Circadian Neurons Research Articles

Light and dopamine impact two circadian neurons to promote morning wakefulness

In both mammals and flies, circadian brain neurons orchestrate physiological oscillations and behaviors like wake and sleep-these neurons can be subdivided by morphology and by gene expression patterns. Recent single-cell sequencing studies identified 17 Drosophila circadian neuron groups. One of these includes only two lateral neurons (LNs), which are marked by the expression of the neuropeptide ion transport peptide (ITP). Although these two ITP+ LNs have long been grouped with five other circadian evening activity cells, inhibiting the two neurons alone strongly reduces morning activity, indicating that they also have a prominent morning function. As dopamine signaling promotes activity in Drosophila, like in mammals, we considered that dopamine might influence this morning activity function. Moreover, the ITP+ LNs express higher mRNA levels than other LNs of the type 1-like dopamine receptor Dop1R1. Consistent with the importance of Dop1R1, cell-specific CRISPR-Cas9 mutagenesis of this receptor in the two ITP+ LNs renders flies significantly less active in the morning, and exvivo live imaging shows Dop1R1-dependent cyclic AMP (cAMP) responses to dopamine in these two neurons. Notably, the response is more robust in the morning, reflecting higher morning Dop1R1 mRNA levels in the two neurons. As mRNA levels are not elevated in constant darkness, this suggests light-dependent upregulation of morning Dop1R1 transcript levels. Taken together with the enhanced morning cAMP response to dopamine, the data indicate how light and dopamine promote morning wakefulness in flies, mimicking the important effect of light on morning wakefulness in humans.

Evo-devo applied to sleep research: an approach whose time has come.

Sleep occurs in all animals but its amount, form, and timing vary considerably between species and between individuals. Currently, little is known about the basis for these differences, in part, because we lack a complete understanding of the brain circuitry controlling sleep-wake states and markers for the cell types which can identify similar circuits across phylogeny. Here, I explain the utility of an "Evo-devo" approach for comparative studies of sleep regulation and function as well as for sleep medicine. This approach focuses on the regulation of evolutionary ancient transcription factors which act as master controllers of cell-type specification. Studying these developmental transcription factor cascades can identify novel cell clusters which control sleep and wakefulness, reveal the mechanisms which control differences in sleep timing, amount, and expression, and identify the timepoint in evolution when different sleep-wake control neurons appeared. Spatial transcriptomic studies, which identify cell clusters based on transcription factor expression, will greatly aid this approach. Conserved developmental pathways regulate sleep in mice, Drosophila, and C. elegans. Members of the LIM Homeobox (Lhx) gene family control the specification of sleep and circadian neurons in the forebrain and hypothalamus. Increased Lhx9 activity may account for increased orexin/hypocretin neurons and reduced sleep in Mexican cavefish. Other transcription factor families specify sleep-wake circuits in the brainstem, hypothalamus, and basal forebrain. The expression of transcription factors allows the generation of specific cell types for transplantation approaches. Furthermore, mutations in developmental transcription factors are linked to variation in sleep duration in humans, risk for restless legs syndrome, and sleep-disordered breathing. This paper is part of the "Genetic and other molecular underpinnings of sleep, sleep disorders, and circadian rhythms including translational approaches" collection.

Dissecting neuron-specific functions of circadian genes using modified cell-specific CRISPR approaches

