Action potential firing rhythms in the suprachiasmatic nucleus of the diurnal grass rat, Arvicanthis niloticus.

In mammals, daily physiological activities are regulated by a central circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Recently, an increasing number of studies have used diurnal grass rats to analyze neuronal mechanisms regulating diurnal behavior. However, spontaneous action potential firing rhythms in SCN neurons have not been demonstrated clearly in diurnal grass rats. Therefore, the present study examined extracellular single-unit recordings from SCN neurons in acute hypothalamic slices of Arvicanthis niloticus (Nile grass rats). The results of this study found that circadian firing rhythms with the highest frequency occurred at dusk (6.4 Hz at zeitgeber time (ZT)10-12), while the secondary peak occurred at dawn (5.6 Hz at ZT0-2), and the lowest frequency took place in the middle of the night (3.6 Hz at ZT14-16). Locomotor activity recordings from a separate group of animals demonstrated that the Nile grass rats of the laboratory colony used in this study displayed diurnal behaviors that coincided with large crepuscular peaks under 12:12 h light-dark cycles and bimodal rhythms under constant dim red light. Thus, a positive correlation between SCN firing frequencies and locomotor activity levels was observed in the Nile grass rats. Previously, behavioral coupling of action potential firings in SCN neurons has been suggested by in vivo recordings while the present study demonstrates that the sustenance of bimodal firing rhythms in grass rat SCN neurons can last at least one day in vitro.

Mitochondrial LETM1 drives ionic and molecular clock rhythms in circadian pacemaker neurons.

The mechanisms that generate robust ionic oscillation in circadian pacemaker neurons are under investigation. Here, we demonstrate critical functions of the mitochondrial cation antiporter leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1), which exchanges K+/H+ in Drosophila and Ca2+/H+ in mammals, in circadian pacemaker neurons. Letm1 knockdown in Drosophila pacemaker neurons reduced circadian cytosolic H+ rhythms and prolonged nuclear PERIOD/TIMELESS expression rhythms and locomotor activity rhythms. In rat pacemaker neurons in the hypothalamic suprachiasmatic nucleus (SCN), circadian rhythms in cytosolic Ca2+ and Bmal1 transcription were dampened by Letm1 knockdown. Mitochondrial Ca2+ uptake peaks late during the day were also observed in rat SCN neurons following photolytic elevation of cytosolic Ca2+. Since cation transport by LETM1 is coupled to mitochondrial energy synthesis, we propose that LETM1 integrates metabolic, ionic, and molecular clock rhythms in the central clock system in both invertebrates and vertebrates.

Intracellular interplay between cholecystokinin and leptin signalling for satiety control in rats.

Cholecystokinin (CCK) and leptin are satiety-controlling peptides, yet their interactive roles remain unclear. Here, we addressed this issue using in vitro and in vivo models. In rat C6 glioma cells, leptin pre-treatment enhanced Ca2+ mobilization by a CCK agonist (CCK-8s). This leptin action was reduced by Janus kinase inhibitor (AG490) or PI3-kinase inhibitor (LY294002). Meanwhile, leptin stimulation alone failed to mobilize Ca2+ even in cells overexpressing leptin receptors (C6-ObRb). Leptin increased nuclear immunoreactivity against phosphorylated STAT3 (pSTAT3) whereas CCK-8s reduced leptin-induced nuclear pSTAT3 accumulation in these cells. In the rat ventromedial hypothalamus (VMH), leptin-induced action potential firing was enhanced, whereas nuclear pSTAT3 was reduced by co-stimulation with CCK-8s. To further analyse in vivo signalling interplay, a CCK-1 antagonist (lorglumide) was intraperitoneally injected in rats following 1-h restricted feeding. Food access was increased 3-h after lorglumide injection. At this timepoint, nuclear pSTAT3 was increased whereas c-Fos was decreased in the VMH. Taken together, these results suggest that leptin and CCK receptors may both contribute to short-term satiety, and leptin could positively modulate CCK signalling. Notably, nuclear pSTAT3 levels in this experimental paradigm were negatively correlated with satiety levels, contrary to the generally described transcriptional regulation for long-term satiety via leptin receptors.

Nighttime activities and peripheral clock oscillations depend on Wolbachia endosymbionts in flies.

