Temporal phasing of locomotor activity, heart rate rhythmicity, and core body temperature is disrupted in VIP receptor 2-deficient mice

Neurons of the brain's biological clock located in the hypothalamic suprachiasmatic nucleus (SCN) generate circadian rhythms of physiology (core body temperature, hormone secretion, locomotor activity, sleep/wake, and heart rate) with distinct temporal phasing when entrained by the light/dark (LD) cycle. The neuropeptide vasoactive intestinal polypetide (VIP) and its receptor (VPAC2) are highly expressed in the SCN. Recent studies indicate that VIPergic signaling plays an essential role in the maintenance of ongoing circadian rhythmicity by synchronizing SCN cells and by maintaining rhythmicity within individual neurons. To further increase the understanding of the role of VPAC2 signaling in circadian regulation, we implanted telemetric devices and simultaneously measured core body temperature, spontaneous activity, and heart rate in a strain of VPAC2-deficient mice and compared these observations with observations made from mice examined by wheel-running activity. The study demonstrates that VPAC2 signaling is necessary for a functional circadian clock driving locomotor activity, core body temperature, and heart rate rhythmicity, since VPAC2-deficient mice lose the rhythms in all three parameters when placed under constant conditions (of either light or darkness). Furthermore, although 24-h rhythms for three parameters are retained in VPAC2-deficient mice during the LD cycle, the temperature rhythm displays markedly altered time course and profile, rising earlier and peaking ～4-6 h prior to that of wild-type mice. The use of telemetric devices to measure circadian locomotor activity, temperature, and heart rate, together with the classical determination of circadian rhythms of wheel-running activity, raises questions about how representative wheel-running activity may be of other behavioral parameters, especially when animals have altered circadian phenotype.     

Circadian periodicity of cerebral blood flow revealed by laser-Doppler flowmetry in awake rats: relation to blood pressure and activity

Cardiovascular parameters such as arterial blood pressure (ABP) and heart rate display pronounced circadian variation. The present study was performed to detect whether there is a circadian periodicity in the regulation of cerebral perfusion. Normotensive Sprague-Dawley rats (SDR, approximately 15 wk old) and hypertensive (mREN2)27 transgenic rats (TGR, approximately 12 wk old) were instrumented in the abdominal aorta with a blood pressure sensor coupled to a telemetry system for continuous recording of ABP, heart rate, and locomotor activity. After 5-12 days, a laser-Doppler flow (LDF) probe was attached to the skull by means of a guiding device to measure changes in brain cortical blood flow (CBF). After the animals recovered from anesthesia, measurements were taken for 3-4 days. The time series were analyzed with respect to the midline estimating statistic of rhythm (i.e., mean value of a periodic event after fit to a cosine function), amplitude, and acrophase (i.e., phase angle that corresponds to the peak of a given period) of the 24-h period. The LDF signal displayed a significant circadian rhythm, with the peak occurring at around midnight in SDR and TGR, despite inverse periodicity of ABP in TGR. This finding suggests independence of LDF periodicity from ABP regulation. Furthermore, the acrophase of the LDF was consistently found before the acrophase of the activity. From the present data, it is concluded that there is a circadian periodicity in the regulation of cerebral perfusion that is independent of circadian changes in ABP and probably is also independent of locomotor activity. The presence of a circadian periodicity in CBF may have implications for the occurrence of diurnal alterations in cerebrovascular events in humans.     

Complete suprachiasmatic lesions eliminate circadian rhythmicity of body temperature and locomotor activity in golden hamsters

The effects of suprachiasmatic and control lesions on the circadian rhythms of locomotor activity and body temperature were studied in golden hamsters (Mesocricetus auratus) maintained in constant light as well as constant darkness. Large suprachiasmatic lesions, but not control lesions, eliminated circadian rhythmicity in locomotor activity as well as in body temperature. Analysis of the robustness of the rhythms of locomotor activity and body temperature in unlesioned and lesioned animals suggests that, because body temperature rhythmicity is more robust than locomotor rhythmicity, lesions that spare a small number of suprachiasmatic cells might abolish the latter but not the former. Our results do not support the hypothesis that the body temperature rhythm is controlled by a circadian pacemaker distinct from the main pacemaker located in the suprachiasmatic nuclei.     

