Circadian Photoreceptor Research Articles

A role for the circadian photoreceptor CRYPTOCHROME in regulating triglyceride metabolism in Drosophila.

The biological rhythms generated by the endogenous circadian clocks across the tree of life regulate numerous behavioral, metabolic, and physiological processes. Although evidence from various studies in Drosophila melanogaster indicates the importance of the core circadian clock genes in the intricate interplay between the circadian clock and metabolism, little is known about the contribution of the circadian photoreceptor/s in this process. The deep brain circadian photoreceptor CRYPTOCHROME (CRY) is essential for resetting the clock in response to light and is also highly expressed in metabolically active tissues in Drosophila. In this study, we sought to explore the possible roles played by CRY in triglyceride (TG) metabolism. We observed that the cry mutant (cry01) flies exhibited increased starvation resistance and TG levels under both 12-hour (h) light:12-h dark cycle (LD) and under constant light compared with the control w1118 flies. We also observed that cry01 flies had significantly increased food intake, glycogen concentrations, and lifespan under LD. In addition, cryptochrome seemed to affect TG levels in adult flies in response to calorie-restricted and high-fat diets. These results suggest a role for the circadian photoreceptor CRY in TG metabolism in Drosophila.

Reorganization of circadian activity and the pacemaker circuit under novel light regimes.

Many environmental features are cyclic, with predictable changes across the day, seasons and latitudes. Additionally, anthropogenic, artificial-light-induced changes in photoperiod or shiftwork-driven novel light/dark cycles also occur. Endogenous timekeepers or circadian clocks help organisms cope with such changes. The remarkable plasticity of clocks is evident in the waveforms of behavioural and molecular rhythms they govern. Despite detailed mechanistic insights into the functioning of the circadian clock, practical means to manipulate activity waveform are lacking. Previous studies using a nocturnal rodent model showed that novel light regimes caused locomotor activity to bifurcate such that mice showed two bouts of activity restricted to the dimly lit phases. Here, we explore the generalizability of these findings and leverage the genetic toolkit of Drosophila melanogaster to obtain mechanistic insights into this unique phenomenon. We find that dim scotopic illumination of specific durations induces circadian photoreceptor CRYPTOCHROME-dependent activity bifurcation in male flies. We show circadian reorganization of the pacemaker circuit, wherein the 'evening' neurons regulate the timing of both bouts of activity under novel light regimes. Our findings indicate that such environmental regimes can be exploited to design light cycles, which can ease the circadian waveform into synchronizing with challenging conditions.

OpsinLW2 serves as a circadian photoreceptor in the entrainment of circadian locomotor rhythm of a firebrat

Photic entrainment is an essential function of the circadian clock, which enables organisms to set the appropriate timing of daily behavioral and physiological events. Recent studies have shown that the mechanisms of the circadian clock and photic entrainment vary among insect species. This study aimed to elucidate the circadian photoreceptors necessary for photic entrainment in firebrats Thermobia domestica, one of the most primitive apterygote insects. A homology search of publicly available RNA sequence (RNA-seq) data from T. domestica exhibited a cryptochrome 2 (cry2) gene and three opsin genes, opsin long wavelength 1 (opLW1), opLW2, and opUV, as candidate circadian photoreceptors. We examined the possible involvement of these genes in photic entrainment of firebrat locomotor rhythms. Firebrats had the highest entrainability to the light–dark cycle of green light. Treatment with dsRNA of the candidate genes strongly downregulated the respective targeted genes, and in the case of opsin genes, other untargeted genes were occasionally downregulated to various degrees. Under constant light, most control firebrats became arrhythmic, whereas a fraction of those treated with double RNAi of the two opLWs remained rhythmic. Behavioral experiments revealed that the transient cycles necessary for re-entrainment to shifted light cycles were lengthened when opLW2 expression was reduced. These results suggest that opLW2 is involved in the photic entrainment of circadian rhythm in firebrats.

Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells.

Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.

Do sleep and circadian characteristics predict alcohol use in adult drinkers?

