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The chi-square periodogram (CSP), developed over 40 years ago, continues to be one of the most popular methods to estimate the period of circadian (circa 24-h) rhythms. Previous work has indicated the CSP is sometimes less accurate than other methods, but understanding of why and under what conditions remains incomplete. Using simulated rhythmic time-courses, we found that the CSP is prone to underestimating the period in a manner that depends on the true period and the length of the time-course. This underestimation bias is most severe in short time-courses (e.g., 3 days), but is also visible in longer simulated time-courses (e.g., 12 days) and in experimental time-courses of mouse wheel-running and ex vivo bioluminescence. We traced the source of the bias to discontinuities in the periodogram that are related to the number of time-points the CSP uses to calculate the observed variance for a given test period. By revising the calculation to avoid discontinuities, we developed a new version, the greedy CSP, that shows reduced bias and improved accuracy. Nonetheless, even the greedy CSP tended to be less accurate on our simulated time-courses than an alternative method, namely the Lomb-Scargle periodogram. Thus, although our study describes a major improvement to a classic method, it also suggests that users should generally avoid the CSP when estimating the period of biological rhythms.

Using optogenetic manipulations and bioluminescence imaging of suprachiasmatic nucleus (SCN) firing rate, this study examines the interaction between molecular, electrical and behavioral circadian rhythms in mice. The study shows that alteration of clock neuron firing can reset molecular and behavioral circadian rhythms, and this effect required neuronal network interaction within the SCN. Thus, clock neuron spiking is fundamental to circadian pacemaking as both an input to and output of the neuronal network responsible for circadian behavior. To examine the interaction between molecular, electrical and behavioral circadian rhythms, we combined optogenetic manipulation of suprachiasmatic nucleus (SCN) firing rate with bioluminescence imaging and locomotor activity monitoring. Manipulating firing rate reset circadian rhythms both ex vivo and in vivo , and this resetting required spikes and network communication. This suggests that SCN firing rate is fundamental to circadian pacemaking as both an input to and output of the molecular clockworks.

Honey bees are critical pollinators in ecosystems and agriculture, but their numbers have significantly declined. Declines in pollinator populations are thought to be due to multiple factors including habitat loss, climate change, increased vulnerability to disease and parasites, and pesticide use. Neonicotinoid pesticides are agonists of insect nicotinic cholinergic receptors, and sub-lethal exposures are linked to reduced honey bee hive survival. Honey bees are highly dependent on circadian clocks to regulate critical behaviors, such as foraging orientation and navigation, time-memory for food sources, sleep, and learning/memory processes. Because circadian clock neurons in insects receive light input through cholinergic signaling we tested for effects of neonicotinoids on honey bee circadian rhythms and sleep. Neonicotinoid ingestion by feeding over several days results in neonicotinoid accumulation in the bee brain, disrupts circadian rhythmicity in many individual bees, shifts the timing of behavioral circadian rhythms in bees that remain rhythmic, and impairs sleep. Neonicotinoids and light input act synergistically to disrupt bee circadian behavior, and neonicotinoids directly stimulate wake-promoting clock neurons in the fruit fly brain. Neonicotinoids disrupt honey bee circadian rhythms and sleep, likely by aberrant stimulation of clock neurons, to potentially impair honey bee navigation, time-memory, and social communication.

Though the seasonal response of organisms to changing day lengths is a phenomenon that has been scientifically reported for nearly a century, significant questions remain about how photoperiod is encoded and effected neurobiologically. In mammals, early work identified the master circadian clock, the suprachiasmatic nuclei (SCN), as a tentative encoder of photoperiodic information. Here, we provide an overview of research on the SCN as a coordinator of photoperiodic responses, the intercellular coupling changes that accompany that coordination, as well as the SCN’s role in a putative brain network controlling photoperiodic input and output. Lastly, we discuss the importance of photoperiodic research in the context of tangible benefits to human health that have been realized through this research as well as challenges that remain.

