Toward dissection of diverse neural components in the suprachiasmatic nucleus (SCN) pacemaker network

Neurons of the suprachiasmatic nucleus (SCN), located in the ventromedial hypothalamus, comprise the central mammalian circadian pacemaker ([Reppert and Weaver 2002][1]). In dispersed culture, these neurons can generate autonomous circadian (ca. 24 h) oscillations in spontaneous firing rate ([Welsh

The suprachiasmatic nucleus (SCN) is composed of functionally distinct subpopulations of GABAergic neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons, and which aspects result from neuronal interaction within a network. Here, we utilize invivo miniaturized microscopy to measure fluorescent GCaMP-reported calcium dynamics in arginine vasopressin (AVP)-expressing neurons in the intact SCN of awake, behaving mice. We report that SCN AVP neurons exhibit periodic, slow calcium waves which we demonstrate, using invivo electrical recordings, likely reflect burst firing. Further, we observe substantial heterogeneity of function in that AVP neurons exhibit unstable rhythms, and relatively weak rhythmicity at the population level. Network analysis reveals that correlated cellular behavior, or coherence, among neuron pairs also exhibited stochastic rhythms with about 33% of pairs rhythmic at any time. Unlike single-cell variables, coherence exhibited a strong rhythm at the population level with time of maximal coherence among AVP neuronal pairs at CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity for the SCN as a whole. These results demonstrate robust circadian variation in the coordination between stochastically rhythmic neurons and that interactions between AVP neurons in the SCN may be more influential than single-cell activity in the regulation of circadian rhythms. Furthermore, they demonstrate that cells in this circuit, like those in many other circuits, exhibit profound heterogenicity of function over time and space.

Synchronization and Maintenance of Timekeeping in Suprachiasmatic Circadian Clock Cells by Neuropeptidergic Signaling

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in the mammalian brain, responsible for coordinating circadian (ca 24 h) rhythms throughout the body. When individual SCN neurons are dispersed in low density culture, they generate independent circadian oscillations in neuronal firing rate. This observation has led to the widely accepted conclusion that single SCN neurons are autonomous circadian oscillators. More recent studies using bioluminescence imaging to monitor rhythms of clock gene expression have indicated that fibroblasts, too, are autonomous circadian oscillators. But does this mean that the fibroblast should “replace the SCN as the in vitro model of choice”? On the contrary, the SCN is much more than a population of independent cellular oscillators. SCN function depends on important network interactions that allow for cell synchrony, enhanced pacemaker precision, reinforcement of rhythm amplitude, and robustness against genetic perturbations. The distribution of phases of cells within the SCN network also permits variously phased rhythmic output signals, as well as the encoding of seasonal changes in photoperiod and other features of environmental lighting history. Thus, unlike fibroblasts, SCN neurons are designed to be “team players,” and SCN network interactions are integral to the normal function of the SCN as a circadian pacemaker.

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.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of a central circadian clock that orchestrates overt rhythms of physiology and behavior. Circadian timekeeping requires intercellular communication among SCN neurons, and multiple signaling pathways contribute to SCN network coupling. Gamma-aminobutyric acid (GABA) is produced by virtually all SCN neurons, and previous work demonstrates that this transmitter regulates coupling in the adult SCN but is not essential for the nucleus to sustain overt circadian rhythms. Here, we show that the deletion of the gene that codes for the GABA vesicular transporter Vgat from neuromedin-S (NMS)+ neurons-a subset of neurons critical for SCN function-causes arrhythmia of locomotor activity and sleep. Further, NMS-Vgat deletion impairs intrinsic clock gene rhythms in SCN explants cultured ex vivo. Although vasoactive intestinal polypeptide (VIP) is critical for SCN function, Vgat deletion from VIP-expressing neurons did not lead to circadian arrhythmia in locomotor activity rhythms. Likewise, adult SCN-specific deletion of Vgat led to mild impairment of behavioral rhythms. Our results suggest that while the removal of GABA release from the adult SCN does not affect the pacemaker's ability to sustain overt circadian rhythms, its removal from a critical subset of neurons within the SCN throughout development removes the nucleus ability to sustain circadian rhythms. Our findings support a model in which SCN GABA release is critical for the developmental establishment of intercellular network properties that define the SCN as a central pacemaker.

The central circadian clock of the suprachiasmatic nucleus as an ensemble of multiple oscillatory neurons

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

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.

Circadian periods of single suprachiasmatic neurons in rats

The circadian system regulates a range of behavioural, physiological and cellular rhythms that allow organisms to anticipate changes in their physical environment, such as cycles of day and night. These predictive near-24-h oscillations persist in the absence of all environmental cues and are driven by the endogenous master pacemaker located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus in mammals (Stephan and Zucker 1972; Ralph et al. 1990; Edgar et al. 1993). Within individual SCN neurons, cyclic core clock genes and proteins establish a molecular basis of circadian control through transcriptional, translational and post-translational feedback loops (Reppert and Weaver 2002). This intracellular clock is not only found in the SCN, but in nearly all peripheral tissues of the mammalian body where it establishes tissue-specific gene expression to temporally coordinate physiology. Signals from the central SCN are relayed throughout the brain and body to synchronize these peripheral clocks to appropriate phases through neuronal, hormonal and physiological mechanisms (Buhr and Takahashi 2013). In this manner, the SCN regulates many diverse processes, including daily patterns of sleep, endocrine secretion, glucose homeostasis and core body temperature.