While sleep and circadian rhythms are recognized contributors to the risk for alcohol use and related problems, few studies have examined whether objective sleep and circadian measures can predict future alcohol use in humans, and no such studies have been conducted in adults. This study examined whether any baseline sleep and/or circadian characteristics of otherwise healthy adults predicted their alcohol use over the subsequent 12 months. Participants (21-42 years) included 28 light and 50 heavy drinkers. At baseline, a comprehensive range of self-reported and objective sleep/circadian measures was assessed via questionnaires, wrist actigraphy, and measurement of dim light melatonin onset and circadian photoreceptor responsivity. Following this, the number of alcoholic drinks per week and binge drinking episodes per month were assessed quarterly over the subsequent 12 months. Anticipated effects of alcohol (stimulation, sedation, and rewarding aspects) were also assessed quarterly over the 12 months. Analyses included generalized linear mixed-effects models and causal mediation analysis. Across the range of measures, only self-reported insomnia symptoms and a longer total sleep time at baseline predicted more drinks per week and binges per month (ps <0.02). There was a trend for the anticipated alcohol effect of wanting more alcohol at the 6-month timepoint to mediate the relationship between insomnia symptoms at baseline and drinks per week at 12 months (p = 0.069). These results suggest that in otherwise healthy adults, insomnia symptoms, even if subclinical, are a significant predictor of future drinking, and appear to outweigh the influence of circadian factors on future drinking, at least in otherwise healthy adults. Insomnia symptoms may be a modifiable target for reducing the risk of alcohol misuse.

Circadian photoreception impacts thoughts of self

Circadian photoreception impacts thoughts of self

Circadian entrainment to red-light Zeitgebers and action spectrum for entrainment in the jewel wasp Nasonia vitripennis

Light is the most important environmental cue for the circadian system of most organisms to stay synchronized to daily environmental changes. Like many other insects, the wasp Nasonia vitripennis has trichromatic compound eye-based colour vision and is sensitive to the light spectrum ranging from UV to green. We recently described a red-sensitive, ocelli-based photoreceptor, but its contribution to circadian entrainment remains unclear. In this study, we investigated the possibility of Nasonia circadian light entrainment under long-wavelength red LED light–dark cycles and characterized the strength of red light as a potential Zeitgeber. Additionally, we measured the possibility of entrainment under various light intensities (from 5·1012 to 4·1015 photons·cm−2·s−1) and a broader range of wavelengths (455—656 nm) to construct corresponding action spectra for characterizing all circadian photoreceptors involved in photic entrainment. We also conducted electroretinogram (ERG) recordings for each wavelength in the compound eyes. Our findings demonstrate that Nasonia can entrain under red light dark cycles, and the sensory pathway underlying the red-light Zeitgeber response may reside in the ocelli. Combined with findings from previous research, we pose that blue- and green-sensitive rhodopsin photoreceptor cells function as the major circadian photoreceptors in both circadian entrainment by light–dark cycles and circadian phase shifts by light pulses, whereas the red-sensitive photoreceptor cell requires higher light intensity for its role in circadian entrainment by light–dark cycles.

Pigment-dispersing factor is present in circadian clock neurons of pea aphids and may mediate photoperiodic signalling to insulin-producing cells.

The neuropeptide pigment-dispersing factor (PDF) plays a pivotal role in the circadian clock of most Ecdysozoa and is additionally involved in the timing of seasonal responses of several photoperiodic species. The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species with an annual life cycle tightly coupled to the seasonal changes in day length. Nevertheless, PDF could not be identified in A. pisum so far. In the present study, we identified a PDF-coding gene that has undergone significant changes in the otherwise highly conserved insect C-terminal amino acid sequence. A newly generated aphid-specific PDF antibody stained four neurons in each hemisphere of the aphid brain that co-express the clock protein Period and have projections to the pars lateralis that are highly plastic and change their appearance in a daily and seasonal manner, resembling those of the fruit fly PDF neurons. Most intriguingly, the PDF terminals overlap with dendrites of the insulin-like peptide (ILP) positive neurosecretory cells in the pars intercerebralis and with putative terminals of Cryptochrome (CRY) positive clock neurons. Since ILP has been previously shown to be crucial for seasonal adaptations and CRY might serve as a circadian photoreceptor vital for measuring day length, our results suggest that PDF plays a critical role in aphid seasonal timing.

