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1.  Mushroom body output neurons encode valence and guide memory-based action selection in Drosophila 
eLife  null;3:e04580.
Animals discriminate stimuli, learn their predictive value and use this knowledge to modify their behavior. In Drosophila, the mushroom body (MB) plays a key role in these processes. Sensory stimuli are sparsely represented by ∼2000 Kenyon cells, which converge onto 34 output neurons (MBONs) of 21 types. We studied the role of MBONs in several associative learning tasks and in sleep regulation, revealing the extent to which information flow is segregated into distinct channels and suggesting possible roles for the multi-layered MBON network. We also show that optogenetic activation of MBONs can, depending on cell type, induce repulsion or attraction in flies. The behavioral effects of MBON perturbation are combinatorial, suggesting that the MBON ensemble collectively represents valence. We propose that local, stimulus-specific dopaminergic modulation selectively alters the balance within the MBON network for those stimuli. Our results suggest that valence encoded by the MBON ensemble biases memory-based action selection.
eLife digest
An animal's survival depends on its ability to respond appropriately to its environment, approaching stimuli that signal rewards and avoiding any that warn of potential threats. In fruit flies, this behavior requires activity in a region of the brain called the mushroom body, which processes sensory information and uses that information to influence responses to stimuli.
Aso et al. recently mapped the mushroom body of the fruit fly in its entirety. This work showed, among other things, that the mushroom body contained 21 different types of output neurons. Building on this work, Aso et al. have started to work out how this circuitry enables flies to learn to associate a stimulus, such as an odor, with an outcome, such as the presence of food.
Two complementary techniques—the use of molecular genetics to block neuronal activity, and the use of light to activate neurons (a technique called optogenetics)—were employed to study the roles performed by the output neurons in the mushroom body. Results revealed that distinct groups of output cells must be activated for flies to avoid—as opposed to approach—odors. Moreover, the same output neurons are used to avoid both odors and colors that have been associated with punishment. Together, these results indicate that the output cells do not encode the identity of stimuli: rather, they signal whether a stimulus should be approached or avoided. The output cells also regulate the amount of sleep taken by the fly, which is consistent with the mushroom body having a broader role in regulating the fly's internal state.
The results of these experiments—combined with new knowledge about the detailed structure of the mushroom body—lay the foundations for new studies that explore associative learning at the level of individual circuits and their component cells. Given that the organization of the mushroom body has much in common with that of the mammalian brain, these studies should provide insights into the fundamental principles that underpin learning and memory in other species, including humans.
PMCID: PMC4273436  PMID: 25535794
mushroom body; memory; behavioral valence; sleep; population code; action selection; D. melanogaster
2.  A tarantula-venom peptide that antagonises the TRPA1 nociceptor ion channel by binding to the S1-S4 gating domain 
Current biology : CB  2014;24(5):473-483.
The venoms of predators such as spiders, scorpions, cone snails, sea anemones, and snakes, have been an excellent source of pharmacological diversity for drug discovery and as pharmacological tools for elucidating the structure, function, and physiological properties of ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1).
We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by co-expression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. Interestingly, ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. To identify the surfaces of ProTx-I by which it binds to these distinct ion channel types, we constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels, and whose specific chemical features explain its higher affinity for NaV1.2 than for TRPA1. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2, but not both. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site.
These studies establish screening of t-toxin libraries of native and mutated toxins, which we term “toxineering”, as a generally applicable method for isolation of novel ion channel modifiers and for design of ion channel modifiers with altered target selectivity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing the biophysical mechanisms of TRPA1 gating, the physiology and pathophysiology of TRPA1 function in nociceptors, and for potential clinical application in the context of pain and inflammation.
PMCID: PMC3949122  PMID: 24530065
3.  Genetically Targeted Optical Electrophysiology in Intact Neural Circuits 
Cell  2013;154(4):10.1016/j.cell.2013.07.027.
Nervous systems process information by integrating the electrical activity of neurons in complex networks. This motivates the long-standing interest in using optical methods to simultaneously monitor the membrane potential of multiple genetically targeted neurons via expression of genetically encoded fluorescent voltage indicators (GEVIs) in intact neural circuits. No currently available GEVIs have demonstrated robust signals in intact brain tissue that enable reliable recording of individual electrical events simultaneously in multiple neurons. Here, we show that the recently developed “ArcLight” GEVI robustly reports both subthreshold events and action potentials in genetically targeted neurons in the intact Drosophila fruit fly brain and reveals electrical signals in neurite branches. In the same way that genetically encoded fluorescent sensors have revolutionized the study of intracellular Ca2+ signals, ArcLight now enables optical measurement in intact neural circuits of membrane potential, the key cellular parameter that underlies neuronal information processing.
