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 daily oscillations of Ca2+ concentration and PTTH levels. PTTH signaling is required at the end of metamorphosis and transmits time information to the PG through changes in 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 contributes 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.

Mutations in trpγ, the homologue of TRPC6 autism candidate gene, causes autism-like behavioral deficits in Drosophila.

Autism Spectrum Disorder (ASD) is characterized by impaired social communication, restricted interests, and repetitive and stereotyped behaviors. The TRPC6 (transient receptor potential channel 6) represents an ASD candidate gene under an oligogenic/multifactorial model based on the initial description and cellular characterization of an individual with ASD bearing a de novo heterozygous mutation disrupting TRPC6, together with the enrichment of disruptive TRPC6 variants in ASD cases as compared to controls. Here, we perform a clinical re-evaluation of the initial non-verbal patient, and also present eight newly reported individuals ascertained for ASD and bearing predicted loss-of-function mutations in TRPC6. In order to understand the consequences of mutations in TRPC6 on nervous system function, we used the fruit fly, Drosophila melanogaster, to show that null mutations in transient receptor gamma (trpγ; the fly gene most similar to TRPC6), cause a number of behavioral defects that mirror features seen in ASD patients, including deficits in social interactions (based on courtship behavior), impaired sleep homeostasis (without affecting the circadian control of sleep), hyperactivity in both young and old flies, and defects in learning and memory. Some defects, most notably in sleep, differed in severity between males and females and became normal with age. Interestingly, hyperforin, a TRPC6 agonist and the primary active component of the St. John's wort antidepressant, attenuated many of the deficits expressed by trpγ mutant flies. In summary, our results provide further evidence that the TRPC6 gene is a risk factor for ASD. In addition, they show that the behavioral defects caused by mutations in TRPC6 can be modeled in Drosophila, thereby establishing a paradigm to examine the impact of mutations in other candidate genes.

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 frequency of motoneuron firing. 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-based model capable of simulating many features of the observed neural dynamics. 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 ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed us to identify a new post-ecdysis phase.

The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis.

The daily rhythm of adult emergence of holometabolous insects is one of the first circadian rhythms to be studied. In these insects, the circadian clock imposes a daily pattern of emergence by allowing or stimulating eclosion during certain windows of time and inhibiting emergence during others, a process that has been described as "gating." Although the circadian rhythm of insect emergence provided many of the key concepts of chronobiology, little progress has been made in understanding the bases of the gating process itself, although the term "gating" suggests that it is separate from the developmental process of metamorphosis. Here, we follow the progression through the final stages of Drosophila adult development with single-animal resolution and show that the circadian clock imposes a daily rhythmicity to the pattern of emergence by controlling when the insect initiates the final steps of metamorphosis itself. Circadian rhythmicity of emergence depends on the coupling between the central clock located in the brain and a peripheral clock located in the prothoracic gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone. Here, we show that the clock exerts its action by regulating not the levels of ecdysone but that of its actions mediated by the ecdysone receptor. Our findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoids are produced in mammals, which result from the coupling between the central clock in the suprachiasmatic nucleus and a peripheral clock located in the suprarenal gland.

Reciprocal Relationship Between Calcium Signaling and Circadian Clocks: Implications for Calcium Homeostasis, Clock Function, and Therapeutics.

