Miyazaki Kenichi

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Miyazaki
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Kenichi
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  • Preprint
    Synaptically activated Ca2+ waves and NMDA spikes locally suppress voltage-dependent Ca2+ signalling in rat pyramidal cell dendrites
    ( 2011-08) Manita, Satoshi ; Miyazaki, Kenichi ; Ross, William N.
    Synaptically activated changes in dendritic [Ca2+]i affect many important physiological processes including synaptic plasticity and gene expression. The location, magnitude, and time course of these changes can determine which mechanisms are affected. Therefore, it is important to understand the processes that control and modulate these changes. One important source is Ca2+ entering through voltage gated Ca2+ channels opened by action potentials backpropagating over the dendrites (bAPs). Here we examine how [Ca2+]i changes, caused by regenerative Ca2+ release from internal stores (Ca2+ waves) or by regenerative Ca2+ entry through NMDA receptors (NMDA spikes) affect subsequent bAP evoked [Ca2+]i changes. These large [Ca2+]i increases suppressed the bAP signals in the regions where the preceding [Ca2+]i increases were largest. The suppression was proportional to the magnitude of the large [Ca2+]i change and was insensitive to kinase and phosphatase inhibitors, consistent with suppression due to Ca2+ dependent inhibition of Ca2+ channels.
  • Article
    Improvements in simultaneous sodium and calcium imaging
    ( 2019-01-08) Miyazaki, Kenichi ; Lisman, John E. ; Ross, William N.
    High speed imaging of ion concentration changes in neurons is an important and growing tool for neuroscientists. We previously developed a system for simultaneously measuring sodium and calcium changes in small compartments in neurons (Miyazaki and Ross, 2015). We used this technique to analyze the dynamics of these ions in individual pyramidal neuron dendritic spines (Miyazaki and Ross, 2017). This system is based on high speed multiplexing of light emitting diodes (LEDs) and classic organic indicators. To improve this system we made additional changes, primarily incorporating lasers in addition to the LEDs, more sophisticated imaging protocols, and the use of newer sodium and calcium indicators. This new system generates signals with higher signal to noise ratio (S/N), less background fluorescence, and less photodynamic damage. In addition, by using longer wavelength indicators instead of indicators sensitive in the UV range, it allows for the incorporation of focal uncaging along with simultaneous imaging, which should extend the range of experiments.
  • Article
    Simultaneous sodium and calcium imaging from dendrites and axons
    (Society for Neuroscience, 2015-10-14) Miyazaki, Kenichi ; Ross, William N.
    Dynamic calcium imaging is a major technique of neuroscientists. It can reveal information about the location of various calcium channels and calcium permeable receptors, the time course, magnitude, and location of intracellular calcium concentration ([Ca2+]i) changes, and indirectly, the occurrence of action potentials. Dynamic sodium imaging, a less exploited technique, can reveal analogous information related to sodium signaling. In some cases, like the examination of AMPA and NMDA receptor signaling, measurements of both [Ca2+]i and [Na+]i changes in the same preparation may provide more information than separate measurements. To this end, we developed a technique to simultaneously measure both signals at high speed and sufficient sensitivity to detect localized physiologic events. This approach has advantages over sequential imaging because the preparation may not respond identically in different trials. We designed custom dichroic and emission filters to allow the separate detection of the fluorescence of sodium and calcium indicators loaded together into a single neuron in a brain slice from the hippocampus of Sprague-Dawley rats. We then used high-intensity light emitting diodes (LEDs) to alternately excite the two indicators at the appropriate wavelengths. These pulses were synchronized with the frames of a CCD camera running at 500 Hz. Software then separated the data streams to provide independent sodium and calcium signals. With this system we could detect [Ca2+]i and [Na+]i changes from single action potentials in axons and synaptically evoked signals in dendrites, both with submicron resolution and a good signal-to-noise ratio (S/N).
