The Interactive Fly

Zygotically transcribed genes

Hormones and neuropeptides

Neuropeptides and Circadian Rhythms

Neuropeptides and the Heart

Neuropeptides and Locomotion

Neuropeptides, Metabolism, Feeding, and Growth

Neuropeptides and Reproduction

Neuropeptides and Stress

Neuropeptides, Neural Development, Neurons, and the Synapse

Neuropeptides, Water Homeostasis and Chill Tolerance

Development of hormone-secreting cells

Neuropeptides and other peptide hormones

Non-peptide hormones

Enzymes involved in processing hormones

Genes regulating neuroendocrine cell differentiation

Genes regulating response to hormones

Conserved hormones detected by immune localization and biochemical techniques

Many of the hormones listed above have only been characterized immunologically in Drosophila; for one or two there is no evidence as yet that they even exist in Drosophila. The list is generated based on the presence of similar hormones in other insect species and is meant to reflect the complexity of the repertoire of hormones in insects in general.

Evidence for presence in Drosophila of some of the hormones listed above is based on the finding of immuno-crossreactive antigens in Drosophila neurons. Antibodies generated against bombyxin and prothoracicotropic hormone (PTTH: see Bombyx and Manduca prothoracicotropic hormone) of Bombyx mori and allatotropin, allatostatin, and diuretic hormone (DH) of Manduca sexta react with distinct sets of cells in the central nervous system of Drosophila larvae, pupae, and adults. Brain neurons immunoreactive with antibodies to bombyxin, PTTH, and DH are in strikingly similar positions to their lepidopteran counterparts, indicating that at least some Drosophila neuroendocrine cells are homologous to those in lepidoptera. Allatotropin and allatostatin-immunopositive neurons in Drosophila differ from those in lepidoptera, but many of them are identical to neurons that express the FMRFamide gene. Antibodies to bombyxin, PTTH, allatostatin, and DH also stain axons and axon terminals in the neurohemal part of the ring gland; all tested antibodies, except those against bombyxin, show positive reaction in the neurohemal area of the ventral ganglion.

Although immunoreactivity with all antibodies is variable during development, the highest levels of staining are found at developmental stages when the neuropeptides would be expected to be functioning. A genetic analysis of neuropeptide expression and function has been initiated by analyzing immunoreactivity in mutants that have prolonged larval life associated with imaginal disc overgrowth. Two of these mutants, dlg and dco, show abnormally strong immunoreactivity for allatotropin during the extended larval period; dlg mutants also show increased staining with the PTTH antibody. The reduced ecdysteroid titer and delayed or blocked metamorphosis in the mutants may be a result of altered neuropeptide production, which is probably secondary to the imaginal disc overgrowth (Zitnan, 1993).

Pacap-like activity has been detected in larvae and neuromuscular junctions that function in the adenylyl cyclase second messenger system. Vertebrate PACAP-38 triggers two muscular responses in Drosophila: an immediate depolarization and a late enhancement (Zhong, 1995b). Antibody to vertebrate PACAP-38 stains segmentally repeated larval CNS neurons as well as motor nerve terminals (Zhong, 1996). It has long been thought that the neuromuscular synapse may be a good model for the synaptic basis of learning. Binding of a PACAP-like peptide to its receptors leads to activation of Rutabaga-adenylyl cyclase by the Galpha subunit and of Ras1/Raf by the Gbeta-gamma complex: the pathways then converge to modulate potassium ion-channel activity (Zhong, 1995a and Zhong, 1996).

Pigment-dispersing hormones (PDH) are a family of octadecapeptides that have been isolated from several crustacean species. An antiserum against the crustacean PDH was used to identify PDH-immunoreactive neurons in the developing nervous systems of wild type Drosophila and the brain mutant disconnected. Particular attention was paid to a group of PDH-immunoreactive neurons at the anterior margin of the medulla, known as the pigment-dispersing factor-containing neurons close to the medulla (PDFMe neurons). This group of neurons seems to be involved in the control of adult circadian rhythms. In adults, this group consists of four to six neurons with large somata (large PDFMe neurons) and four neurons with small somata (small PDFMe neurons). Both the small and the large PDFMe neurons are identical to the ventral lateral neurons, a group of neurons containing the Period protein. Both subgroups are usually absent in adults of behaviorally arrhythmic disconnected (disco) mutants. The compound eyes of these mutants are usually disconnected from the optic lobes due to a severe defect in optic lobe development. disco mutants, as a result, have either very tiny rudiments of optic lobes if no connections are made at all (unconnected phenotype) or, if some connections are established (connected phenotype), the optic lobes have an almost normal size but are grossly disorganized. disco mutants are behaviorally arrhythmic, and the lateral neurons are generally absent in adults. In the wild type, PDH immunoreactivity is seen first in the small PDFMe neurons of 4 hour old first-instar larvae. The small PDFMe neurons persist unchanged into adulthood, whereas the large ones seem to develop halfway through metamorphosis. Beside the PDFMe neurons, three other clusters of PDH-immunoreactive neurons stain in the developing nervous systems of Drosophila and are described in detail. Two of them are located in the brain, and the third is located in the abdominal neuromeres of the thoracic nervous system. In the mutant disconnected, the larval and the adult set of PDFMe neurons are absent. The other clusters of PDH-immunoreactive neurons seemed to develop normally. The present results are consistent with the hypothesis that the PDFMe neurons are circadian pacemaker neurons that may control rhythmic processes in larvae, pupae, and adults (Helfrich-Forster, 1997).

Mating elicits two well-defined reactions in the sexually matured female of many insect species: reduction of receptivity and increased oviposition. These post-mating responses have been shown to be induced by factors synthesized in the reproductive tract of the adult male and transferred in the seminal fluid to the female during copulation. One of these factors, named Accessory gland peptide 70A (sex-peptide or SP), has been identified in Drosophila. Sex peptide, more formally termed 'Accessory gland peptide 70A', encodes a 36-amino-acid peptide that is synthesized in the accessory gland and is transferred to the female where it represses female sexual receptivity and stimulates oviposition. The peptide contains a high concentration of basic amino acids, tryptophan and hydroxyproline as well as an unique residue of unknown nature that is encoded by a leucine codon. Using an in vitro radiochemical assay, it has been shown that synthetic sex-peptide considerably activates juvenile hormone III-bisepoxide (JHB3) synthesis in the corpus allatum (CA) excised from days 3 and 4 post-eclosion virgin females. Base levels are significantly lower at emergence (day 0) than on subsequent days, and only weak stimulation is obtained on day 1, while none is obtained on day 2, when maximal basal synthesis occurs. The CA of mated females cannot be stimulated further for at least 7 days, but regains responsiveness by day 10 after mating. Synthesis of JHB3 stimulated by SP in vitro persists for at least 4 h after removal of the peptide. Development of responsiveness of the CA to SP in vitro is compared with development of the post-mating reactions of sex-peptide injected virgin females. These results suggest that the CA is a direct target for SP in vivo and that sexual maturity is established separately for the two post-mating reactions (Moshitzky, 1996).

In many sexually mature insects, egg production and oviposition are regulated as consequence of copulation. Sex-Peptide (SP) is a 36-amino-acid peptide synthesized in the accessory glands of Drosophila melanogaster males and is transferred to the female during copulation. Sex-Peptide stimulates vitellogenic oocyte progression through a putative control point at about stage 9 of oogenesis. Application of the juvenile hormone (JH) analog methoprene mimics the Sex-Peptide-mediated stimulation of vitellogenic oocyte progression in sexually mature virgin females. Apoptosis is induced by 20-hydroxyecdysone in nurse cells of stage 9 egg chambers at physiological concentrations [10(-7) M]. 20-Hydroxyecdysone thus acts as an antagonist of early vitellogenic oocyte development. However, simultaneous application of JH analog protects early vitellogenic oocytes from 20-hydroxyecdysone-induced resorption. These results suggest that the balance of these hormones in the hemolymph regulates whether oocytes will progress through the control point at stage 9 or undergo apoptosis. These data are further supported by a molecular analysis of the regulation of yolk protein synthesis and uptake into the ovary by the two hormones. It is concluded that JH is a downstream component in the Sex-Peptide response cascade and acts by stimulating vitellogenic oocyte progression and inhibiting apoptosis. Since juvenile hormone analogue does not elicit increased oviposition and reduced receptivity, Sex-Peptide must have an additional, separate effect on these two postmating responses (Soller, 1999).

SP stimulates JH biosynthesis in corpus allatum complexes isolated from sexually mature virgin females in vitro. Consistent with this finding, JH application stimulates progression of oocytes through the control point at stage 9, involving an increased uptake of from the hemolymph and an increased synthesis of yolk proteins in the ovary. JH also protects early vitellogenic oocytes from ecdysone-mediated resorption. Thus, after mating, ecdysone-mediated oocyte resorption in virgins is relieved due to the increase of JH levels. The corpus allatum is likely to be a target organ for SP action. Since application of JH neither induces a reduction in receptivity nor elicits complex behavioral change, neuronal tissues have to be considered further targets of SP (Soller, 1999 and references).

neuropeptide F (NPF) was isolated from Drosophila, based on a radioimmunoassay for a gut peptide from the corn earworm, Helicoverpa zea. A partial sequence was obtained from the fly peptide, and a genomic sequence coding for NPF was cloned after inverse polymerase chain reaction and shown to exist as a single genomic copy. The encoded, putative prepropeptide can be processed into an amidated NPF with 36 residues that is related to invertebrate NPF's and the neuropeptide Y family of vertebrates. In situ hybridization and immunocytochemistry showed that Drosophila NPF was expressed in the brain and midgut of fly larvae and adults (Brown, 1999).

Drosophila adipokinetic hormone (DAKH) is an eight amino acid member of a large arthropod neuropeptide family. The gene encoding the peptide precursor has been identified and sequenced providing an inferred precursor structure of 79 amino acids including a 46 amino acid carboxy-terminal fragment of unknown function. In situ hybridization identifies sites of DAKH synthesis towards the base of the third larval instar ring gland. Like other RPCH (red pigment concentrating hormone)/AKH family peptides, DAKH can act as a cardioaccelerator at least in prepupae. Peptide levels measured in wildtype and mutant flies possessing one or three copies of the DAKH gene suggest that the amount of neuropeptide per fly is tightly regulated (Noyes, 1995).

CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates Malpighian tubule fluid secretion via cGMP, which in turn stimulates the nitric oxide signaling pathway. Liquid chromatography analysis of adult Drosophila reveals the presence of a CAP2b-like peptide, that coelutes with Manduca sexta CAP2b and synthetic CAP2b and that has CAP2b-like effects on the M. sexta heart. CAP2b accelerates fluid secretion in Drosophila Malpighian tubules stimulated by cyclic AMP but has no effect on tubules stimulated by cyclic GMP, implying that it acts through the latter pathway. CAP2b stimulation elevates tubule cGMP levels but not those of cAMP. Both CAP2b and cGMP increase the transepithelial potential difference, suggesting that stimulation of vacuolar ATP action underlies the corresponding increases in fluid secretion (Davies, 1995).

Other hormones likely to be involved in Malpighian tubule function are the leucokinins. Leucokinins are a group of widespread insect hormones. In tubules, their major action is to raise chloride permeability through stellate cells by binding to receptors on the basolateral membrane, and so ultimately to enhance fluid secretion (Julian Dow, personal communication).

For more information about Malpighian tubule function, see The Drosophila melanogaster Malpighian tubule WWW page, maintained by Julian Dow at the University of Glasgow. In addition, the Dow laboratory maintains a sensitive map illustrating the physiology of Malpighian tubule secretory cells.

Larval salivary gland chromosomes undergo a sequential process of gene activation that can be visualized directly. Gene activation is accompanied by the process of puffing. Puffing is initiated by Ecdysone receptor activation of target genes. For information about the regulation of this process, see Polytene chromosomes, endoreduplication and puffing.

Juvenile hormones (JH), a sesquiterpenoid group of ligands that regulate developmental transitions in insects, bind to the nuclear receptor Ultraspiracle (USP). In fluorescence-based binding assays, USP protein binds JH III and JH III acid with specificity, adopting for each ligand a different final conformational state. JH III treatment of Saccharomyces cerevisiae expressing a LexA-USP fusion protein stabilizes an oligomeric association containing this protein, as detected by formation of a protein-DNA complex, and induces USP-dependent transcription in a reporter assay. Juvenile hormone acid induces a different Ultraspiracle conformation than does binding of Juvenile hormone ester. The results strongly support the inference that JH III promotes at least homodimerization of USP. It is proposed that regulation of morphogenetic transitions in invertebrates involves binding of JH or JH-like structures to USP. The demonstration that JH ester and JH acid each induce different conformational states to USP raises the possibility that these two different conformational states may confer different transcriptional activities to USP (Jones, 1997).

Juvenile hormone analog (JHA) insecticides are relatively nontoxic to vertebrates and offer effective control of certain insect pests. Recent reports of resistance in whiteflies and mosquitoes demonstrate the need to identify and understand genes for resistance to this class of insect growth regulators. Mutants of the Methoprene-tolerant (Met) gene in Drosophila melanogaster show resistance to both JHAs and JH, and previous biochemical studies have demonstrated a mechanism of resistance involving an intracellular JH binding-protein that has reduced ligand affinity in Met flies. Met flies are resistant to the toxic and morphogenetic effects of JH and several JHAs, but not to other classes of insecticide. Biochemical studies reveal a target-site resistance mechanism, that of reduced JH binding in cytosolic extracts from either of two JH target tissues in Met flies. This property of reduced JH binding was cytogenetically localized to the Met region on the X chromosome and can account for the resistance. Possible identities for this binding protein include either an accessory JH-binding protein in the cytoplasm, similar to the cellular retinoic acid-binding protein in vertebrates, or a JH receptor protein involved in the action of JH (Ashok, 1998).

The Met+ gene has been cloned by transposable P-element tagging and reduced transcript level has been found in several mutant alleles, showing that underproduction of the normal gene product can lead to insecticide resistance. Transformation of Met flies with a Met+ cDNA results in susceptibility to methoprene, indicating that the cDNA encodes a functional Met+ protein. Met shows homology to the basic helix-loop-helix (bHLH)-PAS family of transcriptional regulators, implicating Met in the action of JH at the gene level in insects. This family also includes the vertebrate dioxin receptor, a transcriptional regulator known to bind a variety of environmental toxicants. Met shows three regions of homology to members of a family of transcriptional activators known as bHLH-PAS proteins. Met generally has higher homology to the vertebrate bHLH-PAS proteins than to those identified in D. melanogaster. A D. melanogaster ARNT-like gene has recently been cloned, and DARNT, known as Tango, has higher homology to vertebrate ARNT than does Met, suggesting that DARNT, not Met, may function like ARNT in flies. Met homology to these proteins includes the bHLH region that is involved in DNA binding (30-38% identity), the PAS-A region (28-40%), and the PAS-B region (22-35%). The arrangement of these domains in the Met gene is the same as for other bHLH-PAS genes (Ashok, 1998).

Neuropeptides in interneurons of the insect brain

Optic lobe: lamina, medulla, lobula complex

Although the optic lobe may contain as many as 75% of all interneurons in the insect brain, the number of peptidergic neurons is relatively small, and several peptides appear to be completely absent in this part of the brain (at least in some insect species). Others are present in single or a few cell types only. A notable exception is the accessory medulla, a small appendage of the medulla proper with a role in circadian rhythmicity. This is a major site of neuropeptide expression within the brain and is, therefore, treated separately from the major optic lobe neuropils: the lamina, medulla, and lobula (Nässel, 2006).

Peptide immunostaining has been detected in all neuropils of the optic lobe. In the lamina of palaeopteran and polyneopteran species (ranging from mayflies to cockroaches) and at least one paraneopteran species (a cicada), clusters of cells near the dorsal and ventral edge of the lamina contain neuropeptides related to PDF. These neurons have wide diffuse ramifications throughout the lamina and may extend processes toward the medulla in some species, suggesting a neuromodulatory role. Studies of cockroach and locust brains have shown that clusters of adjacent and partly identical cells contain FMRFamide-related peptides (FaRPs), CRF-like diuretic hormone, a baratin-related peptide, and locustamyoinhibin. Interestingly, no corresponding staining has been found in holometabolous insects, and in flies, no evidence has been obtained indicating that neurons of corresponding morphology actually exist (Nässel, 2006).

In the locust S. gregaria, but not in the cockroach L. maderae or the moths M. sexta and Heliothis virescens, one type of lamina monopolar neurons is labeled by antisera against M. sexta allatotropin. Furthermore, in locust and cockroach, centrifugal neurons from the medulla and/or accessory medulla with wide processes throughout the lamina contain PDFs, sulfakinins, and other FaRPs, TKRPs, CCAP, orcokinins, and myoinhibitory peptide. Centrifugal PDF-immunoreactive (IR), RFamide-IR, and allatostatin A-IR projections to the lamina are also present in the moths M. sexta and H. virescens, but in flies, in the honeybee, and in the bug Triatoma infestans, they are highly reduced or even completely absent (Nässel, 2006).

FaRPs, allatostatin-A type peptides, Mas-ATs, TKRPs and leucokinin-related peptides, CCAP, sulfakinins, orcokinins, and MIPs have been detected in the medulla of various species. Only a few of the immunostained neurons have been identified anatomically, but the position of cell bodies and the staining pattern in the medulla provide hints regarding the types of neurons. (1) Cell bodies around the second optic chiasm and primary neurites toward the inner face of the medulla are often immunostained. In the locust, they react with antisera against CCAP, Mas-AT, allatostatin-A, and FMRFamide. These neurons give rise to immunostaining in certain layers of the medulla but lack centrally projecting axons and are, therefore, intrinsic neurons of the medulla, most probably types of amacrine cells. RFamide-IR neurons in a similar position have also been reported in flies. (2) Many peptide antisera stain small numbers of cell bodies along the anterior edge of the medulla in various species. Subtypes of these neurons have primary processes in the accessory medulla and additional projections to the medulla, lamina, and/or central brain, but others lack ramifications in the accessory medulla and ramify solely in the medulla. MeRF-1 neurons in the medulla of C. vomitoria and M. domestica are axonless amacrine neurons of this type, whereas MeRF-2 neurons are tangential cells with axonal projections to the median protocerebrum. Both cell types contain FaRPs. Corresponding neurons in D. melanogaster are the OL1 and OL2 neurons. Similar amacrine and tangential RFamide-IR neurons are also present in the optic lobe of locusts, cockroaches, and the sphinx moth but have been analyzed in less detail. (3) Finally, columnar neurons are more rarely immunostained. In the locust, Mas-AT-IR cell bodies around the first optic chiasm and individual fiber trajectories through the medulla appear to be transmedullary neurons that give rise to additional staining in specific layers and subunits of the lobula complex. Approximately 2000 RFamide-IR cell bodies in a similar location have been described in C. vomitoria and M. domestica. Other columnar neurons in the locust, probably T-type cells, contain TKRPs. They have somata around the second optic chiasm and innervate unspecified layers in the medulla and lobula complex (Nässel, 2006).

Immunostaining in the lobula complex has been studied in locusts, flies, and several other species, but few details of the cellular identity of the immunoreactive neurons are available; TKRPs, leucokinin, CCAP, FaRPs, allatostatin-A, Mas-AT, MIP, and orcokinin have been demonstrated, as in the medulla. Again, all principal types of interneuron may contain neuropeptides, including columnar neurons from the medulla with terminals in the lobula, medulla/lobula, and lobula tangentials with axonal projections to the midbrain, or centrifugal neurons from the midbrain with axonal projections to the lobula Nässel, 2006).

Optic lobe: accessory medulla

The accessory medulla is a small anterior appendage of the medulla proper. It is prominent in orthopteroid insects, cockroaches, and moths but is largely fused with the anterior edge of the medulla in flies and bees. In the fly D. melanogaster, the cockroach L. maderae, and probably other insect species, the accessory medulla and its neurons constitute an important part of the master circadian clock controlling circadian behavior. In D. melanogaster, two clusters of PDF-containing lateral neurons (l-LNv, s-LNv) with dendrites in the accessory medulla are important components of the circadian system. These neurons and others are associated with the circadian clock (Nässel, 2006).

In the cockroach L. maderae, the accessory medulla, which has been demonstrated to house the circadian pacemaker, shows strong immunostaining for a variety of neuropeptides, including PDF, FaRPs, orcokinin, allatostatin A, Mas-AT, MIP, leucokinin, and corazonin. Based on their morphology, the immunostained neurons can be classified as local neurons of the accessory medulla (Mas-AT), neurons with projections to the midbrain, medulla, and lamina (PDF, FaRPs, leucokinin), and neurons interconnecting both accessory medullae (PDF, orcokinin). Whereas PDF acts as an output and a coupling signal of the pacemaker, Mas-AT and orcokinin appear to be involved in light entrainment, as suggested by light-like phase shifts in circadian locomotor activity following Mas-allatotropin and orcokinin injections into the brain. Recent electrophysiological experiments have demonstrated that PDF has inhibitory effects on accessory medulla neurons; in excised pacemaker tissue, PDF synchronizes and phase-locks different, regularly firing neurons. It is hypothesized that PDF-dependent phase control is used to gate circadian outputs to locomotor control centers (Nässel, 2006).

All neuropeptides found in L. maderae have also been detected immunocytochemically in the accessory medulla of S. gregaria but an involvement in circadian functions has not been studied in this species. PDF-IR neurons of the accessory medulla have been mapped in a large number of insect species; they show striking variations in projection sites in the central brain and in the medulla and lamina and with respect to contralateral projections. Injections of PDF performed in a cricket indicate that it plays a role in the circadian system. In contrast to the situation in D. melanogaster, the co-localization of the clock genes per and tim in PDF-containing neurons of the accessory medulla has not been found in the honeybee or sphinx moth and appears unlikely in several other insect species, suggesting either that the internal clock in the accessory medulla has a different molecular basis or that clock functions have shifted to other brain areas (Nässel, 2006).

Antennal lobe

The AL is the primary olfactory intregration center in the insect brain. In all insect species, it is organized into a species-specific number of spheroidal neuropil condensations termed olfactory glomeruli. Primary olfactory afferent axons from the antenna enter the AL through the antennal nerve and make synaptic contacts with AL interneurons in the glomeruli. Several types of interneurons have been distinguished in the AL: local interneurons are amacrine cells and interconnect many or all glomeruli but lack axonal projections to other brain areas; projection neurons innervate single or several glomeruli and project axons through one of about five antenno-cerebral tracts to second-order processing sites in the protocerebrum; centrifugal neurons are a diverse group of neuronal elements and comprise neurons with dendritic ramifications in other areas of the nervous system and axonal projections into the AL (Nässel, 2006).

