Extensive soma-soma plate-like contact sites (ephapses) connect suprachiasmatic nucleus neurons.

The hypothalamic suprachiasmatic nucleus (SCN) is the central pacemaker for mammalian circadian rhythms. As such, this ensemble of cell-autonomous neuronal oscillators with divergent periods must maintain coordinated oscillations. To investigate ultrastructural features enabling such synchronization, 805 coronal ultrathin sections of mouse SCN tissue were imaged with electron microscopy and aligned into a volumetric stack, from which selected neurons within the SCN core were reconstructed in silico. We found that clustered SCN core neurons were physically connected to each other via multiple large soma-to-soma plate-like contacts. In some cases, a sliver of a glial process was interleaved. These contacts were large, covering on average ∼21% of apposing neuronal somata. It is possible that contacts may be the electrophysiological substrate for synchronization between SCN neurons. Such plate-like contacts may explain why the synchronization of SCN neurons is maintained even when chemical synaptic transmission or electrical synaptic transmission via gap junctions is blocked. Such ephaptic contact-mediated synchronization among nearby neurons may therefore contribute to the wave-like oscillations of circadian core clock genes and calcium signals observed in the SCN.

Three­dimensional reconstruction of SCN tissue via serial electron microscopy revealed a novel structural feature of SCN neurons that may account for interneuronal synchronization that persists even when the predominant mechanisms of neuronal communication are blocked. We found that SCN core neurons are connected by multiple soma­soma contact specializations, ultrastructural elements that could enable synchronization of tightly packed neurons organized in clustered networks. This extensive network of plate­like soma­soma contacts among clustered SCN neurons may provide insight into how ∼20,000 autonomous neuronal oscillators with a broad range of intrinsic periods remain synchronized in the absence of ordinary communication modalities, thereby conferring the resilience required for the SCN to function as the mammalian circadian pacemaker.

In vivo recording of the circadian calcium rhythm in Prokineticin 2 neurons of the suprachiasmatic nucleus.

Prokineticin 2 (Prok2) is a small protein expressed in a subpopulation of neurons in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals. Prok2 has been implicated as a candidate output molecule from the SCN to control multiple circadian rhythms. Genetic manipulation specific to Prok2-producing neurons would be a powerful approach to understanding their function. Here, we report the generation of Prok2-tTA knock-in mice expressing the tetracycline transactivator (tTA) specifically in Prok2 neurons and an application of these mice to in vivo recording of Ca2+ rhythms in these neurons. First, the specific and efficient expression of tTA in Prok2 neurons was verified by crossing the mice with EGFP reporter mice. Prok2-tTA mice were then used to express a fluorescent Ca2+ sensor protein to record the circadian Ca2+ rhythm in SCN Prok2 neurons in vivo. Ca2+ in these cells showed clear circadian rhythms in both light-dark and constant dark conditions, with their peaks around midday. Notably, the hours of high Ca2+ nearly coincided with the rest period of the behavioral rhythm. These observations fit well with the predicted function of Prok2 neurons as a candidate output pathway of the SCN by suppressing locomotor activity during both daytime and subjective daytime.

Kv12-encoded K+ channels drive the day-night switch in the repetitive firing rates of SCN neurons.

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K+) channels regulate daily oscillations in the spontaneous firing rates of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K+ conductance(s) driving these daily rhythms in the repetitive firing rates of SCN neurons, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K+ channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1-/-) or Kcnh3 (Kv12.2-/-) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1-/- and Kv12.2-/- than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1-/-, Kv12.2-/-, and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1-/- and Kv12.2-/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, the pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of the nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1-/-, Kv12.2-/-, and Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.

Rhythmic cilia changes support SCN neuron coherence in circadian clock.

The suprachiasmatic nucleus (SCN) drives circadian clock coherence through intercellular coupling, which is resistant to environmental perturbations. We report that primary cilia are required for intercellular coupling among SCN neurons to maintain the robustness of the internal clock in mice. Cilia in neuromedin S-producing (NMS) neurons exhibit pronounced circadian rhythmicity in abundance and length. Genetic ablation of ciliogenesis in NMS neurons enabled a rapid phase shift of the internal clock under jet-lag conditions. The circadian rhythms of individual neurons in cilia-deficient SCN slices lost their coherence after external perturbations. Rhythmic cilia changes drive oscillations of Sonic Hedgehog (Shh) signaling and clock gene expression. Inactivation of Shh signaling in NMS neurons phenocopied the effects of cilia ablation. Thus, cilia-Shh signaling in the SCN aids intercellular coupling.

