Pigment-dispersing hormone (PDH)-immunoreactive neurons form a direct coupling pathway between the bilaterally symmetric circadian pacemakers of the cockroach Leucophaea maderae

Circadian locomotor activity rhythms of the cockroach Leucophaea maderae are driven by two bilaterally paired and mutually coupled pacemakers that reside in the optic lobes of the brain. Transplantation studies have shown that this circadian pacemaker is located in the accessory medulla (AMe), a small neuropil of the medulla of the optic lobe. The AMe is densely innervated by about 12 anterior pigment-dispersing-hormone-immunoreactive (PDH-ir) medulla (PDHMe) neurons. PDH-ir neurons are circadian pacemaker candidates in the fruitfly and cockroach. A subpopulation of these neurons also appears to connect both optic lobes and may constitute at least one of the circadian coupling pathways. To determine whether PDHMe neurons directly connect both accessory medullae, we injected rhodamine-labeled dextran as neuronal tracer into one AMe and performed PDH immunocytochemistry. Double-labeled fibers in the anterior, shell, and internodular neuropil of the AMe contralaterally to the injection site showed that PDH-ir fibers directly connect both accessory medullae. This connection is formed by three anterior PDHMe neurons of each optic lobe, which, thus, fulfill morphological criteria for a direct circadian coupling pathway. Our double-label studies also showed that all except one of the midbrain projection areas of anterior PDHMe neurons were innervated ipsilaterally and contralaterally. Thus, anterior PDHMe neurons seem to play multiple roles in generating circadian rhythms. They also deliver timing information output and perform mutual pacemaker coupling in L. maderae. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants STE 531/7-1, 2, 3, and Human Science Frontier     

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 fly's 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. (Chronobiology International, 15(6), 567-594, 1998)     

Membrane electrical excitability is necessary for the free-running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free-running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell-autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K(+) channel stops the free-running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free-running larval pacemaker clock-like that of the adult pacemaker neurons they give rise to-requires membrane electrical activity to oscillate. In contrast to the free-running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light-dark) conditions. This specific disruption of the free-running clock caused by targeted K(+) channel expression likely reflects a specific cell-autonomous clock-membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology.     

Membrane electrical excitability is necessary for the free‐running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free‐running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell‐autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K⁺ channel stops the free‐running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free‐running larval pacemaker clock—like that of the adult pacemaker neurons they give rise to—requires membrane electrical activity to oscillate. In contrast to the free‐running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light–dark) conditions. This specific disruption of the free‐running clock caused by targeted K⁺ channel expression likely reflects a specific cell‐autonomous clock‐membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Spontaneous synchronization of coupled circadian oscillators

In mammals, the circadian pacemaker, which controls daily rhythms, is located in the suprachiasmatic nucleus (SCN). Circadian oscillations are generated in individual SCN neurons by a molecular regulatory network. Cells oscillate with periods ranging from 20 to 28 h, but at the tissue level, SCN neurons display significant synchrony, suggesting a robust intercellular coupling in which neurotransmitters are assumed to play a crucial role. We present a dynamical model for the coupling of a population of circadian oscillators in the SCN. The cellular oscillator, a three-variable model, describes the core negative feedback loop of the circadian clock. The coupling mechanism is incorporated through the global level of neurotransmitter concentration. Global coupling is efficient to synchronize a population of 10,000 cells. Synchronized cells can be entrained by a 24-h light-dark cycle. Simulations of the interaction between two populations representing two regions of the SCN show that the driven population can be phase-leading. Experimentally testable predictions are: 1), phases of individual cells are governed by their intrinsic periods; and 2), efficient synchronization is achieved when the average neurotransmitter concentration would dampen individual oscillators. However, due to the global neurotransmitter oscillation, cells are effectively synchronized.     

Neurobiology of the fruit fly's circadian clock

Studying the fruit fly Drosophila melanogaster has revealed mechanisms underlying circadian clock function. Rhythmic behavior could be assessed to the function of several clock genes that generate circadian oscillations in certain brain neurons, which finally modulate behavior in a circadian manner. This review outlines how individual circadian pacemaker neurons in the fruit fly's brain control rhythm in locomotor activity and eclosion.     

果蝇昼夜节律的分子机制研究进展

果蝇由于遗传易操作性而成为一个研究昼夜节律分子机制的理想模式生物 . 到目前为止,通过遗传学和生物化学方法已经鉴定到 10 多个时钟基因 (clock genes) 和许多时钟相关基因,包括时钟输入基因和钟控基因 . 这些时钟基因以及它们的相应产物组成两个互相依赖的转录 / 翻译反馈环路,从而调节行为和生理的昼夜节律 . 果蝇这种核心钟的工作原理同样见于哺乳动物 .     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

Circadian control of eclosion: interaction between a central and peripheral clock in Drosophila melanogaster

Drosophila melanogaster display overt circadian rhythms in rest:activity behavior and eclosion. These rhythms have an endogenous period of approximately 24 hr and can adjust or "entrain" to environmental inputs such as light. Circadian rhythms depend upon a functioning molecular clock that includes the core clock genes period and timeless (reviewed in and ). Although we know that a clock in the lateral neurons (LNs) of the brain controls rest:activity rhythms, the cellular basis of eclosion rhythms is less well understood. We show that the LN clock is insufficient to drive eclosion rhythms. We establish that the prothoracic gland (PG), a tissue required for fly development, contains a functional clock at the time of eclosion. This clock is required for normal eclosion rhythms. However, both the PG clock function and eclosion rhythms require the presence of LNs. In addition, we demonstrate that pigment-dispersing factor (PDF), a neuropeptide secreted from LNs, is necessary for the PG clock and eclosion rhythms. Unlike other clocks in the fly periphery, the PG is similar to mammalian peripheral oscillators because it depends upon input, including PDF, from central pacemaker cells. This is the first report of a peripheral clock necessary for a circadian event.     

Insect photoperiodism and circadian clocks: models and mechanisms

Photoperiodic clocks allow organisms to predict the coming season. In insects, the seasonal adaptive response mainly takes the form of diapause. The extensively studied photoperiodic clock in insects was primarily characterized by a "black-box" approach, resulting in numerous cybernetic models. This is in contrast with the circadian clock, which has been dissected pragmatically at the molecular level, particularly in Drosophila. Unfortunately, Drosophila melanogaster, the favorite model organism for circadian studies, does not demonstrate a pronounced seasonal response, and consequently molecular analysis has not progressed in this area. In the current article, the authors explore different ways in which identified molecular components of the circadian pacemaker may play a role in photoperiodism. Future progress in understanding the Drosophila circadian pacemaker, particularly as further output components are identified, may provide a direct link between the clock and photoperiodism. In addition, with improved molecular tools, it is now possible to turn to other insects that have a more dramatic photoperiodic response.