Persistence of circadian variation in arterial blood pressure in beta1/beta2-adrenergic receptor-deficient mice

The beta-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in beta1/beta2-adrenergic receptor-deficient mice (beta1/beta2ADR-/-) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four-hour recordings of MAP, heart rate (HR), and locomotor activity were made in conscious 16- to 17-wk-old mice [wild-type, (WT), n = 7; beta1/beta2ADR-/-, n = 10] by telemetry. Both WT and beta1/beta2ADR-/- mice demonstrated robust circadian variation in MAP and HR, although 24-h mean MAP was 10% lower (102.02 +/- 1.81 vs. 92.11 +/- 2.62 mmHg) in beta1/beta2ADR-/- than WT, HR was 16% lower and day-night differences reduced. Both WT and beta1/beta2ADR-/- mice adapted to changed salt intake without changed MAP. However, the beta1/beta2ADR-/- mice demonstrated a striking reduction in locomotor activity in light and dark phases of the day. In WT mice, MAP was markedly affected by locomotor activity, resulting in bimodal distributions in both light and dark. When MAP was analyzed using only intervals without locomotor activity, bimodality and circadian differences were reduced, and there was no significant difference between the two genotypes. The results indicate that there is no direct effect or role for the beta-adrenergic system in circadian variation of arterial pressure in mice, aside from the indirect consequences of altered locomotor activity. Our results also confirm that locomotor activity contributes strongly to circadian variation in blood pressure in mice.     

Entraining signals initiate behavioral circadian rhythmicity in larval zebrafish

The authors show that a circadian clock that regulates locomotor activity in larval zebrafish develops gradually over the first 4 days of life and that exposure to entraining signals late in embryonic development is necessary for initiation of robust behavioral rhythmicity. When zebrafish larvae were transferred from a light-dark (LD) cycle to constant darkness (DD) on the third or fourth day postfertilization, the locomotor activity of almost all fish was rhythmic on days 5 to 9 postfertilization, with peak activity occurring during the subjective day. Rhythm amplitude was higher after four LD cycles than after three LD cycles. When embryos were transferred from LD to DD on the second day postfertilization, only about half of the animals later displayed statistically significant activity rhythms. These rhythms were noisier and of lower amplitude, but phased normally. When zebrafish were raised in DD beginning at 14 h postfertilization, only 22% of them expressed significant circadian rhythmicity as larvae. These rhythms were of low amplitude and phase-locked to the time of handling on the third day rather than to the maternal LD cycle. These results show that behavioral rhythmicity in zebrafish is regulated by a pacemaking system that is sensitive to light by the second day of embryogenesis but continues to develop into the fourth day. This pacemaking system requires environmental signals to initiate or synchronize circadian rhythmicity.     

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 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.     

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.     

Biological clock function is linked to proactive and reactive personality types

Background

Many physiological processes in our body are controlled by the biological clock and show circadian rhythmicity. It is generally accepted that a robust rhythm is a prerequisite for optimal functioning and that a lack of rhythmicity can contribute to the pathogenesis of various diseases. Here, we tested in a heterogeneous laboratory zebrafish population whether and how variation in the rhythmicity of the biological clock is associated with the coping styles of individual animals, as assessed in a behavioural assay to reliably measure this along a continuum between proactive and reactive extremes.

