Perturbing Dynamin Reveals Potent Effects on the Drosophila Circadian Clock

BACKGROUND: Transcriptional feedback loops are central to circadian clock function. However, the role of neural activity and membrane events in molecular rhythms in the fruit fly Drosophila is unclear. To address this question, we expressed a temperature-sensitive, dominant negative allele of the fly homolog of dynamin called shibire(ts1) (shi(ts1)), an active component in membrane vesicle scission. PRINCIPAL FINDINGS: Broad expression in clock cells resulted in unexpectedly long, robust periods (>28 hours) comparable to perturbation of core clock components, suggesting an unappreciated role of membrane dynamics in setting period. Expression in the pacemaker lateral ventral neurons (LNv) was necessary and sufficient for this effect. Manipulation of other endocytic components exacerbated shi(ts1)'s behavioral effects, suggesting its mechanism is specific to endocytic regulation. PKA overexpression rescued period effects suggesting shi(ts1) may downregulate PKA pathways. Levels of the clock component PERIOD were reduced in the shi(ts1)-expressing pacemaker small LNv of flies held at a fully restrictive temperature (29 degrees C). Less restrictive conditions (25 degrees C) delayed cycling proportional to observed behavioral changes. Levels of the neuropeptide PIGMENT-DISPERSING FACTOR (PDF), the only known LNv neurotransmitter, were also reduced, but PERIOD cycling was still delayed in flies lacking PDF, implicating a PDF-independent process. Further, shi(ts1) expression in the eye also results in reduced PER protein and per and vri transcript levels, suggesting that shibire-dependent signaling extends to peripheral clocks. The level of nuclear CLK, transcriptional activator of many core clock genes, is also reduced in shi(ts1) flies, and Clk overexpression suppresses the period-altering effects of shi(ts1). CONCLUSIONS: We propose that membrane protein turnover through endocytic regulation of PKA pathways modulates the core clock by altering CLK levels and/or activity. These results suggest an important role for membrane scission in setting circadian period

Neuroarchitecture of Peptidergic Systems in the Larval Ventral Ganglion of Drosophila melanogaster

Recent studies on Drosophila melanogaster and other insects have revealed important insights into the functions and evolution of neuropeptide signaling. In contrast, in- and output connections of insect peptidergic circuits are largely unexplored. Existing morphological descriptions typically do not determine the exact spatial location of peptidergic axonal pathways and arborizations within the neuropil, and do not identify peptidergic in- and output compartments. Such information is however fundamental to screen for possible peptidergic network connections, a prerequisite to understand how the CNS controls the activity of peptidergic neurons at the synaptic level. We provide a precise 3D morphological description of peptidergic neurons in the thoracic and abdominal neuromeres of the Drosophila larva based on fasciclin-2 (Fas2) immunopositive tracts as landmarks. Comparing the Fas2 “coordinates” of projections of sensory or other neurons with those of peptidergic neurons, it is possible to identify candidate in- and output connections of specific peptidergic systems. These connections can subsequently be more rigorously tested. By immunolabeling and GAL4-directed expression of marker proteins, we analyzed the projections and compartmentalization of neurons expressing 12 different peptide genes, encoding approximately 75% of the neuropeptides chemically identified within the Drosophila CNS. Results are assembled into standardized plates which provide a guide to identify candidate afferent or target neurons with overlapping projections. In general, we found that putative dendritic compartments of peptidergic neurons are concentrated around the median Fas2 tracts and the terminal plexus. Putative peptide release sites in the ventral nerve cord were also more laterally situated. Our results suggest that i) peptidergic neurons in the Drosophila ventral nerve cord have separated in- and output compartments in specific areas, and ii) volume transmission is a prevailing way of peptidergic communication within the CNS. The data can further be useful to identify colocalized transmitters and receptors, and develop peptidergic neurons as new landmarks

Circadian Consequence of Socio-Sexual Interactions in Fruit Flies Drosophila melanogaster