Most studies have demonstrated peptide immunostaining in small groups of local AL interneurons, typically consisting of fewer than 10 to as many as 100 individual cells. Members of the TKRP, FaRP, allatostatin A, and allatotropin (Mas-AT-like) families of peptides have been demonstrated in local AL neurons of many species studied ranging from cockroaches to flies. Most recently, orcokinin immunostaining has been demonstrated in local AL neurons in species of silverfish, cockroach, and locust but not in holometabolous insects, where orcokinin immunostaining is completely absent. In situ hybridization studies have demonstrated the expression of the F10 FLRFamide gene in AL neurons of M. sexta and TKRP gene transcripts in the ALs of D. melanogaster and Apis mellifera (Nässel, 2006).

No reports suggest the presence of neuropeptides in olfactory afferents, but FMRFamide and allatostatin A have been mapped immunocytochemically to small numbers of projection and/or centrifugal neurons in a species-specific manner, without evidence of a common pattern or particular type of labeled neuron in these species. In contrast, an antiserum against leucokinin has revealed a pair of descending protocerebral neurons with efferent side-branches in the AL in a variety of orthopteroid species but not in a fly, a mosquito or the honeybee. These data suggest a conserved peptidergic phenotype of these neurons in orthopteroids and considerable modification or loss of antigen or neuronal cell type in holometabolous species (Nässel, 2006).

Despite the prominent and largely conserved cellular distribution of neuropeptides in the AL, only one study has so far addressed the physiological role of neuropeptides (TKRPs) in the AL of D. melanogaster. The prominent presence of neuropeptides in local, presumably γ aminobutyric acid (GABA)-ergic, AL neurons suggests that peptides largely act as co-transmitters with GABA to shape the olfactory responses of projection neurons. Projection neurons respond with characteristic odor-dependent temporal patterns of excitation and inhibition and oscillatory synchronization of activity across neurons in certain species. The way that TKRPs and other neuropeptides contribute together with GABA to shaping the olfactory profiles of projection neurons will be an exciting subject for future research (Nässel, 2006).

Mushroom bodies

The mushroom bodies are second-order olfactory neuropils and are involved in olfactory learning, memory formation, and other cognitive functions . They are primarily formed by a large number of intrinsic neurons termed Kenyon cells with tightly packed cell bodies in the superior protocerebrum. Their primary neurites give rise to dendritic arborizations in one or a pair of cup-shaped calyces. Axons enter a common stalk or pedunculus and typically bifurcate. One collateral enters the vertical lobe (α-lobe) and the other, the medial lobe (β-lobe and γ-lobe; reviewed by Fahrbach, 2006). The most prominent afferents to the mushroom body are projection neurons from the AL. Their axons enter the calyces and make synaptic contacts with Kenyon cells. Other inputs may originate from the optic lobe (Hymenoptera) or the glomerular lobe (Orthopteroidea) and enter specific layers in the calyx or a distinct neuropil, the accessory calyx. In the medial and vertical lobes, Kenyon cell axons make contacts with output neurons to other brain areas (Nässel, 2006).

Projection neurons from the AL to the calyces appear to be cholinergic, and peptide co-transmitters appear to be generally absent, at least in the most prominent uniglomerular type of projection neuron. In contrast, inputs from the glomerular lobe, recently studied in the desert locust, give rise to distinct immunostaining for Mas-AT throughout the accessory calyx. Evidence has been obtained that glutamate and/or aspartate are the principal transmitters of Kenyon cells. In addition, members of three peptide families have been detected immunocytochemically in Kenyon cells. FMRFamide immunostaining has been reported in subpopulations of Kenyon cells in numerous insect species, including S. gregaria, L. maderae, honeybee, sphinx moth, various blowflies, and D. melanogaster. In the honeybee, but not in several other species, a pattern of immunostained strata different from that with FMRFamide antisera has been obtained with antisera against gastrin/cholecystokinin. Orcokinin immunostaining has been found in Kenyon cells in the silverfish, L. maderae, and S. gregaria but not in holometabolous insects. In all of these studies, Kenyon cell somata are devoid of immunostaining, and likewise, labeling in the calyx neuropil is weak or absent. Staining intensity gradually increases along the pedunculus and is highest in the lobes, suggesting that peptides are transported to axonal release sites in the lobes and may even be processed along the way. Usually, a particular subpopulation of Kenyon cell axons is immunostained and is often organized in strata or layers. Interestingly, RFamide immunostaining in the vertical lobe of the honeybee mushroom body has not been clearly attributed to dense-core vesicles but may have a non-vesicular distribution. Initial evidence for the functional significance of these data has been provided in honeybees. Injections of FMRFamide and cholecystokinin-8 (CCK-8) into the mushroom bodies of bees lead to a change in the field potentials of the mushroom body and to altered proboscis responses to antennal stimulations. Finally, in honeybees, in situ hybridization and cDNA microarrays have demonstrated the predominant expression of the prepro-tachykinin gene in mushroom body Kenyon cells and TKRP immunolabeling has been demonstrated in specific patterns, distinct from FMRFamide-IR labeling, in the mushroom body lobes. This finding is surprising and probably unique to Hymenoptera, since tachykinin transcripts or peptides have not been detected in the mushroom bodies of other insects (Nässel, 2006).

Arborizations from small numbers of extrinsic neurons are immunoreactive with a variety of peptide antisera. These neurons may provide input to the calyces and/or invade the lobes and pedunculus. Among these is the giant feedback neuron of the mushroom body in the locust, a neuron that is immunoreactive with antisera against Mas-AT. In D. melanogaster, a single neuron, viz., the dorsal paired medial neuron (DPM), innervates the lobes of the mushroom body. The DPM neuron is believed to release neuropeptide gene products encoded by the amnesiac (amn) gene.

Central complex

The central complex is a group of neuropils in the center of the insect brain. It consists of several substructures including the protocerebral bridge, the upper and lower divisions of the central body (also termed fan-shaped and ellipsoid bodies), and a pair of spheroidal noduli. The whole structure has a highly regular internal neuroarchitecture. In simplified form, the central complex can be described as a stack of layers, each composed of an array of 16 columns with topographic interhemispheric connections between columns, both within and between the different layers. The most prominent classes of neurons are tangential neurons, columnar neurons, and pontine neurons, each class having a multitude of different subtypes. Tangential neurons innervate particular layers throughout all of their columns. In general, they have dendritic arborizations in various brain areas surrounding the central complex. Columnar neurons, in contrast, interconnect single or a few columns of different layers and provide output signals to the adjacent lateral accessory lobes, where contacts with descending neurons and projections to other brain areas are likely to be present. Corresponding to the 16-fold columnar organization of the system, each type of columnar neuron occurs as a set of 16 isomorphic neuronal elements. Pontine neurons, finally, are intrinsic neurons of the central body and interconnect columns of the right and left hemispheres of the structure (Nässel, 2006).

Neuropeptides appear to be prominent neuromediators in the central complex. Distinct patterns of immunostained layers of the central body have been reported with a large variety of peptide antisera in flies, cockroaches, locusts, and a few other taxa, but the anatomical identification of the immunostained neurons has been attempted in a few studies only, most notably in the desert locust S. gregaria. Immunostaining suggests that the following peptides or peptides families are present in the locust central complex: allatostatin A, Mas-AT, baratin, CCAP, FaRPs, MIP, orcokinin, and TKRPs. Usually, several types of neuron are immunostained with a given antiserum and all principal classes of neuron may be labeled. In the desert locust, particular systems of tangential neurons have been identified with antisera against allatostatin-A, baratin, CCAP, orcokinin, TKRPs, and leucokinin; sets of columnar neurons show immunostaining for TKRPs, allatostatin A, and MIPs, and one or two types of pontine neurons have been revealed by the CCAP antiserum. In D. melanogaster, proctolin and neuropeptide F may also be present in the fan-shaped body (Nässel, 2006).

Functional analyses of the central complex suggest a role in spatial orientation and visual pattern recognition. In S. gregaria and a cricket, distinct cell types are sensitive to polarized light, implicating this complex in sky compass orientation. Recent molecular genetic studies in D. melanogaster have provided compelling evidence for memory traces for two parameters of visual pattern recognition in the fan-shaped body (upper division) of the central body. The possible function of neuropeptides within this framework has not been addressed. Double-labeling studies have shown substantial co-localization of neuropeptides with several classical transmitters and biogenic amines, suggesting that neuropeptides may be co-transmitters in these neurons (Nässel, 2006).

Intersegmental interneurons

Specific types of intersegmental interneurons have been revealed through immunostaining with antisera against several neuropeptides. These are either ascending or descending neurons. Groups of tritocerebral neurons with ascending projections in the median bundle of the brain and wide ramifications in the superior protocerebrum show immunostaining for locustamyotropin (Lom-MT; pyrokinin family) in locusts, cockroaches, and the fly Sarcophaga bullata, but not in D. melanogaster and various Lepidoptera. Apparently, the same neurons are also immunoreactive for periviscerokinin-1 in the cockroach. Judged by their projection patterns, these neurons are suited for the provision of signals from the stomatogastric system (tritocerebrum) to neurosecretory cells of the brain that arborize profoundly in the superior median protocerebrum (Nässel, 2006).

Antisera against sulfakinins have revealed a single bilateral pair of descending neurons in the brain of insects ranging from the apteryogote Lepisma saccharina to the fruitfly D. melanogaster. The neurons have cell bodies in the superior median protocerebrum, wide arborizations in coarse neuropil of the brain, and contralaterally descending axons that extend throughout the length of the ventral nerve cord. The functional role of these neurons and their released sulfakinins has still to be uncovered (Nässel, 2006).

A pair of descending neurons with ramifications in the AL is leucokinin-IR in locusts and cockroaches. In L. maderae, these neurons ramify in several olfactory brain areas, including the calyces of the mushroom body and the lateral horn and superior protocerebrum. Their arborizations in the AL are varicose suggesting output synapses. Interestingly, these neurons have not been detected in an extensive study of cockroach descending neurons through backfills of cervical connectives. This suggests that the neurons terminate in the subesophageal ganglion (Nässel, 2006).

Two pairs of descending neurons with cell bodies in the protocerebrum, described in D. melanogaster, express the peptide SIFamide. In adult flies, these neurons supply processes to many neuropil regions of the brain and axons throughout the length of the ventral nerve cord. These highly extensive neurons are spectacular, since they seem to be the only ones expressing SIFamide, both in larvae and adults and, thus, the function(s) of this peptide may be mediated solely by two bilateral pairs of neurons (Nässel, 2006).

A number of further examples of ascending and descending peptidergic neurons have been described in D. melanogaster and other insects. Indeed, most neuropeptides seem to be expressed in at least a few intersegmental neurons, suggesting an important role of neuropeptides in intersegmental communication. In most cases, the functional roles played by these neurons and their peptide neuromediators remain unclear (Nässel, 2006).

Other brain interneurons and neurosecretory cells

Other interneurons that have the majority of their processes in neuropils other than the specific ones discussed above. Many neuropeptides can be found in such neurons and clearly play important roles in integrating different brain circuits. Most neuropeptides have also been identified in brain neurosecretory cells, but with a few notable exceptions, including neuropeptide F, PDF, TKRPs in most insects, and SIFamide. Neurosecretory cells have axon terminations in neurohemal release sites in contact with the circulation. In addition, however, they commonly also have varicose processes in the brain and these could act as interneuronal segments. Thus, the possibility that neurosecretory cells also subserve peptidergic signaling functions within the brain should be considered (Nässel, 2006).

Co-localization of neuropeptides with other transmitters

Double-immunolabeling experiments have shown that neuropeptides are often co-localized with GABA, biogenic amines, nitric oxide, and other unrelated peptides in certain brain interneurons. Ultrastructural data from the accessory medulla and the central body have revealed that neurons with dense-core vesicles indicative of neuropeptides usually also contain clear synaptic vesicles. This suggests that neuropeptides largely or even exclusively occur as co-transmitters together with conventional fast-acting transmitters in insect brain interneurons. In most experiments performed so far, double-immunolabeling has only been demonstrated in neuronal cell bodies, but whether the co-release of a neuropeptide and another substance actually occurs has not been studied in insect interneurons (Nässel, 2006).

Co-localization of neuropeptides with GABA has been documented for local AL interneurons, feedback neurons of the mushroom body, tangential neurons of the central body, amacrine neurons of the medulla, and neurons of the accessory medulla. In all of these cases, subpopulations of cells from a large cluster of GABA-IR neurons appear to contain a peptide co-transmitter. In a particularly well-studied brain area, viz., the AL of M. sexta, Mas-AT, A-type allatostatins, FaRPs, and TKRPs are present in partly overlapping subpopulations of GABA-IR local interneurons (Nässel, 2006).

The co-localization of neuropeptides with biogenic amines appears to be less common but has been demonstrated in the central complex of the locust (serotonin/A-type allatostatin and in lamina tangential neurons of the cockroach (serotonin/PDH). Unrelated neuropeptides are commonly co-localized, especially in brain areas that are heavily innervated by peptidergic neurons such as the AL and the accessory medulla. In both brain areas, partly overlapping cell populations show immunostaining for two or more different neuropeptides (Nässel, 2006).

The functional role of peptide co-transmission in the insect brain has been given little attention so far. Peptide co-transmitters are likely to add considerable flexibility to neuronal circuits by acting on separate targets, by cooperative effects on the same targets, and by release of the peptide co-transmitter only at particular firing patterns or firing rates. The common presence of neuropeptides in subpopulations of GABAergic lateral and feedback circuitries (a wide-spread phenomenon in the insect brain) might allow for dynamic and differential reconfigurations of these networks depending on the neuropeptides in action (Nässel, 2006).

D. melanogaster as a model for neuropeptide signaling

Representatives of most of the characterized neuropeptide precursors of D. melanogaster have been localized to brain neurons by in situ hybridization and/or specific antisera. Thus, the general distribution patterns of endogenous peptides are known, and some information is available on the distribution of several peptide GPCRs. The major advantage with D. melanogaster is the availability of powerful molecular genetics technology that has improved the understanding of peptide signaling in the brain. Some neuropeptides and their receptors in the brain have been studied by gene-specific or cell-specific interference by using the GAL4-UAS system and other molecular techniques: PDF, neuropeptide F, TKRPs, and amnesiac (amn) gene product(s) (Nässel, 2006).

Neuropeptides in the circadian clock system of D. melanogaster

One of the exciting areas in neuropeptide research in the D. melanogaster brain involves the clock system. Circadian clocks are endogenous timekeepers that help organize daily rhythms in behavior, physiology, and metabolism. D. melanogaster represents one of the major organisms in studies of the circadian clock. In D. melanogaster, the cellular clock consists of two interlocked molecular feedback loops, employing specific clock genes, that drive circadian oscillations of gene and protein expression, leading to cycling levels of mRNA and proteins. The clock can be reset by sensory inputs, notably by light. In D. melanogaster, the master circadian clock is located in the brain and consists of bilateral sets of pacemaker neurons expressing the specific clock genes. In the adult brain, the clock-gene-expressing neurons are: three sets of lateral neurons designated s-LNv, l-LNv, and LNd, and three sets of dorsal neurons, DN1-3. Four of the s-LNvs and all the l-LNvs express the neuropeptide PDF. The output pathways from the D. melanogaster clock have not yet been delineated, but the brain neuroendocrine system is probably one target, with brain neurons controlling locomotor activity being another. Evidence is available that PDF is the main output factor of the LNv clock neurons, necessary for maintaining the circadian locomotor activity and eclosion rhythm under constant dark conditions and for synchronizing pacemaker activity. Following the recent identification of the D. melanogaster PDF receptor, neurons that are targets of PDF signaling have been identified. Antisera to the PDF receptor label several of the clock neurons: the l-LNvs, up to seven DN1s, a few DN3s, and one LNd. These findings support the notion that PDF is important for synchronization of clock neurons. Other neurons also express PDF receptor, as shown by immunolabeling, in the ventral portions of the brain, but these have not been revealed in sufficient detail to establish the circuits of which they are part. Surprisingly, no neurosecretory cells have been reported among the neurons expressing the PDF receptor. This indicates that connections between clock neurons and neurosecretory cells are either indirect via further interneurons or mediated by signals other than PDF. Alternatively, the receptor expression is too low to be detected in some neurons. In D. melanogaster, no further neuropeptides have so far been implicated in clock neurons, it has been demonstrated that as yet unknown, amidated neuropeptides participate in the control of daily activity rhythms (Nässel, 2006).

TKRPs modulate chemosensory processing and locomotor activity

Tachykinin-related peptides (TKRPs) have been identified in numerous interneurons in the D. melanogaster brain. The effects have been studied of the elimination of TKRP signaling on olfactory and locomotor behavior in D. melanogaster (Winther, 2006). The tk gene (TKRP precursor gene) has been targeted by RNA interference by using GAL4-UAS technology, and the adult flies were tested for behavioral responses to four different odorants over a range of concentrations in a T-maze. The behavioral patterns of tk knockdown flies were altered in response to three of these: they were less repelled by higher concentrations of benzaldehyde and butanol than were control flies, whereas the response to isoamyl acetate was altered over a wider range of concentrations. For all three odorants, the altered response indicated a loss of sensitivity in perception. The involvement of DTKs in olfactory information processing is further supported by the presence of TKRP immunoreactivity in local interneurons of the Drosophila ALs and the abundant presence of one of the tachykinin receptors, DTKR, in antennal glomeruli (Nässel, 2006).

The tk-deficient flies were also found to display a significantly higher walking activity between two opposing vertical black stripes in an arena. Two of three parameters were changed, viz., the percentage of active time and distance walked, but not the speed of walking. These findings were correlated with the presence of tachykinins and their receptor DTKR in neuropils of the central body: the initiation and maintenance of locomotion is thought to be controlled by the central complex in the brain, whereas walking speed may be controlled by thoracic motoneurons (Nässel, 2006).

Amnesiac and 'PACAP'signaling in Drosophila

A functional Amnesiac gene (amn) is necessary for normal memory retention in Drosophila. Mutant flies show normal acquisition of an olfactory operant avoidance response, but the memory decays within 30 min of training. Cloning of the amn gene has revealed that it codes for a neuropeptide precursor-like protein that could theoretically generate up to three different neuropeptides. One of the putative peptides has limited sequence similarities to mammalian pituitary adenylatecyclase-activating peptide (PACAP). Although a peptide related to PACAP or the other amn gene products has not yet been identified in Drosophila or any other insect, an antiserum to mammalian PACAP38 labels interneurons and motoneurons in the fly brain and ventral nerve cord. In contrast to the mammalian PACAPs, however, the PACAP-like sequence in the Drosophila precursor-like protein contains four cysteines. This suggests that the peptide, if processed from the precursor, could form internal disulphide bridges and thus attain a conformation different from mammalian PACAPs. Nevertheless, the amn product(s) has been suggested to be secreted and to play a role in memory consolidation. Two large neurons (DPM) express AMN immunoreactivity and are identified by P-element markers. The large DPM neurons are extrinsic to the mushroom bodies and supply extensively arborizing branches to the medial and vertical lobes. By using amn-GAL4 drivers to express temperature-sensitive alleles of shibire, vesicle recycling activity in the DPM can be shut off and restored, and a negative effect on memory consolidation similar to that of the amn mutation can be demonstrated. This does not necessarily implicate the blockage of the release of amn product(s) from the DPMs but might reflect impaired synaptic release of some other neurotransmitter in these neurons. Indeed, the DPMs have recently been suggested to use acetylcholine as an additional neurotransmitter. Another study has shown a role of amn in age-related memory impairment. Flies mutant in amn do not exhibit age-dependent decreases in memory, in contrast to other memory mutants. Finally, amn has been implicated in alcohol sensitivity; amn mutants are more sensitive to ethanol, and this effect can be reversed by short-term restoration of amn activity. Clearly, the product(s) of the amn gene should be identified as soon as possible (Nässel, 2006).

Neuropeptide F signaling in feeding behavior and alcohol tolerance

A signaling system that appears well conserved through evolution is that of vertebrate neuropeptide Y (NPY) and the invertebrate neuropeptide F (NPF). NPF in Drosophila consists of 36 residues and is often referred to as long NPF. The NPF receptor (NPFR1) of Drosophila displays critical sequence similarities to mammalian NPY receptors. NPYs are multifunctional in mammals but play a major role in appetite behavior and feeding regulation. The various reports describing the importance of NPF and NPFR1 in foraging and feeding in Drosophila are therefore of interest (Nässel, 2006).

Only four neurons in the D. melanogaster brain express NPF. Both in larvae and adults, the four NPF neurons have cell bodies and extensive processes in the brain and contralateral descending axons running down the ventral nerve cord. The receptor (NPFR1) distribution partly matches that of the peptide. Transgenes interfering with the expression of NPF or its receptor NPFR1 have revealed a number of intriguing behavioral phenotypes in larval and adult Drosophila shedding light on the roles of peptide signaling in feeding, foraging, social behavior, and alcohol tolerance (Nässel, 2006).

Initial experiments demonstrated a role of NPF signaling during the behavioral switch from continuous feeding to wandering, food aversion, clumping, and cooperative burrowing in third instar larvae. Younger feeding larvae displayed strong expression of NPF mRNA, whereas older non-feeding third instar larvae exhibited low levels. Deficiencies in NPF signaling were produced by targeted interference with NPF-expressing neurons or neurons expressing the receptor NPFR1, by using the GAL4-UAS system to drive toxins or specific RNA interference. Larvae with a deficiency in NPF signaling precociously displayed food aversion and social behaviors normally only seen in older non-feeding larvae. In contrast, during over-expression of NPF or its receptor, the opposite was seen; larvae exhibited prolonged feeding activity and suppressed social behavior of older larvae. Further experiments demonstrated that the NPF system was required to sustain larval foraging activity under adverse feeding conditions: NPF knockdown larvae were less motivated to feed on food mixed in hard Agar, whereas they fed readily on normal yeast paste (Nässel, 2006).

NPF and insulin-like peptide (DILP) signaling converge in the regulation of motivated food ingestion. Food-deprived Drosophila larvae were more likely to feed on noxious (quinine-adulterated) or less accessible (high agarose content) food. Over-expression of NPFR1 caused non-deprived (fed) larvae to feed on non-favorable food, whereas knockdown of the receptor led to the opposite. The neural activity in NPFR1-expressing neurons seemed to be directly regulated by DILP signaling: over-expression of DILP signaling components in NPFR1 neurons led to a decreased feeding response to noxious food, whereas down-regulation produced the opposite phenotype. One of the components studied was the Drosophila p70 ribosomal S6 kinase (dS6K), a cell-autonomous effector of pathways sensing nutrient levels. Knockdown of dS6K in NPFR1 neurons led to larvae feeding on noxious food, even when not food-deprived, and vice versa for upregulated dS6K. Thus, dS6k may be critical for transducing hunger stimuli in NPFR1-expressing interneurons. In Drosophila, dS6K is found downstream of the receptor (dInR) of Drosophila insulin-like peptides (DILPs). Down-regulation of dInR in NPFR1-expressing neurons led to increased feeding on noxious food in non-deprived larvae and upregulation to the opposite. Thus, the dInR pathway seems to regulate averse responses to noxious food, whereas the DILP/NPF pathway may regulate the willingness to acquire foods of lower quality and be part of a mechanism mediating hunger-induced changes in food preference. Finally, disruption of NPF/NPFR1 signaling or the knockdown of npfr1 activity in adult Drosophila has been shown to decrease sensitivity to ethanol sedation, whereas over-expression of NPF increases alcohol sensitivity (Nässel, 2006).

