Fast Synaptically Activated Calcium and Sodium Kinetics in Hippocampal Pyramidal Neuron Dendritic Spines
Kenichi Miyazaki, William N. Ross
eneuro, 9, 6, ENEURO.0396, 22.2022, Society for Neuroscience, 2022年11月15日, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）, Abstract

An accurate assessment of the time course, components, and magnitude of postsynaptic currents is important for a quantitative understanding of synaptic integration and signaling in dendritic spines. These parameters have been studied in some detail in previous experiments, primarily using two-photon imaging of [Ca²⁺]_ichanges and two-photon uncaging of glutamate. However, even with these revolutionary techniques, there are some missing pieces in our current understanding, particularly related to the time courses of synaptically evoked [Ca²⁺]_iand [Na⁺]_ichanges. In new experiments, we used low-affinity, linear Na⁺and Ca²⁺indicators, laser fluorescence stimulation, and a sensitive camera-based detection system, combined with electrical stimulation and two-photon glutamate uncaging, to extend measurements of these spine parameters. We found that (1) almost all synaptically activated Na⁺currents in CA1 hippocampal pyramidal neuron spines in slices from mice of either sex are through AMPA receptors with little Na⁺entry through voltage-gated sodium channels (VGSCs) or NMDA receptor channels; (2) a spectrum of sodium transient decay times was observed, suggesting a spectrum of spine neck resistances, even on the same dendrite; (3) synaptically activated [Ca²⁺]_ichanges are very fast and are almost entirely because of Ca²⁺entry through NMDA receptors at the time when the Mg²⁺block is relieved by the fast AMPA-mediated EPSP; (4) the [Ca²⁺]_ichanges evoked by uncaging glutamate are slower than the changes evoked by synaptic release, suggesting that the relative contribution of Ca²⁺entering through NMDA receptors at rest following uncaging is higher than following electrical stimulation.

Mechanism of ArcLight derived GEVIs involves electrostatic interactions that can affect proton wires
Bok Eum Kang, Lee Min Leong, Yoonkyung Kim, Kenichi Miyazaki, William N. Ross, Bradley J. Baker
Biophysical Journal, 120, 10, 1916, 1926, Elsevier BV, 2021年05月, ［査読有り］
研究論文（学術雑誌）

Improvements in Simultaneous Sodium and Calcium Imaging
Kenichi Miyazaki, John E. Lisman, William N. Ross
Frontiers in Cellular Neuroscience, 12, Frontiers Media SA, 2019年01月08日, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）

Sodium Dynamics in Pyramidal Neuron Dendritic Spines: Synaptically Evoked Entry Predominantly through AMPA Receptors and Removal by Diffusion
Kenichi Miyazaki, William N. Ross
The Journal of Neuroscience, 37, 41, 9964, 9976, Society for Neuroscience, 2017年09月13日, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）, 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⁺]_ichanges during single EPSPs are ∼5 mm. Stronger electrical stimulation evoked small plateau potentials that had significant longer-lasting localized [Na⁺]_iincreases mediated through NMDA receptors.

SIGNIFICANCE STATEMENTDendritic spines, small structures that are difficult to investigate, are important elements in the fundamental processes of synaptic integration and plasticity. The main tool for examining these structures has been calcium imaging. However, the kinds of information that calcium imaging reveals is limited. We used newly developed, high-speed, simultaneous sodium and calcium imaging to examine ion dynamics in spines in hippocampal pyramidal neurons. We found that following single subthreshold synaptic activation most sodium entry was through AMPA receptors and not through NMDA receptors or through voltage-gated sodium channels and that the spine neck is not a significant resistance barrier. Most spine mechanisms are linear. However, regenerative NMDA conductances can be activated with stronger stimulation.