In fruit flies Drosophila melanogaster, courtship is an elaborate ritual comprising chasing, dancing and singing by males to lure females for mating. Courtship interactions peak in the night and heterosexual couples display enhanced nighttime activity. What we do not know is if such socio-sexual interactions (SSI) leave long-lasting after-effects on circadian clock(s). Here we report the results of our study aimed at examining the after-effects of SSI (as a result of co-habitation of males and females in groups) between males and females on their circadian locomotor activity rhythm. Males undergo reduction in the evening activity peak and lengthening of circadian period, while females show a decrease in overall activity. Such after-effects, at least in males, require functional circadian clocks during SSI as loss-of-function clock mutants and wild type flies interacting under continuous light (LL), do not display them. Interestingly, males with electrically silenced Pigment Dispersing Factor (PDF)-positive ventral lateral (LNv) clock neurons continue to show SSI mediated reduction in evening activity peak, suggesting that the LNv clock neurons are dispensable for SSI mediated after-effects on locomotor activity rhythm. Such after-effects in females may not be clock-dependent because clock manipulated females with prior exposure to males show decrease in overall activity, more or less similar to rhythmic wild type females. The expression of SSI mediated after-effects requires a functional olfactory system in males because males with compromised olfactory ability do not display them. These results suggest that SSI causes male-specific, long-lasting changes in the circadian clocks of Drosophila, which requires the presence of functional clocks and intact olfactory ability in males

Glutamate, GABA and Acetylcholine Signaling Components in the Lamina of the Drosophila Visual System

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila we combined Gal4 driven green fluorescent protein in specific lamina neurons with antisera to γ-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABAB type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. We suggest that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABAB receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABAA receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in α-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types

Insulin Signaling, Lifespan and Stress Resistance Are Modulated by Metabotropic GABA Receptors on Insulin Producing Cells in the Brain of Drosophila

Insulin-like peptides (ILPs) regulate growth, reproduction, metabolic homeostasis, life span and stress resistance in worms, flies and mammals. A set of insulin producing cells (IPCs) in the Drosophila brain that express three ILPs (DILP2, 3 and 5) have been the main focus of interest in hormonal DILP signaling. Little is, however, known about factors that regulate DILP production and release by these IPCs. Here we show that the IPCs express the metabotropic GABAB receptor (GBR), but not the ionotropic GABAA receptor subunit RDL. Diminishing the GBR expression on these cells by targeted RNA interference abbreviates life span, decreases metabolic stress resistance and alters carbohydrate and lipid metabolism at stress, but not growth in Drosophila. A direct effect of diminishing GBR on IPCs is an increase in DILP immunofluorescence in these cells, an effect that is accentuated at starvation. Knockdown of irk3, possibly part of a G protein-activated inwardly rectifying K+ channel that may link to GBRs, phenocopies GBR knockdown in starvation experiments. Our experiments suggest that the GBR is involved in inhibitory control of DILP production and release in adult flies at metabolic stress and that this receptor mediates a GABA signal from brain interneurons that may convey nutritional signals. This is the first demonstration of a neurotransmitter that inhibits insulin signaling in its regulation of metabolism, stress and life span in an invertebrate brain

Circadian pacemaker coupling by multi-peptidergic neurons in the cockroach Leucophaea maderae

Lesion and transplantation studies in the cockroach, Leucophaea maderae, have located its bilaterally symmetric circadian pacemakers necessary for driving circadian locomotor activity rhythms to the accessory medulla of the optic lobes. The accessory medulla comprises a network of peptidergic neurons, including pigment-dispersing factor (PDF)-expressing presumptive circadian pacemaker cells. At least three of the PDF-expressing neurons directly connect the two accessory medullae, apparently as a circadian coupling pathway. Here, the PDF-expressing circadian coupling pathways were examined for peptide colocalization by tracer experiments and double-label immunohistochemistry with antisera against PDF, FMRFamide, and Asn13-orcokinin. A fourth group of contralaterally projecting medulla neurons was identified, additional to the three known groups. Group one of the contralaterally projecting medulla neurons contained up to four PDF-expressing cells. Of these, three medium-sized PDF-immunoreactive neurons coexpressed FMRFamide and Asn13-orcokinin immunoreactivity. However, the contralaterally projecting largest PDF neuron showed no further peptide colocalization, as was also the case for the other large PDF-expressing medulla cells, allowing the easy identification of this cell group. Although two-thirds of all PDF-expressing medulla neurons coexpressed FMRFamide and orcokinin immunoreactivity in their somata, colocalization of PDF and FMRFamide immunoreactivity was observed in only a few termination sites. Colocalization of PDF and orcokinin immunoreactivity was never observed in any of the terminals or optic commissures. We suggest that circadian pacemaker cells employ axonal peptide sorting to phase-control physiological processes at specific times of the day

