Charlotte Helfrich-Förster

Drosophila CRY is a deep brain circadian photoreceptor.

cry (cryptochrome) is an important clock gene, and recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cry(b), inhibits circadian photoresponses. Restricting CRY expression to specific fly tissues shows that CRY expression is needed in a cell-autonomous fashion for oscillators present in different locations. CRY overexpression in brain pacemaker cells increases behavioral photosensitivity, and this restricted CRY expression also rescues all circadian defects of cry(b) behavior. As wild-type pacemaker neurons express CRY, the results indicate that they make a striking contribution to all aspects of behavioral circadian rhythms and are directly light responsive. These brain neurons therefore contain an identified deep brain photoreceptor, as well as the other circadian elements: a central pace-maker and a behavioral output system.

The Circadian Clock of Fruit Flies Is Blind after Elimination of All Known Photoreceptors

Circadian rhythms are entrained by light to follow the daily solar cycle. We show that Drosophila uses at least three light input pathways for this entrainment: (1) cryptochrome, acting in the pacemaker cells themselves, (2) the compound eyes, and (3) extraocular photoreception, possibly involving an internal structure known as the Hofbauer-Buchner eyelet, which is located underneath the compound eye and projects to the pacemaker center in the brain. Although influencing the circadian system in different ways, each input pathway appears capable of entraining circadian rhythms at the molecular and behavioral level. This entrainment is completely abolished in glass(60j) cry(b) double mutants, which lack all known external and internal eye structures in addition to being devoid of cryptochrome.

ROBUST CIRCADIAN RHYTHMICITY OF DROSOPHILA MELANOGASTER REQUIRES THE PRESENCE OF LATERAL NEURONS : A BRAIN-BEHAVIORAL STUDY OF DISCONNECTED MUTANTS

Abstract Mutations at the disconnected (disco) locus of Drosophila melanogaster disrupt neural cell patterning in the visual system, leading to the loss of many optic lobe neurons. Drosophilas presumptive circadian pacemaker neurons – the dorsal and ventral lateral neurons – are usually among the missing cells, and most disco flies are behaviorally arrhythmic. In this study, I show that ventral lateral neurons (LNvs) are occasionally present and provoke robust circadian rhythmicity in disco mutants. Of 357 individual disco flies four animals with robust circadian rhythmicity were found. All four retained LNvs together with terminals in the superior protocerebrum. Residual or bi-circadian rhythmicity was found in about 20% of all flies; the remaining flies were completely arrhythmic. One of the flies with residual rhythmicity and two of the arrhythmic flies also had some LNvs stained. However, these flies lacked the LNv fibers in the superior protocerebrum. The results suggest that the presence of single LNvs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LNv terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LNvs indicates that also other cells contribute to the rhythmic control of locomotor activity.

Organization of the Circadian System in Insects

The circadian systems of different insect groups are summarized and compared. Emphasis is placed on the anatomical identification and characterization of circadian pacemakers, as well as on their entrainment, coupling, and output pathways. Cockroaches, crickets, beetles, and flies possess bilaterally organized pacemakers in the optic lobes that appear to be located in the accessory medulla, a small neuropil between the medulla and the lobula. Neurons that are immunoreactive for the peptide pigment-dispersing hormone (PDH) arborize in the accessory medulla and appear to be important components of the optic lobe pacemakers. The neuronal architecture of the accessory medulla with associated PDH-immunoreactive neurons is best characterized in cockroaches, while the molecular machinery of rhythm generation is best understood in fruit flies. One essential component of the circadian clock is the period protein (PER), which colocalizes with PDH in about half of the fruit flys presumptive pacemaker neurons. PER is also found in the presumptive pacemaker neurons of beetles and moths, but appears to have different functions in these insects. In moths, the pacemakers are situated in the central brain and are closely associated with neuroendocrine functions. In the other insects, neurons associated with neuroendocrine functions also appear to be closely coupled to the optic lobe pacemakers. Some crickets and flies seem to possess central brain pacemakers in addition to their optic lobe pacemakers. With respect to neuronal organization, the circadian systems of insects show striking similarities to the vertebrate circadian system.

Targeted ablation of CCAP neuropeptide-containing neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior.

