The Role of Wheel Running in the Coupling of Two Simultaneous Circadian Rhythms of Motor Activity in the Rat

Motor activity is among the non-photic stimuli that act on the internal clock. We have tested the role of motor activity in the circadian pattern of rats under conditions near the lower limits of entrainment, that induce circadian rhythm dissociation. Three groups of 8 rats each were used: a) rats kept individually in 25×25×15 cm cages, b) rats in 50×25×15 cm cages, and c) rats in 50×25×15 cm cages with access to a running wheel. All the rats were kept under light-dark cycles of 22 hours (T22, 11L:11D) for 50 days, after which they were transferred to constant darkness. All the rats without a running wheel showed a motor activity pattern with two statistically significant circadian rhythms in the periodogram of Sokolove and Bushell: one circadian component entrained by the LD cycle, and another free-running. The rats with access to a running wheel showed several patterns: 5 rats showed only one rhythm entrained to the LD cycle, 2 rats showed circadian rhythm dissociation, and 1 showed only a free running rhythm. We believe that the simultaneous manifestation of two circadian components reflects the functional dissociation of the oscillators population that constitutes the circadian pacemaker, of the rat. Physical exercise acts on the pacemaker reinforcing the strongest group of oscillators, which, depending on the structure of the circadian system of the rat, is usually the one entrained to the LD cycle. This study supports the hypothesis that motor activity couples the oscillators that form the circadian system of the rat.     

Dopaminergic Regulation of Circadian Food Anticipatory Activity Rhythms in the Rat

Circadian activity rhythms are jointly controlled by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) and by food-entrainable circadian oscillators (FEOs) located elsewhere. The SCN mediates synchrony to daily light-dark cycles, whereas FEOs generate activity rhythms synchronized with regular daily mealtimes. The location of FEOs generating food anticipation rhythms, and the pathways that entrain these FEOs, remain to be clarified. To gain insight into entrainment pathways, we developed a protocol for measuring phase shifts of anticipatory activity rhythms in response to pharmacological probes. We used this protocol to examine a role for dopamine signaling in the timing of circadian food anticipation. To generate a stable food anticipation rhythm, rats were fed 3h/day beginning 6-h after lights-on or in constant light for at least 3 weeks. Rats then received the D2 agonist quinpirole (1 mg/kg IP) alone or after pretreatment with the dopamine synthesis inhibitor α-methylparatyrosine (AMPT). By comparison with vehicle injections, quinpirole administered 1-h before lights-off (19h before mealtime) induced a phase delay of activity onset prior to the next meal. Delay shifts were larger in rats pretreated with AMPT, and smaller following quinpirole administered 4-h after lights-on. A significant shift was not observed in response to the D1 agonist SKF81297. These results provide evidence that signaling at D2 receptors is involved in phase control of FEOs responsible for circadian food anticipatory rhythms in rats.     

Effects of Pregnancy and Parturition on Free-Running Circadian Activity Rhythms in the Rat

Alterations in circadian rhythms have previously been associated with estrous and seasonal changes in reproductive state. In the present study we explored the effects of the reproductive events of pregnancy and parturition on free-running circadian activity rhythms in the rat. Free-running rhythms were monitored before mating, during pregnancy, and following parturition and removal of pups. Systematic and long-lasting alterations of the period of the free-running activity rhythm were seen following parturition. The effects of estrous, seasonal, and gestational reproductive states on circadian rhythms may be mediated by the endocrine events which accompany these states.     

Effects of Pregnancy and Parturition on Free-Running Circadian Activity Rhythms in the Rat

Alterations in circadian rhythms have previously been associated with estrous and seasonal changes in reproductive state. In the present study we explored the effects of the reproductive events of pregnancy and parturition on free-running circadian activity rhythms in the rat. Free-running rhythms were monitored before mating, during pregnancy, and following parturition and removal of pups. Systematic and long-lasting alterations of the period of the free-running activity rhythm were seen following parturition. The effects of estrous, seasonal, and gestational reproductive states on circadian rhythms may be mediated by the endocrine events which accompany these states.     

