A key biological function of circadian clocks is to encode local environmental time, and the seasons, through interactions with the daily light cycle. Most organisms live in a rhythmic environment where daily environmental changes occur corresponding with solar time, and their endogenous 24 hr timing mechanism, or the circadian clock, enables adjustment of their physiology and behavior accordingly. In mammals, the central clock—the suprachiasmatic nucleus (SCN) of the hypothalamus—represents solar time to synchronize peripheral tissue clocks in the rest of the body, and it drives the expression of daily and seasonal behaviors in tune with the temporal structure of the environment.

Classical behavioral studies performed by manipulating light cycles have revealed fundamental principles in how circadian rhythms are reset and synchronized by external time cues at the level of behavioral outputs (Pittendrigh and Daan, 1976a; Pittendrigh and Daan, 1976b), but the molecular basis of clock setting and synchronization remains to be fully explained. Entrainment or alignment of circadian locomotor behavior with light cycles is achieved based on differential sensitivity of the circadian rhythm to the timing of light exposure – phase delays result from light exposure in the early circadian night, phase advances from light in the late night, and there is a ‘dead zone’ in the mid-day where light does not reset the rhythm (Pittendrigh and Daan, 1976b). At the molecular level, the basis of the mammalian circadian clock is self-sustained circadian oscillations of core clock genes arranged in an autoregulatory transcription-translation feedback loop, with transcription factors Clock and Bmal1 driving the circadian expression of Period (Per) and Cryptochrome (Cry) genes that mediate negative feedback within the clockworks (Takahashi, 2017). Light stimulation results in acute induction of Per1/2 (Takahashi, 2017). Conventional time-point clock gene expression profiling from animals under different light cycles has provided a snapshot of how the clock gene rhythms in the SCN are influenced by various lighting conditions (Messager et al., 2000; Schwartz et al., 2011), but with limited temporal resolution that does not fully capture dynamic changes in the clock gene rhythms induced by different light cycles.

The advent of clock gene reporters (e.g. Per1::GFP, PER2::LUC) (Kuhlman et al., 2000; Yoo et al., 2004) enabled assaying the motion of the circadian clock in real time and with low variability. However, to date the fundamental question of how clock genes in the SCN encode different lighting conditions has been primarily approached by manipulating light exposure in vivo and subsequently explanting the SCN into slice culture in constant darkness to retrospectively infer the in vivo entrained state, due to technical challenges in mimicking retinal light input ex vivo. While much progress has been made with this paradigm, this approach can have significant limitations. The SCN activity observed in a free-running condition reflects relaxation of the explanted SCN network back toward baseline, rather than active encoding of entrainment (Rohr et al., 2019), and explantation can disrupt expression of the in vivo state (Pendergast et al., 2009).

Here, combining long-term organotypic explant culture, cyclic red light optogenetic stimulation, and the PER2 bioluminescent reporter, we assess how the clock gene rhythms in the ex vivo SCN change in real time to achieve entrainment to light cycles. We have instituted an ex vivo optogenetic experimental system that provides precise timing, duration, and intensity of recurring input stimulation to the explanted SCN while tracking clock gene rhythms at high temporal resolution. Acute optogenetic channelrhodopsin-2 (ChR2) stimulation of SCN neurons with 470 nm blue light to make them fire at light-driven spike frequency (e.g. 8–15 Hz, Jones et al., 2015; Mazuski et al., 2018), as does retinal light input, has been shown to be effective to reset circadian rhythms in vivo and ex vivo. However, long-term blue light illumination in culture (without opsins) results in phototoxicity, reducing cell viability (470 nm, 1 Hz, Stockley et al., 2017) and degrading many biological processes, including cell growth (470 nm, continuous, Ohara et al., 2002) and respiration (425~500 nm, continuous, Robertson et al., 2013). Notably, red light is more tolerable in vitro (488 nm vs 558 or 640 nm, continuous, Wäldchen et al., 2015) and in vivo (440~500 nm vs. 540~660 or 655~695 nm, continuous, De Magalhaes Filho et al., 2018). Recent development of optogenetic actuators responding to red light (Klapoetke et al., 2014; Lin et al., 2013) prompted us to test whether red light can be utilized for long-term optogenetic stimulation, and to study how core clock gene PER2 rhythms in the ex vivo SCN are dynamically altered and reset by repeated light stimulation to achieve light entrainment.

