Hugin+ neurons provide a link between sleep homeostat and circadian clock neurons

Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. We show here that hugin⁺ neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin⁺ neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin⁺ neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin⁺ locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin⁺ neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide–dependent fashion. We propose that hugin⁺ neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep.

Sleep is regulated by two processes, circadian and homeostatic (1). The endogenous circadian clock, together with its downstream pathways, is synchronized to external day–night cycles and determines the timing of sleep to produce 24-h rhythms in sleep and wake. The homeostatic process tracks sleep:wake history and generates sleep drive, based on the extent of wakefulness. Overtly, sleep homeostasis can be seen as an increase in sleep duration and depth after prolonged wakefulness. Generally, circadian and homeostatic processes are studied as separate mechanisms that regulate sleep, but they clearly intersect and are coordinated in a daily cycle to promote the onset and maintenance of sleep at night. Following sleep deprivation, the homeostatic system can drive sleep at the wrong time of day, but even under these conditions, interactions between the two systems determine the timing and duration of sleep. However, the neuronal mechanisms by which circadian and homeostatic pathways signal to each other are largely unknown.The functions and regulation of sleep are extensively studied in model organisms, such as Drosophila melanogaster (2). In the Drosophila brain, the circadian clock is expressed in ∼150 clock neurons that are organized into neuroanatomical groups, of which the ventral and dorsal lateral neurons are the most important for driving rhythms of locomotor activity (3, 4). The small ventrolateral neurons (s-LNvs) link to other brain regions through different circuits. One of those circuits connects the s-LNvs to the site of the motor ganglion, the thoracic nerve cord: s-LNvs → DN1s → Dh44⁺ neurons → hugin⁺ neurons→ ventral nerve cord (5). Dh44-expressing neurons in the pars intercerebralis regulate locomotor activity rhythms in part through the signaling of DH44 neuropeptide to hugin-expressing neurons in the subesophageal zone (SEZ) (6, 7). Dh44⁺ and hugin⁺ circadian output neurons do not contain canonical molecular clocks, but display cycling in neuronal activity or peptide release, likely under control of upstream circadian signals (7–9). Links between these neurons and loci regulating sleep homeostasis have not been identified yet.Regulation of sleep homeostasis in flies involves the central complex and mushroom body (10–13). Of particular importance is a group of sleep-promoting neurons labeled by the 23E10-GAL4 driver that projects to the dorsal fan-shaped body (dFB) neuropil in the central complex. These dFB neurons promote sleep when activated (14, 15), and they are required for normal sleep rebound after deprivation (16). 23E10⁺ neurons receive input signals from R5 ellipsoid body neurons, which track sleep need (17).As hugin⁺ neurons are significantly downstream of central clock neurons and close to behavioral outputs, we asked whether they also have a role in sleep. We find that hugin⁺ neurons are dispensable for determining daily sleep amount, but they show decreases in activity following sleep deprivation. They also receive projections from the dFB and counter sleep-promoting effects of 23E10⁺ neurons, such that ablation of hugin⁺ neurons enhances sleep driven by 23E10⁺ cells. Further supporting a role in sleep, mutations in Hugin peptides affect recovery sleep after deprivation and enhance sleep driven by 23E10⁺ cells. hugin⁺ neurons target PDF⁺ s-LNv clock neurons, which also show decreases in intracellular Ca²⁺ levels following sleep deprivation. Thus hugin⁺ neurons serve as an integrations site of signals from both the sleep homeostat and the circadian clock.     

Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock

In mammals, multiple physiological, metabolic, and behavioral processes are subject to circadian rhythms, adapting to changing light in the environment. Here we analyzed circadian rhythms in the fecal microbiota of mice using deep sequencing, and found that the absolute amount of fecal bacteria and the abundance of Bacteroidetes exhibited circadian rhythmicity, which was more pronounced in female mice. Disruption of the host circadian clock by deletion of Bmal1, a gene encoding a core molecular clock component, abolished rhythmicity in the fecal microbiota composition in both genders. Bmal1 deletion also induced alterations in bacterial abundances in feces, with differential effects based on sex. Thus, although host behavior, such as time of feeding, is of recognized importance, here we show that sex interacts with the host circadian clock, and they collectively shape the circadian rhythmicity and composition of the fecal microbiota in mice.The composition of intestinal microbiota is influenced by host genetics (1), aging (2), antibiotic exposure (3), lifestyle (4), diet (5), pet ownership (6), and concomitant disease (7, 8). The impact of diet in shaping the composition of the microbiota has been well established in both humans and mice (9, 10). The type of food consumed and also the feeding behavior of the host influence the microbiota. For example, a 24-h fast increases the abundance of Bacteroidetes and reduces that of Firmicutes in mouse cecum, without altering the communal microbial diversity (11). Bacteroidetes are also dominant in the microbiota of the fasted Burmese python, whereas ingestion of a meal shifts the intestinal composition toward Firmicutes (12).The rotation of the earth results in the oscillation of light during the 24-h cycle. Organisms adapted to this cycle by developing a circadian rhythm, an endogenous and entrainable mechanism that times daily events such as feeding, temperature, sleep-wakefulness, hormone secretion, and metabolic homeostasis (13, 14). In mammals, this rhythm is controlled by a master clock that resides in the suprachiasmatic nucleus of the hypothalamus. It responds to the changing light cycle and signals this information to peripheral clocks in most tissues (15). The core mammalian clock is comprised of activators BMAL1 and CLOCK as well as repressors PERIOD (PER) and CRYPTOCHROME (CRY), forming an interlocked regulatory loop (14).Circadian rhythms also exist in fungi and cyanobacteria (16). For example, a pacemaker in cyanobacteria transduces the oscillating daylight signal to regulate gene expression and to time cell division (17, 18). Hence, the synchronization of endogenous circadian rhythms with the environment is crucial for the survival of the bacteria as well as metazoa.Recent studies show that the intestinal microbiota undergo diurnal oscillation under the control of host feeding time, and that ablation of the host molecular clock Per genes causes dysbiosis (19, 20). Here, we report that microbial composition and its oscillation are influenced by the host clock, including the Bmal1-dependent forward limb of the signaling pathway. We also find that rhythmicity is conditioned by the sex of the host, being more pronounced in females than in males.     

Dim light in the evening causes coordinated realignment of circadian rhythms,sleep, and short-term memory

Light provides the primary signal for entraining circadian rhythms to the day/night cycle. In addition to rods and cones, the retina contains a small population of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). Concerns have been raised that exposure to dim artificial lighting in the evening (DLE) may perturb circadian rhythms and sleep patterns, and OPN4 is presumed to mediate these effects. Here, we examine the effects of 4-h, 20-lux DLE on circadian physiology and behavior in mice and the role of OPN4 in these responses. We show that 2 wk of DLE induces a phase delay of ∼2 to 3 h in mice, comparable to that reported in humans. DLE-induced phase shifts are unaffected in Opn4^−/− mice, indicating that rods and cones are capable of driving these responses in the absence of melanopsin. DLE delays molecular clock rhythms in the heart, liver, adrenal gland, and dorsal hippocampus. It also reverses short-term recognition memory performance, which is associated with changes in preceding sleep history. In addition, DLE modifies patterns of hypothalamic and cortical cFos signals, a molecular correlate of recent neuronal activity. Together, our data show that DLE causes coordinated realignment of circadian rhythms, sleep patterns, and short-term memory process in mice. These effects are particularly relevant as DLE conditions―due to artificial light exposure―are experienced by the majority of the populace on a daily basis.

