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     

Female conspecifics restore rhythmic singing behaviour in arrhythmic male zebra finches

The present study investigated whether pairing with a conspecific female would restore rhythmicity in the singing behaviour of arrhythmic male songbirds. We recorded the singing and, as the circadian response indicator, monitored the activity–rest pattern in male zebra finches (Taeniopygia guttata) housed without or with a conspecific female under 12 h light: 12 h darkness (12L:12D) or constant bright light (LL_bright). Both unpaired and paired birds exhibited a significant daily rhythm in the singing and activity behaviour, but paired birds, under 12L:12D, showed a ~2 h extension in the evening. Exposure to LL_bright decayed rhythmicity, but the female presence restored rhythmic patterns without affecting the 24 h song output. In the acoustic features, we found a significant difference in the motif duration between unpaired and paired male songs. Overall, these results demonstrated for the first time the role of the female in restoring the circadian phenotype of singing behaviour in male songbirds with disrupted circadian functions, although how interaction between sexes affects the circadian timing of male singing is not understood yet. It is suggested that social cues rendered by a conspecific female could improve the circadian performance by restoring rhythmicity in the biological functions of the cohabiting arrhythmic male partner.     

Epigenetic Maternal Effects on Endogenous Rhythms in Precocial Birds

Development involves interactions between genetic and environmental influences. Vertebrate mothers are generally the first individuals to encounter and interact with young animals. Thus, their role is primordial during ontogeny. The present study evaluated non‐genomic effects of mothers on the development of rhythms of precocial Japanese quail (Coturnix c. japonica). First, we investigated the influence of mothering on the ontogeny of endogenous rhythms of young. We compared circadian and ultradian rhythms of feeding activity of quail reared with or without adoptive mothers. More brooded than non‐brooded quail presented a circadian and/or an ultradian rhythm. Thus, the presence of the mother during the normal brooding period favors, in the long term, expression of rhythms in the young. Second, we investigated the influence of rhythmic phenotype of the mother on the development of endogenous rhythms of young by comparing quail brooded by circadian‐rhythmic adoptive mothers (R) to quail brooded by circadian‐arrhythmic adoptive mothers (A). More R‐brooded than A‐brooded quail expressed circadian rhythmicity, and circadian rhythm clarities were greater in R‐brooded than A‐brooded quail. Ultradian rhythmicity did not differ between R‐ and A‐brooded quail, nor between R and A adoptive mothers. Thus, the rhythmic phenotypes of quail mothers influence the rhythmic phenotypes of their young. Our results demonstrate that mothers of precocial birds influence epigenetically the ontogeny of endogenous rhythms of the young they raise.     

Daily melatonin administration synchronizes circadian patterns of brain metabolism and behavior in pinealectomized house sparrows,Passer domesticus

Recent research in our laboratory has indicated that in sparrows the visual suprachiasmatic nucleus (vSCN) is metabolically rhythmic such that 2-deoxy[¹⁴C]glucose (2DG) uptake and specific binding of 2[¹²⁵I]iodomelatonin (IMEL) are high during subjective day for up to 10 circadian cycles in constant darkness (DD). These rhythms damp to arrhythmicity in pinealectomized birds (PINX). The present study was designed to test the hypothesis that exogenous melatonin rhythmically applied can restore disrupted behavioral and cerebral rhythmicity. Pinealectomized house sparrows were placed in constant dim light and allowed to become arrhythmic. Experimental birds received 0.86 mM melatonin in 0.01% ethanol (ETOH) to drink for 12 of every 24 h for 14 days. Control birds received 0.01% ETOH only. Behavioral rhythmicity was restored by melatonin but not by ETOH. Birds were injected with 2DG 6 or 18 h following the beginning of melatonin (for experimental birds: MT06 and MT18 respectively) or ETOH (for control birds: ET06 and ET18 respectively) administration, allowed to survive 1 h and killed for 2DG and IMEL autoradiography. The data indicated 2DG rhythmicity such that uptake was high at MT18 in vSCN and several visual, auditory and limbic system structures in birds receiving melatonin but not in birds receiving ETOH. Similarly, IMEL binding rhythms were restored in vSCN and other visual, auditory and limbic system structures in birds receiving melatonin but not in those receiving ETOH. These data indicate that melatonin cycles are responsible for generating and/or driving a wide array of cerebral metabolic rhythms and that this influence is inhibitory.     