Wolbachia are ubiquitous bacterial endosymbionts of arthropods and affect host gene expression. Although Wolbachia infections were suggested to modulate sleep in flies, their influence on the circadian clock remained obscure. Here, we screened bacterial symbionts in a laboratory Drosophila melanogaster colony, and observed widespread infections of wMel strain Wolbachia. We established a Wolbachia-free strain from a clock gene reporter strain, period-luciferase (per-luc). Temperature (19-29 °C)-compensated free-running periods were detected regardless of infections which may reflect the lack of wMel infections in central circadian pacemaker neurons. However, locomotor activity levels during the night or subjective night were significantly amplified in uninfected flies. Moreover, the behavioral phenotype of F1 offspring of an uninfected female and infected male resembled that of uninfected flies. This trait is consistent with maternal transmission of Wolbachia infection. Interestingly, per-luc activities in headless bodies, as an index of peripheral circadian oscillators, were severely damped in uninfected flies. Additionally, circadian amplitudes of PER immunoreactivities in Malpighian tubules were reduced in uninfected flies. These results demonstrate that Wolbachia boost fly peripheral clock oscillations and diurnal behavioral patterns. Genetic mechanisms underlying behavioral rhythms have been widely analyzed using mutant flies whereas screening of Wolbachia will be necessary for future studies.

Histamine Regulates Molecular Clock Oscillations in Human Retinal Pigment Epithelial Cells via H1 Receptors.

Vertebrate eyes are known to contain circadian clocks, but their regulatory mechanisms remain largely unknown. To address this, we used a cell line from human retinal pigment epithelium (hRPE-YC) with stable coexpression of reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). We observed concentration-dependent increases in cytosolic Ca2+ concentrations after treatment with histamine (1-100 µM) and complete suppression of histamine-induced Ca2+ mobilizations by H1 histamine receptor (H1R) antagonist d-chlorpheniramine (d-CPA) in hRPE-YC cells. Consistently, real-time RT-PCR assays revealed that H1R showed the highest expression among the four subtypes (H1-H4) of histamine receptors in hRPE-YC cells. Stimulation of hRPE-YC cells with histamine transiently increased nuclear localization of phosphorylated Ca2+/cAMP-response element-binding protein that regulates clock gene transcriptions. Administration of histamine also shifted the Bmal1-luciferase rhythms with a type-1 phase-response curve, similar to previous results with carbachol stimulations. Treatment of hRPE-YC cells with d-CPA or with more specific H1R antagonist, ketotifen, blocked the histamine-induced phase shifts. Furthermore, an H2 histamine receptor agonist, amthamine, had little effect on the Bmal1-luciferase rhythms. Although the function of the in vivo histaminergic system within the eye remains obscure, the present results suggest histaminergic control of the molecular clock via H1R in retinal pigment epithelial cells. Also, since d-CPA and ketotifen have been widely used (e.g., to treat allergy and inflammation) in our daily life and thus raise a possible cause for circadian rhythm disorders by improper use of antihistamines.

Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca2+ signaling in human retinal pigment epithelial cells.

Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca2+-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca2+ signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks.

Bimodal modulation of store-operated Ca2+ channels by clozapine in astrocytes.

Clozapine (Clz) and olanzapine (Olz) are second generation (atypical) antipsychotics, used widely for treating schizophrenia and bipolar disorder. These drugs share multiple sites of actions, however their mechanisms remain incompletely understood. Here, we analyzed the effects of these drugs on primary cultures of rat cortical astrocytes and C6 glioma cells using fura-2-based Ca2+ imaging. C6 cells, but not cortical astrocytes, express the serotonin 2A receptor subtype, which couples to phospholipase C. Clz (1µM) significantly blocked serotonin-induced Ca2+ transients in C6 cells, consistent with known antagonistic actions of Clz. Interestingly, at higher concentrations (>10µM), Clz but not Olz increased intracellular Ca2+ concentrations in both cortical astrocytes and C6 cells. This Clz-induced Ca2+ increase was concentration-dependent and completely blocked by removal of extracellular Ca2+ using ethylene glycol tetraacetic acid (EGTA). Furthermore, 2-aminoethyl diphenylborinate or SKF-96365, blockers for store-operated Ca2+ channels, significantly inhibited the Clz-induced Ca2+ increase. Therefore, we analyzed the effects of Clz and Olz during Ca2+ re-entry through store-operated Ca2+ channels, which was maximized following depletion of internal Ca2+ stores by thapsigargin and EGTA. The results demonstrated that Clz decreased Ca2+ re-entry through store-operated Ca2+ channels in cortical astrocytes and C6 cells whereas Olz failed to modulate the Ca2+ re-entry. These results suggest Clz-specific bimodal actions via store-operated Ca2+ channels in astrocytic cells. Since intracellular Ca2+ homeostasis in astrocytes is an important determinant for neighboring synaptic signal transmission, our results may explain Clz-specific adverse effects or differential actions between Clz and Olz reported in the treatment of psychiatric disorders.

