Dirk Rieger

Development and morphology of the clock-gene-expressing lateral neurons of Drosophila melanogaster.

The clock‐gene‐expressing lateral neurons are essential for the locomotor activity rhythm of Drosophila melanogaster. Traditionally, these neurons are divided into three groups: the dorsal lateral neurons (LNd), the large ventral lateral neurons (l‐LNv), and the small ventral lateral neurons (s‐LNv), whereby the latter group consists of four neurons that express the neuropeptide pigment‐dispersing factor (PDF) and a fifth PDF‐negative neuron. So far, only the l‐LNv and the PDF‐positive s‐LNv have been shown to project into the accessory medulla, a small neuropil that contains the circadian pacemaker center in several insects. We show here that the other lateral neurons also arborize in the accessory medulla, predominantly forming postsynaptic sites. Both the l‐LNv and LNd are anatomically well suited to connect the accessory medullae. Whereas the l‐LNv may receive ipsilateral photic input from the Hofbauer‐Buchner eyelet, the LNd invade mainly the contralateral accessory medulla and thus may receive photic input from the contralateral side. Both the LNd and the l‐LNv differentiate during midmetamorphosis. They do so in close proximity to one another and the fifth PDF‐negative s‐LNv, suggesting that these cell groups may derive from common precursors. J. Comp. Neurol. 500:47–70, 2007.

Functional analysis of circadian pacemaker neurons in Drosophila melanogaster

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, a short-period component originated not exclusively from the morning peak but more prominently from the evening peak. This reveals an interesting deviation from the original Pittendrigh and Daan (1976) model and suggests that a subgroup of the ventral subset of the small LN acts as “main” oscillator controlling M and E activity bouts in Drosophila.

Moonlight shifts the endogenous clock of Drosophila melanogaster

The ability to be synchronized by light–dark cycles is a fundamental property of circadian clocks. Although there are indications that circadian clocks are extremely light-sensitive and that they can be set by the low irradiances that occur at dawn and dusk, this has not been shown on the cellular level. Here, we demonstrate that a subset of Drosophilas pacemaker neurons responds to nocturnal dim light. At a nighttime illumination comparable to quarter-moonlight intensity, the flies increase activity levels and shift their typical morning and evening activity peaks into the night. In parallel, clock protein levels are reduced, and clock protein rhythms shift in opposed direction in subsets of the previously identified morning and evening pacemaker cells. No effect was observed on the peripheral clock in the eye. Our results demonstrate that the neurons driving rhythmic behavior are extremely light-sensitive and capable of shifting activity in response to the very low light intensities that regularly occur in nature. This sensitivity may be instrumental in adaptation to different photoperiods, as was proposed by the morning and evening oscillator model of Pittendrigh and Daan. We also show that this adaptation depends on retinal input but is independent of cryptochrome.

Glutamate and its metabotropic receptor in Drosophila clock neuron circuits

Identification of the neurotransmitters in clock neurons is critical for understanding the circuitry of the neuronal network that controls the daily behavioral rhythms in Drosophila. Except for the neuropeptide pigment‐dispersing factor, no neurotransmitters have been clearly identified in the Drosophila clock neurons. Here we show that glutamate and its metabotropic receptor, DmGluRA, are components of the clock circuitry and modulate the rhythmic behavior pattern of Drosophila. The dorsal clock neurons, DN1s in the larval brain and some DN1s and DN3s in the adult brain, were immunolabeled with antibodies against Drosophila vesicular glutamate transporter (DvGluT), suggesting that they are glutamatergic. Because the DN1s may communicate with the primary pacemaker neurons, s‐LNvs, we tested glutamate responses of dissociated larval s‐LNvs by means of calcium imaging. Application of glutamate dose dependently decreased intracellular calcium in the s‐LNvs. Pharmacology of the response suggests the presence of DmGluRA on the s‐LNvs. Antibodies against DmGluRA labeled dissociated s‐LNvs and the LNv dendrites in the intact larval and adult brain. The role of metabotropic glutamate signaling was tested in behavior assays in transgenic larvae and flies with altered DmGluRA expression in the LNvs and other clock neurons. Larval photophobic behavior was enhanced in DmGluRA mutants. For adults, we could induce altered activity patterns in the dark phase under LD conditions and increase the period during constant darkness by knockdown of DmGluRA expression in LNvs. Our results suggest that a glutamate signal from some of the DNs modulates the rhythmic behavior pattern via DmGluRA on the LNvs in Drosophila. J. Comp. Neurol. 505:32–45, 2007.

The lateral and dorsal neurons of Drosophila melanogaster: New insights about their morphology and function

This chapter summarizes our present knowledge about the master clock of the fruit fly at the neuronal level. The clock is organized in distinct groups of interconnected pacemaker neurons with different functions. All of these neurons appear to communicate with one another in order to produce the species-specific activity rhythm, which is organized in morning (M) and evening (E) activity bouts. These two activity components are differentially influenced by distinct groups of pacemaker neurons reminiscent of the Pittendrigh-Daan dual oscillator model. In the original work (Grima et al. 2004; Stoleru et al. 2004), the ventrolateral (LN(v)) and dorsolateral (LN(d)) plus some dorsal groups (DN) of clock neurons have been defined as M and E cells, respectively. We further specify that the clock neurons belong to the M and E oscillators and define a more complex picture of the Drosophila brain clock.

