Development of the circadian clock in the German cockroach, Blattella germanica

The cell distribution and immunoreactivity (ir) against period (PER), pigment dispersing factor (PDF) and corazonin (CRZ), were compared between adults and nymphs in the central nervous system of the German cockroach. Although PER-ir cells in the optic lobes (OL) were expressed in the nymphs from the first instar, the links between major clock cells became more elaborated after second/third instar. A circadian rhythm of locomotion was initiated at the fourth/fifth instar. The results suggest that the clock was running from hatching, but the control network needed more time to develop. In addition, the putative downstream regulators, PDF-ir and CRZ-ir, are co-localized in various regions of the brain, indicating potential output routes of the circadian clock. CRZ-ir cells with typical morphology of neurosecretory cells in the dorsolateral protocerebrum send out three neural fibers to reach the ipsilateral corpora cardiaca (CC), the antennal lobe and two hemispheres of the protocerebrum. Based on co-localization with some PER-ir/PDF-ir cells, the CRZ-ir cells have the potential to serve as a bridge between circadian neural signals and endocrine regulation. Based on PDF's role in the regulation of locomotion, our results support the finding that the locomotor circadian rhythm is possibly controlled by a hormonal route.     

Mapping the cellular network of the circadian clock in two cockroach species

The German cockroach, Blattella germanica, and the double-striped cockroach, B. bisignata, are sibling species with a similar period sequence but a distinctive circadian rhythm in locomotion. The cell distribution of immunoreactivity (ir) against three clock-related proteins, Period (PER), Pigment Dispersing Factor (PDF), and Corazonin (CRZ), was compared between the species. The PER-ir cells tend to form clusters and are sprayed out in the central nervous system. Three major PER-ir cells are located in the optic lobes, which are the sites of the major circadian clock. They are interconnected with PER-ir axon bundles. Interestingly, the potential output signal of the circadian clock, PDF, is co-localized with PER in all three groups of cells. However, only two CRZ-ir cells and their axons are found in the optic lobes and they are not co-localized with PER-ir or PDF-ir cells and axons. Since only one circadian rhythm is expressed in locomotion, the time signals from both major clocks in optic lobes are coupled by connection with PDF-ir axons. A group of 3-4 PER-ir cells in the protocerebrum display typical characteristics of neurosecretary cells. In addition, there are numerous, small PER-ir and PDF-ir co-localized cells in the pars intercerebralis (PI), which have direct connections with the neurohemoorgan, corpora cardiaca, through PER-ir and PDF-ir axons. Based on these findings, the cellular connection shows a circadian control through the endocrine route. For the rest of central nervous system, only a few PER-ir and PDF-ir cells or axons are detected. This finding implies the circadian clock for locomotion is not located in subesophageal ganglion, thoracic or abdominal ganglia, but may use other neural messengers to pass on circadian signals. Since the overall distribution pattern of the clock cells are the same for B. germanica and B. bisignata, the possible explanation for the different expressions of locomotion between the species depends on genes downstream of per, pdf, and crz.     

Neurohormones as putative circadian clock output signals in the central nervous system of two cricket species

Antisera to the neuropeptides corazonin (Crz) and crustacean cardioactive peptide (CCAP) and to the diapause hormone (DH) react with small sets of neurones in the cephalic ganglia of the crickets Dianemobius nigrofasciatus and Allonemobius allardi. The distribution of their immunoreactivities is similar in the two species and overlaps with the locations of presumed circadian clock components in the optic lobes, protocerebrum, tritocerebrum, suboesophageal ganglion (SOG) and frontal ganglion. D. nigrofasciatus contains two Crz-immunoreactive (Crz-ir) cells in each optic lobe, six cell groups in the protocerebrum, four in the tritocerebrum, and one in SOG, whereas A. allardi harbours only five Crz-ir groups in the protocerebrum and four in the tritocerebrum. CCAP immunoreactivity occurs in both species in four protocerebrum cell clusters, four tritocerebrum cell clusters, four SOG cell clusters, one frontal ganglion cell cluster, and two optic lobe cell clusters; D. nigrofasciatus possesses two additional cells with unique links to the lamina in the optic lobe. DH-related antigens are present in four cell clusters in the optic lobe, six (D. nigrofasciatus) or eight (A. allardi) in the protocerebrum, four in the tritocerebrum, and three (A. allardi) or five (D. nigrofasciatus) in the SOG. Some of the detected cells also react with antibody to the clock protein Period (PER) or lie close to PER-ir cells. Crickets reared at two different photoperiods do not differ in the distribution and intensity of immunoreactivities. No changes have been detected during the course of diurnal light/dark cycles, possibly because the antisera react with persistent prohormones, whereas circadian fluctuations may occur at the level of their processing or of hormone release. The projection of immunoreactive fibres to several brain regions, the stomatogastric nervous system and the neurohaemal organs indicates multiple functions of the respective hormones. The work was supported by the “Research for Future” program of the Japan Society for the Promotion of Science (JSPS, 99L01205) and by the JSPS Postdoctoral Fellowship for Foreign Researchers (no. P 04197).     