Light exerts profound effects on physiology and behavior, synchronizing biological rhythms to the light/dark cycle (LD) as well as directly modulating alertness and sleep (1, 2). In mammals, light detected by the eye is the primary time cue synchronizing circadian rhythms of activity and rest, a process termed entrainment. Exposure to light at dawn and dusk plays a key role, adjusting the phase of the master circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) (3–5). Studies on the photoreceptors mediating circadian entrainment led to the identification of a distinct photoreceptor system consisting of a subset of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4) (6, 7). These cells have a peak sensitivity at ∼480 nm (8, 9), hence differing from the classical visual system, which in humans is most sensitive to light at ∼555 nm, corresponding to the red and green cones of the fovea (10). In addition to modulating image-forming responses via local retinal circuitry, OPN4-expressing pRGC axons project to the SCN and different brain areas, setting the circadian clock and driving nonvisual responses to light (5, 7, 11).How does the mammalian brain adapt to changes in daylength? In humans, exposure to long-day photoperiods delays melatonin onset but advances melatonin offset, hence compressing the internal biological night, relative to short-day photoperiods; this is observed in laboratory studies (12, 13) as well as under naturalistic conditions (14, 15). In laboratory mice, the onset and offset of wheel-running activity change dynamically in response to daylength (16). Long-day photoperiods also cause functional reorganization in the SCN. In vivo multiunit recording in mice shows that 16-h light/8-h dark cycles (16:8 LD) weakens phase clustering of SCN neurons (17). Similarly, PERIOD2::LUCIFERASE bioluminescence signals in the mouse dorsal versus ventral SCN are dissociated after 20:4 LD (18). Weakened intercellular coupling in the SCN reflects a form of plasticity, enhancing adaptability of the circadian system to an increase in daylength (19). In addition, 19:5 LD reduces the number of dopamine neurons in the hypothalamus, increasing behavioral immobility and decreasing exploratory activity in rats (20) and mice (21); seasonal variation in photoperiod is also associated with changes in dopamine levels in the human midbrain (22). In the mouse hippocampus, molecular rhythms such as Per1,2 and Cry1,2 are blunted under 20:4 LD (23); however, the consequence is complex: it improves object and spatial discrimination in the spontaneous recognition memory task but disrupts context discrimination in the fear conditioning task (23).Aberrant lighting at night may lead to disrupted circadian rhythms and sleep, which are associated with many adverse health outcomes, including impaired concentration and performance, mood disturbances, metabolic diseases, cardiovascular and neurological disorders, and cancer (24–26). Numerous studies have characterized the disruptive effects of dim light at night (DLAN) on metabolic and mood-related processes in rodents. In these studies, animals were exposed to dim light for the entire night (27–35). As such, DLAN is highly relevant to conditions in which low-level light exposure continues throughout the night, such as light pollution. However, DLAN is somewhat different from exposure to artificial electrical lighting as experienced by the majority of the populace, who typically experience higher light levels during the day (though lower than natural daylight) but dim light for a short period in the evening (DLE) (14, 15, 36, 37). In humans, DLE exposure delays melatonin rhythms and sleep timing (14, 15, 37) and reduces alertness on the subsequent day (36); these phase-delaying effects of DLE on the circadian system are found under both natural summer (14) and winter photoperiods (15). As such, DLE combines features of both long-day photoperiods and DLAN. While similar to a long-day photoperiod, the extended light phase is of a lower light intensity and may exert different effects in comparison with the higher light levels during the day. Conversely, unlike DLAN, under DLE the evening light exposure only occurs at the start of the biological night when the circadian system is most sensitive to light-induced phase delays (38).Although the effects of long-day photoperiods (12–23) and DLAN (27–35) on circadian physiology and behavior have been extensively studied, the effects of DLE—as produced by artificial light exposure—have received less attention. Here, we investigate the effects of 2 wk of DLE in laboratory mice and the role of OPN4 in mediating these responses. Our choice of dim-light level and duration was based upon human studies conducted in nonlaboratory settings (14, 15, 36, 37), which reported that ∼3 to 4 h of ∼20 to 30 lx artificial lighting exposure increased alertness before bedtime, delayed melatonin timing and sleep onset, and increased sleepiness in the morning. Despite their nocturnality, the mouse phase response curve (PRC) is broadly similar to the human PRC: in both species, light presented during the early night delays circadian rhythms, whereas light presented later at night or early in the morning causes phase advances (5, 38, 39). Our DLE protocol comprises a 12-h light phase at 200 lx, a 4-h evening light period at 20 lx, and an 8-h dark phase. Here, we characterize the effects of 4-h, 20-lux DLE on a) locomotor activity rhythms, b) sleep patterns, c) molecular clocks in peripheral tissues, d) short-term memory process, and e) brain cFos signals.     