Circadian behavioral rhythms in Drosophila melanogaster are regulated by about 75 pairs of brain neurons. They all express the core clock genes but have distinct functions and gene expression profiles. To understand the importance of these distinct molecular programs, neuron-specific gene manipulations are essential. Although RNAi based methods are standard to manipulate gene expression in a cell-specific manner, they are often ineffective, especially in assays involving smaller numbers of neurons or weaker Gal4 drivers. We and others recently exploited a neuron-specific CRISPR-based method to mutagenize genes within circadian neurons. Here, we further explore this approach to mutagenize three well-studied clock genes: the transcription factor gene vrille, the photoreceptor gene Cryptochrome (cry), and the neuropeptide gene Pdf (pigment dispersing factor). The CRISPR-based strategy not only reproduced their known phenotypes but also assigned cry function for different light-mediated phenotypes to discrete, different subsets of clock neurons. We further tested two recently published methods for temporal regulation in adult neurons, inducible Cas9 and the auxin-inducible gene expression system. The results were not identical, but both approaches successfully showed that the adult-specific knockout of the neuropeptide Pdf reproduces the canonical loss-of-function mutant phenotypes. In summary, a CRISPR-based strategy is a highly effective, reliable, and general method to temporally manipulate gene function in specific adult neurons.

Alcohol sensitivity and tolerance encoding in sleep regulatory circadian neurons in Drosophila.

Alcohol tolerance is a simple form of behavioural and neural plasticity that occurs with the first drink. Neural plasticity in tolerance is likely a substrate for longer term adaptations that can lead to alcohol use disorder. Drosophila develop tolerance with characteristics similar to vertebrates, and it is a useful model for determining the molecular and circuit encoding mechanisms in detail. Rapid tolerance, measured after the first alcohol exposure is completely metabolized, is localized to specific brain regions that are not interconnected in an obvious way. We used a forward neuroanatomical screen to identify three new neural sites for rapid tolerance encoding. One of these was composed of two groups of neurons, the DN1a and DN1p glutamatergic neurons, that are part of the Drosophila circadian clock. We localized rapid tolerance to the two DN1a neurons that regulate arousal by light at night, temperature-dependent sleep timing, and night-time sleep. Two clock neurons that regulate evening activity, LNd6 and the 5th LNv, are postsynaptic to the DN1as, and they promote rapid tolerance via the metabotropic glutamate receptor. Thus, rapid tolerance to alcohol overlaps with sleep regulatory neural circuitry, suggesting a mechanistic link.

Restoration of Sleep and Circadian Behavior by Autophagy Modulation in Huntington's Disease.

Circadian and sleep defects are well documented in Huntington's disease (HD). Modulation of the autophagy pathway has been shown to mitigate toxic effects of mutant Huntingtin (HTT) protein. However, it is not clear whether autophagy induction can also rescue circadian and sleep defects. Using a genetic approach, we expressed human mutant HTT protein in a subset of Drosophila circadian neurons and sleep center neurons. In this context, we examined the contribution of autophagy in mitigating toxicity caused by mutant HTT protein. We found that targeted overexpression of an autophagy gene, Atg8a in male flies, induces autophagy pathway and partially rescues several HTT-induced behavioral defects, including sleep fragmentation, a key hallmark of many neurodegenerative disorders. Using cellular markers and genetic approaches, we demonstrate that indeed the autophagy pathway is involved in behavioral rescue. Surprisingly, despite behavioral rescue and evidence for the involvement of the autophagy pathway, the large visible aggregates of mutant HTT protein were not eliminated. We show that the rescue in behavior is associated with increased mutant protein aggregation and possibly enhanced output from the targeted neurons, resulting in the strengthening of downstream circuits. Overall, our study suggests that, in the presence of mutant HTT protein, Atg8a induces autophagy and improves the functioning of circadian and sleep circuits.SIGNIFICANCE STATEMENT Defects in sleep and circadian rhythms are well documented in Huntington's disease. Recent literature suggests that circadian and sleep disturbances can exacerbate neurodegenerative phenotypes. Hence, identifying potential modifiers that can improve the functioning of these circuits could greatly improve disease management. We used a genetic approach to enhance cellular proteostasis and found that overexpression of a crucial autophagy gene, Atg8a, induces the autophagy pathway in the Drosophila circadian and sleep neurons and rescues sleep and activity rhythm. We demonstrate that the Atg8a improves synaptic function of these circuits by possibly enhancing the aggregation of the mutant protein in neurons. Further, our results suggest that differences in basal levels of protein homeostatic pathways is a factor that determines selective susceptibility of neurons.

