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.     

Relationships Between Behavioral Rhythms,Plasma Corticosterone and Hypothalamic Circadian Rhythms

Circadian rhythms in physiological processes and behaviors were compared with hypothalamic circadian rhythms in norepinephrine (NE) metabolites, adrenergic transmitter receptors, cAMP, cGMP and suprachiasmatic nucleus (SCN) arginine vasopressin (AVP) in a single population of rats under D: D conditions. Eating, drinking and locomotor activity were high during the subjective night (the time when lights were out in L: D) and low during the subjective day (the time when lights were on in L: D). Plasma corticosterone concentration rose at subjective dusk and remained high until subjective dawn. Binding to hypothalamic α₁- and β-adrenergic receptors also peaked during the subjective night. Cyclic cGMP concentration was elevated throughout the 24-hr period except for a trough at dusk, whereas DHPG concentration peaked at dawn. Arginine vasopressin levels in the suprachiasmatic nucleus peaked in the middle of the day. No rhythm was found either in binding to the α₂-adrenergic receptor, or in MHPG or cAMP concentration. Behavioral and corticosterone rhythms, therefore, are parallel to rhythms in hypothalamic α₁-and β-receptor binding and NE-release. Cyclic GMP falls only at dusk, suggesting the possibility that cGMP inhibits activity much of the day and that at dusk the inhibition of nocturnal activity is removed. SCN AVP, on the other hand, peaking at 1400 hr, may play a role in the pacemaking function of the SCN that drives these other rhythms.     

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 Rhythms of Oxidative Phosphorylation: Effects of Rotenone and Melatonin on Isolated Rat Brain Mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.     

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.     

Influence of Light Intensity on Plasma Melatonin and Locomotor Activity Rhythms in Tench

Melatonin production by the pineal organ is influenced by light intensity, as has been described in most vertebrate species, in which melatonin is considered a synchronizer of circadian rhythms. In tench, strict nocturnal activity rhythms have been described, although the role of melatonin has not been clarified. In this study we investigated daily activity and melatonin rhythms under 12∶12 light‐dark (LD) conditions with two different light intensities (58.6 and 1,091 µW/cm²), and the effect of 1 h broad spectrum white light pulses of different intensities (3.3, 5.3, 10.5, 1,091.4 µW/cm²) applied at middarkness (MD) on nocturnal circulating melatonin. The results showed that plasma melatonin in tench under LD 12∶12 and high light conditions displayed rhythmic variation, where values at MD (255.8±65.9 pg/ml) were higher than at midlight (ML) (70.7±31.9 pg/ml). Such a difference between MD and ML values was reduced in animals exposed to LD 12∶12 and low light intensity. The application of 1 h light pulses at MD lowered plasma melatonin to 111.6±3.2 pg/ml (in the 3.3–10.5 µW/cm² range) and to 61.8±18.3 pg/ml (with the 1,091.4 µW/cm² light pulse) and totally suppressed nocturnal locomotor activity. These results show that melatonin rhythms persisted in tench exposed to low light intensity although the amplitude of the rhythm is affected. In addition, it was observed that light pulses applied at MD affected plasma melatonin content and locomotor activity. Such a low threshold suggests that the melatonin system is capable of transducing light even under dim conditions, which may be used by this nocturnal fish to synchronize to weak night light signals (e.g., moonlight cycles).     

Reduced Neurophysin Immunoreactivity in Rat Suprachiasmatic Nucleus Parallels Dissociation of Circadian Feeding Rhythm in Constant Light

Several distinct neuronal populations can be outlined in the suprachiasmatic nucleus (SCN) by employing immunohistochemistry. Understanding their interaction may serve as the key to the processes involved in the generation of circadian rhythms by the SCN. 15 adult rats were exposed to constant dim light (LL) and 3 animals as controls to an LD 12:12 light schedule over 140 days. When sacrificed 10 of the LL-animals had lost their circadian feeding rhythm while 5 were free-running and the controls kept an entrained rhythm. The brains were immunohistochemically stained for myelin basic protein, neurophysin (NPH), vasoactive intestinal peptide, neuropeptide Y, synaptophysin and the leucocyte epitopes FAL and HNK-1. Demarcation of intensely and very intensely stained NPH-positive areas by subjective gray-level-discrimination and computerized area measurement revealed that in rhythmic rats (n=8) the areas containing the stained material were twice as large (0.06 ± 0.03 mm2 vs. 0.028 ± 0.027 mm2; p=0.05) than in arrhythmic animals. It is hypothesized that low NPH-contents in arrhythmic animals reflect arrest of the ‘clockwork’ in the SCN at circadian time 12:00.     

Phosphorylation of mCRY2 at Ser557 in the Hypothalamic Suprachiasmatic Nucleus of the Mouse

Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557-phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase-3β (GSK-3β), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557-phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.     

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)     

Fractal Stochastic Modeling of Spiking Activity in Suprachiasmatic Nucleus Neurons

Individual neurons in the suprachiasmatic nucleus (SCN), the master biological clock in mammals, autonomously produce highly complex patterns of spikes. We have shown that most (~90%) SCN neurons exhibit truly stochastic interspike interval (ISI) patterns. The aim of this study was to understand the stochastic nature of the firing patterns in SCN neurons by analyzing the ISI sequences of 150 SCN neurons in hypothalamic slices. Fractal analysis, using the periodogram, Fano factor, and Allan factor, revealed the presence of a 1/f-type power-law (fractal) behavior in the ISI sequences. This fractal nature was persistent after the application of the GABA_A receptor antagonist bicuculline, suggesting that the fractal stochastic activity is an intrinsic property of individual SCN neurons. Based on these physiological findings, we developed a computational model for the stochastic SCN neurons to find that their stochastic spiking activity was best described by a gamma point process whose mean firing rate was modulated by a fractal binomial noise. Taken together, we suggest that SCN neurons generate temporal spiking patterns using the fractal stochastic point process.Action Editor: Carson C. Chow     

Circadian Rhythms, Jet Lag, and Chronobiotics: An Overview

This overview considers the origins of jet lag in terms of altered circadian rhythmicity. The properties required of a chronobiotic—an agent to cause phase adjustment of the body clock—are discussed, and an account is given of the major candidates at the present time: light, melatonin, activity, and benzodiazepines. It is concluded that current knowledge indicates that a combination of factors is likely to be most effective.     

