Bridget C. Lear

A G Protein-Coupled Receptor, groom-of-PDF, Is Required for PDF Neuron Action in Circadian Behavior

The neuropeptide Pigment-Dispersing Factor (PDF) plays a critical role in mediating circadian control of behavior in Drosophila. Here we identify mutants (groom-of-PDF; gop) that display phase-advanced evening activity and poor free-running rhythmicity, phenocopying pdf mutants. In gop mutants, a spontaneous retrotransposon disrupts a coding exon of a G protein-coupled receptor, CG13758. Disruption of the receptor is accompanied by phase-advanced oscillations of the core clock protein PERIOD. Moreover, effects on circadian timing induced by perturbation of PDF neurons require gop. Yet PDF oscillations themselves remain robust in gop mutants, suggesting that GOP acts downstream of PDF. gop is expressed most strongly in the dorsal brain in regions that lie in proximity to PDF-containing nerve terminals. Taken together, these studies implicate GOP as a PDF receptor in Drosophila.

DN1p Circadian Neurons Coordinate Acute Light and PDF Inputs to Produce Robust Daily Behavior in Drosophila

BACKGROUND Daily behaviors in animals are determined by the interplay between internal timing signals from circadian clocks and environmental stimuli such as light. How these signals are integrated to produce timely and adaptive behavior is unclear. The fruit fly Drosophila exhibits clock-driven activity increases that anticipate dawn and dusk and free-running rhythms under constant conditions. Flies also respond to the onset of light and dark with acute increases in activity. RESULTS Mutants of a novel ion channel, narrow abdomen (na), lack a robust increase in activity in response to light and show reduced anticipatory behavior and free-running rhythms, providing a genetic link between photic responses and circadian clock function. We used tissue-specific rescue of na to demonstrate a role for approximately 16-20 circadian pacemaker neurons, a subset of the posterior dorsal neurons 1 (DN1(p)s), in mediating the acute response to the onset of light as well as morning anticipatory behavior. Circadian pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) are especially important for morning anticipation and free-running rhythms and send projections to the DN1(p)s. We also demonstrate that DN1(p)Pdfr expression is sufficient to rescue, at least partially, Pdfr morning anticipation defects as well as defects in free-running rhythms, including those in DN1 molecular clocks. Additionally, these DN1 clocks in wild-type flies are more strongly reset to timing changes in PDF clocks than other pacemaker neurons, suggesting that they are direct targets. CONCLUSIONS Taking these results together, we demonstrate that the DN1(p)s lie at the nexus of PDF and photic signaling to produce appropriate daily behavior.

The Neuropeptide PDF Acts Directly on Evening Pacemaker Neurons to Regulate Multiple Features of Circadian Behavior

Animals use distinct sets of clock neurons to time behaviors in the morning and evening. In this article, the direct neural targets for morning neurons and the neuropeptide pigment dispersing factor are revealed in the fruit fly.

The ion channel narrow abdomen is critical for neural output of the Drosophila circadian pacemaker.

Circadian clocks consist of transcriptional feedback loops housed in interdependent pacemaker neurons. Yet little is known about the neuronal output components essential for rhythmic behavior. Drosophila mutants of a putative ion channel, narrow abdomen (na), exhibit poor circadian rhythms and suppressed daylight activity. We find that NA is expressed in pacemaker neurons and induced expression within circadian neurons is sufficient to rescue these mutant phenotypes. Selective na rescue in distinct pacemaker neurons influences rhythmicity and timing of behavior. Oscillations of the clock protein PERIOD are intact in na mutants, indicating an output role. Pore residues are required for robust rescue consistent with NA action as an ion channel. In na mutants, expression of potassium currents and the key neuropeptide PDF are elevated, the latter consistent with reduced release. These data implicate NA and the pacemaker neural network in controlling phase and rhythmicity.

An unusual cation channel mediates photic control of locomotion in Drosophila.

A unique family of putative ion channels that are related to voltage-gated sodium and calcium channels has been identified in genomic and cDNA studies of metazoans. Aside from evidence for expression of family members in the nervous system, little is known about the operation of the channel or its functional significance. In the present study, this conserved familys sole Drosophila member, a gene known both as CG1517 and as Dmalpha1U, is shown to correspond to the narrow abdomen (na) gene and is the locus of a set of mutations that affect sensitivity to anesthetics. Immunohistochemistry of adult heads reveals that the channel is expressed in the neuropil of the central complex and optic lobe; expression is severely depressed in the mutants. In addition to previously described defects, the mutant phenotype is demonstrated here to include dysfunction in the coupling between light and locomotor behavior. Most dramatically, mutant flies have an inversion of relative locomotor activity in light versus dark. The involvement of the channel in daily rhythms of the fruit fly is especially provocative because the human ortholog lies in a candidate region linked to bipolar disorder, a disease frequently associated with altered diurnal behavior.