Neural mechanisms of parasite-induced summiting behavior in 'zombie' Drosophila.

For at least two centuries, scientists have been enthralled by the "zombie" behaviors induced by mind-controlling parasites. Despite this interest, the mechanistic bases of these uncanny processes have remained mostly a mystery. Here, we leverage the Entomophthora muscae-Drosophila melanogaster "zombie fly" system to reveal the mechanistic underpinnings of summit disease, a manipulated behavior evoked by many fungal parasites. Using a high-throughput approach to measure summiting, we discovered that summiting behavior is characterized by a burst of locomotion and requires the host circadian and neurosecretory systems, specifically DN1p circadian neurons, pars intercerebralis to corpora allata projecting (PI-CA) neurons and corpora allata (CA), the latter being solely responsible for juvenile hormone (JH) synthesis and release. Using a machine learning classifier to identify summiting animals in real time, we observed that PI-CA neurons and CA appeared intact in summiting animals, despite invasion of adjacent regions of the "zombie fly" brain by E. muscae cells and extensive host tissue damage in the body cavity. The blood-brain barrier of flies late in their infection was significantly permeabilized, suggesting that factors in the hemolymph may have greater access to the central nervous system during summiting. Metabolomic analysis of hemolymph from summiting flies revealed differential abundance of several compounds compared to non-summiting flies. Transfusing the hemolymph of summiting flies into non-summiting recipients induced a burst of locomotion, demonstrating that factor(s) in the hemolymph likely cause summiting behavior. Altogether, our work reveals a neuro-mechanistic model for summiting wherein fungal cells perturb the fly's hemolymph, activating a neurohormonal pathway linking clock neurons to juvenile hormone production in the CA, ultimately inducing locomotor activity in their host.

A Subset of Circadian Neurons Expressing dTRPA1 Enables Appropriate Phasing of Activity Rhythms in Drosophila melanogaster Under Warm Temperatures.

Under conditions of prolonged durations of warmth, flies counter potential temperature stress by shifting their locomotor activity from day into night when the conditions are likely to be less harsh. Modulation of a rhythmic behavior such as this in response to the environment would require interaction between at least 2 neuronal systems: (1) a sensory system to receive input from the environment, and (2) the internal clock to correctly time rhythmic activity in response to this thermosensory input. Our previous studies found that a thermosensory mutant of the ion channel Drosophila Transient Receptor Potential-A1 (dTRPA1) failed to shift activity into the dark like control flies do and also identified the role of a specific cluster of the dTRPA1-expressing neurons, the dTRPA1sh+neurons necessary for this. In this study, we extended our previous findings and characterized the identity of these dTRPA1sh+ neurons based on their overlap with circadian neurons. Utilizing various genetic manipulations, we asked whether the overlapping neurons could be potential points of intersection between the 2 circuits that modulate behavior under warm temperature, meaning whether they function as both-sensory and clock neurons. We found that the molecular clock within the dTRPA1sh+ cluster was not necessary, but the expression of dTRPA1 in a subset of circadian neurons, the small ventrolateral neurons (sLNvs), was necessary in modulating phasing of behavior under warm temperature. Furthermore, attempting to identify the neuronal circuit, we were able to uncover the potential roles of serotonin and acetylcholine in modulating this temperature-dependent behavior. Finally, we also discuss possible parallel neuronal pathways that may exist to give rise to this modulation of behavior under warm temperature, thereby supporting and expanding the knowledge of the field about circuits that control temperature-mediated behavioral outcomes.

Neural connectivity molecules best identify the heterogeneous clock and dopaminergic cell types in the Drosophila adult brain.

