A Network of (Autonomic) Clock Outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each dependent on the cell‐autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock that produces a coherent output capable of timing all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre‐autonomic and neuro‐endocrine target neurons is controlled by differentially timed waves of vasopressin, GABA, and glutamate release from SCN terminals, among other factors. Together our data indicate that, with regard to the timing of their main release period within the LD cycle, at least four subpopulations of SCN neurons should be discernible. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of four differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure, i.e., the SCN seems to contain neurons that specifically target the liver, pineal gland, and adrenal gland.     

A Network of (Autonomic) Clock Outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each of which is dependent on the cell‐autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock producing a coherent output that is able to time all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre‐autonomic and neuro‐endocrine target neurons is controlled by differentially timed waves of, among others, vasopressin, GABA, and glutamate release from SCN terminals. Together our data indicate that, with regard to the timing of their main release period within the light‐dark (LD) cycle, at least 4 subpopulations of SCN neurons should be discerned. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of 4 differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure; i.e., the SCN seems to contain neurons that specifically target the liver, pineal, and adrenal.     

A network of (autonomic) clock outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each of which is dependent on the cell-autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock producing a coherent output that is able to time all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre-autonomic and neuro-endocrine target neurons is controlled by differentially timed waves of, among others, vasopressin, GABA, and glutamate release from SCN terminals. Together our data indicate that, with regard to the timing of their main release period within the light-dark (LD) cycle, at least 4 subpopulations of SCN neurons should be discerned. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of 4 differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure; i.e., the SCN seems to contain neurons that specifically target the liver, pineal, and adrenal.     

SCN outputs and the hypothalamic balance of life

The circadian clock in the suprachiasmatic nucleus (SCN) is composed of thousands of oscillator neurons, each dependent on the cell-autonomous action of a defined set of circadian clock genes. Still, the major question remains how these individual oscillators are organized into a biological clock producing a coherent output able to time all the different daily changes in behavior and physiology. In the present review, the authors discuss the anatomical connections and neurotransmitters used by the SCN to control the daily rhythms in hormone release. The efferent SCN projections mainly target neurons in the medial hypothalamus surrounding the SCN. The activity of these preautonomic and neuroendocrine target neurons is controlled by differentially timed waves of, among others, vasopressin, GABA, and glutamate release from SCN terminals. Together, the data on the SCN control of neuroendocrine rhythms provide clear evidence not only that the SCN consists of phenotypically (i.e., according to neurotransmitter content) different subpopulations of neurons but also that subpopulations should be distinguished (within phenotypically similar groups of neurons) based on the acrophase of their (electrical) activity. Moreover, the specialization of the SCN may go as far as a single body structure, that is, the SCN seems to contain neurons that specifically target the liver, pineal, and adrenal.     

Synchronization of the molecular clockwork by light- and food-related cues in mammals

The molecular clockwork in mammals involves various clock genes with specific temporal expression patterns. Synchronization of the master circadian clock located in the suprachiasmatic nucleus (SCN) is accomplished mainly via daily resetting of the phase of the clock by light stimuli. Phase shifting responses to light are correlated with induction of Per1, Per2 and Dec1 expression and a possible reduction of Cry2 expression within SCN cells. The timing of peripheral oscillators is controlled by the SCN when food is available ad libitum. Time of feeding, as modulated by temporal restricted feeding, is a potent 'Zeitgeber' (synchronizer) for peripheral oscillators with only weak synchronizing influence on the SCN clockwork. When restricted feeding is coupled with caloric restriction, however, timing of clock gene expression is altered within the SCN, indicating that the SCN function is sensitive to metabolic cues. The components of the circadian timing system can be differentially synchronized according to distinct, sometimes conflicting, temporal (time of light exposure and feeding) and homeostatic (metabolic) cues.     

Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus (SCN) of the brain and subsidiary oscillators in most peripheral cell types. While oscillators in SCN neurons are known to function in a self-sustained fashion, peripheral oscillators have been thought to damp rapidly when disconnected from the control exerted by the SCN. Using two reporter systems, we monitored circadian gene expression in NIH3T3 mouse fibroblasts in real time and in individual cells. In conjunction with mathematical modeling and cell co-culture experiments, these data demonstrated that in vitro cultured fibroblasts harbor self-sustained and cell-autonomous circadian clocks similar to those operative in SCN neurons. Circadian gene expression in fibroblasts continues during cell division, and our experiments unveiled unexpected interactions between the circadian clock and the cell division clock. Specifically, the circadian oscillator gates cytokinesis to defined time windows, and mitosis elicits phase shifts in circadian cycles.     

Neural correlates of individual differences in circadian behaviour

Daily rhythms in mammals are controlled by the circadian system, which is a collection of biological clocks regulated by a central pacemaker within the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Changes in SCN function have pronounced consequences for behaviour and physiology; however, few studies have examined whether individual differences in circadian behaviour reflect changes in SCN function. Here, PERIOD2::LUCIFERASE mice were exposed to a behavioural assay to characterize individual differences in baseline entrainment, rate of re-entrainment and free-running rhythms. SCN slices were then collected for ex vivo bioluminescence imaging to gain insight into how the properties of the SCN clock influence individual differences in behavioural rhythms. First, individual differences in the timing of locomotor activity rhythms were positively correlated with the timing of SCN rhythms. Second, slower adjustment during simulated jetlag was associated with a larger degree of phase heterogeneity among SCN neurons. Collectively, these findings highlight the role of the SCN network in determining individual differences in circadian behaviour. Furthermore, these results reveal novel ways that the network organization of the SCN influences plasticity at the behavioural level, and lend insight into potential interventions designed to modulate the rate of resynchronization during transmeridian travel and shift work.     

GABA synchronizes clock cells within the suprachiasmatic circadian clock

The master clock in the suprachiasmatic nuclei (SCN) is composed of multiple, single-cell circadian clocks. We test the postulate that these individual "clock cells" can be synchronized to each other by the inhibitory transmitter gamma-aminobutyric acid (GABA). For these experiments, we monitored the firing rate rhythm of individual clock cells on fixed multielectrode plates in culture and tested the effects of GABA. The results show that the daily variation in responsiveness of the SCN to phase-shifting agents is manifested at the level of individual neurons. Moreover, GABA, acting through A-type receptors, can both phase shift and synchronize clock cells. We propose that GABA is an important synchronizer of SCN neurons in vivo.     

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.     

Modeling the spontaneous activity in suprachiasmatic nucleus neurons: Role of cation single channels

A population of interconnected neurons of the mammalian suprachiasmatic nuclei (SCN) controls circadian rhythms in physiological functions. In turn, a circadian rhythm of individual neurons is driven by intracellular processes, which via activation of specific membrane channels, produce circadian modulation of electrical firing rate. Yet the membrane target(s) of the cellular clock have remained enigmatic. Previously, subthreshold voltage-dependent cation (SVC) channels have been proposed as the membrane target of the cellular clock responsible for circadian modulation of the firing rate in SCN neurons. We tested this hypothesis with computational modeling based on experimental results from on-cell recording of SVC channel openings in acutely isolated SCN neurons and long-term continuous recording of activity from dispersed SCN neurons in a multielectrode array dish (MED). The model reproduced the circadian behavior if the number of SVC channels or their kinetics were modulated in accordance with protein concentration in a model of the intracellular clock (Scheper et al., 1999. J. Neurosci. 19, 40-47). Such modulation changed the average firing rate of the model neuron from zero (“subjective-night” silence) up to 18 Hz (“subjective-day” peak). Furthermore, the variability of interspike intervals (ISI) and the circadian pattern of firing rate (i.e. silence-to-activity ratio and shape of circadian peaks) are in reasonable agreement with experimental data obtained in dispersed SCN neurons in MED. These results suggest that the variability of ISI in intact SCN neurons is mostly due to stochastic single-channel openings, and that the circadian pattern of the firing rate is specified by threshold properties of dependence of the spontaneous firing rate on the number of single channels (R-N relationship). This plausible mathematical modeling supports the hypothesis that SVC channels could be a critical element in circadian modulation of firing rate in SCN neurons.     

