Phospholipase C-β4 Is Essential for the Progression of the Normal Sleep Sequence and Ultradian Body Temperature Rhythms in Mice

BACKGROUND: THE SLEEP SEQUENCE: i) non-REM sleep, ii) REM sleep, and iii) wakefulness, is stable and widely preserved in mammals, but the underlying mechanisms are unknown. It has been shown that this sequence is disrupted by sudden REM sleep onset during active wakefulness (i.e., narcolepsy) in orexin-deficient mutant animals. Phospholipase C (PLC) mediates the signaling of numerous metabotropic receptors, including orexin receptors. Among the several PLC subtypes, the beta4 subtype is uniquely localized in the geniculate nucleus of thalamus which is hypothesized to have a critical role in the transition and maintenance of sleep stages. In fact, we have reported irregular theta wave frequency during REM sleep in PLC-beta4-deficient mutant (PLC-beta4-/-) mice. Daily behavioral phenotypes and metabotropic receptors involved have not been analyzed in detail in PLC-beta4-/- mice, however. METHODOLOGY/PRINCIPAL FINDINGS: Therefore, we analyzed 24-h sleep electroencephalogram in PLC-beta4-/- mice. PLC-beta4-/- mice exhibited normal non-REM sleep both during the day and nighttime. PLC-beta4-/- mice, however, exhibited increased REM sleep during the night, their active period. Also, their sleep was fragmented with unusual wake-to-REM sleep transitions, both during the day and nighttime. In addition, PLC-beta4-/- mice reduced ultradian body temperature rhythms and elevated body temperatures during the daytime, but had normal homeothermal response to acute shifts in ambient temperatures (22 degrees C-4 degrees C). Within the most likely brain areas to produce these behavioral phenotypes, we found that, not orexin, but group-1 metabotropic glutamate receptor (mGluR)-mediated Ca(2+) mobilization was significantly reduced in the dorsal lateral geniculate nucleus (LGNd) of PLC-beta4-/- mice. Voltage clamp recordings revealed that group-1 mGluR-mediated currents in LGNd relay neurons (inward in wild-type mice) were outward in PLC-beta4-/- mice. CONCLUSIONS/SIGNIFICANCE: These lines of evidence indicate that impaired LGNd relay, possibly mediated via group-1 mGluR, may underlie irregular sleep sequences and ultradian body temperature rhythms in PLC-beta4-/- mice

Recent Results from LHD Experiment with Emphasis on Relation to Theory from Experimentalist’s View

he Large Helical Device (LHD) has been extending an operational regime of net-current free plasmas towardsthe fusion relevant condition with taking advantage of a net current-free heliotron concept and employing a superconducting coil system. Heating capability has exceeded 10 MW and the central ion and electron temperatureshave reached 7 and 10 keV, respectively. The maximum value of β and pulse length have been extended to 3.2% and 150 s, respectively. Many encouraging physical findings have been obtained. Topics from recent experiments, which should be emphasized from the aspect of theoretical approaches, are reviewed. Those are (1) Prominent features in the inward shifted configuration, i.e., mitigation of an ideal interchange mode in the configuration with magnetic hill, and confinement improvement due to suppression of both anomalous and neoclassical transport, (2) Demonstration ofbifurcation of radial electric field and associated formation of an internal transport barrier, and (3) Dynamics of magnetic islands and clarification of the role of separatrix

Localization of PLC-β families in the brain.

<p>A. Immunostaining with an anti-PLC-β4 antibody in sagittal brain sections from representative wild-type (left) and PLC-β4−/− (right) mice. Robust staining was observed in the thalamus (Th), superior colliculus (Sc), and cerebellum (Cb) in wild-type mice, but not in PLC-β4−/− mice. B. Immunostaining (orange-brown) of the thalamic geniculate nucleus and hippocampus of a wild-type mouse with PLC-β1-4 antibodies. No immunoreactivity was found for PLC-β2 and the section exhibits only cells counter-stained with cresyl violet (blue). Robust immunoreactivity for PLC-β4 was observed in the medial geniculate nucleus (MGN) and the dorsal lateral geniculate nucleus (LGNd). The ventral lateral geniculate nucleus (LGNv) exhibited relatively less immunoreactivity. DG, dentate gyrus; CA3, CA3 region of the hippocampus; PT, pretectal nucleus.</p

The metabotropic receptors linked to PLC-β4 in the LGNd were analyzed using Ca²⁺ imaging techniques.