Effects of NALCN-Encoded Na+ Leak Currents on the Repetitive Firing Properties of SCN Neurons Depend on K+-Driven Rhythmic Changes in Input Resistance.

Neurons in the suprachiasmatic nucleus (SCN) generate circadian changes in the rates of spontaneous action potential firing that regulate and synchronize daily rhythms in physiology and behavior. Considerable evidence suggests that daily rhythms in the repetitive firing rates (higher during the day than at night) of SCN neurons are mediated by changes in subthreshold potassium (K+) conductance(s). An alternative "bicycle" model for circadian regulation of membrane excitability in clock neurons, however, suggests that an increase in NALCN-encoded sodium (Na+) leak conductance underlies daytime increases in firing rates. The experiments reported here explored the role of Na+ leak currents in regulating daytime and nighttime repetitive firing rates in identified adult male and female mouse SCN neurons: vasoactive intestinal peptide-expressing (VIP+), neuromedin S-expressing (NMS+) and gastrin-releasing peptide-expressing (GRP+) cells. Whole-cell recordings from VIP+, NMS+, and GRP+ neurons in acute SCN slices revealed that Na+ leak current amplitudes/densities are similar during the day and at night, but have a larger impact on membrane potentials in daytime neurons. Additional experiments, using an in vivo conditional knockout approach, demonstrated that NALCN-encoded Na+ currents selectively regulate daytime repetitive firing rates of adult SCN neurons. Dynamic clamp-mediated manipulation revealed that the effects of NALCN-encoded Na+ currents on the repetitive firing rates of SCN neurons depend on K+ current-driven changes in input resistances. Together, these findings demonstrate that NALCN-encoded Na+ leak channels contribute to regulating daily rhythms in the excitability of SCN neurons by a mechanism that depends on K+ current-mediated rhythmic changes in intrinsic membrane properties.SIGNIFICANCE STATEMENT Elucidating the ionic mechanisms responsible for generating daily rhythms in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, is an important step toward understanding how the molecular clock controls circadian rhythms in physiology and behavior. While numerous studies have focused on identifying subthreshold K+ channel(s) that mediate day-night changes in the firing rates of SCN neurons, a role for Na+ leak currents has also been suggested. The results of the experiments presented here demonstrate that NALCN-encoded Na+ leak currents differentially modulate daily rhythms in the daytime/nighttime repetitive firing rates of SCN neurons as a consequence of rhythmic changes in subthreshold K+ currents.

Sex-specific differences in the circadian pattern of action potential firing by rat suprachiasmatic nucleus vasopressin neurons.

The suprachiasmatic nucleus (SCN) of the hypothalamus serves as the master circadian clock in mammals. Most SCN neurons express the inhibitory neurotransmitter GABA (gamma amino butyric acid) along with a peptide cotransmitter. Notably, the neuropeptides vasopressin (VP) and vasoactive intestinal peptide (VIP) define two prominent clusters within the SCN: those located in the ventral core (VIP) and those forming the dorsomedial "shell" of the nucleus (VP). Axons emerging from VP neurons in the shell are thought to mediate much of the SCN's output to other brain regions as well as VP release into the cerebrospinal fluid (CSF). Previous work has shown that VP release by SCN neurons is activity dependent and SCN VP neurons fire action potentials at a higher rate during the light phase. Accordingly, CSF VP levels are higher during daytime. Interestingly, the amplitude of the CSF VP rhythm is greater in males than females, suggesting the existence of sex differences in the electrical activity of SCN VP neurons. Here we investigated this hypothesis by performing cell-attached recordings from 1070 SCN VP neurons across the entire circadian cycle in both sexes of transgenic rats that express green fluorescent protein (GFP) driven by the VP gene promoter. Using an immunocytochemical approach we confirmed that >60% of SCN VP neurons display visible GFP. Recordings in acute coronal slices revealed that VP neurons display a striking circadian pattern of action potential firing, but the characteristics of this activity cycle differ in males and females. Specifically, neurons in males reached a significantly higher peak firing frequency during subjective daytime compared to females and the acrophase occurred ~1 h earlier in females. Peak firing rates in females were not significantly different at various phases of the estrous cycle.

Regulation of CRE-Dependent Transcriptional Activity in a Mouse Suprachiasmatic Nucleus Cell Line.

We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output.

Female fertility does not require Bmal1 in suprachiasmatic nucleus neurons expressing arginine vasopressin, vasoactive intestinal peptide, or neuromedin-S.