In mammals, the central clock in the suprachiasmatic nucleus (SCN) controls physiological and behavioral circadian rhythms and is entrained to the external light-dark cycle. The ability of the SCN to entrain can be measured by exposing the animal to a light-dark cycle with a duration that deviates from 24 h (T-cycles); a wider entrainment range reflects a higher ability to entrain. The neurons of the SCN are either light responsive or light unresponsive and are mutually synchronized. The coupling and synchronization between individual SCN neurons and between groups of neurons within the SCN influence the SCN's ability to entrain. Some studies suggest that enhanced coupling decreases the entrainment range, whereas others suggest that enhanced coupling increases the entrainment range. The latter results are surprising, as they are not consistent with the prevalent assumption that the SCN is a limit cycle oscillator that has larger phase shifts when the amplitude is smaller. Here, we used the Poincaré and Goodwin models to test entrainment properties using various proportions of neurons that are responsive to an external stimulus. If all neurons receive external input, the SCN shows limit cycle behavior in all conditions. If all neurons do not receive light input, we found that the entrainment range of the SCN was positively related to coupling strength when coupling was weak. When coupling strength was stronger and above a critical value, the entrainment range was negatively correlated with coupling strength. The results obtained from our simulations were confirmed by analytical studies. Thus, the limit cycle behavior of the SCN appears to be critically dependent on the coupling strength among the neurons and the proportion of neurons that respond to the entraining stimulus.

Evaluation of suprachiasmatic nucleus in Alzheimer’s disease with non-invasive magnetic resonance methods

Adenosine modulation of calcium channel currents and synaptic gamma-aminobutyrate (GABA) release was investigated with whole cell voltage-clamp recordings in rat suprachiasmatic nucleus (SCN) and arcuate nucleus cultures (n = 94). In SCN cultures, approximately 70% of the neurons showed a reversible inhibition of whole cell barium currents on the application of adenosine or its analogues. Adenosine at 1 microM reduced the amplitude of the barium currents by approximately 27%. In contrast to the significant reduction in the amplitude, the rising and decaying phases of the barium currents, and the inverted bell shape of the current-voltage curve of the barium currents, were not changed by adenosine. The adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA; 100 nM) and the adenosine A2 receptor agonist N6-[2-(3,5-dimethoxyphenyl)-ethyl]adenosine (DPMA; 100 nM) inhibited the barium currents by 21% and 16%, respectively, in SCN neurons, indicating both A1 and A2 receptor actions. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM) significantly reduced the effect of CPA but did not change the effect of DPMA on the barium currents. In the presence of tetrodotoxin to block action potentials, the frequency, but not the amplitude, of miniature inhibitory postsynaptic currents was significantly reduced (46%) by 1 microM adenosine, suggesting a presynaptic mechanism of adenosine action. In support of this suggestion, the postsynaptic GABA receptor responses were not influenced by 1 microM adenosine in the majority of SCN neurons. Most solitary self-innervating SCN neurons in microisland cultures were GABAergic. In these cells, the evoked autaptic GABA release (inhibitory postsynaptic current) was significantly inhibited by adenosine (37%), CPA (27%), and DPMA (28%), indicating that both A1 and A2 receptors were present in presynaptic axons. Similar to the effect in SCN neurons, adenosine inhibited both barium currents and GABA release in arcuate neurons. The reduction of whole cell barium currents by adenosine (1 microM), CPA (100 nM), and DPMA (100 nM) was 24, 17, and 19%, respectively. In solitary self-innervating arcuate neurons, adenosine inhibited the evoked GABA release (inhibitory postsynaptic current) by approximately 48%. We conclude that both adenosine A1 and A2 receptors are present in the SCN and arcuate nucleus of the hypothalamus. Adenosine inhibits calcium currents and presynaptically reduces inhibitory GABA neurotransmission.

The influence of circadian rhythms on memory has long been studied; however, the molecular prerequisites for their interaction remain elusive. The hippocampus, which is a region of the brain important for long-term memory formation and temporary maintenance, shows circadian rhythmicity in pathways central to the memory-consolidation process. As neuronal plasticity is the translation of numerous inputs, illuminating the direct molecular links between circadian rhythms and memory consolidation remains a daunting task. However, the elucidation of how clock genes contribute to synaptic plasticity could provide such a link. Furthermore, the idea that memory training could actually function as a zeitgeber for hippocampal neurons is worth consideration, based on our knowledge of the entrainment of the circadian clock system. The integration of many inputs in the hippocampus affects memory consolidation at both the cellular and the systems level, leaving the molecular connections between circadian rhythmicity and memory relatively obscure but ripe for investigation.