Takashi Sugiyama

Circadian Dynamics of Cytosolic and Nuclear Ca2+ in Single Suprachiasmatic Nucleus Neurons

Intracellular free Ca(2+) regulates diverse cellular processes, including membrane potential, neurotransmitter release, and gene expression. To examine the cellular mechanisms underlying the generation of circadian rhythms, nucleus-targeted and untargeted cDNAs encoding a Ca(2+)-sensitive fluorescent protein (cameleon) were transfected into organotypic cultures of mouse suprachiasmatic nucleus (SCN), the primary circadian pacemaker. Circadian rhythms in cytosolic but not nuclear Ca(2+) concentration were observed in SCN neurons. The cytosolic Ca(2+) rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode array, with a mean advance in phase of 4 hr. Tetrodotoxin blocked MUA, but not Ca(2+) rhythms, while ryanodine damped both Ca(2+) and MUA rhythms. These results demonstrate cytosolic Ca(2+) rhythms regulated by the release of Ca(2+) from ryanodine-sensitive stores in SCN neurons.

Brain oxidation is an initial process in sleep induction.

CNS activity is generally coupled to the vigilance state, being primarily active during wakefulness and primarily inactive during deep sleep. During periods of high neuronal activity, a significant volume of oxygen is used to maintain neuronal membrane potentials, which subsequently produces cytotoxic reactive oxygen species (ROS). Glutathione, a major endogenous antioxidant, is an important factor protecting against ROS-mediated neuronal degeneration. Glutathione has also been proposed to be a sleep-promoting substance, yet the relationship between sleep and cerebral oxidation remains unclear. Here we report that i.c.v. infusion of the organic peroxide t-butyl-hydroperoxide at a concentration below that triggering neurodegeneration (0.1 micromol/100 microl/10 h) promotes sleep in rats. Also, microinjection (2 nmol, 2 microl) or microdialysis (100 microM, 20 min) of t-butyl-hydroperoxide into the preoptic/anterior hypothalamus (POAH) induces the release of the sleep-inducing neuromodulators, nitric oxide and adenosine, without causing neurodegeneration. Nitric oxide and adenosine release was inhibited by co-dialysis of the N-methyl-D-aspartate receptor antagonist, d(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 mM), suggesting that glutamate-induced neuronal excitation mediates the peroxide-induced release of nitric oxide and adenosine. Indeed, Ca2+ release from mitochondria and delayed-onset Ca2+ influx via N-methyl-D-aspartate receptors was visualized during peroxide exposure using Ca2+ indicator proteins (YC-2.1 and mitochondrial-targeted Pericam) expressed in organotypic cultures of the POAH. In the in vitro models, t-butyl-hydroperoxide (50 microM) causes dendritic swelling followed by the intracellular Ca2+ mobilization, and D-AP5 (100 microM) or glutathione (500 microM) inhibited t-butyl-hydroperoxide-induced intracellular Ca2+ mobilization and protected POAH neurons from oxidative stress. These data suggest that low-level subcortical oxidation under the control of an antioxidant system may trigger sleep via the Ca(2+)-dependent release of sleep-inducing neuromodulators in the POAH, and thus we propose that a moderate increase of ROS during wakefulness in the neuronal circuits regulating sleep may be an initial trigger in sleep induction.

Three-dimensional characterization of interior structures of exocytotic apertures of nerve cells using atomic force microscopy

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques. Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells.

Impaired delay but normal trace eyeblink conditioning in PLCbeta4 mutant mice.

To elucidate the functional role of phospholipase Cβ4 (PLCβ4), which is highly expressed in the Purkinje cells of the rostral cerebellum, cerebellar long-term depression (LTD) and delay and trace eyeblink conditioning were investigated in PLCβ4-deficient mice. Rostral cerebellar LTD and delay eyeblink conditioning were severely impaired, whereas trace eyeblink conditioning was not. These results indicate that PLCβ4 is essential for LTD in the rostral cerebellum and delay conditioning, but not trace conditioning. Rostral cerebellar LTD may be required as a neural substrate for delay conditioning, but is not required for trace conditioning.

Analysis for Distinctive Activation Patterns of Pain and Itchy in the Human Brain Cortex Measured Using Near Infrared Spectroscopy (NIRS)

Pain and itch are closely related sensations, yet qualitatively quite distinct. Despite recent advances in brain imaging techniques, identifying the differences between pain and itch signals in the brain cortex is difficult due to continuous temporal and spatial changes in the signals. The high spatial resolution of positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) has substantially advanced research of pain and itch, but these are uncomfortable because of expensiveness, importability and the limited operation in the shielded room. Here, we used near infrared spectroscopy (NIRS), which has more conventional usability. NIRS can be used to visualize dynamic changes in oxygenated hemoglobin and deoxyhemoglobin concentrations in the capillary networks near activated neural circuits in real-time as well as fMRI. We observed distinct activation patterns in the frontal cortex for acute pain and histamine-induced itch. The prefrontal cortex exhibited a pain-related and itch-related activation pattern of blood flow in each subject. Although it looked as though that activation pattern for pain and itching was different in each subject, further cross correlation analysis of NIRS signals between each channels showed an overall agreement with regard to prefrontal area involvement. As a result, pain-related and itch-related blood flow responses (delayed responses in prefrontal area) were found to be clearly different between pain (τu200a=u200a+18.7 sec) and itch (τu200a=u200a+0.63 sec) stimulation. This is the first pilot study to demonstrate the temporal and spatial separation of a pain-induced blood flow and an itch-induced blood flow in human cortex during information processing.

