknown. eLife digest Light, whether natural or artificial, affects our everyday lives in several ways.
Exposure to light impacts on our health and well-being. It plays a crucial but indirect role in helping to align our internal body clock with the 24-hour cycle of day and night, and a burst of bright light in the middle of the night can wake us up from sleep.
Decades of research have revealed the circuitry that controls the indirect effects of light on the body's internal clock. A tiny set of cells in the base of the brain called the suprachiasmatic nucleus (SCN for short) generates the body's daily or "circadian" rhythm. A small group of nerve cells in the retina of the eye called intrinsically photosensitive retinal ganglion cells (ipRGCs) connect with the SCN. These ipRGCs relay information about light to the SCN to ensure that daily rhythms happen at the appropriate times of day. But scientists do not yet know if the same brain circuits regulate the direct effects of light on alertness.
Mice are often used in studies of circadian rhythms but, unlike humans, mice are normally active at night and sleep throughout the day. This means that a burst of bright light in the middle of the night causes mice to become less alert. Now, in experiments with mice, Rupp et al. show there are two separate circuits from the retina to the brain that influence wakefulness. In the experiments, some mice were genetically engineered to only have ipRGCs that connect with the SCN and to lack those that connect with other brain areas. These mice lived in cages with a normal day/night cycle and their body temperature and sleep-related brain activity were monitored as Rupp et al. sporadically exposed them to bright light at night. These mice continued their normal routines and were unaffected by the bursts of light. In a second set of experiments, ipRGCs that do not connect with the SCN were activated in other mice. This caused an immediate and sustained drop in the body temperature of the mice, which is linked to them becoming less alert.
The experiments suggest that the circuit that connects ipRGCs to the SCN to align the body's circadian rhythm with light does not control the direct effect of light on wakefulness. Instead, a separate circuit that extends from ipRGCs to an unknown part of the brain area influences wakefulness. Better understanding this second circuit could allow scientists to develop ways to keep people like emergency personnel or overnight shift workers awake and alert at night while avoiding harmful disruptions to their circadian rhythms.
It is currently unknown whether distinct ipRGC subpopulations mediate both the acute and circadian effects of light, and two major possibilities exist for how this occurs: (1) ipRGCs mediate both acute and circadian light responses through their innervation of the SCN or (2) ipRGCs mediate circadian photoentrainment through the SCN, but send collateral projections elsewhere in the brain to mediate acute light responses. To date, the predominant understanding has centered on a role for the SCN in both acute and circadian responses to light (Muindi et al., 2014;Morin, 2015;Bedont et al., 2017). However, this model has been controversial due to complications associated with SCN lesions (Redlin and Mrosovsky, 1999) and alternative models proposing a role for direct ipRGC input to other central targets (Redlin and Mrosovsky, 1999;Lupi et al., 2008;Tsai et al., 2009;Hubbard et al., 2013;Muindi et al., 2014). Here, we sought to address the question of how environmental light information-through ipRGCs-mediates both the circadian and acute regulation of physiology. To do so, we investigated the ipRGC subpopulations and coding mechanisms that mediate body temperature and sleep regulation by light. We find that a molecularly distinct subset of ipRGCs is required for the acute, but not circadian, effects of light on thermoregulation and sleep. These findings suggest that, contrary to expectations, functional input to the SCN is not sufficient to drive the acute effects of light on these behaviors. These findings provide new insight into the circuits through which light regulates behavior and physiology.

