Developmental emergence of sleep rhythms enables long-term memory in Drosophila.

In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day. The mechanism through which sleep rhythms emerge, and consequent advantage conferred to a juvenile animal, is unknown. In the second-instar Drosophila larvae (L2), like in human infants, sleep is not under circadian control. We identify the precise developmental time point when the clock begins to regulate sleep in Drosophila, leading to emergence of sleep rhythms in early third-instars (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousal-promoting Dh44 neurons, bringing arousal under clock control to drive emergence of circadian sleep. Last, we demonstrate that L3 but not L2 larvae exhibit long-term memory (LTM) of aversive cues and that this LTM depends upon deep sleep generated once sleep rhythms begin. We propose that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.

Inducible Reporter Lines for Tissue-specific Monitoring of Drosophila Circadian Clock Transcriptional Activity.

Organisms track time of day through the function of cell-autonomous molecular clocks. In addition to a central clock located in the brain, molecular clocks are present in most peripheral tissues. Circadian clocks are coordinated within and across tissues, but the manner through which this coordination is achieved is not well understood. We reasoned that the ability to track in vivo molecular clock activity in specific tissues of the fruit fly, Drosophila melanogaster, would facilitate an investigation into the relationship between different clock-containing tissues. Previous efforts to monitor clock gene expression in single flies in vivo have used regulatory elements of several different clock genes to dictate expression of a luciferase reporter enzyme, the activity of which can be monitored using a luminometer. Although these reporter lines have been instrumental in our understanding of the circadian system, they generally lack cell specificity, making it difficult to compare molecular clock oscillations between different tissues. Here, we report the generation of several novel lines of flies that allow for inducible expression of a luciferase reporter construct for clock gene transcriptional activity. We find that these lines faithfully report circadian transcription, as they exhibit rhythmic luciferase activity that is dependent on a functional molecular clock. Furthermore, we take advantage of our reporter lines' tissue specificity to demonstrate that peripheral molecular clocks are able to retain rhythmicity for multiple days under constant environmental conditions.

Central and Peripheral Clock Control of Circadian Feeding Rhythms.

Many behaviors exhibit ~24-h oscillations under control of an endogenous circadian timing system that tracks time of day via a molecular circadian clock. In the fruit fly, Drosophila melanogaster, most circadian research has focused on the generation of locomotor activity rhythms, but a fundamental question is how the circadian clock orchestrates multiple distinct behavioral outputs. Here, we have investigated the cells and circuits mediating circadian control of feeding behavior. Using an array of genetic tools, we show that, as is the case for locomotor activity rhythms, the presence of feeding rhythms requires molecular clock function in the ventrolateral clock neurons of the central brain. We further demonstrate that the speed of molecular clock oscillations in these neurons dictates the free-running period length of feeding rhythms. In contrast to the effects observed with central clock cell manipulations, we show that genetic abrogation of the molecular clock in the fat body, a peripheral metabolic tissue, is without effect on feeding behavior. Interestingly, we find that molecular clocks in the brain and fat body of control flies gradually grow out of phase with one another under free-running conditions, likely due to a long endogenous period of the fat body clock. Under these conditions, the period of feeding rhythms tracks with molecular oscillations in central brain clock cells, consistent with a primary role of the brain clock in dictating the timing of feeding behavior. Finally, despite a lack of effect of fat body selective manipulations, we find that flies with simultaneous disruption of molecular clocks in multiple peripheral tissues (but with intact central clocks) exhibit decreased feeding rhythm strength and reduced overall food intake. We conclude that both central and peripheral clocks contribute to the regulation of feeding rhythms, with a particularly dominant, pacemaker role for specific populations of central brain clock cells.

Dorsal clock neurons in Drosophila sculpt locomotor outputs but are dispensable for circadian activity rhythms.