PMCID: PMC3874294  PMID: 23932121
4.  Synchronized Bilateral Synaptic Inputs to Drosophila melanogaster Neuropeptidergic Rest/Arousal Neurons 
Neuropeptide Pigment-Dispersing Factor (PDF)-secreting large ventrolateral neurons (lLNvs) in the Drosophila brain regulate daily patterns of rest and arousal. These bilateral wake-promoting neurons are light-responsive and integrate information from the circadian system, sleep circuits, and light environment. In order to begin to dissect the synaptic circuitry of the circadian neural network, we performed simultaneous dual whole-cell patch clamp recordings of pairs of lLNvs. Both ipsilateral and contralateral pairs of lLNvs exhibit synchronous rhythmic membrane activity with a periodicity of about 5 to 10 seconds. This rhythmic lLNv activity is blocked by tetrodotoxin (TTX) voltage-gated sodium blocker, or α-bungarotoxin (α-BuTX) nicotinic acetylcholine receptor antagonist, indicating that action potential-dependent cholinergic synaptic connections are required for rhythmic lLNv activity. Since injecting current into one neuron of the pair had no effect on the membrane activity of the other neuron of the pair, this suggests that the synchrony is due to bilateral inputs and not coupling between the pairs of lLNvs. To further elucidate the nature of these synaptic inputs to lLNvs, we blocked or activated a variety of neurotransmitter receptors and measured effects on network activity and ionic conductances. These measurements indicate the lLNvs possess excitatory nicotinic ACh receptors, inhibitory ionotropic GABAA receptors, and inhibitory ionotropic glutamate-gated chloride (GluCl) receptors. We demonstrate that cholinergic input, but not GABAergic input, is required for synchronous membrane activity, while GABA can modulate firing patterns. We conclude that neuropeptidergic lLNvs that control rest and arousal receive synchronous synaptic inputs mediated by ACh.
PMCID: PMC3125135  PMID: 21632940
synaptic activity; clock neuron; nicotinic acetylcholine receptor; GABAA receptor; glutamate-gated Cl− channel; synchronous activity
5.  Pigment-Dispersing Factor Modulates Pheromone Production in Clock Cells that Influence Mating in Drosophila 
Neuron  2013;79(1):54-68.
Social cues contribute to the circadian entrainment of physiological and behavioral rhythms. These cues supplement the influence of daily and seasonal cycles in light and temperature. In Drosophila, the social environment modulates circadian mechanisms that regulate sex pheromone production and mating behavior. Here we demonstrate that a neuroendocrine pathway, defined by the neuropeptide Pigment-Dispersing Factor (PDF), couples the central nervous system (CNS) to the physiological output of peripheral clock cells that produce pheromones, the oenocytes. PDF signaling from the CNS modulates the phase of the oenocyte clock. Despite its requirement for sustaining free-running locomoter activity rhythms, PDF is not necessary to sustain molecular rhythms in the oenocytes. Interestingly, disruption of the PDF signaling pathway reduces male sex pheromones and results in sex-specific differences in mating behavior. Our findings highlight the role of neuropeptide signaling and the circadian system in synchronizing the physiological and behavioral processes which govern social interactions.
PMCID: PMC3955580  PMID: 23849197
6.  Miniature Neurotransmission Regulates Drosophila Synaptic Structural Maturation 
Neuron  2014;82(3):618-634.
Miniature neurotransmission is the transsynaptic process where single synaptic vesicles spontaneously released from presynaptic neurons induce miniature postsynaptic potentials. Since their discovery over 60 years ago, miniature events have been found at every chemical synapse studied. However, the in vivo necessity for these small-amplitude events has remained enigmatic. Here, we show that miniature neurotransmission is required for the normal structural maturation of Drosophila glutamatergic synapses in a developmental role that is not shared by evoked neurotransmission. Conversely, we find that increasing miniature events is sufficient to induce synaptic terminal growth. We show that miniature neurotransmission acts locally at terminals to regulate synapse maturation via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Our results establish that miniature neurotransmission, a universal but often-overlooked feature of synapses, has unique and essential functions in vivo.