Circadian rhythms of finches under steadily changing light intensity: Are self-sustaining circadian rhythms self-excitatory?

Summary Finches (Chloris chloris, Fringilla montifringilla) showed clear freerunning circadian rhythms when exposed to constant dim light. Increasing the light intensity by doubling it each day made them become arrhythmic at a certain threshold intensity of illumination, showing continuous locomotor activity. When the light intensity was decreased steadily at the reversed rate, the finches became rhythmic again. 7 out of 8 finches had a clear start in their rhythms, from one day to the next, at light intensities about 4 times higher than the point where they had become arrhythmic. The last finch started its freerunning circadian rhythm gradually, a few days after the light intensity had reached a constant dim illumination (0.2 lux).The results of all birds are taken as proof of the self-excitatory capacity of the circadian system. This means, it characterizes the dynamics of the system that the clock mechanism is continuously in operation, and not only after a passive reaction to external stimuli exceeds any threshold. Simultaneously, the results of all but one bird allow the evaluation of the contribution of proportional and differential effects of light in the control of circadian rhythmicity. A relative change in light intensity by 100% in the course of one day is nearly equivalent to a change of 100% in the absolute intensity of illumination.     

Ultradian Rhythm of Activity in Japanese Quail Groups under Semi-Natural Conditions during Ontogeny: Functional Aspects and Relation to Circadian Rhythm

The function of ultradian rhythms is not yet clearly elucidated. In particular, short-term rhythms are expressed during early ontogeny, especially in broods of precocial birds. We investigated the relationship between the clarity of the ultradian rhythm of the activity/rest cycle of a group of young Japanese quail (Coturnix japonica) and the level of social synchronisation and spatial cohesion between the birds within that group. The subjects were descended from two lines selected for either very pronounced rhythmic or arrhythmic circadian activity. We found a positive relationship between the clarity of the ultradian rhythm of the activity/rest cycle when birds were young and the clarity of the circadian rhythm of feeding activity when birds were older, but still immature. The temporal organisation of the behaviour of the chicks from these two lines was observed in outdoor aviaries, when they were 4, 8, 12 and 15 days old. The mean ultradian period expressed by groups of 12 chicks was variable, with a minimum of 6 minutes. The ultradian period lengthened regularly as chicks grew older, and reached approximately 40 min on day 15. The clarity of the ultradian rhythmicity of group activity was linked to the level of inter-individual social synchronisation and of spatial cohesion; the more pronounced the ultradian rhythms of a group, the greater the temporal and spatial cohesion of the chicks within the group. Moreover, these characteristics varied with the age of the chicks. Finally, chicks in the less rhythmic groups weighed less. These results stress the adaptive value of this temporal organisation strategy under natural conditions.     

Comparative effects of inbreeding and outbreeding on the ontogeny of Heliconius erato phyllis (Lepidoptera: Nymphalidae)

Inbreeding depression has been reported in various groups of organisms, including insects. Estimates of inbreeding consequences were obtained by comparing 12 life‐history and morphological traits among nine inbred families (F = 0.25) and 16 outbred families (F = 0) of the Neotropical butterfly Heliconius erato phyllis. A Student's t‐test showed statistically significant differences for pupal weight and right forewing area, both in males and in females, between inbred and outbred families. Survival during development, from egg hatching to adulthood, also differed significantly between inbred and outbred families. The average number of haploid lethal equivalents was 0.17 for pupal weight, 0.15 for forewing area and 0.71 for survival from hatching to adulthood. The results of this study confirm that the consequences of inbreeding are more deleterious to life history traits than to morphological ones.     