Ca(2+) spiking activity caused by the activation of store-operated Ca(2+) channels mediates TNF-α release from microglial cells under chronic purinergic stimulation.

Cytokines released from microglia mediate defensive responses in the brain, but the underlying mechanisms are obscure. One proposed process is that nucleotide leakage or release from surrounding cells is sensed by metabotropic (P2Y) and ionotropic (P2X) purinergic receptors, which may trigger long-term intracellular Ca(2+) flux and tumor necrosis factor α (TNF-α) release. Indeed, 3h of exposure to ATP was required to evoke TNF-α release from a murine microglial cell line (MG5). A Ca(2+) chelator, ethylene glycol tetraacetic acid (EGTA), reduced ATP-induced TNF-α release, suggesting that intracellular Ca(2+) is important in this response. Therefore, Ca(2+) sensor genes (YC3.6) were transfected into MG5 cells to investigate the Ca(2+) dynamics underlying ATP-induced TNF-α release. The results demonstrated ATP-induced biphasic Ca(2+) mobilization mediated by P2Y (~5min) and P2X7 receptors (5-30min). Moreover, Ca(2+) spiking activity in cell processes progressively increased with a reduction in P2X7 receptor-mediated Ca(2+) elevation during 3-h ATP stimulation. Increased Ca(2+) spiking activity paralleled the reduction in thapsigargin-sensitive internal Ca(2+) stores, dendrite extension, and expression of macrophage scavenger receptors with collagenous structure. The Ca(2+) spiking activity was enhanced by a P2X7 receptor antagonist (A438079), but inhibited by a store-operated channel antagonist (SKF96365) or by co-transfection of small interference ribonucleic acid (siRNA) targeted on the channel component (Orai1). Furthermore, ATP-induced TNF-α release was enhanced by A438079 but was inhibited by SKF96365. Because store-operated channels (Stim1/Orai1) were expressed both in MG5 and primary microglial cultures, we suggest that P2X7 receptor signaling inhibits store-operated channels during ATP stimulation, and disinhibition of this process gates TNF-α release from microglial cells.

A novel function of synapsin II in neurotransmitter release

Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-II-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells.

PLC β4-independent Ca2+ rise via muscarinic receptors in the mouse suprachiasmatic nucleus

Cholinergic regulation of the suprachiasmatic nucleus (SCN) has been extensively studied although the intracellular signaling mechanisms are not well understood. We examined immunostaining for phospholipase C-β (PLC-β) families that couple to muscarinic acetylcholine receptors (mAChR) and demonstrated the expression of PLC-β1 and β4 in the mouse SCN. Ca2+ imaging analysis indicated that the M1-mAChR antagonist, pirenzepine blocked carbachol-induced Ca2+ elevation in the SCN and the response was equivalent between the wild type and the PLC-β4-knockout mice. In addition, the knockout mice displayed locomotor and temperature rhythms coupling to 24 h light/dark cycles. Therefore, it was proposed that PLC-β1 but not PLC-β4 was involved in the mAChR-mediated Ca2+ signaling in the SCN.

Bioluminescence microscopy using a short focal-length imaging lens.

Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low‐light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo‐toxicity associated with fluorescence imaging and permits the long‐term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy.

Hydrogen-Deuterium Exchange Effects on β-Endorphin Release from AtT20 Murine Pituitary Tumor Cells

Abundant evidences demonstrate that deuterium oxide (D2O) modulates various secretory activities, but specific mechanisms remain unclear. Using AtT20 cells, we examined effects of D2O on physiological processes underlying β-endorphin release. Immunofluorescent confocal microscopy demonstrated that 90% D2O buffer increased the amount of actin filament in cell somas and decreased it in cell processes, whereas β-tubulin was not affected. Ca2+ imaging demonstrated that high-K+-induced Ca2+ influx was not affected during D2O treatment, but was completely inhibited upon D2O washout. The H2O/D2O replacement in internal solutions of patch electrodes reduced Ca2+ currents evoked by depolarizing voltage steps, whereas additional extracellular H2O/D2O replacement recovered the currents, suggesting that D2O gradient across plasma membrane is critical for Ca2+ channel kinetics. Radioimmunoassay of high-K+-induced β-endorphin release demonstrated an increase during D2O treatment and a decrease upon D2O washout. These results demonstrate that the H2O-to-D2O-induced increase in β-endorphin release corresponded with the redistribution of actin, and the D2O-to-H2O-induced decrease in β-endorphin release corresponded with the inhibition of voltage-sensitive Ca2+ channels. The computer modeling suggests that the differences in the zero-point vibrational energy between protonated and deuterated amino acids produce an asymmetric distribution of these amino acids upon D2O washout and this causes the dysfunction of Ca2+ channels.