Ongoing Projects

Molecular Dynamics of Entrainment

To study molecular and network aspects of SCN entrainment in real time we developed a temporally precise entrainment system for the ex vivo SCN by building on our previous work with SCN optogenetics and circadian reporters which we call EX vivo CIrcadian Timing and Entrainment (EXCITE). We switched to red-light optogenetic stimulation with ChrimsonR to minimize the potential for photodynamic damage and use SCN tissue from younger animals (P11-P14), as well as optimized culture media to greatly extend the viability of cultures. EXCITE can be used for bioluminescence photometry of the whole SCN (Lumicycle), and for single cell and SCN network bioluminescence imaging (Olympus LV200).

EXCITE ex vivo entrainment mimics in vivo light input by using optogenetic stimulation to directly induce SCN neuron membrane depolarization and increase spike rate and Ca++ influx. EXCITE demonstrates that light-like phase shifts and entrainment of the ex vivo SCN clock involve striking changes in the waveform of the negative feedback arm of the TTFL, and regional acute vs plastic responses, as read out by the circadian waveform and timing of a PERIOD2 luciferase chimeric protein (PER2::LUC).

Summary of main results with EXCITE. Top, regionalization of phase shifts and period response. Bottom, waveform changes in the molecular clockworks under entrainment. Graphic created by Suil Kim.

Honey Bee Clock Entrainment

The remarkable time memory (Zeitgedachtnis) of honey bees (Apis mellifera) was one of the first lines of evidence for the importance of circadian clocks in animal behavior. Using their circadian clocks as a time base, honey bees synchronize their foraging with daily rhythms in flowering. Furthermore, forager bees use their circadian clocks for time-compensated sun-compass orientation and waggle dance communication of navigation to food resources to hive-mates. Honey bee circadian rhythms are also important for social organization of the honey bee colony, and honey bees exhibit strong social entrainment. Despite its remarkable impact on bee behavior and the foundation role in circadian rhythms research the functional organization of the honey bee brain circadian clock is still not well defined. Intriguingly, the honey bee clock gene network uniquely combines molecular characteristics of both the fruit fly and mouse clockworks and virtually nothing is known about its mechanisms of entrainment. The honey bee lacks two genetic components from the Drosophila clockworks that critically mediate light entrainment,

Timeless1 and the light-sensitive cryptochrome Cry-d. Instead, honey bees express a non-light sensitive mammalian-type Cry- m cryptochrome (amCry). In addition, as in mammals, the honey bee Cycle gene (amCyc, a homolog of mammalian Bmal1), forms a secondary rhythmic molecular loop in the clockworks. The remarkable molecular genetic variation of the honey bee clock presents a unique opportunity to reveal fundamental principles of molecular clock entrainment – how is this unique clock entrained?

Molecular Genetic Mechanisms of Photoperiod-Induced Plasticity

Circadian entrainment theory predicts that plasticity in light-induced resetting is critical to stable photoperiodic entrainment. What then are the critical molecular genetic mechanisms of the clock photoperiod response? To reveal additional molecular mechanisms of SCN photoperiod plasticity it is important to comprehensively define what are the changes in the circadian transcriptome of the SCN following photoperiod entrainment and to gain insight into how evolutionary adaptation to highly photoperiodic environments has shaped circadian genes. We have found striking changes in the expression of key SCN light signaling and neuropeptide signaling genes across photoperiods (preprint), and evidence for adaptive changes in human clock genes through Neanderthal genetic introgression as compelling candidates for molecular genetic mechanisms for photoperiod plasticity. We will test with the EXCITE platform the roles of specific mouse and human clock associated genes in plasticity and photoperiod entrainment.

Convergent Effects of Light and Cholinergic Agonists on Honeybee Circadian Rhythms

Our work on light entrainment and honeybee circadian neurobiology was initially stimulated by a topical question: Does circadian disruption play a role in the decline in bee populations sometimes characterized as colony collapse disorder (CCD)? Neonicotinoid pesticides are potent agonists of nicotinic acetylcholine receptors throughout the insect nervous system. In the fruit fly there are nicotinic cholinergic inputs to the clock neurons from light sensing organs, and thus we hypothesized that non-lethal doses of neonicotinoids may disrupt the bee clock by abnormal stimulation of clock light input pathways. We indeed found that neonicotinoids and light exposure have convergent effects on the honeybee circadian behavior that alter entrainment and can lead to complete disruption of behavioral rhythms.

To extend this analysis to the cellular and molecular level we have undertaken the development of novel circadian research tools for the honey bee, genetic markers and reporters of honey bee clock neurons. The neurohormone Pigment dispersing factor (Pdf) is a marker of clock neurons in the honey bee as well as Drosophila and many other insects. As proof of principle, we have cloned the honey bee Pdf promoter and constructed a GFP reporter plasmid which we have successfully validated in cultured bee nuerons to mark Pdf-expressing neurons. We will use this to study the mechanisms of entrainment.

Sex-Dependent Effects of Photoperiod on Mood and Motivated Behaviors

A fundamental question in neurobiology is how environmental signals – both developmental and ongoing– induce plasticity in neural circuits and networks to shape behavior. Circadian photoperiod, the proportion of daylight in a solar day, is a pervasive environmental signal that varies substantially with latitude and season, and drives acute and long- term effects on mood regulation in humans and in animal models. The associations of the molecular circadian clock and photoperiod with mood disorders are clear, but the neurobiological mechanisms remain incompletely understood.

We have recently found intriguing sex-dependent photoperiodic regulation of dopamine uptake and release downstream in the NAc of female mice, indicating photoperiodic impact on circuitry for motivation and reward that mirrors the reported female bias of Seasonal Affective Disorder (SAD) in humans.

In a collaborative project with Brad Grueter of VUMC we are investigating further the mechanistic basis of photoperiodic programming of 5-HT neurons involving TREK-1, and downstream effects of this programming on positive valence systems, including NAc dopamine release and uptake, NAc synaptic plasticity, and NAc- driven motivation behavior to enhance understanding of key neurobiological mechanisms underlying photoperiodic regulation of mood, motivation, and reinforcement.

Human Genetic Evolution of Entrainment and Photoperiod Adaptation

When the ancestors of modern Eurasians migrated out of Africa and interbred with Eurasian archaic hominins - Neanderthals and Denisovans - DNA of archaic ancestry integrated into the genomes of anatomically modern humans. This process potentially accelerated adaptation to Eurasian environmental factors, including increased variation in seasonal light dynamics and entrainment. In a recent collaborative study with Prof. Tony Capra, we tested for genetic differences in circadian genes and their regulatory elements between humans and Neanderthals and found that introgressed archaic alleles are enriched for effects on circadian gene regulation, consistently increase the propensity for morningness chronotype, and show latitudinal clines similar to clock gene variants in fruit flies. These results expand our understanding of how the genomes of humans and our closest relatives responded to environments with different light/dark cycles and demonstrate a contribution of admixture to human chronotype that is consistent with adaptation to higher latitudes. We are extending this collaboration to test the function of archaic alleles on human cellular rhythms and entrainment using EXCITE.

Check out the Washington Post article written about this research!