Restoration of self-sustained circadian rhythmicity by the mutant clock allele in mice in constant illumination

Mice mutant for the Clock gene display abnormal circadian behavior characterized by long circadian periods and a tendency to become rapidly arrhythmic in constant darkness (DD). To investigate whether this result is contingent on the absence of light, the authors studied the circadian behavior of homozygous Clock mutant mice under conditions of both constant light and DD. Fourteen of 15 Clock/Clock mice stayed rhythmic in constant light of 70 to 170 lux, where 10 of 15 wild-type mice became arrhythmic. In contrast, only 5 of 15 Clock/ Clock mice and 15 of 15 wild-type mice remained rhythmic after 60 cycles when released in DD (dim red light of < 1.5 lux) after 8 days of entrainment. The restoration of self-sustained rhythmicity by the Clock allele cannot be attributed to reduced sensitivity of the system to light It underscores the fact that self-sustainment is not a secure guide to functional organization.     

Circadian locomotor rhythms in the desert iguana

Summary Desert iguanas, Dipsosaurus dorsalis, displaying freerunning circadian locomotor rhythms in conditions of constant darkness and temperature received electrolytic lesions to the hypothalamus. The locomotor activity of those lizards (N = 9) which sustained 80% or more damage to the suprachiasmatic nucleus (SCN) became arrhythmic whereas all animals that sustained less than 35% damage to the SCN remained rhythmic, even though they sustained significant damage to nearby regions of the hypothalamus and preoptic area. These results suggest strongly that the SCN plays a role in the regulation of circadian rhythms in the desert iguana. Taken together with other evidence, they support the view that this structure is homologous to the mammalian SCN, which acts as a pacemaker in the circadian system.Abbreviations SCN suprachiasmatic nucleus - freerunning circadian period     

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.     

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.     

Homeostatic regulation of sleep in arrhythmic Siberian hamsters

Sleep is regulated by independent yet interacting circadian and homeostatic processes. The present study used a novel approach to study sleep homeostasis in the absence of circadian influences by exposing Siberian hamsters to a simple phase delay of the photocycle to make them arrhythmic. Because these hamsters lacked any circadian organization, their sleep homeostasis could be studied in the absence of circadian interactions. Control animals retained circadian rhythmicity after the phase shift and re-entrained to the phase-shifted photocycle. These animals displayed robust daily sleep-wake rhythms with consolidated sleep during the light phase beginning about 1 h after light onset. This marked sleep-wake pattern was circadian in that it persisted in constant darkness. The distribution of sleep in the arrhythmic hamsters over 24 h was similar to that in the light phase of rhythmic animals. Therefore, daily sleep amounts were higher in arrhythmic animals compared with rhythmic ones. During 2- and 6-h sleep deprivations (SD), it was more difficult to keep arrhythmic hamsters awake than it was for rhythmic hamsters. Because the arrhythmic animals obtained more non-rapid eye movement sleep (NREMS) during the SD, they showed a diminished compensatory response in NREMS EEG slow-wave activity during recovery sleep. When amounts of sleep during the SD were taken into account, there were no differences in sleep homeostasis between experimental and control hamsters. Thus loss of circadian control did not alter the homeostatic response to SD. This supports the view that circadian and homeostatic influences on sleep regulation are independent processes.     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.     

A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila

A fundamental property of circadian rhythms is their ability to persist under constant conditions. In Drosophila, the ventral Lateral Neurons (LNvs) are the pacemaker neurons driving circadian behavior under constant darkness. Wild-type flies are arrhythmic under constant illumination, but flies defective for the circadian photoreceptor CRY remain rhythmic. We found that flies overexpressing the pacemaker gene per or the morgue gene are also behaviorally rhythmic under constant light. Unexpectedly, the LNvs do not drive these rhythms: they are molecularly arrhythmic, and PDF--the neuropeptide they secrete to synchronize behavioral rhythms under constant darkness--is dispensable for rhythmicity in constant light. Molecular circadian rhythms are only found in a group of Dorsal Neurons: the DN1s. Thus, a subset of Dorsal Neurons shares with the LNvs the ability to function as pacemakers for circadian behavior, and its importance is promoted by light.     

