Abstract
Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LNvs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LNv-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LNv-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf01 mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf01 mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LNvs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LNvs alone are insufficient to support the circadian program. Based on the damping results with Pdf01 mutant flies, we propose that LNvs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.
Highlights
Circadian rhythms of diverse organisms are based on similar intracellular molecular feedback loops (Dunlap 1999; Allada et al 2001; Panda et al 2002)
ventral lateral neuron (LNv) Cannot Support Circadian Behavior Independently To test whether the LNvs can support free-running circadian locomotor activity rhythms independently of other functional clock cells, we restricted pacemaker activity to these few pigment-dispersing factor (PDF)-expressing cells
We considered that robust molecular cycling under extended constant darkness conditions might be a good criterion for identifying these cell groups, because prior biochemical studies showed that some head and brain locations undergo damping of molecular oscillations under free-running conditions (Hardin 1994; Stanewsky et al 1997)
Summary
Circadian rhythms of diverse organisms are based on similar intracellular molecular feedback loops (Dunlap 1999; Allada et al 2001; Panda et al 2002). Damping Transcriptional Rhythms micity in DD (Renn et al 1999); and (4) the s-LNvs maintain robust molecular oscillations for at least for 2 days in DD (Yang and Sehgal 2001; Shafer et al 2002), in contrast to at least some other brain neurons and nonneuronal tissues This final property suggests that these cells might fulfill the selfsustaining criterion for the ‘‘core pacemaker.’’ the sLNvs have been proposed to the primary pacemaker cells that generate locomotor activity rhythms (Helfrich-Forster 1997; Renn et al 1999; Emery et al 2000). Our pursuit of the self-sustaining ‘‘core pacemaker’’ of the Drosophila circadian system began with a test of the s-LNv cell-autonomous clock hypothesis
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