In animals, circadian clocks impose a daily rhythmicity to many behaviors and physiological processes. At the molecular level, circadian rhythms are driven by intracellular transcriptional/translational feedback loops (TTFL). Interestingly, emerging evidence indicates that they can also be modulated by multiple signaling pathways. Among these, Ca2+ signaling plays a key role in regulating the molecular rhythms of clock genes and of the resulting circadian behavior. In addition, the application of in vivo imaging approaches has revealed that Ca2+ is fundamental to the synchronization of the neuronal networks that make up circadian pacemakers. Conversely, the activity of circadian clocks may influence Ca2+ signaling. For instance, several genes that encode Ca2+ channels and Ca2+-binding proteins display a rhythmic expression, and a disruption of this cycling affects circadian function, underscoring their reciprocal relationship. Here, we review recent advances in our understanding of how Ca2+ signaling both modulates and is modulated by circadian clocks, focusing on the regulatory mechanisms described in Drosophila and mice. In particular, we examine findings related to the oscillations in intracellular Ca2+ levels in circadian pacemakers and how they are regulated by canonical clock genes, neuropeptides, and light stimuli. In addition, we discuss how Ca2+ rhythms and their associated signaling pathways modulate clock gene expression at the transcriptional and post-translational levels. We also review evidence based on transcriptomic analyzes that suggests that mammalian Ca2+ channels and transporters (e.g., ryanodine receptor, ip3r, serca, L- and T-type Ca2+ channels) as well as Ca2+-binding proteins (e.g., camk, cask, and calcineurin) show rhythmic expression in the central brain clock and in peripheral tissues such as the heart and skeletal muscles. Finally, we discuss how the discovery that Ca2+ signaling is regulated by the circadian clock could influence the efficacy of pharmacotherapy and the outcomes of clinical interventions.

The impact of the gut microbiome on memory and sleep in Drosophila.

The gut microbiome has been proposed to influence diverse behavioral traits of animals, although the experimental evidence is limited and often contradictory. Here, we made use of the tractability of Drosophila melanogaster for both behavioral analyses and microbiome studies to test how elimination of microorganisms affects a number of behavioral traits. Relative to conventional flies (i.e. with unaltered microbiome), microbiologically sterile (axenic) flies displayed a moderate reduction in memory performance in olfactory appetitive conditioning and courtship assays. The microbiological status of the flies had a small or no effect on anxiety-like behavior (centrophobism) or circadian rhythmicity of locomotor activity, but axenic flies tended to sleep for longer and displayed reduced sleep rebound after sleep deprivation. These last two effects were robust for most tests conducted on both wild-type Canton S and w1118 strains, as well for tests using an isogenized panel of flies with mutations in the period gene, which causes altered circadian rhythmicity. Interestingly, the effect of absence of microbiota on a few behavioral features, most notably instantaneous locomotor activity speed, varied among wild-type strains. Taken together, our findings demonstrate that the microbiome can have subtle but significant effects on specific aspects of Drosophila behavior, some of which are dependent on genetic background.

Late Frontal Negativity Discriminates Outcomes and Intentions in Trust-Repayment Behavior.

Altruism (a costly action that benefits others) and reciprocity (the repayment of acts in kind) differ in that the former expresses preferences about the outcome of a social interaction, whereas the latter requires, in addition, ascribing intentions to others. Interestingly, an individual's behavior and neurophysiological activity under outcome- versus intention-based interactions has not been compared directly using different endowments in the same subject and during the same session. Here, we used a mixed version of the Dictator and the Investment games, together with electroencephalography, to uncover a subject's behavior and brain activity when challenged with endowments of different sizes in contexts that call for an altruistic (outcome-based) versus a reciprocal (intention-based) response. We found that subjects displayed positive or negative reciprocity (reciprocal responses greater or smaller than that for altruism, respectively) depending on the amount of trust they received. Furthermore, a subject's late frontal negativity differed between conditions, predicting responses to trust in intentions-based trials. Finally, brain regions related with mentalizing and cognitive control were the cortical sources of this activity. Thus, our work disentangles the behavioral components present in the repayment of trust, and sheds light on the neural activity underlying the integration of outcomes and perceived intentions in human economic interactions.

The tanning hormone, bursicon, does not act directly on the epidermis to tan the Drosophila exoskeleton.