•Miniature, but not evoked, neurotransmission is required for synapse development•Miniature neurotransmission bidirectionally regulates synaptic terminal maturation•Miniature events signal locally through the GEF Trio and the GTPase Rac1•Miniature neurotransmission has unique and essential functions in vivo
Miniature events (or “minis”) are a universal feature of all chemical synapses, but their function in vivo has remained enigmatic. Here, Choi et al. show that miniature neurotransmission is essential for synaptic terminal maturation in a role not shared by evoked neurotransmission.
PMCID: PMC4022839  PMID: 24811381
7.  O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination 
Cell metabolism  2013;17(2):303-310.
Circadian clocks are coupled to metabolic oscillations through nutrient-sensing pathways. Nutrient flux into the hexosamine biosynthesis pathway triggers covalent protein modification by O-linked β-D-N-acetylglucosamine (O-GlcNAc). Here we show that the hexosamine/O-GlcNAc pathway modulates peripheral clock oscillation. O-GlcNAc transferase (OGT) promotes expression of BMAL1/CLOCK target genes and affects circadian oscillation of clock genes in vitro and in vivo. Both BMAL1 and CLOCK are rhythmically O-GlcNAcylated and this protein modification stabilizes BMAL1 and CLOCK by inhibiting their ubiquitination. In vivo analysis of genetically modified mice with perturbed hepatic OGT expression shows aberrant circadian rhythms of glucose homeostasis. These results establish the counteraction between O-GlcNAcylation and ubiquitination as a key mechanism that regulates the circadian clock and suggest a crucial role for O-GlcNAc signaling in transducing nutritional signals to the core circadian timing machinery.
PMCID: PMC3647362  PMID: 23395176
8.  Circadian control of membrane excitability in Drosophila melanogaster lateral ventral clock neurons 
Drosophila circadian rhythms are controlled by a neural circuit containing ∼150 clock neurons. While much is known about mechanisms of autonomous cellular oscillation, the connection between cellular oscillation and functional outputs that control physiological and behavioral rhythms is poorly understood. To address this issue, we performed whole-cell patch-clamp recordings on lateral ventral clock neurons (LNvs), including large and small LNvs (lLNvs, sLNvs), in situ in adult fly whole-brain explants. We found two distinct sizes of action potentials (APs) in more than 50% of lLNvs that fire APs spontaneously, and determined that large APs originate in the ipsilateral optic lobe and small APs in the contralateral. lLNv resting membrane potential (RMP), spontaneous AP firing rate, and membrane resistance are cyclically regulated as a function of time-of-day in 12hr:12hr light:dark conditions (LD). lLNv RMP becomes more hyperpolarized as time progresses from dawn to dusk with a concomitant decrease in spontaneous AP firing rate and membrane resistance. From dusk to dawn, lLNv RMP becomes more depolarized, with spontaneous AP firing rate and membrane resistance remaining stable. In contrast, circadian defective per0 null mutant lLNv membrane excitability is nearly constant in LD. Over 24hr in constant darkness (DD), wild-type lLNv membrane excitability is not cyclically regulated, although RMP gradually becomes slightly more depolarized. sLNv RMP is most depolarized around lights-on, with substantial variability centered around lights-off in LD. Our results indicate that LNv membrane excitability encodes time-of-day via a circadian clock-dependent mechanism, and likely plays a critical role in regulating Drosophila circadian behavior.
PMCID: PMC2680300  PMID: 18562620
circadian rhythms; Drosophila; membrane excitabiliy; time-of-day; clock neuron; cellular oscillation
9.  Peptide neuromodulation in invertebrate model systems 
Neuron  2012;76(1):82-97.
Neuropeptides modulate neural circuits controlling adaptive animal behaviors and physiological processes, such as feeding/metabolism, reproductive behaviors, circadian rhythms, central pattern generation, and sensorimotor integration. Invertebrate model systems have enabled detailed experimental analysis using combined genetic, behavioral, and physiological approaches. Here we review selected examples of neuropeptide modulation in crustaceans, mollusks, insects, and nematodes, with a particular emphasis on the genetic model organisms Drosophila melanogaster and Caenorhabditis elegans, where remarkable progress has been made. On the basis of this survey, we provide several integrating conceptual principles for understanding how neuropeptides modulate circuit function, and also propose that continued progress in this area requires increased emphasis on the development of richer, more sophisticated behavioral paradigms.
