A Conserved Bicycle Model for Circadian Clock Control of Membrane Excitability

SummaryCircadian 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 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 Narrow Abdomen Ion Channel Complex Is Highly Stable and Persists from Development into Adult Stages to Promote Behavioral Rhythmicity

The sodium leak channel NARROW ABDOMEN (NA)/ NALCN is an important component of circadian pacemaker neuronal output. In Drosophila, rhythmic expression of the NA channel regulator Nlf-1 in a subset of adult pacemaker neurons has been proposed to contribute to circadian regulation of channel localization or activity. Here we have restricted expression of Drosophila NA channel subunits or the Nlf-1 regulator to either development or adulthood using the temperature-inducible tubulin-GAL80ts system. Surprisingly, we find that developmental expression of endogenous channel subunits and Nlf-1 is sufficient to promote robust rhythmic behavior in adults. Moreover, we find that channel complex proteins produced during development persist in the Drosophila head with little decay for at least 5–7 days in adults. In contrast, restricting either endogenous or transgenic gene expression to adult stages produces only limited amounts of the functional channel complex. These data indicate that much of the NA channel complex that functions in adult circadian neurons is normally produced during development, and that the channel complex is very stable in most neurons in the Drosophila brain. Based on these findings, we propose that circadian regulation of NA channel function in adult pacemaker neurons is mediated primarily by post-translational mechanisms that are independent of Nlf-1

UNC79 and UNC80, Putative Auxiliary Subunits of the NARROW ABDOMEN Ion Channel, Are Indispensable for Robust Circadian Locomotor Rhythms in <i>Drosophila</i>

<div><p>In the fruit fly <i>Drosophila melanogaster,</i> 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 <i>Drosophila</i>, <i>unc79 (aka dunc79)</i> and <i>unc80 (aka CG18437).</i> We have generated novel <i>unc79</i> and <i>unc80</i> mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from <i>na</i> 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 <i>na, unc79,</i> or <i>unc80</i> 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 <i>unc79</i> and <i>unc80</i> 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 <i>Drosophila</i> brain. Taken together, these data suggest that <i>Drosophila</i> NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.</p></div

The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms

Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. Here we reveal a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, we identified mutants of Transport and Golgi organization 10 (Tango10) with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless. Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. We propose that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms

Transgenic rescue of <i>unc79</i> and <i>unc80</i> rhythmicity phenotypes.

1<p>Few flies survived to the end of DD; refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078147#pone-0078147-g006" target="_blank">Figures 6B,E</a> for LD phenotype.</p

RNAi knockdown of <i>na, unc79,</i> or <i>unc80</i> in circadian pacemaker neurons decreases DD rhythmicity.

<p>RNAi knockdown of <i>na, unc79,</i> or <i>unc80</i> in circadian pacemaker neurons decreases DD rhythmicity.</p

Transgenic <i>unc79, unc80</i> or <i>na</i> expression produces increased protein levels in wild-type and mutant backgrounds.

<p>(A-C) Western blot analyses were used to label UNC79, UNC80, and NA proteins from adult head extracts; representative blots are shown. For all blots shown, lane 1  =  <i>elav</i>GAL4/+ (no UAS); lanes 3,5,7  =  <i>UAS/+</i> (no GAL4); lanes 2,4,6,8  =  <i>elavGAL4; UAS/+.</i> (A) Pan-neuronal expression of <i>UAS-unc79MYC 23-24</i> in wild-type (lane 2), <i>na^har</i> (lane 4), <i>unc79^x25</i> (lane 6), or <i>unc80^x42</i> (lane 8) backgrounds. (B) Expression of <i>UAS-HAunc80 1M</i> in wild-type (lane 2), <i>na^har</i> (lane 4), <i>unc79^x25</i> (lane 6), or <i>unc80^x42</i> (lane 8) backgrounds. (C) Expression of <i>UAS-na U3</i> in wild-type (lane 2), <i>na^har</i> (lane 4), <i>unc79^x25</i> (lane 6), or <i>unc80^x42</i> (lane 8) backgrounds.</p