Our recent single-cell sequencing of most adult Drosophila circadian neurons indicated notable and unexpected heterogeneity. To address whether other populations are similar, we sequenced a large subset of adult brain dopaminergic neurons. Their gene expression heterogeneity is similar to that of clock neurons, i.e., both populations have two to three cells per neuron group. There was also unexpected cell-specific expression of neuron communication molecule messenger RNAs: G protein-coupled receptor or cell surface molecule (CSM) transcripts alone can define adult brain dopaminergic and circadian neuron cell type. Moreover, the adult expression of the CSM DIP-beta in a small group of clock neurons is important for sleep. We suggest that the common features of circadian and dopaminergic neurons are general, essential for neuronal identity and connectivity of the adult brain, and that these features underlie the complex behavioral repertoire of Drosophila.

Single-cell transcriptome profiles of Drosophila fruitless-expressing neurons from both sexes.

Drosophila melanogaster reproductive behaviors are orchestrated by fruitless neurons. We performed single-cell RNA-sequencing on pupal neurons that produce sex-specifically spliced fru transcripts, the fru P1-expressing neurons. Uniform Manifold Approximation and Projection (UMAP) with clustering generates an atlas containing 113 clusters. While the male and female neurons overlap in UMAP space, more than half the clusters have sex differences in neuron number, and nearly all clusters display sex-differential expression. Based on an examination of enriched marker genes, we annotate clusters as circadian clock neurons, mushroom body Kenyon cell neurons, neurotransmitter- and/or neuropeptide-producing, and those that express doublesex. Marker gene analyses also show that genes that encode members of the immunoglobulin superfamily of cell adhesion molecules, transcription factors, neuropeptides, neuropeptide receptors, and Wnts have unique patterns of enriched expression across the clusters. In vivo spatial gene expression links to the clusters are examined. A functional analysis of fru P1 circadian neurons shows they have dimorphic roles in activity and period length. Given that most clusters are comprised of male and female neurons indicates that the sexes have fru P1 neurons with common gene expression programs. Sex-specific expression is overlaid on this program, to build the potential for vastly different sex-specific behaviors.

Phosphorylation Promotes the Accumulation of PERIOD Protein Foci.

Circadian clock drives the 24-h rhythm in our behavior and physiology. The molecular clock consists of a series of transcriptional/translational feedback loops operated by a number of clock genes. A very recent study reported that the clock protein PERIOD (PER) is organized into discrete foci at the nuclear envelope in fly circadian neurons, which is believed to be important for controlling the subcellular localization of clock genes. Loss of inner nuclear membrane protein lamin B receptor (LBR) leads to disruption of these foci, but how they are regulated is yet unknown. Here, we found that PER foci are likely phase-separated condensates, the formation of which is mediated by intrinsically disordered region in PER. Phosphorylation promotes the accumulation of these foci. Protein phosphatase 2A, which is known to dephosphorylate PER, hampers the accumulation of the foci. On the other hand, the circadian kinase DOUBLETIME (DBT) which phosphorylates PER enhances the accumulation of the foci. LBR likely facilitates PER foci accumulation by destabilizing the catalytic subunit of protein phosphatase 2A, MICROTUBULE STAR (MTS). In conclusion, here, we demonstrate a key role for phosphorylation in promoting the accumulation of PER foci, while LBR modulates this process by impinging on the circadian phosphatase MTS.