PMCID: PMC3466441  PMID: 23040808
10.  Tethering toxins and peptide ligands for modulation of neuronal function 
Tethering genetically encoded peptide toxins or ligands close to their point of activity at the cell plasma membrane provides a new approach to the study of cell networks and neuronal circuits, as it allows selective targeting of specific cell populations, enhances the working concentration of the ligand or blocker peptide, and permits the engineering of a large variety of t-peptides (e.g., including use of fluorescent markers, viral vectors and point mutation variants). This review describes the development of tethered toxins and peptides derived from the identification of the cell surface nAChR modulator lynx1, the existence of related endogenous cell surface modulators of nAChR and AMPA receptors, and the application of the t-toxin and t-neuropeptide technology to the dissection of neuronal circuits in metazoans.
PMCID: PMC3294089  PMID: 22119144
11.  Autoreceptor Modulation of Peptide/Neurotransmitter Co-release from PDF Neurons Determines Allocation of Circadian Activity in Drosophila 
Cell reports  2012;2(2):332-344.
Drosophila melanogaster flies concentrate behavioral activity around dawn and dusk. This organization of daily activity is controlled by central circadian clock neurons, including the lateral ventral pacemaker neurons (LNvs) that secrete the neuropeptide PDF (Pigment Dispersing Factor). Previous studies have demonstrated the requirement for PDF signaling to PDF receptor (PDFR)-expressing dorsal clock neurons in organizing circadian activity. While LNvs also express functional PDFR, the role of these autoreceptors has remained enigmatic. Here we show that (1) PDFR activation in LNvs shifts the balance of circadian activity from evening to morning, similar to behavioral responses to summer-like environmental conditions and (2) this shift is mediated by stimulation of the Ga,s-cAMP pathway and a consequent change in PDF/neurotransmitter co-release from the LNvs. These results suggest a novel mechanism for environmental control of the allocation of circadian activity and provide new general insight into the role of neuropeptide autoreceptors in behavioral control circuits.
PMCID: PMC3432947  PMID: 22938867
12.  Circadian expression of clock genes in mouse macrophages, dendritic cells, and B cells 
Brain, behavior, and immunity  2011;26(3):407-413.
In mammals, circadian and daily rhythms influence nearly all aspects of physiology, ranging from behavior to gene expression. Functional molecular clocks have been described in the murine spleen and splenic NK cells. The aim of our study was to investigate the existence of molecular clock mechanisms in other immune cells. Therefore, we measured the circadian changes in gene expression of clock genes (Per1, Per2, Bmal1, and Clock) and clock-controlled transcription factors (Rev-erbα and Dbp) in splenic enriched macrophages, dendritic cells, and B cells in both mice entrained to a light-dark cycle and under constant environmental conditions. Our study reveals the existence of functional molecular clock mechanisms in splenic macrophages, dendritic cells, and B cells.
PMCID: PMC3336152  PMID: 22019350
Mouse splenic macrophages; dendritic cells; B cells possess functional circadian molecular clocks
13.  Cellular dissection of circadian peptide signals using genetically encoded membrane-tethered ligands 
Current biology : CB  2009;19(14):1167-1175.
Neuropeptides regulate a broad range of physiological and behavioral processes. Elucidation of neuropeptide function requires identifying the cells that respond to neuropeptide signals and determining the molecular, cellular, physiological, and behavioral consequences of activation of their cognate GPCRs in those cells. As a novel tool for answering these questions, we have developed genetically encoded neuropeptides covalently tethered to a glycosylphosphatidyl inositol (GPI) glycolipid anchor on the extracellular leaflet of the plasma membrane (“t-peptides”).
We show that t-peptides cell-autonomously induce activation of their cognate GPCRs in cells that express both the t-peptide and its receptor. In the neural circuit controlling circadian rest-activity rhythms in Drosophila melanogaster, rhythmic secretion of the neuropeptide Pigment Dispersing Factor (PDF) and activation of its GPCR (PDFR) are important for intercellular communication of phase information and coordination of cellular oscillations of multiple circadian clock neurons. Broad expression of t-PDF in the circadian control circuit overcomes arrhythmicity induced by pdf01 null mutation, most likely due to activation of PDFR in PDFR-expressing clock neurons that do not themselves secrete PDF. More restricted cellular expression of t-PDF suggests that activation of PDFR accelerates cellular timekeeping in some clock neurons, while decelerating others.
