Scheduled Feeding Alters the Timing of the Suprachiasmatic Nucleus Circadian Clock in Dexras1-Deficient Mice

Restricted feeding (RF) schedules are potent zeitgebers capable of entraining metabolic and hormonal rhythms in peripheral oscillators in anticipation of food. Behaviorally, this manifests in the form of food anticipatory activity (FAA) in the hours preceding food availability. Circadian rhythms of FAA are thought to be controlled by a food-entrainable oscillator (FEO) outside of the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. Although evidence suggests that the FEO and the SCN are capable of interacting functionally under RF conditions, the genetic basis of these interactions remains to be defined. In this study, using dexras1-deficient (dexras1^?/?) mice, the authors examined whether Dexras1, a modulator of multiple inputs to the SCN, plays a role in regulating the effects of RF on activity rhythms and gene expression in the SCN. Daytime RF under 12L:12D or constant darkness (DD) resulted in potentiated (but less stable) FAA expression in dexras1^?/? mice compared with wild-type (WT) controls. Under these conditions, the magnitude and phase of the SCN-driven activity component were greatly perturbed in the mutants. Restoration to ad libitum (AL) feeding revealed a stable phase displacement of the SCN-driven activity component of dexras1^?/? mice by ～2?h in advance of the expected time. RF in the late night/early morning induced a long-lasting increase in the period of the SCN-driven activity component in the mutants but not the WT. At the molecular level, daytime RF advanced the rhythm of PER1, PER2, and pERK expression in the mutant SCN without having any effect in the WT. Collectively, these results indicate that the absence of Dexras1 sensitizes the SCN to perturbations resulting from restricted feeding. (Author correspondence: haiying.cheng@utoronto.ca)     

The Role of the Suprachiasmatic Nucleus in Cardiac Autonomic Control during Sleep

Background

The suprachiasmatic nucleus (SCN) may play an important role in central autonomic control, since its projections connect to (para)sympathetic relay stations in the brainstem and spinal cord. The cardiac autonomic modifications during nighttime may therefore not only result from direct effects of the sleep-related changes in the central autonomic network, but also from endogenous circadian factors as directed by the SCN. To explore the influence of the SCN on autonomic fluctuations during nighttime, we studied heart rate and its variability (HRV) in a clinical model of SCN damage.

Methods

Fifteen patients in follow-up after surgical treatment for nonfunctioning pituitary macroadenoma (NFMA) compressing the optic chiasm (8 females, 26–65 years old) and fifteen age-matched healthy controls (5 females, 30–63 years) underwent overnight ambulatory polysomnography. Eleven patients had hypopituitarism and received adequate replacement therapy. HRV was calculated for each 30-second epoch and corrected for sleep stage, arousals, and gender using mixed effect regression models.

Results

Compared to controls, patients spent more time awake after sleep onset and in NREM1-sleep, and less in REM-sleep. Heart rate, low (LF) and high frequency (HF) power components and the LF/HF ratio across sleep stages were not significantly different between groups.

Conclusions

These findings suggest that the SCN does not play a dominant role in cardiac autonomic control during sleep.     

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.     

Cell Cultures of Rat Suprachiasmatic Nucleus: Circadian Oscillation of Vasopressin Release

A dissociated cell culture system has been developed for the suprachiasmatic nucleus (SCN), in which release of vasopressin showed a clear circadian oscillation. The oscillation peaked at early subjective day and appeared within one day in culture. This system may provide a valuable model for the study of cellular and molecular mechanisms of the mammalian circadian pacemaker.     

The Circadian Clock Gene Period1 Connects the Molecular Clock to Neural Activity in the Suprachiasmatic Nucleus

The neural activity patterns of suprachiasmatic nucleus (SCN) neurons are dynamically regulated throughout the circadian cycle with highest levels of spontaneous action potentials during the day. These rhythms in electrical activity are critical for the function of the circadian timing system and yet the mechanisms by which the molecular clockwork drives changes in the membrane are not well understood. In this study, we sought to examine how the clock gene Period1 (Per1) regulates the electrical activity in the mouse SCN by transiently and selectively decreasing levels of PER1 through use of an antisense oligodeoxynucleotide. We found that this treatment effectively reduced SCN neural activity. Direct current injection to restore the normal membrane potential partially, but not completely, returned firing rate to normal levels. The antisense treatment also reduced baseline [Ca²⁺]i levels as measured by Fura2 imaging technique. Whole cell patch clamp recording techniques were used to examine which specific potassium currents were altered by the treatment. These recordings revealed that the large conductance [Ca²⁺]i-activated potassium currents were reduced in antisense-treated neurons and that blocking this current mimicked the effects of the anti-sense on SCN firing rate. These results indicate that the circadian clock gene Per1 alters firing rate in SCN neurons and raise the possibility that the large conductance [Ca²⁺]i-activated channel is one of the targets.     

