Clock Neurons Research Articles

Bilateral enucleation at birth modifies calcium spike amplitude, but not frequency, in neurons of the somatosensory thalamus and cortex: Implications for developmental cross-modal plasticity

Bilateral eye enucleation at birth (BE) leads to an expansion of the primary somatosensory cortex (S1) in rat pups. Although increased growth of the somatosensory thalamo-cortical afferents (STCAs) in part explains S1 expansion, timing mechanisms governing S1 formation are also involved. In this work, we begin the search of a developmental clock by intending to document the existence of putative clock neurons in the somatosensory thalamus (VPM) and S1 based upon changes of spontaneous spike amplitude; a biophysical property sensitive to circadian regulation; the latter known to be shifted by enucleation. In addition, we also evaluated whether STCAs growth rate and segregation timing were modified, as parameters the clock might time. We found that spontaneous spike amplitude transiently, but significantly, increased or decreased in VPM and S1 neurons of BE rat pups, respectively, as compared to their control counterparts. The growth rate and segregation timing of STCAs was, however, unaffected by BE. These results support the existence of a developmental clock that ticks differently in the VPM and S1 after BE. This observation, together with the fact that STCAs growth rate and segregation timing is unchanged, suggests that S1 expansion in BE rats may in part be controlled at the cortical level.

A circadian output center controlling feeding:fasting rhythms in Drosophila.

Circadian rhythms allow animals to coordinate behavioral and physiological processes with respect to one another and to synchronize these processes to external environmental cycles. In most animals, circadian rhythms are produced by core clock neurons in the brain that generate and transmit time-of-day signals to downstream tissues, driving overt rhythms. The neuronal pathways controlling clock outputs, however, are not well understood. Furthermore, it is unclear how the central clock modulates multiple distinct circadian outputs. Identifying the cellular components and neuronal circuitry underlying circadian regulation is increasingly recognized as a critical step in the effort to address health pathologies linked to circadian disruption, including heart disease and metabolic disorders. Here, building on the conserved components of circadian and metabolic systems in mammals and Drosophila melanogaster, we used a recently developed feeding monitor to characterize the contribution to circadian feeding rhythms of two key neuronal populations in the Drosophila pars intercerebralis (PI), which is functionally homologous to the mammalian hypothalamus. We demonstrate that thermogenetic manipulations of PI neurons expressing the neuropeptide SIFamide (SIFa) as well as mutations of the SIFa gene degrade feeding:fasting rhythms. In contrast, manipulations of a nearby population of PI neurons that express the Drosophila insulin-like peptides (DILPs) affect total food consumption but leave feeding rhythms intact. The distinct contribution of these two PI cell populations to feeding is accompanied by vastly different neuronal connectivity as determined by trans-Tango synaptic mapping. These results for the first time identify a non-clock cell neuronal population in Drosophila that regulates feeding rhythms and furthermore demonstrate dissociable control of circadian and homeostatic aspects of feeding regulation by molecularly-defined neurons in a putative circadian output hub.

Dissection of central clock function in Drosophila through cell-specific CRISPR-mediated clock gene disruption.

In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called 'master pacemakers' because they are essential for circadian rhythms. A subset of Pdf+ neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf+ neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf+ neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.

Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity.

Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, we assayed behavior as well as molecular rhythms within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. We also generated CRISPR-mediated neuron-specific circadian clock knockouts. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light.

Ataxin2 functions via CrebA to mediate Huntingtin toxicity in circadian clock neurons.

Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington’s disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, we discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. Our results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis.

Direct effects of the light environment on daily neuroendocrine control.