A large population of diverse neurons in the Drosophila central nervous system expresses short neuropeptide F, suggesting multiple distributed peptide functions

<p>Abstract</p> <p>Background</p> <p>Insect neuropeptides are distributed in stereotypic sets of neurons that commonly constitute a small fraction of the total number of neurons. However, some neuropeptide genes are expressed in larger numbers of neurons of diverse types suggesting that they are involved in a greater diversity of functions. One of these widely expressed genes, <it>snpf</it>, encodes the precursor of short neuropeptide F (sNPF). To unravel possible functional diversity we have mapped the distribution of transcript of the <it>snpf </it>gene and its peptide products in the central nervous system (CNS) of <it>Drosophila </it>in relation to other neuronal markers.</p> <p>Results</p> <p>There are several hundreds of neurons in the larval CNS and several thousands in the adult <it>Drosophila </it>brain expressing <it>snpf </it>transcript and sNPF peptide. Most of these neurons are intrinsic interneurons of the mushroom bodies. Additionally, sNPF is expressed in numerous small interneurons of the CNS, olfactory receptor neurons (ORNs) of the antennae, and in a small set of possibly neurosecretory cells innervating the corpora cardiaca and aorta. A sNPF-Gal4 line confirms most of the expression pattern. None of the sNPF immunoreactive neurons co-express a marker for the transcription factor DIMMED, suggesting that the majority are not neurosecretory cells or large interneurons involved in episodic bulk transmission. Instead a portion of the sNPF producing neurons co-express markers for classical neurotransmitters such as acetylcholine, GABA and glutamate, suggesting that sNPF is a co-transmitter or local neuromodulator in ORNs and many interneurons. Interestingly, sNPF is coexpressed both with presumed excitatory and inhibitory neurotransmitters. A few sNPF expressing neurons in the brain colocalize the peptide corazonin and a pair of dorsal neurons in the first abdominal neuromere coexpresses sNPF and insulin-like peptide 7 (ILP7).</p> <p>Conclusion</p> <p>It is likely that sNPF has multiple functions as neurohormone as well as local neuromodulator/co-transmitter in various CNS circuits, including olfactory circuits both at the level of the first synapse and at the mushroom body output level. Some of the sNPF immunoreactive axons terminate in close proximity to neurosecretory cells producing ILPs and adipokinetic hormone, indicating that sNPF also might regulate hormone production or release.</p

Neuroarchitecture of Aminergic Systems in the Larval Ventral Ganglion of Drosophila melanogaster

Biogenic amines are important signaling molecules in the central nervous system of both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, biogenic amines take part in the regulation of various vital physiological processes such as feeding, learning/memory, locomotion, sexual behavior, and sleep/arousal. Consequently, several morphological studies have analyzed the distribution of aminergic neurons in the CNS. Previous descriptions, however, did not determine the exact spatial location of aminergic neurite arborizations within the neuropil. The release sites and pre-/postsynaptic compartments of aminergic neurons also remained largely unidentified. We here used gal4-driven marker gene expression and immunocytochemistry to map presumed serotonergic (5-HT), dopaminergic, and tyraminergic/octopaminergic neurons in the thoracic and abdominal neuromeres of the Drosophila larval ventral ganglion relying on Fasciclin2-immunoreactive tracts as three-dimensional landmarks. With tyrosine hydroxylase- (TH) or tyrosine decarboxylase 2 (TDC2)-specific gal4-drivers, we also analyzed the distribution of ectopically expressed neuronal compartment markers in presumptive dopaminergic TH and tyraminergic/octopaminergic TDC2 neurons, respectively. Our results suggest that thoracic and abdominal 5-HT and TH neurons are exclusively interneurons whereas most TDC2 neurons are efferent. 5-HT and TH neurons are ideally positioned to integrate sensory information and to modulate neuronal transmission within the ventral ganglion, while most TDC2 neurons appear to act peripherally. In contrast to 5-HT neurons, TH and TDC2 neurons each comprise morphologically different neuron subsets with separated in- and output compartments in specific neuropil regions. The three-dimensional mapping of aminergic neurons now facilitates the identification of neuronal network contacts and co-localized signaling molecules, as exemplified for DOPA decarboxylase-synthesizing neurons that co-express crustacean cardioactive peptide and myoinhibiting peptides