The circadian system is comprised three components: a network of core clock cells in the brain that keeps time, input pathways that entrain clock cells to the environment, and output pathways that use this information to ensure appropriate timing of physiological and behavioral processes throughout the day. Core clock cells can be divided into molecularly distinct populations that likely make unique functional contributions. Here we clarify the role of the dorsal neuron 1 (DN1) population of clock neurons in the transmission of circadian information by the Drosophila core clock network. Using an intersectional genetic approach that allowed us to selectively and comprehensively target DN1 cells, we show that suppressing DN1 neuronal activity alters the magnitude of daily activity and sleep without affecting overt rhythmicity. This suggests that DN1 cells are dispensable for both the generation of circadian information and the propagation of this information across output circuits.

Slowpoke functions in circadian output cells to regulate rest:activity rhythms.

The circadian system produces ~24-hr oscillations in behavioral and physiological processes to ensure that they occur at optimal times of day and in the correct temporal order. At its core, the circadian system is composed of dedicated central clock neurons that keep time through a cell-autonomous molecular clock. To produce rhythmic behaviors, time-of-day information generated by clock neurons must be transmitted across output pathways to regulate the downstream neuronal populations that control the relevant behaviors. An understanding of the manner through which the circadian system enacts behavioral rhythms therefore requires the identification of the cells and molecules that make up the output pathways. To that end, we recently characterized the Drosophila pars intercerebralis (PI) as a major circadian output center that lies downstream of central clock neurons in a circuit controlling rest:activity rhythms. We have conducted single-cell RNA sequencing (scRNAseq) to identify potential circadian output genes expressed by PI cells, and used cell-specific RNA interference (RNAi) to knock down expression of ~40 of these candidate genes selectively within subsets of PI cells. We demonstrate that knockdown of the slowpoke (slo) potassium channel in PI cells reliably decreases circadian rest:activity rhythm strength. Interestingly, slo mutants have previously been shown to have aberrant rest:activity rhythms, in part due to a necessary function of slo within central clock cells. However, rescue of slo in all clock cells does not fully reestablish behavioral rhythms, indicating that expression in non-clock neurons is also necessary. Our results demonstrate that slo exerts its effects in multiple components of the circadian circuit, including PI output cells in addition to clock neurons, and we hypothesize that it does so by contributing to the generation of daily neuronal activity rhythms that allow for the propagation of circadian information throughout output circuits.

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.

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.

Chronic circadian misalignment results in reduced longevity and large-scale changes in gene expression in Drosophila.

BACKGROUND: Circadian clocks are found in nearly all organisms, from bacteria to mammals, and ensure that behavioral and physiological processes occur at optimal times of day and in the correct temporal order. It is becoming increasingly clear that chronic circadian misalignment (CCM), such as occurs in shift workers or as a result of aberrant sleeping and eating schedules common to modern society, has profound metabolic and cognitive consequences, but the proximate mechanisms connecting CCM with reduced organismal health are unknown. Furthermore, it has been difficult to disentangle whether the health effects are directly induced by misalignment or are secondary to the alterations in sleep and activity levels that commonly occur with CCM. Here, we investigated the consequences of CCM in the powerful model system of the fruit fly, Drosophila melanogaster. We subjected flies to daily 4-h phase delays in the light-dark schedule and used the Drosophila Activity Monitoring (DAM) system to continuously track locomotor activity and sleep while simultaneously monitoring fly lifespan. RESULTS: Consistent with previous results, we find that exposing flies to CCM leads to a ~ 15% reduction in median lifespan in both male and female flies. Importantly, we demonstrate that the reduced longevity occurs independent of changes in overall sleep or activity. To uncover potential molecular mechanisms of CCM-induced reduction in lifespan, we conducted whole body RNA-sequencing to assess differences in gene transcription between control and misaligned flies. CCM caused progressive, large-scale changes in gene expression characterized by upregulation of genes involved in response to toxic substances, aging and oxidative stress, and downregulation of genes involved in regulation of development and differentiation, gene expression and biosynthesis. CONCLUSIONS: Many of these gene expression changes mimic those that occur during natural aging, consistent with the idea that CCM results in premature organismal decline, however, we found that genes involved in lipid metabolism are overrepresented among those that are differentially regulated by CCM and aging. This category of genes is also among the earliest to exhibit CCM-induced changes in expression, thus highlighting altered lipid metabolism as a potentially important mediator of the negative health consequences of CCM.

