|
所属 |
大学院医学系研究科（医学専攻等） 医学専攻 病態制御医学系 細胞生理学講座 |
職務経歴（学外） 【 表示 ／ 非表示 】
-
2018年04月-2023年11月
Institute of Science and Technology Austria Cellular Neuroscience ポスドク
学位論文 【 表示 ／ 非表示 】
-
Distinct modes of endocytotic presynaptic membrane and protein retrieval at the calyx of held terminal
Yuji Okamoto
2018年03月
単著
研究等業績 【 表示 ／ 非表示 】
-
Olena Kim, Yuji Okamoto, Walter A Kaufmann, Nils Brose, Ryuichi Shigemoto, Peter Jonas
PLoS Biology ( Public Library of Science (PLoS) ) 22 ( 11 ) e3002879 - e3002879 2024年11月 [査読有り]
研究論文（学術雑誌） 国際共著
It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the rodent hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size and vesicular release probability by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the distance between Munc13-1 and Ca<sub>V</sub>2.1 Ca<sup>2+</sup> channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones. We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse.
-
Takafumi Miki, Yuji Okamoto, Miyuki Ueno-Umegai, Rio Toyofuku, Shun Hattori, Takeshi Sakaba
Proceedings of the National Academy of Sciences of the United States of America ( Proceedings of the National Academy of Sciences of the United States of America ) 121 ( 43 ) e2402152121 2024年10月 [査読有り]
研究論文（学術雑誌） 国内共著
Synaptic-vesicle (SV) recruitment is thought to maintain reliable neurotransmitter release during high-frequency signaling. However, the mechanism underlying the SV reloading for sustained neurotransmission at central synapses remains unknown. To elucidate this, we performed direct observations of SV reloading and mobility at a single-vesicle level near the plasma membrane in cerebellar mossy fiber terminals using total internal reflection fluorescence microscopy, together with simultaneous recordings of membrane fusion by capacitance measurements. We found that actin disruption abolished the rapid SV recruitment and reduced sustained release. In contrast, induction of actin polymerization and stabilization did not affect vesicle recruitment and release, suggesting that the presence of actin filaments, rather than actin dynamics, was required for the rapid recruitment. Single-particle tracking experiments of quantum dot-labeled vesicles, which allows nanoscale resolution of vesicle mobility, revealed that actin disruption caused vesicles to diffuse more rapidly. Hidden Markov modeling with Bayesian inference revealed that SVs had two diffusion states under normal conditions: free-diffusing and trapped. After disruption of the actin filament, vesicles tended to have only the free-diffusing state. F-actin staining showed that actin filaments were localized outside the active zones (AZs) and surrounded some SV trajectories. Perturbation of SV mobility, possibly through interference with biomolecular condensates, also suggested that the restricted diffusion state determined the rate of SV recruitment. We propose that actin filaments confined SVs near the AZ to achieve rapid and efficient recruitment followed by priming and sustained synaptic transmission.
-
Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses
David Vandael, Yuji Okamoto, Carolina Borges-Merjane, Victor Vargas-Barroso, Benjamin A Suter, Peter Jonas
Nature Protocols 16 ( 6 ) 2947 - 2967 2021年06月 [査読有り]
研究論文（学術雑誌） 国際共著
Rigorous investigation of synaptic transmission requires analysis of unitary synaptic events by simultaneous recording from presynaptic terminals and postsynaptic target neurons. However, this has been achieved at only a limited number of model synapses, including the squid giant synapse and the mammalian calyx of Held. Cortical presynaptic terminals have been largely inaccessible to direct presynaptic recording, due to their small size. Here, we describe a protocol for improved subcellular patch-clamp recording in rat and mouse brain slices, with the synapse in a largely intact environment. Slice preparation takes ~2 h, recording ~3 h and post hoc morphological analysis 2 d. Single presynaptic hippocampal mossy fiber terminals are stimulated minimally invasively in the bouton-attached configuration, in which the cytoplasmic content remains unperturbed, or in the whole-bouton configuration, in which the cytoplasmic composition can be precisely controlled. Paired pre-postsynaptic recordings can be integrated with biocytin labeling and morphological analysis, allowing correlative investigation of synapse structure and function. Paired recordings can be obtained from mossy fiber terminals in slices from both rats and mice, implying applicability to genetically modified synapses. Paired recordings can also be performed together with axon tract stimulation or optogenetic activation, allowing comparison of unitary and compound synaptic events in the same target cell. Finally, paired recordings can be combined with spontaneous event analysis, permitting collection of miniature events generated at a single identified synapse. In conclusion, the subcellular patch-clamp techniques detailed here should facilitate analysis of biophysics, plasticity and circuit function of cortical synapses in the mammalian central nervous system.
-
Transsynaptic modulation of presynaptic short-term plasticity in hippocampal mossy fiber synapses
David Vandael, Yuji Okamoto, Peter Jonas
Nature Communications 12 ( 1 ) 2912 - 2912 2021年05月 [査読有り]
研究論文（学術雑誌） 国際共著
The hippocampal mossy fiber synapse is a key synapse of the trisynaptic circuit. Post-tetanic potentiation (PTP) is the most powerful form of plasticity at this synaptic connection. It is widely believed that mossy fiber PTP is an entirely presynaptic phenomenon, implying that PTP induction is input-specific, and requires neither activity of multiple inputs nor stimulation of postsynaptic neurons. To directly test cooperativity and associativity, we made paired recordings between single mossy fiber terminals and postsynaptic CA3 pyramidal neurons in rat brain slices. By stimulating non-overlapping mossy fiber inputs converging onto single CA3 neurons, we confirm that PTP is input-specific and non-cooperative. Unexpectedly, mossy fiber PTP exhibits anti-associative induction properties. EPSCs show only minimal PTP after combined pre- and postsynaptic high-frequency stimulation with intact postsynaptic Ca2+ signaling, but marked PTP in the absence of postsynaptic spiking and after suppression of postsynaptic Ca2+ signaling (10 mM EGTA). PTP is largely recovered by inhibitors of voltage-gated R- and L-type Ca2+ channels, group II mGluRs, and vacuolar-type H+-ATPase, suggesting the involvement of retrograde vesicular glutamate signaling. Transsynaptic regulation of PTP extends the repertoire of synaptic computations, implementing a brake on mossy fiber detonation and a "smart teacher" function of hippocampal mossy fiber synapses.
-
Distinct modes of endocytotic presynaptic membrane and protein uptake at the calyx of Held terminal of rats and mice
Yuji Okamoto, Noa Lipstein, Yunfeng Hua, Kun-Han Lin, Nils Brose, Takeshi Sakaba, Mitsuharu Midorikawa
eLife 2016年05月 [査読有り]
研究論文（学術雑誌） 国際共著
◆原著論文【 表示 ／ 非表示 】
科研費（文科省・学振）獲得実績 【 表示 ／ 非表示 】
-
単粒子追跡法によるシナプス小胞リサイクリング経路の解明
若手研究
研究期間: 2025年04月 - 2027年03月
-
シナプス前終末におけるシナプス小胞サイクリング機構の解明
特別研究員奨励費
研究期間: 2017年04月 - 2019年03月 代表者: 岡本 悠志