Free-running Rhythms Research Articles

Magnetic Fluctuations Entrain the Circadian Rhythm of Locomotor Activity in Zebrafish: Can Cryptochrome Be Involved?

Simple SummaryMost physiological processes are subject to biological circadian rhythms maintained by a complex cascade of biochemical events. The circadian rhythmicity of behavior allows organisms to use energy and resources optimally under changing environmental conditions. To that end, endogenous circadian rhythms are synchronized with external pacemakers (zeitgebers), especially daily changes in illumination. In the 1960s, it was assumed that, in addition to this primary photic cue, animals can use diurnal geomagnetic variation as a secondary zeitgeber. Earlier research found that slow magnetic fluctuations can affect some behavioral endpoints of circadian rhythms by modulating an organism’s physiological state. However, no direct experiments to test such an entrainment of biological clocks by artificial magnetic fields were performed due to the technical difficulty of eliminating natural geomagnetic variation. For the first time, we carried out such tests in a fully controlled magnetic environment using zebrafish as a research model. The experimental treatments included various light/dark cycles and continuous illumination coupled with pre-recorded natural geomagnetic variations. The obtained results indicate that slow magnetic fluctuations can entrain endogenous rhythmical activity in vertebrates. Probably, cryptochromes play a key role in this process. This research provides promising opportunities for the magnetic control of circadian processes, e.g., correcting circadian dysfunctions.In the 1960s, it was hypothesized that slow magnetic fluctuations could be a secondary zeitgeber for biological circadian rhythms. However, no comprehensive experimental research has been carried out to test the entrainment of free-running circadian rhythms by this zeitgeber. We studied the circadian patterns of the locomotor activity of zebrafish (Danio rerio) under different combinations of light regimes and slow magnetic fluctuations, based on a record of natural geomagnetic variation. A rapid synchronization of activity rhythms to an unusual 24:12 light/dark cycle was found under magnetic fluctuations with a period of 36 h. Under constant illumination, significant locomotor activity rhythms with 26.17 h and 33.07 h periods were registered in zebrafish exposed to magnetic fluctuations of 26.8 h and 33.76 h, respectively. The results reveal the potential of magnetic fluctuations for entrainment of circadian rhythms in zebrafish and genuine prospects to manipulate circadian oscillators via magnetic fields. The putative mechanisms responsible for the entrainment are discussed, including the possible role of cryptochromes.

Human ARHGEF9 intellectual disability syndrome is phenocopied by a mutation that disrupts collybistin binding to the GABAA receptor α2 subunit

Intellectual disability (ID) is a common neurodevelopmental disorder that can arise from genetic mutations ranging from trisomy to single nucleotide polymorphism. Mutations in a growing number of single genes have been identified as causative in ID, including ARHGEF9. Evaluation of 41 ARHGEF9 patient reports shows ubiquitous inclusion of ID, along with other frequently reported symptoms of epilepsy, abnormal baseline EEG activity, behavioral symptoms, and sleep disturbances. ARHGEF9 codes for the Cdc42 Guanine Nucleotide Exchange Factor 9 collybistin (Cb), a known regulator of inhibitory synapse function via direct interaction with the adhesion molecule neuroligin-2 and the α2 subunit of GABAA receptors. We mutate the Cb binding motif within the large intracellular loop of α2 replacing it with the binding motif for gephyrin from the α1 subunit (Gabra2-1). The Gabra2-1 mutation causes a strong downregulation of Cb expression, particularly at cholecystokinin basket cell inhibitory synapses. Gabra2-1 mice have deficits in working and recognition memory, as well as hyperactivity, anxiety, and reduced social preference, recapitulating the frequently reported features of ARHGEF9 patients. Gabra2-1 mice also have spontaneous seizures during postnatal development which can lead to mortality, and baseline abnormalities in low-frequency wavelengths of the EEG. EEG abnormalities are vigilance state-specific and manifest as sleep disturbance including increased time in wake and a loss of free-running rhythmicity in the absence of light as zeitgeber. Gabra2-1 mice phenocopy multiple features of human ARHGEF9 mutation, and reveal α2 subunit-containing GABAA receptors as a druggable target for treatment of this complex ID syndrome.

