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This study characterizes a subset of clock neurons known as large lateral-ventral neurons and their dopaminergic/octopaminergic input circuitry in balancing light-mediated wakefulness in Drosophila. How animals maintain proper amounts of sleep yet remain flexible to changes in environmental conditions remains unknown. We found that environmental light suppressed the wake-promoting effects of dopamine in fly brains. The ten large lateral-ventral neurons (l-LNvs), a subset of clock neurons, are wake-promoting and respond to dopamine, octopamine and light. Behavioral and imaging analyses suggested that dopamine is a stronger arousal signal than octopamine. Notably, light exposure not only suppressed l-LNv responses, but also synchronized responses of neighboring l-LNvs. This regulation occurred by distinct mechanisms: light-mediated suppression of octopamine responses was regulated by the circadian clock, whereas light regulation of dopamine responses occurred by upregulation of inhibitory dopamine receptors. Plasticity therefore alters the relative importance of diverse cues on the basis of the environmental mix of stimuli. The regulatory mechanisms described here may contribute to the control of sleep stability while still allowing behavioral flexibility.

To compare circadian gene expression within highly discrete neuronal populations, we separately purified and characterized two adjacent but distinct groups of Drosophila adult circadian neurons: the 8 small and 10 large PDF-expressing ventral lateral neurons (s-LNvs and l-LNvs, respectively). The s-LNvs are the principal circadian pacemaker cells, whereas recent evidence indicates that the l-LNvs are involved in sleep and light-mediated arousal. Although half of the l-LNv–enriched mRNA population, including core clock mRNAs, is shared between the l-LNvs and s-LNvs, the other half is l-LNv– and s-LNv–specific. The distribution of four specific mRNAs is consistent with prior characterization of the four encoded proteins, and therefore indicates successful purification of the two neuronal types. Moreover, an octopamine receptor mRNA is selectively enriched in l-LNvs, and only these neurons respond to in vitro application of octopamine. Dissection and purification of l-LNvs from flies collected at different times indicate that these neurons contain cycling clock mRNAs with higher circadian amplitudes as well as at least a 10-fold higher fraction of oscillating mRNAs than all previous analyses of head RNA. Many of these cycling l-LNv mRNAs are well expressed but do not cycle or cycle much less well elsewhere in heads. The results suggest that RNA cycling is much more prominent in circadian neurons than elsewhere in heads and may be particularly important for the functioning of these neurons.

SummaryCircadian systems are entrained and phase shifted by light. In Drosophila, the model of light-mediated phase shifting begins with photon capture by CRYPTOCHROME (CRY) followed by rapid TIMELESS (TIM) degradation. In this study, we focused on phase delays and assayed TIM degradation within individual brain clock neurons in response to light pulses in the early night. Surprisingly, there was no detectable change in TIM staining intensity within the eight pacemaker s-LNvs. This indicates that TIM degradation within s-LNvs is not necessary for phase delays, and similar assays in other genotypes indicate that it is also not sufficient. In contrast, more dorsal circadian neurons appear light-sensitive in the early night. Because CRY is still necessary within the s-LNvs for phase shifting, the results challenge the canonical cell-autonomous molecular model and raise the question of how the pacemaker neuron transcription-translation clock is reset by light in the early night.

The neural circuits that regulate sleep and arousal as well as their integration with circadian circuits remain unclear, especially in Drosophila . This issue intersects with that of photoreception, because light is both an arousal signal in diurnal animals and an entraining signal for the circadian clock. To identify neurons and circuits relevant to light-mediated arousal as well as circadian phase-shifting, we developed genetic techniques that link behavior to single cell-type resolution within the Drosophila central brain. We focused on the unknown function of the 10 PDF-containing large ventral lateral neurons (l-LNvs) of the Drosophila circadian brain network and show here that these cells function in light-dependent arousal. They also are important for phase shifting in the late-night (dawn), indicating that the circadian photoresponse is a network property and therefore non-cell-autonomous. The data further indicate that the circuits underlying light-induced arousal and circadian photoentrainment intersect at the l-LNvs and then segregate.