Nuclear receptor corepressors (NCoRs) function in multiprotein complexes containing histone deacetylase 3 (HDAC3) to alter transcriptional output primarily through repressive chromatin remodelling at target loci 1 – 5 . In the liver, loss of HDAC3 causes a marked hepatosteatosis largely because of de-repression of genes involved in lipid metabolism 6 , 7 ; however, the individual roles and contribution of other complex members to hepatic and systemic metabolic regulation are unclear. Here we show that adult loss of both NCoR1 and NCoR2 (double knockout (KO)) in hepatocytes phenocopied the hepatomegalic fatty liver phenotype of HDAC3 KO. In addition, double KO livers exhibited a dramatic reduction in glycogen storage and gluconeogenic gene expression that was not observed with hepatic KO of individual NCoRs or HDAC3, resulting in profound fasting hypoglycaemia. This surprising HDAC3-independent activation function of NCoR1 and NCoR2 is due to an unexpected loss of chromatin accessibility on deletion of NCoRs that prevented glucocorticoid receptor binding and stimulatory effect on gluconeogenic genes. These studies reveal an unanticipated, non-canonical activation function of NCoRs that is required for metabolic health. Hauck et al. show that during fasting, nuclear receptor corepressors 1 and 2 act together to activate the transcription of target genes, which is critical for the physiological response to fasting in mice.

The biological response to changes in seasonal light has been the subject of scientific interest for nearly 100 years. Over time, the significant role of the circadian pacemaker in mediating the mammalian response to seasonal light changes has become clearer. Despite knowledge of a role in transducing this light signal into a physiological response, the exact mechanism of that transduction and the specific components of the light signal that induce those changes have yet to be fully defined. In this dissertation, the distinct components of seasonal light (duration, timing, onset-offset interval) are examined for their ability to induce persistent changes in two circadian behavioral properties implicated in seasonal responses: locomotor activity duration and free-running period. We find that the onset-offset interval is critical for determining activity duration, while net phase shift direction and light interval are critical for determining period. Further, we find that a change to phase distribution underlies changes to activity duration but not to free-running period. To interrogate the cellular mechanism of these changes, the role of a particular set of circadian clock neurons, the VIPergic neurons of the SCN, in inducing proximal changes in activity duration is examined. We find that VIPergic neuron activation by optogenetics induces proximal changes in activity duration. Lastly, a set of techniques for the assessment of relevant circadian properties both in vivo and ex vivo are presented. These include a more precise and accurate period-independent method to measure phase shifts, a higher-throughput computational approach to calculating activity duration, and a strategy for quantifying phase dispersal across bioluminescent SCN cultures. The results and techniques presented in this dissertation represent an advance in our understanding of the underlying induction of seasonal responses (both in cues and in cellular mechanism) as well as a step forward in the accurate quantification of relevant properties.

Abstract The chi-square periodogram (CSP), developed over 40 years ago, continues to be one of the most popular methods to estimate the period of circadian (circa 24-h) rhythms. Previous work has indicated the CSP is sometimes less accurate than other methods, but understanding of why and under what conditions remains incomplete. Using simulated rhythmic time-courses, we found that the CSP is prone to underestimating the period in a manner that depends on the true period and the length of the time-course. This underestimation bias is most severe in short time-courses (e.g., 3 days), but is also visible in longer simulated time-courses (e.g., 12 days) and in experimental time-courses of mouse wheel-running and ex vivo bioluminescence. We traced the source of the bias to discontinuities in the periodogram that are related to the number of time-points the CSP uses to calculate the observed variance for a given test period. By revising the calculation to avoid discontinuities, we developed a new version, the greedy CSP, that shows reduced bias and improved accuracy. Nonetheless, even the greedy CSP tended to be less accurate on our simulated time-courses than an alternative method, namely the Lomb-Scargle periodogram. Thus, although our study describes a major improvement to a classic method, it also suggests that users should generally avoid the CSP when estimating the period of biological rhythms. Competing Interest Statement The authors have declared no competing interest. Footnotes * The major additions are: - analysis of simulated time-courses with lower temporal resolution - analysis of ex vivo PER2::LUCIFERASE SCN recordings - analysis of simulated time-courses with Poisson distributions of noise - plotted summary statistics for several figures - mathematical formula and explanation in the Methods Overall, these revisions strengthen our findings, particularly in demonstrating that the issues with the chi-square periodogram are not specific to sampling interval or data type. * https://doi.org/10.6084/m9.figshare.12805082 * https://spectr.hugheylab.org

