The Interactive Fly

Zygotically transcribed genes

Hormones and neuropeptides

  • Conserved hormones detected by immune localization and biochemical techniques
  • Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-ß-Arrestin2 interactions
  • Neuropeptides in interneurons of the insect brain
  • Nutrient sensor in the brain directs the action of the brain-gut axis in Drosophila
  • The nutrient-responsive hormone CCHamide-2 controls growth by regulating Insulin-like peptides in the brain of Drosophila melanogaster
  • Variability in the number of abdominal leucokinergic neurons in adult Drosophila melanogaster
  • Systemic corazonin signalling modulates stress responses and metabolism in Drosophila
  • SIFamide translates hunger signals into appetitive and feeding behavior in Drosophila
  • Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles
    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).

    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).

    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).


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