<p>A. Orexin-A (300 nM) and orexin-B (300 nM) failed to mobilize intracellular Ca²⁺ in the LGNd of wild-type mice. Intracellular Ca²⁺ levels in LGNd from 3 different animals were plotted in this graph. B. The group-1 mGluR agonist, DHPG (100 µM), and the nonspecific mGluR agonist, 1<i>S</i>,3<i>R</i>-ACPD (100 µM), induced increased the intracellular Ca2+ in the LGNd (solid lines) of wild-type mice (left column). In PLC-β4−/− mice (right column), DHPG induced a significantly smaller Ca²⁺ increase while 1<i>S</i>,3<i>R</i>-ACPD decreased in Ca²⁺ in the LGNd. Control responses to 50 mM high-K⁺ stimulation (middle row) and in the dentate gyrus/CA3 region of hippocampus (broken lines) are also shown. C. Corresponding virtual color images of intracellular Ca²⁺ levels in the LGNd (approximate area is outlined in red in the top images) and dentate gyrus (DG)/CA3 region of the hippocampus were superimposed onto the transmitted light images shown at the top. Increasingly warmer colors indicate higher Ca²⁺ levels. Baseline was taken from the first frame of experiments. Frames of DHPG, high K⁺, and 1<i>S</i>,3<i>R</i>-ACPD were obtained from the peak Ca²⁺ response times in the dentate gyrus/CA3 region. All experiments were conducted in the presence of 1 µM tetrodotoxin.</p

Responses of LGNd relay neurons to DHPG were examined using whole-cell voltage-clamp recordings.

<p>DHPG (200 µM) induced inward currents in LGNd relay-neurons in wild-type mice (A) but produced outward currents at the same holding membrane potential (−70 mV) in PLC-β4−/− mice (B). Stimulation of the same neurons with AMPA (10 µM) induced large inward currents in both wild-type (A) and PLC-β4−/− mice (B). C. The mean current amplitude was calculated and compared (error bars denote S.E.M.) n = 13–15 for each group. **<i>P</i><0.01 compared with the wild-type group by Student's <i>t</i>-test. All experiments were conducted in the presence of 1 µM tetrodotoxin.</p

Reduced ultradian body temperature rhythms in PLC-β4−/− mice.

<p>Abdominal temperatures were measured in wild-type mice (A) and PLC-β4−/− mice (B) using a telemetry system. Representative abdominal temperature rhythms in 3 animals were plotted for each genotype. Although circadian rhythms of the temperature rhythm were manifest in both the wild-type and knockout mice, ultradian (i.e., 2–3 hour) oscillations were reduced in the knockout mice. White and black bars at the top denote light and dark periods. C. The temperature rhythm (±S.E.M.) was calculated for wild-type and PLC-β4−/− mice (n = 5 each). The daytime body temperature was significantly higher in the knockout mice than in the wild-type mice. *<i>P</i><.05; **<i>P</i><.01 compared with the corresponding wild-type group by Student's <i>t</i>-test. D. The ambient temperature was reduced from 22°C to 4°C for 2 hours to analyze the homeostatic thermal regulation in mice. No significant differences in the adaptive responses were found between the wild-type (n = 4, closed circle) and knockout (n = 4, open triangle) mice.</p

Sleep abnormality in PLC-β4−/− mice.

<p>A. The time courses of non-REM sleep (NREMS) and REM sleep (REMS) were plotted in 2 hour intervals for wild-type mice (n = 6, closed circles) and PLC-β4−/− mice (n = 6, open triangles). There was no difference in the time course of non-REM sleep except for a significant loss of sleep at the onset of darkness in PLC-β4−/− mice. The circadian time course of REM sleep was eliminated in PLC-β4−/− mice. *<i>P</i><.05; **<i>P</i><.01 compared with the corresponding wild-type group by Student's <i>t</i>-test. B. Hypnographic analysis also indicated abnormal REM sleep episodes in PLC-β4−/− mice. REM sleep regularly occurred after non-REM sleep in wild-type mice (the regular onset of REM sleep is marked by green arrows) whereas REM sleep episodes with frequent bouts of wakefulness were observed in PLC-β4−/− mice (REM sleep onset after wakefulness is marked by red arrows). C. <i>Upper</i>. An example sleep polygraph (combination of EEG and EMG) during regular REM sleep in a wild type mouse. <i>Lower</i>. An example sleep polygraph showing direct REM sleep transition from wakefulness in a PLC-β4−/− mouse. Squared 3-second EEG waves (a–d) were enlarged on the top. Typical theta waves were observed during both regular (a,b) and irregular (d) REM sleep. Estimated sleep stages are presented in 12-second bins. W: wakefulness, R: REM sleep.</p