Endocrine systems function as key mediators of adaptive responses to the external environment. As a reliable predictor of many salient variations in the external world, the light environment thus constitutes an influential source of control over neuroendocrine function. Accordingly, the vast majority of endocrine systems display 24hr variations in activity that are aligned to daily changes in external illumination. While the neural mechanisms responsible for driving these rhythms are still incompletely understood, circadian and light-dependent signals relayed via the suprachiasmatic nucleus of the hypothalamus (SCN) play a key role. Retinal projections to the SCN provide information from rods, cones and melanopsin, which, together, encode variations in the amount and spectral content of ambient light over the solar day. This sensory input, in turn, drives acute modulations in SCN cellular activity and aligns daily rhythms in the electrophysiological output of individual clock neurons. Neural outputs from the SCN can therefore convey both rapid and longer-term information about the light environment to other hypothalamic nuclei responsible for neuroendocrine control. In this review we summarises current understanding of the specific neural pathways by which the light environment influences key neuroendocrine axes, with a particular focus on the retinal and SCN-dependent circuits involved and their known sensory properties.

Light-Mediated Circuit Switching in the Drosophila Neuronal Clock Network.

The circadian clock is a timekeeper but also helps adapt physiology to the outside world. This is because an essential feature of clocks is their ability to adjust (entrain) to the environment, with light beingthe most important signal. Whereas cryptochrome-mediated entrainment is well understood in Drosophila, integration of light information via the visual system lacks a neuronal or molecular mechanism. Here, we show that a single photoreceptor subtype is essential for long-day adaptation. These cells activate key circadian neurons, namely the large ventral-lateral neurons (lLNvs), which release the neuropeptide pigment-dispersing factor (PDF). RNAi and rescue experiments show that PDF from these cells is necessary and sufficient for delaying the timing of the evening (E) activity in long-day conditions. This contrasts to PDF that derives from the small ventral-lateral neurons (sLNvs), which are essential for constant darkness (DD) rhythmicity. Using a cell-specific CRISPR/Cas9 assay, we show that lLNv-derived PDF directly interacts with neurons important for E activity timing. Interestingly, this pathway is specific for long-day adaptation and appears to be dispensable in equinox or DD conditions. The results therefore indicate that external cues cause a rearrangement of neuronal hierarchy, which contributes to behavioral plasticity.

Locomotor Behaviour and Clock Neurons Organisation in the Agricultural Pest Drosophila suzukii.

Drosophila suzukii (Matsumara) also called Spotted Wing Drosophila (SWD), is an invasive pest species originally from Asia that has now spread widely across Europe and North America. The majority of drosophilids including the best known Drosophila melanogaster only breed on decaying fruits. On the contrary, the presence of a strong serrated ovipositor and behavioural and metabolic adaptations allow D. suzukii to lay eggs inside healthy, ripening fruits that are still on the plant. Here we present an analysis of the rhythmic locomotor activity behaviour of D. suzukii under several laboratory settings. Moreover, we identify the canonical clock neurons in this species by reporting the expression pattern of the major clock proteins in the brain. Interestingly, a fundamentally similar organisation of the clock neurons network between D. melanogaster and D. suzukii does not correspond to similar characteristics in rhythmic locomotor activity behaviour.

Spatiotemporal expression pattern of PERIOD 1 and PERIOD 2 in the mouse SCN is dependent on VIP receptor 2 signaling.

Neurons of the hypothalamic suprachiasmatic nucleus (SCN) express clock genes, which regulate their own transcription and generate daily output signals driving circadian rhythmic behavior and physiology. The neuropeptide VIP and its specific receptor, the VPAC2 receptor, are important for synchronization of clock neurons. In the present study, we characterized PER1 and PER2 expressing neurons in wild-type and VPAC2-deficient mice. We found evidence for distinct spatiotemporal circadian oscillation in the expression of the PER genes in two separate clusters of SCN neurons. In wild-type mice corresponding to the SCN shell and ventral core, high expression of PER was found at lights-off most likely representing an evening clock (E-clock). In another smaller cluster of neurons located in the central core of the SCN, PER expression peaks in antiphase at lights-on and could represent a morning clock (M-clock). BMAL1 immunoreactivity was found to be expressed in antiphase to PER in M and E neurons, respectively. PER was found in 98% of neurons expressing vasopressin (AVP) and in 92% of VIP neurons. The chemotype of M neurons was not identified. M but not E cells were responsive to long but not short photoperiods. The expression of the VPAC2 receptor was found in both M and E cells, and VPAC2-deficient mice displayed markedly blunted PER expression in both cell clusters of the SCN. Conclusion: These observations support the existence of M and E clocks involved in circadian and seasonal adaptation, which seem dependent on intact VIP/VPAC2 signaling in the SCN.

