Circadian-and light-dependent regulation of resting membrane potential and spontaneous action potential firing of Drosophila circadian pacemaker neurons (original) (raw)
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A Self-Sustaining, Light-Entrainable Circadian Oscillator in the Drosophila Brain
Current Biology, 2003
negative feedback loop (reviewed in [1]). In flies the most prominent biological rhythm con-Institut fü r Zoologie Lehrstuhl fü r Entwicklungsbiologie trolled by the circadian clock is the rest-activity cycle. Under free-running conditions of constant darkness and 93040 Regensburg Germany constant temperature (DD), this rhythm persists for at least five weeks (e.g., [2]). In stark contrast to these 2 Department of Biology Brandeis University robust behavioral rhythms, studies of clock-gene expression under such free-running conditions revealed a Waltham, Massachusetts 02454 rapid dampening of molecular oscillations within 2-4 days [1]. It is therefore not proven if cycling gene products are required for generating behavioral rhythmicity. Summary Several arguments have been put forward in order to explain the observed discrepancies. (1) Molecular rhythms Background: The circadian clock of Drosophila is able are usually measured after extraction of mRNA or protein to drive behavioral rhythms for many weeks in continufrom many individuals (typically 30-50) at a given time of ous darkness (DD). The endogenous rhythm generator day. Since the internal free-running periods vary slightly is thought to be generated by interlocked molecular between the different animals, this will lead to an overall feedback loops involving circadian transcriptional and amplitude dampening the longer the flies are kept in posttranscriptional regulation of several clock genes, DD (e.g., [3, 4]). (2) Many tissues within one fly contain including period. However, all attempts to demonstrate circadian clocks (e.g., [5, 6]). Without entrainment cues sustained rhythms of clock gene expression in DD have they could internally desynchronize, resulting in damped failed, making it difficult to link the molecular clock modmolecular rhythms when all such tissues are monitored els with the circadian behavioral rhythms. Here we resimultaneously; the same could also apply for the clock stricted expression of a novel period-luciferase transcells within a given tissue. (3) There is a qualitative differgene to certain clock neurons in the Drosophila brain, ence between "pacemaker oscillators" (e.g., those drivpermitting us to monitor reporter gene activity in these ing robust behavioral rhythms) and "peripheral oscillacells in real-time. tors" (e.g., the fly's eyes, in which clock gene expression Results: We show that only a subset of the previously has been analyzed for the majority of chronomolecular described pacemaker neurons is able to sustain PERIOD studies); only bona-fide pacemakers are able to maintain protein oscillations after 5 days in constant darkness. molecular oscillations in DD. Establishment of luciferase In addition, we identified a sustained and autonomous as a real-time reporter gene helped to rule out the first molecular oscillator in a group of clock neurons in the possibility; recordings from individual transgenic perdorsal brain with heretofore unknown function. We luc flies also showed rapid dampening in DD [7]. Tofound that these "dorsal neurons" (DNs) can synchrogether with the finding that isolated Drosophila body nize behavioral rhythms and that light input into these parts and organs contain circadian clocks (e.g., [5, 6, 8, cells involves the blue-light photoreceptor cryptochrome. 9]), this made the second possibility seem likely. But the Conclusions: Our results suggest that the DNs play a fact that transcriptional rhythms in individually cultured prominent role in controlling locomotor behavior when body parts and organs also rapidly dampen in DD favors flies are exposed to natural light-dark cycles. Analysis the third argument [5, 6, 9]. Therefore, true circadian of similar "stable mosaic" transgenes should help to molecular oscillations could be a unique feature of a set reveal the function of the other clock neuronal clusters of brain neurons known to control behavioral rhythmicity within the fly brain. (e.g., [10, 11]). Little is known about the features of molecular oscilla
Functional analysis of circadian pacemaker neurons in Drosophila melanogaster
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2006
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,...
PLoS Biology, 2007
Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest-activity rhythms and relies upon different groups of PERIOD (PER)-expressing neurons. Two distinct oscillators have been functionally characterized under lightdark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day-night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2006
Coupling of autonomous cellular oscillators is an essential aspect of circadian clock function but little is known about its circuit requirements. Functional ablation of the pigment-dispersing factor-expressing lateral ventral subset (LNV) of Drosophila clock neurons abolishes circadian rhythms of locomotor activity. The hypothesis that LNVs synchronize oscillations in downstream clock neurons was tested by rendering the LNVs hyperexcitable via transgenic expression of a low activation threshold voltage-gated sodium channel. When the LNVs are made hyperexcitable, free-running behavioral rhythms decompose into multiple independent superimposed oscillations and the clock protein oscillations in the dorsal neuron 1 and 2 subgroups of clock neurons are phase-shifted. Thus, regulated electrical activity of the LNVs synchronize multiple oscillators in the fly circadian pacemaker circuit.