A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat

Small poikilotherms such as the fruit fly Drosophila depend on absolute temperature measurements to identify external conditions that are above (hot) or below (cold) their preferred range and to react accordingly. Hot and cold temperatures have a different impact on fly activity and sleep, but the circuits and mechanisms that adjust behavior to specific thermal conditions are not well understood. Here, we use patch-clamp electrophysiology to show that internal thermosensory neurons located within the fly head capsule (the AC neurons1) function as a thermometer active in the hot range. ACs exhibit sustained firing rates that scale with absolute temperature-but only for temperatures above the fly's preferred ∼25°C (i.e., "hot" temperature). We identify ACs in the fly brain connectome and demonstrate that they target a single class of circadian neurons, the LPNs.2 LPNs receive excitatory drive from ACs and respond robustly to hot stimuli, but their responses do not exclusively rely on ACs. Instead, LPNs receive independent drive from thermosensory neurons of the fly antenna via a new class of second-order projection neurons (TPN-IV). Finally, we show that silencing LPNs blocks the restructuring of daytime "siesta" sleep, which normally occurs in response to persistent heat. Our previous work described a distinct thermometer circuit for cold temperature.3 Together, the results demonstrate that the fly nervous system separately encodes and relays absolute hot and cold temperature information, show how patterns of sleep and activity can be adapted to specific temperature conditions, and illustrate how persistent drive from sensory pathways can impact behavior on extended temporal scales.

Mosquito cryptochromes expressed in Drosophila confer species-specific behavioral light responses

Cryptochrome (CRY) is a short-wavelength light-sensitive photoreceptor expressed in a subset of circadian neurons and eyes in Drosophila that regulates light-evoked circadian clock resetting. Acutely, light evokes rapid electrical excitation of the ventral lateral subset of circadian neurons and confers circadian-modulated avoidance behavioral responses to short-wavelength light. Recent work shows dramatically different avoidance versus attraction behavioral responses to short-wavelength light in day-active versus night-active mosquitoes and that these behavioral responses are attenuated by CRY protein degradation by constant light exposure in mosquitoes. To determine whether CRY1s mediate species-specific coding for behavioral and electrophysiological light responses, we used an "empty neuron" approach and transgenically expressed diurnal Aedes aegypti (AeCRY1) versus nocturnal Anopheles gambiae (AgCRY1) in a cry-null Drosophila background. AeCRY1 is much less light sensitive than either AgCRY1 or DmCRY as shown by partial behavioral rhythmicity following constant light exposure. Remarkably, expression of nocturnal AgCRY1 confers low survival to constant white light as does expression of AeCRY1 to a lesser extent. AgCRY1 mediates significantly stronger electrophysiological cell-autonomous responses to 365nm ultraviolet (UV) light relative to AeCRY1. AgCRY1 expression mediates electrophysiological sensitivity to 635nm red light, whereas AeCRY1 does not, consistent with species-specific mosquito red light responses. AgCRY1 and DmCRY mediate intensity-dependent avoidance behavior to UV light at different light intensity thresholds, whereas AeCRY1 does not, thus mimicking mosquito and fly behaviors. These findings highlight CRY as a key non-image-forming visual photoreceptor that mediates physiological and behavioral light responses in a species-specific fashion.

Circadian and Neuroendocrine Basis of Photoperiodism Controlling Diapause in Insects and Mites: A Review.