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

RNAi of the circadian clock gene period disrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket

The clock mechanism for circatidal rhythm has long been controversial, and its molecular basis is completely unknown. The mangrove cricket, Apteronemobius asahinai, shows two rhythms simultaneously in its locomotor activity: a circatidal rhythm producing active and inactive phases as well as a circadian rhythm modifying the activity intensity of circatidal active phases. The role of the clock gene period (per), one of the key components of the circadian clock in insects, was investigated in the circadian and circatidal rhythms of A. asahinai using RNAi. After injection of double-stranded RNA of per, most crickets did not show the circadian modulation of activity but the circatidal rhythm persisted without a significant difference in the period from controls. Thus, per is functionally involved in the circadian rhythm but plays no role, or a less important role, in the circatidal rhythm. We conclude that the circatidal rhythm in A. asahinai is controlled by a circatidal clock whose molecular mechanism is different from that of the circadian clock.     

Serotonin- and two putative serotonin receptors-like immunohistochemical reactivities in the ground crickets Dianemobius nigrofasciatus and Allonemobius allardi

Serotonin (5-hydroxytryptamine; 5-HT)- and two putative serotonin receptors, 5-HT1A- and 5-HT1B-like, immunohistochemical reactivities were investigated in the cephalic ganglia of two ground crickets, Dianemobius nigrofasciatus and Allonemobius allardi. 5-HT-ir was strongly expressed in the central body, accessory medulla region of the optic lobe, frontal ganglion, posterior cortex of the protocerebrum, dorsolateral region of the protocerebrum, and the suboesphageal ganglion (SOG) in both crickets. However, 5-HT1A-ir and 5-HT1B-ir showed quite mutually distinct patterns that were also distinct from 5-HT-ir. 5-HT1A-ir was located in the pars intercerebralis, dorsolateral region of the protocerebrum, optic tract, optic lobe, and the midline of the SOG in both crickets. 5-HT1B-ir was located in the pars intercerebralis and dorsolateral region of the protocerebrum, and detected weakly in the optic lobe, tritocerebrum, and the midline of the SOG in both crickets. Interspecific differences were observed with 5-HT1A-ir. 5-HT1A-ir was expressed weakly in two neurons in the mandibular neuromere of the SOG in D. nigrofasciatus, while it was expressed strongly in the tritocerebrum, mandibular neuromere, and maxillary neuromere of the SOG in A. allardi and co-localized with CLOCK-ir (CLK-ir). 5HT-1B-ir was co-localized with CLK-ir in the tritocerebrum, mandibular neuromere, and maxillary neuromere of the SOG when double-labeling was conducted in both crickets. These results indicated that 5-HT and both types of 5-HT receptors may regulate circadian photo-entrainment or photoperiodism in A. allardi, while only 5-HT1B may be involved in circadian photo-entrainment or photoperiodism in D. nigrofasciatus.     

RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.     

