Suprachiasmatic Nucleus Neuronal Activity Research Articles

Stochastic simulation of the circadian rhythmicity in the SCN neuronal network

The suprachiasmatic nucleus (SCN) of the hypothalamus is the mammalian circadian pacemaker that plays a dominant role in the generation and control of daily physiological and behavioral rhythms. The coupling of the SCN neuronal activity with the solar day–night cycle and the intercellular communication between SCN neurons are the most important factors that ensure the precise 24 h timing of the circadian rhythmicity. In individual cells, however, the timekeeping is not precise because of the inherent randomness in the biochemical reactions. In order to investigate the impact of molecular noise and intercellular connectivity on the circadian network synchronization, we develop a stochastic multicellular model of the SCN. The circadian pacemaker is modeled as a realistic small-world network of neurons and was found to be robust to rather high levels of intrinsic fluctuations. We show that the SCN neuronal network synchronizes well with the photic entraining even at very large levels of stochasticity, whereby intercellular coupling is the required agent that ensures coherent oscillations. Furthermore, we show that the neuronal activity patterns form localized clusters, which are more pronounced in short photoperiods than in long days. The localized synchronization behavior was also found to be affected by the SCN network structure.

Disrupted reproduction, estrous cycle, and circadian rhythms in female mice deficient in vasoactive intestinal peptide.

The female reproductive cycle is gated by the circadian timing system and may be vulnerable to disruptions in the circadian system. Prior work suggests that vasoactive intestinal peptide (VIP)-expressing neurons in the suprachiasmatic nucleus (SCN) are one pathway by which the circadian clock can influence the estrous cycle, but the impact of the loss of this peptide on reproduction has not been assessed. In the present study, we first examine the impact of the genetic loss of the neuropeptide VIP on the reproductive success of female mice. Significantly, mutant females produce about half the offspring of their wild-type sisters even when mated to the same males. We also find that VIP-deficient females exhibit a disrupted estrous cycle; that is, ovulation occurs less frequently and results in the release of fewer oocytes compared with controls. Circadian rhythms of wheel-running activity are disrupted in the female mutant mice, as is the spontaneous electrical activity of dorsal SCN neurons. On a molecular level, the VIP-deficient SCN tissue exhibits lower amplitude oscillations with altered phase relationships between the SCN and peripheral oscillators as measured by PER2-driven bioluminescence. The simplest explanation of our data is that the loss of VIP results in a weakened SCN oscillator, which reduces the synchronization of the female circadian system. These results clarify one of the mechanisms by which disruption of the circadian system reduces female reproductive success.

Caffeine increases light responsiveness of the mouse circadian pacemaker.

Caffeine is the most commonly used psychoactive stimulant worldwide. It reduces sleep and sleepiness by blocking access to the adenosine receptor. The level of adenosine increases during sleep deprivation, and is thought to induce sleepiness and initiate sleep. Light-induced phase shifts of the rest-activity circadian rhythms are mediated by light-responsive neurons of the suprachiasmatic nucleus (SCN) of the hypothalamus, where the circadian clock of mammals resides. Previous studies have shown that sleep deprivation reduces circadian clock phase-shifting capacity and decreases SCN neuronal activity. In addition, application of adenosine agonists and antagonists mimics and blocks, respectively, the effect of sleep deprivation on light-induced phase shifts in behaviour, suggesting a role for adenosine. In the present study, we examined the role of sleep deprivation in and the effect of caffeine on light responsiveness of the SCN. We performed in vivo electrical activity recordings of the SCN in freely moving mice, and showed that the sustained response to light of SCN neuronal activity was attenuated after 6 h of sleep deprivation prior to light exposure. Subsequent intraperitoneal application of caffeine was able to restore the response to light. Finally, we performed behavioural recordings in constant conditions, and found enhanced period lengthening during chronic treatment with caffeine in drinking water in constant light conditions. The data suggest that increased homeostatic sleep pressure changes circadian pacemaker functioning by reducing SCN neuronal responsiveness to light. The electrophysiological and behavioural data together provide evidence that caffeine enhances clock sensitivity to light.

