Orie T. Shafer

PDF Receptor Signaling in Drosophila Contributes to Both Circadian and Geotactic Behaviors

The neuropeptide Pigment-Dispersing Factor (PDF) is a principle transmitter regulating circadian locomotor rhythms in Drosophila. We have identified a Class II (secretin-related) G protein-coupled receptor (GPCR) that is specifically responsive to PDF and also to calcitonin-like peptides and to PACAP. In response to PDF, the PDF receptor (PDFR) elevates cAMP levels when expressed in HEK293 cells. As predicted by in vivo studies, cotransfection of Neurofibromatosis Factor 1 significantly improves coupling of PDFR to adenylate cyclase. pdfr mutant flies display increased circadian arrhythmicity, and also display altered geotaxis that is epistatic to that of pdf mutants. PDFR immunosignals are expressed by diverse neurons, but only by a small subset of circadian pacemakers. These data establish the first synapse within the Drosophila circadian neural circuit and underscore the importance of Class II peptide GPCR signaling in circadian neural systems.

Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging.

The neuropeptide PDF is released by sixteen clock neurons in Drosophila and helps maintain circadian activity rhythms by coordinating a network of approximately 150 neuronal clocks. Whether PDF acts directly on elements of this neural network remains unknown. We address this question by adapting Epac1-camps, a genetically encoded cAMP FRET sensor, for use in the living brain. We find that a subset of the PDF-expressing neurons respond to PDF with long-lasting cAMP increases and confirm that such responses require the PDF receptor. In contrast, an unrelated Drosophila neuropeptide, DH31, stimulates large cAMP increases in all PDF-expressing clock neurons. Thus, the network of approximately 150 clock neurons displays widespread, though not uniform, PDF receptivity. This work introduces a sensitive means of measuring cAMP changes in a living brain with subcellular resolution. Specifically, it experimentally confirms the longstanding hypothesis that PDF is a direct modulator of most neurons in the Drosophila clock network.

Development and morphology of the clock-gene-expressing lateral neurons of Drosophila melanogaster.

The clock‐gene‐expressing lateral neurons are essential for the locomotor activity rhythm of Drosophila melanogaster. Traditionally, these neurons are divided into three groups: the dorsal lateral neurons (LNd), the large ventral lateral neurons (l‐LNv), and the small ventral lateral neurons (s‐LNv), whereby the latter group consists of four neurons that express the neuropeptide pigment‐dispersing factor (PDF) and a fifth PDF‐negative neuron. So far, only the l‐LNv and the PDF‐positive s‐LNv have been shown to project into the accessory medulla, a small neuropil that contains the circadian pacemaker center in several insects. We show here that the other lateral neurons also arborize in the accessory medulla, predominantly forming postsynaptic sites. Both the l‐LNv and LNd are anatomically well suited to connect the accessory medullae. Whereas the l‐LNv may receive ipsilateral photic input from the Hofbauer‐Buchner eyelet, the LNd invade mainly the contralateral accessory medulla and thus may receive photic input from the contralateral side. Both the LNd and the l‐LNv differentiate during midmetamorphosis. They do so in close proximity to one another and the fifth PDF‐negative s‐LNv, suggesting that these cell groups may derive from common precursors. J. Comp. Neurol. 500:47–70, 2007.

Functional analysis of circadian pacemaker neurons in Drosophila melanogaster

The molecular mechanisms of circadian rhythms are well known, but how multiple clocks within one organism generate a structured rhythmic output remains a mystery. Many animals show bimodal activity rhythms with morning (M) and evening (E) activity bouts. One long-standing model assumes that two mutually coupled oscillators underlie these bouts and show different sensitivities to light. Three groups of lateral neurons (LN) and three groups of dorsal neurons govern behavioral rhythmicity of Drosophila. Recent data suggest that two groups of the LN (the ventral subset of the small LN cells and the dorsal subset of LN cells) are plausible candidates for the M and E oscillator, respectively. We provide evidence that these neuronal groups respond differently to light and can be completely desynchronized from one another by constant light, leading to two activity components that free-run with different periods. As expected, a long-period component started from the E activity bout. However, a short-period component originated not exclusively from the morning peak but more prominently from the evening peak. This reveals an interesting deviation from the original Pittendrigh and Daan (1976) model and suggests that a subgroup of the ventral subset of the small LN acts as “main” oscillator controlling M and E activity bouts in Drosophila.

Reevaluation of Drosophila melanogaster's Neuronal Circadian Pacemakers Reveals New Neuronal Classes

In the brain of the fly Drosophila melanogaster, ∼150 clock‐neurons are organized to synchronize and maintain behavioral rhythms, but the physiological and neurochemical bases of their interactions are largely unknown. Here we reevaluate the cellular properties of these pacemakers by application of a novel genetic reporter and several phenotypic markers. First, we describe an enhancer trap marker called R32 that specifically reveals several previously undescribed aspects of the flys central neuronal pacemakers. We find evidence for a previously unappreciated class of neuronal pacemakers, the lateral posterior neurons (LPNs), and establish anatomical, molecular, and developmental criteria to establish a subclass within the dorsal neuron 1 (DN1) group of pacemakers. Furthermore, we show that the neuropeptide IPNamide is specifically expressed by this DN1 subclass. These observations implicate IPNamide as a second candidate circadian transmitter in the Drosophila brain. Finally, we present molecular and anatomical evidence for unrecognized phenotypic diversity within each of four established classes of clock neurons. J. Comp. Neurol. 498:180–193, 2006.

A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling.