Coupled Flip-Flop Model for REM Sleep Regulation in the Rat

Recent experimental studies investigating the neuronal regulation of rapid eye movement (REM) sleep have identified mutually inhibitory synaptic projections among REM sleep-promoting (REM-on) and REM sleep-inhibiting (REM-off) neuronal populations that act to maintain the REM sleep state and control its onset and offset. The control mechanism of mutually inhibitory synaptic interactions mirrors the proposed flip-flop switch for sleep-wake regulation consisting of mutually inhibitory synaptic projections between wake- and sleep-promoting neuronal populations. While a number of synaptic projections have been identified between these REM-on/REM-off populations and wake/sleep-promoting populations, the specific interactions that govern behavioral state transitions have not been completely determined. Using a minimal mathematical model, we investigated behavioral state transition dynamics dictated by a system of coupled flip-flops, one to control transitions between wake and sleep states, and another to control transitions into and out of REM sleep. The model describes the neurotransmitter-mediated inhibitory interactions between a wake- and sleep-promoting population, and between a REM-on and REM-off population. We proposed interactions between the wake/sleep and REM-on/REM-off flip-flops to replicate the behavioral state statistics and probabilities of behavioral state transitions measured from experimental recordings of rat sleep under ad libitum conditions and after 24 h of REM sleep deprivation. Reliable transitions from REM sleep to wake, as dictated by the data, indicated the necessity of an excitatory projection from the REM-on population to the wake-promoting population. To replicate the increase in REM-wake-REM transitions observed after 24 h REM sleep deprivation required that this excitatory projection promote transient activation of the wake-promoting population. Obtaining the reliable wake-nonREM sleep transitions observed in the data required that activity of the wake-promoting population modulated the interaction between the REM-on and REM-off populations. This analysis suggests neuronal processes to be targeted in further experimental studies of the regulatory mechanisms of REM sleep.     

Modeling the Seasonal Adaptation of Circadian Clocks by Changes in the Network Structure of the Suprachiasmatic Nucleus

The dynamics of circadian rhythms needs to be adapted to day length changes between summer and winter. It has been observed experimentally, however, that the dynamics of individual neurons of the suprachiasmatic nucleus (SCN) does not change as the seasons change. Rather, the seasonal adaptation of the circadian clock is hypothesized to be a consequence of changes in the intercellular dynamics, which leads to a phase distribution of electrical activity of SCN neurons that is narrower in winter and broader during summer. Yet to understand this complex intercellular dynamics, a more thorough understanding of the impact of the network structure formed by the SCN neurons is needed. To that effect, we propose a mathematical model for the dynamics of the SCN neuronal architecture in which the structure of the network plays a pivotal role. Using our model we show that the fraction of long-range cell-to-cell connections and the seasonal changes in the daily rhythms may be tightly related. In particular, simulations of the proposed mathematical model indicate that the fraction of long-range connections between the cells adjusts the phase distribution and consequently the length of the behavioral activity as follows: dense long-range connections during winter lead to a narrow activity phase, while rare long-range connections during summer lead to a broad activity phase. Our model is also able to account for the experimental observations indicating a larger light-induced phase-shift of the circadian clock during winter, which we show to be a consequence of higher synchronization between neurons. Our model thus provides evidence that the variations in the seasonal dynamics of circadian clocks can in part also be understood and regulated by the plasticity of the SCN network structure.     

Fractal Patterns of Neural Activity Exist within the Suprachiasmatic Nucleus and Require Extrinsic Network Interactions

The mammalian central circadian pacemaker (the suprachiasmatic nucleus, SCN) contains thousands of neurons that are coupled through a complex network of interactions. In addition to the established role of the SCN in generating rhythms of ∼24 hours in many physiological functions, the SCN was recently shown to be necessary for normal self-similar/fractal organization of motor activity and heart rate over a wide range of time scales—from minutes to 24 hours. To test whether the neural network within the SCN is sufficient to generate such fractal patterns, we studied multi-unit neural activity of in vivo and in vitro SCNs in rodents. In vivo SCN-neural activity exhibited fractal patterns that are virtually identical in mice and rats and are similar to those in motor activity at time scales from minutes up to 10 hours. In addition, these patterns remained unchanged when the main afferent signal to the SCN, namely light, was removed. However, the fractal patterns of SCN-neural activity are not autonomous within the SCN as these patterns completely broke down in the isolated in vitro SCN despite persistence of circadian rhythmicity. Thus, SCN-neural activity is fractal in the intact organism and these fractal patterns require network interactions between the SCN and extra-SCN nodes. Such a fractal control network could underlie the fractal regulation observed in many physiological functions that involve the SCN, including motor control and heart rate regulation.     