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.     

Sympathetic Regulation of Circadian Rhythm of Serotonin N-Acetyltransferase Activity in Pineal Gland of Infant Rat

Abstract: The circadian rhythms of serotonin N -acetyltransferase activity in the pineal glands of infant and adult rats were compared. The nighttime increase of N -acetyltransferase activity in the pineals of infant rats was blocked by removal of superior cervical ganglion or by pretreatment with reserpine, l -propranolol, and cycloheximide. Injection of isoproterenol to infant rats markedly elevated pineal N -acetyltransferase activity. When the pineal glands of infant rats were organ-cultured, N -acetyltransferase activity spontaneously increased 7–12 h after the rats were killed. When infant rats were previously denervated or pretreated with reserpine and their pineals were cultured, this spontaneous elevation of N -acetyltransferase activity was abolished, indicating that the transient increase of the enzyme activity in organ culture was due to a liberation of catecholamine from degenerating nerve endings. Additional illumination until midnight prevented the nighttime increase of N -acetyltransferase activity in intact infant rats but not in blinded infant rats. These observations are taken to indicate that N -acetyltransferase rhythm in immature rat pineals is regulated by the sympathetic nerves in the same manner as in adult rat pineals, that the immature rat pineal does not contain a time-keeping system, and that there is no extraretinal light perception in infant rats as far as N -acetyltransferase rhythm is concerned.     

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.     

A Model for the Fast Synchronous Oscillations of Firing Rate in Rat Suprachiasmatic Nucleus Neurons Cultured in a Multielectrode Array Dish

When dispersed and cultured in a multielectrode dish (MED), suprachiasmatic nucleus (SCN) neurons express fast oscillations of firing rate (FOFR; fast relative to the circadian cycle), with burst duration ∼10 min, and interburst interval varying from 20 to 60 min in different cells but remaining nevertheless rather regular in individual cells. In many cases, separate neurons in distant parts of the 1 mm recording area of a MED exhibited correlated FOFR. Neither the mechanism of FOFR nor the mechanism of their synchronization among neurons is known. Based on recent data implicating vasoactive intestinal polypeptide (VIP) as a key intercellular synchronizing agent, we built a model in which VIP acts as both a feedback regulator to generate FOFR in individual neurons, and a diffusible synchronizing agent to produce coherent electrical output of a neuronal network. In our model, VIP binding to its (VPAC₂) receptors acts through G_s G-proteins to activate adenylyl cyclase (AC), increase intracellular cAMP, and open cyclic-nucleotide-gated (CNG) cation channels, thus depolarizing the cell and generating neuronal firing to release VIP. In parallel, slowly developing homologous desensitization and internalization of VPAC₂ receptors terminates elevation of cAMP and thereby provides an interpulse silent interval. Through mathematical modeling, we show that this VIP/VPAC₂/AC/cAMP/CNG-channel mechanism is sufficient for generating reliable FOFR in single neurons. When our model for FOFR is combined with a published model of synchronization of circadian rhythms based on VIP/VPAC₂ and Per gene regulation synchronization of circadian rhythms is significantly accelerated. These results suggest that (a) auto/paracrine regulation by VIP/VPAC₂ and intracellular AC/cAMP/CNG-channels are sufficient to provide robust FOFR and synchrony among neurons in a heterogeneous network, and (b) this system may also participate in synchronization of circadian rhythms.     

Plasticity of Circadian Activity and Body Temperature Rhythms in Golden Spiny Mice

Most animals can be categorized as nocturnal, diurnal, or crepuscular. However, rhythms can be quite plastic in some species and vary from one individual to another within a species. In the golden spiny mouse (Acomys russatus), a variety of rhythm patterns have been seen, and these patterns can change considerably as animals are transferred from the field into the laboratory. We previously suggested that these animals may have a circadian time‐keeping system that is fundamentally nocturnal and that diurnal patterns seen in their natural habitat reflect mechanisms operating outside of the basic circadian time‐keeping system (i.e., masking). In the current study, we further characterized plasticity evident in the daily rhythms of golden spiny mice by measuring effects of lighting conditions and access to a running wheel on rhythms in general activity (GA) and body temperature (Tb). Before the wheel was introduced, most animals were active mainly during the night, though there was considerable inter‐individual variability and patterns were quite plastic. The introduction of the wheel caused an increase in the level of nighttime activity and Tb in most individuals. The periods of the rhythms in constant darkness (DD) were very similar, and even slightly longer in this study (24.1±0.2 h) than in an earlier one in which animals had not been provided with running wheels. We found no correlation between the distance animals ran in their wheels and the period of their rhythms in DD. Re‐entrainment after phase delays of the LD cycle occurred more rapidly in the presence than absence of the running wheel. The characteristics of the rhythms of golden spiny mice seen in this study may be the product of natural selection favoring plasticity of the circadian system, perhaps reflecting what can happen during an evolutionary transition as animals move from a nocturnal to a diurnal niche.