The suprachiasmatic nucleus is part of a neural feedback circuit adapting blood pressure response

The suprachiasmatic nucleus (SCN) is typically considered our autonomous clock synchronizing behavior with physiological parameters such as blood pressure (BP), just transmitting time independent of physiology. Yet several studies show that the SCN is involved in the etiology of hypertension. Here, we demonstrate that the SCN is incorporated in a neuronal feedback circuit arising from the nucleus tractus solitarius (NTS), modulating cardiovascular reactivity. Tracer injections into the SCN of male Wistar rats revealed retrogradely filled neurons in the caudal NTS, where BP information is integrated. These NTS projections to the SCN were shown to be glutamatergic and to terminate in the ventrolateral part of the SCN where light information also enters. BP elevations not only induced increased neuronal activity as measured by c-Fos in the NTS but also in the SCN. Lesioning the caudal NTS prevented this activation. The increase of SCN neuronal activity by hypertensive stimuli suggested involvement of the SCN in counteracting BP elevations. Examining this possibility we observed that elevation of BP, induced by α1-agonist infusion, was more than twice the magnitude in SCN-lesioned animals as compared to in controls, indicating indeed an active involvement of the SCN in short-term BP regulation. We propose that the SCN receives BP information directly from the NTS enabling it to react to hemodynamic perturbations, suggesting the SCN to be part of a homeostatic circuit adapting BP response. We discuss how these findings could explain why lifestyle conditions violating signals of the biological clock may, in the long-term, result in cardiovascular disease.

Single unit activity of the suprachiasmatic nucleus and surrounding neurons during the wake–sleep cycle in mice

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains a circadian clock for timing of diverse neuronal, endocrine, and behavioral rhythms, such as the cycle of sleep and wakefulness. Using extracellular single unit recordings, we have determined, for the first time, the discharge activity of individual SCN neurons during the complete wake–sleep cycle in non-anesthetized, head restrained mice. SCN neurons (n=79) were divided into three types according to their regular (type I; n=38) or irregular (type II; n=19) discharge activity throughout the wake–sleep cycle or their quiescent activity during waking and irregular discharge activity during sleep (type III; n=22). The type I and II neurons displayed a long-duration action potential, while the type III neurons displayed either a short-duration or long-duration action potential. The type I neurons discharged exclusively as single isolated spikes, whereas the type II and III neurons fired as single isolated spikes, clusters, or bursts. The type I and II neurons showed wake-active, wake/paradoxical (or rapid eye movement) sleep-active, or state-unrelated activity profiles and were, respectively, mainly located in the ventral or dorsal region of the SCN. In contrast, the type III neurons displayed sleep-active discharge profiles and were mainly located in the lateral region of the SCN. The majority of type I and II neurons tested showed an increase in discharge rate following application of light to the animal’s eyes. Of the 289 extra-SCN neurons recorded, those displaying sleep-active discharge profiles were mainly located dorsal to the SCN, whereas those displaying wake-active discharge profiles were mainly located lateral or dorsolateral to the SCN. This study shows heterogeneity of mouse SCN and surrounding anterior hypothalamic neurons and suggests differences in their topographic organization and roles in mammalian circadian rhythms and the regulation of sleep and wakefulness.

Vasoactive intestinal peptide produces long-lasting changes in neural activity in the suprachiasmatic nucleus

The neuropeptide vasoactive intestinal peptide (VIP) is expressed at high levels in the neurons of the suprachiasmatic nucleus (SCN). While VIP is known to be important to the input and output pathways from the SCN, the physiological effects of VIP on electrical activity of SCN neurons are not well known. Here the impact of VIP on firing rate of SCN neurons was investigated in mouse slice cultures recorded during the night. The application of VIP produced an increase in electrical activity in SCN slices that lasted several hours after treatment. This is a novel mechanism by which this peptide can produce long-term changes in central nervous system physiology. The increase in action potential frequency was blocked by a VIP receptor antagonist and lost in a VIP receptor knockout mouse. In addition, inhibitors of both the Epac family of cAMP binding proteins and cAMP-dependent protein kinase (PKA) blocked the induction by VIP. The persistent increase in spike rate following VIP application was not seen in SCN neurons from mice deficient in Kv3 channel proteins and was dependent on the clock protein PER1. These findings suggest that VIP regulates the long-term firing rate of SCN neurons through a VIPR2-mediated increase in the cAMP pathway and implicate the fast delayed rectifier (FDR) potassium currents as one of the targets of this regulation.