The photoperiodic system is concealed in the highly complex black-box, comprising four functional subunits: 1) a photo/thermo-sensitive input unit, 2) a photoperiodic clock based on a circadian system, 3) a condenser unit counting the number of inductive signals, and 4) a neuroendocrine switch that triggers a phenotypic shift. This review aims to summarize the research history and current reach of our understanding on this subject to connect it with the molecular mechanism of the circadian clock rapidly being unveiled. The review also focuses on the mode of intersubunit information transduction. It will scan the recent advancement in research on each functional subunit, but special attention will be given to the circadian clock–endocrine conjunct and the role of melatonin signaling in the regulation of insect photoperiodism. Prothoracicotropic hormone (PTTH) probably plays the most crucial role in the regulation of pupal diapause, which is the simplest model system of diapause regulation by hormones investigated so far, particularly in the Chinese oak silkmoth (Antheraea pernyi). A search for the trigger to release the PTTH found some candidates, that is, indoleamines. Indolamine metabolism is controlled by arylalkylamine N-acetyltransferase (aaNAT). Indolamine dynamics and aaNAT enzymatic activity changed according to photoperiods. aaNAT activity and melatonin content in the brain showed not only a photoperiodic response but also a circadian fluctuation. aaNAT had multiple E-boxes, suggesting that it is a clock-controlled gene (ccg), which implies that cycle (cyc, or brain–muscle Arnt-like 1 = Bmal1)/Clock (Clk) heterodimer binds to E-box and stimulates the transcription of aaNAT, which causes the synthesis of melatonin. RNAi against transcription modulators, cyc, or Clk downregulated aaNAT transcription, while RNAi against repressor of cyc/Clk, per upregulated aaNAT transcription. Immunohistochemical localization showed that the circadian neurons carry epitopes of melatonin-producing elements such as aaNAT, the precursor serotonin, HIOMT, and melatonin as well as clock gene products such as cyc-ir, Per-ir, and dbt-ir, while PTTH-producing neurons juxtaposed against the clock neurons showed hMT2-ir in A. pernyi brain. Melatonin probably binds to the putative melatonin receptor (MT) that stimulates Ca2+ influx, which in turn activates PKC. This induces Rab 8 phosphorylation and exocytosis of PTTH, leading to termination of diapause. All the PTTH-expressing neurons have PKC-ir, and Rab8-ir. When diapause is induced and maintained under short days, serotonin binding to 5HTR1B suppresses PTTH release in a yet unknown way. RNAi against this receptor knocked out photoperiodism; short day response is blocked and diapause was terminated even under the short day condition. The result showed that a relatively simple system controls both induction and termination in pupal diapause of A. pernyi: the circadian system regulates the transcription of aaNAT as a binary switch, the enzyme produces a melatonin rhythm that gates PTTH release, and 5HTR1B and MT are probably also under photoperiodic regulation. Finally, we listed the remaining riddles which need to be resolved, to fully understand this highly complex system in future studies.

Recurrent circadian circuitry regulates central brain activity to maintain sleep

Animal brains have discrete circadian neurons, but little is known about how they are coordinated to influence and maintain sleep. Here, through a systematic optogenetic screening, we identified a subtype of uncharacterized circadian DN3 neurons that is strongly sleep promoting in Drosophila. These anterior-projecting DN3s (APDN3s) receive signals from DN1 circadian neurons and then output to newly identified noncircadian "claw" neurons (CLs). CLs have a daily Ca2+ cycle, which peaks at night and correlates with DN1 and DN3 Ca2+ cycles. The CLs feedback onto a subset of DN1s to form a positive recurrent loop that maintains sleep. Using trans-synaptic photoactivatablegreen fluorescent protein (PA-GFP) tracing and functional invivo imaging, we demonstrated that the CLs drive sleep by interacting with and releasing acetylcholine onto the mushroom body γ lobe. Taken together, the data identify a novel self-reinforcing loop within the circadian network and a new sleep-promoting neuropile that are both essential for maintaining normal sleep.

Astrocytic GABA transporter controls sleep by modulating GABAergic signaling in Drosophila circadian neurons

A precise balance between sleep and wakefulness is essential to sustain a good quality of life and optimal brain function. GABA is known to play a key and conserved role in sleep control, and GABAergic tone should, therefore, be tightly controlled in sleep circuits. Here, we examined the role of the astrocytic GABA transporter (GAT) in sleep regulation using Drosophila melanogaster. We found that a hypomorphic gat mutation (gat33-1) increased sleep amount, decreased sleep latency, and increased sleep consolidation at night. Interestingly, sleep defects were suppressed when gat33-1 was combined with a mutation disrupting wide-awake (wake), a gene that regulates the cell-surface levels of the GABAA receptor resistance to dieldrin (RDL) in the wake-promoting large ventral lateral neurons (l-LNvs). Moreover, RNAi knockdown of rdl and its modulators dnlg4 and wake in these circadian neurons also suppressed gat33-1 sleep phenotypes. Brain immunohistochemistry showed that GAT-expressing astrocytes were located near RDL-positive l-LNv cell bodies and dendritic processes. We concluded that astrocytic GAT decreases GABAergic tone and RDL activation in arousal-promoting LNvs, thus determining proper sleep amount and quality in Drosophila.