Here, we uncover that PER2 rhythms in the ex vivo SCN under entrainment to optogenetic light cycles show contraction of the rising phase and elongation of the falling phase depending on the timing of light exposure, and reveal ex vivo SCN plasticity at the clock gene level similar to canonical features of light-induced plasticity in circadian behavior. Aspects of circadian plasticity to light entrainment and regional heterogeneity of light responsiveness are apparently intrinsic to the SCN clockworks.

In this study, we assessed at high-resolution the temporal and spatial dynamics of core clock gene rhythms in the ex vivo SCN following single optogenetic light exposures and various forms of optogenetic light cycles to gain further insights into light resetting and entrainment of clock gene rhythms in the SCN. Using real-time bioluminescent recording of PER2 expression combined with recurring optogenetic stimulation, we confirmed that the isolated SCN strikingly recapitulates many of the canonical features of circadian clock entrainment in intact animals, including (1) phase responses with period after-effects, (2) period matching and phase angle differences to stimuli of non-24-hour cycles, and (3) differential entrainment to skeleton photoperiods with a minimum tolerable night. Entrainment is expressed as transformation of the ongoing rhythmic waveform of the clock protein we monitored, PER2, from approximately sinusoidal in free-running conditions to highly asymmetrical trajectories with accelerated synthetic (rising) phases, and extended degradative (falling) phases of PER2 protein abundance mediating clock advances and delays, respectively. Spatiotemporal analysis revealed intrinsic regional differences in light resetting within the SCN neural network, with the magnitude of acute phase shifts that underlie ongoing waveform changes being greatest in the ventrolateral SCN core, while the magnitude of subsequent period after-effects is greatest in the dorsomedial SCN shell.

A key question addressed in our study was how exactly light input changes the clock gene rhythms in the SCN to drive phase shifts and to achieve entrainment to external light cycles. Previous studies (Messager et al., 2000; Schwartz et al., 2011) have assayed the clock gene rhythms under lighting conditions by time-point sampling clock gene expression from populations of animals for each time point at low temporal resolution (e.g. 4 hr sampling intervals), making it difficult to fully capture dynamic waveform changes in the rhythms induced by light input. Using bioluminescent recording of PER2 expression at an interval of minutes, while optogenetically providing the SCN explant with simulated light signals, we directly showed that discrete light input induces acute induction of PER2 expression and differential waveform changes in PER2 rhythms in a phase-dependent manner, leading to a time-dependent phase shifts: high PER2 induction by early-night light exposure induces phase delays by elongating the falling phase of the molecular clockworks, while late-night light induces phase advances by accelerating the rising phase of the clockworks, and mid-day light does not affect the rhythm phase due to low PER2 induction. These differential waveform changes in specific PER2 phases differentiate PER2 waveforms under light cycles from those in free-running conditions. PER2 rhythms express typical sinusoidal waveforms in free-running conditions, whereas under light entrainment they show asymmetric waveforms with elongated falling phases, abbreviated rising phases and dramatic waveform changes aligned with light stimulation events. Given that a pulse of retinal light input can induce PER gene expression in the SCN and PER induction is required for the SCN clock resetting (Shigeyoshi et al., 1997; Tischkau et al., 2003), our results suggest that the SCN clock rhythms such as neural activity rhythms can phase-shift to acute light exposure and entrain to a light cycle via single and regular events of the aforementioned waveform changes, respectively, as a result of light induction of PER gene expression.

As daily phase shifts underlie adjustment of the endogenous period of circadian behavior to different day-night cycle lengths, daily waveform acceleration in the rising phase of PER2 aligned with dawn, and daily elongation in the falling phase of PER2 abundance aligned with dusk, express the entrainment of circadian PER2 rhythm to light cycle periods. Stable alignment of the SCN clock with repeated light stimulations at a phase-delaying circadian time achieved circadian entrainment to a longer-than-24 hr cycle mainly via repeated lengthening of the falling phase of PER2, whereas repeated shortening of the rising phase at a stable phase angle accompanied short T-cycle entrainment. For photoperiodic encoding, as brief light pulses defining dawn and dusk can entrain circadian behavior, daily brief optogenetic simulations of light transitions at dawn and dusk shortened PER2 rising phase and lengthened the falling phase in the SCN, respectively, thereby matching PER2 rhythms to a 24 hr period. PER2 waveform widths were different between equinox and long skeleton photoperiods. Previous studies suggested that long photoperiod induces Per waveform broadening as high Per gene expression gets extended throughout long light periods (Messager et al., 1999; Schaap et al., 2003). However, our results from skeleton photoperiods suggest that light-dark transitions at dawn and dusk are sufficient to alter PER2 waveform width. Light exposures at earlier dawns and later dusks for long photoperiods drive more PER2 induction than do light exposures in equinox photoperiods, thus expanding PER2 waveform. This is consistent with waveform broadening of neural activity rhythms in animals under a long photoperiod (VanderLeest et al., 2007).