The Effect of Circadian Photoreceptors Stimulation on the Stress Response of Subjects with High Anxiety: A Pilot Study

The circadian and stress-realizing systems are interconnected, and the balance of their interaction determines the state of human health. The objective of this study was to investigate the modulating effect of activating the circadian system on heart rate variability in female medical students with high anxiety while performing a cognitive task. After 20 min of adequate stimulation of circadian photoreceptors with a monochromatic blue light, the cognitive task performance resulted in a decrease in sympathetic impact. During the session of monochromatic blue light exposure, a trend of increasing heart rate variability was observed in a state of relative rest (especially in the first 5 min). A comparative analysis of the parameters of the letter cancellation test before and after light exposure revealed a statistically significant increase in the indexes reflecting mental productivity, work accuracy, and concentration of attention. The results suggest an essential physiological role of the human circadian system in modulating the autonomic and psycho-emotional conditions, as well as cognitive functions of individuals with high anxiety. Our findings indicate the possibility of quickly correcting the balance in human systemic regulatory mechanisms using the activation of retinal circadian photoreceptors by blue light.

Visual and circadian regulation of Drosophila BDBT and BDBT effects on DBT and PER localization

BRIDE OF DOUBLETIME (BDBT) interacts with the circadian kinase DOUBLETIME (DBT) and accumulates in eye foci during the dark of a light:dark cycle. BDBT foci are shown here to be broadly expressed in constant dark and low in constant light. Analysis of circadian photoreceptor cry and visual photoreceptor ninaE mutantsshowed that disappearance of eye BDBT foci requires both the CRYPTOCHROME and the RHODOPSIN-1 pathways. The arr1 and arr2 mutants, which affect rhodopsin quenching, eliminated BDBT foci under dark conditions. arr1 and arr2 mutants also caused increased nuclear PER protein. The changesin BDBT foci do not result from altered BDBT levels in the eye but from changes in its immunodetection. Knockdown of BDBT specifically in theeye produced constitutively nuclear PER and constitutively cytosolic DBT. The results show that BDBT is necessary for co-transport of DBT and PER into the nucleus and suggest that this process is regulated by a light-dependent mechanism.

17.1 Habitual Heavy Alcohol Drinking in Healthy Adults Is Associated With Reduced Circadian Photoreceptor Responsivity to Light

Habitual alcohol consumption and circadian timing are connected. Studies have found that heavy alcohol use is associated with eveningness. Only 2 studies have assessed dim light melatonin onset (DLMO) in the context of habitual alcohol use, and both reported that a shorter DLMO-midsleep interval was associated with heavier alcohol use. A gap in this research is the potential impact of alcohol use on the circadian photoreceptor and the intrinsically photosensitive retinal ganglion cells, which influence circadian timing.

Circadian photoreceptors are required for light-dependent maintenance of long-term memory in Drosophila

In the fruit fly Drosophila melanogaster, environmental light is required for maintaining long-term memory (LTM). Furthermore, the Pigment dispersing factor (Pdf), which is a circadian neuropeptide, and the neuronal activity of Pdf neurons are essential for light-dependent maintenance of courtship LTM. Since Pdf neurons can sense light directly via circadian photoreceptors [Rhodopsin 7 (Rh7) and Cryptochrome (Cry)], it is possible that Rh7 and Cry in Pdf neurons are involved in the maintenance of LTM. In this study, using a courtship conditioning assay, we demonstrated that circadian photoreceptors in Pdf neurons are required for maintaining courtship LTM.

Room-temperature serial synchrotron crystallography of Drosophila cryptochrome.