Short NPFs (sNPFs) have been localized to Drosophila brain interneurons with processes in optic lobe and median protocerebrum. Gain-of-function (UAS-snpf) and loss-of-function (UAS-RNAi) constructs have been generated to interfere with sNPF expression. Flies and non-wandering larvae over-expressing sNPF increase feeding, and knockdowns decrease feeding. However, wandering larvae are not affected by interference with sNPF: thus, in contrast to the over-expression of NPF (long) signaling, which prolongs feeding in wandering-stage larvae, sNPF over-expression does not prolong feeding in wandering larvae. It has also been found that over-expression of sNPF leads to increased appetite in adults (Nässel, 2006).

Neuroarchitecture of peptidergic systems in the larval ventral ganglion of Drosophila melanogaster

Recent studies on Drosophila melanogaster and other insects have revealed important insights into the functions and evolution of neuropeptide signaling. In contrast, in- and output connections of insect peptidergic circuits are largely unexplored. Existing morphological descriptions typically do not determine the exact spatial location of peptidergic axonal pathways and arborizations within the neuropil, and do not identify peptidergic input and output compartments. Such information is however fundamental to screen for possible peptidergic network connections, a prerequisite to understand how the CNS controls the activity of peptidergic neurons at the synaptic level. This study provide a precise 3D morphological description of peptidergic neurons in the thoracic and abdominal neuromeres of the Drosophila larva based on fasciclin-2 (Fas2) immunopositive tracts as landmarks. Comparing the Fas2 "coordinates" of projections of sensory or other neurons with those of peptidergic neurons, it is possible to identify candidate input and output connections of specific peptidergic systems. These connections can subsequently be more rigorously tested. By immunolabeling and GAL4-directed expression of marker proteins, this study analyzed the projections and compartmentalization of neurons expressing 12 different peptide genes, encoding approximately 75% of the neuropeptides chemically identified within the Drosophila CNS. Results are assembled into standardized plates which provide a guide to identify candidate afferent or target neurons with overlapping projections. In general, this study found that putative dendritic compartments of peptidergic neurons are concentrated around the median Fas2 tracts and the terminal plexus. Putative peptide release sites in the ventral nerve cord were also more laterally situated. The results suggest that peptidergic neurons in the Drosophila ventral nerve cord have separated in- and output compartment. The lack of a strict segmentally reiterated pattern throughout the thoracic and abdominal neuromeres suggests that the restricted and differential distribution of peptidergic neurons reflects neuromere-specific functional connections. Other larval neuron types or circuits that match the observed peptidergic distribution patterns have not been characterized (Santos, 2007).

The last two abdominal neuromeres a8/9 have a unique pattern of peptidergic somata and projections (e.g. FMRFa, MIP or PDF neurons, and show the least serial homology to the more anterior neuromeres of the ventral ganglion. This finding also extends to descending processes. Descending axons may stop before or when reaching the border to a8 (HUG and DTK neurons), form extensive varicose ramifications within the neuropil of a8 (AST, corazonin or branch extensively in the terminal plexus of a9 (FMRFa-, leucokinin-, MIP and PDF-neurons. Belonging to the tail region, the segments a8/9 differ from the homomeric segments a1-7 with respect to the organization of muscles and sensory neurons. Furthermore, several unique structures such as the spiracles or the anal pads belong to these terminal segments. Unlike other segmental nerves, the segmental nerve of a9 innervates the hindgut musculature. The unique pattern of peptidergic neurons in a8/9 might thus, at least partially, reflect a segment-specific function related to e.g. control of spiracles or intestinal functions. For example, the PDF neurons innervate the hindgut, but their exact function is so far unknown. Similar segmental differences between a8 and the rest of the abdominal neuromeres have been found for neurons expressing biogenic amines (Santos, 2007).

The fusion construct syb.egfp has been developed as a presynaptic marker. Since synaptobrevin (SYB) is an integral membrane protein of small synaptic vesicles and large peptide-containing vesicles alike, SYB.EGFP also labels peptide vesicles and hence peptide accumulation and release sites (varicosities), which typically do not spatially coincide with synapses. Concomitantly, it is assumed that purely dendritic compartments of peptidergic neurons do not contain vesicles and show no or only weak SYB.EGFP labeling. These assumptions are supported by results obtained for PDF neurons in the brain, and the Tv and Va neurons that innervate neurohemal organs. SYB.EGFP was only found in the cell bodies (where the protein is made) and in the terminals in the neurohemal organs. The axonal projections as well as the arborizations within the VNC were unlabeled. Nevertheless, when interpreting the SYB.EGFP distribution, it has to be kept in mind that SYB.EGFP might also label presynaptic sites if the peptidergic neurons contain colocalized classical neurotransmitters (Santos, 2007).

The haemagglutinin-tagged GABAA receptor subunit RDL.HA has been shown to be a useful specific postsynaptic marker in motor neurons. Since The GABAA receptor subunit RDL is involved in mediating GABAergic postsynaptic currents, attempts were made to see whether ectopic RDL.HA expression indicates postsynaptic sites (dendrites) of peptidergic neurons also. The general expression level of RDL.HA was very weak, and only discernible labeling intensities were obtained with two different GAL4-drivers: Ccap- and c929-GAL4. Nevertheless, the labeling was spatially very confined to neuron compartments that showed no varicosities or only weak SYB.EGFP fluorescence. This suggests that RDL.HA labeled postsynaptic sites in peptidergic neurons (Santos, 2007).

Arborizations around the median DM and VM tracts turned out to be a prominent feature of most characterized peptidergic neurons with somata in the ventral ganglion, including the AST, CCAP, corazonin, FMRFa, MIP and PDF neurons. In contrast to motor neurons, the prominent midline arborizations of peptidergic neurons were rather short, and did not occupy large areas in the more lateral neuropils between the median and lateral tracts. For the CCAP neurons, ectopically expressed RDL.HA localized exclusively to these median arborizations. In contrast, SYB.EGFP as well as peptide-immunoreactivity was absent or relatively low in these arborizations. Also in the general peptidergic c929-GAL4-line, SYB.EGFP expression was low in the median compared to lateral fascicles. This might suggest that the median arborizations represent peptidergic dendrites. Descending processes of CCAP, EH, HUG and leucokinin neurons (originating from somata in the suboesophageal ganglion or in the brain) all have putative release sites around the DM and VM tracts. Of the peptidergic neurons with cell bodies in the VNC, only those expressing corazonin were found to have varicosities indicative of release sites around the DM and VM tracts (Santos, 2007).

Taken together, these findings suggest that the arborizations around the dorsomedial (DM) and ventromedial (VM) tracts are mainly input compartments for peptidergic VNC neurons, and point to this midline region as a main site for synaptic inputs onto peptidergic neurons including the CCAP neurons. The different putative sites of in- and outputs to peptidergic neurons in the VNC are summarized (see Assignment of putative main compartment identities as suggested by morphology, immunolabeling intensities and distribution of synaptic markers). Peptides released from varicosities of leucokinin, CCAP, HUG-, EH and corazonin neurites along the DM tract may modulate synaptic transmission around the DM tracts, or might represent direct input signals to peptidergic neurons. Also, the dorsal ap-let neurons with somata in the ventral ganglion expressing the peptide precursor Nplp1 appear to have their output sites along the DM tracts as indicated by strong peptide immunoreactivity. Unlike any of the peptidergic neurons characterized here, the dorsal ap-let neurons seem to have extensive arborizations within the neuropil of each hemineuromere, which appear to contain no or only little peptide immunoreactive material and hence might represent dendritic regions. Also the leucokinin neurons with somata in the ventral ganglion do not send projections towards the midline. Since leucokinin release is likely to occur at peripheral release sites on body wall muscles, it is possible that a synaptic input region is located along the VL tract, the only projection site of abdominal leucokinin neurons within the CNS neuropil (Santos, 2007).

Nutrient sensor in the brain directs the action of the brain-gut axis in Drosophila

Animals can detect and consume nutritive sugars without the influence of taste. However, the identity of the taste-independent nutrient sensor and the mechanism by which animals respond to the nutritional value of sugar are unclear. This study reports that six neurosecretory cells in the Drosophila brain that produce Diuretic hormone 44 (Dh44), a homolog of the mammalian corticotropin-releasing hormone (CRH), are specifically activated by nutritive sugars. Flies in which the activity of these neurons or the expression of Dh44 is disrupted fail to select nutritive sugars. Manipulation of the function of Dh44 receptors has a similar effect. Notably, artificial activation of Dh44 receptor-1 neurons results in proboscis extensions and frequent episodes of excretion. Conversely, reduced Dh44 activity leads to decreased excretion. Together, these actions facilitate ingestion and digestion of nutritive foods. The study proposes that the Dh44 system directs the detection and consumption of nutritive sugars through a positive feedback loop (Dus, 2015).

The nutrient-responsive hormone CCHamide-2 controls growth by regulating Insulin-like peptides in the brain of Drosophila melanogaster

In Drosophila melanogaster, the fat body (adipose tissue) has been suggested to play an important role in coupling growth with nutritional status. This study shows that the peripheral tissue-derived peptide hormone CCHamide-2 (CCHa2) acts as a nutrient-dependent regulator of Drosophila insulin-like peptides (Dilps). A BAC-based transgenic reporter revealed strong expression of CCHa2 receptor (CCHa2-R) in insulin-producing cells (IPCs) in the brain. Calcium imaging of brain explants and IPC-specific CCHa2-R knockdown demonstrated that peripheral-tissue derived CCHa2 directly activates IPCs. Interestingly, genetic disruption of either CCHa2 or CCHa2-R caused almost identical defects in larval growth and developmental timing. Consistent with these phenotypes, the expression of dilp5, and the release of both Dilp2 and Dilp5, were severely reduced. Furthermore, transcription of CCHa2 is altered in response to nutritional levels, particularly of glucose. These findings demonstrate that CCHa2 and CCHa2-R form a direct link between peripheral tissues and the brain, and that this pathway is essential for the coordination of systemic growth with nutritional availability. A mammalian homologue of CCHa2-R, Bombesin receptor subtype-3 (Brs3), is an orphan receptor that is expressed in the islet beta-cells; however, the role of Brs3 in insulin regulation remains elusive. This genetic approach in Drosophila melanogaster provides the first evidence that bombesin receptor signaling with its endogenous ligand promotes insulin production (Sano, 2015).

Variability in the number of abdominal leucokinergic neurons in adult Drosophila melanogaster

Developmental plasticity allows individuals with the same genotype to show different phenotypes in response to environmental changes. An example of this is how neuronal diversity is protected at the expense of neuronal number under sustained undernourishment during the development of the Drosophila optic lobe. In the development of the Drosophila central nervous system, neuroblasts go through two phases of neurogenesis separated by a period of mitotic quiescence. Although during embryonic development much evidence indicates that both cell number and the cell fates generated by each neuroblast are very precisely controlled in a cell autonomous manner, after quiescence extrinsic factors control the reactivation of neuroblast proliferation in a poorly understood manner. Moreover, there is very little information about whether environmental changes affect lineage progression during postembryonic neurogenesis. Using as a model system the pattern of abdominal leucokinergic neurons (ABLKs), this study analysed how changes in a set of environmental factors affect the number of ABLKs generated during postembryonic neurogenesis. The variability in ABLK number between individuals and between hemiganglia of the same individual is described and, by genetic analysis, the Bithorax-Complex genes and the Ecdysone hormone were identified as critical factors in these differences. The possible adaptive roles involved in this process were explored (Alvarez-Rivero, 2016).

Systemic corazonin signalling modulates stress responses and metabolism in Drosophila

Stress triggers cellular and systemic reactions in organisms to restore homeostasis. Mammalian gonadotropin-releasing hormone (GnRH) and its insect orthologue, adipokinetic hormone (AKH), are known for their roles in modulating stress-related behaviour. This study shows that corazonin (Crz), a peptide homologous to AKH/GnRH, also alters stress physiology in Drosophila. The Crz receptor (CrzR) is expressed in salivary glands and adipocytes of the liver-like fat body, and CrzR knockdown targeted simultaneously to both these tissues increases the fly's resistance to starvation, desiccation and oxidative stress, reduces feeding, alters expression of transcripts of Drosophila insulin-like peptides (DILPs), and affects gene expression in the fat body. Furthermore, in starved flies, CrzR-knockdown increases circulating and stored carbohydrates. Thus, these findings indicate that elevated systemic Crz signalling during stress coordinates increased food intake and diminished energy stores to regain metabolic homeostasis. This study suggests that an ancient stress-peptide in Urbilateria evolved to give rise to present-day GnRH, AKH and Crz signalling systems (Kubrak, 2016).

SIFamide translates hunger signals into appetitive and feeding behavior in Drosophila

Animal behavior is, on the one hand, controlled by neuronal circuits that integrate external sensory stimuli and induce appropriate motor responses. On the other hand, stimulus-evoked or internally generated behavior can be influenced by motivational conditions, e.g., the metabolic state. Motivational states are determined by physiological parameters whose homeostatic imbalances are signaled to and processed within the brain, often mediated by modulatory peptides. This study investigate the regulation of appetitive and feeding behavior in the fruit fly, Drosophila melanogaster. Four neurons in the fly brain that release SIFamide were found to be integral elements of a complex neuropeptide network that regulates feeding. SIFamidergic cells integrate feeding stimulating (orexigenic) and feeding suppressant (anorexigenic) signals to appropriately sensitize sensory circuits, promote appetitive behavior, and enhance food intake. This study advances the cellular dissection of evolutionarily conserved signaling pathways that convert peripheral metabolic signals into feeding-related behavior (Martelli, 2017).

Animals have interlaced neuronal and endocrine systems to control feeding behavior by integrating internal information about metabolic needs and external stimuli signaling the availability and quality of nutrition. In mammals, various internal sensors monitor the metabolic state and convey endocrine and neuronal signals to peripheral organs and the brain, e.g., through the release of peptides, such as leptin, ghrelin, insulin, and peptide YY, or through the neuronal activity of the sensory vagus nerve afferents. The hypothalamus (HT) represents a main integrator of these signals and contains neuronal circuits regulating energy homeostasis. Antagonistically acting populations of neurons in the arcuate nucleus that express neuropeptide Y (NPY), agouti-related peptide (AgRP), peptides derived from the precursors pro-opiomelanocortin (POMC), or cocaine- and amphetamine-regulated transcript (CART), respectively, integrate these peripheral signals. Activating NPY/AgRP-releasing and orexin-releasing neurons, or injection of these peptides, enhances food intake, whereas activating POMC- and CART-expressing neurons or injection of these peptides decreases it. How exactly these peptides modulate neuronal circuits that control feeding-related behavior remains unclear (Martelli, 2017).

The brain of the fruit fly, Drosophila melanogaster, is much simpler in terms of cell numbers when compared to the mammalian brain. Its often individually identifiable neurons can be genetically targeted and manipulated or monitored using DNA-encoded Ca2+ sensors. Feeding-related behavior ranging from odor-guided foraging to food uptake has been exceedingly well described in Drosophila and other flies. Neural circuits controlling distinct aspects of feeding, e.g., the detection of gustatory and olfactory food stimuli, internal sensing of hemolymph sugar concentration, motor control of proboscis extension, food intake, and feeding-induced suppression of alternative behaviors like locomotion, have been characterized. Also in flies, peptidergic neurons modulate feeding behavior. The release of short neuropeptide F (sNPF) increases appetitive odor-guided behavior and food uptake. Conversely, drosulfakinin, a cholecystokinin homolog, allatostatin A (AstA), and myosin inhibitory peptide (MIP) reduce food intake. However, a function for the neuropeptide SIFamide in feeding-related behavior remains unclear. The SIFamide amino acid sequence is largely conserved across the arthropod lineage and has been implicated in courtship behavior and sleep in Drosophila, aggression in a freshwater prawn, as well as in various feeding-related physiological processes, e.g., the modulation of the stomatogastric ganglion in lobsters or the control of salivary glands in blood-sucking ticks. The SIFamide receptor (SIFaR) is a homolog of the vertebrate gonadotropin inhibitory hormone receptor (GnIHR), although their respective ligands, SIFamide and GnIH, are not sequence related. GnIHR regulates food intake and reproductive behavior in opposite directions, thereby promoting feeding behavior over alternative behavioral tasks in periods of metabolic needs. However, it remains unclear whether the functions of the SIFamide- and GnIH-signaling pathways, respectively, are conserved across phyla (Martelli, 2017).

This study used Drosophila to study the role of SIFamide in feeding behavior. Thermogenetic activation of SIFamidergic neurons was shown to enhance appetitive behavior evoked by gustatory and olfactory stimuli, as well as food intake. Second, it was shown that release of SIFamide sensitizes olfactory signaling in the antennal lobe (AL). Third, it was demonstrated that orexigenic as well as anorexigenic peptidergic neurons interact anatomically and functionally with SIFamidergic cells in the brain. These findings together identify SIFamide neurons as an interface between intrinsic metabolic signals and sensory neuronal circuits mediating appetitive behavior and food intake (Martelli, 2017).

A nutrient-responsive hormonal circuit mediates an inter-tissue program regulating metabolic homeostasis in adult Drosophila

Animals maintain metabolic homeostasis by modulating the activity of specialized organs that adjust internal metabolism to external conditions. However, the hormonal signals coordinating these functions are incompletely characterized. This study shows that six neurosecretory cells in the Drosophila central nervous system respond to circulating nutrient levels by releasing Capa hormones, homologs of mammalian neuromedin U, which activate the Capa receptor (CapaR) in peripheral tissues to control energy homeostasis. Loss of Capa/CapaR signaling causes intestinal hypomotility and impaired nutrient absorption, which gradually deplete internal nutrient stores and reduce organismal lifespan. Conversely, increased Capa/CapaR activity increases fluid and waste excretion. Furthermore, Capa/CapaR inhibits the release of glucagon-like adipokinetic hormone from the corpora cardiaca, which restricts energy mobilization from adipose tissue to avoid harmful hyperglycemia. These results suggest that the Capa/CapaR circuit occupies a central node in a homeostatic program that facilitates the digestion and absorption of nutrients and regulates systemic energy balance (Koyama, 2021).

Tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding neuropeptides via Lgr3/8 in the brain

In patients with advanced-stage cancer, cancer-associated anorexia affects treatment success and patient survival. However, the underlying mechanism is poorly understood. This study shows that Dilp8, a Drosophila homologue of mammalian insulin-like 3 peptide (INSL3), is secreted from tumour tissues and induces anorexia through the Lgr3 receptor in the brain. Activated Dilp8-Lgr3 signalling upregulated anorexigenic nucleobinding 1 (NUCB1) and downregulated orexigenic short neuropeptide F (sNPF) and NPF expression in the brain. In the cancer condition, the protein expression of Lgr3 and NUCB1 was significantly upregulated in neurons expressing sNPF and NPF. INSL3 levels were increased in tumour-implanted mice and INSL3-treated mouse hypothalamic cells showed Nucb2 upregulation and Npy downregulation. Food consumption was significantly reduced in intracerebrospinal INSL3-injected mice. In patients with pancreatic cancer, higher serum INSL3 levels increased anorexia. These results indicate that tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding hormones through the Lgr3/Lgr8 receptor in Drosophila and mammals (Yeom, 2021).

Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles

Synaptic neuropeptide and neurotrophin stores are maintained by constitutive bidirectional capture of dense-core vesicles (DCVs) as they circulate in and out of the nerve terminal. Activity increases DCV capture to rapidly replenish synaptic neuropeptide stores following release. However, it is not known whether this is due to enhanced bidirectional capture. Experiments at the Drosophila neuromuscular junction, where DCVs contain neuropeptides and a bone morphogenic protein, show that activity-dependent replenishment of synaptic neuropeptides following release is evident after inhibiting the retrograde transport with the dynactin disruptor mycalolide B or photobleaching DCVs entering a synaptic bouton by retrograde transport. In contrast, photobleaching anterograde transport vesicles entering a bouton inhibits neuropeptide replenishment after activity. Furthermore, tracking of individual DCVs moving through boutons shows that activity selectively increases capture of DCVs undergoing anterograde transport. Finally, upregulating fragile X mental retardation 1 protein (Fmr1, also called FMRP) acts independently of futsch/MAP-1B to abolish activity-dependent, but not constitutive, capture. Fmr1 also reduces presynaptic neuropeptide stores without affecting activity-independent delivery and evoked release. Therefore, presynaptic motoneuron neuropeptide storage is increased by a vesicle capture mechanism that is distinguished from constitutive bidirectional capture by activity dependence, anterograde selectivity, and Fmr1 sensitivity. These results show that activity recruits a separate mechanism than used at rest to stimulate additional synaptic capture of DCVs for future release of neuropeptides and neurotrophins (Cavolo, 2016).

Synapses are supplied by anterograde axonal transport from the soma, the site of synthesis of synaptic vesicle proteins and dense-core vesicles (DCVs) that contain neuropeptides and neurotrophins. Delivery to synapses was thought to be based on a one-way anterograde trip until it was discovered that DCVs are subject to sporadic capture while traveling bidirectionally through en passant boutons as part of long-distance vesicle circulation (Wong, 2012). Interestingly, constitutive DCV capture occurs both during fast anterograde and retrograde transport, which are mediated by different motors (i.e., the kinesin 3 family member unc-104/Kif1A and the dynein/dynactin complex, respectively). Balanced capture in both directions is advantageous because DCVs are distributed equally among en passant boutons (Wong, 2012). In principle, bidirectional capture could occur by parallel regulation of anterograde and retrograde motors or by modification of the microtubules that both anterograde and retrograde DCV motors travel on (Cavolo, 2016).

Before the discovery of bidirectional capture of circulating vesicles, activity was shown to replenish the presynaptic neuropeptide pool following release by inducing Ca2+-dependent capture of DCVs being transported through boutons. This result and subsequent experiments (Bulgari, 2014) established that capture, rather than delivery or DCV turnover, limits synaptic neuropeptide stores. Activity-dependent capture was first described with a GFP-tagged neuropeptide in the Drosophila neuromuscular junction (NMJ), but also occurs with neurotrypsin, wnt/wingless, and brain-derived neurotrophic factor. Mechanistically, activity-dependent capture was characterized in terms of the rebound in presynaptic GFP-tagged peptide content following release and correlated with decreased retrograde transport. However, it is now evident that the reduction in retrograde flux could be caused by enhanced bidirectional capture as DCVs travel back and forth through the terminal as part of vesicle circulation (Wong, 2012). Therefore, prior studies support the hypothesis that there is only one synaptic capture mechanism, which is bidirectional and facilitated by activity (Cavolo, 2016).