The activation of PDFR in pdf01 null-mutant flies—and thus the absence of PDF-mediated intercellular transfer of phase information—induces strong rhythmicity in constant darkness, thus establishing a distinct role for PDF signaling in the circadian control circuit independent of the intercellular communication of temporal phase information. The t-peptide technology we have developed and validated should provide a useful tool for cellular dissection of bioactive peptide signaling in a variety of organisms and physiological contexts.
PMCID: PMC2719018  PMID: 19592252
14.  Electrical Hyperexcitation of Lateral Ventral Pacemaker Neurons Desynchronizes Downstream Circadian Oscillators in the Fly Circadian Circuit and Induces Multiple Behavioral Periods 
Coupling of autonomous cellular oscillators is an essential aspect of circadian clock function but little is known about its circuit requirements. Functional ablation of the pigment-dispersing factor-expressing lateral ventral subset (LNV ) of Drosophila clock neurons abolishes circadian rhythms of locomotor activity. The hypothesis that LNVs synchronize oscillations in downstream clock neurons was tested by rendering the LNVs hyperexcitable via transgenic expression of a low activation threshold voltage-gated sodium channel. When the LNVs are made hyperexcitable, free-running behavioral rhythms decompose into multiple independent superimposed oscillations and the clock protein oscillations in the dorsal neuron 1 and 2 subgroups of clock neurons are phase-shifted. Thus, regulated electrical activity of the LNVs synchronize multiple oscillators in the fly circadian pacemaker circuit.
PMCID: PMC2597197  PMID: 16407545
arrhythmia; behavior; circadian rhythms; desynchronization; Drosophila; sodium channel
15.  Phase Coupling of a Circadian Neuropeptide With Rest/Activity Rhythms Detected Using a Membrane-Tethered Spider Toxin 
PLoS Biology  2008;6(11):e273.
Drosophila clock neurons are self-sustaining cellular oscillators that rely on negative transcriptional feedback to keep circadian time. Proper regulation of organismal rhythms of physiology and behavior requires coordination of the oscillations of individual clock neurons within the circadian control network. Over the last decade, it has become clear that a key mechanism for intercellular communication in the circadian network is signaling between a subset of clock neurons that secrete the neuropeptide pigment dispersing factor (PDF) and clock neurons that possess its G protein-coupled receptor (PDFR). Furthermore, the specific hypothesis has been proposed that PDF-secreting clock neurons entrain the phase of organismal rhythms, and the cellular oscillations of other clock neurons, via the temporal patterning of secreted PDF signals. In order to test this hypothesis, we have devised a novel technique for altering the phase relationship between circadian transcriptional feedback oscillation and PDF secretion by using an ion channel–directed spider toxin to modify voltage-gated Na+ channel inactivation in vivo. This technique relies on the previously reported “tethered-toxin” technology for cell-autonomous modulation of ionic conductances via heterologous expression of subtype-specific peptide ion channel toxins as chimeric fusion proteins tethered to the plasma membrane with a glycosylphosphatidylinositol (GPI) anchor. We demonstrate for the first time, to our knowledge, the utility of the tethered-toxin technology in a transgenic animal, validating four different tethered spider toxin ion channel modifiers for use in Drosophila. Focusing on one of these toxins, we show that GPI-tethered Australian funnel-web spider toxin δ-ACTX-Hv1a inhibits Drosophila para voltage-gated Na+ channel inactivation when coexpressed in Xenopus oocytes. Transgenic expression of membrane-tethered δ-ACTX-Hv1a in vivo in the PDF-secreting subset of clock neurons induces rhythmic action potential bursts and depolarized plateau potentials. These in vitro and in vivo electrophysiological effects of membrane-tethered δ-ACTX-Hv1a are consistent with the effects of soluble δ-ACTX-Hv1a purified from venom on Na+ channel physiological and biophysical properties in cockroach neurons. Membrane-tethered δ-ACTX-Hv1a expression in the PDF-secreting subset of clock neurons induces an approximately 4-h phase advance of the rhythm of PDF accumulation in their terminals relative to both the phase of the day:night cycle and the phase of the circadian transcriptional feedback loops. As a consequence, the morning anticipatory peak of locomotor activity preceding dawn, which has been shown to be driven by the clocks of the PDF-secreting subset of clock neurons, phase advances coordinately with the phase of the PDF rhythm of the PDF-secreting clock neurons, rather than maintaining its phase relationship with the day:night cycle and circadian transcriptional feedback loops. These results (1) validate the tethered-toxin technology for cell-autonomous modulation of ion channel biophysical properties in vivo in transgenic Drosophila, (2) demonstrate that the kinetics of para Na+ channel inactivation is a key parameter for determining the phase relationship between circadian transcriptional feedback oscillation and PDF secretion, and (3) provide experimental support for the hypothesis that PDF-secreting clock neurons entrain the phase of organismal rhythms via the temporal patterning of secreted PDF signals.