Circadian Activity Rhythms and Phase-Shifting of Cultured Neurons of the Rat Suprachiasmatic Nucleus

The mammalian suprachiasmatic nucleus (SCN) is the major endogenous pacemaker that coordinates various daily rhythms including locomotor activity and autonomous and endocrine responses, through a neuronal and humoral influence. In the present study we examined the behavior of dispersed individual SCN neurons obtained from 1- to 3-day-old rats cultured on multi-microelectrode arrays (MEAs). SCN neurons were identified by immunolabeling for the neuropeptides arginine-vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). Single SCN neurons cultured at low density onto an MEA can express firing rate patterns with different circadian phases. In these cultures we observed rarely synchronized firing patterns on adjacent electrodes. This suggests that, in cultures of low cell densities, SCN neurons function as independent pacemakers. To investigate whether individual pacemakers can be influenced independently by phase-shifting stimuli, we applied melatonin (10 pM to 100 nM) for 30 min at different circadian phases and continuously monitored the firing rate rhythms. Melatonin could elicit phase-shifting responses in individual clock cells which had no measurable input from other neurons. In several neurons, phase-shifts occurred with a long delay in the second or third cycle after melatonin treatment, but not in the first cycle. Phase-shifts of isolated SCN neurons were also observed at times when the SCN showed no sensitivity to these phase-shifting stimuli in recordings from brain slices. This finding suggests that the neuronal network plays an essential role in the control of phase-shifts.     

Circadian Rhythms of Rat Pancreatic Acinar Cells

On the basis of the circadian oscillations of the rat’s exocrine pancreatic function and previous reports on concomitant ultrastructural changes in the pancreatic tissue, we analysed stereologically the circadian rhythmicity in the structure of this organ. Twenty-four male Wistar rats, four and a half months old, were singly housed two months before the experiment in a lighting regimen LD=12:12, constant environmental temperature and relative humidity, with food and water ad libitum. The experiment was performed in winter. The rats were randomly divided into 6 balanced groups and killed under ether anesthesia at 6 equidistant time points in 24 hours. The pancreatic tissue was fixed in glutaraldehyde and osmium and embedded in Epon. 1 µm thick sections were examined by light microscopy for the evaluation by stereological methods of: a) volume fractions of the different parenchymal components of the exocrine pancreas; b) surface fractions of acinar cell faces; c) size distribution of acinar cell nuclei, their number per unit tissue volume and their mean diameter. Single cosinor method analysis of the data demonstrated statistically significant circadian rhythms for the volume fraction of the cytoplasm of acinar cells and the volume fractions of pancreatic acini and acinar cells. The volume fraction of the cytoplasm of the rat pancreatic acinar cells undergoes circadian oscillations with the highest values at the end of the light span; this rise precedes the well-known physiological nocturnal surge of pancreatic digestive enzymes. Our findings further support the hypothesis of a close relationship between pancreatic cell structure and its function.     

Circadian Rhythms of Rat Pancreatic Acinar Cells

On the basis of the circadian oscillations of the rat's exocrine pancreatic function and previous reports on concomitant ultrastructural changes in the pancreatic tissue, we analysed stereologically the circadian rhythmicity in the structure of this organ. Twenty-four male Wistar rats, four and a half months old, were singly housed two months before the experiment in a lighting regimen LD=12:12, constant environmental temperature and relative humidity, with food and water ad libitum. The experiment was performed in winter. The rats were randomly divided into 6 balanced groups and killed under ether anesthesia at 6 equidistant time points in 24 hours. The pancreatic tissue was fixed in glutaraldehyde and osmium and embedded in Epon. 1 µm thick sections were examined by light microscopy for the evaluation by stereological methods of: a) volume fractions of the different parenchymal components of the exocrine pancreas; b) surface fractions of acinar cell faces; c) size distribution of acinar cell nuclei, their number per unit tissue volume and their mean diameter. Single cosinor method analysis of the data demonstrated statistically significant circadian rhythms for the volume fraction of the cytoplasm of acinar cells and the volume fractions of pancreatic acini and acinar cells. The volume fraction of the cytoplasm of the rat pancreatic acinar cells undergoes circadian oscillations with the highest values at the end of the light span; this rise precedes the well-known physiological nocturnal surge of pancreatic digestive enzymes. Our findings further support the hypothesis of a close relationship between pancreatic cell structure and its function.     

