A single pair of leucokinin neurons are modulated by feeding state and regulate sleep-metabolism interactions.

Dysregulation of sleep and feeding has widespread health consequences. Despite extensive epidemiological evidence for interactions between sleep and metabolic function, little is known about the neural or molecular basis underlying the integration of these processes. D. melanogaster potently suppress sleep in response to starvation, and powerful genetic tools allow for mechanistic investigation of sleep-metabolism interactions. We have previously identified neurons expressing the neuropeptide leucokinin (Lk) as being required for starvation-mediated changes in sleep. Here, we demonstrate an essential role for Lk neuropeptide in metabolic regulation of sleep. The activity of Lk neurons is modulated by feeding, with reduced activity in response to glucose and increased activity under starvation conditions. Both genetic silencing and laser-mediated microablation localize Lk-dependent sleep regulation to a single pair of Lk neurons within the Lateral Horn (LHLK neurons). A targeted screen identified a role for 5' adenosine monophosphate-activated protein kinase (AMPK) in starvation-modulated changes in sleep. Knockdown of AMPK in Lk neurons suppresses sleep and increases LHLK neuron activity in fed flies, phenocopying the starvation state. Further, we find a requirement for the Lk receptor in the insulin-producing cells (IPCs), suggesting LHLK-IPC connectivity is critical for sleep regulation under starved conditions. Taken together, these findings localize feeding-state-dependent regulation of sleep to a single pair of neurons within the fruit fly brain and provide a system for investigating the cellular basis of sleep-metabolism interactions

Modulation of Drosophila post-feeding physiology and behavior by the neuropeptide leucokinin.

Behavior and physiology are orchestrated by neuropeptides acting as central neuromodulators and circulating hormones. An outstanding question is how these neuropeptides function to coordinate complex and competing behaviors. In Drosophila, the neuropeptide leucokinin (LK) modulates diverse functions, but mechanisms underlying these complex interactions remain poorly understood. As a first step towards understanding these mechanisms, we delineated LK circuitry that governs various aspects of post-feeding physiology and behavior. We found that impaired LK signaling in Lk and Lk receptor (Lkr) mutants affects diverse but coordinated processes, including regulation of stress, water homeostasis, feeding, locomotor activity, and metabolic rate. Next, we sought to define the populations of LK neurons that contribute to the different aspects of this physiology. We find that the calcium activity in abdominal ganglia LK neurons (ABLKs), but not in the two sets of brain neurons, increases specifically following water consumption, suggesting that ABLKs regulate water homeostasis and its associated physiology. To identify targets of LK peptide, we mapped the distribution of Lkr expression, mined a brain single-cell transcriptome dataset for genes coexpressed with Lkr, and identified synaptic partners of LK neurons. Lkr expression in the brain insulin-producing cells (IPCs), gut, renal tubules and chemosensory cells, correlates well with regulatory roles detected in the Lk and Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) and transcripts in IPCs and increased starvation resistance. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, our data suggest that the three sets of LK neurons have different targets, but modulate the establishment of post-prandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating feeding-related neuroendocrine regulation of vital behavior and physiology

Ade2 Functions in the Drosophila Fat Body To Promote Sleep

Metabolic state is a potent modulator of sleep and circadian behavior, and animals acutely modulate their sleep in accordance with internal energy stores and food availability. Across phyla, hormones secreted from adipose tissue act in the brain to control neural physiology and behavior to modulate sleep and metabolic state. Growing evidence suggests the fat body is a critical regulator of complex behaviors, but little is known about the genes that function within the fat body to regulate sleep. To identify molecular factors functioning in non-neuronal tissues to regulate sleep, we performed an RNAi screen selectively knocking down genes in the fat body. We found that knockdown of Phosphoribosylformylglycinamidine synthase/Pfas (Ade2), a highly conserved gene involved the biosynthesis of purines, sleep regulation and energy stores. Flies heterozygous for multiple Ade2 mutations are also short sleepers and this effect is partially rescued by restoring Ade2 to the Drosophila fat body. Targeted knockdown of Ade2 in the fat body does not alter arousal threshold or the homeostatic response to sleep deprivation, suggesting a specific role in modulating baseline sleep duration. Together, these findings suggest Ade2 functions within the fat body to promote both sleep and energy storage, providing a functional link between these processes