Results

Using RNA sequencing on brain samples, we demonstrated a prominent difference in the expression level of genes involved in the biological clock between proactive and reactive individuals. Subsequently, we tested whether this correlation between gene expression and coping style was due to a consistent change in the level of clock gene expression or to a phase shift or to altered amplitude of the circadian rhythm of gene expression. Our data show a remarkable individual variation in amplitude of the clock gene expression rhythms, which was also reflected in the fluctuating concentrations of melatonin and cortisol, and locomotor activity. This variation in rhythmicity showed a strong correlation with the coping style of the individual, ranging from robust rhythms with large amplitudes in proactive fish to a complete absence of rhythmicity in reactive fish. The rhythmicity of the proactive fish decreased when challenged with constant light conditions whereas the rhythmicity of reactive individuals was not altered.

Conclusion

These results shed new light on the role of the biological clock by demonstrating that large variation in circadian rhythmicity of individuals may occur within populations. The observed correlation between coping style and circadian rhythmicity suggests that the level of rhythmicity forms an integral part of proactive or reactive coping styles.

     

Emergence of circadian and photoperiodic system level properties from interactions among pacemaker cells

Daily patterns of behavior and physiology in animals in temperate zones often differ substantially between summer and winter. In mammals, this may be a direct consequence of seasonal changes of activity of the suprachiasmatic nucleus (SCN). The purpose of this study was to understand such variation on the basis of the interaction between pacemaker neurons. Computer simulation demonstrates that mutual electrical activation between pacemaker cells in the SCN, in combination with cellular electrical activation by light, is sufficient to explain a variety of circadian phenomena including seasonal changes. These phenomena are: self-excitation, that is, spontaneous development of circadian rhythmicity in the absence of a light-dark cycle; persistent rhythmicity in constant darkness, and loss of circadian rhythmicity in pacemaker output in constant light; entrainment to light-dark cycles; aftereffects of zeitgeber cycles with different periods; adjustment of the circadian patterns to day length; generation of realistic phase response curves to light pulses; and relative independence from day-to-day variation in light intensity. In the model, subsets of cells turn out to be active at specific times of day. This is of functional importance for the exploitation of the SCN to tune specific behavior to specific times of day. Thus, a network of on-off oscillators provides a simple and plausible construct that behaves as a clock with readout for time of day and simultaneously as a clock for all seasons.     

Effect of long-term exposure to constant dim light on the circadian system of rats

The newly discovered multi-oscillatory nature of the mammalian circadian clock system and the cloning of the genes involved in the molecular mechanism that generates circadian rhythmicity have opened new approaches for understanding how mammals are temporally organized and how the mammalian circadian system reacts to the lack of normal synchronization cues. In the present study we investigated the effects of long-term exposure to constant red dim light on the pattern of the expression of Period 1 in the suprachiasmatic nuclei of the hypothalamus and of Arylalkylamine N-acetyltransferase(Aa-nat) in the retina and pineal gland. Our data demonstrate that Period 1 mRNA expression in the suprachiasmatic nuclei of the hypothalamus was not affected by exposure to constant red dim light for 60 days, whereas Aa-nat mRNA expression in the retina and in the pineal gland was significantly affected, since in some animals (20-30%) Aa-nat mRNA levels were found to be higher during the subjective day. A circadian rhythm of serum melatonin and locomotor activity was present in all the animals tested. In 4 animals serum melatonin levels were high during the subjective day. Our data suggest that long-term exposure to constant red dim light may induce desynchronization between the circadian rhythm of locomotor activity and serum melatonin levels.     

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.     

Seasonal crayfish activity as influenced by fluctuating water levels and presence of a fish predator

The diel rhythm of juvenile (0+) Astacus astacus was studied experimentally with constant and dielly fluctuating water levels. In the first case the diel rhythm was mainly crepescular, but with tendencies of a phase shift towards diurnal rhythm during low light conditions. The locomotor activity increased with temperature and with the length of the dark period. In presence of a crepescular predator the crayfish decreased their light period activity.
With fluctuating water levels the activity increased especially when the water level changes occurred during normal resting periods. Crayfish did not show any adaptation to fluctuating water levels by preferring covers constantly situated beneath the surface. The relative effect of the water level fluctuations increased with temperature.
As the main part of the juvenile crayfish population is concentrated to the litoral zone, diel water level regulations are thought to be detrimental to crayfish populations even if the regulation amplitude is small.     