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.

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.

Development of pigment-dispersing hormone-immunoreactive neurons in the nervous system of Drosophila melanogaster

An antiserum against the crustacean pigment‐dispersing hormone (PDH) was used to identify PDH‐immunoreactive neurons in the developing nervous systems of wild type Drosophila melanogaster and the brain mutant disconnected. Particular attention was paid to a group of PDH‐immunoreactive neurons at the anterior margin of the medulla—the pigment‐dispersing factor‐containing neurons close to the medulla (PDFMe neurons)—that seem to be involved in the control of adult circadian rhythmicity. In adults, this group consists of four to six neurons with large somata (large PDFMe neurons) and of four neurons with small somata (small PDFMe neurons). Both subgroups were usually absent in adults of behaviorally arrhythmic mutants of disconnected. In the wild type, PDH immunoreactivity was seen first in the small PDFMe neurons of 4 hour old first‐instar larvae. The small PDFMe neurons were found to persist unchanged into adulthood, whereas the large ones seemed to develop halfway through metamorphosis. Beside the PDFMe neurons, three other clusters of PDH‐immunoreactive neurons were stained in the developing nervous systems of Drosophila and are described in detail. Two of them were located in the brain, and the third was located in the abdominal neuromeres of the thoracic nervous system. In the mutant disconnected, the larval and the adult set of PDFMe neurons were absent. The other clusters of PDH‐immunoreactive neurons seemed to develop normally. The present results are consistent with the hypothesis that the PDFMe neurons are circadian pacemaker neurons that may control rhythmic processes in larvae, pupae, and adults. J. Comp. Neurol. 380:335–354, 1997.

Differential Control of Morning and Evening Components in the Activity Rhythm of Drosophila melanogaster—Sex-Specific Differences Suggest a Different Quality of Activity

The rhythms of locomotor activity of male and virgin or mated female flies were compared in the Drosophila melanogaster wild-type strains Canton S, Berlin, and Oregon R . Under light-dark conditions, most flies showed a bimodal activity pattern with a morning peak around lights-on and an evening peak before lights-off. For all strains, a distinct sexual dimorphism was observed in the phase of the morning peak. Males had a significantly earlier morning peak than females and consequently a larger phase angle between morning and evening peak ([.Psi]m, e). Under constant dark conditions, the morning component merged with the evening component to a unimodal activity band in about half of the flies. In those flies who maintained bimodality, the sex-specific difference in [.Psi]m, e disappeared. Other sex-specific differences were now apparent: Males showed a shorter free running period than females, and in two of the three strains, females were more active than males. Morning and evening components seem to contribute to the free-running period. Spontaneous or externally provoked change in [.Psi]m, e were correlated with period changes. In some flies, the morning and the evening components showed splitting, indicating that they are the output of two different oscillators. The sexual dimorphism in the phase of the morning peak under LDconditions suggests that the function of activity during morning and evening peak might be different, for example, during the morning peak, males are active to find females. Overall, the results underline the multioscillatory nature of Drosophilas circadian system.

Cryptochrome Is Present in the Compound Eyes and a Subset of Drosophila's Clock Neurons

Cryptochrome (CRY) is intimately associated with the circadian clock of many organisms. In the fruit fly Drosophila melanogaster, CRY seems to be involved in photoreception as well as in the core clockwork. In spite of the critical role of CRY for the clock of Drosophila, it was not quite clear whether CRY is expressed in every clock cell. With the help of a new antibody and a mutant that lacks CRY, we show here that CRY is expressed in specific subsets of Drosophilas pacemaker neurons and in the photoreceptor cells of the compound eyes. In the pacemaker neurons, CRY levels and kinetics under light‐dark cycles are quite different from each other. High‐amplitude oscillations are observed in only three groups of clock neurons, suggesting that these three groups are strongly receptive to light. The different CRY kinetics may account for phase differences in oscillations of the clock proteins observed in these three groups in earlier studies. The molecular clock of the neurons that contain lower CRY levels or are completely CRY negative can still be synchronized by light, probably via intercellular communication with the CRY‐positive neurons as well as via external photoreceptors. J. Comp. Neurol. 508:952–966, 2008.