This study tested the above hypothesis by investigating the directionality of activity-dependent capture. Experiments were performed with multiple approaches, including inhibiting retrograde transport, particle tracking, and simultaneous photobleaching and imaging (SPAIM; Wong, 2012). Furthermore, the effect of fragile X retardation protein (Fmr1, also called FMRP) was examined because it is known to affect bouton size and neuropeptide release. Together, these studies establish that different mechanisms mediate synaptic capture at rest and in response to activity (Cavolo, 2016).

Until recently, it was thought that presynaptic neuropeptide stores were set by controlling synthesis and delivery by fast one-way axonal transport of DCVs. However, studies of the Drosophila NMJ have shown that there is an excess of DCVs delivered to type-I boutons by long-distance vesicle circulation. Therefore, because DCV delivery is not limiting, the presynaptic neuropeptide pool is determined by capture, which was found to be bidirectional (Wong, 2012). However, in addition to constitutive capture, activity induces Ca2+-dependent capture. This is advantageous because tapping into the circulating vesicle pool removes delays associated with synthesis and transport, which can take days in humans, to rapidly replace released peptides. Surprisingly, experiments presented in this study demonstrate that activity-dependent capture is unidirectional and selectively sensitive to a genetic perturbation (i.e., Fmr1 overexpression). Therefore, activity does not simply enhance constitutive bidirectional capture that operates at rest, but instead stimulates an independent synaptic capture mechanism (Cavolo, 2016).

Previously, it was not possible to genetically block activity-dependent capture to determine its contribution to steady-state presynaptic stores. However, this study documented inhibition of activity-dependent capture by Fmr1 overexpression. As this was accompanied by a dramatic decrease in presynaptic DCV number, it is concluded that activity-dependent capture makes a large contribution to steady-state presynaptic peptide stores and hence the capacity for future release. At the Drosophila NMJ, DCVs contain a bone morphogenic protein and neuropeptides. Thus, it is possible that activity-dependent capture affects development and acute synaptic function (Cavolo, 2016).

Capture efficiency measurements revealed that the previously detected decrease in retrograde traffic following activity was an indirect effect of vesicle circulation; activity-induced capture of only anterograde DCVs at each en passant bouton simply leaves fewer DCVs for the retrograde trip back into the axon without changing retrograde capture. Of interest, anterograde selectivity for activity-induced capture rules out mechanisms that would perturb transport in both directions (e.g., microtubule breaks). DCV anterograde transport is mediated by the unc-104/Kif1A motor, which also transports SSV proteins and is required for formation of boutons. Therefore, activity-dependent capture may regulate unc-104/Kif1A to affect synaptic release of both small-molecule transmitters and peptides. However, alternative targets could be involved, including proteins that mediate DCV interaction with this anterograde motor or alter the DCV itself (e.g., its phosphoinositides, which may bind to the unc-104/Kif1A pleckstrin homology domain) (Cavolo, 2016).

The thirsty fly: Ion transport peptide (ITP) is a novel endocrine regulator of water homeostasis in Drosophila

Animals need to continuously adjust their water metabolism to the internal and external conditions. Homeostasis of body fluids thus requires tight regulation of water intake and excretion, and a balance between ingestion of water and solid food. This study investigated how these processes are coordinated in Drosophila melanogaster. The first thirst-promoting and anti-diuretic hormone of Drosophila was identified, encoded by the gene Ion transport peptide (ITP). This endocrine regulator belongs to the CHH (crustacean hyperglycemic hormone) family of peptide hormones. Using genetic gain- and loss-of-function experiments, this study showed that ITP signaling acts analogous to the human vasopressin and renin-angiotensin systems; expression of ITP is elevated by dehydration of the fly, and the peptide increases thirst while repressing excretion, promoting thus conservation of water resources. ITP responds to both osmotic and desiccation stress, and dysregulation of ITP signaling compromises the fly's ability to cope with these stressors. In addition to the regulation of thirst and excretion, ITP also suppresses food intake. Altogether, this work identifies ITP as an important endocrine regulator of thirst and excretion, which integrates water homeostasis with feeding of Drosophila (Galikova, 2018).

Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance

For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization, and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of Capability (CAPA) (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been previously demonstrated to cause anti-diuresis in insects, including dipterans. This study provides evidence that low (fM) and high (microM) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K(+) clearance rates and reduce chill tolerance, high doses facilitate K(+) clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, the maximum achievable hormonal titres of CAPA was estimated, and evidence was further found that CAPA may function as an anti-diuretic hormone in Drosophila melanogaster. Evidence is provided of a neuropeptide that can negatively affect cold tolerance in an insect, and further evidence of CAPA functioning as an anti-diuretic peptide in this ubiquitous insect model (MacMillan, 2018).

Specification of the Drosophila Orcokinin A neurons by combinatorial coding

The central nervous system contains a daunting number of different cell types. Understanding how each cell acquires its fate remains a major challenge for neurobiology. The developing embryonic ventral nerve cord (VNC) of Drosophila melanogaster has been a powerful model system for unraveling the basic principles of cell fate specification. This pertains specifically to neuropeptide neurons, which typically are stereotypically generated in discrete subsets, allowing for unambiguous single-cell resolution in different genetic contexts. The specification of the OrcoA-LA neurons, characterized by the expression of the neuropeptide Orcokinin A and located laterally in the A1-A5 abdominal segments of the VNC, was studied. The progenitor neuroblast (NB; NB5-3) and the temporal window (castor/grainyhead) that generate the OrcoA-LA neurons were identified. The role of the Ubx, abd-A, and Abd-B Hox genes in the segment-specific generation of these neurons was studied. Additionally, these results indicate that the OrcoA-LA neurons are "Notch Off" cells, and neither programmed cell death nor the BMP pathway appears to be involved in their specification. Finally, a targeted genetic screen was performed of 485 genes known to be expressed in the CNS and nab, vg, and tsh were identified as crucial determinists for OrcoA-LA neurons. This work provides a new neuropeptidergic model that will allow for addressing new questions related to neuronal specification mechanisms in the future (Rubio-Ferrera, 2023).

Specification of Drosophila neuropeptidergic neurons by the splicing component brr2

During embryonic development, a number of genetic cues act to generate neuronal diversity. While intrinsic transcriptional cascades are well-known to control neuronal sub-type cell fate, the target cells can also provide critical input to specific neuronal cell fates. Such signals, denoted retrograde signals, are known to provide critical survival cues for neurons, but have also been found to trigger terminal differentiation of neurons. One salient example of such target-derived instructive signals pertains to the specification of the Drosophila FMRFamide neuropeptide neurons, the Tv4 neurons of the ventral nerve cord. Tv4 neurons receive a BMP signal from their target cells, which acts as the final trigger to activate the FMRFa gene. A recent FMRFa-eGFP genetic screen identified several genes involved in Tv4 specification, two of which encode components of the U5 subunit of the spliceosome: brr2 (l(3)72Ab) and Prp8. This study focused on the role of RNA processing during target-derived signaling. brr2 and Prp8 were found to play crucial roles in controlling the expression of the FMRFa neuropeptide specifically in six neurons of the VNC (Tv4 neurons). Detailed analysis of brr2 revealed that this control is executed by two independent mechanisms, both of which are required for the activation of the BMP retrograde signaling pathway in Tv4 neurons: (1) Proper axonal pathfinding to the target tissue in order to receive the BMP ligand. (2) Proper RNA splicing of two genes in the BMP pathway: the thickveins (tkv) gene, encoding a BMP receptor subunit, and the Medea gene, encoding a co-Smad. These results reveal involvement of specific RNA processing in diversifying neuronal identity within the central nervous system (Monedero, 2018).

Identification and in vivo characterisation of cardioactive peptides in Drosophila melanogaster

Neuropeptides and peptide hormones serve as critical regulators of numerous biological processes, including development, growth, reproduction, physiology, and behaviour. In mammals, peptidergic regulatory systems are complex and often involve multiple peptides that act at different levels and relay to different receptors. To improve the mechanistic understanding of such complex systems, invertebrate models in which evolutionarily conserved peptides and receptors regulate similar biological processes but in a less complex manner have emerged as highly valuable. Drosophila melanogaster represents a favoured model for the characterisation of novel peptidergic signalling events and for evaluating the relevance of those events in vivo. The present study analysed a set of neuropeptides and peptide hormones for their ability to modulate cardiac function in semi-intact larval Drosophila melanogaster. Numerous peptides were identifed that significantly affected heart parameters such as heart rate, systolic and diastolic interval, rhythmicity, and contractility. Thus, peptidergic regulation of the Drosophila heart is not restricted to chronotropic adaptation but also includes inotropic modulation. By specifically interfering with the expression of corresponding peptides in transgenic animals, the in vivo relevance of the respective peptidergic regulation was assessed. Based on the functional conservation of certain peptides throughout the animal kingdom, the identified cardiomodulatory activities may be relevant not only to proper heart function in Drosophila, but also to corresponding processes in vertebrates, including humans (Schiemann, 2018).

Loss of angiotensin-converting enzyme-related (ACER) peptidase disrupts behavioural and metabolic responses to diet in Drosophila melanogaster

Drosophila Acer (Angiotensin-converting enzyme-related) encodes a member of the angiotensin-converting enzyme (ACE) family of metallopeptidases that in mammals play roles in the endocrine regulation of blood homeostasis. ACE is also expressed in adipose tissue, where it is thought to play a role in metabolic regulation. Drosophila ACER is expressed in the adult fat body of the head and abdomen and is secreted into the haemolymph. Acer null mutants have previously been found to have reduced night-time sleep and greater sleep fragmentation. ACER may thus be part of a signalling system linking metabolism with sleep. To further understand the role of ACER in response to diet, sleep and other nutrient-responsive phenotypes were measured in Acer null flies under different dietary conditions. Loss of Acer disrupted the normal response of sleep to changes in nutrition. Other nutrient-sensitive phenotypes, including survival and glycogen storage, were also altered in the Acer mutant but lipid storage was not. Although the physiological substrate of the ACER peptidase has not been identified, an alteration of the normal nutrient-dependent control of Drosophila insulin-like peptide 5 protein in the Acer mutant suggests insulin/IGF-like signalling as a candidate pathway modulated by ACER in the nutrient-dependent control of sleep, survival and metabolism (Glover, 2019).

Imaging neuropeptide release at synapses with a genetically engineered reporter

Research on neuropeptide function has advanced rapidly, yet there is still no spatio-temporally resolved method to measure the release of neuropeptides in vivo. This study introduce Neuropeptide Release Reporters (NPRRs): novel genetically-encoded sensors with high temporal resolution and genetic specificity. Using the Drosophila larval neuromuscular junction (NMJ) as a model, evidence is provided that NPRRs recapitulate the trafficking and packaging of native neuropeptides, and report stimulation-evoked neuropeptide release events as real-time changes in fluorescence intensity, with sub-second temporal resolution (Ding, 2019).

Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate

Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, this study addressed the role of carboxypeptidase D (dCPD) in global neuropeptide processing and selected peptide-regulated behaviours in Drosophila. A deficiency in dCPD results in C-terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD-encoding gene silver in the larva causes lethality, and leads to deficits in starvation-induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. These results show that dCPD is a key enzyme for neuropeptide processing and peptide-regulated behaviour in Drosophila. dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE (Pauls, 2019).

Activity-evoked and spontaneous opening of synaptic fusion pores

Synaptic release of neuropeptides packaged in dense-core vesicles (DCVs) regulates synapses, circuits, and behaviors including feeding, sleeping, and pain perception. This study imaged synaptic DCV fusion pore openings without interference from cotransmitting small synaptic vesicles (SSVs) with the use of a fluorogen-activating protein (FAP). Activity-evoked kiss and run exocytosis opens synaptic DCV fusion pores away from active zones that readily conduct molecules larger than most native neuropeptides (i.e., molecular weight [MW] up to, at least, 4.5 kDa). Remarkably, these synaptic fusion pores also open spontaneously in the absence of stimulation and extracellular Ca(2+) SNARE perturbations demonstrate different mechanisms for activity-evoked and spontaneous fusion pore openings with the latter sharing features of spontaneous small molecule transmitter release by active zone-associated SSVs. Fusion pore opening at resting synapses provides a mechanism for activity-independent peptidergic transmission (Bulgari, 2019).

Drosulfakinin signaling in fruitless circuitry antagonizes P neurons to regulate sexual arousal in Drosophila

Animals perform or terminate particular behaviors by integrating external cues and internal states through neural circuits. Identifying neural substrates and their molecular modulators promoting or inhibiting animal behaviors are key steps to understand how neural circuits control behaviors. This study identified the Cholecystokinin-like peptide Drosulfakinin (DSK) that functions at single-neuron resolution to suppress male sexual behavior in Drosophila. Dsk neurons physiologically interact with male-specific P neurons, part of a command center for male sexual behaviors, and function oppositely to regulate multiple arousal-related behaviors including sex, sleep and spontaneous walking. This study further found that the DSK- peptide functions through its receptor CCKLR-17D to suppress sexual behaviors in flies. Such a neuropeptide circuit largely overlaps with the fruitless-expressing neural circuit that governs most aspects of male sexual behaviors. Thus DSK/CCKLR signaling in the sex circuitry functions antagonistically with P neurons to balance arousal levels and modulate sexual behaviors (Wu, 2019).

Male courtship in Drosophila melanogaster is one of the best-understood innate behaviors, and largely controlled by the fruitless (fru) gene and doublesex (dsx) gene, which encode sex-specific transcription factors (FRUM and DSXM in males and DSXF in females). FRUM is responsible for most aspects of male courtship, and DSXM is important for the experience-dependent acquisition of courtship in the absence of FRUM, and courtship intensity and sine song production in the presence of FRUM. FRUM is expressed in a dispersed subset of ca. 2000 neurons including sensory neurons, interneurons, and motor neurons that are potentially interconnected to form a sex circuitry controlling sexual behaviors. In contrast, DSXM is expressed in ca. 700 neurons in males, the majority of which also express FRUM, and are crucial for male courtship. Recently, substantial progress has been made into how external sensory cues are perceived and integrated by fruM and/or dsxM neurons to initiate male courtship, in particular, how a subset of male-specific fruM- and dsxM-expressing P1 neurons integrate olfactory and gustatory cues from female or male targets to initiate or terminate courtship. Such a neuronal pathway is also conserved in other Drosophila species (Wu, 2019).

Behavioral decisions depend on both excitatory and inhibitory modulations. P1 neurons represent an excitatory center that integrates multiple (both excitatory and inhibitory) sensory cues and initiates courtship. However, whether there is an inhibitory counterpart that operates against P1 neurons to balance sexual activity is still unknown. Indeed, males do not absolutely court virgin females even if these females may provide the same visual, olfactory, and gustatory cues, depending on the male's internal states and past experiences. It has been previously shown that neuropeptide SIFamide acts on fruM-positive neurons and inhibits male-male but not male-female courtship, and SIFamide neurons also integrate multiple peptidergic neurons to orchestrate feeding behaviors, but whether SIFamide inhibits internal arousal states for sexual behaviors is not clear. Recently, it was found that sleep and sex circuitries interact mutually and demonstrate how DN1 neurons in the sleep circuitry and P1 neurons in the courtship circuitry function together to coordinate behavioral choices between sleep and sex. However, very little is known about the inhibitory pathway(s) that may represent internal arousal states and inhibit courtship toward females (Wu, 2019).

This study sets out to identify courtship inhibitory neurons that express neuropeptides in Drosophila, as neuropeptides play key roles in adjusting animal behaviors based on environmental cues and internal needs. This study identifies that the neuropeptide Drosulfakinin (DSK), the fly ortholog of Cholecystokinin (CCK) in mammals, functions through its receptor CCKLR-17D3 in the fruM-expressing sex circuitry to inhibit male courtship toward females. It is further demonstrate dthat Dsk neurons and P1 neurons interact and oppositely regulate male sexual behaviors (Wu, 2019).

The results identify, at single-neuron resolution, four pairs of fruM-expressing Dsk neurons (MP1 and MP3) that suppress male and female sexual behaviors. The suppression of male and female sexual behaviors depends on the secretion of the neuropeptide DSK-2, which then acts on one of its receptors CCKLR-17D3 that is expressed in many fruM neurons including P1 neurons and the mushroom bodies. Dsk neurons function antagonistically with courtship promoting P1 neurons to co-regulate male courtship, as well as sleep and spontaneous walking (Wu, 2019).

Cholecystokinin (CCK) signaling appears well conserved over evolution and modulates multiple behaviors. In Drosophila, the CCK-like sulfakinin (DSK) is multifunctional and has been reported to be involved in regulating aspects of food ingestion and satiety, aggression, as well as escape-related locomotion and synaptic plasticity during neuromuscular junction development. In mammals, CCK generated from the intestine acts on its receptors in the nucleus of the solitary tract of the brain to transmit satiety signaling and thus inhibit feeding. Furthermore, CCK signaling in the nucleus accumbens modulates dopaminergic influences on male sexual behaviors in rats. CCK is also involved in nociception, learning and memory, aggression and depressive-like behaviors (Wu, 2019).

Despite its significant and conserved roles in modulating multiple innate and learned behaviors, how CCK or DSK signaling responds to environment and/or internal changes and acts on specific neurons expressing its receptors to modulate multiple behaviors, is still rarely known. The finding that DSK/CCKLR signaling functions in the fruM- and/or dsx-expressing sex circuitry to inhibit male courtship is an effort to use Drosophila as a model to investigate how this conserved signaling modulates animal behaviors (Wu, 2019).

The results uncovered a functional circuitry from many dsx neurons (including courtship-promoting P1 neurons) to four pairs of Dsk MP neurons via direct synaptic transmission, and these MP neurons then modulate CCKLR-17D3 neurons including many fruM and/or dsx neurons via secretion of DSK peptides. It is of particular interest to reveal how the four pairs of MP neurons integrate sensory information (in any), physiological states and past experiences in the future to better understand how this neuropeptide signaling modulate arousal states. It is still not known if these MP neurons receive sensory inputs, but since they receive inputs from many dsx neurons, including P1 neurons that integrate multiple chemosensory information, these sensory inputs will at least relay to Dsk MP neurons via P1. Whether there are other pathways from sensory inputs to MP neurons awaits further study. It is also noted that multiple physiological changes including feeding states, aging and sleep deprivation, as well as past housing conditions affect expression of DSK/CCKLR-17D3, but how they affect DSK signaling and behaviors is still unclear. This study showed that male-male group-housing increases DSK expression and thereby reduces male courtship at least under a restricted condition, and previous findings also revealed opposite effects of group-housing on the excitability of P1 neurons in males that have fruM function or lack fruM function. Such male-male housing experience may mildly reduce sexual arousal in a persistent manner, perhaps by increasing DSK expression, but how such housing condition affects physiological roles of Dsk MP neurons and P1 neurons awaits further functional imaging studies on a potential P1-Dsk-P1 functional loop (and a much complex dsx-Dsk-17D3 pathway) with sensory stimulation under different physiological states. In terms of the time scale that DSK functions to inhibit male courtship, the results indicate an immediate behavioral effect upon Dsk neuronal activation. It is also noted that activation of Dsk neurons inhibits male courtship and lasts for minutes, and previous findings also showed that activation of P1 neurons promoted wing extension and aggression and lasts for minutes. These persistent behavioral effects may represent persistent arousal states regulated by Dsk and P1 neurons in this study; however, how this persistency is generated both in the circuit level and behavioral level still needs further investigation (Wu, 2019).

Dsk mutants do not have obvious courtship abnormality under the courtship assays. There are at least two possibilities: (1) DSK may function only in specific conditions (e.g., group-housing) that increase its expression to inhibit courtship; and (2) There are redundant inhibitory signals for courtship, such as another neuropeptide SIFamide that acts on fruM neurons, although they specifically inhibit male-male courtship. Further studies on how DSK/CCKLR signaling is activated under certain conditions, as well as how DSK, SIFamide and other inhibitory signals (if any) jointly modulate male courtship are needed to fully understand this. Nevertheless, that courtship inhibition by activation of Dsk neurons depends on DSK/CCKLR-17D3 signaling, and increasing such signaling through Cas9 activators in an otherwise wild-type male efficiently inhibited courtship, unambiguously reveal the role of DSK/CCKLR-17D3 signaling in suppressing sexual behaviors (Wu, 2019).

As DSK signaling modulates multiple behaviors, one may argue that its role in male courtship is not specific, e.g., activation of Dsk neurons may drive a competing behavior that phenotypically shunting male courtship. Although such possibility cannot be excluded, a number of evidences are listed to support DSK's role with specificity in courtship inhibition: (1) DSK functions in four pairs of fruM-expressing neurons to inhibit courtship; (2) males with activated Dsk neurons rarely court virgin females, while they follow rotating visual objects normally; (3) Dsk neurons receive synaptic transmission from courtship promoting P1 neurons (and many other dsx-expressing neurons) in an experience-dependent manner; (4) Dsk and P1 neurons antagonistically modulate sexual behaviors and wakefulness; and (5) DSK receptor CCKLR-17D3 inhibits male courtship and expresses in many fruM and/or dsx neurons including P1 neurons. It is noted that CCKLR-17D3 is expressed broadly in the CNS including not only P1 neurons, but also mushroom bodies that regulate a range of behaviors including learning, locomotion and sleep. That DSK signaling is multifunctional is possibly due to broad expression of its receptors, and further studies on dissection of CCKLR function in subsets of neurons will help to understand how DSK/CCKLR signaling modulates multiple behaviors (Wu, 2019).

The decision for male flies to court or not depends on not only environmental cues such as availability and suitability of potential mates (males, virgin females, or mated females), but also their internal states (e.g., thirsty or sleepy). It is proposed that there are at least four factors affecting such a decision: (1) external cues that inhibit courtship, referred to as Ex-In factor, such as the male-specific pheromone cVA4; (2) external cues that are excitatory for courtship, referred to as Ex-Ex factor, such as courtship song; (3) internal states that inhibit courtship, referred to as In-In factor; and (4) internal states that are excitatory for courtship, referred to as In-Ex factor. These factors dynamically change and jointly determine males' decision to court or not (Wu, 2019).

Substantial progress has been made on how Ex-In and Ex-Ex factors jointly modulate the activity of male-specific P1 neurons, which is crucial for courtship initiation. In contrast, much less is understood on In-In and In-Ex factors. Recently, Zhang (2016; 2018) found that dopaminergic modulation of P1 neurons drives male courtship not only by desensitizing P1 to inhibition, but also by promoting recurrent P1 stimulation, thus may act as an In-Ex factor for male courtship. Note that all the three factors mentioned above converge on P1 neurons, making them a decision-making center for male courtship. The DSK/CCKLR signaling identified in this study is of particular interest, as it is likely to act as an In-In factor for male courtship, and above all, it does not simply act on P1 neurons like three other factors, but instead forms a potential functional loop with P1 neurons and antagonizes P1 function in modulating male courtship and wakefulness. That Dsk neurons receive synaptic transmission from P1 neurons and other dsx-expressing neurons in an experience-dependent manner further highlights a central role that the DSK/CCKLR signaling plays. These factors, excitatory vs. inhibitory, external vs. internal, jointly control appropriate performance of sexual behaviors, and further studies will reveal how P1 and other dsx-expressing neurons physiologically interact with Dsk neurons to balance behavioral output (Wu, 2019).