Hypocretin/Orexin Peptides Alter Spike Encoding by Serotonergic Dorsal Raphe Neurons through Two Distinct Mechanisms That Increase the Late Afterhyperpolarization
Masaru Ishibashi, Iryna Gumenchuk, Kenichi Miyazaki, Takafumi Inoue, William N. Ross, Christopher S. Leonard
The Journal of Neuroscience, 36, 39, 10097, 10115, Society for Neuroscience, 2016年09月28日, ［査読有り］
研究論文（学術雑誌）, Orexins (hypocretins) are neuropeptides that regulate multiple homeostatic processes, including reward and arousal, in part by exciting serotonergic dorsal raphe neurons, the major source of forebrain serotonin. Here, using mouse brain slices, we found that, instead of simply depolarizing these neurons, orexin-A altered the spike encoding process by increasing the postspike afterhyperpolarization (AHP) via two distinct mechanisms. This orexin-enhanced AHP (oeAHP) was mediated by both OX₁and OX₂receptors, required Ca²⁺influx, reversed near E_K, and decayed with two components, the faster of which resulted from enhanced SK channel activation, whereas the slower component decayed like a slow AHP (sAHP), but was not blocked by UCL2077, an antagonist of sAHPs in some neurons. Intracellular phospholipase C inhibition (U73122) blocked the entire oeAHP, but neither component was sensitive to PKC inhibition or altered PKA signaling, unlike classical sAHPs. The enhanced SK current did not depend on IP3-mediated Ca²⁺release but resulted from A-current inhibition and the resultant spike broadening, which increased Ca²⁺influx and Ca²⁺-induced-Ca²⁺release, whereas the slower component was insensitive to these factors. Functionally, the oeAHP slowed and stabilized orexin-induced firing compared with firing produced by a virtual orexin conductance lacking the oeAHP. The oeAHP also reduced steady-state firing rate and firing fidelity in response to stimulation, without affecting the initial rate or fidelity. Collectively, these findings reveal a new orexin action in serotonergic raphe neurons and suggest that, when orexin is released during arousal and reward, it enhances the spike encoding of phasic over tonic inputs, such as those related to sensory, motor, and reward events.

SIGNIFICANCE STATEMENTOrexin peptides are known to excite neurons via slow postsynaptic depolarizations. Here we elucidate a significant new orexin action that increases and prolongs the postspike afterhyperpolarization (AHP) in 5-HT dorsal raphe neurons and other arousal-system neurons. Our mechanistic studies establish involvement of two distinct Ca²⁺-dependent AHP currents dependent on phospholipase C signaling but independent of IP3 or PKC. Our functional studies establish that this action preserves responsiveness to phasic inputs while attenuating responsiveness to tonic inputs. Thus, our findings bring new insight into the actions of an important neuropeptide and indicate that, in addition to producing excitation, orexins can tune postsynaptic excitability to better encode the phasic sensory, motor, and reward signals expected during aroused states.

Simultaneous Sodium and Calcium Imaging from Dendrites and Axons
Kenichi Miyazaki, William N. Ross
eneuro, 2, 5, ENEURO.0092, 15.2015, Society for Neuroscience, 2015年09月, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）, Abstract

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⁺]_iand [Na⁺]_ichanges 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⁺]_iand [Na⁺]_ichanges 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).

Imaging with organic indicators and high-speed charge-coupled device cameras in neurons: some applications where these classic techniques have advantages
William N. Ross, Kenichi Miyazaki, Marko A. Popovic, Dejan Zecevic
Neurophotonics, 2, 2, 021005, 021005, SPIE-Intl Soc Optical Eng, 2014年12月22日, ［査読有り］
研究論文（学術雑誌）

Ca²⁺Sparks and Puffs Are Generated and Interact in Rat Hippocampal CA1 Pyramidal Neuron Dendrites
Kenichi Miyazaki, William N. Ross
The Journal of Neuroscience, 33, 45, 17777, 17788, Society for Neuroscience, 2013年11月06日, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）, 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 IP₃increased 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 IP₃and Ca²⁺released by RyRs. The actions of IP₃appear to be confined to the main apical dendrite because uncaging IP₃in the oblique dendrites has no effect on the time course of localized events or backpropagating action potential-evoked Ca²⁺signals in this region.

Developmental profile of localized spontaneous Ca2+ release events in the dendrites of rat hippocampal pyramidal neurons
Kenichi Miyazaki, Satoshi Manita, William N. Ross
Cell Calcium, 52, 6, 422, 432, Elsevier BV, 2012年12月, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）

Synaptically activated Ca²⁺waves and NMDA spikes locally suppress voltage-dependent Ca²⁺signalling in rat pyramidal cell dendrites
Satoshi Manita, Kenichi Miyazaki, William N. Ross
The Journal of Physiology, 589, 20, 4903, 4920, Wiley, 2011年10月14日, ［査読有り］
研究論文（学術雑誌）