Temperature synchronization of the Drosophila circadian clock protein PERIOD is controlled by the TRPA channel PYREXIA

Circadian clocks are endogenous molecular oscillators that temporally organize behavioral activity thereby contributing to the fitness of organisms. To synchronize the fly circadian clock with the daily fluctuations of light and temperature, these environmental cues are sensed both via brain clock neurons, and by light and temperature sensors located in the peripheral nervous system. Here we demonstrate that the TRPA channel PYREXIA (PYX) is required for temperature synchronization of the key circadian clock protein PERIOD. We observe a molecular synchronization defect explaining the previously reported defects of pyx mutants in behavioral temperature synchronization. Surprisingly, surgical ablation of pyx-mutant antennae partially rescues behavioral synchronization, indicating that antennal temperature signals are modulated by PYX function to synchronize clock neurons in the brain. Our results suggest that PYX protects antennal neurons from faulty signaling that would otherwise interfere with temperature synchronization of the circadian clock neurons in the brain.

Decoding Drosophila circadian pacemaker circuit.

Drosophila circadian circuit is one of the best described neural circuits but is complex enough to obscure our understanding of how it actually works. Animals' rhythmic behavior, the seemingly simple outcome of their internal clocks, relies on the interaction of heterogeneous clock neurons that are spread across the brain. Direct observations of their coordinated network interactions can bring us forward in understanding the circuit. The current challenge is to observe activity of each of these neurons over a long span of time - hours to days - in live animals. Here we review the progress in circadian circuit interrogation powered by in vivo calcium imaging.

Analysing the evolutional and functional differentiation of four types of Daphnia magna cryptochrome in Drosophila circadian clock

Cryptochrome (CRY) plays an important role in the input of circadian clocks in various species, but gene copies in each species are evolutionarily divergent. Type I CRYs function as a photoreceptor molecule in the central clock, whereas type II CRYs directly regulate the transcriptional activity of clock proteins. Functions of other types of animal CRYs in the molecular clock remain unknown. The water flea Daphnia magna contains four Cry genes. However, it is still difficult to analyse these four genes. In this study, we took advantage of powerful genetic resources available from Drosophila to investigate evolutionary and functional differentiation of CRY proteins between the two species. We report differences in subcellular localisation of each D. magna CRY protein when expressed in the Drosophila clock neuron. Circadian rhythm behavioural experiments revealed that D. magna CRYs are not functionally conserved in the Drosophila molecular clock. These findings provide a new perspective on the evolutionary conservation of CRY, as functions of the four D. magna CRY proteins have diverse subcellular localisation levels. Furthermore, molecular clocks of D. magna have been evolutionarily differentiated from those of Drosophila. This study highlights the extensive functional diversity existing among species in their complement of Cry genes.

Alzheimer's disease-associated tau alters Drosophila circadian activity, sleep and clock neuron electrophysiology

Alzheimer's disease (AD) is the most common cause of dementia, which is associated with an enormous personal, social and economic burden worldwide. However, there are few current treatments with none of them targeting the underlying causes of the disease. Sleep and circadian rhythm defects are not only distressing symptoms of AD and other tauopathies and are a leading cause of care home admission but are also thought to accelerate AD pathology. Despite this, little is understood about the underlying causes of these behavioural changes. Expression of the 0N4R isoform of tau has been associated with AD pathology and we show that expressing it in the Drosophila clock network gives rise to circadian and sleep phenotypes which closely match the behavioural changes seen in human AD patients. Tauopathic flies exhibited greater locomotor activity throughout the day and night and displayed a loss of sleep, particularly at night. Under constant darkness, the locomotor behaviour of tau-expressing flies was less rhythmic than controls indicating a defect in their intrinsic circadian rhythm. Current clamp recordings from wake-promoting, pigment dispersing factor (PDF)-positive large lateral ventral clock neurons (l-LNvs) revealed elevated spontaneous firing throughout the day and night which likely underlies the observed hyperactive circadian phenotype. Interestingly, expression of tau in only the PDF-positive pacemaker neurons, which are thought to be the most important for behaviour under constant conditions, was not sufficient or even necessary to affect circadian rhythmicity. This work establishes Drosophila as a model to investigate interactions between human pathological versions of tau and the machinery that controls neuronal excitability, allowing the identification of underlying mechanisms of disease that may reveal new therapeutic targets.