Circadian Activity Rhythms and Phase‐Shifting of Cultured Neurons of the Rat Suprachiasmatic Nucleus

The mammalian suprachiasmatic nucleus (SCN) is the major endogenous pacemaker that coordinates various daily rhythms including locomotor activity and autonomous and endocrine responses, through a neuronal and humoral influence. In the present study we examined the behavior of dispersed individual SCN neurons obtained from 1‐ to 3‐day‐old rats cultured on multi‐microelectrode arrays (MEAs). SCN neurons were identified by immunolabeling for the neuropeptides arginine‐vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). Single SCN neurons cultured at low density onto an MEA can express firing rate patterns with different circadian phases. In these cultures we observed rarely synchronized firing patterns on adjacent electrodes. This suggests that, in cultures of low cell densities, SCN neurons function as independent pacemakers. To investigate whether individual pacemakers can be influenced independently by phase‐shifting stimuli, we applied melatonin (10 pM to 100 nM) for 30 min at different circadian phases and continuously monitored the firing rate rhythms. Melatonin could elicit phase‐shifting responses in individual clock cells which had no measurable input from other neurons. In several neurons, phase‐shifts occurred with a long delay in the second or third cycle after melatonin treatment, but not in the first cycle. Phase‐shifts of isolated SCN neurons were also observed at times when the SCN showed no sensitivity to these phase‐shifting stimuli in recordings from brain slices. This finding suggests that the neuronal network plays an essential role in the control of phase‐shifts.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Circadian Discrimination of Reward: Evidence for Simultaneous Yet Separable Food- and Drug-Entrained Rhythms in the Rat

A unique extra-suprachiasmatic nucleus (SCN) oscillator, operating independently of the light-entrainable oscillator, has been hypothesized to generate feeding and drug-related rhythms. To test the validity of this hypothesis, sham-lesioned (Sham) and SCN-lesioned (SCNx) rats were housed in constant dim-red illumination (LL_red) and received a daily cocaine injection every 24?h for 7 d (Experiment 1). In a second experiment, rats underwent 3-h daily restricted feeding (RF) followed 12 d later by the addition of daily cocaine injections given every 25?h in combination with RF until the two schedules were in antiphase. In both experiments, body temperature and total activity were monitored continuously. Results from Experiment 1 revealed that cocaine, but not saline, injections produced anticipatory increases in temperature and activity in SCNx and Sham rats. Following withdrawal from cocaine, free-running temperature rhythms persisted for 2–10 d in SCNx rats. In Experiment 2, robust anticipatory increases in temperature and activity were associated with RF and cocaine injections; however, the feeding periodicity (23.9?h) predominated over the cocaine periodicity. During drug withdrawal, the authors observed two free-running rhythms of temperature and activity that persisted for >14 d in both Sham and SCNx rats. The periods of the free-running rhythms were similar to the feeding entrainment (period?=?23.7 and 24.0?h, respectively) and drug entrainment (period?=?25.7 and 26.1?h, respectively). Also during withdrawal, the normally close correlation between activity and temperature was greatly disrupted in Sham and SCNx rats. Taken together, these results do not support the existence of a single oscillator mediating the rewarding properties of both food and cocaine. Rather, they suggest that these two highly rewarding behaviors can be temporally isolated, especially during drug withdrawal. Under stable dual-entrainment conditions, food reward appears to exhibit a slightly greater circadian influence than drug reward. The ability to generate free-running temperature rhythms of different frequencies following combined food and drug exposures could reflect a state of internal desynchrony that may contribute to the addiction process and drug relapse. (Author correspondence: heiko@vetmed.wsu.edu)     

Simultaneous Recording of Circadian Rhythms of Brain and Intraperitoneal Temperatures and Locomotor and Drinking Activities in the Rat

In order to investigate the circadian oscillatory system, the present study performed simultaneous and continuous recordings of brain and intraperitoneal temperatures, drinking and locomotion in rats under light-dark (LD) cycles and continuous dim illumination (dim LL) for a total period of 16 days. Compared to circadian amplitudes under LD, those under dim LL were significantly reduced by 34% for drinking and 50% for locomotion, but were not for brain and intraperitoneal temperatures. On the other hand, means of steady circadian periods during last 10 days under dim LL were all within a close range between 24.2 and 24.3 h in these rhythms. Besides the steady periods, one rat exhibited weak circadian period of 23.7 and 24.6 h, but these multiple frequencies were also equally observed in the four rhythms. The similarity in the periodicities suggests that these temperature and activity rhythms might be driven by a common oscillatory system. Therefore differential reductions in the amplitudes of drinking and loc omotor rhythms might be caused by a masking effect of dim LL on their rhythm output-pathways. Hence rats may temporally coordinate various physiological and behavioral functions by such clock system under time cue free environment.     