Another important point of this study is that plasticity in circadian behavior following alteration of lighting conditions is intrinsic to the SCN clockworks. Key principles of circadian entrainment and plasticity in mammals have been discovered by classical behavioral studies that assessed circadian locomotor behavior under different lighting conditions. Circadian behavior, however, is a product of multiple oscillators involving different brain areas, and the SCN receives both extensive feed-forward and feedback from other brain nuclei in situ (Dibner et al., 2010). Our results from ex vivo entrainment of isolated SCN strongly suggest that many of the classically studied forms of circadian plasticity and properties of entrainment apparently reside intrinsically within the SCN molecular and neural network themselves. As previously reported (Jones et al., 2015), differential phase responses of circadian rhythms depending on the timing of light input is evident even though the SCN neurons are directly stimulated, rather than them receiving retinal input. Furthermore, as entrainment of circadian behavior to different T-cycles is achieved by different phase angles of entrainment, the SCN clock itself establishes different entrained phase angles to the T-cycles, consistent with non-parametric model of entrainment (Pittendrigh and Daan, 1976b). Daily light stimulations fall in the phase-advancing zone (late falling PER2 phase) and the phase-delaying zone (late rising PER2 phase) during short and long T cycles, respectively. Also, the SCN clock itself can indeed be entrained directly by the light-dark transitions in skeleton photoperiods as mimicked by optogenetic stimulation, and a higher order aspect of entrainment, such as the bias of the circadian system to resolve ambiguous skeleton light cycles in favor of short-day entrainment (Pittendrigh and Daan, 1976b), also resides in the SCN clockworks.

We also uncovered whether and how the SCN clock itself expresses light-induced after-effects on the endogenous clock period. Acute phase shifts produce after-effects on the endogenous period of the SCN clock itself, and interestingly, the magnitude of the period after-effects (up to several hours) is larger than that at the level of behavioral rhythms (less than an hour) (Pittendrigh and Daan, 1976a). This suggests that the after-effects on circadian behavior following single light pulses become diminished as light signals are transmitted downstream of the SCN, or that the plasticity of the in vivo SCN is constrained by interactions with extra-SCN oscillators or circuits. In the case of entrainment to non-24 hr periods (T-cycles), surprisingly, we did not detect significant period after-effects in the SCN clock, although T-cycle entrainment produces most significant period after-effects in circadian behavior (Pittendrigh and Daan, 1976b). It is an unexpected finding that repeated stimulation does not produce large period after-effects, while single light stimulation does. Period after-effects of circadian locomotor behavior are expressed after more than a month of exposure to different T-cycles (Azzi et al., 2014; Schwartz et al., 2011), suggesting that our one-week entrainment paradigm might not be sufficient to drive T-cycle period aftereffects. Notably, previous findings indicate that the clock period of SCN explanted from animals entrained to T-cycles does not represent circadian behavioral period of those animals (Aton et al., 2004; Azzi et al., 2017; Ciarleglio et al., 2014; Molyneux et al., 2008), suggesting the possibility that extra-SCN clocks are a critical influence on the period after-effects of T-cycles. Our results from direct entrainment of isolated SCN to T cycles also suggest that there may be additional non-SCN influences underlying the T cycle period after-effects at the behavioral level.