BRAIN PHOTORECEPTOR PATHWAYS CONTRIBUTING TO CIRCADIAN RHYTHMICITY IN CRAYFISH

Freshwater crayfish have three known photoreceptive systems: the compound eyes, extraretinal brain photoreceptors, and caudal photoreceptors. The primary goal of the work described here was to explore the contribution of the brain photoreceptors to circadian locomotory activity and define some of the underlying neural pathways. Immunocytochemical studies of the brain photoreceptors in the parastacid (southern hemisphere) crayfish Cherax destructor reveal their expression of the blue light-sensitive photopigment cryptochrome and the neurotransmitter histamine. The brain photoreceptors project to two small protocerebral neuropils, the brain photoreceptor neuropils (BPNs), where they terminate among fibers expressing the neuropeptide pigment-dispersing hormone (PDH), a signaling molecule in arthropod circadian systems. Comparable pathways are also described in the astacid (northern hemisphere) crayfish Procambarus clarkii. Despite exhibiting markedly different diurnal locomotor activity rhythms, removal of the compound eyes and caudal photoreceptors in both C. destructor and P. clarkii (leaving the brain photoreceptors intact) does not abolish the normal light/dark activity cycle in either species, nor prevent the entrainment of their activity cycles to phase shifts of the light/dark period. These results suggest, therefore, that crayfish brain photoreceptors are sufficient for the entrainment of locomotor activity rhythms to photic stimuli, and that they can act in the absence of the compound eyes and caudal photoreceptors. We also demonstrate that the intensity of PDH expression in the BPNs varies in phase with the locomotor activity rhythm of both crayfish species. Together, these findings suggest that the brain photoreceptor cells can function as extraretinal circadian photoreceptors and that the BPN represents part of an entrainment pathway synchronizing locomotor activity to environmental light/dark cycles, and implicating the neuropeptide PDH in these functions. (Author correspondence: bbeltz@wellesley.edu)     

Influences of phase delay shifts of light and food restriction on blood pressure and heart rate in telemetry monitored rats

Effects of phase delay shifts (PDS) of light in combination with moderately increased salt intake (SL) (2%) or time restriction of food (FR) during the light-time (passive phase) on heart rate (HR), blood pressure (BP) and locomotor activity (LA) in radiotelemetry-measured rats were evaluated. PDS decreased amplitude and spectral power of circadian oscillations of HR, BP and LA. Moderately increased SL did not interfere with the circadian rhythmicity of HR, BP or LA. A prominent decrease in amplitude and spectral power of circadian oscillations was observed if food was available during the lighttime. Combination of PDS with FR split cardiovascular and behavioural parameters. In conclusion, food availability during the light-time in combination with PDS decreased amplitude and spectral power of circadian oscillations of BP, HR and LA more than PDS only. Different response of cardiovascular and behavioural parameters to photic and non-photic stimuli can have consequences for shift workers.     

Circadian locomotor rhythms, but not photoperiodic responses, survive surgical isolation of the SCN in hamsters.

