Disruption of synaptic transmission or clock-gene-product oscillations in circadian pacemaker cells of Drosophila cause abnormal behavioral rhythms.

To study the function of clock-gene-expressing neurons, the tetanus-toxin light chain (TeTxLC), which blocks chemical synaptic transmission, was expressed under the control of promoters of the clock genes period (per) and timeless (tim), each fused to GAL4-encoding sequences. Although TeTxLC did not affect cycling of a clock-gene product at the gross level, it disrupted the rhythmic behavior of adult Drosophila. In constant darkness, the proportion of rhythmic flies was reduced in flies expressing active TeTxLC compared to controls, including those expressing inactive toxin. The behavior of TeTxLC-expressing flies was less synchronized to light:dark cycles than that of controls. To determine which neurons are responsible for these effects on behavior, the toxin was also expressed in restricted subsets of per/tim-expressing, laterally located pacemaker neurons by expressing TeTxLC under the control of a driver in which GAL4-encoding sequences are fused to the promoter of the pigment dispersing factor (pdf) gene. pdf-gal4-driven TeTxLC expression had relatively little effect on behavioral rhythms, implying that per/tim neurons other than pdf-expressing lateral neurons participate in the generation of rhythmic behavior. In another set of experiments, period gene products were expressed under the control of per-gal4 or tim-gal4. This resulted in an increased level of PER protein in many brain cells and reduction of bioluminescence cycling reported by a per-luciferase transgene, especially in the case of per expression affected by tim-gal4. This indicates a disruption of the transcriptional feedback loop that is a part of the oscillatory mechanism underlying Drosophila's circadian rhythms. Consistent with this molecular defect, the proportion of rhythmic individuals in constant darkness was subnormal in flies expressing PER under the control of tim-gal4, and their behavior in light:dark cycles was abnormal.

Kaneko M ，Park JH ，Cheng Y ，Hardin PE ，Hall JC ... -  《journal of neurobiology》

Defining the role of Drosophila lateral neurons in the control of circadian rhythms in motor activity and eclosion by targeted genetic ablation and PERIOD protein overexpression.

Blanchardon E ，Grima B ，Klarsfeld A ，Chélot E ，Hardin PE ，Préat T ，Rouyer F ... -  《EUROPEAN JOURNAL OF NEUROSCIENCE》

Neuroanatomy of cells expressing clock genes in Drosophila: transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections.

Subsets of brain neurons expressing the clock genes period (per) and timeless (tim) are involved in the generation of circadian behavioral rhythms. However, current knowledge of projection patterns of these neurons is limited to those immunoreactive to an antibody against a crustacean neuropeptide. The GAL4-expression system was utilized to visualize neuronal processes from all per and tim-expressing neurons in the central nervous system. Each of two types of GAL4-driver fusion genes, per-gal4 or tim-gal4, was combined in transgenic flies with marker genes-lacZ, and sequences encoding green fluorescent protein or TAU protein-under the control of the GAL4-responsive element UAS. This allowed visualization of the cytoplasm of GAL4-expressing cells. Thus, neurites of clock neurons in the adult brain as well as those of larvae and pupae were revealed. Among the anatomical patterns revealed by per-gal4- or tim-gal4-driven marker expression were a previously unknown, dorsally located neuronal cluster, along with the projections of these cells and of other dorsal neurons characterized in earlier studies only by the location of their perikarya. The similarity of projections from PER- or TIM-containing neurons during development to those in the adult implies that these features of mature clock neurons are established by the larval stages. Neurons that have never been identified as PER- or TIM-immunoreactive were also visualized in this assay system, indicating promoter activity of the clock genes in these cells and suggesting that their products cannot accumulate to detectable levels in certain neurons.

Kaneko M ，Hall JC 《JOURNAL OF COMPARATIVE NEUROLOGY》

The Drosophila Receptor Protein Tyrosine Phosphatase LAR Is Required for Development of Circadian Pacemaker Neuron Processes That Support Rhythmic Activity in Constant Darkness But Not during Light/Dark Cycles.