Effects of light on the sleep-wakefulness cycle of mice mediated by intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are able to synthesize the photosensitive protein melanopsin, which is involved in the regulation of circadian rhythms, the papillary light reflex and other nonimaging visual functions. To investigate whether ipRGCs are involved in mediating the light modulation of sleep-wakefulness in rodents, melanopsin knockout mice (MKO), melanopsin-only mice (MO) and coneless, rodless, melanopsin knockout mice (TKO) were used in this study to record electroencephalogram and electromyography variations in the normal 12:12 h light:dark cycle, and 1 h and 3 h light pulses were administered at 1 h after the light was turned off. In the normal 12:12 h light-dark cycle, the WT, MKO and MO mice had a regular day-night rhythm and no significant difference in wakefulness, rapid eye movement (REM) or nonrapid eye movement (NREM) sleep. However, TKO mice could not be entrained according to the light-dark cycle and exhibited a free-running rhythm. Extending the light pulse durations significantly changed the sleep and wakefulness activities of the WT and MO mice but did not have an effect on the MKO mice. These results indicate that melanopsin significantly affects REM and NREM sleep and that ipRGCs play an important role in light-induced sleep in mice.

ВЛИЯНИЕ МАГНИТНЫХ ФЛУКТУАЦИЙ НА ЦИРКАДНУЮ СТРУКТУРУ ДВИГАТЕЛЬНОЙ АКТИВНОСТИ У DANIO RERIO (ПРЕДВАРИТЕЛЬНЫЕ ДАННЫЕ)

The locomotor activity of zebrafish (Danio rerio) has a pronounced, well-studied circadian rhythm. Under constant illumination, the period of free-running locomotor activity in this species usually becomes less than 24 hours. To evaluate the entraining capabilities of slow magnetic variations, zebrafish locomotor activity was evaluated at constant illumination and fluctuating magnetic field with a period of 26.8 hours. Lomb-Scargle periodogram revealed significant free-running rhythms of locomotor activity and related behavioral endpoints with a period close to 27 hours. Obtained results reveal the potential of slow magnetic fluctuations for entrainment of the circadian rhythms in zebrafish. The putative mechanisms responsible for the entrainment are discussed, including the possible role of cryptochromes.

Role of heterozygous and homozygous alleles in cryptochrome-deficient mice

The circadian rhythms of physiology and behavior are based on molecular systems at the cellular level, which are regulated by clock genes, including cryptochrome genes, Cry1 and Cry2. In mammals, the circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus maintains the circadian rhythms throughout the body. Cry1 and Cry2 play distinct roles in regulating the circadian rhythm. However, the different effects of manipulating clock genes in heterozygous and homozygous alleles, Cry1 and Cry2, remain unclear. Therefore, this study aimed to understand the haplosufficiency of cryptochrome genes in regulating the circadian system. We examined wheel-running activity rhythms and PER2::LUC expression rhythms in SCN slices and pituitary explants in mice. Compared with wild-type mice, Cry1−/− or Cry2−/− mice had shortened or lengthened periods in free-running behavioral rhythms and PER2::LUC expression in the SCN and pituitary gland. Cry1+/− mice had similar circadian rhythms as wild-type mice, although Cry2+/− mice had lengthened periods. The amplitude of PER2::LUC expression exhibited faster damping in Cry1−/− mice. Therefore, Cry1 deficiency affects the circadian period length and stability of the circadian system. A single allele of Cry2 deficiency affects the circadian rhythm, whereas that of Cry1 deficit is compensated.

Spontaneous Recovery of Circadian Organization in Mice Lacking a Core Component of the Molecular Clockwork.

Circadian rhythms are generated by interlocked transcriptional-translational feedback loops of circadian clock genes and their protein products. Mice homozygous for a functional deletion in the Period-2 gene (Per2m/m mice) exhibit short free-running circadian periods and eventually lose behavioral circadian rhythmicity in constant darkness (DD). We investigated Per2m/m mice in DD for several months and identified a categorical sex difference in the dependence on Per2 for maintenance of circadian rhythms. Nearly all female Per2m/m mice became circadian arrhythmic in DD, whereas free-running rhythms persisted in 37% of males. Remarkably, with extended testing, Per2m/m mice did not remain arrhythmic in DD, but after varying intervals spontaneously recovered robust, free-running circadian rhythms, with periods shorter than those expressed prior to arrhythmia. Spontaneous recovery was strikingly sex-biased, occurring in 95% of females and 33% of males. Castration in adulthood resulted in male Per2m/m mice exhibiting female-like levels of arrhythmia in DD, but did not affect spontaneous recovery. The circadian pacemaker of many gonad-intact males, but not females, can persist in DD for long intervals without a functional PER2 protein; their circadian clocks may be in an unstable equilibrium, incapable of sustaining persistent coherent circadian organization, resulting in transient cycles of circadian organization and arrhythmia.