A prominent feature of the neuronal control of male and female sexual behaviors in Drosophila is that, despite large similarity in sensory systems, central integrative neurons are sex-specific in the two sexes, with dsx-expressing pC1 neurons integrating olfactory and auditory cues and promoting receptivity to courting males, and fruM-expressing P1 neurons (largely overlapped with dsx-expressing pC1) integrating olfactory, gustatory, and auditory cues and promoting courtship to females. In contrast, the four pairs of Dsk-expressing MP neurons investigated in this study are sexually monomorphic and inhibit both male courtship and female receptivity. Thus DSK/CCKLR signaling may inhibit sexual behaviors in response to physiological changes that are common to both sexes, while sex-promoting central neurons integrating distinct sensory cues are sexually dimorphic. Interestingly, these Dsk neurons common to both sexes receive synaptic transmission from sexually dimorphic dsx neurons in both males and females, providing a simple solution to link sex-specific excitatory and sexually non-specific inhibitory control of sexual behaviors in males and females (Wu, 2019).

The cellular diversity and transcription factor code of Drosophila enteroendocrine cells

Enteroendocrine cells (EEs) in the intestinal epithelium have important endocrine functions, yet this cell lineage exhibits great local and regional variations that have hampered detailed characterization of EE subtypes. Through single-cell RNA-sequencing analysis, combined with a collection of peptide hormone and receptor knockin strains, this study provides a comprehensive analysis of cellular diversity, spatial distribution, and transcription factor (TF) code of EEs in adult Drosophila midgut. Ten major EE subtypes were identified that totally produced approximately 14 different classes of hormone peptides. Each EE on average co-produces approximately 2-5 different classes of hormone peptides. Functional screen with subtype-enriched TFs suggests a combinatorial TF code that controls EE cell diversity; class-specific TFs Mirr and Ptx1 respectively define two major classes of EEs, and regional TFs such as Esg, Drm, Exex, and Fer1 further define regional EE identity. These single-cell data should greatly facilitate Drosophila modeling of EE differentiation and function (Guo, 2019).

Apart from the function in food digestion and absorption, the gastrointestinal tract is also considered as the largest endocrine organ due to the resident enteroendocrine cells (EEs). In mice and humans, EEs are scattered throughout the intestinal epithelium and take up only 1% of total intestinal cells, yet they produce more than 20 types of hormones that regulate a diverse of physiological processes, such as appetite, metabolism, and gut motility. There are at least 12 major subtypes of EEs based on hormones that they produce, and due to their great regional and local cellular diversity, the complete characterization of EE specification and diversification still remains as a challenge (Guo, 2019).

The adult Drosophila midgut has become an attractive model system for the understanding of EE cell diversity and their regulatory mechanisms. The EEs are scattered along the epithelium of the entire midgut, including anterior midgut (regions R1 and R2), middle midgut (the gastric region, R3) and posterior midgut (regions R4 and R5). They have important roles in regulating local stem cell division and lipid metabolism, as well as feeding and mating behaviors. Approximately 10 peptide hormone genes are found to be expressed in EEs, yielding more than 20 different peptide hormones. Studies using RNA in situ hybridization, antibody staining, and gene reporter tools have provided a glimpse of regional EE diversity in terms of peptide hormones that they produce. However, due to limited availability of antibodies against all these hormones and a limit in the number of hormones that can be simultaneously analyzed, the detailed characterization of EE subtypes and peptide profiles is still lacking (Guo, 2019).

As in mammals, EEs in the fly midgut are derived from multipotent intestinal stem cells (ISCs). The initial fate determination between absorptive enterocyte versus secretory EEs is controlled by Notch signaling and appears to be regulated by the antagonistic activities of E(spl)-C genes and achaete-scute complex genes. This is also analogous to the antagonistic activities between Hes1 (orthologous to E(spl)) and Math1 (paralogous to AS-C) in mammalian ISCs that control the initial cell fate decision. The committed EE progenitor cell usually divides one more time to yield a pair of EEs. Interestingly, the two EEs within each pair produce distinct hormone peptides as a result of differentially acquired Notch activity, suggesting that, at least in the posterior midgut, differential Notch signaling defines two major subtypes of EEs. The specification and commitment of EE fate requires the homeodomain transcription factor (TF) Prospero (Pros), and the maturation of peptide hormones in EEs requires a Neuro D family bHLH TF Dimmed (Dimm). Besides these general TFs that promote EE specification and function, little is known regarding the TFs that participate in EE subtype specification and regional EE identity (Guo, 2019).

Single-cell RNA-sequencing (scRNA-seq) has emerged as an efficient tool for revealing cell heterozygosity in different tissues and organisms. By using scRNA-seq and a collection of recently generated peptide and receptor knockin lines, this study provides a comprehensive analysis of EE cell diversity, peptide profiling, and regional distribution along the entire length of the fly midgut at single-cell resolution. In addition, TF enrichment analysis followed by genetic screen allowed thew identification of class and region EE regulators. These results suggest a TF code composed of class-specific and region-specific TFs generates EE cell diversity (Guo, 2019).

Using single-cell transcriptomics in combination with a collection of reporter lines, this study has provided a comprehensive characterization of the EE population in the entire midgut of adult Drosophila. In addition to the two major classes of EEs that respectively produce TK and AstC peptide hormones, a third class of EEs was identified that reside only in the anterior midgut (R2) and produce sNPF and CCHa2. Ten EE subtypes were identified that generally show region-specific distributions. In addition, functional screens with subtype-specific TFs have revealed class- and region-specific TFs that regulate subtype specification. These single-cell data should serve as an important resource for further understanding the differentiation, regulation, and function of EEs using Drosophila midgut as a genetic model system (Guo, 2019).

The single-cell data reveal 14 classes of peptide hormone genes that are expressed in EEs, compared to the previously known 10 classes. The midgut expression patterns of all these peptide hormones, including several peptide hormones whose gut expression patterns have not been clearly defined, such as Gbp5, ITP, and Nplp2 as well as sNPF, are also determined. As EEs perform their endocrine function by secreting various peptide hormones, the types of peptide hormones that they produced are usually used to classify EE subtypes in mammals. Indeed, the exclusive expression pattern of Tk and AstC is sufficient to distinguish between class I EEs and class II EEs. However, although different EE subtypes show distinct peptide hormone expression profiles, the types of peptide hormone expressed and the EE subtypes are not strictly correlated. In fact, the peptide hormone co-expression patterns are highly variable among individual EEs, even for EEs that belong to the same cluster or subtype. For example, for the II-m (C4) subtype, although they commonly produce Tk and NPF, their expression for Mip, Nplp2, and CCAP is highly variable. The external stimuli, such as stress and microbiota, may have an impact on the expression status of these variable peptide hormone genes. Alternatively, EEs could be plastic and change their peptide hormone expression profiles with age. Recent studies demonstrate that the mammalian EEs are plastic and can switch their hormone profiles as they differentiate and migrate upward along the crypt-villus axis (Guo, 2019).

One major limit associated with the scRNA-seq technology is that the spatial information of the cells is lost during tissue dissociation. In a way to overcome this limit, this study has developed a RSGE algorithm based on the region- and cell-type-specific transcriptome database from flygut-seq. As confirmed, for the various peptide hormone markers, including GAL4 knockin lines and antibodies, this algorithm has allowed generation of a reliable distribution map a for all the EE subtype clusters along the length of the midgut. The determination of the spatial distribution of EE subtypes should greatly facilitate the understanding of their regulation and function. For instance, DH31 and ITP expressing EEs are found be located in the posterior-most region of the midgut, and their location is clearly consistent with their known function: DH31 is known to regulate fluid secretion in Malpighian tubules, and ITP is known to regulate ion transport in hindgut. As regional difference for a common cell type is likely a general phenomenon in diverse tissues of many organisms, the algorithm in this study could provide an example of possible approaches for acquiring the lost spatial information of cells when conducting this type of single-cell analysis (Guo, 2019).

By analyzing the TF code for the EE subtypes followed by functional screen, this study has identified a number of TFs that participate in the specification of EE subtypes, including the class-I- and class-II-specific TFs Mirr and Ptx1 for the two major classes of EEs and region-specific TFs such as Esg, Drm, Fer1, and Sug that define regional EE identity. Previous studies in the posterior midgut have revealed that class I and II EEs are specified by differential Notch signaling. In this study, cell-type specific manipulating of Notch activity allows the conclusion that Notch must function transiently at the progenitor stage, between the two immediate daughters of an EEP, to define the two classes of EEs. As Mirr and Ptx1 are expressed only in differentiated EEs, the sequential activity of Notch and Mirr/Ptx1 indicates that these two TFs act downstream of Notch to specify class I versus class II EE type. The regional diversity of EEs is then further specified by region-specific TFs and possibly impacted by other environmental factors. It is proposed that EE cellular diversity is generated by a combination of class-specific and region-specific TFs, with class-specific TFs regulated by Notch signaling and region-specific TFs determined by anterior-posterior body planning during early development. The local EE diversity could also be regulated by environmental changes and age-related cell plasticity, possibilities that remain to be explored in the future (Guo, 2019).

Collectively, these single-cell data have provided a comprehensive characterization of EE cell diversity and their peptide hormone expression profiles. The TF code analysis also provides insights into EE diversity mechanisms. This data should greatly facilitate functional annotations of EE subtypes and gut peptide hormones under diverse physiological and pathological conditions, such as mating, starvation, bacterial infection, and so on. The Perrimon lab recently conducted single-cell transcriptomes for all types of midgut cells using the inDrop method. As EEs only represent a small fraction of total cells analyzed, their analysis primarily focused on progenitor cells and enterocytes (Hung, 2018). Therefore, the data and their scRNA-seq data should serve as complementary resources for understanding Drosophila gut cells. An online searchable database has been established to facilitate the use of these single-cell data (Guo, 2019).

Temporal groups of lineage-related neurons have different neuropeptidergic fates and related functions in the Drosophila melanogaster CNS

The central nervous system (CNS) of Drosophila is comprised of the brain and the ventral nerve cord (VNC). which are the homologous structures of the vertebrate brain and the spinal cord, respectively. Neurons of the CNS arise from neural stem cells called neuroblasts (NBs). Each neuroblast gives rise to a specific repertory of cell types whose fate is unknown in most lineages. A combination of spatial and temporal genetic cues defines the fate of each neuron. The origin and specification was studied of a group of peptidergic neurons present in several abdominal segments of the larval VNC that are characterized by the expression of the neuropeptide GPB5, the GPB5-expressing neurons (GPB5-ENs). The data reveal that the progenitor NB that generates the GPB5-ENs also generates the abdominal leucokinergic neurons (ABLKs) in two different temporal windows. This study also shows that these two set of neurons share the same axonal projections in larvae and in adults and, as previously suggested, may both function in hydrosaline regulation. Tenetic analysis of potential specification determinants reveals that Klumpfuss (klu) and huckebein (hkb) are involved in the specification of the GPB5 cell fate. Additionally, GPB5-ENs have a role in starvation resistance and longevity; however, their role in desiccation and ionic stress resistance is not as clear. It is hypothesize that the neurons arising from the same neuroblast lineage are both architecturally similar and functionally related (Diaz-de-la-Pena, 2020).

Cellular metabolic reprogramming controls sugar appetite in Drosophila

Cellular metabolic reprogramming is an important mechanism by which cells rewire their metabolism to promote proliferation and cell growth. This process has been mostly studied in the context of tumorigenesis, but less is known about its relevance for nonpathological processes and how it affects whole-animal physiology. This study shows that metabolic reprogramming in Drosophila female germline cells affects nutrient preferences of animals. Egg production depends on the upregulation of the activity of the pentose phosphate pathway in the germline, which also specifically increases the animal's appetite for sugar, the key nutrient fuelling this metabolic pathway. Functional evidence is provided that the germline alters sugar appetite by regulating the expression of the fat-body-secreted satiety factor Fit. These findings demonstrate that the cellular metabolic program of a small set of cells is able to increase the animal's preference for specific nutrients through inter-organ communication to promote specific metabolic and cellular outcomes (Carvalho-Santos, 2020).

The Drosophila melanogaster Neprilysin Nepl15 is involved in lipid and carbohydrate storage

The prototypical M13 peptidase, human Neprilysin, functions as a transmembrane "ectoenzyme" that cleaves neuropeptides that regulate e.g. glucose metabolism, and has been linked to type 2 diabetes. The M13 family has undergone a remarkable, and conserved, expansion in the Drosophila genus. This study describes the function of Drosophila melanogaster Neprilysin-like 15 (Nepl15). Nepl15 is likely to be a secreted protein, rather than a transmembrane protein. Nepl15 has changes in critical catalytic residues that are conserved across the Drosophila genus and likely renders the Nepl15 protein catalytically inactive. Nevertheless, a knockout of the Nepl15 gene reveals a reduction in triglyceride and glycogen storage, with the effects likely occurring during the larval feeding period. Conversely, flies overexpressing Nepl15 store more triglycerides and glycogen. Protein modeling suggests that Nepl15 is able to bind and sequester peptide targets of catalytically active Drosophila M13 family members, peptides that are conserved in humans and Drosophila, potentially providing a novel mechanism for regulating the activity of neuropeptides in the context of lipid and carbohydrate homeostasis (Banerjee, 2021).

Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission

Many neurons influence their targets through co-release of neuropeptides and small molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release, as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both STED super-resolution and transmission electron microscopy. Ptp4E also controls presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content (Tao, 2019).

Synaptic complexity is enhanced by co-transmission with small molecules and bioactive peptides. The two transmitter classes differ in their postrelease distances traveled and durations of action, thus providing mechanisms for rapid point-to-point control and slow neuromodulation of circuits, development and behavior. Furthermore, from a cell biology perspective, transmission by small molecules and neuropeptides is distinguished by different vesicular loading mechanisms. The genetic results presented here are remarkable because (a) they reveal increased transmitter packaging, when past genetic screens have only yielded mutants that reduce vesicular packaging; (b) control of vesicular packaging varied between neuron subtypes based on differential Rptp expression, which represents a new mechanism for generating variation in co-transmission in the nervous system. Furthermore, this result is intriguing in the context of monoaminergic neurons because Ptp4E interacts genetically with α-synuclein toxicity in Drosophila. Given that synuclein is implicated in Parkinson's disease, DCV fusion pore dynamics and the early secretory pathway, the results here suggest that the mechanistic relationship between Rptps and synuclein may be broader than previously recognized; and (c) presynaptic abundance of a small-molecule vesicular membrane transporter and luminal neuropeptides are regulated in parallel. This shows that regulation of co-transmission is not limited to control of activity-induced vesicle exocytosis. Instead, an Rptp regulates vesicular packaging of both small-molecule and peptide neurotransmitters that underlies co-release (Tao, 2019).

How can a single Rptp simultaneously modify vesicular loading of both monoamines and neuropeptides? Peptidergic neurotransmission relies on packaging of neuropeptides in the soma, where they condense in the TGN and are sorted into DCVs. There is little DCV circulation in octopamine terminals because of their extensive axonal arbors and numerous boutons. Therefore, Rptp regulation of neuropeptide content of individual DCVs likely originates prior to axonal transport. VMAT is also processed in the TGN to be sorted into DCVs and small synaptic vesicles, rather than proceeding through the constitutive secretory pathway. A recent study found that knockdown of the TGN protein HID-1 reduces DCV luminal cargo and VMAT in DCVs by affecting sorting and DCV production. The coordinated effects on neuropeptides and VMAT are reminiscent of the results presented in this study, but the Ptp4E effect on packaging was not associated with a change in DCV number or transport. Therefore, the uncoupling of DCV number from packaging is indicative of a novel cell biological mechanism. With this in mind, a possible explanation for the effect of inhibiting Ptp4E is that the tyrosine phosphorylation stimulates TGN sorting of luminal and vesicle membrane content without changing DCV number or size. By this mechanism, Rptp regulation of vesicular packaging in the soma could scale co-release at the distal synaptic ending (Tao, 2019).

These results pose the question of the site of Ptp4E function. Rptps often mediate signaling triggered by cell-cell contacts. For example, the presynaptic Rptp Lar is activated by muscle Syndecan during development of the NMJ. By analogy, it is possible that presynaptic Ptp4E governs retrograde signaling (e.g., by interrupting tyrosine kinase-dependent mechanisms). In favor of this hypothesis, the closely related Rptp Ptp10D is found on axons in the embryo, where it is positioned to regulate axonal guidance during development. However, the potential involvement of vesicle biogenesis and the unknown localization of Ptp4E raise the possibility that somatic Ptp4E is responsible for synaptic effects. Novel tools to differentially control of Rptp activity by compartment (i.e. soma versus terminal) will be needed to distinguish between these possibilities. Regardless of the cellular location of Ptp4E signaling, the mechanism discovered here (i.e. coincident control of packaging of neuropeptides and small-molecule transmitters) represents a previously unknown cell biological strategy for regulating synaptic co-transmission (Tao, 2019).

Previous experiments have shown that increased VMAT leads to enlargement of vesicles and greater vesicular monoamine storage, but this effect was not seen in this study. Notably, the mechanistic basis of the VMAT expression effect on vesicle size is not understood because thermodynamics with a simple system suggests that maximal vesicular monoamine concentration should be reached even with one VMAT per vesicle. Therefore, to explain the previously observed effect on vesicle size, some other factor, such as monoamine leakage or membrane flexibility, must come into play. It is suggested that these parameters might differ in the synaptic terminals examined in this study. Alternatively, in contrast to the spherical vesicles studied previously, the DCVs in octopamine neurons are ovoid. Therefore, the current analysis of largest dimension cannot exclude that the narrow axis of these DCVs increased. According to the latter scenario, increases in vesicular volume and membrane surface area could have been undetected with the methodology used in the current study (Tao, 2019).

What would the expected consequences be of upregulating VMAT and neuropeptides in vesicles? Upregulating VMAT will increase the speed of vesicle loading when there is exocytosis-endocytosis cycling or kiss-and-run release. Hence, increased VMAT will affect release more when vesicle emptying by release is most marked. In Drosophila, the effect of increased VMAT on behavior has not been examined. However, the dopamine precursor L-DOPA increases vesicular monoamine content and ameliorates Parkinsonian symptoms in humans. Thus, by analogy, it is suggested that octopaminergic signaling would be boosted by increased vesicular octopamine packaging induced by synaptic VMAT upregulation. Of course, increased co-transmission by neuropeptides could further alter octopamine action. Therefore, it would be interesting to explore how Rptps in the brain affect octopamine-dependent fly behaviors such as feeding and egg laying (Tao, 2019).

Enteric neurons increase maternal food intake during reproduction

Reproduction induces increased food intake across females of many animal species, providing a physiologically relevant paradigm for the exploration of appetite regulation. By examining the diversity of enteric neurons in Drosophila melanogaster, this study identified a key role for gut-innervating neurons with sex and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. These findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain (Hadjieconomou, 2020).

Internal state has profound effects on brain function. Despite increasingly recognized roles for the gut-brain axis in maintaining energy balance, links between internal state and gastrointestinal innervation remain poorly characterized. Progress has been hindered by neuroanatomical complexity, which is only beginning to be parsed in mammals. The simpler-yet physiologically complex-intestine of Drosophila provides an alternative entry point into the study of gastrointestinal innervation (Hadjieconomou, 2020).

Innervation of the main digestive portion of the adult fly intestine, which encompasses the anterior midgut and the crop and central neurons of the pars intercerebralis (PI) in the brain. PI neurons directly innervate the anterior midgut and the crop, and include insulin-producing neurons and other peptidergic subtypes. The crop is further populated by processes that emanate from cells of the corpora cardiaca, which produce the glucagon-like adipokinetic hormone and are adjacent to the hypocerebral ganglion (HCG). Also adjacent to both the HCG and the corpora cardiaca are the corpus allatum cells, which produce juvenile hormone and extend short local projections. The thoracico-abdominal ganglion of the central nervous system might not innervate these gut regions (Hadjieconomou, 2020).

The crop-an expandable structure found in the intestines of insects-might be disregarded as a passive food store, but several observations suggest active regulation of its physiology. Refeeding flies after starvation results in enlarged, food-filled crops, pointing to modulation of food ingression into and out of the crop. Live imaging or temporal dissections of flies revealed that food always enters the crop before proceeding to the midgut. Additionally, food transit through the crop is dependent on both its palatability and its nutritional value. Therefore, in adult flies, all food transits through the crop, which is nutrient-sensitive and shows chemically and anatomically diverse innervation (Hadjieconomou, 2020).

The crop and anterior midgut are innervated by myosuppressin (Ms)-positive neurons located in the PI and the HCG. PI Ms neurons are distinct from known neuronal subsets, with the exception of eight Ms neurons that co-express the Taotie-GAL4 marker. Two PI Ms neuron populations can be distinguished by cell size: one comprises 18 large cells and another comprises 12 smaller cells. Single-cell clones of large Ms neurons reveal a single process that bifurcates into a longer, probably axonal projection to the gut-which arborizes in the HCG and extends further to innervate the crop-and a shorter, probably dendritic process that reaches the suboesophageal zone, where the axons of peripheral gustatory sensory neurons terminate. A subset of HCG Ms-expressing neurons also innervates the crop, whereas another subset projects locally. This study confirmed the expression of Ms using an endogenously tagged Ms reporter (Ms-GFP) and in situ hybridization Ms innervation was also observed of the hindgut, the rectal ampulla and the heart, and a subset of peripheral Ms-positive neurons innervating the female reproductive tract (Hadjieconomou, 2020).

This study selectively activated or silenced Ms neurons in adult flies. Activation resulted in greatly enlarged crops in flies that were fed ad libitum, consistent with the relaxant properties of Ms on insect muscles ex vivo. By contrast, silencing of Ms neurons prevented crop enlargement in a starved-refed condition in which the crop normally expands. Genetic downregulation or mutation of Ms (using a new mutant) prevented crop enlargement, albeit to a lesser extent than Ms neuron silencing. This could be due to another Ms-neuron-derived neurotransmitter or neuropeptide contributing to crop enlargement, or to loss of the Ms peptide during development in these experiments, resulting in adaptations that render the crop more active than it would be in response to acute loss of the Ms peptide. A Gal4 insertion into the Ms locus was generated that disrupts Ms production (MsTGEM). In contrast to the crop enlargement resulting from TrpA1-mediated activation from Ms-Gal4, TrpA1 expression from this (Ms mutant) MsTGEM-Gal4 driver failed to induce crop enlargement, further confirming a requirement for Ms. Ms neuron subtype-specific downregulations and activations enabled establishing that the PI Ms neurons (in particular, the Taotie-Gal4-positive subset of large PI Ms neurons) induce, and are indispensable for, crop enlargement through their production of Ms neuropeptide (Hadjieconomou, 2020).