Brn3b-positive ipRGCs are required for light's acute effects on thermoregulation
To identify mechanisms of acute thermoregulation, we maintained mice on a 12 hr/12 hr light/dark cycle and then presented a 3 hr light pulse two hours into the night (Zeitgeber time 14, ZT14) while measuring core body temperature ( Figure 1A). The nocturnal light pulse paradigm is well-established for studying acute regulation of sleep and wheel-running activity Mrosovsky and Hattar, 2003;Altimus et al., 2008;Lupi et al., 2008). We focused first on body temperature because of its critical role in cognition and alertness (Wright et al., 2002;Darwent et al., 2010), sleep induction and quality (Kräuchi et al., 1999), metabolic control (Kooijman et al., 2015), and circadian resetting (Buhr et al., 2010).
Body temperature photoentrains to the light/dark cycle with peaks during the night and troughs during the day ( Figure 1B). Both rodents and humans utilize ocular light detection to acutely adjust body temperature in response to a nocturnal light pulse (Dijk et al., 1991;Cajochen et al., 2005), though how this body temperature change is initiated by the retina and relayed to the brain is unknown. When we presented wildtype mice with a nocturnal light pulse, we observed a decrease in both body temperature and general activity compared to the previous night ( Figure 1C). The decrease in body temperature and activity was sustained for the entire 3 hr stimulus, with moderate rundown ( Figure 1C).
We observed that acute body temperature regulation only occurred at relatively bright light intensities (>100 lux) (Figure 1-figure supplement 1). This, in combination with previous reports that body temperature regulation is most sensitive to short-wavelength light (Cajochen et al., 2005), suggested that it might be mediated by the insensitive and blue-shifted melanopsin phototransduction (Lucas et al., 2001;Do et al., 2009). To test this, we measured body temperature in mice lacking either functional rods and cones (melanopsin-only: Gnat1 -/-; Gnat2 -/-) or lacking melanopsin (melanopsin KO: Opn4 -/-). Both genotypes photoentrained their body temperature ( Figure 1D,E), with an amplitude indistinguishable from wildtype ( Figure 1F). However, we found that acute body temperature decrease to a nocturnal light pulse was present in melanopsin-only mice (Gnat1 -/-; Gnat2 -/-) ( Figure 1G,H and . This indicates that melanopsin is critical for light's ability to drive acute body temperature decreases, as it is for acute sleep induction Lupi et al., 2008;Tsai et al., 2009). These results suggest that ipRGCs are the only retinal cells that are necessary and sufficient for acute thermoregulation by light.
ipRGCs comprise multiple subtypes (M1-M6) with distinct gene expression profiles, light responses, and central projections (Schmidt et al., 2011;Quattrochi et al., 2019), prompting us to  ask which subtypes mediate acute thermoregulation. ipRGCs can be molecularly subdivided based on whether they express the transcription factor Brn3b. Brn3b(+) ipRGCs project to many structures including the olivary pretectal nucleus (OPN) and dorsal lateral geniculate nucleus (dLGN), but largely avoid the SCN Li and Schmidt, 2018). In contrast, Brn3b(-) ipRGCs project extensively to the SCN and intergeniculate leaflet (IGL), while avoiding the OPN and dLGN . Non-M1 (i.e. M2-M6) ipRGC subtypes express Brn3b, along with the majority of M1 ipRGCs. Interestingly, just 200 (out of 700-800) M1 ipRGCs lack any Brn3b expression . Ablation of Brn3b(+) ipRGCs using melanopsin-Cre and a Cre-dependent diphtheria toxin knocked into the Brn3b locus (Brn3b-DTA: Opn4 Cre/+ ;Brn3b zDTA/+ ) removes virtually all ipRGC input to brain areas aside from the SCN and IGL Li and Schmidt, 2018), and these mice lack a pupillary light reflex and show deficits in contrast sensitivity, but retain circadian photoentrainment of wheel-running activity Schmidt et al., 2014). When we measured body temperature in Brn3b-DTA mice, we found that their body temperature was photoentrained with a similar amplitude to controls (Figure 2A-C). However, despite the presence of melanopsin in the Brn3b(-) ipRGCs of Brn3b-DTA mice (Opn4 Cre/+ ;Brn3b zDTA/+ ), they did not acutely decrease body temperature in response to a nocturnal light pulse ( Figure 2F,G). Importantly, melanopsin heterozygous littermate controls (Opn4 Cre/+ ) displayed normal acute thermoregulation by light ( Figure 2D,E), indicating that halving melanopsin gene dosage is not the cause of the impaired body temperature decrease in Brn3b-DTA mice. Additionally, when we compared the change in body temperature of Control to Brn3b-DTA mice during that light pulse, we found that Control mice showed a significantly larger decrease in body temperature (Figure 2-figure supplement 1). These results demonstrate that Brn3b(+) ipRGCs are required for acute thermoregulation by light but not photoentrainment of body temperature and reveal that light information to the SCN is sufficient for circadian photoentrainment of body temperature, but not its acute regulation.