The microtubule-associated protein Tau suppresses the axonal distribution of PDF neuropeptide and mitochondria in circadian clock neurons.

Disrupted circadian rhythms are a prominent feature of multiple neurodegenerative diseases. Yet mechanisms linking Tau to rhythmic behavior remain unclear. Here, we find that expression of a phosphomimetic human Tau mutant (TauE14) in Drosophila circadian pacemaker neurons disrupts free-running rhythmicity. While cell number and oscillations of the core clock protein PERIOD are unaffected in the small LNv (sLNv) neurons important for free running rhythms, we observe a near complete loss of the major LNv neuropeptide pigment dispersing factor (PDF) in the dorsal axonal projections of the sLNvs. This was accompanied by a ~50% reduction in the area of the dorsal terminals and a modest decrease in cell body PDF levels. Expression of wild-type Tau also reduced axonal PDF levels but to a lesser extent than TauE14. TauE14 also induces a complete loss of mitochondria from these sLNv projections. However, mitochondria were increased in sLNv cell bodies in TauE14 flies. These results suggest that TauE14 disrupts axonal transport of neuropeptides and mitochondria in circadian pacemaker neurons, providing a mechanism by which Tau can disrupt circadian behavior prior to cell loss.

Paclitaxel chemotherapy disrupts behavioral and molecular circadian clocks in mice

Cancer patients experience circadian rhythm disruptions in activity cycles and cortisol release that correlate with poor quality of life and decreased long-term survival rates. However, the extent to which chemotherapy contributes to altered circadian rhythms is poorly understood. In the present study, we examined the extent to which paclitaxel, a common chemotherapy drug, altered entrained and free-running circadian rhythms in wheel running behavior, circulating corticosterone, and circadian clock gene expression in the brain and adrenal glands of tumor-free mice. Paclitaxel injections delayed voluntary wheel running activity onset in a light–dark cycle (LD) and lengthened the free-running period of locomotion in constant darkness (DD), indicating an effect on inherent suprachiasmatic nucleus (SCN) pacemaker activity. Paclitaxel attenuated clock gene rhythms in multiple brain regions in LD and DD. Furthermore, paclitaxel disrupted circulating corticosterone rhythms in DD by elevating its levels across a 24-hour cycle, which correlated with blunted amplitudes of Arntl, Nr1d1, Per1, and Star rhythms in the adrenal glands. Paclitaxel also shortened SCN slice rhythms, increased the amplitude of adrenal gland oscillations in PER2::luciferase cultures, and increased the concentration of pro-inflammatory cytokines and chemokines released from the SCN. These findings indicate that paclitaxel disrupts clock genes and behavior driven by the SCN, other brain regions, and adrenal glands, which were associated with chemotherapy-induced inflammation. Together, this preclinical work demonstrates that chemotherapy disrupts both central and peripheral circadian rhythms and supports the possibility that targeted circadian realignment therapies may be a novel and non-invasive way to improve patient outcomes after chemotherapy.

Gap junction protein Innexin2 modulates the period of free-running rhythms in Drosophila melanogaster

SummaryA neuronal circuit of ∼150 neurons modulates rhythmic activity-rest behavior of Drosophila melanogaster. While it is known that coherent ∼24-hr rhythms in locomotion are brought about when 7 distinct neuronal clusters function as a network due to chemical communication amongst them, there are no reports of communication via electrical synapses made up of gap junctions. Here, we report that gap junction proteins, Innexins play crucial roles in determining the intrinsic period of activity-rest rhythms in flies. We show the presence of Innexin2 in the ventral lateral neurons, wherein RNAi-based knockdown of its expression slows down the speed of activity-rest rhythm along with alterations in the oscillation of a core-clock protein PERIOD and the output molecule pigment dispersing factor. Specifically disrupting the channel-forming ability of Innexin2 causes period lengthening, suggesting that Innexin2 may function as hemichannels or gap junctions in the clock circuit.

Behaviorally and environmentally induced non-24-hour sleep-wake rhythm disorder in sighted patients.