Simultaneous Recording of Circadian Rhythms of Brain and Intraperitoneal Temperatures and Locomotor and Drinking Activities in the Rat

In order to investigate the circadian oscillatory system, the present study performed simultaneous and continuous recordings of brain and intraperitoneal temperatures, drinking and locomotion in rats under light-dark (LD) cycles and continuous dim illumination (dim LL) for a total period of 16 days. Compared to circadian amplitudes under LD, those under dim LL were significantly reduced by 34% for drinking and 50% for locomotion, but were not for brain and intraperitoneal temperatures. On the other hand, means of steady circadian periods during last 10 days under dim LL were all within a close range between 24.2 and 24.3 h in these rhythms. Besides the steady periods, one rat exhibited weak circadian period of 23.7 and 24.6 h, but these multiple frequencies were also equally observed in the four rhythms. The similarity in the periodicities suggests that these temperature and activity rhythms might be driven by a common oscillatory system. Therefore differential reductions in the amplitudes of drinking and loc omotor rhythms might be caused by a masking effect of dim LL on their rhythm output-pathways. Hence rats may temporally coordinate various physiological and behavioral functions by such clock system under time cue free environment.     

The Effect of Old Age on the Free-Running Period of Circadian Rhythms in Rat

The free-running period is regarded to be an exclusive feature of the endogenous circadian clock. Changes during aging in the free-running period may therefore reflect age-related changes in the internal organization of this clock. However, the literature on alterations in the free-running period in aging is not unequivocal. In the present study, with various confounding factors kept to a minimum, it was found that the free-running periods for active wakefulness, body temperature, and drinking behavior were significantly shorter (by 12-17 min) in old than in young rats. In addition, it was found that the day-to-day stability of the different sleep states was reduced in old rats, whereas that of the drinking rhythm was enhanced. Transient cycles were not observed, nor were there any age-related differences in daily totals of the various sleep-wake states. The amplitudes of the circadian rhythms of active wakefulness, quiet sleep, and temperature were reduced, whereas those of paradoxical sleep and quiet wakefulness remained unchanged.     

Circadian Rhythms of Locomotor Activity in the Nile Tilapia Oreochromis niloticus

Behavioral rhythms of the Nile tilapia were investigated to better characterize its circadian system. To do so, the locomotor activity patterns of both male and female tilapia reared under a 12:12 h light-dark (LD) cycle were studied, as well as in males the existence of endogenous rhythmicity under free-running conditions (DD and 45 min LD pulses). When exposed to an LD cycle, the daily pattern of activity differed between individuals: some fish were diurnal, some nocturnal, and a few displayed an arrhythmic pattern. This variability would be typical of the plastic circadian system of fish. Moreover, reproductive events clearly affected the behavioral rhythms of female tilapia, a mouth-brooder teleost species. Under DD, 50% (6 of 12) of male fish showed circadian rhythms with an average period (τ) of 24.1±0.2 h, whereas under the 45 min LD pulses, 58% (7 of 12) of the fish exhibited free-running activity rhythms with an average τ of 23.9±0.5 h. However, interestingly in this case, activity was always confined to the dark phase. Furthermore, when the LD cycle was reversed, a third of the fish showed gradual resynchronization to the new phase, taking 7–10 days to be completely re-entrained. Taken together, these results suggest the existence of an endogenous circadian oscillator that controls the expression of locomotor activity rhythms in the Nile tilapia, although its anatomical localization remains unknown.     

Secondary Rhythms of Cardiac Activity in Rat Ontogeny

Endogenous periodic oscillations of the heart beat rate are described in rat pups aged 3–4, 7–8, 10–11, 13–14, 21–22 days and 1.5 month after birth. These oscillations have all characteristic features established earlier for the secondary rhythms of endogenous contractile activity in the wall of various regions of the gastrointestinal tract and for bursts of spontaneous somatomotor excitation in the early postnatal ontogeny of rats: a multi-stage organization, inconstancy, irregularity of components. In frequency spectra of secondary oscillations of the heart rate obtained by means of fast Fourier transform of R–R intervals of the periodogram, age-related changes of the spectral frequency power are demonstrated in 4 ranges, 0.01–0.03, 0.03–0.1, 0.1–1.0, and 1.0–2.5 Hz, which correspond to the about-one-minute, decasecond, and about-one-second waves of the heart rhythm oscillations and to sinus arrhythmia. It is shown that the dominating frequencies of the secondary rhythms in each range do not have regular age-related changes, which is characteristic of all endogenous secondary rhythms.     