Peptidergic signaling from clock neurons regulates reproductive dormancy in Drosophila melanogaster.

With the approach of winter, many insects switch to an alternative protective developmental program called diapause. Drosophila melanogaster females overwinter as adults by inducing a reproductive arrest that is characterized by inhibition of ovarian development at previtellogenic stages. The insulin producing cells (IPCs) are key regulators of this process, since they produce and release insulin-like peptides that act as diapause-antagonizing hormones. Here we show that in D. melanogaster two neuropeptides, Pigment Dispersing Factor (PDF) and short Neuropeptide F (sNPF) inhibit reproductive arrest, likely through modulation of the IPCs. In particular, genetic manipulations of the PDF-expressing neurons, which include the sNPF-producing small ventral Lateral Neurons (s-LNvs), modulated the levels of reproductive dormancy, suggesting the involvement of both neuropeptides. We expressed a genetically encoded cAMP sensor in the IPCs and challenged brain explants with synthetic PDF and sNPF. Bath applications of both neuropeptides increased cAMP levels in the IPCs, even more so when they were applied together, suggesting a synergistic effect. Bath application of sNPF additionally increased Ca2+ levels in the IPCs. Our results indicate that PDF and sNPF inhibit reproductive dormancy by maintaining the IPCs in an active state.

Effects of MUL1 and PARKIN on the circadian clock, brain and behaviour in Drosophila Parkinson\u2019s disease models

BackgroundMutants which carry mutations in genes encoding mitochondrial ligases MUL1 and PARKIN are convenient Drosophila models of Parkinson’s disease (PD). In several studies it has been shown that in Parkinson’s disease sleep disturbance occurs, which may be the result of a disturbed circadian clock.ResultsWe found that the ROS level was higher, while the anti-oxidant enzyme SOD1 level was lower in mul1A6 and park1 mutants than in the white mutant used as a control. Moreover, mutations of both ligases affected circadian rhythms and the clock. The expression of clock genes per, tim and clock and the level of PER protein were changed in the mutants. Moreover, expression of ATG5, an autophagy protein also involved in circadian rhythm regulation, was decreased in the brain and in PDF-immunoreactive large ventral lateral clock neurons. The observed changes in the molecular clock resulted in a longer period of locomotor activity rhythm, increased total activity and shorter sleep at night. Finally, the lack of both ligases led to decreased longevity and climbing ability of the flies.ConclusionsAll of the changes observed in the brains of these Drosophila models of PD, in which mitochondrial ligases MUL1 and PARKIN do not function, may explain the mechanisms of some neurological and behavioural symptoms of PD.

Busted! A Dope Ring with Activity Clocked at Dawn and Dusk

Clock neurons generate circadian rhythms in behavioral activity, but the relevant pathways remain poorly understood. In this issue of Neuron, Liang etal. (2019) show that distinct clock neurons independently drive movement-promoting "ring neurons" in Drosophila through dopaminergic relays to support morning and evening locomotor activity.

Temporal Control of Appetite by AgRP Clocks

Neuronal control of diurnal appetitive drive is not well understood. In this issue, a study by Cedernaes etal. (2019) elucidates a key role of AgRP neuronal clocks and delineates the cell-type-specific transcriptional landscape in the temporal regulation of hunger.

Neuronal Activity in Non-LNv Clock Cells Is Required to Produce Free-Running Rest:Activity Rhythms in Drosophila.