Brn3b-positive ipRGCs are sufficient for acute thermoregulation
Our data thus far suggest that there are two functionally distinct populations of ipRGCs that regulate thermoregulation: (1) Brn3b(-) ipRGCs that project to the SCN to mediate circadian photoentrainment of body temperature and (2) Brn3b(+) ipRGCs that project elsewhere in the brain and are necessary to mediate acute thermoregulation. If Brn3b(+) ipRGCs are not just necessary, but also sufficient, for acute thermoregulation, then activation of this population at ZT14 should result in a body temperature decrease. To test if Brn3b(+) ipRGCs are sufficient for acute thermoregulation, we expressed a chemogenetic activator in Brn3b(+) RGCs ( Figure 3A, Brn3b Cre/+ with intravitreal AAV2-hSyn-DIO-hM3Dq-mCherry, we refer to these mice as Brn3b-hM3Dq). As a control, we also injected this virus into Control (Brn3b +/+ ) littermates. We then injected both genotypes first with PBS at ZT14 on the first night, and CNO at ZT14 on the second night. This technique allowed for statistical within animal comparisons of body temperature changes in response to PBS versus CNO injection. Importantly, CNO did not cause a significant decrease in body temperature in the absence of hM3Dq (Figure 3-figure supplement 1). This technique allowed us to acutely activate the Brn3b(+) RGCs with the DREADD agonist clozapine N-oxide (CNO) (Armbruster et al., 2007). We found that after intravitreal viral delivery, many RGCs were infected, including melanopsin-expressing ipRGCs ( Figure 3A and  The body temperature of Brn3b-hM3Dq mice photoentrained to a normal light/dark cycle ( Figure 3B). Following CNO administration to Brn3b-hM3Dq mice at ZT14 to depolarize Brn3b(+) RGCs, we observed a robust decrease in body temperature that lasted at least 6 hr ( Figure 3D). Importantly, PBS administration in Brn3b-hM3Dq mice ( Figure 3C) and nocturnal CNO administration in wildtype control mice (Figure 3-figure supplement 2) had no measurable effect on body temperature, while CNO administration significantly decreased body temperature in Brn3b-hM3Dq compared to pre-injection temperature (Figure 3-figure supplement 2). Together, these results demonstrate that Brn3b(+) ipRGCs mediate the acute effects of light on body temperature though extra-SCN projection(s), while Brn3b(-) ipRGCs mediate circadian photoentrainment of body temperature by projections to the SCN and/or IGL. day, with only one 30 min bin at ZT12 (light offset) showing a significant difference between Control and Brn3b-DTA animals ( Figure 4A,B). This is consistent with previous reports of normal circadian photoentrainment of daily activity rhythms in Brn3b-DTA mice . Control and Brn3b-DTA mice also showed similar total percent time awake or asleep across an entire day ( Figure 4C), though Brn3b-DTA mice showed a small, but significant, increase in the proportion of total sleep that was classified as NREM and decrease in the proportion of total sleep that was classified as REM (Figure 4-figure supplement 1A).

Control (Opn4
We hypothesized that this small difference in sleep at lights-off in Brn3b-DTA mice could be due to a defect in their acute response to light for sleep modulation. To test this, we subjected mice to a 3 hr light pulse from ZT14-17 , when the homeostatic drive for sleep is low and Control and Brn3b-DTA animals display similar amounts of sleep ( Figure 4A,B). We found that in Control mice, a light pulse decreased time awake and increased time asleep relative to baseline (previous day) ( Figure 4C,D), while in Brn3b-DTA mice a light pulse caused no change in total percent time asleep or awake ( Figure 4F,G), but moderately increased sleep in the first 30 min bin ( Figure 4F). Importantly, when we compared the time spent asleep during the light pulse between control and Brn3b-DTA animals, the control mice slept significantly more (Figure 4-figure supplement 2). Neither Control nor Brn3b-DTA animals showed any change in proportion of non-REM or REM sleep in response to the light pulse (Figure 4-figure supplement 1B,C). These data show that Brn3b(+) ipRGCs are necessary for the acute light induction of sleep. Consistent with our body temperature data, although Brn3b-DTA mice have apparently normal input to the SCN and show normal circadian photoentrainment of wheel-running activity , body temperature (Figure 2), and sleep (Figure 4), this ipRGC innervation of the SCN is not sufficient to drive the normal light induction of sleep. These disruptions in light's acute effects on thermoregulation and sleep are circuit specific effects because Brn3b-DTA mice showed robust inhibition of wheel running behavior to a 3 hr light pulse delivered from ZT14-17 (Figure 4-figure supplement 3).