Age-Related Unstructured Spike Patterns and Molecular Localization in Drosophila Circadian Neurons.

Aging decreases sleep quality by disrupting the molecular machinery that regulates the circadian rhythm. However, we do not fully understand the mechanism that underlies this process. In Drosophila, sleep quality is regulated by precisely timed patterns of spontaneous firing activity in posterior DN1 (DN1p) circadian clock neurons. How aging affects the physiological function of DN1p neurons is unknown. In this study, we found that aging altered functional parameters related to neural excitability and disrupted patterned spike sequences in DN1p neurons during nighttime. We also characterized age-associated changes in intrinsic membrane properties related to spike frequency adaptations and synaptic properties, which may account for the unstructured spike patterns in aged DN1p neurons. Because Slowpoke binding protein (SLOB) and the Na+/K+ ATPase β subunit (NaKβ) regulate clock-dependent spiking patterns in circadian networks, we compared the subcellular organization of these factors between young and aged DN1p neurons. Young DN1p neurons showed circadian cycling of HA-tagged SLOB and myc-tagged NaKβ targeting the plasma membrane, whereas aged DN1p neurons showed significantly disrupted subcellular localization patterns of both factors. The distribution of SLOB and NaKβ signals also showed greater variability in young vs. aged DN1p neurons, suggesting aging leads to a loss of actively formed heterogeneity for these factors. These findings showed that aging disrupts precisely structured molecular patterns that regulate structured neural activity in the circadian network, leading to age-associated declines in sleep quality. Thus, it is possible to speculate that a recovery of unstructured neural activity in aging clock neurons could help to rescue age-related poor sleep quality.

DBT affects sleep in both circadian and non-circadian neurons.

Sleep is a very important behavior observed in almost all animals. Importantly, sleep is subject to both circadian and homeostatic regulation. The circadian rhythm determines the daily alternation of the sleep-wake cycle, while homeostasis mediates the rise and dissipation of sleep pressure during the wake and sleep period. As an important kinase, dbt plays a central role in both circadian rhythms and development. We investigated the sleep patterns of several ethyl methanesulfonate-induced dbt mutants and discuss the possible reasons why different sleep phenotypes were shown in these mutants. In order to reduce DBT in all neurons in which it is expressed, CRISPR-Cas9 was used to produce flies that expressed GAL4 in frame with the dbt gene at its endogenous locus, and knock-down of DBT with this construct produced elevated sleep during the day and reduced sleep at night. Loss of sleep at night is mediated by dbt loss during the sleep/wake cycle in the adult, while the increased sleep during the day is produced by reductions in dbt during development and not by reductions in the adult. Additionally, using targeted RNA interference, we uncovered the contribution of dbt on sleep in different subsets of neurons in which dbt is normally expressed. Reduction of dbt in circadian neurons produced less sleep at night, while lower expression of dbt in noncircadian neurons produced increased sleep during the day. Importantly, independently of the types of neurons where dbt affects sleep, we demonstrate that the PER protein is involved in DBT mediated sleep regulation.

TRPM4 Contributes to Subthreshold Membrane Potential Oscillations in Multiple Mouse Pacemaker Neurons.