The contributions of myosuppressin receptors 1 and 2 (MsR1 and MsR2) were explored. MsR1 expression was observed in crop muscles, in subsets of neurons including the PI and HCG Ms-positive neurons and neurons innervating the ovary and heart; no MsR1 expression was detected in ovarian or heart muscles. Expression of MsR2 was also detected in crop muscles. To investigate the function of the Ms receptor, MsR1 was downregulated specifically in adult crop muscles using two independent driver lines (vm-Gal4 and MsR1crop-Gal4). Both genetic manipulations led to reduced crop enlargement in a starvation-refeeding assay, comparable to that observed for Ms neuron silencing or Ms mutation. Downregulation of MsR2 did not affect crop enlargement. A role for MsR1 in mediating crop enlargement was confirmed using a MsR1TGEM mutant. MsR1 is therefore identified as the crop muscle receptor through which Ms signals to modulate crop enlargement (Hadjieconomou, 2020).

The physiological regulation of crop enlargement was explored and found that it is dependent on sex and on reproductive status: the crops of mated females fed ad libitum (which were used for all the experiments described above) were consistently more expanded than those of virgin female or mated male flies fed ad libitum. Because post-mating changes were not seen in Ms neuron projections, it was asked whether post-mating crop enlargement might result from the release of Ms preferentially in mated females. Ms peptide levels were lower in the PI neuron cell bodies of females only after mating. In the absence of Ms transcriptional changes this observation is consistent with a post-mating increase in the secretion of Ms peptide in females. This effect of mating on Ms levels was specific to mating: nutrient availability did not affect intracellular Ms levels. It was also observed that the Ms neurons of mated females had higher cumulative calcium levels and a reduced amplitude of calcium oscillations compared to virgin females, as detected both by in vivo GCaMP6 calcium imaging and by the calcium-sensitive reporter CaLexA, in which GFP expression is proportional to cumulative neuronal activity. Physiologically, and in contrast to observations in mated females, a reduction of Ms signalling in males or in virgin female flies failed to impair crop enlargement. Consequently, when Ms signalling to crop muscles was prevented, the size of the crop of mated females no longer differed from that of virgin females. Collectively, these findings support the idea that, in female flies, the activity of PI Ms neurons changes after mating to promote Ms release (Hadjieconomou, 2020).

Levels of the steroid hormone ecdysone, which promotes egg production and intestinal stem-cell proliferation, increase after mating. The ecdysone receptor (EcR) is expressed by all PI Ms neurons, which suggests that they might be sensitive to circulating ecdysone. Expression of a dominant-negative EcR-which targets all EcR isoforms-confined to the Ms neurons of adult flies was found to increase intracellular Ms levels in the Ms PI neuron cell bodies of mated females to the levels observed in virgin females, whereas it had no effect on virgin females. Downregulation of EcR (using RNA interference lines that target all isoforms or the B1 isoform specifically) produced comparable results. In both experiments, the amplitude of in vivo calcium oscillations in Ms neurons was increased to levels seen in virgin females. Compromising EcR signalling in adult Ms neurons significantly reduced crop enlargement preferentially in mated females; this phenotype was also apparent when the PI Ms neurons were targeted using Taotie-Gal4. Ecdysone therefore communicates mating status to Ms neurons through its B1 receptor (Hadjieconomou, 2020).

Previous work showed that the adult intestine is resized and metabolically remodelled after mating (Reiff, 2016), but did not investigate possible effects on its hormone-producing enteroendocrine cells. This study now observe a post-mating increase in the number of enteroendocrine cells, including a subset that expresses the hormone bursicon α (Burs), which is known to signal to adipose tissue through an unidentified neuronal relay. An endogenous protein reporter for the Burs receptor Rickets (Rk, also known as Lgr2) revealed its expression in subsets of neurons including all PI Ms neurons (including the Taotie-Gal4-positive subset) and in projections terminating in the HCG. Expression in a subset of the HCG Ms neurons was observed only sporadically (Hadjieconomou, 2020).

Consistent with the regulation of Ms neurons by the increase in Burs derived from enteroendocrine cells after mating, adult-specific downregulation of the Burs receptor gene rk in Ms neurons reverted intracellular Ms levels in the PI Ms neurons of mated females to levels observed in virgin females; there was no effect in virgin females. Like EcR downregulation, rk downregulation in Ms neurons also increased the amplitude of in vivo calcium oscillations in the Ms neuron cell bodies of mated females to values similar to those observed in virgin females. Functionally, both the downregulation of Burs in intestinal enteroendocrine cells and the adult-specific rk downregulation in Ms neurons-either in all neurons or in the Taotie-Gal4-positive subset in the PI-preferentially reduced crop enlargement in mated females. Conversely, stimulating the intestinal release of enteroendocrine hormones-including Burs-from enteroendocrine cells resulted in reduced Ms levels in the Ms neuron cell bodies of virgin females, similar to those observed in mated females, and greatly enlarged crops (Hadjieconomou, 2020).

Thus, steroid and enteroendocrine hormones communicate mating status to the brain. Acting through their receptors in the PI Ms neurons, these hormones change Ms neuronal activity, promoting the release of Ms after mating (Hadjieconomou, 2020).

To investigate the importance of Ms neuron modulation after mating, post-mating crop enlargement was selectively prevented by downregulating MsR1 in adult crop muscles using two independent strategies. This had no discernible effects in males or virgin females, but specifically prevented the increase in food intake that is normally observed in female flies after mating. Comparable results were obtained by blocking the post-mating ecdysone and Burs inputs into the Ms neurons. Downregulation of MsR2 had no such effect. The post-mating change in crop expandability, mediated by Ms and MsR1 signalling, thus causes the increased food intake observed in females after mating (Hadjieconomou, 2020).

The negative pressures that have been reported in the crops of larger insects suggest that the crop may draw food in by generating suction. The increased crop expandability enabled by Ms release after mating could therefore increase food intake through changes in suction. It was observed that mated females ingest more food per sip than virgin females, which is consistent with mated females needing to generate a higher suction pressure to facilitate bigger sips. Crop enlargement was therefore modeled using the Poiseuille equation for incompressible fluid flow in a pipe and found that the crop would need a suction pressure of the order of -1 kPa to achieve the previously reported intake volume per sip. This is in reasonable agreement with previously reported values measured in cockroach crops of between -0.5 and -1 kPa. The model predicts that mated flies would require a modest increase in suction pressure to -1.3kPa in order to facilitate the increased sip size (Hadjieconomou, 2020).

In the model, the change in crop volume drives food intake through increased suction. A crop that cannot enlarge, or a persistently enlarged crop, should therefore result in a comparable reduction in food intake by preventing the generation of suction. This was tested by persistently preventing crop enlargement (using crop-muscle-specific MsR1 knockdown) or by persistently inducing it (using TrpA1-mediated Ms neuron activation from Ms-Gal4 or Taotie-Gal4), after which the diet of these flies was switched from an undyed to a dye-laced food source to assess food intake. As predicted, both genetic manipulations reduced food intake. Conversely, increasing the rate at which the crop expands should increase food intake. This was tested by activating the Ms neurons as in the previous experiment, but this time the dye-laced food source was provided, and its intake was monitored at the same time as the neurons were activated (that is, as inducing greater crop expansion was being induced) rather than after a persistent activation (when the crop is already maximally expanded). Increased food intake was observed under these conditions in the absence of changes in the number of meals. Although further work will be required to elucidate the full dynamics of crop enlargement, filling and emptying, these experiments support the idea that the Ms-induced enlargement of the crop after mating increases food intake at least partly by increasing the suction power of the crop (Hadjieconomou, 2020).

Finally, given the links between nutrient intake and fecundity, it is proposed that the Ms-driven crop enlargement after mating might be adaptive and support reproduction. Crop enlargement was prevented selectively after mating by downregulating MsR1 from crop muscles, as in previous experiments. This resulted in reduced egg production, and the eggs that were produced had reduced viability. It is therefore conclude that the crop and its Ms innervation sustain the increase in food intake after mating, maximising female fecundity (Hadjieconomou, 2020).

These findings lead to a proposal that the maternal increase in food intake during reproduction is adaptive, that the crop is a key reproductive organ, and that Ms is a major effector of post-mating responses. In support of these ideas, the crop is absent in larvae-the juvenile stage of insects-and other Diptera have co-opted it for reproductive behaviours such as the regurgitation of nuptial gifts or the secretion of male pheromones. Ms receptors are also closely related to the Sex peptide receptor (the 'mating sensor' of female flies), and both diverged after duplication of an ancestral receptor that might have responded to the Myoinhibitory peptide (Mip) in the last common ancestor of protostomes. It will be interesting to explore possible links between Ms and Sex peptide signalling, and whether and how these mating signals affect recently described crop mechanosensing mechanisms that restrain ingestion as the crop expands in order to terminate large meals (Hadjieconomou, 2020).

This study has provided evidence for a gut-to-brain axis in Drosophila by identifying central Ms neurons as targets of the gut-derived hormone Burs. These central neurons innervate the gut, 'closing' a gut-brain-gut loop that connects midgut enteroendocrine signals to the crop, a more anterior gut region. This might allow for the functional coordination of different gut portions, while enabling central modulation by sensory cues (for example, gustatory). This study also identified the Ms neurons as the neural targets of ecdysone, which has been shown to promote food intake. Reproduction has pronounced, and in some cases lasting, effects on the human female brain; Ms neurons provide a tractable and physiologically relevant neural substrate for the investigation of the mechanisms involved (Hadjieconomou, 2020).

The human digestive system might be similarly modulated by reproductive cues to affect food intake. In mammals, enteric neurons express sex and reproductive-hormone receptors, and enteroendocrine hormone levels change during reproduction. It is suggested that pregnancy and lactation represent an attractive and relatively unexplored physiological adaptation for the investigation of nutrient intake regulation, organ remodelling and metabolic plasticity-mechanisms that might eventually be leveraged to curb appetite and/or weight gain (Hadjieconomou, 2020).

Cholecystokinin-like peptide mediates satiety by inhibiting sugar attraction

Feeding is essential for animal survival and reproduction and is regulated by both internal states and external stimuli. However, little is known about how internal states influence the perception of external sensory cues that regulate feeding behavior. This study investigated the neuronal and molecular mechanisms behind nutritional state-mediated regulation of gustatory perception in control of feeding behavior in the brown planthopper and Drosophila. Feeding was found to increase the expression of the cholecystokinin-like peptide, sulfakinin (SK), and the activity of a set of SK-expressing neurons. Starvation elevates the transcription of the sugar receptor Gr64f and SK negatively regulates the expression of Gr64f in both insects. Interestingly, it was found that one of the two known SK receptors, CCKLR-17D3, is expressed by some of Gr64f-expressing neurons in the proboscis and proleg tarsi. Thus, this study has identified SK as a neuropeptide signal in a neuronal circuitry that responds to food intake, and regulates feeding behavior by diminishing gustatory receptor gene expression and activity of sweet sensing GRNs. The findings demonstrate one nutritional state-dependent pathway that modulates sweet perception and thereby feeding behavior, but the experiments cannot exclude further parallel pathways. Importantly, it was shown that the underlying mechanisms are conserved in the two distantly related insect species (Guo, 2021).

Ion transport peptide regulates energy intake, expenditure, and metabolic homeostasis in Drosophila

In mammals, energy homeostasis is regulated by the antagonistic action of hormones insulin and glucagon. However, in contrast to the highly conserved insulin, glucagon is absent in most invertebrates. Although there are several endocrine regulators of energy expenditure and catabolism (such as the Adipokinetic hormone), no single invertebrate hormone with all of the functions of glucagon has been described so far. This study used genetic gain- and loss-of-function experiments to show that the Drosophila gene Ion transport peptide (ITP) codes for a novel catabolic regulator that increases energy expenditure, lowers fat and glycogen reserves, and increases glucose and trehalose. Intriguingly, ITP has additional functions reminiscent of glucagon, such as inhibition of feeding and transit of the meal throughout the digestive tract. Furthermore, ITP interacts with the well-known signaling via the Adipokinetic hormone (AKH); ITP promotes the pathway by stimulating AKH secretion and transcription of the receptor AkhR. The genetic manipulations of ITP on standard and AKH deficient backgrounds showed that the AKH peptide mediates the hyperglycemic and hypertrehalosemic effects of ITP, while the other metabolic functions of ITP seem to be AKH-independent. In addition, ITP is necessary for critical processes such as development, starvation-induced foraging, reproduction, and average lifespan. Altogether, this work describes a novel master regulator of fly physiology with functions closely resembling mammalian glucagon (Galikova, 2022).

A hierarchical transcription factor cascade regulates enteroendocrine cell diversity and plasticity in Drosophila

Enteroendocrine cells (EEs) represent a heterogeneous cell population in intestine and exert endocrine functions by secreting a diverse array of neuropeptides. Although many transcription factors (TFs) required for specification of EEs have been identified in both mammals and Drosophila, it is not understood how these TFs work together to generate this considerable subtype diversity. This study shows that EE diversity in adult Drosophila is generated via an 'additive hierarchical TF cascade'. Specifically, a combination of a master TF, a secondary-level TF and a tertiary-level TF constitute a "TF code" for generating EE diversity. A high degree of post-specification plasticity of EEs was found, as changes in the code-including as few as one distinct TF-allow efficient switching of subtype identities. This study thus reveals a hierarchically-organized TF code that underlies EE diversity and plasticity in Drosophila, which can guide investigations of EEs in mammals and inform their application in medicine (Guo, 2022).

The neuropeptide allatostatin C from clock-associated DN1p neurons generates the circadian rhythm for oogenesis

The link between the biological clock and reproduction is evident in most metazoans. The fruit fly Drosophila melanogaster, a key model organism in the field of chronobiology because of its well-defined networks of molecular clock genes and pacemaker neurons in the brain, shows a pronounced diurnal rhythmicity in oogenesis. Still, it is unclear how the circadian clock generates this reproductive rhythm. A subset of the group of neurons designated "posterior dorsal neuron 1" (DN1p), which are among the ~150 pacemaker neurons in the fly brain, produces the neuropeptide allatostatin C (AstC-DN1p). This study reports that six pairs of AstC-DN1p neurons send inhibitory inputs to the brain insulin-producing cells, which express two AstC receptors, star1 and AICR2. Consistent with the roles of insulin/insulin-like signaling in oogenesis, activation of AstC-DN1p suppresses oogenesis through the insulin-producing cells. This study shows evidence that AstC-DN1p activity plays a role in generating an oogenesis rhythm by regulating juvenile hormone and vitellogenesis indirectly via insulin/insulin-like signaling. AstC is orthologous to the vertebrate neuropeptide somatostatin (SST). Like AstC, SST inhibits gonadotrophin secretion indirectly through gonadotropin-releasing hormone neurons in the hypothalamus. The functional and structural conservation linking the AstC and SST systems suggest an ancient origin for the neural substrates that generate reproductive rhythms (Zhang, 2021).

Six pairs of DN1p neurons were discovered that are part of the circadian pacemaker neuron network in the brain and make functional inhibitory connections to the brain IPCs. The IPCs are endocrine sensors that link the organism's nutritional status with anabolic processes, such as those associated with growth in developmental stages and with reproduction in adults. In juvenile stages, activation of insulin and insulin-like growth factor (IGF) signaling (IIS) through the InR results in larger flies, whereas inhibition of this pathway produces smaller flies. Consistent with this, it was also found that forced activation of the AstC-DN1p (i.e., CNMa-Gal4/UAS-NaChBac) during development resulted in 12% smaller adults, confirming their role as a negative regulator of the IPCs. In adults, the IPCs are associated with many physiological and behavioral processes, such as feeding, glycaemic homeostasis, sleep, lifespan, and stress resistance. As such, the IPCs receive a variety of modulatory inputs from both central and peripheral sources, such as sNPF, corazonin, tachykinins, limostatin, allatostatin A, adipokinetic hormone, GABA, serotonin, and octopamine. Regarding reproduction, IIS directed by the IPCs stimulates GSC proliferation and vitellogenesis. The results also indicate that AstC from AstC-DN1p suppresses the secretory activity of the IPCs and juvenile hormone (JH)-dependent oocyte development (i.e., vitellogenesis). Indeed, it was found that the JH mimic methoprene can rescue the suppression of oogenesis induced by AstC-DN1p activation. From these results it is concluded that IPCs are inhibited by AstC released by AstC-DN1p. A similar link between IIS and the circadian clock has also been reported in mammals, but the mechanism remains unclear (Zhang, 2021).

Although the genetic evidence supporting the inhibitory action of AstC-DN1p on IPCs is compelling, it is also puzzling because a previous study found forced activation of 8 to 10 pairs of DN1p neurons (i.e., Clk4.1-LexA+ neurons) induced Ca2+ transients in IPCs. This study also found that, under LD 12:12 conditions, the IPCs showed electrical activity early in the morning when DN1p neurons are also active. The same study, however, reported that, under DD conditions, the IPCs showed no bursting activity in the morning (i.e., CT0 to -4). Instead, they showed bursting activity in the late afternoon (i.e., CT8 to -12) when DN1 activity falls. Furthermore, DN1p activation evokes varying levels of Ca2+ transients from individual IPCs, some of which produce no detectable Ca2+ transient. Thus, like mammalian pancreatic β-cells, the IPCs in Drosophila seem to comprise a heterogeneous cell population. It is noted that individual IPCs show highly variable AstC-R1 expression, which would also lead to individual IPCs showing variable responses to AstC (Zhang, 2021).

In D. melanogaster, the LD cycle generates an egg-laying rhythm by influencing oogenesis and oviposition. Oviposition depends on light cues, whereas oogenesis cycles with the circadian rhythm that itself continues to run in DD conditions. In live-brain Ca2+ imaging experiments, DN1 neurons show a circadian Ca2+ activity rhythm that peaks around CT19 and reaches its lowest point between CT6 and CT8. This DN1 activity rhythm correlates well with the rhythm of vitellogenesis initiation observed in this study. In this model, the lowest point in DN1 Ca2+ activity between CT6 and CT8 leads to a significant attenuation of AstC secretion. This leads to a derepression of IPC activity, which eventually induces JH biosynthesis and vitellogenesis initiation. The 6-h delay required for previtellogenic stage 7 follicles to develop into vitellogenic stage 8 follicles would result in a peak in the number of stage 8 follicles between CT12 and CT14. Notably, the ovaries of the AstC-deficient mutant showed similar numbers of stage 8 oocytes at all examined circadian time points, indicating that any other JH- or vitellogenesis-regulating factors play only minor roles in producing the circadian vitellogenesis rhythm (Zhang, 2021).

Like the IPCs, the DN1p cluster is also heterogeneous. A subset of the DN1p neurons is most active at dawn and promotes wakefulness. Another subset of the DN1p cluster (also known as, spl-gDN1) promotes sleep. The DN1p cluster comprises two morphologically distinct subpopulations, a-DN1p and vc-DN1p. The a-DN1p subcluster promotes wakefulness by inhibiting sleep promoting neurons, whereas the vc-DN1p subcluster resembles the sleep-promoting spl-gDN1. These results indicate AstC-DN1p are a-DN1p neurons that project to the anterior optic tubercle (AOTU. Although the possibility cannot be ruled out that AstC-DN1p is also heterogeneous and includes some vc-DN1p neurons, the wake-promoting role of a-DN1p aligns well with the circadian vitellogenesis rhythm that requires the secretory activity of AstC-DN1p to be lowest in the afternoon and highest at dawn. Furthermore, AstC-DN1p neurons express Dh31. Dh31-expressing DN1 clock neurons are intrinsically wake-promoting and Dh31-DN1p activity in the late night or early morning suppresses sleep. Again, this is consistent with the observation that AstC-DN1p are also wake-promoting a-DN1p. It is speculated Dh31 plays a limited role in oogenesis regulation, because unlike AstC, RNAi-mediated knockdown of Dh31 had a negligible impact on female fecundity (Zhang, 2021).

Besides AstC-DN1p, the female brain has many additional AstC neurons. However, it seems unlikely that other AstC neurons contribute to the circadian vitellogenesis rhythm. This is because restoring AstC expression specifically in AstC-DN1p almost completely restored the vitellogenesis rhythm in AstC-deficient mutants. It is feasible, however, that other AstC neurons contribute to different aspects of female reproduction. Indeed, a sizable difference was noted in the final oogenesis outcome between AstC-Gal4 neuron activation and brain-specific AstC-Gal4 neuron activation. This suggests AstC cells outside of the brain also regulate oogenesis probably in other physiological contexts, such as the postmating responses (Zhang, 2021).

AstC receptors are orthologous to mammalian SST receptors (sstr1-5). SST is a brain neuropeptide that was originally identified as an inhibitor of growth hormone (GH) secretion in the anterior pituitary. Thus, the observation that AstC inhibits IIS from IPCs, a major endocrine signal that promotes growth in Drosophila, suggests remarkable structural and functional conservation between the invertebrate AstC and vertebrate SST systems. In addition, SST inhibits the hypothalamic neuropeptide GnRH, which stimulates the anterior pituitary's production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH stimulates clutches of immature follicles to initiate follicular development, while LH stimulates ovulation. Thus, both AstC and SST regulate the secretion of gonadotropins (JH in insects, FSH and LH in mammals) indirectly through the IPCs in insects and through the hypothalamic GnRH neurons in mammals. This functional conservation between AstC and SST is also evident in the immune system. AstC inhibits the innate immune system in insects, while SST inhibits inflammation in mammals (Zhang, 2021).

In many seasonal breeders, the changing photoperiod as the seasons progress acts as an environmental cue for the biological clock system, which would then direct any necessary physiological changes. During the winter, Drosophila females enter a form of reproductive dormancy characterized by a pronounced suppression of vitellogenesis. A winter-like condition (i.e., short-day length, low temperature, and food shortage) down-regulates neural activity in the IPCs. But the IPCs are not equipped with a cell-autonomous clock, so they must receive seasonal information from the brain clock neuron network. Indeed, two clock related neuropeptides~pigment dispersing factor and short neuropeptide F~from circadian morning pacemaker or M-cells have been implicated in regulating reproductive dormancy. Intriguingly, AstC-DN1p neurons are the DN1p subset that receives pigment dispersing factor signals from these M-cells. Furthermore, DN1p can process light and temperature information for the circadian regulation of behavior. Finally, the finding that AstC-DN1p generates the circadian vitellogenesis rhythm via the IPCs makes AstC-DN1p neurons the prime candidates for integrating the seasonal cues that control the entrance, maintenance, or exit from reproductive dormancy. Considering the functional and structural conservation between the AstC and SST systems, the SST system may also link the brain clock, GnRH, and/or its downstream reproductive pathways in controlling seasonal reproductive patterns in vertebrates (Zhang, 2021).

