Circadian dysfunction reduces lifespan in Drosophila melanogaster

Circadian clocks regulate physiological and behavioral processes in a wide variety of organisms, and any malfunction in these clocks can cause significant health problems. In this paper, we report the results of our study on the physiological consequences of circadian dysfunction (malfunctioning of circadian clocks) in two wild-type populations of fruit flies (Drosophila melanogaster). We assayed locomotor activity behavior and lifespan among adult flies kept under constant dark (DD) conditions of the laboratory, wherein they were categorized as rhythmic if their activity/rest schedules followed circadian (approximately 24 h) patterns, and as arrhythmic if their activity/rest schedules did not display any pattern. The rhythmic flies from both populations lived significantly longer than the arrhythmic ones. Based on these results, we conclude that circadian dysfunction is deleterious, and proper functioning of circadian clocks is essential for the physiological well being of D. melanogaster.     

Behavioral and molecular analyses suggest that circadian output is disrupted by disconnected mutants in D. melanogaster.

Mutations in the disconnected (disco) gene act to disrupt neural cell patterning in the Drosophila visual system. These mutations also affect adult locomotor activity rhythms, as disco flies are arrhythmic under conditions of constant darkness (DD). To determine the state of the circadian pacemaker in disco mutants, we constructed with pers double mutants (a short period allele of the period gene) and assayed their behavioral rhythms in light-dark cycles (LD), and their biochemical rhythms of period gene expression under both LD and DD conditions. The results demonstrate that disco flies are rhythmic, indicating that they have an active circadian pacemaker that can be entrained by light. They also suggest that disco mutants block or interfere with elements of the circadian system located between the central pacemaker and its outputs that mediate overt rhythms.     

Circadian phenotypes of Drosophila fragile x mutants in alternative genetic backgrounds

Drosophila FMR1 mutants are models of human fragile X syndrome. They show a loss of locomotor activity rhythm and severe degradation of eclosion timing. We analyzed the circadian behavior of FMR1 mutants (dfmr1(B55)) in two genetic backgrounds, yellow white (yw) and Canton S (CS). The arrhythmic phenotype of circadian locomotor activity in constant darkness (DD) did not significantly change in either genetic background. Surprisingly, eclosion timing was completely restored by backcrossing dfmr1(B55) with yw or CS flies. Morphological analysis of the small ventrally located lateral neurons of FMR1 mutants revealed that the dorsal-projection area was significantly larger in arrhythmic than rhythmic flies. In addition, dfmr1(B55) mutants in both genetic backgrounds had a significantly lower evening peak in the light-dark (LD) cycle. These results indicate that lack of FMR1 does not affect eclosion timing, but alters locomotor activity patterns in both LD and DD conditions by affecting the arborization of small ventrally located lateral neurons. Thus, the FMR1 gene may regulate the circadian-related locomotor activity of Drosophila.     

Circadian Dysfunction Reduces Lifespan in Drosophila melanogaster

Circadian clocks regulate physiological and behavioral processes in a wide variety of organisms, and any malfunction in these clocks can cause significant health problems. In this paper, we report the results of our study on the physiological consequences of circadian dysfunction (malfunctioning of circadian clocks) in two wild‐type populations of fruit flies (Drosophila melanogaster). We assayed locomotor activity behavior and lifespan among adult flies kept under constant dark (DD) conditions of the laboratory, wherein they were categorized as rhythmic if their activity/rest schedules followed circadian (approximately 24 h) patterns, and as arrhythmic if their activity/rest schedules did not display any pattern. The rhythmic flies from both populations lived significantly longer than the arrhythmic ones. Based on these results, we conclude that circadian dysfunction is deleterious, and proper functioning of circadian clocks is essential for the physiological well being of D. melanogaster.     

Localization of the circadian pacemakers of Hemideina thoracica (Orthoptera; Stenopelmatidae)

The location of the circadian pacemakers of the orthopteran Hemideina thoracica (White) has been investigated through observation of the effects of surgical removal of brain tissues (principally optic lobes and tracts) on free-running and entrained locomotor rhythms. Bilobectomy and severance of optic tracts invariably resulted in arrhythmicity, whereas rhythmicity was sustained following unilateral lobectomy, generally with increases in the free-running period (FRP) and decreases in both the active-phase lengths and activity-to-rest ratios of the rhythm. Bilobectomized subjects could be entrained by temperature cycles, but exhibited no transients or residual rhythmicity, indicating that temperature brought about a direct response or masking effect. These results support the hypothesis that the circadian locomotor pacemakers of Hemideina are located within each optic lobe, and that there are no extraoptic centers for the control of the timing of locomotor activity. Although confirmation of the pacemaker role of the optic lobes requires transplantation of the tissues, the conclusion may be drawn by inference from other studies (e.g., Leucophaea maderae--Page, 1983; Gryllus bimaculatus--Tomioka and Chiba, 1986). Light entrainment continued after surgical binding and blackening of the compound eyes and ocelli, supporting the view that direct illumination of neural tissue through the cuticle may be one possible pathway for light entrainment.     