Causes and Consequences of Hyperexcitation in Central Clock Neurons

Hyperexcited states, including depolarization block and depolarized low amplitude membrane oscillations (DLAMOs), have been observed in neurons of the suprachiasmatic nuclei (SCN), the site of the central mammalian circadian (∼24-hour) clock. The causes and consequences of this hyperexcitation have not yet been determined. Here, we explore how individual ionic currents contribute to these hyperexcited states, and how hyperexcitation can then influence molecular circadian timekeeping within SCN neurons. We developed a mathematical model of the electrical activity of SCN neurons, and experimentally verified its prediction that DLAMOs depend on post-synaptic L-type calcium current. The model predicts that hyperexcited states cause high intracellular calcium concentrations, which could trigger transcription of clock genes. The model also predicts that circadian control of certain ionic currents can induce hyperexcited states. Putting it all together into an integrative model, we show how membrane potential and calcium concentration provide a fast feedback that can enhance rhythmicity of the intracellular circadian clock. This work puts forward a novel role for electrical activity in circadian timekeeping, and suggests that hyperexcited states provide a general mechanism for linking membrane electrical dynamics to transcription activation in the nucleus.

Fast synchronous oscillations of firing rate in cultured rat suprachiasmatic nucleus neurons: Possible role in circadian synchronization in the intact nucleus

The coherent circadian rhythm of the brain's master circadian pacemaker, the suprachiasmatic nucleus (SCN), is a result of synchronization of electrical activity of many SCN neurons possessing their own circadian oscillators. However, how the activity of these neurons is synchronized is not precisely known. By plotting the electrical firing rates of dispersed rat SCN neurons in multi-electrode array dishes with 20-s averaging of action-potential activity, we have investigated a novel phenomenon: fast (relative to the circadian cycle) oscillations of firing rate (FOFR) with duration of bursts ∼10min and interburst interval varying in a range from 20 to 60min in different cells, remaining nevertheless rather regular in individual cells. In many cases, separate neurons in distant parts of the 1mm recording area of an array exhibited correlated FOFR. FOFR of individual cells were positively or negatively correlated with those of other cells in a functioning neural network. Intriguingly, in occasional neuron pairs, transformation of their irregular firing to circadian peaks was accompanied by appearance of FOFR and an increase in the magnitude of firing correlation. We hypothesize that this FOFR observed in cultured SCN neurons contribute to synchronization of the circadian rhythm in the intact SCN.

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.

Disruption of circadian rhythmicity and suprachiasmatic action potential frequency in a mouse model with constitutive activation of glycogen synthase kinase 3

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in psychiatric diseases, neurodevelopment, and circadian regulation. Both GSK3 isoforms, α and β, exhibit a 24-h variation of inhibitory phosphorylation within the suprachiasmatic nucleus (SCN), the primary circadian pacemaker. We examined the hypothesis that rhythmic GSK3 activity is critical for robust circadian rhythmicity using GSK3α21A/21A/β9A/9A knock-in mice with serine–alanine substitutions at the inhibitory phosphorylation sites, making both forms constitutively active. We monitored wheel-running locomotor activity of GSK3 knock-in mice and used loose-patch electrophysiology to examine the effect of chronic GSK3 activity on circadian behavior and SCN neuronal activity. Double transgenic GSK3α/β knock-in mice exhibit disrupted behavioral rhythmicity, including significantly decreased rhythmic amplitude, lengthened active period, and increased activity bouts per day. This behavioral disruption was dependent on chronic activation of both GSK3 isoforms and was not seen in single transgenic GSK3α or GSK3β knock-in mice. Underlying the behavioral changes, SCN neurons from double transgenic GSK3α/β knock-in mice exhibited significantly higher spike rates during the subjective night compared to those from wild-type controls, with no differences detected during the subjective day. These results suggest that constitutive activation of GSK3 results in the loss of the typical day/night variation of SCN neuronal activity. Together, these results implicate GSK3 activity as a critical regulator of circadian behavior and neurophysiological rhythms. Because GSK3 has been implicated in numerous pathologies, understanding how GSK3 modulates circadian rhythms and neurophysiological activity may lead to novel therapeutics for pathological disorders and circadian rhythm dysfunction.

Amplitude of the SCN Clock Enhanced by the Behavioral Activity Rhythm

Circadian rhythms are regulated by the suprachiasmatic nucleus (SCN), a small structure at the base of the hypothalamus. While light effects on the SCN are well established, little is known of behavioral effects. This study elucidates direct modulating action of behavioral activity on the SCN by use of in vivo electrophysiology recordings, assessments of general locomotor behavior, and video-tracking of mice. The results show suppression of SCN neuronal activity by spontaneous behavior, the magnitude being dependent on the intensity, duration and type of behavioral activity. The suppression was moderate (32% of circadian amplitude) for low-intensity behavior and considerable (59%) for locomotor activity. Mild manipulation of the animals had reversed effects on the SCN indicating that different mechanisms are involved in the regulatory effect of spontaneous versus induced activity. The results indicate that exercise at the proper time of the cycle can boost the amplitude of the rhythm of the SCN clock itself. This has potentially beneficial effects for other rhythmic functions that are under the control of the SCN.