Juvenile hormone drives the maturation of spontaneous mushroom body neural activity and learned behavior

Mature behaviors emerge from neural circuits sculpted by genetic programs and spontaneous and evoked neural activity. However, how neural activity is refined to drive maturation of learned behavior remains poorly understood. This study explored how transient hormonal signaling coordinates a neural activity state transition and maturation of associative learning. Spontaneous, asynchronous activity was identified in a Drosophila learning and memory brain region, the mushroom body. This activity declines significantly over the first week of adulthood. Moreover, this activity is generated cell-autonomously via Cacophony voltage-gated calcium channels in a single cell type, α'/β' Kenyon cells. Juvenile hormone, a crucial developmental regulator, acts transiently in α'/β' Kenyon cells during a young adult sensitive period to downregulate spontaneous activity and enable subsequent enhanced learning. Hormone signaling in young animals therefore controls a neural activity state transition and is required for improved associative learning, providing insight into the maturation of circuits and behavior (Leinwand, 2021).

Genetic programs and experience in the form of neural activity refine neural circuits, sculpting cognitive function over time. Activity state transitions in neural circuits are widespread during normal development. Achieving the mature activity state is correlated with the emergence of adult behavioral outputs. For example, periodic waves of spontaneous neural activity occur throughout immature visual, somatosensory, and motor brain regions in perinatal critical periods, before distinct, less correlated sensory-evoked or locomotion-related activity patterns emerge in older animals. Periodic bursts of spontaneous activity also occur in the hippocampus, specifically in early mammalian post-natal development, in a brief period prior to development of robust long-term potentiation. Although temporal evolution of spontaneous neural activity patterns is prevalent in developing circuits, the molecular mechanisms that control the timing of neural activity state transitions and coordinate maturation of behavioral outputs in young animals are largely unknown (Leinwand, 2021).

Hormones regulate multiple aspects of the maturation of the nervous system. Systemic hormonal signaling controls neural differentiation, remodeling, physiology, and other key events for the refinement of neural circuits. For example, sex steroid hormones act transient in a critical prenatal window to regulate the development of neural circuits for sexually dimorphic behaviors, producing enduring changes in the brain. Furthermore, many hormone receptors directly alter transcription and consequently have direct or indirect effects on ion channels, synapses, and neurotransmission. Gonadal hormone signaling accelerates the maturation of inhibitory neurotransmission in cortical circuits, with correlated effects on behavior. Moreover, thyroid hormones regulate synaptic transmission in the hippocampus in young animals, with clear implications for memory. Juvenile hormone (JH) is an insect hormone with functional similarities to mammalian thyroid hormones. JH circulates widely and acts on diverse neural circuits in young animals to regulate metamorphosis, reproduction, and courtship. Across species, hormonal signaling is therefore well poised to coordinate key transitions in the maturation of the nervous system and behavior at particular stages of animal development (Leinwand, 2021).

A mechanistic understanding of how the nervous system achieves activity state transitions will provide insight into the origins of mature behaviors. Critically, evaluating the role of hormones in neural activity and behavior maturation requires isolating their effects on specific cells in known circuits and at particular developmental times. The fruit fly Drosophila melanogaster system offers powerful genetic tools to causally link in vivo neural activity with behavior and to manipulate gene expression, with single-cell resolution. Recently, high levels of spontaneous neural activity were observed in the developing Drosophila visual system, illustrating that activity maturation occurs in invertebrate systems, as well as vertebrates. In examining activity in the adult fly brain, this study observed high levels of activity in the Drosophila mushroom body (MB) brain region that declined rapidly with age. The MB is critical for learned behavior. Physiological, molecular, behavioral, and anatomical studies, including a complete connectome, have provided a uniquely rich understanding of the neuronal architecture and function of the MB. Discovery of an immature to mature activity state transition in this well-described system offers an entry point to rigorously examine how neural activity in young animals drives refinement and maturation of behavior (Leinwand, 2021).

This study employed in vivo functional imaging and powerful genetic tools to describe a high spontaneous activity state in the Drosophila MB learning and memory brain center of young animals and its crucial role in the maturation of learned behavior. Spontaneous, asynchronous activity was identified specifically in one MB cell type, the α'/β' Kenyon cells (KCs), in young animals, that unexpectedly declines over the first week of adulthood. Cacophony (Cac) voltage-gated calcium channels mediate this young animal spontaneous activity. JH, a crucial regulator of insect development similar to vertebrate thyroid hormones, signaling specifically in α'/β' KCs during a sensitive period in early adulthood coordinates the maturation of neural activity states and is required for mature associative learning (Leinwand, 2021).

This study shows that JH acts on the α'/β' KCs of young adults to downregulate spontaneous activity and enhance associative learning in older animals. Specifically, it was found that α'/β' KCs exhibit sensorimotor-independent, TTX-insensitive asynchronous activity in young animals that is mediated by Cac voltage-gated calcium channels. JH signaling in a young animal sensitive period, when the titer of JH circulating is high, is required to achieve the mature, low KC activity state and enhance learned behavior. The discovery that a hormone triggers a neural activity state transition essential for robust learning provides a model for mechanistically probing the maturation of learning circuits and behavior (Leinwand, 2021).

Many animals are born with immature learning capabilities. In mammals, periodic giant depolarizing potentials occur in the immature hippocampus. Because multiple hippocampus-dependent learned behaviors are poor at the time of the giant depolarizing potentials and mature slowly over the first post-natal month, a correlation between this pattern of spontaneous activity and learning is apparent. However, causal links between hippocampal activity patterns and maturation of learned behavioral outputs are lacking, despite their profound implications for the plasticity to form new associations throughout adulthood. These studies demonstrate that Drosophila associative learning improves over the first week of adulthood and that appropriately regulated activity state transitions in higher-order brain regions are necessary for this learning maturation (Leinwand, 2021).

These studies reveal that the spontaneous activity generated in young α'/β' KCs is critical for honing the neural circuits that subsequently produce mature learning. Notably, the activity in young KCs is asynchronous and unpatterned, unlike the propagating waves of activity in immature visual and somatosensory areas or the rhythmic alternations in motor regions. In contrast with these sensorimotor systems, the MB and many higher-order brain regions are not topographically organized, and neighboring neurons do not respond to similar stimulus features. Instead, connectivity between KCs and their presynaptic partners is stochastic, and sensory-evoked responses are sparse and unpatterned. It is proposed that transient unpatterned activity in young animals is a necessary precursor to the unordered and spatially distributed sensory-evoked responses seen in adults, providing a substrate for subsequent adult experiences (Leinwand, 2021).

This study describes age-dependent spontaneous activity that is restricted to a single cell type within the learning circuit, the α'/β' KCs. Although multi-parallel and distributed processing in MB circuit modules gives rise to associative learning, the precise role of α'/β' KCs in learned behavior remains less well understood than other MB cell types. Sparse activity in α'/β' KCs may encode sensory information and information about reward or punishment. Behaviorally, α'/β' KCs are required for the acquisition and consolidation of appetitive and aversive olfactory and gustatory associative memories. Because KC activity coincident with salient stimuli are key elements to form associative memories, the poor learning performance of young animals and of older animals manipulated to aberrantly retain high levels of α'/β' KC activity is unexpected. The results suggest that high α'/β' KC activity is a necessary feature of immature circuits but may acutely interfere with robust learning. Activity state transitions in young animals may refine responses to conditioned stimuli in mature animals. Whether high α'/β' KC activity in young animals organizes or is permissive for the subsequent role of α'/β' KCs in memory acquisition and consolidation remains to be investigated (Leinwand, 2021).

Among the MB cell types, only α'/β' KCs undergo a high- to low-activity state transition. α'/β' KCs are not uniquely able to directly transduce JH, because the JH receptors Met and Gce are highly expressed throughout the MB. Cac voltage-gated calcium channels are also highly expressed in the entire MB. Nevertheless, α'/β' KCs were found to have the lowest firing threshold and, correspondingly, the highest rate of baseline and odor sensory-evoked spiking of the three KC classes. Although these physiological properties were not studied in the context of age, the current studies reveal a change in baseline activity states in the first week of adulthood. It is therefore speculated that α'/β' KCs are intrinsically more excitable due to a unique gene expression profile. Specific ligand- or voltage-gated ion channels or ion pumps may display α'/β' KC-biased expression and may undergo changes in expression in early adulthood that directly contribute to cellular excitability. α'/β' KCs may have distinct plasticity rules that derive from these age-dependent gene expression and excitability changes (Leinwand, 2021).

Hormone signaling regulates α'/β' KC physiology with age. Although Cac channels mediate young α'/β' KC spontaneous activity and JH signaling controls the neural activity state transition, a direct JH-to-Cac channel connection is unlikely. Cac mRNA expression in α'/β' KCs does not change over the first week of adulthood, consistent with the absence of evidence that Met and Gce hormone receptors directly target Cac channels. It is therefore hypothesized that JH may indirectly influence Cac channel function. The finding that the high activity retained in α'/β' KCs in mature flies with Met and Gce receptors knocked down was sensitive to the voltage-gated calcium channel antagonist PLTX supports an indirect link between JH and these channels. Thus, it is speculated that JH signaling normally produces transcriptional changes in young animals that influence the overall physiology and resting membrane potential of α'/β' KCs. These changes in α'/β' KC membrane potential may then reduce Cac channel opening and calcium flux over the first week of adulthood. Future investigation of how the direct targets of JH signaling ultimately influence the membrane potential and Cac function will provide new insights into the underlying circuit maturation mechanisms (Leinwand, 2021).

This study found that transient hormonal signaling is critically necessary to impart stable changes in neural activity and learned behavior. JH, acting on KCs of young animals, coordinates the decrease in spontaneous activity and the maturation of adult learned behavior. When JH signaling is disrupted transiently in α'/β' KCs during a sensitive period in young animals, older animals retain high levels of spontaneous KC activity and poor learned behavior, mimicking the activity and behavior of young animals. Thus, hormone signaling is essential for learning circuits to transition from an immature to a mature state capable of robust learning. The JH receptors Met and Gce, like many hormone receptors, can directly alter transcription. Gene expression changes downstream of these hormone receptors likely directly or indirectly modulate ion channels, synapses, and neurotransmission, thereby sculpting learning circuits. It is speculated that structural refinement of learning circuits underlies the maturation of learned behavior; therefore, further investigation of hormone-triggered molecular changes affecting neurotransmission may provide new entry points for investigating these fundamental age-dependent processes. Together, these studies provide insight into the maturation of activity states and learned behaviors and a platform to examine how hormonally evoked cellular changes enhance the acquisition and maintenance of learned associations (Leinwand, 2021).

Temporally and spatially partitioned neuropeptide release from individual clock neurons

Neuropeptides control rhythmic behaviors, but the timing and location of their release within circuits is unknown. Imaging in the brain shows that synaptic neuropeptide release by Drosophila clock neurons is diurnal, peaking at times of day that were not anticipated by prior electrical and Ca(2+) data. Furthermore, hours before peak synaptic neuropeptide release, neuropeptide release occurs at the soma, a neuronal compartment that has not been implicated in peptidergic transmission. The timing disparity between release at the soma and terminals results from independent and compartmentalized mechanisms for daily rhythmic release: consistent with conventional electrical activity-triggered synaptic transmission, terminals require Ca(2+) influx, while somatic neuropeptide release is triggered by the biochemical signal IP(3). Upon disrupting the somatic mechanism, the rhythm of terminal release and locomotor activity period are unaffected, but the number of flies with rhythmic behavior and sleep-wake balance are reduced. These results support the conclusion that somatic neuropeptide release controls specific features of clock neuron-dependent behaviors. Thus, compartment-specific mechanisms within individual clock neurons produce temporally and spatially partitioned neuropeptide release to expand the peptidergic connectome underlying daily rhythmic behaviors (Klose, 2021).

FAP imaging revealed synaptic neuropeptide release from LNv clock neurons that does not conform to predictions from previously used indirect methods. Earlier neuropeptide-content measurements could not resolve whether somatic changes were due to release or traffic and did not detect l-LNv rhythmic neuropeptide release, likely because it is relatively modest and/or obscured by DCV capture that replenishes synaptic neuropeptide stores. Furthermore, Ca2+ measured at the soma was not reflective of release at terminals likely because of somatic IP3 signaling. Thus, presynaptic Ca2+ may be more predictive of release by LNv termini. Finally, somatic electrical recording does not take into account regulation by presynaptic inputs. Thus, direct live imaging of neuropeptide release is essential for monitoring peptidergic transmission in the brain (Klose, 2021).

Indeed, this approach demonstrates that central clock neurons release neuropeptide from terminals and the soma, with each compartment operating with different mechanisms and timing. Release from LNv clock neuron terminals is conventional (i.e., mediated by extracellular Ca2+ influx); because cell specific genetic Ca2+-channel inhibition was not used, the contributions of Ca2+ channels in LNv neurons and their presynaptic inputs was not determined. In contrast, somatic neuropeptide release is triggered by IP3 signaling that operates in the absence of action potential-induced Ca2+ influx. This shows that the two compartments use different mechanisms. It also raises the possibility that release by the two compartments differ in cell autonomy. Most importantly, different release mechanisms allow for multiphasic temporal control of neuropeptide release from separate compartments of the same neuron, each of which releases onto different parts of the clock circuit, thereby providing separate output avenues to independently influence different parameters of behavior (Klose, 2021).

Axonal chemokine-like orion induces astrocyte infiltration and engulfment during mushroom body neuronal remodeling

The remodeling of neurons is a conserved fundamental mechanism underlying nervous system maturation and function. Astrocytes can clear neuronal debris and they have an active role in neuronal remodeling. Developmental axon pruning of Drosophila memory center neurons occurs via a degenerative process mediated by infiltrating astrocytes. However, how astrocytes are recruited to the axons during brain development is unclear. Using an unbiased screen, the gene requirement of orion/CG2206, encoding for a chemokine-like protein, was identified in the developing mushroom bodies. Functional analysis shows that Orion is necessary for both axonal pruning and removal of axonal debris. Orion performs its functions extracellularly and bears some features common to chemokines, a family of chemoattractant cytokines. It is proposed that Orion is a neuronal signal that elicits astrocyte infiltration and astrocyte-driven axonal engulfment required during neuronal remodeling in the Drosophila developing brain (Boulanger, 2021).

Neuronal remodeling is a widely used developmental mechanism, across the animal kingdom, to refine dendrite and axon targeting necessary for the maturation of neural circuits. Importantly, similar molecular and cellular events can occur during neurodevelopmental disorders or after nervous system injury. A key role for glial cells in synaptic pruning and critical signaling pathways between glia and neurons has been identified. In Drosophila, the mushroom body (MB), a brain memory center, is remodeled at metamorphosis and MB γ neuron pruning occurs by a degenerative mechanism. Astrocytes surrounding the MB have an active role in the process: blocking their infiltration into the MBs prevents remodeling. MB γ neuron remodeling relies on two processes: axon fragmentation and the subsequent clearance of axonal debris. Importantly, it has been shown that astrocytes are involved in these two processes and that these two processes can be decoupled. Altering the ecdysone signaling in astrocytes, during metamorphosis, results both in a partial axon pruning defect, visualized as either some individual larval axons or as thin bundles of intact larval axons remaining in the adults, and also in a strong defect in clearance of debris, visualized by the presence of clusters of axonal debris. Astrocytes have only a minor role in axon severing as evidenced by the observation that most of the MB γ axons are correctly pruned when ecdysone signaling is altered in these cells. When astrocyte function is blocked, the γ axon-intrinsic fragmentation process remains functional and the majority of axons degenerate (Boulanger, 2021).

It has been widely proposed that a 'find-me/eat-me' signal emanating from the degenerating γ neurons is necessary for astrocyte infiltration and engulfment of the degenerated larval axons. However, the nature of this glial recruitment signal is unclear (Boulanger, 2021).

This study has identified a gene (orion), not previously described, by screening for viable ethyl methanesulfonate (EMS)-induced mutations and not for lethal mutations in MB clones as was done previously. This allowed the identification of genes involved in glial cell function by directly screening for defects in MB axon pruning. It was found that orion1, a viable X-chromosome mutation, is necessary for both the pruning of some γ axons and removal of the resulting debris. orion is secreted from the neurons, remains near the axon membranes where it associates with infiltrating astrocytes, and is necessary for astrocyte infiltration into the γ bundle. This implies a role for an as-yet-undefined orion receptor on the surface of the astrocytes. orion bears some chemokine features, for example, a CX3C motif, three glycosaminoglycan (GAG) binding consensus sequences that are required for its function. Altogether, these results identify a neuron-secreted extracellular messenger, which is likely to be the long-searched-for signal responsible for astrocyte infiltration and engulfment of the degenerated larval axons and demonstrate its involvement for neuronal remodeling (Boulanger, 2021).

Adult orion1 individuals showed a clear and highly penetrant MB axon pruning phenotype as revealed by the presence of some adult unpruned vertical γ axons as well as the strong presence of debris (100% of mutant MBs). Astrocytes, visualized with alrm-GAL4, are the major glial subtype responsible for the clearance of the MB axon debris. The presence of γ axon debris is a landmark of defective astrocyte function, as has been described, and is also further shown in this study. The unpruned axon phenotype was particularly apparent during metamorphosis. At 24 h after puparium formation (APF), although γ axon branches were nearly completely absent in the wild-type control, they persisted in the orion1 mutant brains, where a significant accumulation of debris was also observed. The number of unpruned axons at this stage is lower in orion1 than in Hr39C13 where the γ axon-intrinsic process of pruning is blocked. In addition, the MB dendrite pruning was clearly affected in orion1 individuals (Boulanger, 2021).

The orion1 EMS mutation was localized by standard duplication and deficiency mapping as well as by whole-genome sequencing. The orion gene (CG2206) encodes two putatively secreted proteins: Orion-A [664 amino acid (a.a.)] and Orion-B (646 a.a.), whose messenger RNAs (mRNAs) arise from two different promoters. These two proteins differ in their N-terminal domains and are identical in the remainder of their sequences. The EMS mutation is a G to C nucleotide change inducing the substitution of the glycine (at position 629 for Orion-A and 611 for Orion-B) into an aspartic acid. The mutation lies in the common shared part and therefore affects both Orion-A and -B functions. Both isoforms display a signal peptide at their N termini, suggesting that they are secreted. Interestingly, a CX3C chemokine signature is present in the orion common region. Chemokines are a family of chemoattractant cytokines, characterized by a CC, CXC, or CX3C motif, promoting the directional migration of cells within different tissues. Mammalian CX3CL1 (also known as fractalkine) is involved in, among other contexts, neuron-glia communication. Mammalian Fractalkines display conserved intramolecular disulfide bonds that appear to be conserved with respect to their distance from the CX3C motif present in both orion isoforms. Fractalkine and its receptor, CX3CR1, have been recently shown to be required for post-trauma cortical brain neuron microglia-mediated remodeling in a mouse whisker lesioning paradigm. This study observed that the change of the CX3C motif into CX4C or AX3C blocked the orion function necessary for the MB pruning. Similarly, the removal of the signal peptide also prevented pruning. These two results indicate that the orion isoforms likely act as secreted chemokine-like molecules. Three CRISPR/Cas9-mediated mutations in the orion gene were generated that either delete the common part (orionΔC), the A-specific part (orionΔA), or the B-specific part (orionΔB). Noticeably, orionΔC displayed the same MB pruning phenotype as orion1, which is also the same in orion1/Deficiency females, indicating that orion1 and orionΔC are likely null alleles for this phenotype. In contrast, orionΔA and orionΔB have no MB phenotype by themselves, indicating the likelihood of functional redundancy between the two proteins in the pruning process (Boulanger, 2021).

Using the GAL4/UAS system, this study found that expression of wild-type orion in the orion1 MB γ neurons (201Y-GAL4) fully rescued the MB mutant phenotype (100% of wild-type MBs n = 387), although wild-type orion expression in the astrocytes (alrm-GAL4) did not rescue. repo-GAL4 could not be used because of lethality when combined with UAS-orion. This supports the hypothesis that orion is produced by axons and, although necessary for astrocyte infiltration, not by astrocytes. Both UAS-orion-A and UAS-orion-B rescued the orion1 pruning phenotype indicating again a likely functional redundancy between the two proteins at least in the pruning process. Complementary to the rescue results, this study found that the expression of an orion-targeting RNA interference (RNAi) in the MBs produced unpruned axons similar to that in orion1, although the debris is not apparent likely due to an incomplete inactivation of the gene expression by the RNAi. The expression of the same RNAi in the glia had no effect. Using the mosaic analysis with a repressible cell marker (MARCM), it was found that orion1 homozygous mutant neuroblast clones of γ neurons, in orion1/+ phenotypically wild-type individuals, were normally pruned. Therefore, orion1 is a non-cell-autonomous mutation that is expected if the orion proteins are secreted. orion proteins secreted by the surrounding wild-type axons rescue the pruning defects in the orion mutant clones (Boulanger, 2021).

From genetic data, orion expression is expected in the γ neurons. The fine temporal transcriptional landscape of MB γ neurons was recently described and a corresponding resource is freely accessible. Noteworthy, orion is transcribed at 0 h APF and dramatically decreases at 9 h APF with a peak at 3 h APF. The nuclear receptor EcR-B1 and its target Sox14 are key transcriptional factors required for MB neuronal remodeling. orion was found to be a likely transcriptional target of EcR-B1 and Sox14. This is also consistent with earlier microarray analysis observations. Noticeably, forced expression of UAS-EcR-B1 in the MBs did not rescue the orion mutant phenotype and EcR-B1 expression, in the MB nuclei, and is not altered in orion1 individuals. Furthermore, the unpruned axon phenotype produced by orion RNAi is rescued by forced expression of EcR-B1 in the MBs. Therefore, the genetic interaction analyses support orion being downstream of EcR-B1 (Boulanger, 2021).

Further molecular and cellular work focused on Orion-B alone since a functional redundancy between the two isoforms was apparent. The Orion-B protein was expressed in the γ neurons using an UAS-orion-B-Myc insert and the 201Y-GAL4 driver. Orion-B was present along the MB lobes and extracellularly present as visualized by anti-Myc staining. Indeed, anti-Myc staining was particularly strong at the tip of the lobes indicating the presence of extracellular Orion-B. Synaptic terminals are condensed in the γ axon varicosities that disappear progressively during remodeling and hole-like structures corresponding to the vestiges of disappeared varicosities can be observed at 6 h APF. The presence of Myc-labeled Orion-B was noticed inside these hole-like structures. The secretion of the Orion proteins should be under the control of their signal peptide and, therefore, Orion proteins lacking their signal peptide (ΔSP) should not show this 'extracellular' phenotype. When UAS-orion-B-Myc-ΔSP was expressed, Orion-B was not observed outside the axons or in the hole-like structures. The possibility that this 'extracellular' phenotype was due to some peculiarities of the Myc labeling was excluded by using a UAS-drl-Myc construct. Drl is a membrane-bound receptor tyrosine kinase and Drl-Myc staining, unlike Orion-B, was not observed outside the axons or in the hole-like structures. In addition, the presence of Myc-labeled Orion-B protein not associated with green fluorescent protein (GFP)-labeled axon membranes can be observed outside the γ axon bundle in 3D reconstructing images. Nevertheless, these signals are possibly located inside the glial compartments and not as freely diffusing orion protein. Finally, supporting the hypothesis that orion acts as a secreted protein, it has been reported to be present in biochemically purified exosomes, indicating that it may act on the glia via its presence on or in exosomes (Boulanger, 2021).