Circadian rhythm of the locomotor activity in Drosophila melanogaster and its mutants 'sine oculis' and 'small optic lobes'

ABSTRACT. The locomotor activity patterns of wildtype Drosophila melanogaster and the mutants so (sine oculis) and sol (small optic lobes) were investigated. In all strains the proportions showing circadian rhythmicity, arrhythmicity and more complex patterns which could not be thus classified were similar. The occurrence of abnormal activity patterns is thus not a property of the mutation as previously claimed (Mack & Engelmann, 1981). In flies with a distinct circadian rhythmicity, the period lengths (τ) varied between strains, τ for wildtype Italy and the mutant so was longer than for wildtype Berlin and the mutant sol. As different τ's have been reported by others, τ does not seem to be closely determined for Drosophila melanogaster. Many flies exhibited two rhythms simultaneously, one with τ shorter and one with τ longer than 24 h, apparently implying two-oscillator control of the locomotor activity. The eyeless so mutants were entrained by LD cycles, so the compound eyes are not necessary, and so must possess the relevant photoreceptor(s) elsewhere. This may therefore also be true for the wildtype. Histology of the so adults revealed no correlation between the degree of reduction in the medulla and the occurrence of abnormal activity patterns. Since the only structures common to the medulla of so and sol are known to be large tangential cells, it is concluded that either they are of importance for the rhythmic system, or the oscillator(s) controlling locomotor activity is (are) not located in the medulla.     

Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: a brain-behavioral study of disconnected mutants

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. Drosophila's 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 (LN_vs) 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 LN_vs 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 LN_vs stained. However, these flies lacked the LN_v fibers in the superior protocerebrum. The results suggest that the presence of single LN_vs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LN_v terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LN_vs indicates that also other cells contribute to the rhythmic control of locomotor activity. Accepted: 17 September 1997     

A case for multiple oscillators controlling different circadian rhythms in Drosophila melanogaster

A population of the fruit fly Drosophila melanogaster was raised in periodic light/dark (LD) cycles of 12:12 h for about 35 generations. Eclosion, locomotor activity, and oviposition were found to be rhythmic in these flies, when assayed in constant laboratory conditions where the light intensity, temperature, humidity and other factors which could possibly act as time cue for these flies, were kept constant. These rhythms also entrained to a LD cycle of 12:12 h in the laboratory with each of them adopting a different temporal niche. The free-running periods (tau) of the eclosion, locomotor activity and oviposition rhythms were significantly different from each other. The peak of eclosion and the onset of locomotor activity occurred during the light phase of the LD cycle, whereas the peak of oviposition was found to occur during the dark phase of the LD cycle. Based on these results, we conclude that different circadian oscillators control the eclosion, locomotor activity and oviposition rhythms in the fruit fly D. melanogaster.     

Unequal coupling between locomotor pacemakers of the German cockroach,Blattella germanica (L.)

Male adult German cockroaches, Blattella germanica (L.), expressed robust locomotor circadian rhythmicity under 28 degrees C and constant darkness (DD) conditions. By surgically severing the connections between the optic lobes and midbrain and their subsequent regeneration, we demonstrated that the locomotor circadian pacemaker was located in the optic lobes and that it controlled the locomotor circadian rhythm through neural pathways. From the results that unilaterally optic tract severed males still showed locomotor circadian rhythmicity (93.1%, n=29) without significantly changing the circadian period (tau) or level of motor activity, we concluded that the right and left optic lobes each contain a circadian pacemaker competent to drive the locomotor circadian rhythm. These two pacemakers were strongly coupled since only one rhythm was expressed when the pacemakers were independently exposed to opposite lighting conditions (DD or LL) at the same time. However, an unequal contribution of each pacemaker in determining the overt circadian period was found under constant dim light (10 lux) conditions, revealing a major-minor coupling relationship between these two pacemakers, so that the unilaterally blinded male expressed either a LL-rhythm with a circadian period of 24.27+/-0.21 h (41.7%, n=24) or a DD-rhythm with a circadian period of 23.43+/-0.19 h (58.3%, n=24). However, higher intensity of photic information (200-300 lux) could overpower this relationship and cause the minor pacemaker to lead the rhythm.     