Circadian control of the daily plasma glucose rhythm: an interplay of GABA and glutamate

The mammalian biological clock, located in the hypothalamic suprachiasmatic nuclei (SCN), imposes its temporal structure on the organism via neural and endocrine outputs. To further investigate SCN control of the autonomic nervous system we focused in the present study on the daily rhythm in plasma glucose concentrations. The hypothalamic paraventricular nucleus (PVN) is an important target area of biological clock output and harbors the pre-autonomic neurons that control peripheral sympathetic and parasympathetic activity. Using local administration of GABA and glutamate receptor (ant)agonists in the PVN at different times of the light/dark-cycle we investigated whether daily changes in the activity of autonomic nervous system contribute to the control of plasma glucose and plasma insulin concentrations. Activation of neuronal activity in the PVN of non-feeding animals, either by administering a glutamatergic agonist or a GABAergic antagonist, induced hyperglycemia. The effect of the GABA-antagonist was time dependent, causing increased plasma glucose concentrations only when administered during the light period. The absence of a hyperglycemic effect of the GABA-antagonist in SCN-ablated animals provided further evidence for a daily change in GABAergic input from the SCN to the PVN. On the other hand, feeding-induced plasma glucose and insulin responses were suppressed by inhibition of PVN neuronal activity only during the dark period. These results indicate that the pre-autonomic neurons in the PVN are controlled by an interplay of inhibitory and excitatory inputs. Liver-dedicated sympathetic pre-autonomic neurons (responsible for hepatic glucose production) and pancreas-dedicated pre-autonomic parasympathetic neurons (responsible for insulin release) are controlled by inhibitory GABAergic contacts that are mainly active during the light period. Both sympathetic and parasympathetic pre-autonomic PVN neurons also receive excitatory inputs, either from the biological clock (sympathetic pre-autonomic neurons) or from non-clock areas (para-sympathetic pre-autonomic neurons), but the timing information is mainly provided by the GABAergic outputs of the biological clock.     

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 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.     

Early Chronotype and Tissue-Specific Alterations of Circadian Clock Function in Spontaneously Hypertensive Rats

Malfunction of the circadian timing system may result in cardiovascular and metabolic diseases, and conversely, these diseases can impair the circadian system. The aim of this study was to reveal whether the functional state of the circadian system of spontaneously hypertensive rats (SHR) differs from that of control Wistar rat. This study is the first to analyze the function of the circadian system of SHR in its complexity, i.e., of the central clock in the suprachiasmatic nuclei (SCN) as well as of the peripheral clocks. The functional properties of the SCN clock were estimated by behavioral output rhythm in locomotor activity and daily profiles of clock gene expression in the SCN determined by in situ hybridization. The function of the peripheral clocks was assessed by daily profiles of clock gene expression in the liver and colon by RT-PCR and in vitro using real time recording of Bmal1-dLuc reporter. The potential impact of the SHR phenotype on circadian control of the metabolic pathways was estimated by daily profiles of metabolism-relevant gene expression in the liver and colon. The results revealed that SHR exhibited an early chronotype, because the central SCN clock was phase advanced relative to light/dark cycle and the SCN driven output rhythm ran faster compared to Wistar rats. Moreover, the output rhythm was dampened. The SHR peripheral clock reacted to the dampened SCN output with tissue-specific consequences. In the colon of SHR the clock function was severely altered, whereas the differences are only marginal in the liver. These changes may likely result in a mutual desynchrony of circadian oscillators within the circadian system of SHR, thereby potentially contributing to metabolic pathology of the strain. The SHR may thus serve as a valuable model of human circadian disorders originating in poor synchrony of the circadian system with external light/dark regime.