Effect of Light Intensity on Circadian Activity in Developing Japanese Quail

The effects of constant light on the expression of the circadian rhythm of feeding activity in Japanese quail, and in particular on the clarity of the rhythm were investigated. We used 46 4-week-old birds (35 females and 11 males) issued from two lines selected for a more (line R: 25 females and 10 males) or less (line A: 10 females and 1 male) clear circadian rhythm of feeding activity. The birds, were placed successively under three light schedules: constant darkness (DD), constant dim green light (LLdim) and constant bright light (LLbright). Schedules were changed every 2 weeks. Feeding activity was recorded continuously, analysed by autocorrelation and spectral analysis, and the ratio of the correlation coefficients and the area of the spectrum peak were used as indexes to quantify the clarity of the circadian rhythm. During the experiment, some birds showed gonadal development. Therefore, we analysed separately birds showing either a high or low degree of sexual development at the end of the experiment. In DD, 35 birds showed a circadian feeding rhythm with a mean period of 22.5 ± 0.1 h, whereas 11 birds showed an arrhythmic activity. In LLdim, 27 birds were rhythmic (22 birds R and 5 birds A), and in LLbright, only 3 birds showed a rhythmic circadian activity. For the R-line birds (for females and males), the rhythm clarity decreased in LLdim compared to DD, except for the not developed females. For the A-line birds (for females), the rhythm clarity of the immature birds increased in LLdim and that of the developed birds remained stable. In LLbright, circadian activity became arrhythmic. In LLdim, the active phases of 12 birds showed two main peaks, with mean periods of 22.7 h and 25.1 h, respectively. Therefore, constant light appeared to have an inhibitory effect on the expression of the circadian rhythm. We postulate that two hierarchically coupled oscillators could control circadian feeding activity, and arrhythmia in LLbright could be the results of internal desynchronization of the pacemakers.     

Effect of Light Intensity on Circadian Activity in Developing Japanese Quail

The effects of constant light on the expression of the circadian rhythm of feeding activity in Japanese quail, and in particular on the clarity of the rhythm were investigated. We used 46 4-week-old birds (35 females and 11 males) issued from two lines selected for a more (line R: 25 females and 10 males) or less (line A: 10 females and 1 male) clear circadian rhythm of feeding activity. The birds, were placed successively under three light schedules: constant darkness (DD), constant dim green light (LLdim) and constant bright light (LLbright). Schedules were changed every 2 weeks. Feeding activity was recorded continuously, analysed by autocorrelation and spectral analysis, and the ratio of the correlation coefficients and the area of the spectrum peak were used as indexes to quantify the clarity of the circadian rhythm. During the experiment, some birds showed gonadal development. Therefore, we analysed separately birds showing either a high or low degree of sexual development at the end of the experiment. In DD, 35 birds showed a circadian feeding rhythm with a mean period of 22.5 ± 0.1 h, whereas 11 birds showed an arrhythmic activity. In LLdim, 27 birds were rhythmic (22 birds R and 5 birds A), and in LLbright, only 3 birds showed a rhythmic circadian activity. For the R-line birds (for females and males), the rhythm clarity decreased in LLdim compared to DD, except for the not developed females. For the A-line birds (for females), the rhythm clarity of the immature birds increased in LLdim and that of the developed birds remained stable. In LLbright, circadian activity became arrhythmic. In LLdim, the active phases of 12 birds showed two main peaks, with mean periods of 22.7 h and 25.1 h, respectively. Therefore, constant light appeared to have an inhibitory effect on the expression of the circadian rhythm. We postulate that two hierarchically coupled oscillators could control circadian feeding activity, and arrhythmia in LLbright could be the results of internal desynchronization of the pacemakers.     

