Åsa M.E. Winther

Drosophila neuropeptides in regulation of physiology and behavior

Studies of neuropeptide and peptide hormone signaling are coming of age in Drosophila due to rapid developments in molecular genetics approaches that overcome the difficulties caused by the small size of the fly. In addition we have genome-wide information on genes involved in peptide signaling, and growing pools of peptidomics data. A large number of different neuropeptides has been identified in a huge variety of neuron types in different parts of the Drosophila nervous system and cells in other locations. This review addresses questions related to peptidergic signaling in the Drosophila nervous system, especially how peptides regulate physiology and behavior during development and in the mature fly. We first summarize novel findings on neuropeptide precursor genes, processed bioactive peptides and their cognate receptors. Thereafter we provide an overview of the physiological and behavioral roles of peptide signaling in Drosophila. These roles include regulation of development, growth, feeding, metabolism, reproduction, homeostasis, and longevity, as well as neuromodulation in learning and memory, olfaction and locomotor control. The substrate of this signaling is the peptide products of about 42 precursor genes expressed in different combinations in a variety of neuronal circuits or that act as circulating hormones. Approximately 45 G-protein-coupled peptide receptors are known in Drosophila and for most of these the ligands have been identified. Functions of some peptides are better understood than others, and much work remains to reveal the spectrum of roles neuropeptides and peptide hormones play in the daily life of a fly.

Molecular Cloning, Genomic Organization, and Expression of a B-Type (Cricket-Type) Allatostatin Preprohormone from Drosophila melanogaster

The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the allatostatins can be subdivided into three different peptide groups: the A-type allatostatins, first discovered in cockroaches, which have the C-terminal sequence Y/FXFGLamide in common; the B-type allatostatins, first discovered in crickets, which all have the C-terminal sequence W(X)(6)Wamide; and the C-type allatostatins, first discovered in the moth Manduca sexta, which have an unrelated and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each of the following putative allatostatins: AWQSLQSSWamide (drostatin-B1), AWKSMNVAWamide (drostatin-B2), <EAQGWNKFRGAWamide (drostatin-B3), EPTWNNLKGMWamide (drostatin-B4), and DQWQKLHGGWamide (drostatin-B5). All five drostatins are novel peptide structures. The DAP-B gene has one intron and two exons and is located at position 74B1 on the left arm of the third chromosome. The gene is expressed in all developmental stages, but weakly in embryos and strongly in larvae. In situ hybridizations of larvae showed that neurons in the brain and abdominal ganglia and endocrine cells in the gut expressed DAP-B. This is the first published report of a B-type allatostatin preprohormone in insects, and the first paper describing the presence of B-type allatostatins in a representative of the insect order Diptera (flies).

Presynaptic peptidergic modulation of olfactory receptor neurons in Drosophila

The role of classical neurotransmitters in the transfer and processing of olfactory information is well established in many organisms. Neuropeptide action, however, is largely unexplored in any peripheral olfactory system. A subpopulation of local interneurons (LNs) in the Drosophila antannal lobe is peptidergic, expressing Drosophila tachykinins (DTKs). We show here that olfactory receptor neurons (ORNs) express the DTK receptor (DTKR). Using two-photon microscopy, we found that DTK applied to the antennal lobe suppresses presynaptic calcium and synaptic transmission in the ORNs. Furthermore, reduction of DTKR expression in ORNs by targeted RNA interference eliminates presynaptic suppression and alters olfactory behaviors. We detect opposite behavioral phenotypes after reduction and over expression of DTKR in ORNs. Our findings suggest a presynaptic inhibitory feedback to ORNs from peptidergic LNs in the antennal lobe.

Tachykinin-related peptides modulate odor perception and locomotor activity in Drosophila

The invertebrate tachykinin-related peptides (TKRPs) constitute a conserved family, structurally related to the mammalian tachykinins, including members such as substance P and neurokinins A and B. Although their expression has been documented in the brains of insects and mammals, their neural functions remain largely unknown, particularly in behavior. Here, we have studied the role of TKRPs in Drosophila. We have analyzed the olfactory perception and the locomotor activity of individuals in which TKRPs are eliminated in the nervous system specifically, by using RNAi constructs to silence gene expression. The perception of specific odorants and concentrations is modified towards a loss of sensitivity, thus resulting in a significant change of the behavioral response towards indifference. In locomotion assays, the TKRP-deficient flies show hyperactivity. We conclude that these peptides are modulators of olfactory perception and locomotion activity in agreement with their abundant expression in the olfactory lobes and central complex. In these brain centers, TKRPs seem to enhance the regulatory inhibition of the neurons in which they are expressed.