Neuropeptide signaling differentially affects phase maintenance and rhythm generation in SCN and extra-SCN circadian oscillators.

Circadian rhythms in physiology and behavior are coordinated by the brain's dominant circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Vasoactive intestinal polypeptide (VIP) and its receptor, VPAC2, play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC2 receptors (Vipr2−/−) express disrupted behavioral and metabolic rhythms and show altered SCN neuronal activity and clock gene expression. Within the brain, the SCN is not the only site containing endogenous circadian oscillators, nor is it the only site of VPAC2 receptor expression; both VPAC2 receptors and rhythmic clock gene/protein expression have been noted in the arcuate (Arc) and dorsomedial (DMH) nuclei of the mediobasal hypothalamus, and in the pituitary gland. The functional role of VPAC2 receptors in rhythm generation and maintenance in these tissues is, however, unknown. We used wild type (WT) and Vipr2−/− mice expressing a luciferase reporter (PER2::LUC) to investigate whether circadian rhythms in the clock gene protein PER2 in these extra-SCN tissues were compromised by the absence of the VPAC2 receptor. Vipr2−/− SCN cultures expressed significantly lower amplitude PER2::LUC oscillations than WT SCN. Surprisingly, in Vipr2−/− Arc/ME/PT complex (Arc, median eminence and pars tuberalis), DMH and pituitary, the period, amplitude and rate of damping of rhythms were not significantly different to WT. Intriguingly, while we found WT SCN and Arc/ME/PT tissues to maintain a consistent circadian phase when cultured, the phase of corresponding Vipr2−/− cultures was reset by cull/culture procedure. These data demonstrate that while the main rhythm parameters of extra-SCN circadian oscillations are maintained in Vipr2−/− mice, the ability of these oscillators to resist phase shifts is compromised. These deficiencies may contribute towards the aberrant behavior and metabolism associated with Vipr2−/− animals. Further, our data indicate a link between circadian rhythm strength and the ability of tissues to resist circadian phase resetting.

Activation of glycine receptor phase‐shifts the circadian rhythm in neuronal activity in the mouse suprachiasmatic nucleus

In mammals, the master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus is composed of numerous synchronized oscillating cells that drive daily behavioural and physiological processes. Several entrainment pathways, afferent inputs to the SCN with their neurotransmitter and neuromodulator systems, can reset the circadian system regularly and also modulate neuronal activity within the SCN. In the present study, we investigated the function of the inhibitory neurotransmitter glycine on neuronal activity in the mouse SCN and on resetting of the circadian clock. The effects of glycine on the electrical activity of SCN cells from C57Bl/6 mice were studied either by patch-clamp recordings from acute brain slices or by long-term recordings from organotypic brain slices using multi-microelectrode arrays(MEA). Voltage-clamp recordings confirmed the existence of glycine-induced, chloride-selective currents in SCN neurons. These currents were reversibly suppressed by strychnine, phenylbenzeneω-phosphono-α-amino acid (PMBA) or ginkgolide B, selective blockers of glycine receptors(GlyRs). Long-term recordings of the spontaneous activity of SCN neurons revealed that glycine application induces a phase advance during the subjective day and a phase delay during the early subjective night. Both effects were suppressed by strychnine or by PMBA. These results suggest that glycine is able to modulate circadian activity by acting directly on its specific receptors in SCN neurons.

Acute Ethanol Disrupts Photic and Serotonergic Circadian Clock Phase-Resetting in the Mouse