SUMMARY The Drosophila orphan G protein-coupled receptor encoded by CG17415 is related to members of the calcitonin receptor-like receptor (CLR) family. In mammals, signaling from CLR receptors depend on accessory proteins, namely the receptor activity modifying proteins (RAMPs) and receptor component protein (RCP). We tested the possibility that this Drosophila CLR might also require accessory proteins for proper function and we report that co-expression of the mammalian or Drosophila RCP or mammalian RAMPs permitted neuropeptide diuretic hormone 31 (DH31) signaling from the CG17415 receptor. RAMP subtype expression did not alter the pharmacological profile of CG17415 activation. CG17415 antibodies revealed expression within the principal cells of Malpighian tubules, further implicating DH31 as a ligand for this receptor. Immunostaining in the brain revealed an unexpected convergence of two distinct DH signaling pathways. In both the larval and adult brain, most DH31 receptor-expressing neurons produce the neuropeptide corazonin, and also express the CRFR-related receptor CG8422, which is a receptor for the neuropeptide diuretic hormone 44 (DH44). There is extensive convergence of CRF and CGRP signaling within vertebrates and we report a striking parallel in Drosophila involving DH44 (CRF) and DH31 (CGRP). Therefore, it appears that both the molecular details as well as the functional organization of CGRP signaling have been conserved.

Mechanisms of clock output in the Drosophila circadian pacemaker system.

Molecular oscillations that underlie the circadian clock are coupled to different output signals by which daily rhythms in downstream events are evoked and/or synchronized. Here the authors review the literature that describes circadian output mechanisms in Drosophila. They begin at the most proximal level, within oscillator cells themselves, by surveying studies of rhythmic gene expression within Drosophila heads. Next the authors describe the several neuron groups that compose the circadian pacemaker network underlying rhythmic locomotor activity, and they detail current models of how that network is organized and coordinated. The authors outline the body of evidence that describes a role for the neuropeptide pigment dispersing factor (PDF) as a circadian transmitter in the fly brain. Finally, in the context of PDF, they consider studies that address mechanisms of signaling from the circadian pacemaker network to downstream neurons and nonneuronal cells that directly control rhythmic outputs.

Flies by night: Effects of changing day length on Drosophila's circadian clock.

In Drosophila, two intersecting molecular loops constitute an autoregulatory mechanism that oscillates with a period close to 24 hr. These loops touch when proteins from one loop, PERIOD (PER) and TIMELESS (TIM), repress the transcription of their parent genes, period (per) and timeless (tim), by blocking positive transcription factors from the other loop. The arrival of PER and TIM into the nucleus of a clock cell marks the timing of this interaction between the two loops; thus, control of PER:TIM nuclear accumulation is a central component of the molecular model of clock function. If a light pulse occurs early in the night as the heterodimer accumulates in the nucleus of clock cells, TIM is degraded, PER is destabilized, and clock time is delayed. Alternatively, if TIM is degraded during the later part of the night, after peak accumulation, clock time advances. Current models state that the effect of a light pulse depends on the state of the PER:TIM oscillation, which turns on the changing levels of TIM. However, previous studies have shown that light:dark (LD) regimes mimicking seasonal changes cause behavioral adjustments while altering clock gene expression. This should be reflected in the adjustment of PER and TIM dynamics. We manipulated LD cycles to assess the effects of altered day length on PER and TIM dynamics in clock cells within the central brain as well as light-induced resetting of locomotor rhythms.

RNA-Interference Knockdown of Drosophila Pigment Dispersing Factor in Neuronal Subsets: The Anatomical Basis of a Neuropeptide's Circadian Functions

Background In animals, neuropeptide signaling is an important component of circadian timekeeping. The neuropeptide pigment dispersing factor (PDF) is required for several aspects of circadian activity rhythms in Drosophila. Methodology/Principal Findings Here we investigate the anatomical basis for PDFs various circadian functions by targeted PDF RNA-interference in specific classes of Drosophila neuron. We demonstrate that PDF is required in the ventro-lateral neurons (vLNs) of the central brain and not in the abdominal ganglion for normal activity rhythms. Differential knockdown of PDF in the large or small vLNs indicates that PDF from the small vLNs is likely responsible for the maintenance of free-running activity rhythms and that PDF is not required in the large vLNs for normal behavior. PDFs role in setting the period of free-running activity rhythms and the proper timing of evening activity under light:dark cycles emanates from both subtypes of vLN, since PDF in either class of vLN was sufficient for these aspects of behavior. Conclusions/Significance These results reveal the neuroanatomical basis PDFs various circadian functions and refine our understanding of the clock neuron circuitry of Drosophila.

Analysis of functional neuronal connectivity in the Drosophila brain

Drosophila melanogaster is a valuable model system for the neural basis of complex behavior, but an inability to routinely interrogate physiologic connections within central neural networks of the fly brain remains a fundamental barrier to progress in the field. To address this problem, we have introduced a simple method of measuring functional connectivity based on the independent expression of the mammalian P2X2 purinoreceptor and genetically encoded Ca(2+) and cAMP sensors within separate genetically defined subsets of neurons in the adult brain. We show that such independent expression is capable of specifically rendering defined sets of neurons excitable by pulses of bath-applied ATP in a manner compatible with high-resolution Ca(2+) and cAMP imaging in putative follower neurons. Furthermore, we establish that this approach is sufficiently sensitive for the detection of excitatory and modulatory connections deep within larval and adult brains. This technically facile approach can now be used in wild-type and mutant genetic backgrounds to address functional connectivity within neuronal networks governing a wide range of complex behaviors in the fly. Furthermore, the effectiveness of this approach in the fly brain suggests that similar methods using appropriate heterologous receptors might be adopted for other widely used model systems.