Neuropeptides in the Drosophila central complex in modulation of locomotor behavior

SUMMARY The central complex is one of the most prominent neuropils in the insect brain. It has been implicated in the control of locomotor activity and is considered as a pre-motor center. Several neuropeptides are expressed in circuits of the central complex, and thus may be modulators of locomotor behavior. Here we have investigated the roles of two different neuropeptides, Drosophila tachykinin (DTK) and short neuropeptide F (sNPF), in aspects of locomotor behavior. In the Drosophila brain, DTK and sNPF are expressed in interneurons innervating the central complex. We have directed RNA interference (RNAi) towards DTK and sNPF specifically in different central complex neurons. We also expressed a temperature-sensitive dominant negative allele of the fly ortholog of dynamin called shibirets1, essential in membrane vesicle recycling and endocytosis, to disrupt synaptic transmission in central complex neurons. The spontaneous walking activity of the RNAi- or shibirets1-expressing flies was quantified by video tracking. DTK-deficient flies displayed drastically increased center zone avoidance, suggesting that DTK is involved in the regulation of spatial orientation. In addition, DTK deficiency in other central complex neurons resulted in flies with an increased number of activity–rest bouts. Perturbations in the sNPF circuit indicated that this peptide is involved in the fine regulation of locomotor activity levels. Our findings suggest that the contribution of DTK and sNPF to locomotor behavior is circuit dependent and associated with particular neuronal substrates. Thus, peptidergic pathways in the central complex have specific roles in the fine tuning of locomotor activity of adult Drosophila.

Neuronal expression of tachykinin-related peptides and gene transcript during postembryonic development of Drosophila

The gene Dtk, encoding the prohormone of tachykinin‐related peptides (TRPs), has been identified from Drosophila. This gene encodes five putative tachykinin‐related peptides (DTK‐1 to 5) that share the C‐terminal sequence FXGXRamide (where X represents variable residues) as well as an extended peptide (DTK‐6) with the C‐terminus FVAVRamide). By mass spectrometry (MALDI‐TOF‐MS), we identified ion signals with masses identical to those of DTK‐1 to 5 in specific brain regions. We have analyzed the distribution of the Dtk transcript and peptides, by in situ hybridization and immunocytochemistry during postembryonic development of the central nervous system (CNS) of Drosophila. Antiserum against a cockroach TRP that cross‐reacts with the DTKs was used for immunocytochemistry. Expression of transcript and peptides was detected from first to third instar larvae, through metamorphosis to adult flies. Throughout postembryonic development, we were able to follow the strong expression of TRPs in a pair of large descending neurons with cell bodies in the brain. The number of TRP‐expressing neuronal cell bodies in the brain and ventral nerve cord increases during larval development. In the early pupa (stage P8), the number of TRP‐expressing cell bodies is lower than in the third instar larvae. The number drastically increases during later pupal development, and in the adult fly about 200 TRP‐expressing neurons can be seen in the CNS. The continuous expression of TRPs in neurons throughout postembryonic development suggests specific functional roles in both larval and imaginal flies and possibly also in some neurons during pupal development. J. Comp. Neurol. 464:180–196, 2003.

Chemical neuroanatomy of the Drosophila central complex: distribution of multiple neuropeptides in relation to neurotransmitters.

The central complex of the insect brain is an integration center, receiving inputs from many parts of the brain. In Drosophila it has been associated with the control of both locomotor and visually correlated behaviors. The central complex can be divided into several substructures and is comprised of a large number of neuronal types. These neurons produce classical neurotransmitters, biogenic amines, and different neuropeptides. However, the distribution of neurotransmitters and neuromodulators in central‐complex circuits of Drosophila is poorly known. By immunolabeling and GAL4‐directed expression of marker proteins, we analyzed the distribution of acetylcholine, glutamate, GABA, monoamines, and eight different neuropeptides; Drosophila tachykinin, short neuropeptide F, myoinhibitory peptide, allatostatin A, proctolin, SIFamide, neuropeptide F, and FMRFamide. All eight neuropeptides were localized to the fan‐shaped body, the largest substructure of the central complex, and were mapped to different layers within this structure. Several populations of peptide‐immunoreactive tangential and columnar neurons were identified, of which some colocalized acetylcholine. Fewer peptides were found to be expressed in the other substructures: the ellipsoid body, the protocerebral bridge, and the noduli. The ellipsoid body and the protocerebral bridge were innervated by extrinsic peptide expressing neurons. Our findings reveal that numerous neuropeptides are expressed in the central complex and that each peptide has a distinct distribution pattern, suggesting important roles for neuropeptides as neuromediators and cotransmitters in this brain area. J. Comp. Neurol. 519:290‐315, 2011.