Surgical isolation of the suprachiasmatic nuclei (SCN) within a hypothalamic island is reported to produce loss of circadian rhythmicity. The results have been interpreted to indicate that SCN efferents are necessary for the expression of circadian rhythms. It is not clear, however, whether the loss of circadian rhythms in behavioral responses following SCN isolation is attributable to transection of efferents, to loss of cells within the island, or to gliosis produced by the knife cut. To explore this issue, we examined locomotor activity and gonadal state of male golden hamsters housed in constant darkness (DD, with a dim red light for maintenance) for at least 10 weeks following isolation of the SCN from the rest of the brain by cuts by means of a Halasz wire microknife. Brain sections were immunocytochemically stained for the peptides vasoactive intestinal polypeptide (VIP), vasopressin (VP) or neurophysin II (NP II), and neuropeptide Y (NPY) to localize the SCN and to assess its viability, and for glial fibrillary acidic protein (GFAP) to delimit the border of the knife cut. Experimental animals with VIP and VP/NP II immunoreactivity in the SCN within the island retained free-running locomotor rhythms following transection of SCN efferents. Animals with cuts that failed to sever SCN efferents, and sham-operated animals (in which the Halasz knife was lowered but not rotated), also maintained circadian rhythmicity. Hamsters sustaining severe damage to the SCN showed disrupted locomotor activity. In those hamsters that retained circadian locomotor rhythmicity following SCN isolation, gonads failed to regress in DD, demonstrating the absence of an appropriate photoperiodic response. The results suggest a multiplicity of SCN coupling mechanisms in the control of circadian rhythms.     

Endogenous cardiac activity rhythms of continental slope Nephrops norvegicus (decapoda: nephropidae)

The endogenous cardiac activity rhythm of the Norway lobster Nephrops norvegicus was studied under constant conditions of darkness by means of a computer-aided monitoring system (CAPMON). Time series recordings of the heart rate (beats min^?1) were obtained from 47 adult males freshly collected from the continental slope (400–430?m) in the western Mediterranean. Periodogram analysis revealed the occurrence of circadian periodicity (of around 24?h) in most cases. A large percentage of animals showed significant ultradian periods (of around 12 and 18?h). The analysis of the circadian time series revealed the occurrence of peaks of heart rate activity during the expected night phase of the cycle. These results are discussed in relation to the emergence and locomotor activity rhythms of the species.     

Aggression and competition for shelter between a native and an introduced crayfish in Europe

1. The introduced North American crayfish, Pacifastacus leniusculus Dana, is expanding its range in Europe and locally often replaces two native crayfish species, Astacus astacus L. and Austropotamobius pallipes Lereboullet. Pacifastacus leniusculus is also expected to invade the habitat of a third native crayfish, the endangered Austropotamobius torrentium Schrank. Interspecific aggressive interactions and competition for shelter were experimentally studied in the laboratory to assess the potential impact of P. leniusculus on A. torrentium .
2. Neither species was inherently dominant in aggressive interactions, but dominance was strongly size-dependent, favouring the larger and faster growing species, P. leniusculus .
3. Access to limited shelter was generally determined by aggressive dominance, although species-specific preference also influenced the outcome of competition for shelter. Austropotamobius torrentium had a higher preference for experimental shelters and often defended these even against larger P. leniusculus .
4. In accordance with theoretical models of animal conflicts, agonistic interactions between equally sized contestants were more severe than between animals of different size.
5. The P. leniusculus used in the present experiment were infected with the crayfish plague, Aphanomyces astaci , to which the animals are resistant. The crayfish transmitted the disease to non-resistant A. torrentium which died ≈ 2 weeks after contact with P. leniusculus .     

Interaction of endogenous and exogenous factors controlling locomotor activity rhythms in Carcinus exposed to tidal salinity cycles

The shore crab Carcinus maenas (L.) reported hitherto not to express endogenous circatidal rhythmicity in winter, is shown not to lose the ability to express such rhythmicity. Crabs maintained in constant reduced salinity in winter exhibit circatidal and circadian rhythms similar to the normal endogenous rhythms of summer caught crabs.In sinusoidal changes of salinity of tidal periodicity, reductions of salinity and increases to ambient sea water induced increased locomotor activity. The former were purely exogenous responses but the latter were also observed to entrain the underlying endogenously controlled circatidal pattern of behaviour.The occurrence of separate exogenous and endogenous responses to different phases of imposed salinity cycles has implications when seeking to understand rhythmic locomotor activity of crabs on the shore and in the search for components of the underlying physiological clock mechanism.