A Peptidergic Circuit Links the Circadian Clock to Locomotor Activity.

The mechanisms by which clock neurons in the Drosophila brain confer an ∼24-hr rhythm onto locomotor activity are unclear, but involve the neuropeptide diuretic hormone 44 (DH44), an ortholog of corticotropin-releasing factor. Here we identified DH44 receptor 1 as the relevant receptor for rest:activity rhythms and mapped its site of action to hugin-expressing neurons in the subesophageal zone (SEZ). We traced a circuit that extends from Dh44-expressing neurons in the pars intercerebralis (PI) through hugin+ SEZ neurons to the ventral nerve cord. Hugin neuropeptide, a neuromedin U ortholog, also regulates behavioral rhythms. The DH44 PI-Hugin SEZ circuit controls circadian locomotor activity in a daily cycle but has minimal effect on feeding rhythms, suggesting that the circadian drive to feed can be separated from circadian locomotion. These findings define a linear peptidergic circuit that links the clock to motor outputs to modulate circadian control of locomotor activity.

The Drosophila Circadian Clock Gates Sleep through Time-of-Day Dependent Modulation of Sleep-Promoting Neurons.

STUDY OBJECTIVES: Sleep is under the control of homeostatic and circadian processes, which interact to determine sleep timing and duration, but the mechanisms through which the circadian system modulates sleep are largely unknown. We therefore used adult-specific, temporally controlled neuronal activation and inhibition to identify an interaction between the circadian clock and a novel population of sleep-promoting neurons in Drosophila. METHODS: Transgenic flies expressed either dTRPA1, a neuronal activator, or Shibire(ts1), an inhibitor of synaptic release, in small subsets of neurons. Sleep, as determined by activity monitoring and video tracking, was assessed before and after temperature-induced activation or inhibition using these effector molecules. We compared the effect of these manipulations in control flies and in mutant flies that lacked components of the molecular circadian clock. RESULTS: Adult-specific activation or inhibition of a population of neurons that projects to the sleep-promoting dorsal Fan-Shaped Body resulted in bidirectional control over sleep. Interestingly, the magnitude of the sleep changes were time-of-day dependent. Activation of sleep-promoting neurons was maximally effective during the middle of the day and night, and was relatively ineffective during the day-to-night and night-to-day transitions. These time-ofday specific effects were absent in flies that lacked functional circadian clocks. CONCLUSIONS: We conclude that the circadian system functions to gate sleep through active inhibition at specific times of day. These data identify a mechanism through which the circadian system prevents premature sleep onset in the late evening, when homeostatic sleep drive is high.

Sleep: a neuropeptidergic wake-up call for flies.

Endogenous circadian rhythms exert strong effects on sleep, but the neuronal mechanisms that produce these effects have remained obscure. New work implicates neuropeptidergic signaling in a subset of circadian clock cells in the regulation of sleep late at night.

Identification of a circadian output circuit for rest:activity rhythms in Drosophila.

Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. We conducted a screen for circadian-relevant neurons in the Drosophila brain and report here that cells of the pars intercerebralis (PI), a functional homolog of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GFP reconstitution across synaptic partners (GRASP) analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin-releasing factor (CRF) homolog, DH44, as a circadian output molecule that is specifically expressed by PI neurons and is required for normal rest:activity rhythms. Notably, selective activation or ablation of just six DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms.

The modality-specific contribution of peptidergic and non-peptidergic nociceptors is manifest at the level of dorsal horn nociresponsive neurons.