Summary Circadian clocks play key roles in how organisms respond to and even anticipate seasonal change in day length, or photoperiod. In mammals, photoperiod is encoded by the central circadian pacemaker in the brain, the suprachiasmatic nucleus (SCN). The subpopulation of SCN neurons that secrete the neuropeptide VIP mediate the transmission of light information within the SCN neural network, suggesting a role for these neurons in circadian plasticity in response to light information that has yet to be directly tested. Here, we used in vivo optogenetic stimulation of VIPergic SCN neurons followed by ex vivo PERIOD 2::LUCIFERASE (PER2::LUC) bioluminescent imaging to test whether activation of this SCN neuron sub-population can induce SCN network changes that are hallmarks of photoperiodic encoding. We found that optogenetic stimulation designed to mimic a long photoperiod indeed altered subsequent SCN entrained phase, increased the phase dispersal of PER2 rhythms within the SCN network, and shortened SCN free-running period – similar to the effects of a true extension of photoperiod. Optogenetic stimulation also induced analogous changes on related aspects of locomotor behavior in vivo. Thus, selective activation of VIPergic SCN neurons induces photoperiodic network plasticity in the SCN which underpins photoperiodic entrainment of behavior. Competing Interest Statement The authors have declared no competing interest.

Honey bees are critical pollinators in ecosystems and agriculture, but their numbers have significantly declined. Declines in pollinator populations are thought to be due to multiple factors including habitat loss, climate change, increased vulnerability to disease and parasites, and pesticide use. Neonicotinoid pesticides are agonists of insect nicotinic cholinergic receptors, and sub-lethal exposures are linked to reduced honey bee hive survival. Honey bees are highly dependent on circadian clocks to regulate critical behaviors, such as foraging orientation and navigation, time-memory for food sources, sleep, and learning/ memory processes. Because circadian clock neurons in insects receive light input through cholinergic signaling we tested for effects of neonicotinoids on honey bee circadian rhythms and sleep. Neonicotinoid ingestion by feeding over several days results in neonicotinoid accumulation in the bee brain, disrupts circadian rhythmicity in many individual bees, shifts the timing of behavioral circadian rhythms in bees that remain rhythmic, and impairs sleep. Neonicotinoids and light input act synergistically to disrupt bee circadian behavior, and neonicotinoids directly stimulate wake-promoting clock neurons in the fruit fly brain. Neonicotinoids disrupt honey bee circadian rhythms and sleep, likely by aberrant stimulation of clock neurons, to potentially impair honey bee navigation, time-memory, and social communication. Competing Interest Statement The authors have declared no competing interest.

Seasonal light cycles influence multiple physiological functions and are mediated through photoperiodic encoding by the circadian system. Despite our knowledge of the strong connection between seasonal light input and downstream circadian changes, less is known about the specific components of seasonal light cycles that are encoded and induce persistent changes in the circadian system. Using combinations of three T cycles (23, 24, 26 hr.) and two photoperiods per T cycle (Long and Short, with duty cycles scaled to each T cycle), we investigate after-effects of entrainment to these six light cycles on locomotor behavior duration (α), period (τ), and entrained phase angle (ψ) in vivo, and SCN phase distribution (σϕ), τ, and ψ ex vivo in order to refine our understanding of critical light components for influencing particular circadian properties. We find that photoperiod and T cycle length both drive determination of in vivo ψ but differentially influence after-effects in α and τ, with photoperiod driving changes in α and photoperiod length and T cycle length combining to influence τ. Using skeleton photoperiods, we demonstrate that in vivo ψ is determined by both parametric and non-parametric components, while changes in α are driven non-parametrically. Within the ex vivo SCN, we find that ψ and σϕ of the PER2∷LUCIFERASE rhythm follow closely with their likely behavioral counterparts (ψ and α of the locomotor activity rhythm), while also confirming previous reports of τ after-effects of gene expression rhythms showing negative correlations with behavioral τ after-effects in response to T cycles. We demonstrate that within-SCN σϕ changes, thought to underly α changes in vivo, are induced primarily non-parametrically. Taken together, our results demonstrate distinct components of seasonal light input differentially influence ψ, α, and τ, and suggest the possibility of separate mechanisms driving the persistent changes in circadian behaviors mediated by seasonal light. Competing Interest Statement The authors have declared no competing interest.