A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste

<div><p>Fat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. The highly palatable taste of free fatty acids (FAs), one of the building blocks of fat, promotes food consumption, activates reward circuitry, and is thought to contribute to hedonic feeding underlying many metabolism-related disorders. Despite a role in the etiology of metabolic diseases, little is known about how dietary fats are detected by the gustatory system to promote feeding. Previously, we showed that a broad population of sugar-sensing taste neurons expressing Gustatory Receptor 64f (<i>Gr64f</i>) is required for reflexive feeding responses to both FAs and sugars. Here, we report a genetic silencing screen to identify specific populations of taste neurons that mediate fatty acid (FA) taste. We find neurons identified by expression of Ionotropic Receptor 56d (<i>IR56d</i>) are necessary and sufficient for reflexive feeding response to FAs. Functional imaging reveals that IR56d-expressing neurons are responsive to short- and medium-chain FAs. Silencing IR56d neurons selectively abolishes FA taste, and their activation is sufficient to drive feeding responses. Analysis of co-expression with Gr64f identifies two subpopulations of IR56d-expressing neurons. While physiological imaging reveals that both populations are responsive to FAs, IR56d/Gr64f neurons are activated by medium-chain FAs and are sufficient for reflexive feeding response to FAs. Moreover, flies can discriminate between sugar and FAs in an aversive taste memory assay, indicating that FA taste is a unique modality in <i>Drosophila</i>. Taken together, these findings localize FA taste within the <i>Drosophila</i> gustatory center and provide an opportunity to investigate discrimination between different categories of appetitive tastants.</p></div

IR56d neurons are selectively responsive to short and medium-chain FAs.

<p>(A) PER in response to short-chain pentanoic acid (PeA, n = 46), medium-chain octanoic acid (OcA, n = 26), and long-chain oleic acid (n = 46) in TNT and control impTNT flies. Blocking synaptic release in IR56d neurons with TNT significantly decreases PER to octanoic acid (n = 29), but does not affect PER for pentanoic (n = 42) or oleic acid (n = 42). Wilcoxon Rank-Sum Test; ***<i>p</i><0.001. (B) Average peak %ΔF/F for the posterior projections of IR56d neurons in response to water, 1% DMSO, pentanoic acid, octanoic acid, and oleic acid (n = 9, 8, 9, 9, 8, respectively), and (C) the anterior projections (n = 9, 7, 9, 9, 7, respectively). Error bars indicate SEM. One-way ANOVA with Tukey’s HSD; ***<i>p</i><0.001.</p

IR56d anterior projections are dispensable for PER to FAs.

<p>(A) IR56d and Gr64f neurons visualized with IR56d-GAL4 driving RFP and Gr64f-LexA driving GFP. Co-localization is detected in the posterior projections, but is minimal in anterior projections. (B) Driving the GAL4 repressor Gal80 with Gr64f-LexA limits GFP expression to the non-overlapping IR56d anterior projections. (C) Restricting TNT expression to the non-overlapping anterior projection neurons does not significantly impact PER to sugar or HxA (impTNT n = 23; TNT n = 22). Error bars indicate SEM. Wilcoxon Rank Sum Test Sucrose: <i>p</i>>0.98; HxA: <i>p</i>>0.96. (D) Restricting TRPA1 expression to the non-overlapping neurons abolishes PER compared to control in which TRPA1 is driven in all IR56d neurons. Fisher’s Exact Test with Bonferroni correction for multiple comparisons; ***<i>p</i><0.001.</p

Gr64f gustatory receptor neurons respond to sucrose and HxA.

<p>(A) Expression of GFP in Gr64f-GAL4 neurons reveals axon terminals in the sub-esophagael zone (SEZ). Scale bar = 50 μm. (B) Diagram of live-imaging experimental protocol. Cuticle above the SEZ is removed and GCaMP5 fluorescence is recorded while tastant is applied to the proboscis. (C) Average peak %ΔF/F during response to water, 1% HxA, and 10mM sucrose (n = 11, 11, 10 respectively). Error bars indicate SEM. One-way ANOVA with Tukey’s HSD; ***<i>p</i><0.001. (D) Average %ΔF/F traces and representative images of calcium activity in Gr64f neurons responding to water, (E) 1% HxA, and (F) 10mM sucrose. Scale bar = 50 μm. Shaded region of trace indicates +/- SEM.</p

<i>Drosophila</i> discriminates between sucrose and HxA.