Fixed-target serial crystallography allows the high-throughput collection of diffraction data from small crystals at room temperature. This methodology is particularly useful for difficult samples that have sensitivity to radiation damage or intolerance to cryoprotection measures; fixed-target methods also have the added benefit of low sample consumption. Here, this method is applied to the structure determination of the circadian photoreceptor cryptochrome (CRY), previous structures of which have been determined at cryogenic temperature. In determining the structure, several data-filtering strategies were tested for combining observations from the hundreds of crystals that contributed to the final data set. Removing data sets based on the average correlation coefficient among equivalent reflection intensities between a given data set and all others was most effective at improving the data quality and maintaining overall completeness. CRYs are light sensors that undergo conformational photoactivation. Comparisons between the cryogenic and room-temperature CRY structures reveal regions of enhanced mobility at room temperature in loops that have functional importance within the CRY family of proteins. The B factors of the room-temperature structure correlate well with those predicted from molecular-dynamics simulations.

Constant Light, Pdp1, and Tim Exert Influence on Free-Running Period of Locomotor Rhythms in the Cricket Gryllus bimaculatus.

Most insects show circadian rhythms of which the free-running period changes in a light-dependent manner and is generally longer under constant light (LL) than under constant dark conditions in nocturnal animals. However, the mechanism underlying this LL-dependent period change remains unclear. Here, using the cricket Gryllus bimaculatus, we examined the effects of long-term LL exposure on the free-running period of locomotor rhythms. Initially, the free-running period was considerably longer than 24 h but it gradually became shorter during long-term exposure to LL. The initial lengthening and ensuing gradual shortening under long-term LL exposure were observed even after unilateral removal of the optic lobe. Thus, these changes in the free-running period could be attributable to a single optic lobe clock. RNA interference (RNAi)-mediated silencing of the clock genes Par domain protein 1 (Pdp1) and timeless (tim) revealed that the treatments eliminated the initial period lengthening by LL without reducing circadian photoreceptor gene expression. However, they did not affect the period shortening during long-term LL exposure. The slopes of the regression line for the period change during long-term LL for Pdp1RNAi-treated and timRNAi-treated crickets were not different from that of the dsDsRed2-treated control. These results suggest that the initial period lengthening after transfer to LL requires tim and Pdp1, while the ensuing period shortening during long-term LL exposure is caused by a mechanism independent of tim and Pdp1.

Sleep and circadian differences between light and heavy adult alcohol drinkers.

BackgroundNumerous studies have reported that eveningness is associated with increased alcohol consumption. However, biological markers of circadian timing, such as dim light melatonin onset (DLMO) and circadian photoreceptor responsivity (post‐illumination pupil response, PIPR), have rarely been assessed in the context of habitual alcohol consumption. This study aimed to examine sleep, circadian timing, and photoreceptor responsivity in adult alcohol drinkers.MethodsParticipants (21 to 45 years) included 28 light and 50 heavy drinkers. The 8‐day study consisted of a week of ad lib sleep monitored with wrist actigraphy, followed by a 9‐h laboratory session with a photoreceptor responsivity and circadian phase assessment.ResultsThe heavy drinkers obtained on average 28 more minutes of sleep (p = 0.002) and reported more eveningness than the light drinkers (p = 0.029). There was a trend for a shorter DLMO‐midsleep interval (p = 0.059) in the heavy drinkers, reflecting a tendency for them to sleep at an earlier circadian phase. The PIPR in the heavy drinkers was significantly smaller than in the light drinkers (p = 0.032), suggesting reduced circadian photoreceptor responsivity in the heavy drinkers. A larger PIPR was significantly associated with a later DLMO in the light drinkers (r = 0.44, p = 0.019), but this relationship was absent in the heavy drinkers (r = −0.01, p = 0.94).ConclusionsThese results are consistent with earlier reports of more eveningness and a shorter DLMO‐midsleep interval being associated with heavier alcohol drinking. The novel finding of reduced circadian photoreceptor responsivity in heavy drinkers is consistent with prior rodent studies. Future studies should explore the impact of habitual alcohol consumption on other measures of circadian photoreceptor responsivity.

