Ewer, J. | bioRxiv Science

bioRxiv2023

Timed receptor tyrosine kinase signaling couples the central and a peripheral circadian clock in Drosophila

Circadian clocks impose daily periodicities to behavior, physiology, and metabolism. This control is mediated by a central clock and by peripheral clocks, which are synchronized to provide the organism with a unified time through mechanisms that are not fully understood. Here, we characterized in Drosophila the cellular and molecular mechanisms involved in coupling the central clock and the peripheral clock located in the prothoracic gland (PG), which together control the circadian rhythm of emergence of adult flies. The time signal from central clock neurons is transmitted via small neuropeptide F (sNPF) to neurons that produce the neuropeptide Prothoracicotropic Hormone (PTTH), which is then translated into circadian oscillations of Ca2+ concentration and daily changes in PTTH levels. Rhythmic PTTH signaling is required at the end of metamorphosis, and transmits time information to the PG by imposing a daily rhythm to the expression of the PTTH receptor tyrosine kinase (RTK), TORSO, and of ERK phosphorylation, a key component of PTTH transduction. In addition to PTTH, we demonstrate that signaling mediated by other RTKs contribute to the rhythmicity of emergence. Interestingly, the ligand to one of these receptors (Pvf2), plays an autocrine role in the PG, which may explain why both central brain and PG clocks are required for the circadian gating of emergence. Our findings show that the coupling between the central and the PG clock is unexpectedly complex and involves several RTKs that act in concert, and could serve as a paradigm to understand how circadian clocks are coordinated. Significance statementCircadian clocks impose daily periodicities to behavior, physiology, and metabolism, and are synchronized to provide the organism with a unified time through mechanisms that are poorly understood. In holometabolous insects, the circadian control of adult emergence depends on the coupling between the central clock and a peripheral clock located in the prothoracic gland (PG). Here we identify the cellular and molecular mechanism that transmits time information from the central clock to the PG clock. This process is unexpectedly complex and involves a number of receptor tyrosine kinases (RTKs). Such a mechanism may add robustness to the coupling between the 2 clocks and serve as a paradigm for understanding how circadian clocks are coordinated.

neuroscience↗

bioRxiv2021

Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: application to Drosophila ecdysis behavior

Neuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. The interplay between the activity of neuronal circuits, their modulation by neuropeptides, and the resulting behavior, is still poorly understood. Here, we present a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. We analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. We found that the activity of both neuronal populations is tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the faster oscillation of motoneurons. To explore this idea further, we used a probabilistic logistic model to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-base model capable of simulating many of the observed neural dynamics features. Finally, we developed an algorithm to quantify the motor behavior observed in videos of pupal ecdysis, and compared their features to the patterns of neuronal calcium activity recorded ex vivo. We found that the motor activity of the intact animal is more regular than the motoneuronal activity recorded from the ex vivo preparations during fictive ecdysis behavior; the analysis of movement patterns also allowed us to identify a new post-ecdysis phase. Author SummaryRepetitive movements such as walking, swimming, and flying are controlled by networks of neurons known as central patter generators. In many cases, the exact pattern of activity is modulated by neuropeptides, which are small signaling molecules that, unlike neurotransmitters, are broadly released within regions of the nervous system. Because of this mode of action, it can be difficult to discern the relationship between the temporal pattern of firing of peptidergic neurons and the timing of the resulting motor behavior. Here, we developed methods to analyze the patterns of activity of such weakly coupled systems as applied to ecdysis, the stereotyped sequence of behaviors used by insects to shed the remains of their old exoskeleton at the end of every molt. Key actors in this process are motoneurons (MN) and a set of neurons expressing the neuropeptide, Crustacean Cardioactive Peptide (CCAP). Combining real-time calcium imaging, frequency analysis, computational simulations, and image processing, we determined the relationships between the activity of CCAP neurons and the resulting motor output during pupal ecdysis in the fruit fly, Drosophila melanogaster. We found that several temporal features of the activity of CCAP neurons are highly coupled to the patter of activity motoneuronal pattern, suggesting an active role of CCAP neurons during ecdysis. We also developed quantitative approaches that allowed us to identify a new ecdysis sub-phase.

neuroscience↗