Since glial cells are likely directly involved in the orion1 pruning phenotype, their behavior early during the pruning process was examined. At 6 h APF the axon pruning process starts and is complete by 24 h APF, but the presence of glial cells in the vicinity of the wild-type γ lobes is already clearly apparent at 6 h APF9. Glial cells visualized by a membrane-targeted GFP (UAS-mGFP) under the control of repo-GAL4 were examined, and the γ axons were co-stained with anti-Fas2. At 6 h APF, a striking difference was noted between wild-type and orion1 brains. Unlike in the wild-type control, there is essentially no glial cell invasion of the γ bundle in the mutant. Interestingly, glial infiltration as well as engulfment of the degenerated larval axons was not observed in orion1 neither at 12 h APF nor at 24 h APF, suggesting that glial cells never infiltrate MBs in mutant individuals. The possibility that this lack of glial cell activity was due to a lower number of astrocytes in mutant versus wild-type brains was ruled out (Boulanger, 2021).

The proximity was examined between MB Orion-Myc and astrocytes, as inferred from the shape of the glial cells, labeled with the anti-Drpr antibody at 6 h APF. The distribution was examined along the vertical γ lobes (60 μm of distance) of Orion-B-Myc (wild-type protein) and of Orion-B-ΔSP-Myc (not secreted), in an otherwise wild-type background. Quantification was performed only from images where an astrocyte sat on the top of the vertical lobe. A peak of Orion-Myc localization was always found in the axonal region close to the astrocyte (<7 μm) when wild-type Orion-B-Myc was quantified. However, this was not the case (n = 9) when Orion-B-ΔSP-Myc was quantified. This strongly suggests that astrocytic processes may be 'attracted' by secreted orion (Boulanger, 2021).

Moreover, it was observed that extracellularly present orion stays close to axon membranes. Protein (in particular chemokine) localization to membranes is often mediated by GAGs, a family of highly anionic polysaccharides that occurs both at the cell surface and within the extracellular matrix. GAGs, to which all chemokines bind, ensure that these signaling proteins are presented at the correct site and time in order to mediate their functions. Three consensus sequences for GAG linkage were identified in the common part of orion. These sequences were mutated individually, and the mutant proteins were examined for their ability to rescue the orion1 pruning deficit in vivo. The three GAG sites are required for full orion function, although mutating only GAG3 produced a strong mutant phenotype (Boulanger, 2021).

These findings imply a role for an as-yet-undefined orion receptor on the surface of the glial cells. The glial receptor draper (drpr) seemed an obvious candidate, although Drpr ligands unrelated to orion have been identified. The MB remodeling phenotypes in orion1 and drprΔ5 are, however, different with orion mutant phenotype being stronger than the drpr one. The use of an UAS-mGFP driven by 201Y-GAL4, instead of anti-Fas2, where the labeling of αβ axons often masks individual unpruned γ axons, allowed occasionally observation of unpruned axons in drprΔ5 1-week-old post-eclosion brains in addition to uncleared debris. This indicates a certain degree of previously undescribed unpruned axon persistence in the mutant background. Nevertheless, only orion mutant displayed a 100% penetrant phenotype of both unpruned axons and debris (strong category) in adult flies, which are still present in old flies. On the contrary, the weaker drpr mutant phenotype strongly decreases throughout adulthood. This suggests that Drpr is not an, or at least not the sole, orion receptor (Boulanger, 2021).

Independently of the possible role of Drpr as an orion receptor, it was of interest to test if orion could activate the drpr signaling pathway as it is the case for neuron-derived injury released factors and Spätzle ligands, which bind to glial insulin-like receptors and Toll-6, respectively, upregulating in turn the expression of drpr in phagocytic glia. These ligands are necessary for axonal debris elimination and act as a find-me/eat-me signal in injury and apoptosis, as orion is doing for MB pruning. The data indicate that orion does not modify either Drpr expression nor the level of the drpr transcriptional activator STAT92E in astrocytes. Consequently, orion does not seem to induce the Drpr signaling pathway in astrocytes (Boulanger, 2021).

This study has uncovered a neuronally secreted chemokine-like protein acting as a 'find-me/eat-me' signal involved in the neuron-glia crosstalk required for axon pruning during developmental neuron remodeling. Chemokine-like signaling in insects was not described previously and, furthermore, the results point to an unexpected conservation of chemokine CX3C signaling in the modulation of neural circuits. Thus, it is possible that chemokine involvement in neuron/glial cell interaction is an evolutionarily ancient mechanism (Boulanger, 2021).

Spatiotemporal organization of enteroendocrine peptide expression in Drosophila

The digestion of food and absorption of nutrients occurs in the gut. The nutritional value of food and its nutrients is detected by enteroendocrine cells, and peptide hormones produced by the enteroendocrine cells are thought to be involved in metabolic homeostasis, but the specific mechanisms are still elusive. The enteroendocrine cells are scattered over the entire gastrointestinal tract and can be classified according to the hormones they produce. This study followed the changes in combinatorial expression of regulatory peptides in the enteroendocrine cells during metamorphosis from the larva to the adult fruit fly, and re-confirmed the diverse composition of enteroendocrine cell populations. Drosophila enteroendocrine cells appear to differentially regulate peptide expression spatially and temporally depending on midgut region and developmental stage. In the late pupa, Notch activity is known to determine which peptides are expressed in mature enteroendocrine cells of the posterior midgut. This study also found that the loss of Notch activity in the anterior midgut results in classes of enteroendocrine cells distinct from the posterior midgut. These results suggest that enteroendocrine cells that populate the fly midgut can differentiate into distinct subtypes that express different combinations of peptides, which likely leads to functional variety depending on specific needs (Jang, 2021).

Honeybee queen mandibular pheromone induces a starvation response in Drosophila melanogaster

Eusocial insect societies are defined by the reproductive division of labour, a social structure that is generally enforced by the reproductive dominant(s) or 'queen(s)'. Reproductive dominance is maintained through behavioural dominance or production of queen pheromones, or a mixture of both. Queen mandibular pheromone (QMP) is a queen pheromone produced by queen honeybees (Apis mellifera) which represses reproduction in worker honeybees. How QMP acts to repress worker reproduction, the mechanisms by which this repression is induced, and how it has evolved this activity, remain poorly understood. Surprisingly, QMP is capable of repressing reproduction in non-target arthropods. This study showed that in Drosophila melanogaster QMP treatment mimics the starvation response, disrupting reproduction. QMP exposure induces an increase in food consumption and activation of checkpoints in the ovary that reduce fecundity and depresses insulin signalling. The magnitude of these effects is indistinguishable between QMP-treated and starved individuals. As QMP triggers a starvation response in an insect diverged from honeybees, it is proposed that QMP originally evolved by co-opting nutrition signalling pathways to regulate reproduction (Lovegrove, 2023).

Social experience and pheromone receptor activity reprogram gene expression in sensory neurons

Social experience and pheromone signaling in olfactory neurons affect neuronal responses and male courtship behaviors in Drosophila. Previous work has shown that social experience and pheromone signaling modulate chromatin around behavioral switch gene fruitless, which encodes a transcription factor necessary and sufficient for male sexual behaviors. To identify the molecular mechanisms driving social experience-dependent changes in neuronal responses, RNA-seq was performed from antennal samples of mutants in pheromone receptors and fruitless, as well as grouped or isolated wild-type males. Genes affecting neuronal physiology and function, such as neurotransmitter receptors, ion channels, ion and membrane transporters, and odorant binding proteins are differentially regulated by social context and pheromone signaling. While this study found that loss of pheromone detection only has small effects on differential promoter and exon usage within fruitless gene, many of the differentially regulated genes have Fruitless binding sites or are bound by Fruitless in the nervous system. Recent studies showed that social experience and juvenile hormone signaling co-regulate fruitless chromatin to modify pheromone responses in olfactory neurons. Interestingly, genes involved in juvenile hormone metabolism are also misregulated in different social contexts and mutant backgrounds. These results suggest that modulation of neuronal activity and behaviors in response to social experience and pheromone signaling likely arise due to large-scale changes in transcriptional programs for neuronal function downstream of behavioral switch gene function (Deanhardt, 2023).

Orcokinin neuropeptides regulate reproduction in the fruit fly, Drosophila melanogaster

In animals, neuropeptidergic signaling is essential for the regulation of survival and reproduction. In insects, Orcokinins are poorly studied, despite their high level of conservation among different orders. In particular, there are currently no reports on the role of Orcokinins in the experimental insect model, the fruit fly, Drosophila melanogaster. The present work made use of the genetic tools available in this species to investigate the role of Orcokinins in the regulation of different innate behaviors including ecdysis, sleep, locomotor activity, oviposition, and courtship. RNAi-mediated knockdown of the orcokinin gene caused a disinhibition of male courtship behavior, including the occurrence of male to male courtship, which is rarely seen in wildtype flies. In addition, orcokinin gene silencing caused a reduction in egg production. Orcokinin is emerging as an important neuropeptide family in the regulation of the physiology of insects from different orders. In the case of the fruit fly, these results suggest an important role in reproductive success (Silva, 2021).

Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: Application to Drosophila ecdysis behavior

Neuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. This study presents a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. This study analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. The activity of both neuronal populations was found to be tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the frequency of motoneuron firing. To explore this idea further, a probabilistic logistic model was used to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-based model capable of simulating many features of the observed neural dynamics. Finally, an algorithm was developed to quantify the motor behavior observed in videos of pupal ecdysis, and their features were compared to the patterns of neuronal calcium activity recorded ex vivo. The motor activity of the intact animal was found to be more regular than the motoneuronal activity recorded from ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed to identification of a new post-ecdysis phase (Pineiro, 2021).

A neuroendocrine pathway modulating osmotic stress in Drosophila

Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. This study identified a peptidergic pathway that links these types of regulatory functions. The neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, was uncovered as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. Crz was shown to act to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. It is additionally proposed that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila (Zandawala, 2021).

Environmental conditions continuously challenge the physiological homeostasis in animals, thereby evoking stress that can adversely affect the health and lifespan of an individual. For instance, lack of food and water, extreme temperatures, infection and predation can all evoke stress responses. In order to counter this stress and restore homeostasis, animals have evolved a multitude of physiological and behavioral mechanisms, which involve actions of multiple tissues and/or organs. The core of these mechanisms involves hormones and neuropeptides, which orchestrate the actions of various organs to counteract stress and maintain homeostasis. One well-studied mechanism counteracting water-deficit stress is the mammalian anti-diuretic system that involves hypothalamic osmoreceptors stimulating the sensation of thirst that leads to the release of the anti-diuretic hormone vasopressin, which targets multiple organs, including the kidney, to decrease urine output and conserve water. In insects such as the vinegar fly, Drosophila melanogaster, much progress has been made on the mechanisms and factors regulating metabolic homeostasis, nutritional stress and longevity. Several neuropeptides and peptide hormones have been shown to influence responses to nutrient stress via actions on peripheral tissues such as the liver-like fat body. Specifically, these peptide hormones include Drosophila insulin-like peptides (DILPs), adipokinetic hormone (AKH) and corazonin (Crz). In addition, mechanisms regulating the release of these hormones are being unraveled. Hence, the neural circuits and neuroendocrine pathways regulating metabolic homeostasis and nutritional stress are beginning to be understood. However, the circuits and/or pathways that regulate thermal, osmotic and ionic stresses remain largely unexplored. Thus, this study asked what factors and cellular systems constitute the osmoregulatory axis in Drosophila (and other insects). Since nutrient and osmotic homeostasis are inter-dependent, it was hypothesized that regulation of osmotic stress may involve factors that also regulate nutritional stress. This study identified Crz signaling, a paralog of the AKH/Gonadotropin releasing hormone signaling system, as a candidate regulating these stresses. Based on previous research in Drosophila and other insects, it has been hypothesized that Crz modulates responses to stress, especially nutritional stress. Consistent with this, recent work has shown that neurosecretory cells co-expressing Crz and short neuropeptide F (sNPF) are nutrient sensing and modulate nutrient homeostasis through differential actions of the two co-expressed neuropeptides. Whereas sNPF acts on the insulin-producing cells (IPCs) and AKH-producing cells to stimulate DILP release and inhibit AKH release, respectively, systemic Crz signaling modulates feeding and nutritional stress through actions on the fat body (Zandawala, 2021).

Although the role of Crz in regulating responses to nutritional stress is now established, less is known about its role in cold tolerance and ion/water homeostasis. Hence, to address this, the role of Crz in modulating osmotic and ionic stresses was studied and the cellular systems constituting the Crz signaling axis is furthermore outlined. To this end, this study analyzed the effects of manipulating Crz signaling on desiccation tolerance and chill-coma recovery as these two assays are routinely used to assess responses to osmotic/ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Comprehensive mapping of the Crz receptor (CrzR) expression revealed that these effects of Crz are not likely mediated by direct modulation of the osmoregulatory tissues but indirectly via three pairs of neurons (Va neurons) in the ventral nerve cord (VNC), which express diuretic CAPA neuropeptides. Knockdown of the CrzR in Va neurons affects CAPA release and ion/water balance, consequently influencing desiccation tolerance and chill-coma recovery. Crz acts via inhibition of cAMP production in Va neurons to inhibit release of CAPA and thereby reducing water loss during desiccation. These data, taken together with published findings [14] suggest that Crz is released into the hemolymph during nutritional stress and acts on the fat body to mobilize energy for food search to increase food intake. In summary, we propose that Crz acts upstream of CAPA signaling to regulate water and ion balance as well as restore nutrient levels caused by starvation. Thus, in addition to the hormonal actions of Crz on the fat body to maintain metabolic homeostasis and counter nutritional stress (Kubrak, 2021), this peptide also helps maintain osmotic homeostasis (Zandawala, 2021).

This study has found that a peptidergic neuroendocrine pathway in Drosophila, known to restore nutrient deficiency (utilizing Crz), integrates a further peptidergic component (CAPA) to maintain osmotic and ionic homeostasis. The Crz-CAPA signaling thereby also influences tolerance to osmotic and cold stress. An earlier study suggested that Crz is released during nutritional stress to mobilize energy stores from the fat body to fuel food search behavior (Kubrak, 2021). Furthermore, that study suggests that increased Crz signaling compromises resistance to starvation, desiccation and oxidative stress (Kubrak, 2021). This study has confirmed these findings, and also found that Crz inhibits a set of CrzR expressing Va neurons in the abdominal ganglia that produce CAPA peptides, which when released in vivo through optogenetic or thermogenetic control, leads to increased excretion and decreased whole body water content, respectively. Thus, the two peptidergic systems act together to maintain both energy and ion/water homeostasis (Zandawala, 2021).

The Capa gene-derived neuropeptides (CAPA1 and CAPA2) are well established as osmoregulatory factors that act on the Malpighian tubule principal cells (Davies, 2013; Paluzzi, 2012) and perhaps the hindgut, as indicated herein by receptor expression data. Previous ex vivo studies have largely showed that CAPA neuropeptides act as diuretic hormones in Drosophila and other dipterans while anti-diuretic actions have also been reported. This study's in vivo findings are more aligned with the observations supporting a diuretic role of CAPA peptides. Furthermore, this study shows that the CAPA-producing Va neurons are downstream of Crz signaling and it is proposed that under adverse conditions when flies are exposed to dry starvation (desiccation without food) the two signaling systems act in tandem to restore homeostasis. When the fly experiences starvation and nutrients diminish in the fly, nutrient sensors record this deficiency, which triggers release of hormones that act to restore metabolic homeostasis. As indicated above, one such hormone is Crz, known to provide energy for food search and induce feeding. The released Crz also inhibits CAPA release from Va cells during the nutrient shortage and results in diminished excretion (and defecation) during starvation and food search. Additionally, during the revision of this manuscript, a recent paper (Koyama, 2021) reported that CAPA from Va cells also acts on AKH-producing cells in the corpora cardiaca to inhibit AKH release and thereby decreasing lipolysis in adipocytes. Thus, when Crz inhibits the Va neurons, AKH signaling is likely to be elevated and results in increased energy mobilization from the fat body, further facilitating food search. In insects, increased food intake yields water, which requires post-feeding diuresis to be activated to restore water homeostasis. Therefore, the inhibition of CAPA release from Va cells needs to be lifted after successful food search and nutrient intake. This suggests that the Crz action occurs during the starvation/fasting, and thereafter postprandial CAPA action sets in to restore nutrient and water homeostasis. Unfortunately, it was not possible to provide direct evidence for this sequence of events since there is no method sensitive enough to monitor the timing of changes in hemolymph levels of Crz and CAPA in small organisms such as Drosophila. Indirect measurements, like monitoring levels of peptide-immunolabel in neurons of interest are not necessarily accurate, since these levels reflect the 'balance' between peptide release and production and therefore not useful for resolving onset and duration of release with accuracy (Zandawala, 2021).

The evidence for the Crz action on CAPA-producing Va neurons is based on the expression of CrzR in these cells, as well as functional imaging data which shows that Crz inhibits cAMP production in Va neurons (but has no impact on intracellular Ca2+ levels). This led to an investigation of the effects of manipulating Crz signaling and targeted CrzR knockdown in Va neurons on aspects of water and ion homeostasis and cold tolerance, which have been shown earlier to be affected by CAPA signaling.It was observed that global Crz or CrzR knockdown leads to delayed recovery from chill-coma (i.e. jeopardizing cold tolerance) and increased tolerance to ionic stress. Furthermore, experiments that target knockdown of CrzR to Va neurons also affected chill-coma recovery, and tolerance to starvation, desiccation, and ionic stresses. Injecting flies in vivo with Crz in the present study also resulted in effects on chill-coma recovery and survival during desiccation, further strengthening the model in which Crz modulates release of CAPA. However, the effect of CAPA injections on chill-coma recovery shown previously are not directly compatible with the current findings. This may be due to the difficulty in comparing the effects obtained from RNAi manipulations (chronic) and peptide injections (acute). Furthermore, Crz not only affects cold tolerance via its actions on CAPA signaling, it also regulates levels of trehalose, a cryoprotectant, via actions on the fat body [14]. Thus, Crz signaling could modulate cold tolerance via two independent hormonal pathways (Zandawala, 2021).

Previous work in Drosophila showed that not only does global Crz knockdown result in increased resistance to starvation, desiccation and oxidative stress, but also found that CrzR knockdown in the fat body (and salivary glands) led to these same phenotypes. These findings suggest that Crz signaling to the fat body accounts for some of the stress tolerance phenotypes seen following Crz knockdown. Thus, it is possible that the effects observed on desiccation survival following Crz peptide injections in the present study are partly confounded by the actions of this peptide on the fat body and other tissues expressing the CrzR (Zandawala, 2021).

Earlier data show that after CrzR knockdown in the fat body, glucose, trehalose and glycogen levels are elevated in starved flies, but not in normally fed flies. Moreover, starved, but not normally fed flies with Crz knockdown, display increased triacyl glycerides and Crz transcript is upregulated in starved flies with CrzR knockdown in the fat body. This further emphasizes that systemic Crz signaling is critical under nutritional stress. The data on Crz immunolabeling intensity corroborate these earlier findings and suggest that Crz is released to restore metabolic homeostasis by mobilizing energy stores from the fat body to fuel food search behavior. After restoring nutritional and osmotic stress the Crz signaling decreases and thus the proposed inhibition of CAPA release from Va neurons is lifted (Zandawala, 2021).

The question as to how the Crz neurons sense nutrient deficiency or whether they can detect changes in osmolarity. However, it is known that Crz-producing DLPs express an aquaporin, Drip, and the carbohydrate-sensing gustatory receptors, Gr43a and Gr64a. A subset of the Crz neurons also express a glucose transporter (Glut1) that is involved in glucose-sensing. Thus, imbalances in internal nutritional and maybe even osmotic status could either be sensed cell autonomously by the Crz neurons or indirectly by signals relayed to them via other pathways (Zandawala, 2021).

A few other findings might support roles of Crz in ameliorating nutrient stress. This study found that Crz neurons innervate the antennal lobe (AL), and that the CrzR is strongly expressed in local interneurons of the AL. Possibly, Crz modulates odor sensitivity in hungry flies to increase food search, similar to peptides like short neuropeptide F (sNPF), tachykinin and SIFamide. Another peptide hormone, adipokinetic hormone (AKH), has been shown to be critical in initiating locomotor activity and food search in food deprived flies and AKH also affects sensitivity of gustatory neurons to glucose. The effect of AKH on increasing locomotor activity is evident only after 36h of starvation and may correlate with the proposed action of Crz during starvation. Possibly Crz acts in concert with AKH to allocate fuel during metabolic stress. Since it was found that the CrzR is not expressed in AKH-producing cells, it is likely that these two peptides act in parallel rather than in the same circuit/pathway. However, the DLPs also produce sNPF and this peptide is known to act on the AKH producing cells and thereby modulate glucose homeostasis and possibly, sensitivity of gustatory neurons. Thus, the DLPs may act systemically with Crz and by paracrine signaling with sNPF in the corpora cardiaca to act on AKH cells. Thereby the Crz and AKH systems could be linked by sNPF. In addition, while this manuscript was under revision, a paper (Koyama, 2021) was posted that added an interesting angle to the role of AKH signaling. That study revealed that the AKH producing cells (APCs) express the CAPA receptor and that CAPA acts on APCs to decrease AKH release, which diminishes lipolysis in adipocytes. Thus, in the fed fly, CAPA not only induces diuresis, it also diminishes energy mobilization. The same study also showed that the Va cells in flies are active after sucrose feeding or drinking, and that CAPA triggers nutrient uptake and peristalsis in the intestine. This interaction between CAPA-AKH signaling can also explain the direction of the phenotypes seen following CrzR knockdown in Va neurons. For instance, knockdown of CrzR in Va neurons results in increased CAPA release and one would predict decreased desiccation survival if CAPA is exclusively promoting excretion. However, increased CAPA signaling could also result in reduced AKH signaling that in turn promotes starvation survival. Thus, the direction of the phenotypes observed following CrzR knockdown in Va neurons can be explained by CAPA actions on excretion and AKH-release from APCs. Lastly, the possibility was not ruled out that other neuropeptides possibly coexpressed with CAPA could also contribute to the observed phenotypes (Zandawala, 2021).

In conclusion, it is suggested that Crz regulates acute metabolic stress-associated physiology and behavior via the fat body to ensure nutrient allocation to power food search and feeding during prolonged starvation. During food search and feeding, excretion is blocked by Crz acting on the Va cells to inhibit CAPA release. Following food intake and ensuing need for diuresis, Crz signaling ceases and CAPA can be released to ensure restoration of water and ion homeostasis. Taken together, these findings and those of previous studies indicate that Crz acts on multiple neuronal and peripheral targets to coordinate and sustain water, ion and metabolic homeostasis. It might even be possible that an ancient role of the common ancestor of Crz and AKH signaling systems was to modulate stress-associated physiology and that these paralogous signaling systems have sub-functionalized and neo-functionalized over evolution (Zandawala, 2021).


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