Nobiletin, a citrus flavonoid with neurotrophic action, augments protein kinase A-mediated phosphorylation of the AMPA receptor subunit, GluR1, and the postsynaptic receptor response to glutamate in murine hippocampus
Kentaro Matsuzaki, Kenichi Miyazaki, Seiichiro Sakai, Hiromu Yawo, Norihito Nakata, Shigeki Moriguchi, Kohji Fukunaga, Akihito Yokosuka, Yutaka Sashida, Yoshihiro Mimaki, Tohru Yamakuni, Yasushi Ohizumi
European Journal of Pharmacology, 578, 2-3, 194, 200, Elsevier BV, 2008年01月, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）

Optical monitoring of progressive synchronization in dentate granule cells during population burst activities
Masanori Murayama, Kenichi Miyazaki, Yoshihisa Kudo, Hiroyoshi Miyakawa, Masashi Inoue
European Journal of Neuroscience, 21, 12, 3349, 3360, Wiley, 2005年06月, ［査読有り］
研究論文（学術雑誌）

Synapse-to-synapse variation of calcium channel subtype contributions in large mossy fiber terminals of mouse hippocampus
K. Miyazaki, T. Ishizuka, H. Yawo
Neuroscience, 136, 4, 1003, 1014, Elsevier BV, 2005年, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）

Pharmacological dissection of calcium channel subtype-related components of strontium inflow in large mossy fiber boutons of mouse hippocampus
Takashi Tokunaga, Kenichi Miyazaki, Makoto Koseki, Jalal Izadi Mobarakeh, Toru Ishizuka, Hiromu Yawo
Hippocampus, 14, 5, 570, 585, Wiley, 2004年, ［査読有り］
研究論文（学術雑誌）

シナプス伝達を促進するnobiletinはマウスの記憶学習能力を向上する
小野塚 寛, 山國 徹, 中島 晶, 松崎 健太郎, 宮崎 憲一, 酒井 誠一郎, 八尾 寛, 横須賀 章人, 指田 豊, 三巻 祥造, 大泉 康, 日本薬学会年会要旨集, 127年会, 2, 127, 127, 2007年03月
(公社)日本薬学会, 日本語

AMPA受容体の機能を促進するnobiletinはAβ誘発性記憶障害を改善する
松崎 健太郎, 山國 徹, 中田 徳仁, 中島 晶, 宮崎 憲一, 酒井 誠一郎, 八尾 寛, 橋本 道男, 紫藤 治, 横須賀 章人, 指田 豊, 三巻 祥浩, 大泉 康, 日本薬理学雑誌, 129, 1, 8P, 8P, 2007年01月
(公社)日本薬理学会, 日本語

遊離海馬標本における苔状線維または苔状細胞を介した海馬長軸方向への伝達経路の解析
科学研究費助成事業
2007年 - 2008年
宮崎 憲一
本研究の目的は、顆粒細胞から長軸方向CA3a錐体細胞への伝達経路と、苔状細胞そして側頭側の顆粒細胞を介した、CA3a錐体細胞への伝達経路の相互作用を調べることであるが、それぞれの経路を金属電極により電気刺激すると、顆粒細胞または苔状細胞以外の細胞や線維を刺激してしまい、それぞれの経路の応答を記録することが困難であることが判明した。そこで、光感受性カチオンチャネルのキメラであるChannelrhodopsin-WideReceiver(ChWH)をそれぞれの経路の細胞に発現させ、光刺激によって特異的に刺激する方法を開発する。ChWR-venus sindbis virusをin vivoで投与し、十分に発現した後、急性海馬横断面スライスを作成し、青色LED光による光刺激をした。Venusを発現している細胞に全細胞記録にて膜電位を記録したところ、この光刺激は顆粒細胞に十分な脱分極を引き起こし、活動電位を発生させることができることを確認できた。
本研究では、不特定多数の細胞を刺激してしまう金属電極に変わり、光感受性陽イオンチャネルを使用することで、顆粒細胞を特異的に刺激できる方法を確立した。この方法を用いることで、今まで困難であった海馬長軸の伝達経路を研究することができるようになると考える。さらに、この方法は海馬長軸の実験だけでなく、海馬横断面スライスを使用した多くの実験にも使用することができ、より詳細な解析ができるようになると考えられる。
日本学術振興会, 若手研究(B), 東北大学, 19700359