InDrosophila, a transcriptional feedback loop that is activated by CLOCK-CYCLE (CLK-CYC) complexes and repressed by PERIOD-TIMELESS (PER-TIM) complexes keeps circadian time. The timing of CLK-CYC activation and PER-TIM repression is regulated post-translationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Although kinases that control PER, TIM, and CLK levels, activity, and/or subcellular localization have been identified, less is known about phosphatases that control clock protein dephosphorylation. To identify clock-relevant phosphatases, clock-cell-specific RNAi knockdowns ofDrosophilaphosphatases were screened for altered activity rhythms. One phosphatase that was identified, the receptor protein tyrosine phosphatase leukocyte-antigen-related (LAR), abolished activity rhythms in constant darkness (DD) without disrupting the timekeeping mechanism in brain pacemaker neurons. However, expression of the neuropeptide pigment-dispersing factor (PDF), which mediates pacemaker neuron synchrony and output, is eliminated in the dorsal projections from small ventral lateral (sLNv) pacemaker neurons whenLarexpression is knocked down during development, but not in adults. Loss ofLarfunction eliminates sLNvdorsal projections, but PDF expression persists in sLNvand large ventral lateral neuron cell bodies and their remaining projections. In contrast to the defects in lights-on and lights-off anticipatory activity seen in flies that lack PDF,LarRNAi knockdown flies anticipate the lights-on and lights-off transition normally. Our results demonstrate thatLaris required for sLNvdorsal projection development and suggest that PDF expression in LNvcell bodies and their remaining projections mediate anticipation of the lights-on and lights-off transitions during a light/dark cycle. In animals, circadian clocks drive daily rhythms in physiology, metabolism, and behavior via transcriptional feedback loops. Because key circadian transcriptional activators and repressors are regulated by phosphorylation, we screened for phosphatases that alter activity rhythms when their expression was reduced. One such phosphatase, leukocyte-antigen-related (LAR), abolishes activity rhythms, but does not disrupt feedback loop function. Rather,Lardisrupts clock output by eliminating axonal processes from clock neurons that release pigment-dispersing factor (PDF) neuropeptide into the dorsal brain, but PDF expression persists in their cell bodies and remaining projections. In contrast to flies that lack PDF, flies that lackLaranticipate lights-on and lights-off transitions normally, which suggests that the remaining PDF expression mediates activity during light/dark cycles.

Agrawal P ，Hardin PE 《-》

Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster.

Circadian locomotor rhythms of Drosophila melanogaster are controlled by a neuronal circuit composed of approximately 150 clock neurons that are roughly classified into seven groups. In the circuit, a group of neurons expressing pigment-dispersing factor (PDF) play an important role in organizing the pacemaking system. Recent studies imply that unknown chemical neurotransmitter(s) (UNT) other than PDF is also expressed in the PDF-positive neurons. To explore its role in the circadian pacemaker, we examined the circadian locomotor rhythms of pdf-Gal4/UAS-TNT transgenic flies in which chemical synaptic transmission in PDF-positive neurons was blocked by expressed tetanus toxin light chain (TNT). In constant darkness (DD), the flies showed a free-running rhythm, which was similar to that of wild-type flies but significantly different from pdf null mutants. Under constant light conditions (LL), however, they often showed complex rhythms with a short period and a long period component. The UNT is thus likely involved in the synaptic transmission in the clock network and its release caused by LL leads to arrhythmicity. Immunocytochemistry revealed that LL induced phase separation in TIMELESS (TIM) cycling among some of the PDF-positive and PDF-negative clock neurons in the transgenic flies. These results suggest that both PDF and UNT play important roles in the Drosophila circadian clock, and activation of PDF pathway alone by LL leads to the complex locomotor rhythm through desynchronized oscillation among some of the clock neurons.

Umezaki Y ，Yasuyama K ，Nakagoshi H ，Tomioka K ... -  《-》