Alcohol dependence is associated with impaired circadian rhythms and sleep. Ethanol administration disrupts circadian clock phase-resetting, suggesting a mode for the disruptive effect of alcohol dependence on the circadian timing system. In this study, we extend previous work in C57BL/6J mice to: (i) characterize the suprachiasmatic nucleus (SCN) pharmacokinetics of acute systemic ethanol administration, (ii) explore the effects of acute ethanol on photic and nonphotic phase-resetting, and (iii) determine if the SCN is a direct target for photic effects. First, microdialysis was used to characterize the pharmacokinetics of acute intraperitoneal (i.p.) injections of 3 doses of ethanol (0.5, 1.0, and 2.0 g/kg) in the mouse SCN circadian clock. Second, the effects of acute i.p. ethanol administration on photic phase delays and serotonergic ([+]8-OH-DPAT-induced) phase advances of the circadian activity rhythm were assessed. Third, the effects of reverse-microdialysis ethanol perfusion of the SCN on photic phase-resetting were characterized. Peak ethanol levels from the 3 doses of ethanol in the SCN occurred within 20 to 40 minutes postinjection with half-lives for clearance ranging from 0.6 to 1.8 hours. Systemic ethanol treatment dose-dependently attenuated photic and serotonergic phase-resetting. This treatment also did not affect basal SCN neuronal activity as assessed by Fos expression. Intra-SCN perfusion with ethanol markedly reduced photic phase delays. These results confirm that acute ethanol attenuates photic phase-delay shifts and serotonergic phase-advance shifts in the mouse. This dual effect could disrupt photic and nonphotic entrainment mechanisms governing circadian clock timing. It is also significant that the SCN clock is a direct target for disruptive effects of ethanol on photic shifting. Such actions by ethanol could underlie the disruptive effects of alcohol abuse on behavioral, physiological, and endocrine rhythms associated with alcoholism.

Gastrin-releasing peptide modulates fast delayed rectifier potassium current in Per1-expressing SCN neurons.

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) drives and maintains 24-h physiological rhythms, the phases of which are set by the local environmental light-dark cycle. Gastrin-releasing peptide (GRP) communicates photic phase setting signals in the SCN by increasing neurophysiological activity of SCN neurons. Here, the ionic basis for persistent GRP-induced changes in neuronal activity was investigated in SCN slice cultures from Per1::GFP reporter mice during the early night. Recordings from Per1 -fluorescent neurons in SCN slices several hours after GRP treatment revealed a significantly greater action potential frequency, a significant increase in voltage-activated outward current at depolarized potentials, and a significant increase in 4-aminopyridine-sensitive fast delayed rectifier (fDR) potassium currents when compared to vehicle-treated slices. In addition, the persistent increase in spike rate following early-night GRP application was blocked in SCN neurons from mice deficient in Kv3 channel proteins. Because fDR currents are regulated by the clock and are elevated in amplitude during the day, the present results support the model that GRP delays the phase of the clock during the early night by prolonging day-like membrane properties of SCN cells. Furthermore, these findings implicate fDR currents in the ionic basis for GRP-mediated entrainment of the primary mammalian circadian pacemaker.

Interaction between hypothalamic dorsomedial nucleus and the suprachiasmatic nucleus determines intensity of food anticipatory behavior

Food anticipatory behavior (FAA) is induced by limiting access to food for a few hours daily. Animals anticipate this scheduled meal event even without the suprachiasmatic nucleus (SCN), the biological clock. Consequently, a food-entrained oscillator has been proposed to be responsible for meal time estimation. Recent studies suggested the dorsomedial hypothalamus (DMH) as the site for this food-entrained oscillator, which has led to considerable controversy in the literature. Herein we demonstrate by means of c-Fos immunohistochemistry that the neuronal activity of the suprachiasmatic nucleus (SCN), which signals the rest phase in nocturnal animals, is reduced when animals anticipate the scheduled food and, simultaneously, neuronal activity within the DMH increases. Using retrograde tracing and confocal analysis, we show that inhibition of SCN neuronal activity is the consequence of activation of GABA-containing neurons in the DMH that project to the SCN. Next, we show that DMH lesions result in a loss or diminution of FAA, simultaneous with increased activity in the SCN. A subsequent lesion of the SCN restored FAA. We conclude that in intact animals, FAA may only occur when the DMH inhibits the activity of the SCN, thus permitting locomotor activity. As a result, FAA originates from a neuronal network comprising an interaction between the DMH and SCN. Moreover, this study shows that the DMH-SCN interaction may serve as an intrahypothalamic system to gate activity instead of rest overriding circadian predetermined temporal patterns.