Discussion
We show here that for the same physiological outcome, the acute effects of light are relayed through distinct circuitry from that of circadian photoentrainment, despite both processes requiring ipRGCs. Our results suggest that for thermoregulation and sleep, ipRGCs can be genetically and functionally segregated into Brn3b(+) 'acute' cells, and Brn3b(-) 'circadian' cells. Because Brn3b(+) cells largely avoid the SCN, and Brn3b(-) cells preferentially target the SCN, our results discount a critical role for the SCN in acute light responses in these two behaviors, and instead implicate direct ipRGC projections to other brain areas (Gooley et al., 2003;Hattar et al., 2006). Surprisingly, Brn3b(-) cells are sufficient to drive the acute and circadian effects of light on wheel running activity, demonstrating further divergence in the circuits mediating the acute effects of light on behavior, and suggesting that, unlike for thermoregulation and sleep, acute and circadian regulation of activity is driven via the SCN.
Our results indicate that activation of Brn3b(+) RGCs at ZT14 using the Brn3b Cre line in combination with Gq-DREADDs is sufficient to induce a body temperature decrease. Because other (non-ipRGC) RGC types express Brn3b (Badea et al., 2009), this manipulation likely also activated multiple non-ipRGCs in addition to Brn3b(+) ipRGCs. However, our data indicate that melanopsin signaling (Figure 1), and therefore ipRGCs, are required for the acute effects of light on thermoregulation. Moreover, when we ablate Brn3b(+) ipRGCs, this acute effect of light on thermoregulation is also lost (Figure 2), again arguing for a necessity of ipRGCs for this behavior. Therefore, though we are unable to specifically activate only Brn3b(+) ipRGCs using available genetic tools, we think it highly likely that the temperature changes driven by the activation of all Brn3b(+) RGCs is occurring through ipRGCs.
The specific Brnb(+) ipRGC subtypes that mediate the light's acute effects on body temperature and sleep remain a mystery. A majority of all known ipRGC subtypes (M1-M6) are lost in Brn3b-DTA mice , with the exception of a subset of~200 M1 ipRGCs. In agreement with this, ipRGC projections to all minor hypothalamic targets are lost in Brn3b-DTA mice, while innervation of the SCN and part of the IGL remains intact Li and Schmidt, 2018). This suggests that all non-M1 subtypes and a portion of M1 ipRGCs are Brn3b(+). Each subtype has a distinct reliance on melanopsin versus rod/cone phototransduction for light detection (Schmidt and Kofuji, 2009). The necessity and sufficiency of melanopsin in mediating acute effects of light on body temperature ( Figure 1) and sleep Lupi et al., 2008;Tsai et al., 2009) suggests that a subtype with strong melanopsin, but weak rod/cone photodetection is responsible -possibly either M1 or M2 cells. However, experiments to tease this apart will require novel methods to specifically manipulate ipRGC subtypes that are currently unavailable. The brain areas that mediate the acute effects of light on physiology are essentially unknown. There are many candidate areas that both receive direct ipRGC innervation and have been documented to be involved in light's acute effects on physiology, including the preoptic areas (Muindi et al., 2014), the ventral subparaventricular zone (Kramer et al., 2001), and the pretectum/ superior colliculus (Miller et al., 1998). Aside from the SCN, ipRGC projections to the median (MPO) and ventrolateral preoptic (VLPO) areas have been the most widely supported. The preoptic areas are involved in sleep and body temperature regulation (Szymusiak and McGinty, 2008;Nakamura, 2011) and are activated by an acute light pulse (Lupi et al., 2008;Tsai et al., 2009). In support of our behavioral findings, ipRGC projections to each of these areas is lost in Brn3b-DTA animals (Li and Schmidt, 2018). However, ipRGC projections to these areas are sparse (Gooley et al., 2003;Hattar et al., 2006), suggesting their activation by light may be indirect.

Control (Opn4
In contrast, the superior colliculus (SC) and pretectum receive robust innervation from ipRGCs (Hattar et al., 2002;Hattar et al., 2006;Gooley et al., 2003;Ecker et al., 2010), their lesioning blocks light's acute effects on sleep (Miller et al., 1998), and melanopsin knockout mice lose lightinduced cFOS expression in the SC (Lupi et al., 2008). However, the SC and pretectum receive robust innervation from many conventional RGCs, making the requirement for melanopsin and ipRGCs in acute sleep and body temperature regulation difficult to reconcile. It is also possible (and perhaps probable), that multiple ipRGC target regions are involved, with distinct areas mediating distinct physiological responses. Future studies silencing each retinorecipient target while activating Brn3b(+) ipRGCs will be necessary to tease apart the downstream circuits mediating light's acute effects on physiology.
Alternatively, it remains possible that direct ipRGC control of body temperature is the primary and critical step for many acute responses to light that are mediated by ipRGCs. In support of this possibility, changes in body temperature and heat loss can directly influence sleep induction (Kräuchi et al., 1999). This change in sleep is in turn presumably causative of at least some of light's effects on wheel-running and general activity . Further, core body temperature can acutely regulate cognition and alertness (Wright et al., 2002;Darwent et al., 2010). It is therefore possible that ipRGCs can have widespread influence on an animal's basic physiology and cognitive function simply by regulating body temperature.
Together, our identification of the photopigment and the retinal circuits mediating acute body temperature and sleep induction will facilitate better methods to promote or avoid human alertness and cognition at appropriate times of day (Chellappa et al., 2011). Our results support many recent efforts to capitalize on the specific light-detection properties of melanopsin (Lucas et al., 2014), such as its insensitivity and short-wavelength preference, to promote or avoid its activation at different times of day. However, this approach is problematic because acute activation of melanopsin to promote alertness has the unintended effect of shifting the circadian clock (Provencio et al., 1994), thereby making subsequent sleep difficult. Our identification that the Brn3b(+) ipRGCs specifically mediate light's acute effects on body temperature provides a cellular basis for developing targeted methods for promoting acute alertness, while minimizing circadian misalignment. Continued on next page between the control night and the night where the light pulse was given. For CNO experiments, injections were carried out near ZT14, but specific times were recorded for each mouse to align the data to the time of injection. Comparisons of mean body temperature after PBS or CNO utilized the 6 hr following injection. Clozapine-N-oxide (Sigma) was prepared as a 0.1 mg/ml solution in PBS and injected at 1 mg/kg intraperitoneally at ZT14.