A Conserved Bicycle Model for Circadian Clock Control of Membrane Excitability

Circadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. Here, we find that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1, linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, we reveal an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake.

The CRYPTOCHROME Photoreceptor Gates PDF Neuropeptide Signaling to Set Circadian Network Hierarchy in Drosophila

Circadian clocks in the brain are organized as coupled oscillators that integrate seasonal cues such as light and temperature to time daily behaviors. In Drosophila, the PIGMENT DISPERSING FACTOR (PDF) neuropeptide-expressing morning (M) and non-PDF evening (E) cells are coupled cell groups important for morning and evening behavior, respectively. Depending on day length, either M cells (short days) or E cells (long days) dictate both the morning and the evening phase, a phenomenon that we term network hierarchy. To examine the role of PDF in light-dark conditions, we examined flies lacking both the PDF receptor (PDFR) and the circadian photoreceptor CRYPTOCHROME (CRY). We found that subsets of E cells exhibit molecular oscillations antiphase to those of wild-type flies, single cry mutants, or single Pdfr mutants, demonstrating a potent role for PDF in light-mediated entrainment, specifically in the absence of CRY. Moreover, we find that the evening behavioral phase is more strongly reset by PDF(+) M cells in the absence of CRY. On the basis of our findings, we propose that CRY can gate PDF signaling to determine behavioral phase and network hierarchy.

Processing sleep data created with the Drosophila activity monitoring (DAM) system

Adult behavioral assays have been used with great success in Drosophila melanogaster to identify circadian rhythm genes. In particular, the locomotor activity assay can identify altered behavior patterns over the course of several days in small populations, or even individual flies. Sleep is a highly conserved behavior that is required for optimal performance and, in many cases, life of an organism. Drosophila demonstrate a behavioral state that shows traits consistent with sleep: periods of relative behavioral immobility that coincide with an increased arousal threshold after ~5 min of inactivity, regulated by circadian and homeostatic mechanisms. However, because flies do not produce brain waves recordable by electroencephalography, sleep researchers use behavior-based paradigms to infer when a fly is asleep, as opposed to awake but immobile. Data on Drosophila activity can be collected using an automated monitoring system to provide insight into sleep duration, consolidation, and latency, as well as sleep deprivation and rebound. This protocol details the use of Counting Macro, an Excel-based program, to process data created with the Drosophila Activity Monitoring (DAM) System from TriKinetics for sleep analyses. Specifically, it details the steps necessary to convert the raw data created by the DAM System into sleep duration and consolidation data, broken down into the light (L), dark (D), light:dark cycling (LD), and constant darkness (DD) phases of a behavior experiment.

UNC79 and UNC80, putative auxiliary subunits of the NARROW ABDOMEN ion channel, are indispensable for robust circadian locomotor rhythms in Drosophila.

In the fruit fly Drosophila melanogaster, a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA), orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in Drosophila, unc79 (aka dunc79) and unc80 (aka CG18437). We have generated novel unc79 and unc80 mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from na mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for unc79 and unc80 in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain. Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

Physiological, anatomical, and behavioral changes after acoustic trauma in Drosophila melanogaster

Significance Noise-induced hearing loss (NIHL) is an important health issue, yet its mechanisms and potential treatments remain unclear. We present the first study of NIHL in the fruit fly Drosophila, which has many advantages as an NIHL model. We examined auditory function and structure after exposing flies to acoustic trauma. Trauma impairs auditory system function and changes neural mitochondria size, suggesting metabolic stress. In mutant flies with a reduced ability to cope with such stresses, the responses to trauma were more severe and recovery delayed or impaired. Noise-induced hearing loss (NIHL) is a growing health issue, with costly treatment and lost quality of life. Here we establish Drosophila melanogaster as an inexpensive, flexible, and powerful genetic model system for NIHL. We exposed flies to acoustic trauma and quantified physiological and anatomical effects. Trauma significantly reduced sound-evoked potential (SEP) amplitudes and increased SEP latencies in control genotypes. SEP amplitude but not latency effects recovered after 7 d. Although trauma produced no gross morphological changes in the auditory organ (Johnston’s organ), mitochondrial cross-sectional area was reduced 7 d after exposure. In nervana 3 heterozygous flies, which slightly compromise ion homeostasis, trauma had exaggerated effects on SEP amplitude and mitochondrial morphology, suggesting a key role for ion homeostasis in resistance to acoustic trauma. Thus, Drosophila exhibit acoustic trauma effects resembling those found in vertebrates, including inducing metabolic stress in sensory cells. This report of noise trauma in Drosophila is a foundation for studying molecular and genetic sequelae of NIHL.