Disruptions to the circadian system alter reproductive capacity, particularly in females. Mice lacking the core circadian clock gene, Bmal1, are infertile and have evidence of neuroendocrine disruption including the absence of the preovulatory luteinizing hormone (LH) surge and enhanced responsiveness to exogenous kisspeptin. Here, we explore the role of Bmal1 in suprachiasmatic nucleus (SCN) neuron populations known to project to the neuroendocrine axis. We generated four mouse lines using Cre/Lox technology to create conditional deletion of Bmal1 in arginine vasopressin (Bmal1fl/fl:Avpcre ), vasoactive intestinal peptide (Bmal1fl/fl:Vipcre ), both (Bmal1fl/fl:Avpcre+Vipcre ), and neuromedin-s (Bmal1fl/fl:Nmscre ) neurons. We demonstrate that the loss of Bmal1 in these populations has substantial effects on home-cage circadian activity and temperature rhythms. Despite this, we found that female mice from these lines demonstrated normal estrus cycles, fecundity, kisspeptin responsiveness, and inducible LH surge. We found no evidence of reproductive disruption in constant darkness. Overall, our results indicate that while conditional Bmal1 knockout in AVP, VIP, or NMS neurons is sufficient to disrupted locomotor activity, this disruption is insufficient to recapitulate the neuroendocrine reproductive effects of the whole-body Bmal1 knockout.

Long-term SCN calcium signal recording in freely moving mice.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker of the mammalian biological clock. Here, we provide a detailed protocol for long-term recording of calcium signals in SCN neurons of freely moving mice through a multichannel optical fiber recording system. This system can simultaneously collect calcium signals from up to seven animals. The calcium signals can be visualized by the appropriate software and code. This protocol can be used to explore the long-term response of SCN to external environmental stimulation. For complete details on the use and execution of this protocol, please refer to Zhai et al. (2022).

Network rewiring and plasticity promotes synchronization of suprachiasmatic nucleus neurons.

In mammals, circadian rhythms throughout the body are orchestrated by the master clock in the hypothalamic suprachiasmatic nucleus (SCN), where SCN neurons are coupled with neurotransmitters to generate a uniform circadian rhythm. How the SCN circadian rhythm is so robust and flexible is, however, unclear. In this paper, we propose a temporal SCN network model and investigate the effects of dynamical rewiring and flexible coupling due to synaptic plasticity on the synchronization of the neural network in SCN. In networks consisting of simple Poincaré oscillators and complex circadian clocks, we found that dynamical rewiring and coupling plasticity enhance the synchronization in inhomogeneous networks. We verified the effect of enhanced synchronization in different architectures of random, scale-free, and small-world networks. A simple mean-field analysis for synchronization in plastic networks is proposed. Intuitively, the synchronization is greatly enhanced because both the random rewiring and coupling plasticity in the heterogeneous network have effectively increased the coupling strength in the whole network. Our results suggest that a proper network model for the master SCN circadian rhythm needs to take into account the effects of dynamical changes in topology and plasticity in neuron interactions that could help the brain to generate a robust circadian rhythm for the whole body.

Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons.

The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism.

Optogenetic stimulation of VIPergic SCN neurons induces photoperiodic-like changes in the mammalian circadian clock.

Circadian clocks play key roles in how organisms respond to and even anticipate seasonal change in day length, or photoperiod. In mammals, photoperiod is encoded by the central circadian pacemaker in the brain, the suprachiasmatic nucleus (SCN). The subpopulation of SCN neurons that secrete the neuropeptide VIP mediates the transmission of light information within the SCN neural network, suggesting a role for these neurons in circadian plasticity in response to light information that has yet to be directly tested. Here, we used in vivo optogenetic stimulation of VIPergic SCN neurons followed by ex vivo PERIOD 2::LUCIFERASE (PER2::LUC) bioluminescent imaging to test whether activation of this SCN neuron subpopulation can induce SCN network changes that are hallmarks of photoperiodic encoding. We found that optogenetic stimulation designed to mimic a long photoperiod indeed altered subsequent SCN entrained phase, increased the phase dispersal of PER2 rhythms within the SCN network, and shortened SCN free-running period-similar to the effects of a true extension of photoperiod. Optogenetic stimulation also induced analogous changes on related aspects of locomotor behaviour in vivo. Thus, selective activation of VIPergic SCN neurons induces photoperiodic network plasticity in the SCN that underpins photoperiodic entrainment of behaviour.

Light stimulation during postnatal development is not determinant for glutamatergic neurotransmission from the retinohypothalamic tract to the suprachiasmatic nucleus in rats.