BACKGROUND: In insects, continuous growth requires the periodic replacement of the exoskeleton. Once the remains of the exoskeleton from the previous stage have been shed during ecdysis, the new one is rapidly sclerotized (hardened) and melanized (pigmented), a process collectively known as tanning. The rapid tanning that occurs after ecdysis is critical for insect survival, as it reduces desiccation, and gives the exoskeleton the rigidity needed to support the internal organs and to provide a solid anchor for the muscles. This rapid postecdysial tanning is triggered by the "tanning hormone", bursicon. Since bursicon is released into the hemolymph, it has naturally been assumed that it would act on the epidermal cells to cause the tanning of the overlying exoskeleton. RESULTS: Here we investigated the site of bursicon action in Drosophila by examining the consequences on tanning of disabling the bursicon receptor (encoded by the rickets gene) in different tissues. To our surprise, we found that rapid tanning does not require rickets function in the epidermis but requires it instead in peptidergic neurons of the ventral nervous system (VNS). Although we were unable to identify the signal that is transmitted from the VNS to the epidermis, we show that neurons that express the Drosophila insulin-like peptide ILP7, but not the ILP7 peptide itself, are involved. In addition, we found that some of the bursicon targets involved in melanization are different from those that cause sclerotization. CONCLUSIONS: Our findings show that bursicon does not act directly on the epidermis to cause the tanning of the overlying exoskeleton but instead requires an intermediary messenger produced by peptidergic neurons within the central nervous system. Thus, this work has uncovered an unexpected layer of control in a process that is critical for insect survival, which will significantly alter the direction of future research aimed at understanding how rapid postecdysial tanning occurs.

Calcium and cAMP directly modulate the speed of the Drosophila circadian clock.

Circadian clocks impose daily periodicities to animal behavior and physiology. At their core, circadian rhythms are produced by intracellular transcriptional/translational feedback loops (TTFL). TTFLs may be altered by extracellular signals whose actions are mediated intracellularly by calcium and cAMP. In mammals these messengers act directly on TTFLs via the calcium/cAMP-dependent transcription factor, CREB. In the fruit fly, Drosophila melanogaster, calcium and cAMP also regulate the periodicity of circadian locomotor activity rhythmicity, but whether this is due to direct actions on the TTFLs themselves or are a consequence of changes induced to the complex interrelationship between different classes of central pacemaker neurons is unclear. Here we investigated this question focusing on the peripheral clock housed in the non-neuronal prothoracic gland (PG), which, together with the central pacemaker in the brain, controls the timing of adult emergence. We show that genetic manipulations that increased and decreased the levels of calcium and cAMP in the PG caused, respectively, a shortening and a lengthening of the periodicity of emergence. Importantly, knockdown of CREB in the PG caused an arrhythmic pattern of eclosion. Interestingly, the same manipulations directed at central pacemaker neurons caused arrhythmicity of eclosion and of adult locomotor activity, suggesting a common mechanism. Our results reveal that the calcium and cAMP pathways can alter the functioning of the clock itself. In the PG, these messengers, acting as outputs of the clock or as second messengers for stimuli external to the PG, could also contribute to the circadian gating of adult emergence.

Stereotyped responses of Drosophila peptidergic neuronal ensemble depend on downstream neuromodulators.

Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. Here, we focus on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and we show that the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, depends fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. Our findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors.

Regulation of cuticular hydrocarbon profile maturation by Drosophila tanning hormone, bursicon, and its interaction with desaturase activity.