Insect circadian clock outputs

Insects display an impressive variety of daily rhythms, which are most evident in their behaviour. Circadian timekeeping systems that generate these daily rhythms of physiology and behaviour all involve three interacting elements: the timekeeper itself (i.e. the clock), inputs to the clock through which it entrains and otherwise responds to environmental cues such as light and temperature, and outputs from the clock through which it imposes daily rhythms on various physiological and behavioural parameters. In insects, as in other animals, cellular clocks are embodied in clock neurons capable of sustained autonomous circadian rhythmicity, and those clock neurons are organized into clock circuits. Drosophila flies spend their entire lives in small areas near the ground, and use their circadian brain clock to regulate daily rhythms of rest and activity, so as to organize their behaviour appropriately to the daily rhythms of their local environment. Migratory locusts and butterflies, on the other hand, spend substantial portions of their lives high up in the air migrating long distances (sometimes thousands of miles) and use their circadian brain clocks to provide time-compensation to their sun-compass navigational systems. Interestingly, however, there appear to be substantial similarities in the cellular and network mechanisms that underlie circadian outputs in all insects.     

Pigment-dispersing factor affects nocturnal activity rhythms, photic entrainment, and the free-running period of the circadian clock in the cricket gryllus bimaculatus

Pigment-dispersing factor (PDF) is a neuropeptide widely distributed in insect brains and plays important roles in the circadian system. In this study, we used RNA interference to study the role of the pigment-dispersing factor (pdf) gene in regulating circadian locomotor rhythms in the cricket, Gryllus bimaculatus. Injections of pdf double-stranded RNA (dspdf) effectively knocked down the pdf mRNA and PDF peptide levels. The treated crickets maintained the rhythm both under light-dark cycles (LD) and constant darkness (DD). However, they showed rhythms with reduced nocturnal activity with prominent peaks at lights-on and lights-off. Entrainability of dspdf-injected crickets was higher than control crickets as they required fewer cycles to resynchronize to the LD cycles shifted by 6 h. The free-running periods of the dspdf-injected crickets were shorter than those of control crickets in DD. These results suggest that PDF is not essential for the rhythm generation but involved in control of the nocturnality, photic entrainment, and fine tuning of the free-running period of the circadian clock.     

The intrinsic circadian clock within the cardiomyocyte

Circadian clocks are intracellular molecular mechanisms that allow the cell to anticipate the time of day. We have previously reported that the intact rat heart expresses the major components of the circadian clock, of which its rhythmic expression in vivo is consistent with the operation of a fully functional clock mechanism. The present study exposes oscillations of circadian clock genes [brain and arylhydrocarbon receptor nuclear translocator-like protein 1 (bmal1), reverse strand of the c-erbaalpha gene (rev-erbaalpha), period 2 (per2), albumin D-element binding protein (dbp)] for isolated adult rat cardiomyocytes in culture. Acute (2 h) and/or chronic (continuous) treatment of cardiomyocytes with FCS (50% and 2.5%, respectively) results in rhythmic expression of circadian clock genes with periodicities of 20-24 h. In contrast, cardiomyocytes cultured in the absence of serum exhibit dramatically dampened oscillations in bmal1 and dbp only. Zeitgebers (timekeepers) are factors that influence the timing of the circadian clock. Glucose, which has been previously shown to reactivate circadian clock gene oscillations in fibroblasts, has no effect on the expression of circadian clock genes in adult rat cardiomyocytes, either in the absence or presence of serum. Exposure of adult rat cardiomyocytes to the sympathetic neurotransmitter norephinephrine (10 microM) for 2 h reinitiates rhythmic expression of circadian clock genes in a serum-independent manner. Oscillations in circadian clock genes were associated with 24-h oscillations in the metabolic genes pyruvate dehydrogenase kinase 4 (pdk4) and uncoupling protein 3 (ucp3). In conclusion, these data suggest that the circadian clock operates within the myocytes of the heart and that this molecular mechanism persists under standard cell culture conditions (i.e., 2.5% serum). Furthermore, our data suggest that norepinephrine, unlike glucose, influences the timing of the circadian clock within the heart and that the circadian clock may be a novel mechanism regulating myocardial metabolism.     