To determine whether there was evidence of circadian or sleep-regulatory dysfunction in sighted individuals with non-24-hour sleep-wake rhythm disorder. Three sighted individuals with signs and/or symptoms of non-24-hour sleep-wake rhythm disorder were studied. Thirty-five- to 332-day laboratory and home-based assessments of sleep-wake and circadian timing, endogenous circadian period, photic input to the circadian pacemaker, and/or circadian and sleep-wake-dependent regulation of sleep were conducted. No evidence of circadian dysfunction was found in these individuals. Instead, sleep-wake timing appeared to dissociate from the circadian timing system, and/or self-selected sleep-wake and associated light/dark timing shifted the circadian pacemaker later, rather than the circadian pacemaker determining sleep-wake timing. These findings suggest that the etiology of this disorder may be light- and/or behaviorally induced in some sighted people, which has implications for the successful treatment of this disorder. Emens JS, St Hilaire MA, Klerman EB, etal. Behaviorally and environmentally induced non-24-hour sleep-wake rhythm disorder in sighted patients. J Clin Sleep Med. 2022;18(2):453-459.

Diel and Circadian Patterns of Locomotor Activity in the Adults of Diamondback Moth (Plutella xylostella).

The Diamondback Moth (Plutella xylostella) is a highly destructive lepidopteran pest of cruciferous crops. However, there still is relatively little known about the locomotor activities of diel and the circadian patterns in P. xylostella. Here, we present an analysis of the diel locomotion of P. xylostella under several laboratory settings. We established a system for measuring the individual locomotor activities of P. xylostella and found that both males and females showed a nocturnal pattern of activity under 26 or 20 °C LD conditions, with activity peaking immediately after lights off and quickly declining after lights on. In addition, we showed that it is difficult to assess the free-running circadian rhythms of P. xylostella under 26 °C DD conditions. However, we found that males showed a higher power, rhythm index (RI) and rhythmic ratio than females under 20 °C DD conditions, which indicated that males in low-temperature conditions are much more suitable to study the free-running circadian rhythms of P. xylostella. The findings of this study will help us to have a better understanding of the diel activity of P. xylostella and may provide a foundation for the development of an effective pest management strategy.

Effect of periodic social interaction and odour presentation of same and opposite-sex conspecifics on free-running mice

ABSTRACT Social interaction, a non-photic cue, plays a role in the synchronization of circadian rhythm under constant environmental conditions. However, little is known about the effect of sex-specific interaction and sex-specific odor on the free-running circadian clock of mice. Hence, in the present study, we investigated the effect of social interaction and odor presentation of same and opposite-sex conspecifics on the free-running rhythm of mice. In this investigation, stranger and dummy mice (saturated with either male- or female-associated odor) were separately exposed to the same and opposite-sex conspecifics of male and female runners and receivers, respectively for 30 min at CT15 for 10–12 d. The results showed that the daily exposure of runners with same and opposite-sex strangers entrained the free-running locomotor activity rhythm in a gender-specific manner. However, daily exposure of same and opposite-sex conspecific odors masked the circadian rhythm of receiver animals by increasing the activity. Further, social interaction and odor presentation caused lengthened and shortened period length, respectively. These results suggest that periodic social interactions can produce entrainment and conspecific odor can produce only masking on free-running circadian locomotor activity rhythm in mice.

Locomotor activity rhythm in catfish Heteropneustes fossilis as a function of shoal size under different light regimens

ABSTRACT The information on the circadian characteristics of catfish in shoals is scanty. We examined the circadian locomotor activity rhythm of catfish Heteropneustes fossilis either singly housed (SS1) or in shoals of four (SS4) and six (SS6) under different light regimens. We carried out the study sequentially under LD1 (12:12), constant darkness (DD), LD2, continuous light (LL), LD3, and DL (12:12). Each condition was for at least 10–12 consecutive days. We analyzed the time-series data by employing Cosinor rhythmometry to detect circadian rhythm characteristics in locomotor activity at a fixed time window with a τ = 24 h. Results indicated that singly housed or shoals exhibited statistically significant (p < .001) circadian rhythm in locomotor activity under LD conditions with a higher activity level during the dark phase. Further, we observed free-running rhythms in locomotor activity under DD and LL, irrespective of the shoal sizes. In phase inverse DL, both singly housed and shoals demonstrated higher activity in the dark phase. The two-way ANOVA results revealed a significant effect of the factor ‘light regimen’ on amplitude and acrophase; the factor ‘shoal size’ produced a statistically significant impact on the mesor only. Both shoals showed significantly higher mesor than singly housed fish. The circadian amplitude declined under constant conditions of DD and LL. The locomotor activity rhythm exhibited a free-running pattern with a τFR greater than 24 h under both DD and LL conditions. We conclude that light is a more prominent factor for the entrainment of circadian activity in catfish H. fossilis. However, the extent of social aggregation (shoal size) has little or no effects on the characteristics of circadian locomotor activity rhythm in H. fossilis.