We previously demonstrated that genetic and/or pharmacological ablation of the TRPV1+/peptidergic or the MrgprD+/non-peptidergic subset of nociceptors produced selective, modality-specific deficits in the behavioural responses to heat and mechanical stimuli, respectively. To assess whether this modality-specific contribution is also manifest at the level of spinal cord neuron responsiveness, here we made extracellular recordings from lumbar dorsal horn neurons of the mouse in response to graded thermal and mechanical stimulation. We found that, following intrathecal injection of capsaicin to eliminate the central terminals of TRPV1+ nociceptors, neurons in the region of laminae I and V of the spinal cord lost responsiveness to noxious heat (whether generated by a contact heat probe or diode laser), with no change in their response to noxious mechanical stimulation. In contrast, ablation of MrgprD+ afferents did not alter the response to noxious heat, but reduced the firing of superficial dorsal horn nociceptive-specific neurons in response to graded mechanical stimulation and decreased the relative number of wide dynamic range neurons that were exclusively mechanosensitive. Neither ablation procedure reduced the number of dorsal horn neurons that responded to noxious cold. These findings support the conclusion that TRPV1+ nociceptors are necessary and probably sufficient for noxious heat activation of dorsal horn neurons and that, despite their polymodal properties, TRPV1+ and MrgprD+ nociceptors provide modality-specific contributions to the response properties of spinal cord neurons.

Restriction of transient receptor potential vanilloid-1 to the peptidergic subset of primary afferent neurons follows its developmental downregulation in nonpeptidergic neurons.

Primary afferent "pain" fibers (nociceptors) are divided into subclasses based on distinct molecular and anatomical features, and these classes mediate noxious modality-specific contributions to behaviors evoked by painful stimuli. Whether the heat and capsaicin receptor transient receptor potential vanilloid-1 (TRPV1) is expressed heterogeneously across several sensory populations, or is selectively expressed by a unique nociceptor subclass, however, is unclear. Here we used two lines of Trpv1 reporter mice to investigate the primary afferent expression of TRPV1, both during development and in the adult. We demonstrate, using Cre-induced lineage tracing, that during development TRPV1 is transiently expressed in a wide range of dorsal root ganglion neurons, and that its expression is gradually refined, such that TRPV1 transcripts become restricted to a specific subset of peptidergic sensory neurons. Finally, the remarkable sensitivity that is characteristic of these reporter mice revealed an innervation of central and peripheral targets by TRPV1+ primary afferents in the adult that is considerably more extensive than has previously been appreciated.

Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells.

The heat and capsaicin receptor, TRPV1, is required for the detection of painful heat by primary afferent pain fibers (nociceptors), but the extent to which functional TRPV1 channels are expressed in the CNS is debated. Because previous evidence is based primarily on indirect physiological responses to capsaicin, here we genetically modified the Trpv1 locus to reveal, with excellent sensitivity and specificity, the distribution of TRPV1 in all neuronal and non-neuronal tissues. In contrast to reports of widespread and robust expression in the CNS, we find that neuronal TRPV1 is primarily restricted to nociceptors in primary sensory ganglia, with minimal expression in a few discrete brain regions, most notably in a contiguous band of cells within and adjacent to the caudal hypothalamus. We confirm hypothalamic expression in the mouse using several complementary approaches, including in situ hybridization, calcium imaging, and electrophysiological recordings. Additional in situ hybridization experiments in rat, monkey, and human brain demonstrate that the restricted expression of TRPV1 in the CNS is conserved across species. Outside of the CNS, we find TRPV1 expression in a subset of arteriolar smooth muscle cells within thermoregulatory tissues. Here, capsaicin increases calcium uptake and induces vasoconstriction, an effect that likely counteracts the vasodilation produced by activation of neuronal TRPV1.

Pain behavior in the formalin test persists after ablation of the great majority of C-fiber nociceptors.