How Does Light Affect the Body’s Internal Clock?

Have you ever wondered why you are active during the day and sleepy at night? Why are some animals like owls and mice the opposite? A big reason for this is called circadian photoreception, which is the way we synchronize the body’s internal, 24-h clock to the light cycle outside. This process starts in the eye. In this article, we discuss the characteristics of circadian photoreception and the scientists that helped us understand this process. We explore the key parts involved in circadian photoreception: photopigments, which are the pigments in the eye that change when they absorb light and are each found in their own cells. Last, we examine ganglion cells, which are special cells in the eye that send information to the brain.

Morning light treatment for traumatic stress: The role of amygdala reactivity study protocol.

BackgroundExposure to trauma can result in various mental health disorders including anxiety, depression, and posttraumatic stress disorder (PTSD). Although psychotherapies and pharmacotherapies exist for the treatment of these disorders, many individuals fail to receive treatment and among those who do, many remain symptomatic. Therefore, it is critical to continue developing new interventions for traumatic stress that target underlying mechanisms of pathology and offer a safe and acceptable alternative to current treatments. Morning light treatment has good potential as a novel non-invasive, low risk treatment for traumatic stress. Evidence suggests that morning light may improve traumatic stress by reducing reactivity in the amygdala, a brain region implicated in the pathophysiology of PTSD and anatomically linked to circadian photoreceptors in the eye.MethodsIn this study, we aim to establish a significant dose-response relationship between duration of morning light treatment and reduction in amygdala reactivity among individuals with traumatic stress symptoms (NCT# 04117347). Using a transdiagnostic approach, sixty-six individuals with a history of a DSM-5 criterion A trauma and traumatic stress symptoms will be recruited to participate in a 5-week study. Participants will be randomized across three treatment arms based on morning light treatment duration: 15-minutes, 30-minutes, or 60-minutes of light treatment per day for four weeks. To evaluate amygdala activity, participants will undergo fMRI at pre-treatment, mid-treatment, and post-treatment. Participants will also complete clinical assessments and self-report measures of PTSD, depression, and anxiety at pre-treatment, mid-treatment, and post-treatment.DiscussionMorning light therapy may be an acceptable, feasible, and effective treatment for individuals suffering from traumatic stress. Identifying mechanistically relevant targets, and the doses needed to impact them, are critical steps in developing this new treatment approach for the sequelae of traumatic stress.

0212 Habitual Heavy Alcohol Drinking in Healthy Adults is Associated With Reduced Circadian Photoreceptor Responsivity to Light

Abstract Introduction Habitual alcohol consumption and circadian timing are interconnected. Numerous studies have reported that heavy alcohol use is associated with eveningness. Only two studies have assessed the dim light melatonin onset (DLMO) in the context of habitual alcohol use, and both reported a shorter DLMO-midsleep interval was associated with heavier alcohol consumption. A gap in this research is the potential impact of alcohol use on the primary circadian photoreceptor, the intrinsically photosensitive retinal ganglion cells (ipRGCs), which influence circadian timing. This study aimed to compare sleep, circadian timing, and photoreceptor responsivity between light and heavy alcohol drinkers. Methods Participants were healthy (average age 27 years) and included 28 light (average 2.6 drinks/week) and 50 heavy (average 17.9 drinks/week) drinkers. They participated in an 8-day study protocol: 1 week of adlib sleep monitored with wrist actigraphy, followed by a 9-hour laboratory session with a photoreceptor responsivity (post-illumination pupil response, PIPR) and circadian phase assessment. Participants passed a breathalyzer test at the start of the laboratory session. Results Both drinking groups were good sleepers with minimal mood disturbance. Consistent with earlier studies, the heavy drinkers had more eveningness (p=0.029), and a trend for a shorter DLMO-midsleep interval (p=0.059) as compared to the light drinkers. The PIPR in the heavy drinkers was significantly smaller than in the light drinkers (p=0.006), suggesting reduced circadian photoreceptor responsivity in the heavy drinkers. In the light drinkers, a larger PIPR in the evening, reflecting greater sensitivity to phase-delaying light, was significantly associated with a later DLMO (r=0.42, p=0.028). However, this relationship was absent in the heavy drinkers (r=-0.07, p=0.68). Conclusion The findings replicate previous work examining the relationship between chronotype, DLMO, and habitual alcohol consumption. For the first time, circadian photoreceptor responsivity was examined, and found to be significantly reduced in heavy alcohol drinkers as compared to light alcohol drinkers. This finding is consistent with prior rodent studies that found reduced phase shifts to light with acute or chronic alcohol consumption, and suggests that habitual heavy alcohol use may impair the circadian response to light in humans. Support (If Any) Grant awarded from NIAAA R01 AA023839.