The hypothalamic suprachiasmatic nucleus (SCN) is the leading circadian pacemaker in mammals, which synchronizes with environmental light through the retinohypothalamic tract (RHT). Although the SCN regulates circadian rhythms before birth, postnatal synaptic changes are needed for the RHT-SCN pathway to achieve total functional development. However, it is unknown whether visual experience affects developmental maturation. Here, we studied the effects of constant darkness (DD) rearing on the physiology (at pre- and postsynaptic levels) of glutamatergic neurotransmission between RHT and SCN during postnatal development in rats. Upon recording spontaneous and evoked excitatory postsynaptic currents (EPSCs) by electrical stimulation of RHT fibers, we found that DD animals at early postnatal ages (P3-19) exhibited different frequencies of spontaneous EPSCs and lower synaptic performance (short-term depression, release sites, and recruitment of RHT fibers) when compared with their normal light/dark (LD) counterparts. At the oldest age evaluated (P30-35), there was a synaptic response strengthening (probability of release, vesicular re-filling rate, and reduced synaptic depression) in DD rats, which functionally equaled (or surmounted) that of LD animals. Control experiments evaluating EPSCs in ventral SCN neurons of LD rats during day and night revealed no significant differences in spontaneous or evoked EPSCs by high-frequency trains in the RHT at any postnatal age. Our results suggest that DD conditions induce a compensatory mechanism in the glutamatergic signaling of the circadian system to increase the chances of synchronization to light at adult ages, and that the synaptic properties of RHT terminals during postnatal development are not critically influenced by environmental light.

Communicating clocks shape circadian homeostasis.

Circadian clocks temporally coordinate physiology and align it with geophysical time, which enables diverse life-forms to anticipate daily environmental cycles. In complex organisms, clock function originates from the molecular oscillator within each cell and builds upward anatomically into an organism-wide system. Recent advances have transformed our understanding of how clocks are connected to achieve coherence across tissues. Circadian misalignment, often imposed in modern society, disrupts coordination among clocks and has been linked to diseases ranging from metabolic syndrome to cancer. Thus, uncovering the physiological circuits whereby biological clocks achieve coherence will inform on both challenges and opportunities in human health.

Development of serotonergic projections to the suprachiasmatic nucleus in the mouse brain.

Serotonin (5-HT) and its innervation have been implicated in various neural functions including circadian systems. Although classical studies have examined the 5-HT innervation pattern in the adult suprachiasmatic nucleus (SCN), the fine-grained morphological study of the development of pathway and terminal projections to the SCN remains scarce. Here, we utilize transgenic mice expressing GFP under the serotonin transporter (SERT) promoter to subserve our developmental mapping study. We demonstrate that the morphology of 5-HT pathway fibers decussating over the supraoptic commissure that projects to the SCN exhibits two distinct developmental patterns. The punctate fibers at the fetal stage gradually become smooth and filamentous, especially during postnatal one week and remain constant thereafter. The innervation field in the SCN develops properly only during postnatal two weeks. Its ventromedial area remains one of the highest 5-HT innervated areas in the adult brain, whereas the dorsolateral area is less innervated. Thus, we provide novel and specific insights on the developmental map of 5-HT system into the SCN using transgenic mouse.

Circadian VIPergic Neurons of the Suprachiasmatic Nuclei Sculpt the Sleep-Wake Cycle.

Although the mammalian rest-activity cycle is controlled by a "master clock" in the suprachiasmatic nucleus (SCN) of the hypothalamus, it is unclear how firing of individual SCN neurons gates individual features of daily activity. Here, we demonstrate that a specific transcriptomically identified population of mouse VIP+ SCN neurons is active at the "wrong" time of day-nighttime-when most SCN neurons are silent. Using chemogenetic and optogenetic strategies, we show that these neurons and their cellular clocks are necessary and sufficient to gate and time nighttime sleep but have no effect upon daytime sleep. We propose that mouse nighttime sleep, analogous to the human siesta, is a "hard-wired" property gated by specific neurons of the master clock to favor subsequent alertness prior to dawn (a circadian "wake maintenance zone"). Thus, the SCN is not simply a 24-h metronome: specific populations sculpt critical features of the sleep-wake cycle.

Retinal Tropism and Transduction of Adeno-Associated Virus Varies by Serotype and Route of Delivery (Intravitreal, Subretinal, or Suprachoroidal) in Rats.