Interruption of the rat circadian clock by short light-dark cycles

Ninety male Sprague-Dawley rats were exposed to 1:1-h light-dark (LD1:1) cycles for 50-90 days, and then they were released into constant darkness (DD). During LD1:1 cycles, behavioral rhythms were gradually disintegrated, and circadian rhythms of locomotor activity, drinking, and urine 6-sulfatoxymelatonin excretion were eventually abolished. After release into DD, 44 (49%) rats showed arrhythmic behavior for >10 days. Seven (8%) animals that remained arrhythmic for >50 days in DD were exposed to brief light pulses or 12:12-h light-dark cycles, and then they restored their circadian rhythms. These results indicate that the circadian clock was stopped, at least functionally, by LD1:1 cycles and was restarted by subsequent light stimulation.     

Drosophila cryb mutation reveals two circadian clocks that drive locomotor rhythm and have different responsiveness to light

Cryptochrome (CRY) is a blue-light-absorbing protein involved in the photic entrainment of the circadian clock in Drosophila melanogaster. We have investigated the locomotor activity rhythms of flies carrying cryb mutant and revealed that they have two separate circadian oscillators with different responsiveness to light. When kept in constant light conditions, wild-type flies became arrhythmic, while cryb mutant flies exhibited free-running rhythms with two rhythmic components, one with a shorter and the other with a longer free-running period. The rhythm dissociation was dependent on the light intensities: the higher the light intensities, the greater the proportion of animals exhibiting the two oscillations. External photoreceptors including the compound eyes and the ocelli are the likely photoreceptors for the rhythm dissociation, since rhythm dissociation was prevented in so1;cryb and norpAP41;cryb double mutant flies. Immunohistochemical analysis demonstrated that the PERIOD expression rhythms in ventrally located lateral neurons (LNvs) occurred synchronously with the shorter period component, while those in the dorsally located per-expressing neurons showed PER expression most likely related to the longer period component, in addition to that synchronized to the LNvs. These results suggest that the Drosophila locomotor rhythms are driven by two separate per-dependent clocks, responding differentially to constant light.     

Effects of preparation time on phase of cultured tissues reveal complexity of circadian organization

The phases of central (SCN) and peripheral circadian oscillators are held in specific relationships under LD cycles but, in the absence of external rhythmic input, may damp or drift out of phase with each other. Rats exposed to prolonged constant light become behaviorally arrhythmic, perhaps as a consequence of dissociation of phases among SCN cells. The authors asked whether individual central and peripheral circadian oscillators were rhythmic in LL-treated arrhythmic rats and, if rhythmic, what were the phase relationships between them. The authors prepared SCN, pineal gland, pituitary, and cornea cultures from transgenic Period1-luciferaserats whose body temperature and locomotor activity were arrhythmic and from several groups of rhythmic rats held in LD, DD, and short-term LL. The authors measured mPer1gene expression by recording light output with sensitive photomultipliers. Most of the cultures from all groups displayed circadian rhythms. This could reflect persistent rhythmicity in vivo prior to culture or, alternatively, rhythmicity that may have been initiated by the culture procedure. To test this, the authors cultured tissues at 2 different times 12 h apart and asked whether phase of the rhythm was related to culture time. The pineal, pituitary, and SCN cultures showed partial or complete dependence of phase on culture time, while peak phases of the cornea cultures were independent of culture time in rhythmic rats and were randomly distributed regardless of culture time in arrhythmic animals. These results suggest that in behaviorally arrhythmic rats, oscillators in the pineal, pituitary, and SCN had been arrhythmic or severely damped in vivo, while the cornea oscillator was free running. The peak phases of the SCN cultures were particularly sensitive to some aspect of the culture procedure since rhythmicity of SCN cultures from robustly rhythmic LD-entrained rats was strongly influenced when the procedure was carried out at any time except the 2nd half of the day.     