Constant light disrupts the circadian but not the circatidal rhythm in mangrove crickets

Mangrove crickets have a circatidal activity rhythm (~12.6 h cycles) with a circadian modulation under constant darkness (DD), whereby activity levels are higher during subjective night low tides than subjective day low tides. This study explored the locomotor activity rhythm of mangrove crickets under constant light (LL). Under LL, the crickets also exhibited a clear circatidal activity rhythm with a free-running period of 12.6 ± 0.26 h (mean ± SD, n = 6), which was not significantly different from that observed under DD. In contrast, activity levels were almost the same between subjective day and night, unlike those under DD, which were greater during subjective night. The loss of circadian modulation under LL may be explained by the suspension of the circadian clock in these conditions. These results strongly suggest that the circatidal activity rhythm is driven by its own clock system, distinct from the circadian clock.     

GABA synchronizes clock cells within the suprachiasmatic circadian clock

The master clock in the suprachiasmatic nuclei (SCN) is composed of multiple, single-cell circadian clocks. We test the postulate that these individual "clock cells" can be synchronized to each other by the inhibitory transmitter gamma-aminobutyric acid (GABA). For these experiments, we monitored the firing rate rhythm of individual clock cells on fixed multielectrode plates in culture and tested the effects of GABA. The results show that the daily variation in responsiveness of the SCN to phase-shifting agents is manifested at the level of individual neurons. Moreover, GABA, acting through A-type receptors, can both phase shift and synchronize clock cells. We propose that GABA is an important synchronizer of SCN neurons in vivo.     

The circadian clock influences heart performance

Circadian clocks are believed to provide the selective advantage of anticipation, thus allowing organisms to respond efficiently to stimuli at the appropriate moment. Disrupted circadian rhythms have been found to affect a variety of basic physiological processes. However, the importance of the circadian clock in regulating heart performance remains undetermined. We hypothesized that the circadian clock plays a crucial role in heart performance through the anticipation of daily workload. Echocardiography was employed to monitor heart function and structure in mice in a noninvasive, real-time manner. In wild-type mice, both the ejection fraction (EF) and the shortening fraction (FS), two important markers of cardiac function, show diurnal variation. In addition, the amplitude of the EF and the FS enlarges in response to forced exercise in a time-dependent manner. The diurnal variations in EF and FS are altered in mice with disruptions in circadian clock genes and are significantly attenuated under an imposed light regimen. Furthermore, it shows that the overexpression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc1α) under control of the muscle creatine kinase (MCK) promoter inhibited clock gene expression in the heart and muscle and decreased the expression of peroxisome proliferator-activated receptor alpha (Pparα), metabolic genes glucose transporter (Glut4), and acetyl-coA synthetase (Acs1). Pgc1α overexpression abolished the diurnal variation of EF. We thus propose that PGC1α might play an important role in circadian-mediated, impaired cardiac function by regulating the circadian rhythm of metabolic genes.     

The circadian clock starts ticking at a developmentally early stage

Although overt diurnal rhythms of behavior do not begin until well after birth, molecular studies suggest that the circadian clock may begin much earlier at a cellular level: mouse embryonic fibroblasts, for example, already possess robust clocks. By multiple criteria, we found no circadian clock present in mouse embryonic stem cells. Nevertheless, upon their differentiation into neurons, circadian gene expression was observed. In the first steps along the pathway from ES cells to neurons, a neural precursor cell (NPC) line already showed robust circadian oscillations. Therefore, at a cellular level, the circadian clock likely begins at the very earliest stages of mammalian development.     

The circadian clock of cyanobacteria

A circadian clock, with physiological characteristics similar to those of eukaryotes, functions in the photosynthetic prokaryote, cyanobacteria. The molecular mechanism of this clock has been efficiently dissected using a luciferase reporter gene that reports the status of the clock. A circadian clock gene cluster, kaiABC, has been cloned via rhythm mutants of cyanobacterium, Synechococcus, and many clock mutations mapped to the three kai genes. Although kai genes do not share any homology with clock genes so far identified in eukaryotes, analysis of their expression suggests that a negative feedback control of kaiC expression by KaiC generates the circadian oscillation and that KaiA functions as a positive factor to sustain this oscillation. BioEssays 22:10-15, 2000.