Drosophila Clock Neurons under Natural Conditions

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.

Period Gene Expression in Four Neurons Is Sufficient for Rhythmic Activity of Drosophila melanogaster under Dim Light Conditions

The clock gene expressing lateral neurons (LN) is crucial for Drosophila s rhythmic locomotor activity under constant conditions. Among the LN, the PDF expressing small ventral lateral neurons (s-LNv) are thought to control the morning activity of the fly (M oscillators) and to drive rhythmic activity under constant darkness. In contrast, a 5th PDF-negative s-LN v and the dorsal lateral neurons (LNd) appeared to control the flys evening activity (E oscillators) and to drive rhythmic activity under constant light. Here, the authors restricted period gene expression to 4 LN—the 5th s-LNv and 3 LNd— that are all thought to belong to the E oscillators and tested them in low light conditions. Interestingly, such flies showed rather normal bimodal activity patterns under light moonlight and constant moonlight conditions, except that the phase of M and E peaks was different. This suggests that these 4 neurons behave as ″M″ and ″E″ cells in these conditions. Indeed, they found by PER and TIM immunohistochemistry that 2 LNd advanced their phase upon moonlight as predicted for M oscillators, whereas the 5th s-LNv and 1 LNd delayed their activity upon moonlight as predicted for E oscillators. Their results suggest that the M or E characteristic of clock neurons is rather flexible. M and E oscillator function may not be restricted to certain anatomically defined groups of clock neurons but instead depends on the environmental conditions.

Two clocks in the brain: an update of the morning and evening oscillator model in Drosophila.

Circadian clocks play an essential role in adapting the activity rhythms of animals to the day-night cycles on earth throughout the four seasons. In many animals, including the fruit fly Drosophila melanogaster, two separate but mutually coupled clocks in the brain -morning (M) and evening (E) oscillators- control the activity in the morning and evening. M and E oscillators are thought to track dawn and dusk, respectively. This alters the phase-angle between the two oscillators under different day lengths, optimally adapting the animals activity pattern to colder short and warmer long days. Using excellent genetic tools, Drosophila researchers have addressed the neural basis of the two oscillators and could partially track these to distinct clock cells in the brain. Nevertheless, not all data are consistent with each other and many questions remained open. So far, most studies about M and E oscillators focused on the influence of light (photoperiod). Here, we will review the effects of light and temperature on the two oscillators, will update the present knowledge, discuss the limitations of the model, and raise questions that have to be addressed in the future.

The Nocturnal Activity of Fruit Flies Exposed to Artificial Moonlight Is Partly Caused by Direct Light Effects on the Activity Level That Bypass the Endogenous Clock

Artificial moonlight was recently shown to shift the endogenous clock of fruit flies and make them nocturnal. To test whether this nocturnal activity is partly due to masking effects of light, we exposed the clock‐mutants per01, tim01, per01;tim01, cyc01, and ClkJRK to light/dark and light/dim‐light cycles and determined the activity level during the day and night. We found that under moonlit nights, all clock mutants shifted their activity significantly into the night, suggesting that this effect is independent of the clock. We also recorded the flies under continuous artificial moonlight and darkness to judge the effect of dim constant light on the activity level. All mutants, except ClkJRK flies, were significantly more active under artificial moonlight conditions than under complete darkness. Unexpectedly, we found residual rhythmicity of per01 and especially tim01 mutants under these conditions, suggesting that TIM and especially PER retained some activity in the absence of its respective partner. Nevertheless, as even the double mutants and the cyc01 and ClkJRK mutants shifted their activity into the night, we conclude that dim light stimulates the activity of fruit flies in a clock‐independent manner. Thus, nocturnal light has a twofold influence on flies: it shifts the circadian clock, and it increases nocturnal activity independently of the clock. The latter was also observed in some primates by others and might therefore be of a more general validity.

GABA(B) receptors play an essential role in maintaining sleep during the second half of the night in Drosophila melanogaster.

SUMMARY GABAergic signalling is important for normal sleep in humans and flies. Here we advance the current understanding of GABAergic modulation of daily sleep patterns by focusing on the role of slow metabotropic GABAB receptors in the fruit fly Drosophila melanogaster. We asked whether GABAB-R2 receptors are regulatory elements in sleep regulation in addition to the already identified fast ionotropic Rdl GABAA receptors. By immunocytochemical and reporter-based techniques we show that the pigment dispersing factor (PDF)-positive ventrolateral clock neurons (LNv) express GABAB-R2 receptors. Downregulation of GABAB-R2 receptors in the large PDF neurons (l-LNv) by RNAi reduced sleep maintenance in the second half of the night, whereas sleep latency at the beginning of the night that was previously shown to depend on ionotropic Rdl GABAA receptors remained unaltered. Our results confirm the role of the l-LNv neurons as an important part of the sleep circuit in D. melanogaster and also identify the GABAB-R2 receptors as the thus far missing component in GABA-signalling that is essential for sleep maintenance. Despite the significant effects on sleep, we did not observe any changes in circadian behaviour in flies with downregulated GABAB-R2 receptors, indicating that the regulation of sleep maintenance via l-LNv neurons is independent of their function in the circadian clock circuit.