Select neuronal populations display steady rhythmic neuronal firing that provides tonic excitation to drive downstream networks and behaviors. In noradrenergic neurons of the locus coeruleus (LC), circadian neurons of the suprachiasmatic nucleus (SCN), and CO2/H+-activated neurons of the brainstem retrotrapezoid nucleus (RTN), large subthreshold membrane potential oscillations contribute to the pacemaker-like action potential discharge. The oscillations and firing in LC and SCN involve contributions from leak sodium (NALCN) and L-type calcium channels while recent work from RTN suggested an additional pivotal role for a secondary calcium-activated and voltage-gated cationic current sensitive to TRPM4 channel blockers. Here, we tested whether TRPM4 contributes to subthreshold oscillations in mouse LC and SCN. By RNAscope in situ hybridization, Trpm4 transcripts were detected in both cell groups. In whole-cell recordings from acute slice preparations, prominent voltage-dependent membrane potential oscillations were revealed in LC and SCN after blocking action potentials. These oscillations were inhibited by two chemically-distinct blockers of TRPM4, 9-phenanthrol (9-pt) and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). Under whole-cell voltage clamp, inward currents evoked by oscillation voltage waveforms were inhibited in LC by blocking L-type calcium channels and TRPM4. These data implicate TRPM4 in the large subthreshold membrane potential oscillations that underlie tonic action potential discharge in LC and SCN, providing a voltage-dependent and calcium-dependent cationic current to augment the depolarizing inward Na+ and Ca2+ currents previously associated with this distinctive electroresponsive property.

Circadian neurons in the paraventricular nucleus entrain and sustain daily rhythms in glucocorticoids

Signals from the central circadian pacemaker, the suprachiasmatic nucleus (SCN), must be decoded to generate daily rhythms in hormone release. Here, we hypothesized that the SCN entrains rhythms in the paraventricular nucleus (PVN) to time the daily release of corticosterone. In vivo recording revealed a critical circuit from SCN vasoactive intestinal peptide (SCNVIP)-producing neurons to PVN corticotropin-releasing hormone (PVNCRH)-producing neurons. PVNCRH neurons peak in clock gene expression around midday and in calcium activity about three hours later. Loss of the clock gene Bmal1 in CRH neurons results in arrhythmic PVNCRH calcium activity and dramatically reduces the amplitude and precision of daily corticosterone release. SCNVIP activation reduces (and inactivation increases) corticosterone release and PVNCRH calcium activity, and daily SCNVIP activation entrains PVN clock gene rhythms by inhibiting PVNCRH neurons. We conclude that daily corticosterone release depends on coordinated clock gene and neuronal activity rhythms in both SCNVIP and PVNCRH neurons.

Decapentaplegic Acutely Defines the Connectivity of Central Pacemaker Neurons in Drosophila.

Rhythmic rest-activity cycles are controlled by an endogenous clock. In Drosophila, this clock resides in ∼150 neurons organized in clusters whose hierarchy changes in response to environmental conditions. The concerted activity of the circadian network is necessary for the adaptive responses to synchronizing environmental stimuli. Thus far, work was devoted to unravel the logic of the coordination of different clusters focusing on neurotransmitters and neuropeptides. We further explored communication in the adult male brain through ligands belonging to the bone morphogenetic protein (BMP) pathway. Herein we show that the lateral ventral neurons (LNvs) express the small morphogen decapentaplegic (DPP). DPP expression in the large LNvs triggered a period lengthening phenotype, the downregulation of which caused reduced rhythmicity and affected anticipation at dawn and dusk, underscoring DPP per se conveys time-of-day relevant information. Surprisingly, DPP expression in the large LNvs impaired circadian remodeling of the small LNv axonal terminals, likely through local modulation of the guanine nucleotide exchange factor Trio. These findings open the provocative possibility that the BMP pathway is recruited to strengthen/reduce the connectivity among specific clusters along the day and thus modulate the contribution of the clusters to the circadian network.SIGNIFICANCE STATEMENT The circadian clock relies on the communication between groups of so-called clock neurons to coordinate physiology and behavior to the optimal times across the day, predicting and adapting to a changing environment. The circadian network relies on neurotransmitters and neuropeptides to fine-tune connectivity among clock neurons and thus give rise to a coherent output. Herein we show that decapentaplegic, a ligand belonging to the BMP retrograde signaling pathway required for coordinated growth during development, is recruited by a group of circadian neurons in the adult brain to trigger structural remodeling of terminals on a daily basis.