Dysfunctions in circadian behavior and physiology in mouse models of Huntington's disease

Many patients with Huntington's disease (HD) exhibit disturbances in their daily cycle of sleep and wake as part of their symptoms. These patients have difficulty sleeping at night and staying awake during the day, which has a profound impact on the quality of life of the patients and their care-givers. In the present study, we examined diurnal and circadian rhythms of four models of HD including the BACHD, CAG 140 knock-in and R6/2 CAG 140 and R6/2 CAG 250 lines of mice. The BACHD and both R6/2 lines showed profound circadian phenotypes as measured by wheel-running activity. Focusing on the BACHD line for further analysis, the amplitude of the rhythms in the BACHD mice declined progressively with age. In addition, the circadian regulation of heart rate and body temperature in freely behaving BACHD mice were also disrupted. Furthermore, the distribution of sleep as well as the autonomic regulation of heart rate was disrupted in this HD model. To better understand the mechanistic underpinnings of the circadian disruption, we used electrophysiological tools to record from neurons within the central clock in the suprachiasmatic nucleus (SCN). The BACHD mice exhibit reduced rhythms in spontaneous electrical activity in SCN neurons. Interestingly, the expression of the clock gene PERIOD2 was not altered in the SCN of the BACHD line. Together, this data is consistent with the hypothesis that the HD mutations interfere with the expression of robust circadian rhythms in behavior and physiology. The data raise the possibility that the electrical activity within the central clock itself may be altered in this disease.

Electrophysiological effects of melatonin on mouse Per1 and non-Per1 suprachiasmatic nuclei neurones in vitro.

The master circadian pacemaker in the suprachiasmatic nuclei (SCN) regulates the nocturnal secretion of the pineal hormone melatonin. Melatonin, in turn, has feedback effects on SCN neuronal activity rhythms via high affinity G protein-coupled receptors (MT(1) and MT(2) ). However, the precise effects of melatonin on the electrical properties of individual SCN neurones are unclear. In the present study, we investigated the acute effects of exogenous melatonin on SCN neurones using whole-cell patch-clamp recordings in brain slices prepared from Per1::d2EGFP-expressing transgenic mice. In current-clamp mode, bath applied melatonin, at near-physiological concentrations (1 nM), hyperpolarised the majority (63.7%) of SCN neurones tested at all times of the projected light/dark cycle. In addition, melatonin depolarised a small proportion of cells (11.0%). No differences were observed for the effects of melatonin between Per1::GFP or non-Per1::GFP SCN neurones. Melatonin-induced effects were blocked by the MT(1)/MT(2) antagonist, luzindole (1 μM) and the proportion of SCN neurones responsive to melatonin was greatly reduced in the presence of either tetrodotoxin (200 or 500 nM) or gabazine (20 μM). In voltage-clamp recordings, 1 nM melatonin increased the frequency of GABA-mediated currents. These findings indicate, for the first time, that exogenous melatonin can alter neuronal excitability in the majority of SCN neurones, regardless of whether or not they overtly express the core clock gene Per1. The results also suggest that melatonin acts mainly by modulating inhibitory GABAergic transmission within the SCN. This may explain why exogenous application of melatonin has heterogenous effects on individual SCN neurones.

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.

The Mammalian Circadian Clock Exhibits Acute Tolerance to Ethanol

Tolerance to ethanol is observed over a variety of time courses, from minutes to days. Acute tolerance, which develops over 5 to 60 minutes, has been observed for both behavioral and neurophysiological variables and may involve changes in signaling through NMDA, GABA, or other receptors. Previous work has shown that both acute and chronic ethanol treatments modulate photic and nonphotic phase resetting of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Although not specifically tested, the data thus far do not point to the development of chronic tolerance to the modulatory effects of ethanol. Here we investigated whether acute tolerance the ethanol occurs with respect to in vitro phase modulation of the SCN clock. Mouse brain slices containing the SCN were pretreated with ethanol for varying lengths of time, followed by treatment concurrent with either glutamate or the serotonin agonist, 8-hydroxy-DPAT (DPAT). The phase of the SCN circadian clock was assessed the following day through extracellular recordings of SCN neuronal activity. SCN neuronal activity normally peaks during mid-day, and this rhythm can be shifted by treatment with either glutamate or DPAT. While concurrent treatment of SCN-containing brain slices with ethanol and glutamate blocks glutamate-induced phase delays of the SCN clock, pretreating the slices with ethanol for > or =15 minutes prevents this inhibition. Likewise, while concurrent treatment with ethanol and DPAT enhances DPAT-induced phase advances of the SCN clock, pretreating the slices with ethanol for > or =30 minutes prevents this enhancement. Both the inhibiting and enhancing effects of ethanol on in vitro SCN clock phase resetting show acute tolerance. Additional experiments are needed to determine whether more slowly developing forms of tolerance also occur with respect to the SCN circadian clock.