Circadian Locomotor Activity Rhythms in the African Clawed Frog, Xenopus laevis: The Role of the Eye and the Hypothalamus

The effects of hypothalamic lesioning and removal of the eyes on locomotor activity rhythms of African clawed frog, Xenopus laevis were examined under light-dark cycles (LD12:12) and constant conditions. Frogs were kept individually and the activity rhythms at the bottom layer of water tank were recorded by means of the infrared photocells. Intact frogs displayed clear entrained nocturnal activity and expressed freerunning activity rhythms in constant darkness (DD), while some frogs did not freerun under co nstant dim light (dimLL) and constant light (LL). Freerunning periods in intact frogs were significantly shorter in dimLL than in DD. Although freerunning periods were shortened after blinding in same individuals, no significant changes in the freerunning periods were observed after blinding under dimLL and LL. When electrolytic lesions to the hypothalamus were performed, all frogs with more than 70% damage of the SCN abolished freerunning rhythms and in frogs with less than 70% damage, 57% of the animals became arrhythmic. In conclusion, (1) There is a circadian pacemaker somewhere outside the eyes, and it is probably situated in the hypothalamusincluding the SCN. (2) Both the eyes and the SCN are involved in the circadian system of the frogs.     

Circadian Locomotor Activity Rhythms in the African Clawed Frog,Xenopus laevis: The Role of the Eye and the Hypothalamus

The effects of hypothalamic lesioning and removal of the eyes on locomotor activity rhythms of African clawed frog, Xenopus laevis were examined under light-dark cycles (LD12:12) and constant conditions. Frogs were kept individually and the activity rhythms at the bottom layer of water tank were recorded by means of the infrared photocells. Intact frogs displayed clear entrained nocturnal activity and expressed freerunning activity rhythms in constant darkness (DD), while some frogs did not freerun under co nstant dim light (dimLL) and constant light (LL). Freerunning periods in intact frogs were significantly shorter in dimLL than in DD. Although freerunning periods were shortened after blinding in same individuals, no significant changes in the freerunning periods were observed after blinding under dimLL and LL. When electrolytic lesions to the hypothalamus were performed, all frogs with more than 70% damage of the SCN abolished freerunning rhythms and in frogs with less than 70% damage, 57% of the animals became arrhythmic. In conclusion, (1) There is a circadian pacemaker somewhere outside the eyes, and it is probably situated in the hypothalamusincluding the SCN. (2) Both the eyes and the SCN are involved in the circadian system of the frogs.     

On the Role of Histamine Receptors in the Regulation of Circadian Rhythms

Several lines of evidence suggest a regulatory role of histamine in circadian rhythms, but little is known about signaling pathways that would be involved in such a putative role. The aim of this study was to examine whether histamine mediates its effects on the circadian system through Hrh1 or Hrh3 receptors. We assessed both diurnal and free-running locomotor activity rhythms of Hrh1 ^-/- and Hrh3 ^-/- mice. We also determined the expression of Per1, Per2 and Bmal1 genes in the suprachiasmatic nuclei, several areas of the cerebral cortex and striatum under symmetric 24 h light-dark cycle at zeitgeber times 14 and 6 by using radioactive in situ hybridization. We found no differences between Hrh1 ^-/- and wild type mice in the length, amplitude and mesor of diurnal and free-running activity rhythms as well as in expression of Per1, Per2 and Bmal1 genes in any of the examined brain structures. The amplitude of free-running activity rhythm of the Hrh3 ^-/- mice was significantly flattened, whereas the expression of the clock genes in Hrh3 ^-/- mice was similar to the wild type animals in all of the assessed brain structures. Therefore, the knockout of Hrh1 receptor had no effects on the circadian rhythm of spontaneous locomotion, and a knockout of Hrh3 receptor caused a substantial reduction of free-running activity rhythm amplitude, but none of these knockout models affected the expression patterns of the core clock genes in any of the studied brain structures.