<p>(A) Taste memory protocol to determine sucrose and FA discrimination. Flies are trained by pairing HxA or sucrose on tarsi with quinine on proboscis. PER in response to HxA and sucrose is then tested following training to sucrose or HxA in the absence of quinine. In control experiments (naïve), the same procedure is followed, but quinine is not applied to the proboscis. (B) The pairing of sucrose and quinine (dark green circles) results in a significant reduction in PER across all three training trials compared to unpaired naïve flies (light green circles). PER response to sucrose in the test is significantly lower in trained flies compared to naïve flies (n = 7, 11), but no differences in response to HxA (triangles) is detected between the experimental and naïve groups (n = 11, 12). Kruskal-Wallis Test followed by Dunn’s Test (control: w¹¹¹⁸); **<i>p</i><0.01. (C) The pairing of HxA and quinine (dark blue triangles) results in a significant reduction in PER across all three training trials compared to unpaired naïve flies (light green triangles). The test PER response to HxA alone is significantly lower in trained flies compared to naïve flies (n = 13, 16), but no differences in response to sucrose (circles) is detected between the groups (n = 12, 12). Kruskal-Wallis Test followed by Dunn’s Test (control: w¹¹¹⁸); **<i>p</i><0.01. (D) Percent suppression of PER reveals that flies trained and tested to the same tastant (either sucrose or HxA) show significantly reduced PER compared to flies trained and tested with different tastants.</p

IR56d neurons are necessary and sufficient for PER to FAs.

<p>(A) Expression pattern of IR56d neurons visualized with GFP. Distinct regions of projection are seen in (B) posterior and (C) anterior regions of SEZ. Scale bar = 50 μm. (D) Blocking synaptic release by genetic expression of light-chain tetanus toxin (TNT) in IR56d neurons significantly reduces PER to HxA, but not sucrose, compared to control flies expressing an inactive form of tetanus toxin (impTNT). (impTNT n = 26; TNT n = 29). Wilcoxon Rank Sum Test; ***<i>p</i><0.001. (E) Heat activation of IR56d neurons with TRPA1 induces significant PER compared to either transgene alone, and is comparable to PER induced by activation of Gr64f neurons. (n = 49 for all genotypes). Fisher’s Exact Test with Bonferroni correction for multiple comparisons. (F) Targeted knockdown of <i>norpA</i> in IR56d neurons significantly reduces response to HxA, while response to sucrose did not differ from controls flies harboring IR56d-GAL4 alone (N>27 for all genotypes); ***<i>p</i>< 0.001.</p

translin Is Required for Metabolic Regulation of Sleep

Dysregulation of sleep or feeding has enormous health consequences. In humans, acute sleep loss is associated with increased appetite and insulin insensitivity, while chronically sleep-deprived individuals are more likely to develop obesity, metabolic syndrome, type II diabetes, and cardiovascular disease. Conversely, metabolic state potently modulates sleep and circadian behavior; yet, the molecular basis for sleep-metabolism interactions remains poorly understood. Here, we describe the identification of translin (trsn), a highly conserved RNA/DNA binding protein, as essential for starvation-induced sleep suppression. Strikingly, trsn does not appear to regulate energy stores, free glucose levels, or feeding behavior suggesting the sleep phenotype of trsn mutant flies is not a consequence of general metabolic dysfunction or blunted response to starvation. While broadly expressed in all neurons, trsn is transcriptionally upregulated in the heads of flies in response to starvation. Spatially restricted rescue or targeted knockdown localizes trsn function to neurons that produce the tachykinin family neuropeptide Leucokinin. Manipulation of neural activity in Leucokinin neurons revealed these neurons to be required for starvation-induced sleep suppression. Taken together, these findings establish trsn as an essential integrator of sleep and metabolic state, with implications for understanding the neural mechanism underlying sleep disruption in response to environmental perturbation