Viral-mediated gene augmentation offers tremendous promise for the treatment of inherited retinal diseases. The development of effective gene therapy requires an understanding of the vector's tissue-specific behavior, which may vary depending on serotype, route of delivery, or target species. Using an ex vivo organotypic explant system, we previously demonstrated that retinal tropism and transduction of adeno-associated virus type 2 (AAV2) vary significantly depending on serotype in human eyes. However, the ex vivo system has limited ability to assess route of ocular delivery, and relatively little literature exists on tropic differences between serotypes and routes of delivery in vivo. In this study, we demonstrate that retinal tropism and transduction efficiency of five different AAV2 serotypes (AAV2/1, AAV2/2, AAV2/6, AAV2/8, and AAV2/9) expressing enhanced green fluorescent protein driven by a cytomegalovirus promoter vary greatly depending on serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats. With subretinal delivery, all serotypes successfully transduced the retinal pigmented epithelium and outer nuclear layer (ONL), with AAV2/1 displaying the highest transduction efficiency and AAV2/2 and AAV2/6 showing lower ONL transduction. There was minimal transduction of the inner retina through subretinal delivery for any serotype. Tropism by suprachoroidal delivery mirrored that of subretinal delivery for all AAV serotypes but resulted in a wider distribution and greater ONL transduction. With intravitreal delivery, retinal transduction was seen primarily in the inner retina (retinal nerve fiber, ganglion cell, and inner nuclear layers) for AAV2/1 and AAV2/6, with AAV2/6 showing the highest transduction. When compared with data from human explant models, there are substantial differences in tropism and transduction that are important to consider when using rats as preclinical models for the development of ocular gene therapies for humans.

Dual-Color Single-Cell Imaging of the Suprachiasmatic Nucleus Reveals a Circadian Role in Network Synchrony.

The suprachiasmatic nucleus (SCN) acts as a master pacemaker driving circadian behavior and physiology. Although the SCN is small, it is composed of many cell types, making it difficult to study the roles of particular cells. Here we develop bioluminescent circadian reporter mice that are Cre dependent, allowing the circadian properties of genetically defined populations of cells to be studied in real time. Using a Color-Switch PER2::LUCIFERASE reporter that switches from red PER2::LUCIFERASE to green PER2::LUCIFERASE upon Cre recombination, we assess circadian rhythms in two of the major classes of peptidergic neurons in the SCN: AVP (arginine vasopressin) and VIP (vasoactive intestinal polypeptide). Surprisingly, we find that circadian function in AVP neurons, not VIP neurons, is essential for autonomous network synchrony of the SCN and stability of circadian rhythmicity.

Circadian rhythms in Per1, PER2 and Ca2+ of a solitary SCN neuron cultured on a microisland.

Circadian rhythms in Per1, PER2 expression and intracellular Ca2+ were measured from a solitary SCN neuron or glial cell which was physically isolated from other cells. Dispersed cells were cultured on a platform of microisland (100-200 µm in diameter) in a culture dish. Significant circadian rhythms were detected in 57.1% for Per1 and 70.0% for PER2 expression. When two neurons were located on the same island, the circadian rhythms showed desynchronization, indicating a lack of oscillatory coupling. Circadian rhythms were also detected in intracellular Ca2+ of solitary SCN neurons. The ratio of circadian positive neurons was significantly larger without co-habitant of glial cells (84.4%) than with it (25.0%). A relatively large fraction of SCN neurons generates the intrinsic circadian oscillation without neural or humoral networks. In addition, glial cells seem to interrupt the expression of the circadian rhythmicity of intracellular Ca2+ under these conditions.

Role of Intracellular Na+ in the Regulation of [Ca2+]i in the Rat Suprachiasmatic Nucleus Neurons.

Transmembrane Ca2+ influx is essential to the proper functioning of the central clock in the suprachiasmatic nucleus (SCN). In the rat SCN neurons, the clearance of somatic Ca2+ following depolarization-induced Ca2+ transients involves Ca2+ extrusion via Na+/Ca2+ exchanger (NCX) and mitochondrial Ca2+ buffering. Here we show an important role of intracellular Na+ in the regulation of [Ca2+]i in these neurons. The effect of Na+ loading on [Ca2+]i was determined with the Na+ ionophore monensin and the cardiac glycoside ouabain to block Na+/K+-ATPase (NKA). Ratiometric Na+ and Ca2+ imaging was used to measure the change in [Na+]i and [Ca2+]i, and cell-attached recordings to investigate the effects of monensin and ouabain on spontaneous firing. Our results show that in spite of opposite effects on spontaneous firing and basal [Ca2+], both monensin and ouabain induced Na+ loading, and increased the peak amplitude, slowed the fast decay rate, and enhanced the slow decay phase of 20 mM K+-evoked Ca2+ transients. Furthermore, both ouabain and monensin preferentially enhanced nimodipine-insensitive Ca2+ transients. Together, our results indicate that in the SCN neurons the NKA plays an important role in regulating [Ca2+]i, in particular, associated with nimodipine-insensitive Ca2+ channels.