Summary Conflicting views exist of how circuits of the antennal lobe, the insect equivalent of the olfactory bulb, translate input from olfactory receptor neurons (ORNs) into projection-neuron (PN) output. Synaptic connections between ORNs and PNs are one-to-one, yet PNs are more broadly tuned to odors than ORNs. The basis for this difference in receptive range remains unknown. Analyzing a Drosophila mutant lacking ORN input to one glomerulus, we show that some of the apparent complexity in the antennal lobe's output arises from lateral, interglomerular excitation of PNs. We describe a previously unidentified population of cholinergic local neurons (LNs) with multiglomerular processes. These excitatory LNs respond broadly to odors but exhibit little glomerular specificity in their synaptic output, suggesting that PNs are driven by a combination of glomerulus-specific ORN afferents and diffuse LN excitation. Lateral excitation may boost PN signals and enhance their transmission to third-order neurons in a mechanism akin to stochastic resonance.

We have used in vitro mutagenesis and gene replacement to study the function of the nucleotide-binding domain (NBD) of γ-tubulin in Tetrahymena thermophila. In this study, we show that the NBD has an essential function and that point mutations in two conserved residues lead to over-production and mislocalization of basal body (BB) assembly. These results, coupled with previous studies (Dammermann, A., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai, and K. Oegema. 2004. Dev. Cell. 7:815–829; La Terra, S., C.N. English, P. Hergert, B.F. McEwen, G. Sluder, and A. Khodjakov. 2005. J. Cell Biol. 168:713–722), suggest that to achieve the precise temporal and spatial regulation of BB/centriole assembly, the initiation activity of γ-tubulin is normally suppressed by a negative regulatory mechanism that acts through its NBD.

The Cd 2+ -inducible metallothionein ( MTT1 ) gene was cloned from Tetrahymena thermophila . Northern blot analysis showed that MTT1 mRNA is not detectable in the absence of Cd 2+ , is induced within 10 min of its addition, is expressed in proportion to its concentration, and rapidly disappears upon its withdrawal. Similarly, when the neo1 gene coding region flanked by the MTT1 gene noncoding sequences was used to disrupt the MTT1 locus, no transformants were observed in the absence of Cd 2+ , and the number of transformants was proportional to increased Cd 2+ concentration. The neo3 cassette, in which the MTT1 promoter replaced the histone gene HHF1 promoter of the previously used neo2 cassette , transformed cells at much higher frequencies than neo2 and produced germ-line knockouts where neo2 had failed. Rescuing the progeny of a mating of γ-tubulin gene, GTU1, knockout heterokaryons with a GTU1 gene inserted into the MTT1 locus yielded >75 times more transformants than rescuing with the wild-type GTU1 gene itself. When cells rescued with the MTT1-GTU1 chimeric gene were transferred to medium lacking Cd 2+ , they stopped growing and had phenotypic changes indistinguishable from cells containing only disrupted GTU1 genes. Thus, it is now possible to create conditional lethal mutants and study the terminal phenotypes of null mutations for essential genes by replacing the endogenous gene with one under the control of the MTT1 promoter. The MTT1 promoter also resulted in ≈30 times more overexpression of the IAG48[G1] surface antigen gene of the ciliate fish parasite Ichthyophthirius multifiliis than the highly expressed BTU1 promoter, accounting for ≈1% of the total cell protein. Thus, the MTT1 promoter should enable routine over-expression of endogenous and foreign genes in Tetrahymena .

The gene (GTU1) encoding Tetrahymena thermophila gamma-tubulin was cloned and analyzed. GTU1 is a single-copy, essential gene encoding a conventional gamma-tubulin. HA-tagged GTU1p localizes to four microtubule-organizing centers (MTOCs) in vegetative cells: basal bodies (BBs), macronuclear envelopes, micronuclear envelopes, and contractile vacuole pores. gamma-Tubulin function was studied by placing the GTU1 gene under control of an inducible-repressible promoter. Overexpression of GTU1 had no detectable effect on cell growth or morphology. Depletion of gamma-tubulin resulted in marked changes in cell morphology and in MT bundling. MTOCs showed different sensitivities to gamma-tubulin depletion, with BBs being the most sensitive. gamma-Tubulin was required not only for the formation of new BBs but also for maintenance of mature BBs. BBs disappeared in stages, first losing gamma-tubulin and then centrin and glutamylated tubulin. When GTU1 expression was reinduced in depleted cells, BBs reformed rapidly, and the normal, highly organized structure of the Tetrahymena cell cortex was reestablished, indicating that the precise patterning of the cortex can be formed de novo.