Metabolic stress responses in Drosophila are modulated by brain neurosecretory cells that produce multiple neuropeptides.

In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells.

Regulation of insulin-producing cells in the adult Drosophila brain via the tachykinin peptide receptor DTKR.

SUMMARY Drosophila insulin-like peptides (DILPs) play important hormonal roles in the regulation of metabolic carbohydrates and lipids, but also in reproduction, growth, stress resistance and aging. In spite of intense studies of insulin signaling in Drosophilag the regulation of DILP production and release in adult fruit flies is poorly understood. Here we investigated the role of Drosophila tachykinin-related peptides (DTKs) and their receptors, DTKR and NKD, in the regulation of brain insulin-producing cells (IPCs) and aspects of DILP signaling. First, we show DTK-immunoreactive axon terminations close to the presumed dendrites of the IPCs, and DTKR immunolabeling in these cells. Second, we utilized targeted RNA interference to knock down expression of the DTK receptor, DTKR, in IPCs and monitored the effects on Dilp transcript levels in the brains of fed and starved flies. Dilp2 and Dilp3, but not Dilp5, transcripts were significantly affected by DTKR knockdown in IPCs, both in fed and starved flies. Both Dilp2 and Dilp3 transcripts increased in fed flies with DTKR diminished in IPCs whereas at starvation the Dilp3 transcript plummeted and Dilp2 increased. We also measured trehalose and lipid levels as well as survival in transgene flies at starvation. Knockdown of DTKR in IPCs leads to increased lifespan and a faster decrease of trehalose at starvation but has no significant effect on lipid levels. Finally, we targeted the IPCs with RNAi or ectopic expression of the other DTK receptor, NKD, but found no effect on survival at starvation. Our results suggest that DTK signaling, via DTKR, regulates the brain IPCs.

Distribution of metabotropic receptors of serotonin, dopamine, GABA, glutamate, and short neuropeptide F in the central complex of Drosophila

The central complex is a prominent set of midline neuropils in the insect brain, known to be a higher locomotor control center that integrates visual inputs and modulates motor outputs. It is composed of four major neuropil structures, the ellipsoid body (EB), fan-shaped body (FB), noduli (NO), and protocerebral bridge (PB). In Drosophila different types of central complex neurons have been shown to express multiple neuropeptides and neurotransmitters; however, the distribution of corresponding receptors is not known. Here, we have mapped metabotropic, G-protein-coupled receptors (GPCRs) of several neurotransmitters to neurons of the central complex. By combining immunocytochemistry with GAL4 driven green fluorescent protein, we examined the distribution patterns of six different GPCRs: two serotonin receptor subtypes (5-HT(1B) and 5-HT(7)), a dopamine receptor (DopR), the metabotropic GABA(B) receptor (GABA(B)R), the metabotropic glutamate receptor (DmGluR(A)) and a short neuropeptide F receptor (sNPFR1). Five of the six GPCRs were mapped to different neurons in the EB (sNPFR1 was not seen). Different layers of the FB express DopR, GABA(B)R, DmGluR(A,) and sNPFR1, whereas only GABA(B)R and DmGluR(A) were localized to the PB. Finally, strong expression of DopR and DmGluR(A) was detected in the NO. In most cases the distribution patterns of the GPCRs matched the expression of markers for their respective ligands. In some nonmatching regions it is likely that other types of dopamine and serotonin receptors or ionotropic GABA and glutamate receptors are expressed. Our data suggest that chemical signaling and signal modulation are diverse and highly complex in the different compartments and circuits of the Drosophila central complex. The information provided here, on receptor distribution, will be very useful for future analysis of functional circuits in the central complex, based on targeted interference with receptor expression.