A neuropeptide speeds circadian entrainment by reducing intercellular synchrony

Shift work or transmeridian travel can desynchronize the body''s circadian rhythms from local light–dark cycles. The mammalian suprachiasmatic nucleus (SCN) generates and entrains daily rhythms in physiology and behavior. Paradoxically, we found that vasoactive intestinal polypeptide (VIP), a neuropeptide implicated in synchrony among SCN cells, can also desynchronize them. The degree and duration of desynchronization among SCN neurons depended on both the phase and the dose of VIP. A model of the SCN consisting of coupled stochastic cells predicted both the phase- and the dose-dependent response to VIP and that the transient phase desynchronization, or “phase tumbling”, could arise from intrinsic, stochastic noise in small populations of key molecules (notably, Period mRNA near its daily minimum). The model also predicted that phase tumbling following brief VIP treatment would accelerate entrainment to shifted environmental cycles. We tested this using a prepulse of VIP during the day before a shift in either a light cycle in vivo or a temperature cycle in vitro. Although VIP during the day does not shift circadian rhythms, the VIP pretreatment approximately halved the time required for mice to reentrain to an 8-h shifted light schedule and for SCN cultures to reentrain to a 10-h shifted temperature cycle. We conclude that VIP below 100 nM synchronizes SCN cells and above 100 nM reduces synchrony in the SCN. We show that exploiting these mechanisms that transiently reduce cellular synchrony before a large shift in the schedule of daily environmental cues has the potential to reduce jet lag.Circadian rhythms of living organisms entrain (synchronize) to daily environmental cues such as light and dark. Living organisms have not evolved to make large daily adjustments in their circadian timing, so it is a challenge for them to respond to changes such as those that humans are subjected to during shift work and travel across time zones. Long-term misalignment between internal circadian rhythms in mammals and environmental cycles can induce physiological and psychological abnormalities, including depression, cancer, heart problems, obesity, and increased mortality (1, 2).The master circadian pacemaker in mammals, the bilateral suprachiasmatic nucleus (SCN), is composed of ∼20,000 neurons that synchronize their daily rhythms to each other and entrain to ambient light cycles (3, 4). Vasoactive intestinal polypeptide (VIP), released in the SCN as a function of circadian time and light intensity (5–7), plays a critical role in this circadian synchronization. In the absence of VIP or its receptor, VPAC2R, SCN neurons fail to synchronize to each other and consequently many daily rhythms of the organism are lost (8–12). The addition of VIP to SCN cultures induces the production of Period (Per) 1 and 2 (13), two genes implicated in light-induced resetting (14–16), and shifts rhythms in behavior and SCN physiology (17–21). Notably, daily addition of VIP or an agonist to the VIP receptor, VPAC2R, entrains rhythms in SCN explants and Vip^−/− SCN neurons (21, 22). Thus, VIP is thought to play a role in both the synchronization between SCN cells when the organism is in the dark and the entrainment of the SCN when the organism is exposed to a light cycle.Exposure to a brief light pulse has been shown to reduce the amplitude of circadian rhythms in humans (23), insects (24, 25), and cell lines expressing transgenic melanopsin (26, 27). Lower-amplitude oscillations have been associated with larger shifts in behavioral and physiological rhythms (28, 29) and the ability to entrain to a wider range of periods (30, 31). An explanation of the cause of this amplitude reduction was given as phase dispersion among oscillators or damping of individual circadian oscillators or both. However, whether or how circadian cells in a network modulate their amplitudes or synchrony to entrain to the environment has not been fully determined. Several of the studies mentioned here have suggested that reducing synchrony among oscillators might allow them to entrain faster. Following an unexpected finding that VIP rapidly reduced the amplitude of SCN tissue rhythms, we tested the hypotheses that (i) VIP can either increase or decrease synchrony among SCN cells, depending on its concentration and time of release, and (ii) VIP can accelerate entrainment through transient phase dispersion (“phase tumbling”) among SCN cells.     

A functional division of Drosophila sweet taste neurons that is value-based and task-specific

Sucrose is an attractive feeding substance and a positive reinforcer for Drosophila. But Drosophila females have been shown to robustly reject a sucrose-containing option for egg-laying when given a choice between a plain and a sucrose-containing option in specific contexts. How the sweet taste system of Drosophila promotes context-dependent devaluation of an egg-laying option that contains sucrose, an otherwise highly appetitive tastant, is unknown. Here, we report that devaluation of sweetness/sucrose for egg-laying is executed by a sensory pathway recruited specifically by the sweet neurons on the legs of Drosophila. First, silencing just the leg sweet neurons caused acceptance of the sucrose option in a sucrose versus plain decision, whereas expressing the channelrhodopsin CsChrimson in them caused rejection of a plain option that was “baited” with light over another that was not. Analogous bidirectional manipulations of other sweet neurons did not produce these effects. Second, circuit tracing revealed that the leg sweet neurons receive different presynaptic neuromodulations compared to some other sweet neurons and were the only ones with postsynaptic partners that projected prominently to the superior lateral protocerebrum (SLP) in the brain. Third, silencing one specific SLP-projecting postsynaptic partner of the leg sweet neurons reduced sucrose rejection, whereas expressing CsChrimson in it promoted rejection of a light-baited option during egg-laying. These results uncover that the Drosophila sweet taste system exhibits a functional division that is value-based and task-specific, challenging the conventional view that the system adheres to a simple labeled-line coding scheme.