Circadian Rhythms of Locomotor Activity in Lycosa tarentula (Araneae, Lycosidae) and the Pathways of Ocular Entrainment

Lycosa tarentula is a ground-living spider that inhabits a burrow where it awaits the appearance of prey or conspecifics. In this study, circadian rhythms of locomotor activity were examined as well as the ocular pathway of entrainment. Thirty-three adult virgin females were examined under constant darkness (DD); all of them exhibited robust circadian rhythms of locomotor activity with a period averaging 24.1h. Fourteen of these spiders were studied afterwards under an LD 12:12 cycle; they usually entrained to in the first or second day, even when the light intensity was as low as 1 lx. During the LD cycle, locomotor activity was generally restrained to the darkness phase, although several animals showed a small amount of diurnal activity. Ten males were also examined under LD; they were also nocturnal, but were much more active than the females. Seven females were examined under constant light (LL); under this they became arrhythmic. Except for the anterior median eyes (OMAs), all the eyes were capable of entraining the locomotor activity to an LD cycle. These results demonstrate that under laboratory conditions and low light intensities locomotor activity of Lycosa tarentula is circadian and in accordance with Aschoff's 'rule'. Only OMAs are unable to entrain the rhythm; the possible localization of circadian clock is therefore discussed.     

Locomotor Activity Rhythms in Cave Catfishes, Genus Taunayia, from Eastern Brazil (Teleostei: Siluriformes: Heptapterinae)

We studied the locomotor rhythmicity in heptapterine catfishes, genus Taunayia, under free-running conditions (DD) and LD cycles (12:12). Taunayia sp., anophthalmic and depigmented undescribed species from a cave in northeastern Brazil, is the fourth Brazilian troglobitic catfish studied with focus on circadian rhythms. Weak free-running rhythmicity, with absence of significant circadian components, was observed for this species when compared to the epigean, eyed relatives. On the other hand, the studied troglobitic catfishes in general presented significant circadian rhythms under LD cycles, with activity peaks in the night phase probably corresponding to nocturnal activity pattern inherited from their epigean ancestors. However, no residual oscillations were observed after transition from LD to DD. This indicates masking of activity by light-dark cycles. Regression of circadian rhythmicity in the stable, permanently dark subterranean habitat was also observed for other cave fishes. Such regression corroborates the notion that circadian rhythmicity is mainly selected in the epigean environment by ecological factors, namely daily cycles of light and/or temperature.     

Manipulating the light/dark cycle: effects on dopamine levels in optic lobes of the honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) brain

This study examines the relationship between cyclical variations in optic-lobe dopamine levels and the circadian behavioural rhythmicity exhibited by forager bees. Our results show that changing the light–dark regimen to which bees are exposed has a significant impact not only on forager behaviour, but also on the levels of dopamine that can be detected in the optic lobes of the brain. Consistent with earlier reports, we show that foraging behaviour exhibits properties characteristic of a circadian rhythm. Foraging activity is entrained by daily light cycles to periods close to 24 h, it changes predictably in response to phase shifts in light, and it is able to free-run under constant conditions. Dopamine levels in the optic lobes also undergo cyclical variations, and fluctuations in endogenous dopamine levels are influenced significantly by alterations to the light/dark cycle. However, the time course of these changes is markedly different from changes observed at a behavioural level. No direct correlation could be identified between levels of dopamine in the optic lobes and circadian rhythmic activity of the honey bee.     

Circadian rhythms of heart rate in freely moving and restrained American lobsters, Homarus americanus

While circadian rhythms of locomotion have been reported in the American lobster, Homarus americanus, it is unclear whether heart rate is also modulated on a circadian basis. To address this issue, both heart rate and locomotor activity were continuously monitored in light-dark (LD) cycles and constant darkness (DD). Lobsters in running wheels exhibited significant nocturnal increases in locomotor activity and heart rates during LD, and these measures were significantly correlated. In DD, most lobsters exhibited persistent circadian rhythms of both locomotion and heart rate. When heart rate was monitored in restrained lobsters in LD and DD, most animals also demonstrated clear daily and circadian rhythms in heart rate. Overall, this is the first demonstration of circadian rhythms of heart rate in H. americanus, the expression of which does not appear to be dependent on the expression of locomotor activity.     

Circadian oscillations outside the optic lobe in the cricket Gryllus bimaculatus

Although circadian rhythms are found in many peripheral tissues in insects, the control mechanism is still to be elucidated. To investigate the central and peripheral relationships in the circadian organization, circadian rhythms outside the optic lobes were examined in the cricket Gryllus bimaculatus by measuring mRNA levels of period (per) and timeless (tim) genes in the brain, terminal abdominal ganglion (TAG), anterior stomach, mid-gut, testis, and Malpighian tubules. Except for Malpighian tubules and testis, the tissues showed a daily rhythmic expression in either both per and tim or tim alone in LD. Under constant darkness, however, the tested tissues exhibited rhythmic expression of per and tim mRNAs, suggesting that they include a circadian oscillator. The amplitude and the levels of the mRNA rhythms varied among those rhythmic tissues. Removal of the optic lobe, the central clock tissue, differentially affected the rhythms: the anterior stomach lost the rhythm of both per and tim; in the mid-gut and TAG, tim expression became arrhythmic but per maintained rhythmic expression; a persistent rhythm with a shifted phase was observed for both per and tim mRNA rhythms in the brain. These data suggest that rhythms outside the optic lobe receive control from the optic lobe to different degrees, and that the oscillatory mechanism may be different from that of Drosophila.     