The Ganzhou area of Jiangxi Province, southern China is becoming one of the most productive oviraptorosaurian localities in the world. A new oviraptorid dinosaur was unearthed from the uppermost Upper Cretaceous Nanxiong Formation of Ganzhou area. It is characterized by an anterodorsally sloping occiput and quadrate (a feature shared with Citipati), a circular supratemporal fenestra that is much smaller than the lower temporal fenestra and a dentary in which the dorsal margin above the external mandibular fenestra is strongly concave ventrally. The position of the anteroventral corner of the external naris in relation to the posterodorsal corner of the antorbital fenestra provides new insight into the craniofacial evolution of oviraptorosaurid dinosaurs. A phylogenetic analysis recovers the new taxon as closely related to the Mongolian Citipati. Six oviraptorid dinosaurs from the Nanxiong Formation (Ganzhou and Nanxiong) are distributed within three clades of the family. Each of the three clades from the Nanxiong Formation has close relatives in Inner Mongolia and Mongolia and in both places each clade may have had a specific diet or occupied a different ecological niche. Oviraptorid dinosaurs were geographically widespread across Asia in the latest Cretaceous and were an important component of terrestrial ecosystems during this time.

Insulin resistance contributes to a number of metabolic disorders, including type II diabetes, hypertension, and atherosclerosis. Cytokines, such as tumor necrosis factor-α, interleukin-1β, and interleukin-6, and hormones, such as growth hormone, are known to cause insulin resistance, but the mechanisms by which they inhibit the cellular response to insulin have not been elucidated. One mechanism by which these agents could cause insulin resistance is by inducing the expression of cellular proteins that inhibit insulin receptor (IR) signaling. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling pathways, the expression of which is regulated by certain cytokines. SOCS proteins are therefore attractive candidates as mediators of cytokine-induced insulin resistance. We have found that SOCS-1 and SOCS-6 interact with the IR when expressed in human hepatoma cells (HepG2) or in rat hepatoma cells overexpressing the human IR. In SOCS-1-expressing cells, insulin treatment increases the extent of interaction with the IR, whereas in SOCS-6-expressing cells the association with the IR appears to require insulin treatment. SOCS-1 and SOCS-6 do not inhibit insulin-dependent IR autophosphorylation, but both proteins inhibit insulin-dependent activation of ERK1/2 and protein kinase Bin vivo and IR-directed phosphorylation of IRS-1 in vitro. These results suggest that SOCS proteins may be inhibitors of IR signaling and could mediate cytokine-induced insulin resistance and contribute to the pathogenesis of type II diabetes.

SummaryTo advance the understanding of sleep regulation, we screened for sleep-promoting cells and identified neurons expressing neuropeptide Y-like short neuropeptide F (sNPF). Sleep induction by sNPF meets all relevant criteria. Rebound sleep following sleep deprivation is reduced by activation of sNPF neurons, and flies experience negative sleep rebound upon cessation of sNPF neuronal stimulation, indicating that sNPF provides an important signal to the sleep homeostat. Only a subset of sNPF-expressing neurons, which includes the small ventrolateral clock neurons, is sleep promoting. Their release of sNPF increases sleep consolidation in part by suppressing the activity of wake-promoting large ventrolateral clock neurons, and suppression of neuronal firing may be the general response to sNPF receptor activation. sNPF acutely increases sleep without altering feeding behavior, which it affects only on a much longer time scale. The profound effect of sNPF on sleep indicates that it is an important sleep-promoting molecule.

The daily sleep cycle in humans and other mammals is driven by a complex circuit within which GABAergic sleep-promoting neurons oppose arousal systems. The latter includes the circadian system, aminergic/cholinergic systems as well as neurons secreting the peptide orexin/hypocretin, which contribute to sharp behavioral transitions (Lu and Greco, 2006). Drosophila sleep has recently been shown also to be controlled by GABAergic inputs, which act on unknown cells expressing the Rdl GABAA receptor (Agosto et al., 2008). We identify here the relevant Rdl-containing cells as a subset of the well-studied Drosophila circadian clock neurons, the PDF-expressing small and large ventral lateral neurons (LNvs). LNv activity regulates the total amount of sleep as well as the rate of sleep onset, and both large and small LNvs are part of the sleep circuit. Flies mutant for either the pdf gene or its receptor are hypersomnolent, and PDF acts on the LNvs themselves to control sleep. These features of the Drosophila sleep circuit, GABAergic control of sleep onset and maintenance as well as peptidergic control of arousal, support the idea that features of sleep circuit architecture as well as the mechanisms governing the behavioral transitions between sleep and wake are conserved between mammals and insects.