Although the formalin test is a widely used model of persistent pain, the primary afferent fiber types that underlie the cellular and behavioral responses to formalin injection are largely unknown. Here we used a combined genetic and pharmacological approach to investigate the effect of ablating subsets of primary afferent nociceptors on formalin-induced nocifensive behaviors and spinal cord Fos protein expression. Intrathecal capsaicin-induced ablation of the central terminals of TRPV1+neurons greatly reduced the behavioral responses and Fos elicited by low-dose (0.5%) formalin. In contrast, genetic ablation of the MrgprD-expressing subset of non-peptidergic unmyelinated afferents, which constitute a largely non-overlapping population, altered neither the behavior nor the Fos induced by low-dose formalin. Remarkably, nocifensive behavior following high-dose (2%) formalin was unchanged in mice lacking either afferent population, or even in mice lacking both populations, which together make up the great majority of C-fiber nociceptors. Thus, at high doses, which are routinely used in the formalin test, formalin-induced "pain" behavior persists in the absence of the vast majority of C-fiber nociceptors, which points to a contribution of a large spectrum of afferents secondary to non-specific formalin-induced tissue and nerve damage.

Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli.

Behavioral responses to painful stimuli require peripheral sensory neurons called nociceptors. Electrophysiological studies show that most C-fiber nociceptors are polymodal (i.e., respond to multiple noxious stimulus modalities, such as mechanical and thermal); nevertheless, these stimuli are perceived as distinct. Therefore, it is believed that discrimination among these modalities only occurs at spinal or supraspinal levels of processing. Here, we provide evidence to the contrary. Genetic ablation in adulthood of unmyelinated sensory neurons expressing the G protein-coupled receptor Mrgprd reduces behavioral sensitivity to noxious mechanical stimuli but not to heat or cold stimuli. Conversely, pharmacological ablation of the central branches of TRPV1(+) nociceptors, which constitute a nonoverlapping population, selectively abolishes noxious heat pain sensitivity. Combined elimination of both populations yielded an additive phenotype with no additional behavioral deficits, ruling out a redundant contribution of these populations to heat and mechanical pain sensitivity. This double-dissociation suggests that the brain can distinguish different noxious stimulus modalities from the earliest stages of sensory processing.

Accumbal neurons that are activated during cocaine self-administration are spared from inhibitory effects of repeated cocaine self-administration.

Hypoactivity of the accumbens is induced by repeated cocaine exposure and is hypothesized to play a role in cocaine addiction. However, it is difficult to understand how a general hypoactivity of the accumbens, which facilitates multiple types of motivated behaviors, could contribute to the selective increase in drug-directed behavior that defines addiction. Electrophysiological recordings, made during sessions in which rats self-administer cocaine, show that most accumbal neurons that encode events related to drug-directed behavior achieve and maintain higher firing rates during the period of cocaine exposure (Task-Activated neurons) than do other accumbal neurons (Task-Non-Activated neurons). We have hypothesized that this difference in activity makes the neurons that facilitate drug-directed behavior less susceptible than other neurons to the chronic inhibitory effects of cocaine. A sparing of neurons that facilitate drug-directed behavior from chronic hypoactivity might lead to a relative increase in the transmission of neuronal signals that facilitate drug-directed behavior through accumbal circuits and thereby contribute to changes in behavior that characterize addiction (ie differential inhibition hypothesis). A prediction of the hypothesis is that neurons that are activated in relation to task events during cocaine self-administration sessions will show less of a decrease in firing across repeated self-administration sessions than will other neurons. To test this prediction, rats were exposed to 30 daily (6 h/day) cocaine self-administration sessions. Chronic extracellular recordings of single accumbal neurons were made during the second to third session and the 30th session. Between-session comparisons showed that decreases in firing were exhibited by Task-Non-Activated, but not by Task-Activated, neurons. During the day 30 session, the magnitude of the difference in firing rate between the two groups of neurons was positively related to the propensity of animals to seek and take cocaine. The findings of the present study are consistent with a basic prediction of the differential inhibition hypothesis and may be relevant to understanding cocaine addiction.