Two oscillators might control the locomotor activity rhythm of the high-altitude Himalayan strain of Drosophila helvetica

The properties of the pacemaker controlling the adult locomotor activity rhythm of the high-altitude Himalayan (haH) strain (Hemkund Sahib, 4121 m above sea level) of Drosophila helvetica are strikingly different from those of the low-altitude Himalayan (laH) strain (Birahi, 1132 m above sea level) of the same species. The haH strain has a unimodal activity pattern with a delayed peak occurring about 4.5 h after lights-on of the entraining light-dark (LD) cycle, while the laH strain has a bimodal activity pattern with the morning and evening peaks. It is rather unusual for a wild type strain of any Drosophila species to have a unimodal activity pattern during entrainment as observed in the haH strain. The single activity peak of the haH strain is regarded as a consequence of delayed morning peak merging with the evening one. Three experiments were performed to test this hypothesis. The first experiment examined whether the single activity peak could be dissociated into two components by LD cycles in which photoperiods varied from 10 to 16 h per 24 h. The haH strain again exhibited a unimodal activity pattern with a delayed peak in 10, 12, and 14 h photoperiods but a bimodal activity pattern in 16 h photoperiod. The laH strain had bimodality in 10 and 12 h photoperiods, unimodality in a 14 h photoperiod, but complete arrhythmicity in a 16 h photoperiod. In the second experiment, the haH flies were transferred from LD 16:8 to LL at 5 lux to confirm whether the bimodality of this strain in LD 16:8 cycles was not the result of masking by the long photoperiod of 16 h. Bimodality of the haH strain persisted in LL too; moreover, the morning component free-ran with period (tau) <24 h, while the evening component free-ran with tau>24 h. The third experiment examined the LL-induced splitting of activity peak of the haH strain. Flies were transferred from LD 12:12 cycles to LL at 0, 1, 5, and 15 lux. The haH strain was rhythmic in LL at 0 and 1 lux with a unimodal activity pattern. It was also rhythmic in LL at 5 lux, but the single activity peak was split into two discrete components; the morning component free-ran with tau<24 h, while the evening component free-ran with tau>24 h. This strain, however, was completely arrhythmic in LL at 15 lux. The laH strain was uniformly arrhythmic in LL at all levels of light intensity. These results suggest that the single but late activity component of the haH strain during entrainment appears to be the consequence of merging the delayed morning peak with the evening one as an adaptation to the environmental conditions at the altitude of origin of this strain, where these flies begin activity in the forenoon owing to non-permissible low temperature in the morning.     

Neural organization of the circadian system of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac- tive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of γ-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.     

Entrainment Properties of the Locomotor Activity Rhythm of Drosophila melanogaster Under Different Photoperiodic Regimens

In this paper we report the results of an experiment to assess how closely repeated brief light pulses (LPs) mimic the effects of 12:12 h light/dark (LD) cycles (PPc). The locomotor activity rhythm of individual fruit flies from a laboratory population of Drosophila melanogaster was monitored under four different photoperiodic regimens, created using 12 h of light and 12 h of darkness or brief light pulses (LPs). The phase relationship (Ψ) and the stability (precision) of the locomotor activity rhythm during entrainment were estimated in order to compare the state of the circadian clocks under the four different photoperiodic regimens. The flies (n = 72) were subjected to four different LD cycles: (i) 12 h of light and 12 h of darkness (complete photoperiod, PPc); (ii) a single brief LP of 15 min duration presented close to the onset of activity (SLP-1); (iii) a single brief LP of 15 min duration administered close to the offset of activity (SLP-2); and (iv) two brief LPs administered 12 h apart (skeleton photoperiod, PPs). The locomotor activity rhythm of the flies was first monitored under constant darkness (DD) for about 10 days and then under the four different photoperiodic regimens for about 10 days, and finally in DD for the remainder of the experiment. The Ψ of the locomotor activity rhythm and its precision under PPc and PPs did not differ significantly, but they were significantly different from the SLP-1 and SLP-2 conditions. The results provide interesting insights into photoentrainment mechanisms of circadian clocks in D. melanogaster, and suggest that skeleton photoperiods, but not single brief LPs, mimic the actions of complete photoperiods.