Animals (Sleep)
All procedures were conducted in accordance with NIH guidelines and approved by the Institutional Animal Care and Use Committee of Northwestern University. Opn4Cre and Brn3bz-dta were maintained on a mixed C57Bl/6J; 129Sv/J background (Hattar et al., 2002;Hattar et al., 2006;Mu et al., 2005). Male and female littermate Opn4 Cre/+ and Opn4 Cre/+ ; Brn3b z-dta/+ animals between the ages of 2 and 3 months were used for sleep analysis.

Sleep recording
Male and female littermate Opn4 Cre/+ and Opn4 Cre/+ ; Brn3b z-dta/+ mice were used for sleep recordings. Electroencephalogram (EEG) and electromyogram (EMG) electrode implantation was performed simultaneously at 8 weeks of age. Mice were anesthetized with a ketamine/xylazine (98 and10 mg/kg respectively) and a 2-channel EEG and 1-channel EMG implant (Pinnacle Technology) was affixed to the skull. Mice were transferred to the sleep-recording cage 6 days after surgery, tethered with a preamplifier, and allowed 3 days to acclimate to the new cage and tether. Mice were housed in 12:12 light/dark conditions before and after EEG implantation. EEG and EMG recording began simultaneously at the end of the habituation period, which were displayed on a monitor and stored in a computer for analysis of sleep states. The high pass filter setting for both EEG channels was set at 0.5 Hz and low pass filtering was set at 100 Hz. EMG signals were high pass filtered at 10 Hz and subjected to a 100 Hz low pass cutoff. EEG and EMG recordings were collected in PAL 8200 sleep recording software (Pinnacle Technology) and scored, using a previously described, multiple classifier, automatic sleep scoring system, into 10 s epochs as wakefulness, NREM sleep, or REM sleep on the basis of rodent sleep criteria (Gao et al., 2016). Light source for all sleep experiments was a 3000 Kelvin light source at 500 lux.

Wheel-running activity and masking experiment
Mice were placed in cages with a 4.5-inch running wheel, and their activity was monitored with Vital-View software (MiniMitter). Analyses of wheel running activity were calculated with ClockLab (Actimetrics). We used 500 lux light intensity. Mice were initially placed under 12:12 LD masking experiments. Mice were exposed, in their home cage, to a timer-controlled 3 hr light pulse at ZT14-ZT17. Percent activity for each mouse was normalized to its own activity at ZT13 (1 hr before light pulse), and analyzed in 30 min bins.

Tissue staining and imaging
Animals were anesthetized with Avertin and euthanized prior to fresh dissection of retinas in PBS. Retinas were fixed in 4% paraformaldehyde (Sigma) for at least 1 hr on ice. Retinas were then washed in PBS before staining overnight in anti-OPN4 antibody (1:1000, Advanced Targeting Systems) and then washed prior to 2 hr in secondary antibody (1:1000 goat anti-rabbit AlexaFluor 488, Life Technologies). Retinas were then flat-mounted on slides and imaged on a Zeiss LSM 710 confocal microscope.

Statistics
All statistical tests were performed in Graphpad Prism or R 3.4.4. Specific tests are listed in the text and figure legends. Linear mixed models were performed with the R packages lme4 1.1-21 and emmeans 1.3.4.

Data availability
All raw data are linked to this manuscript and available online.