Circadian rhythms in behavior and physiology are produced by central brain clock neurons that can be divided into subpopulations based on molecular and functional characteristics. It has become clear that coherent behavioral rhythms result from the coordinated action of these clock neuron populations, but many questions remain regarding the organizational logic of the clock network. Here we used targeted genetic tools in Drosophila to eliminate either molecular clock function or neuronal activity in discrete clock neuron subsets. We find that neuronal firing is necessary across multiple clock cell populations to produce free-running rhythms of rest and activity. In contrast, such rhythms are much more subtly affected by molecular clock suppression in the same cells. These findings demonstrate that network connectivity can compensate for a lack of molecular oscillations within subsets of clock cells. We further show that small ventrolateral (sLNv) clock neurons, which have been characterized as master pacemakers under free-running conditions, cannot drive rhythms independent of communication between other cells of the clock network. In particular, we pinpoint an essential contribution of the dorsolateral (LNd) clock neurons, and show that manipulations that affect LNd function reduce circadian rhythm strength without affecting molecular cycling in sLNv cells. These results suggest a hierarchical organization in which circadian information is first consolidated among one or more clock cell populations before accessing output pathways that control locomotor activity.

SOX2-Dependent Transcription in Clock Neurons Promotes the Robustness of the Central Circadian Pacemaker

Clock neurons within the mammalian suprachiasmatic nuclei (SCN) encode circadian time using interlocked transcription-translation feedback loops (TTFLs) that drive rhythmic gene expression. However, the contributions of other transcription factors outside of the circadian TTFLs to the functionality of the SCN remain obscure. Here, we report that the stem and progenitor cell transcription factor, sex-determining region Y-box 2 (SOX2), is expressed inadult SCN neurons and positively regulates transcription of the core clock gene, Period2. Mice lacking SOX2 selectively in SCN neurons display imprecise, poorly consolidated behavioral rhythms that do not entrain efficiently to environmental light cycles and that are highly susceptible to constant light-induced arrhythmicity. RNA sequencing revealed that Sox2 deficiency alters the SCN transcriptome, reducing the expression of core clock genes and neuropeptide-receptor systems. By defining the transcriptional landscape within SCN neurons, SOX2 enables the generation of robust, entrainable circadian rhythms that accurately reflect environmental time.

Achilles-Mediated and Sex-Specific Regulation of Circadian mRNA Rhythms in Drosophila.

The circadian clock is an evolutionarily conserved mechanism that generates the rhythmic expression of downstream genes. The core circadian clock drives the expression of clock-controlled genes, which in turn play critical roles in carrying out many rhythmic physiological processes. Nevertheless, the molecular mechanisms by which clock output genes orchestrate rhythmic signals from the brain to peripheral tissues are largely unknown. Here we explored the role of one rhythmic gene, Achilles, in regulating the rhythmic transcriptome in the fly head. Achilles is a clock-controlled gene in Drosophila that encodes a putative RNA-binding protein. Achilles expression is found in neurons throughout the fly brain using fluorescence in situ hybridization (FISH), and legacy data suggest it is not expressed in core clock neurons. Together, these observations argue against a role for Achilles in regulating the core clock. To assess its impact on circadian mRNA rhythms, we performed RNA sequencing (RNAseq) to compare the rhythmic transcriptomes of control flies and those with diminished Achilles expression in all neurons. Consistent with previous studies, we observe dramatic upregulation of immune response genes upon knock-down of Achilles. Furthermore, many circadian mRNAs lose their rhythmicity in Achilles knock-down flies, suggesting that a subset of the rhythmic transcriptome is regulated either directly or indirectly by Achilles. These Achilles-mediated rhythms are observed in genes involved in immune function and in neuronal signaling, including Prosap, Nemy and Jhl-21. A comparison of RNAseq data from control flies reveals that only 42.7% of clock-controlled genes in the fly brain are rhythmic in both males and females. As mRNA rhythms of core clock genes are largely invariant between the sexes, this observation suggests that sex-specific mechanisms are an important, and heretofore under-appreciated, regulator of the rhythmic transcriptome.