Clock-controlled arylalkylamine N-acetyltransferase (aaNAT) regulates circadian rhythms of locomotor activity in the American cockroach, Periplaneta americana, via melatonin/MT2-like receptor.

Melatonin (MEL) orchestrates daily and seasonal rhythms (eg, locomotion, sleep/wake cycles, and migration among other rhythms) in diverse organisms. We investigated the effects of pharmacological doses (0.03-1mM) of exogenous MEL intake in the cockroach, Periplaneta americana, on locomotor activity. As per os MEL concentration increased, cockroach locomotor rhythm in light-dark (LD) cycles became more synchronized. The ratio of night activity to 24-h activity increased and the acrophase (peak) slightly advanced. MEL application also influenced total activity bouts in the free-running rhythm. Since MEL slightly influenced τ in the free-running rhythms, it is not a central element of the circadian pacemaker but must influence mutual coupling of multi-oscillatory system components. Arylalkylamine N-acetyltransferase (aaNAT) regulates enzymatic production of MEL. aaNAT activities vary in circadian rhythms, and the immunoreactive aaNAT (aaNAT-ir) is colocalized with the key clock proteins cycle (CYC)-ir and pigment-dispersing factor (PDF)-ir These are elements of the central pacemaker and its output pathway as well as other circadian landmarks such as the anterior and posterior optic commissures (AOC and POC, respectively). It also partially shares immunohistochemical reactivity with PER-ir and DBT-ir neurons. We analyzed the role of Pamericana aaNAT1 (PaaaNAT1) (AB106562.1) by injecting dsRNAaaNAT1 . qPCR showed a decrease in accumulations of mRNAs encoding PaaaNAT1. The injections led to arrhythmicity in LD cycles and the arrhythmicity persisted in constant dark (DD). Continuous administration of MEL resynchronized the rhythm after arrhythmicity was induced by dsRNAaaNAT1 injection, suggesting that PaaaNAT is the key regulator of the circadian system in the cockroach via MEL production. PaaaNAT1 contains putative E-box regions which may explain its tight circadian control. The receptor that mediates MEL function is most likely similar to the mammalian MT2, because injecting the competitive MT2 antagonist luzindole blocked MEL function, and MEL injection after luzindole treatment restored MT function. Human MT2-ir was localized in the circadian neurons in the cockroach brain and subesophageal ganglion. We infer that MEL and its synthesizing enzyme, aaNAT, constitute at least one circadian output pathway of locomotor activity either as a distinct route or in association with PDF system.

Uncovering the Roles of Clocks and Neural Transmission in the Resilience of Drosophila Circadian Network.

Studies of circadian locomotor rhythms in Drosophila melanogaster gave evidence to the preceding theoretical predictions on circadian rhythms. The molecular oscillator in flies, as in virtually all organisms, operates using transcriptional-translational feedback loops together with intricate post-transcriptional processes. Approximately150 pacemaker neurons, each equipped with a molecular oscillator, form a circuit that functions as the central pacemaker for locomotor rhythms. Input and output pathways to and from the pacemaker circuit are dissected to the level of individual neurons. Pacemaker neurons consist of functionally diverse subclasses, including those designated as the Morning/Master (M)-oscillator essential for driving free-running locomotor rhythms in constant darkness and the Evening (E)-oscillator that drives evening activity. However, accumulating evidence challenges this dual-oscillator model for the circadian circuit organization and propose the view that multiple oscillators are coordinated through network interactions. Here we attempt to provide further evidence to the revised model of the circadian network. We demonstrate that the disruption of molecular clocks or neural output of the M-oscillator during adulthood dampens free-running behavior surprisingly slowly, whereas the disruption of both functions results in an immediate arrhythmia. Therefore, clocks and neural communication of the M-oscillator act additively to sustain rhythmic locomotor output. This phenomenon also suggests that M-oscillator can be a pacemaker or a downstream path that passively receives rhythmic inputs from another pacemaker and convey output signals. Our results support the distributed network model and highlight the remarkable resilience of the Drosophila circadian pacemaker circuit, which can alter its topology to maintain locomotor rhythms.

Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora.

