The Interplay of Light and Melatonin in Biological Timekeeping: Conservation, Divergence, and Translation of the Drosophila Timekeeping Network

Abstract

The central pacemaker for biological rhythms in mammals is located in a 10,000-cell structure known as the suprachiasmatic nucleus (SCN) in the anterior hypothalamus (Hastings_2018, Meijer_1996). Clock cells in the suprachiasmatic nucleus work together to generate a coordinated and synchronous output that results in an intrinsic endogenous “ticking” of the biological clock at a set periodicity (Liu_1997). Circadian rhythms are generated by the daily fluctuation of a transcription-translation feedback loop between clock proteins and genes located in clock cells. Master pacemaker cells in the SCN project to peripheral pacemaker cells in various organs and tissues in the body. All biological rhythms oscillate at a set period known either as the intrinsic or endogenous period and is determined by the oscillatory speed of the transcription-translation feedback loop determined by the individual organism’s genetics (Jin_1999, Liu_1997). The endogenous period is not typically aligned optimally with the external 24-hour period of the earth’s daily rotation about its axis. The amount of time it takes for an animal to return to the same phase of its endogenous rhythm without an external natural or artificial cycle to align to is known as the free running period. Mammals do not have endogenous periods that cycle at exactly 24-hours but generally have free running periods ranging between 22 and 26 hours, with diurnal mammals typically exhibiting free running periods above 24-hours (Aschoff_1981). The difference in cycling between the endogenous period and the external 24-hour photoperiod (the period of light and dark denoting one complete rotation of the earth about its axis), eventually results in a drift and subsequent misalignment of important behaviors, which lead to negative and in some cases fatal health outcomes for living organisms (Aschoff_1981, Lewy_2009). In order to prevent misalignment and optimally time behaviors to the external photoperiod, the biological clock evolved a process known as entrainment. Entrainment of biological rhythms to an external zeitgeber is achieved through advancing or delaying internal rhythms based on the animal’s previous photic history, so that they optimally align with the 24-hour external period (Julius_2019). The first chapter in this section will cover the anatomy and circuitry of the SCN, mechanisms of the mammalian molecular and biological clock, and explore the mechanisms of entrainment for optimally phase aligning biological rhythms to a 24-hour photoperiod. In the second chapter, I will describe the Drosophila biological time keeping network, discuss what mechanisms and functions are conserved or diverge between mammals and Drosophila, and where possible translational overlap between mammals and Drosophila can occur to further our understanding of entrainment of circadian and seasonal rhythms. The final chapter in this section will present published data on a comprehensive seasonal phase response curve (PRC) atlas of phase shifts in the activity rhythms of Drosophila ananassae under different seasonal photoperiods. Timing behavior and physiology to the 24-hour solar cycle has enabled life to persist and thrive on Earth. The most powerful time cue, light, is used for aligning endogenous rhythms to the day and night cycle; however, the clock also heavily relies on another internal rhythm, melatonin secretion, to help adjust circadian but primarily seasonal rhythms cooperatively with light input (Aschoff_1978, Ardent_2009). Melatonin and light work synergistically to entrain endogenous rhythms to the external photoperiod set by the solar cycle (Wood_2013, Wehr_1997). Together, both light and melatonin optimize interconnecting suites of behavior and physiology so that they are expressed during the most conducive parts of the day-and-year to enhance biological fitness. Regarding circadian/seasonal function, it is impossible to talk about melatonin without also understanding (1) the control that ambient light exerts over its secretion and (2) how melatonin feeding back onto the clock can work to adjust oscillatory behavior the clock makes in response to light exposure, especially during the subjective night.Melatonin functions as an important biological imprint of night-length by supplementing daytime light information collected by the brain’s circadian pacemaker. The suprachiasmatic nucleus (SCN) acts as the mammalian biological clock; and the Lateral and Dorsal Nuclei (LN and DN respectively) comprise the Drosophila clock network (Taghert_2006, Ardent_2009). Melatonin has its primordial role as an antioxidant that directly and indirectly – through metabolite cascades – sequesters free-radicals to help prevent oxidative stress (Zhao_2014, Manchester_2015). Melatonin freely circulates throughout the body and passes through lipid membranes and the blood brain barrier (Pardridge_1980, Costa_1995). These two features contribute heavily to its potency as an antioxidant in both the plant and animal kingdoms. Timing of melatonin secretion is important to optimize its antioxidant properties and to phase-lock various biological behaviors to occur at the optimal phase of the day-night cycle. Its enhanced secretion at night tracks closely to periods of high oxidative stress that occur during the day when solar UV radiation and system energy demand are at their highest (Haldar_2010). This is thought to keep oxidative processes to a minimum during the day and time the bulk of them to occur during the evening when there is less demand on the system from UV-radiation and environmental conditions. The history of melatonin’s functionality, from just an antioxidant in simpler species to evolving more of a modulatory role for circadian and seasonal behaviors in higher invertebrates, avian species, and mammals, is an interesting one. It stemmed from melatonin’s ability to orchestrate and participate directly in restorative processes that work best in the absence of light (such as DNA methylation and sequestration of free radicals) (Zhao_2019). More of its evolutionary history will be discussed later, but it is interesting to note that melatonin concentration is highest in mitochondria, where many reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated during the citric acid cycle. Finally, data will be presented on a comprehensive seasonal study on melatonin secretion in Drosophila ananassae across different seasonal photoperiods. This data was collected in an attempt to draw a phylogenetic line between where melatonin took on its circadian and seasonal role and where it acts solely as an antioxidant. This data also sought to understand what, if any, circadian and seasonal properties of melatonin are conserved between the Drosophila and mammalian systems. Using the gold standard of melatonin measurement, mass spectrometry (Markey_1981), and over 18,000 animals, we measured melatonin secretion in 4 photoperiods from high summer (16:8), equatorial (12:12), and high winter (8:16) photoperiods. Animals were also exposed to an extreme arctic summer photoperiod (20:4) to evaluate the potential suppressive effects of light on melatonin secretion in this species in addition to a physiologically extreme photoperiod.