Author Summary
The regulation of the daily fluctuations that characterize an organism's physiology and behavior requires coordination of the cellular oscillations of individual “clock” neurons within the circadian control network. Clock neurons that secrete a neuropeptide called pigment dispersing factor (PDF) calibrate, or entrain, both the phase of organismal rhythms and the cellular oscillations of other clock neurons. In this study, we tested the hypothesis that phase of PDF secretion rhythms entrains phase of non-PDF neurons and locomotor rhythms using the tethered- toxin technique (which affixes toxins to the cell membrane) to express ion channel–specific peptide toxins in PDF neurons. A particular toxin inhibits inactivation of the Drosophila para sodium (Na+) channel. Inhibition of Na+ channel inactivation in PDF neurons of transgenic flies induces phase advance of PDF rhythm, and correlated phase advance of lights-on anticipatory locomotor activity, suggesting that phase of morning activity is determined by phase of PDF oscillation. Therefore, voltage-gated Na+ channels of Drosophila clock neurons play a key role in determining the phase relationship between circadian transcriptional feedback oscillation and PDF secretion, and PDF-secreting clock neurons entrain the phase of organismal rhythms via the temporal patterning of secreted PDF signals.
Cell-autonomous inhibition ofDrosophila para Na+ channel inactivation using a membrane-tethered spider toxin phase shifts circadian neuropeptide output from cellular oscillation, and the phase of morning anticipatory activity is determined by this phase-shifted neuropeptide output.
PMCID: PMC2577701  PMID: 18986214
16.  Functional Dissection of a Neuronal Network Required for Cuticle Tanning and Wing Expansion in Drosophila 
A subset of Drosophila neurons that expresses Crustacean Cardioactive Peptide (CCAP) previously has been shown to make the hormone bursicon, which is required for cuticle tanning and wing expansion after eclosion. Here we present evidence that CCAP-expressing neurons (NCCAP) consist of two functionally distinct groups, one of which releases bursicon into the hemolymph and the other of which regulates its release. The first group, which we call NCCAP-c929, includes 14 bursicon-expressing neurons of the abdominal ganglion, which lie within the expression pattern of the enhancer-trap line c929-Gal4. We show that suppression of activity within this group blocks bursicon release into the hemolymph together with tanning and wing expansion. The second group, which we call NCCAP-R, consists of NCCAP neurons outside the c929-Gal4 pattern. Since suppression of synaptic transmission and PKA activity throughout NCCAP, but not in NCCAP-c929, also blocks tanning and wing expansion, we conclude that neurotransmission and PKA are required in NCCAP-R to regulate bursicon secretion from NCCAP-c929. Enhancement of electrical activity in NCCAP-R by expression of the bacterial sodium channel NaChBac also blocks tanning and wing expansion, and leads to depletion of bursicon from central processes. NaChBac expression in NCCAP-c929 is without effect, suggesting that the abdominal bursicon-secreting neurons are likely to be silent until stimulated to release the hormone. Our results suggest that NCCAP form an interacting neuronal network responsible for the regulation and release of bursicon, and suggest a model in which PKA-mediated stimulation of inputs to normally quiescent bursicon-expressing neurons activates release of the hormone.
PMCID: PMC1857274  PMID: 16407556
Excitability; Network; Circuit; Hormone; Neuropeptide; Drosophila

Results 1-16 (16)