Serotonin-2C receptor involved serotonin-induced Ca²âº mobilisations in neuronal progenitors and neurons in rat suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, undergoes serotonergic regulation, but the underlying mechanisms remain obscure. Here, we generated a subclone of an SCN progenitor cell line expressing Ca(2+) sensors (SCN2.2YC) and compared its 5-HT receptor signalling with that of rat SCN neurons in brain slices. SCN2.2YC cells expressed 5-HT1A/2A/2B/2C, but not 5A/7, while all six subtypes were expressed in SCN tissues. High K(+) or 5-HT increased cytosolic Ca(2+) in SCN2.2YC cells. The 5-HT responses were inhibited by ritanserin and SB-221284, but resistant to WAY-100635 and RS-127445, suggesting predominant involvement of 5-HT2C for Ca(2+) mobilisations. Consistently, Ca(2+) imaging and voltage-clamp electrophysiology using rat SCN slices demonstrated post-synaptic 5-HT2C expression. Because 5-HT2C expression was postnatally increased in the SCN and 5-HT-induced Ca(2+) mobilisations were amplified in differentiated SCN2.2YC cells and developed SCN neurons, we suggest that this signalling development occurs in accordance with central clock maturations.

Infant satiety depends on transient expression of cholecystokinin-1 receptors on ependymal cells lining the third ventricle in mice.

Cholecystokinin (CCK) is a hypothetical controller for suckling and infancy body weight, although the underlying mechanisms remain unclear. Therefore, the present study analysed the mechanisms using mice lacking the CCK-1 receptor (CCK1R-/-). Although CCK1R-/- mice displayed normal weights at birth and adulthood, CCK1R-/- pups had enlarged adipocytes and were overweight from the first to second week after birth, regardless of maternal genotype. The lacZ reporter gene assay and/or calcium imaging analysis demonstrated that CCK-1 receptors were abundant in satiety-controlling regions such as the hypothalamus, brainstem, nodose ganglion and pylorus in adults, whereas these signals were few to lacking at pre-weanling stages. At postnatal day (PD) 6, the increase in cFos expression in the medullary nucleus tractus solitarius was similarly triggered by gastrointestinal milk- or saline filling in both genotypes, further indicating immature CCK-1 receptor function in an ascending satiety-controlling system during infancy. Conversely, third ventricle ependymal tanycyte-like cells expressed CCK-1 receptors with expression peaking at PD6. At PD6, wild-type but not CCK1R-/- mice had increased cFos immunoreactivity in ependymal cells following gastrointestinal milk filling whereas the response became negligible at PD12. In addition, ependymal cFos was not increased by saline filling, indicating that these responses are dependent on CCK-1 receptors, developmental stage and nutrients. Furthermore, body weights of wild-type pups were transiently increased by blocking ependymal CCK receptor function with microinjection of a CCK-1 antagonist, but not a CCK-2 antagonist. Hence, we demonstrate de novo functions of ependymal CCK-1 receptors and reveal a new aspect of infant satiety-controlling mechanisms.

Functional compensation between cholecystokinin-1 and -2 receptors in murine paraventricular nucleus neurons.

Cholecystokinin (CCK) and its receptor subtypes CCK-1 and -2 have diverse homeostatic functions. CCK-1 and -2 receptors share a common phosphatidylinositol signaling pathway, yet little is known regarding their possible functional coupling. We focused on CCK-mediated Ca(2+) signaling in parvocellular paraventricular nucleus (PVN) cells, which control satiety and other autonomic functions. Analysis of mouse hypothalamic slices demonstrated that the general CCK receptor agonist CCK-8s (10 nM) triggered Ca(2+) transients most significantly in the posterior subregion of the PVN (PaPo). This 10 nM CCK-8s-induced response was absent in CCK-1 receptor knock-out (CCK1R(-/-)) slices, showing that the response is mediated by CCK-1 receptors. CCK-8s concentrations higher than 30 nM triggered a Ca(2+) rise similarly in wild-type and CCK1R(-/-) slices. The large CCK-8s (100 nM)-induced Ca(2+) responses in CCK1R(-/-) slices were blocked by a CCK-2 receptor antagonist (CI-988), whereas those in wild-type slices required a mixture of CI-988 and lorglumide (a CCK-1 receptor antagonist) for complete antagonism. Therefore, CCK-1 and -2 receptors may function synergistically in single PaPo neurons and deletion of CCK-1 receptors may facilitate CCK-2 receptor signaling. This hypothesis was supported by results of real-time RT-PCR, immunofluorescence double labeling and Western blotting assays, which indicated CCK-2 receptor overexpression in PaPo neurons of CCK1R(-/-) mice. Furthermore, behavioral studies showed that intraperitoneal injections of lorglumide up-regulated food accesses in wild-type but not in CCK1R(-/-) mice, whereas CI-988 injections up-regulated food accesses in CCK1R(-/-) but not in wild-type mice. Compensatory CCK signaling via CCK-2 receptors in CCK1R(-/-) mice shed light on currently controversial satiety-controlling mechanisms.