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes (frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation (vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δgtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δgtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δgtr2 is defective in entrainment. In all of these assays, Δgtr2 is similar to Δvta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

Infradian Modulation of Circadian Rhythm in Small Mammals and Birds under Constant Light.

The study examined the infradian rhythms in total motor activity and the periods of "freerunning" circadian rhythm in male C57BL/6 mice and European greenfinch Chloris chloris exposed to constant light. In birds and mice, the motor activity was characterized with inphase 4-day-long rhythm. The periods of free-running circadian rhythms in birds and mice were 23 and 25 h, respectively, and they were modulated with 4-day-long periodicity. The maximal deviations in the period of free-running circadian rhythm from the individual mean levels coincided with the acrophase of 4-day-long rhythm of motor activity in both species.

GmLCLs negatively regulate ABA perception and signalling genes in soybean leaf dehydration response.

The circadian clock allows plants to actively adapt to daily environmental changes through temporal regulation of physiological traits. In response to drought stress, circadian oscillators gate ABA signalling, but the molecular mechanisms remain unknown, especially in crops. Here, we investigated the role of soybean circadian oscillators GmLCLa1, GmLCLa2, GmLCLb1 and GmLCLb2 in leaf water stress response. Under dehydration stress, the GmLCL quadruple mutant had decreased leaf water loss. We found that the dehydration treatment delayed the peak expression of GmLCL genes by 4 hr. In addition, the circadian clock in hairy roots also responded to ABA, which led to a free-running rhythm with shortened period. Importantly, in the gmlclq quadruple mutant, diurnal expression phases of several circadian-regulated ABA receptor, ABA catabolism and ABA signalling-related genes were shifted significantly to daytime. Moreover, in the gmlclq mutant leaf, expression of GmPYL17, GmCYP707A, GmABI2 and GmSnRK2s was increased under water dehydration stress. In summary, our results show that GmLCLs act as negative regulators of ABA signalling in leaves during dehydration response.

Entrainment of circadian rhythms of locomotor activity by ambient temperature cycles in the dromedary camel

In the dromedary camel, a well-adapted desert mammal, daily ambient temperature (Ta)-cycles have been shown to synchronize the central circadian clock. Such entrainment has been demonstrated by examining two circadian outputs, body temperature and melatonin rhythms. Locomotor activity (LA), another circadian output not yet investigated in the camel, may provide further information on such specific entrainment. To verify if daily LA is an endogenous rhythm and whether the desert Ta-cycle can entrain it, six dromedaries were first kept under total darkness and constant-Ta. Results showed that the LA rhythm free runs with a period of 24.8–24.9 h. After having verified that the light–dark cycle synchronizes LA, camels were subjected to a Ta-cycle with warmer temperatures during subjective days and cooler temperatures during subjective nights. Results showed that the free-running LA rhythm was entrained by the Ta-cycle with a period of exactly 24.0 h, while a 12 h Ta-cycle phase advance induced an inversion of the LA rhythm and advanced the acrophase by 9 h. Similarly, activity onset and offset were significantly advanced. All together, these results demonstrate that the Ta-cycle is a strong zeitgeber, able to entrain the camel LA rhythm, hence corroborating previous results concerning the Ta non-photic synchronization of the circadian master clock.

A sleep-like state in Hydra unravels conserved sleep mechanisms during the evolutionary development of the central nervous system.

Sleep behaviors are observed even in nematodes and arthropods, yet little is known about how sleep-regulatory mechanisms have emerged during evolution. Here, we report a sleep-like state in the cnidarian Hydra vulgaris with a primitive nervous organization. Hydra sleep was shaped by homeostasis and necessary for cell proliferation, but it lacked free-running circadian rhythms. Instead, we detected 4-hour rhythms that might be generated by ultradian oscillators underlying Hydra sleep. Microarray analysis in sleep-deprived Hydra revealed sleep-dependent expression of 212 genes, including cGMP-dependent protein kinase 1 (PRKG1) and ornithine aminotransferase. Sleep-promoting effects of melatonin, GABA, and PRKG1 were conserved in Hydra However, arousing dopamine unexpectedly induced Hydra sleep. Opposing effects of ornithine metabolism on sleep were also evident between Hydra and Drosophila, suggesting the evolutionary switch of their sleep-regulatory functions. Thus, sleep-relevant physiology and sleep-regulatory components may have already been acquired at molecular levels in a brain-less metazoan phylum and reprogrammed accordingly.