Neuron, 2012
Circadian rhythms offer an excellent opportunity to dissect the neural circuits underlying innate behavior because the genes and neurons involved are relatively well understood. We first sought to understand how Drosophila clock neurons interact in the simple circuit that generates circadian rhythms in larval light avoidance. We used genetics to manipulate two groups of clock neurons, increasing or reducing excitability, stopping their molecular clocks, and blocking neurotransmitter release and reception. Our results revealed that lateral neurons (LN v s) promote and dorsal clock neurons (DN 1 s) inhibit light avoidance, these neurons probably signal at different times of day, and both signals are required for rhythmic behavior. We found that similar principles apply in the more complex adult circadian circuit that generates locomotor rhythms. Thus, the changing balance in activity between clock neurons with opposing behavioral effects generates robust circadian behavior and probably helps organisms transition between discrete behavioral states, such as sleep and wakefulness.
Reevaluation ofDrosophila melanogaster's neuronal circadian pacemakers reveals new neuronal classes
The Journal of Comparative Neurology, 2006
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 fly's 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.
Drosophila Clock Neurons under Natural Conditions
Journal of Biological Rhythms, 2013
The circadian clock modulates the adaptive daily patterns of physiology and behavior and adjusts these rhythms to seasonal changes. Recent studies of seasonal locomotor activity patterns of wild-type and clock mutant fruit flies in quasi-natural conditions have revealed that these behavioral patterns differ considerably from those observed under standard laboratory conditions. To unravel the molecular features accompanying seasonal adaptation of the clock, we investigated Drosophila's neuronal expression of the canonical clock proteins PERIOD (PER) and TIMELESS (TIM) in nature. We find that the profile of PER dramatically changes in different seasons, whereas that of TIM remains more constant. Unexpectedly, we find that PER and TIM oscillations are decoupled in summer conditions. Moreover, irrespective of season, PER and TIM always peak earlier in the dorsal neurons than in the lateral neurons, suggesting a more rapid molecular oscillation in these cells. We successfully reproduced most of our results under simulated natural conditions in the laboratory and show that although photoperiod is the most important zeitgeber for the molecular clock, the flies' activity pattern is more strongly affected by temperature. Our results are among the first to systematically compare laboratory and natural studies of Drosophila rhythms.
PLoS ONE, 2011
To synchronize their molecular rhythms, circadian pacemaker neurons must input both external and internal timing cues and, therefore, signal integration between sensory information and internal clock status is fundamental to normal circadian physiology. We demonstrate the specific convergence of clock-derived neuropeptide signaling with that of a deep brain photoreceptor. We report that the neuropeptide PDF receptor and the circadian photoreceptor CRYPTOCROME (CRY) are precisely co-expressed in a subset of pacemakers, and that these pathways together provide a requisite drive for circadian control of daily locomotor rhythms. These convergent signaling pathways influence the phase of rhythm generation, but also its amplitude. In the absence of both pathways, PER rhythms were greatly reduced in only those specific pacemakers that receive convergent inputs and PER levels remained high in the nucleus throughout the day. This suggested a large-scale dis-regulation of the pacemaking machinery. Behavioral rhythms were likewise disrupted: in light:dark conditions they were aberrant, and under constant dark conditions, they were lost. We speculate that the convergence of environmental and clock-derived signals may produce a coincident detection of light, synergistic responses to it, and thus more accurate and more efficient re-setting properties.
Light-mediated circuit switching in the Drosophila neuronal clock network
2019
SummaryThe circadian clock is a timekeeper but also helps adapt physiology to the outside world. This is because an essential feature of clocks is their ability to adjust (entrain) to the environment, with light being the most important signal. Whereas Cryptochrome-mediated entrainment is well understood in Drosophila, integration of light information via the visual system lacks a neuronal or molecular mechanism. Here we show that a single photoreceptor sub-type is essential for long day adaptation. These cells activate key circadian neurons, namely the lLNvs, which release the neuropeptide PDF. Using a cell-specific CRISPR/Cas9 assay, we show that PDF directly interacts with neurons important for evening (E) activity timing. Interestingly, this pathway is specific for light entrainment and appears to be dispensable in constant darkness (DD). The results therefore indicate that external cues cause a rearrangement of neuronal hierarchy, which is a novel form of plasticity.