Neuronal influence on peripheral circadian oscillators in pupal Drosophila prothoracic glands.

Rhythmic expression of period (per) and timeless (tim) genes in central circadian pacemaker neurons and prothoracic gland cells, part of the peripheral circadian oscillators in flies, may synergistically control eclosion rhythms, but their oscillatory profiles remain unclear. Here we show differences and interactions between peripheral and central oscillators using per-luciferase and cytosolic Ca(2+) reporter (yellow cameleon) imaging in organotypic prothoracic gland cultures with or without the associated central nervous system. Isolated prothoracic gland cells exhibit light-insensitive synchronous per-transcriptional rhythms. In prothoracic gland cells associated with the central nervous system, however, per transcription is markedly amplified following 12-h light exposure, resulting in the manifestation of day-night rhythms in nuclear PER immunostaining levels and spontaneous Ca(2+) spiking. Unlike PER expression, nuclear TIM expression is associated with day-night cycles that are independent of the central nervous system. These results demonstrate that photoreception and synaptic signal transduction in/from the central nervous system coordinate molecular 'gears' in endocrine oscillators to generate physiological rhythms.

Induction of prolonged, continuous slow-wave sleep by blocking cerebral H1 histamine receptors in rats.

BACKGROUND AND PURPOSE: Classic H(1) histamine receptor (H(1)R) antagonists are non-selective for H(1)R and known to produce drowsiness. Modern antihistamines are more selective for H(1)R, and are 'non-drowsy' presumably due to reduced permeability through the blood-brain barrier. To characterize both histaminergic sleep regulation and the central actions of antihistamines, in the present study we analysed the effect of classic and modern antihistamines on rats' sleep using continuous i.c.v. infusions. EXPERIMENTAL APPROACH: Effects of classic (d-chlorpheniramine; d-CPA) and second-generation (cetirizine) antihistamines on sleep were compared after i.p. injections or continuous i.c.v. infusions into rats. Fluorescent cetirizine/DBD-pz was synthesized to trace the approximate distribution of cerebral cetirizine. Furthermore, the effects of H(1) R antagonists on cultured preoptic neurons were examined using calcium imaging. KEY RESULTS: d-CPA 4 mg·kg(-1) i.p. increased non-rapid eye movement (REM) sleep whereas 10-40 mg·kg(-1) d-CPA decreased non-REM sleep at dark onset time. Nocturnal i.c.v. infusions of d-CPA (10 µmol·100 µL(-1)·10 h(-1)) increased drowsiness but not non-REM sleep, whereas the same i.c.v. infusions of cetirizine significantly increased non-REM sleep, abolished REM sleep, and decreased wakefulness for more than 10 h. The medial preoptic area contained the greatest fluorescent labelling after i.c.v. cetirizine/DBD-pz infusions. Histamine-induced Ca(2+) increases in medial preoptic neurons were blocked by d-CPA or cetirizine, whereas d-CPA, but not cetirizine, increased Ca(2+) irrespective of antihistaminergic activity at ≥ 100 µM. CONCLUSION AND IMPLICATIONS: The excitatory action of d-CPA may explain the seemingly inconsistent actions of d-CPA on sleep. Cerebral H(1)R inhibition by cetirizine induces synchronization of cerebral activity and prolonged, continuous slow-wave sleep.

Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

BACKGROUND: Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca(2+) spikes" (i.e., [Ca(2+)](c) transients having a bandwidth of 10 approximately 100 seconds) in SCN neurons, but it is unclear if these SCN Ca(2+) spikes are related to the slow circadian rhythms. METHODOLOGY/PRINCIPAL FINDINGS: We addressed this issue based on a Ca(2+) indicator dye (fluo-4) and a protein Ca(2+) sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca(2+) spikes in 18% of rat SCN cells in acute brain slices, but the Ca(2+) spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca(2+) spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca(2+) spikes was increased to 13 approximately 14%. CONCLUSIONS/SIGNIFICANCE: Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca(2+) spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca(2+) spiking activity is caused by the Ca(2+) chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca(2+)](c) in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca(2+) spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca(2+) spikes in the function of SCN.