Shortly after emergence the exoskeleton (cuticle) of adult insects is rapidly expanded, hardened (sclerotized), and pigmented (melanized). In parallel with this process, the oenocytes, which are large polyploid cells located below the abdominal epidermis, secrete onto the cuticle a cocktail of cuticular hydrocarbons (CHs) and waxes. These improve the waterproofing of the cuticle, and also provide important chemosensory and pheromonal cues linked with gender, age, and species differentiation. The hardening and pigmentation of the new cuticle are controlled by the neurohormone, bursicon, and its receptor, encoded by the DLGR2 receptor, rickets (rk); by contrast, little is known about the timecourse of changes in CH profile and about the role of bursicon in this process. Here we show in Drosophila that rk function is also required for the normal maturation of the fly's CH profile, with flies mutant for rk function showing dramatically elevated levels of CHs. Interestingly, this effect is mostly abrogated by mutations in the Δ9 desaturase encoded by the desaturase1 gene, which introduces a first double bond into elongated fatty-acid chains, suggesting that desaturase1 acts downstream of rk. In addition, flies mutant for rk showed changes in the absolute and relative levels of specific 7-monoenes (in males) and 7,11-dienes (in females). The fact that these differences in CH amounts were obtained using extractions of very different durations suggests that the particular CH profile of flies mutant for rk is not simply due to their unsclerotized cuticle but that bursicon may be involved in the process of CH biosynthesis itself.

Genetic variants associated with neurodegenerative Alzheimer disease in natural models.

The use of transgenic models for the study of neurodegenerative diseases has made valuable contributions to the field. However, some important limitations, including protein overexpression and general systemic compensation for the missing genes, has caused researchers to seek natural models that show the main biomarkers of neurodegenerative diseases during aging. Here we review some of these models-most of them rodents, focusing especially on the genetic variations in biomarkers for Alzheimer diseases, in order to explain their relationships with variants associated with the occurrence of the disease in humans.

Genetic variants associated with neurodegenerative Alzheimer disease in natural models

The use of transgenic models for the study of neurodegenerative diseases has made valuable contributions to the field. However, some important limitations, including protein overexpression and general systemic compensation for the missing genes, has caused researchers to seek natural models that show the main biomarkers of neurodegenerative diseases during aging. Here we review some of these models-most of them rodents, focusing especially on the genetic variations in biomarkers for Alzheimer diseases, in order to explain their relationships with variants associated with the occurrence of the disease in humans.

Genetic analysis of Eclosion hormone action during Drosophila larval ecdysis.

Insect growth is punctuated by molts, during which the animal produces a new exoskeleton. The molt culminates in ecdysis, an ordered sequence of behaviors that causes the old cuticle to be shed. This sequence is activated by Ecdysis triggering hormone (ETH), which acts on the CNS to activate neurons that produce neuropeptides implicated in ecdysis, including Eclosion hormone (EH), Crustacean cardioactive peptide (CCAP) and Bursicon. Despite more than 40 years of research on ecdysis, our understanding of the precise roles of these neurohormones remains rudimentary. Of particular interest is EH; although it is known to upregulate ETH release, other roles for EH have remained elusive. We isolated an Eh null mutant in Drosophila and used it to investigate the role of EH in larval ecdysis. We found that null mutant animals invariably died at around the time of ecdysis, revealing an essential role in its control. Further analyses showed that these animals failed to express the preparatory behavior of pre-ecdysis while directly expressing the motor program of ecdysis. Although ETH release could not be detected, the lack of pre-ecdysis could not be rescued by injections of ETH, suggesting that EH is required within the CNS for ETH to trigger the normal ecdysial sequence. Using a genetically encoded calcium probe, we showed that EH configured the response of the CNS to ETH. These findings show that EH plays an essential role in the Drosophila CNS in the control of ecdysis, in addition to its known role in the periphery of triggering ETH release.

Octodon degus (Molina 1782): a model in comparative biology and biomedicine.

One major goal of integrative and comparative biology is to understand and explain the interaction between the performance and behavior of animals in their natural environment. The Caviomorph, Octodon degu, is a native rodent species from Chile, and represents a unique model to study physiological and behavioral traits, including cognitive and sensory abilities. Degus live in colonies and have a well-structured social organization, with a mostly diurnal-crepuscular circadian activity pattern. More notable is the fact that in captivity, they reproduce and live between 5 and 7 yr and show hallmarks of neurodegenerative diseases (including Alzheimer's disease), diabetes, and cancer.