We also revealed how light exposure alters the network state of the SCN clock to induce phase shifts and period plasticity, and directly showed that the SCN itself has subregional heterogeneity in clock-resetting capacity. Previously, it was assumed that retinal light input is mainly received by the ventral SCN, creating differences in light-induced clock resetting between the ventral and dorsal regions due to differential input (Yan et al., 2007). For example, in the in vivo SCN light induction of Per1 mRNA expression is more prominent to the ventral region, whereas Per2 induction is widespread (Yan and Silver, 2002). More recent studies using intrinsically photosensitive retinal ganglion cell (ipRGC) labeling (Chen et al., 2011; Fernandez et al., 2016), however, showed that retinal projections are widespread across the entire SCN and neuronal activation following light exposure was ubiquitous, suggesting the possibility of intrinsic differences in responsiveness between the SCN subregions. Using pan-neuronal optogenetic stimulation that was synchronous across the entire SCN, we found that the lateral or ventrolateral SCN exhibits large phase responses and small period responses, while the medial or dorsomedial SCN exhibits small phase responses but large period plasticity, revealing an intrinsic nature of the clock-resetting heterogeneity in the SCN. As the VIP and AVP neurons are respectively located in the ventral and dorsal SCN, this suggests that regionally differential phase and period responses in the SCN might be derived from intrinsic differences between the VIP and AVP neuronal clocks. The period response, inversely correlated with the phase response, could serve to help the SCN recover back to its baseline network phase state from decreased synchrony following phase shifts. Decreased phase synchrony among SCN neurons following light exposure has been observed in the case of VIP-induced phase shifts (Hamnett et al., 2019) and entrainment to long photoperiods or non-24hr light cycles (Azzi et al., 2017; Evans et al., 2013). Interestingly, stimulating VIP neurons reset the ensemble SCN phase, while stimulating VPAC2 neurons located in the dorsal SCN do not (Patton et al., 2020), suggesting regionally different phase-resetting capacity. Future studies will be needed to address whether and how the subregional heterogeneity in light responsiveness fulfills encoding of various lighting conditions in the SCN.

Technical limitations of our methods are twofold. We used~ P12 SCN slices to achieve long-term monitoring of real-time clock gene rhythms throughout optogenetic entrainment. Although SCN astrocytes become fully mature by P20-25 and they are recently identified as an important component for entrainment in vivo (Brancaccio et al., 2019; Brancaccio et al., 2017; Tso et al., 2017), SCN maturity reaches near-adult levels at P12 in many aspects including retinal innervation, clock gene rhythmicity, neuropeptide expression profile, and photic responses (Bedont and Blackshaw, 2015). Additionally, our current system is not readily applicable to studying real-time SCN rhythms under a full optogenetic light cycle (e.g. 12 hr of stimulation every day) as excitation light for optogenetics interrupts bioluminescence detection. Moving forward, overcoming such a limitation will further extend the usage to studying SCN-intrinsic mechanisms of entrainment. Interestingly, real-time clock gene rhythms in the in vivo SCN re-entrain to a new light cycle during experimental jet lag (Mei et al., 2018). Future waveform analyses of clock gene rhythms in such an in vivo setup would allow comparison with our observations in the isolated SCN.

Lastly, our study provides technical contributions to studies of SCN entrainment and plasticity and to the study of neural plasticity in general. Our system that employs long-term red optogenetic stimulation and photomultiplier tube-based bioluminescence recording from cultured neural tissues enables ex vivo entrainment of the isolated SCN neural network over intervals of days to weeks in a similar way to light entrainment in animals, and high-resolution assessment of plasticity in the SCN clock gene rhythms throughout entrainment. As the SCN explant is more accessible to genetic or pharmacological manipulations than the in vivo SCN, our method will provide a great opportunity to further study molecular mechanisms of the SCN entrainment. Our system can also be adapted for long-term optogenetic stimulation with other bioluminescent readouts, such as bioluminescence resonance energy transfer (BRET) Ca²⁺ sensors (Suzuki et al., 2016; Yang et al., 2016), and thus it is potentially widely applicable to studying induction of long-term neural plasticity in different regions of the brain.

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 to 6.

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Author details

1. Suil Kim

   Vanderbilt Brain Institute, Vanderbilt University, Nashville, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2739-1238

2. Douglas G McMahon

   1. Vanderbilt Brain Institute, Vanderbilt University, Nashville, United States
   2. Department of Biological Sciences, Vanderbilt University, Nashville, United States

   Contribution

   Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - original draft, Writing - review and editing

   For correspondence

   douglas.g.mcmahon@vanderbilt.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0645-2281

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