Nucleotide Variation in Drosophila cryptochrome Is Linked to Circadian Clock Function: An Association Analysis.

Cryptochrome (CRY) is a conserved protein associated with the circadian clock in a broad range of organisms, including plants, insects, and mammals. In Drosophila, cry is a pleiotropic gene that encodes a blue light-dedicated circadian photoreceptor, as well as an electromagnetic field sensor and a geotaxis behavior regulator. We have generated a panel of nearly-isogenic strains that originated from various wild populations and which carry different natural alleles of cry. Sequencing of these alleles revealed substantial polymorphism, the functional role of which was elusive. To link this natural molecular diversity to gene function, we relied on association mapping. Such analysis revealed two major haplogroups consisting of six linked nucleotides associated with circadian phase (haplotypes All1/All2). We also generated a maximum-likelihood gene-tree that uncovered an additional pair of haplogroups (B1/B2). Behavioral analysis of the different haplotypes indicated significant effect on circadian phase and period, as well on the amount of activity and sleep. The data also suggested substantial epistasis between the All and B haplogroups. Intriguingly, circadian photosensitivity, assessed by light-pulse experiments, did not differ between the genotypes. Using CRISPR-mediated transgenic flies, we verified the effect of B1/B2 polymorphism on circadian phase. The transgenic flies also exhibited substantially different levels of cry transcription. We, moreover, analyzed the geographical distribution of the B1/B2 haplotypes, focusing on a 12 bp insertion/deletion polymorphism that differentiates the two haplotypes. Analysis of cry sequences in wild populations across Europe revealed a geographical cline of B1/B2 indel frequency, which correlated with seasonal bioclimatic variables. This spatial distribution of cry polymorphism reinforces the functional importance of these haplotypes in the circadian system and local adaptation.

Light-dependent reactions of animal circadian photoreceptor cryptochrome.

Circadian rhythms are endogenous autonomous 24-h oscillations that are generated by a transcription-translation feedback loop (TTFL). In the positive arm of the TTFL, two transcription factors activate the expression of two genes of the negative arm as well as circadian clock-regulated genes. The circadian clocks are reset through photoreceptor proteins by sunlight in the early morning to keep synchrony with the geological clock. Among animal circadian photoreceptors, Drosophila Cryptochrome (DmCRY) has some unique properties because Drosophila has a single cryptochrome (CRY) that appears to have functions which are specific to organs or tissues, or even to a subset of cells. In mammals, CRYs are not photoreceptors but function in the TTFL, while insects have a light-insensitive mammalian-like CRY or a Drosophila-like photoreceptor CRY (or both). Here, we postulate that as being just one CRY in Drosophila, DmCRY might play different roles in different tissues/organs in a context-dependent manner. In addition to being a circadian photoreceptor/protein, attributing also a magnetoreception function to DmCRY has increased its workload. Considering that DmCRY senses photons as a photoreceptor but also can regulate many different events in a light-dependent manner, differential protein-protein interactions (PPIs) of DmCRY might play a critical role in the generation of such diverse outputs. Therefore, we need to add novel approaches in addition to the current ones to study multiple and context-dependent functions of DmCRY by adopting recently developed techniques. Successful identification of transient/fast PPIs on a scale of minutes would enhance our understanding of light-dependent and/or magnetoreception-associated reactions.