  • Preprint
    Developmental profile of localized spontaneous Ca2+ release events in the dendrites of rat hippocampal pyramidal neurons
    ( 2012-08) Miyazaki, Kenichi ; Manita, Satoshi ; Ross, William N.
    Recent experiments demonstrate that localized spontaneous Ca2+ release events can be detected in the dendrites of pyramidal cells in the hippocampus and other neurons (J. Neurosci. 29:7833-7845, 2009). These events have some properties that resemble ryanodine receptor mediated “sparks” in myocytes, and some that resemble IP3 receptor mediated “puffs” in oocytes. They can be detected in the dendrites of rats of all tested ages between P3 and P80 (with sparser sampling in older rats), suggesting that they serve a general signaling function and are not just important in development. However, in younger rats the amplitudes of the events are larger than the amplitudes in older animals and almost as large as the amplitudes of Ca2+ signals from backpropagating action potentials (bAPs). The rise time of the event signal is fast at all ages and is comparable to the rise time of the bAP fluorescence signal at the same dendritic location. The decay time is slower in younger animals, primarily because of weaker Ca2+ extrusion mechanisms at that age. Diffusion away from a brief localized source is the major determinant of decay at all ages. A simple computational model closely simulates these events with extrusion rate the only age dependent variable.
  • Article
    Ca2+ sparks and puffs are generated and interact in rat hippocampal CA1 pyramidal neuron dendrites
    (Society for Neuroscience, 2013-11-06) Miyazaki, Kenichi ; Ross, William N.
    1,4,5-Inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) mediate release of Ca2+ from internal stores in many neurons. The details of the spatial and temporal characteristics of these signals and their interactions in dendrites remain to be clarified. We found that localized Ca2+ release events, with no associated change in membrane potential, occurred spontaneously in the dendrites of rat hippocampal CA1 pyramidal neurons. Their rate, but not their amplitude or time course, could be modulated by changes in membrane potential. Together, these results suggest that the spontaneous events are similar to RyR-dependent Ca2+ “sparks” found in cardiac myocytes. In addition, we found that we could generate another kind of localized Ca2+ release event by either a synaptic tetanus in the presence of 3-((R)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid and CNQX or by uncaging IP3. These events had slower rise times and decay times than sparks and were more heterogeneous. These properties are similar to Ca2+ “puffs” found in oocytes. These two localized signals interact. Low-intensity tetanic synaptic stimulation or uncaging of IP3 increased the decay time of spontaneous Ca2+ events without changing their rise time or amplitude. Pharmacological experiments suggest that this event widening is attributable to a delayed IP3R-mediated release of Ca2+ triggered by the synergistic action of IP3 and Ca2+ released by RyRs. The actions of IP3 appear to be confined to the main apical dendrite because uncaging IP3 in the oblique dendrites has no effect on the time course of localized events or backpropagating action potential-evoked Ca2+ signals in this region.
  • Article
    Sodium dynamics in pyramidal neuron dendritic spines : synaptically evoked entry predominantly through AMPA receptors and removal by diffusion
    (Society for Neuroscience, 2017-10-11) Miyazaki, Kenichi ; Ross, William N.
    Dendritic spines are key elements underlying synaptic integration and cellular plasticity, but many features of these important structures are not known or are controversial. We examined these properties using newly developed simultaneous sodium and calcium imaging with single-spine resolution in pyramidal neurons in rat hippocampal slices from either sex. Indicators for both ions were loaded through the somatic patch pipette, which also recorded electrical responses. Fluorescence changes were detected with a high-speed, low-noise CCD camera. Following subthreshold electrical stimulation, postsynaptic sodium entry is almost entirely through AMPA receptors with little contribution from entry through NMDA receptors or voltage-gated sodium channels. Sodium removal from the spine head is through rapid diffusion out to the dendrite through the spine neck with a half-removal time of ∼16 ms, which suggests the neck has low resistance. Peak [Na+]i changes during single EPSPs are ∼5 mm. Stronger electrical stimulation evoked small plateau potentials that had significant longer-lasting localized [Na+]i increases mediated through NMDA receptors.