The taste systems of many animal species are known to possess a dedicated “channel” for detecting sugars, a class of chemicals that is highly nutritious. For example, mice have been shown to encode gustatory receptors that specifically sense sugars, and the taste neurons that express these sugar receptors on their tongues generally do not express receptors that sense chemicals of another taste modality (e.g., bitterness) (1–3). Furthermore, activation of these sugar-sensing taste neurons by artificial means has been shown to be able to drive appetitive sugar-induced innate responses (e.g., licking) and act as a positive reinforcer for learning (3–5). In some recent studies, these properties of the sweet taste neurons have been found to be present in some of their central nervous system (CNS) targets (e.g., taste-sensitive neurons in the insular cortex), too (6, 7). Thus, one school of thought is that taste coding for sweetness in mice may follow the simple “labeled-line” rule: sweet taste neurons, and potentially some of their central targets, are hardwired to detect sugars specifically and drive sugar-induced reinforcing neural signals and appetitive behaviors (1–7).Drosophila melanogaster also possess sugar-detecting taste neurons. Pioneering early studies have shown that sugar-sensing taste neurons in flies are molecularly, anatomically, and functionally distinct from taste neurons that sense bitterness; sweet-sensing and bitter-sensing taste neurons express different gustatory receptors, project axons to different areas in the brain, and are required to promote different (appetitive versus aversive) behaviors (8–12). Moreover, the activation of sweet neurons by artificial means can drive appetitive behaviors and act as a positive reinforcer for learning (10, 13, 14), while artificial activation of bitter-sensing neurons can induce rejection behaviors and be used as a punishment for learning (10, 13, 15). Interestingly, while these results suggest that Drosophila sweet neurons and their mammalian counterparts have some shared properties, subsequent studies suggest that significant differences exist between them, too. First, the Drosophila genome appears to encode many more sweet receptors than mouse genome does (12, 16–23). Second, Drosophila sweet neurons appear to be able to detect some chemicals that belong to another taste modality [e.g., acetic acid (AA)] (24–27). Third, Drosophila sweet neurons can be found on several body parts (e.g., proboscis and legs) (8, 12, 18, 20, 23, 28–30). Interestingly, sweet neurons on different body parts of Drosophila do not promote identical behavioral outputs (8, 20, 23, 24, 28, 29). For example, labellar sweet neurons and esophageal sweet neurons on the proboscis have been shown to promote proboscis extension reflex (PER) and ingestion, respectively, whereas leg sweet neurons have been shown to promote PER and slowing down of locomotion (8, 12, 28, 29). Collectively, these results suggest that in contrast to the apparent homogeneity of sweet neurons in some mammals, a functional division exists among Drosophila sweet neurons, although the different behavioral responses promoted by different Drosophila sweet neurons generally appear appetitive in nature.In this work, we report yet another striking feature of Drosophila sweet neurons that sets them apart from their mammalian counterparts, namely a functional division that is value-based and task-specific. We discovered this by taking advantage of a context-dependent but highly robust sugar rejection behavior exhibited by egg-laying females (31–34). Previous studies have shown that when selecting for egg-laying site in a small enclosure (dimension ∼16 × 10 × 18 mm), Drosophila readily accept a sucrose-containing agarose for egg-laying when it is the sole option but strongly reject it when a plain option is also available (31, 32). Importantly, silencing their sweet neurons causes the females to no longer reject the sucrose option when choosing between the sucrose versus plain options (31, 32). Thus, in addition to promoting appetitive behaviors and acting as a positive reinforcer, activation of sweet neurons on an egg-laying option can also decrease the value of such an option (thereby causing its rejection over an option that does not activate sweet neurons). These observations not only suggest the existence of an apparent “antiappetitive” role of Drosophila sweet neurons when the task of animals is to select for egg-laying sites but also raise a key question as to whether such counterintuitive, value-decreasing property of sweetness detection during egg-laying may be 1) solely an emergent property of specific neurons in the brain that respond similarly to all peripheral sweet neurons but are sensitive to animals’ behavioral goal and context or 2) carried out by specific sweet neurons at the periphery and then transmitted into the brain via a unique neural pathway activated by these neurons. To disambiguate between these possibilities, we genetically targeted different subsets of sweet neurons to assess their circuit properties as well as their behavioral roles as the animals decided in either a regular or a virtual sweet versus plain decision during egg-laying, taking advantage of a high-throughput closed-loop optogenetic stimulation platform we developed recently. Our collective results support the second scenario and suggest that the value-decreasing property of sweetness/sucrose is conveyed specifically by the sweet neurons on the legs of Drosophila—and not by other sweet neurons—and the unique postsynaptic target(s) of the leg sweet neurons that send long-range projections to the superior lateral protocerebrum (SLP) in the brain. These results reveal a previously unappreciated functional and anatomical division of the Drosophila sweet taste neurons that is both task-specific and value-based, pointing to a level of complexity and sophistication that seems unmatched by their mammalian counterparts so far.