Circadian organization in the pigeon,Columba livia: the role of the pineal organ and the eye

Summary The roles of the pineal organ and the eye in the control of circadian locomotor rhythmicity were studied in the pigeon (Columba livia). Neither pinealectomy nor blinding abolished the circadian rhythms in constant dim light conditions (LLdim). All the pinealectomized birds and the blinded birds entrained to light-dark (LD) cycles with no discernible anticipatory activity. However, the birds which had been both pinealectomized and blinded showed no circadian rhythms in prolonged LLdim. These birds entrained to LD cycles with anticipatory activity and showed residual rhythmicity for a while after transfer from LD cycles to LLdim. Continuous administration of melatonin induced suppression of the circadian rhythms and reduced total amount of locomotor activity in LLdim. These results suggest that not only the pineal organ but also the eye (perhaps the retina) is involved in the pigeon's circadian system.Abbreviations NAT N-acetyltransferase - LLdim constant dim light - cadian period - SCN suprachiasmatic nucleus - circadian activity time - LD light-dark     

Circadian organization in the ruin lizard Podarcis sicula: the role of the suprachiasmatic nuclei of the hypothalamus

The effects of electrolytic lesions to the suprachiasmatic nuclei of the hypothalamus (SCN) on circadian rhythms of locomotor activity were examined in ruin lizards Podarcis sicula maintained in constant darkness and constant temperature (29°C). All lizards (N=15) in which the lesion damaged 80% or more of the SCN became behaviorally arrhythmic. On the contrary, locomotor rhythms persisted in all cases (N=11) when the SCN remained intact and lesions were confined to neighbouring regions of the preoptic area. Taken together with previous work which demonstrates that the pineal and the retinae are not essential for the persistence of circadian locomotor rhythmicity in Podarcis sicula and with recent evidence showing the homology between the SCN of lizards and those of mammals the present results strongly support the view that the SCN of Podarcis sicula contain the primary pacemaker(s) for locomotor rhythms.Abbreviations DD constant darkness - LL constant light - SCN suprachiasmatic nuclei of the hypothalamus - PH nucleus periventricularis hypothalami - OC optic chiasm - te length of circadian activity - freerunning circadian period     

The pineal clock affects behavioral circadian rhythms but not photoperiodic induction in the Indian weaver bird (Ploceus philippinus)

We investigated whether pineal is part of the circadian clock system which regulates circadian rhythms of activity and photosensitivity in the Indian weaver bird (Ploceus philippinus). Two experiments were performed. The first experiment examined the induction of testicular growth, and androgen-dependent beak pigmentation and luteinizing hormone (LH)-specific plumage coloration in pinealectomised (pinx) and sham-operated (sham) birds exposed to short day (8 h light: 16 h darkness, 8L:16D) and long day (16L:8D) for 9 months in the late breeding and early regressive phase (October), or the late regressive and preparatory phase (January) of the annual testicular cycle. As expected, short days were non-stimulatory, and long days stimulated testicular growth, beak pigmentation and plumage coloration. There was no difference in the response between pinx and sham birds subjected to short or long days in October, but the response was enhanced in pinx birds that were subjected to long day in January. In the second experiment circadian behavioral rhythms were studied (activity pattern in singly housed birds) in weaver birds first exposed at two different phases of the annual testicular cycle to a stimulatory photoperiod (12L:12D in preparatory phase or 13L:11D in early breeding phase) and then released into dim continuous light (LL_dim). All birds showed synchronization to the light period before and after the pinealectomy; there was no difference in the response between pinx and sham birds. When released into LL_dim, sham birds exhibited circadian rhythmicity continuously, whereas pinx birds lost circadian rhythmicity after some cycles. Considered together, these results suggest that circadian clock residing within the pineal gland regulates the circadian rhythm in activity, but not the circadian rhythm involved in photoperiodic induction of the Indian weaver bird.