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.     

The Timing of the Circadian Clock and Sleep Differ between Napping and Non-Napping Toddlers

The timing of the internal circadian clock shows large inter-individual variability across the lifespan. Although the sleep-wakefulness pattern of most toddlers includes an afternoon nap, the association between napping and circadian phase in early childhood remains unexplored. This study examined differences in circadian phase and sleep between napping and non-napping toddlers. Data were collected on 20 toddlers (34.2±2.0 months; 12 females; 15 nappers). Children followed their habitual napping and non-napping sleep schedules (monitored with actigraphy) for 5 days before an in-home salivary dim light melatonin onset (DLMO) assessment. On average, napping children fell asleep during their nap opportunities on 3.6±1.2 of the 5 days before the DLMO assessment. For these napping children, melatonin onset time was 38 min later (p = 0.044; d = 0.93), actigraphically-estimated bedtime was 43 min later (p = 0.014; d = 1.24), sleep onset time was 59 min later (p = 0.006; d = 1.46), and sleep onset latency was 16 min longer (p = 0.030; d = 1.03) than those not napping. Midsleep and wake time did not differ by napping status. No difference was observed in the bedtime, sleep onset, or midsleep phase relationships with DLMO; however, the wake time phase difference was 47 min smaller for napping toddlers (p = 0.029; d = 1.23). On average, nappers had 69 min shorter nighttime sleep durations (p = 0.006; d = 1.47) and spent 49 min less time in bed (p = 0.019; d = 1.16) than non-nappers. Number of days napping was correlated with melatonin onset time (r = 0.49; p = 0.014). Our findings indicate that napping influences individual variability in melatonin onset time in early childhood. The delayed bedtimes of napping toddlers likely permits light exposure later in the evening, thereby delaying the timing of the clock and sleep. Whether the early developmental trajectory of circadian phase involves an advance associated with the decline in napping is a question necessitating longitudinal data as children transition from a biphasic to monophasic sleep-wakefulness pattern.     

Overnight Fasting Regulates Inhibitory Tone to Cholinergic Neurons of the Dorsomedial Nucleus of the Hypothalamus

The dorsomedial nucleus of the hypothalamus (DMH) contributes to the regulation of overall energy homeostasis by modulating energy intake as well as energy expenditure. Despite the importance of the DMH in the control of energy balance, DMH-specific genetic markers or neuronal subtypes are poorly defined. Here we demonstrate the presence of cholinergic neurons in the DMH using genetically modified mice that express enhanced green florescent protein (eGFP) selectively in choline acetyltransferase (Chat)-neurons. Overnight food deprivation increases the activity of DMH cholinergic neurons, as shown by induction of fos protein and a significant shift in the baseline resting membrane potential. DMH cholinergic neurons receive both glutamatergic and GABAergic synaptic input, but the activation of these neurons by an overnight fast is due entirely to decreased inhibitory tone. The decreased inhibition is associated with decreased frequency and amplitude of GABAergic synaptic currents in the cholinergic DMH neurons, while glutamatergic synaptic transmission is not altered. As neither the frequency nor amplitude of miniature GABAergic or glutamatergic postsynaptic currents is affected by overnight food deprivation, the fasting-induced decrease in inhibitory tone to cholinergic neurons is dependent on superthreshold activity of GABAergic inputs. This study reveals that cholinergic neurons in the DMH readily sense the availability of nutrients and respond to overnight fasting via decreased GABAergic inhibitory tone. As such, altered synaptic as well as neuronal activity of DMH cholinergic neurons may play a critical role in the regulation of overall energy homeostasis.     

Signals from the Brainstem Sleep/Wake Centers Regulate Behavioral Timing via the Circadian Clock

Sleep-wake cycling is controlled by the complex interplay between two brain systems, one which controls vigilance state, regulating the transition between sleep and wake, and the other circadian, which communicates time-of-day. Together, they align sleep appropriately with energetic need and the day-night cycle. Neural circuits connect brain stem sites that regulate vigilance state with the suprachiasmatic nucleus (SCN), the master circadian clock, but the function of these connections has been unknown. Coupling discrete stimulation of pontine nuclei controlling vigilance state with analytical chemical measurements of intra-SCN microdialysates in mouse, we found significant neurotransmitter release at the SCN and, concomitantly, resetting of behavioral circadian rhythms. Depending upon stimulus conditions and time-of-day, SCN acetylcholine and/or glutamate levels were augmented and generated shifts of behavioral rhythms. These results establish modes of neurochemical communication from brain regions controlling vigilance state to the central circadian clock, with behavioral consequences. They suggest a basis for dynamic integration across brain systems that regulate vigilance states, and a potential vulnerability to altered communication in sleep disorders.