@article{13202, abstract = {Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons.}, author = {Eguchi, Kohgaku and Le Monnier, Elodie and Shigemoto, Ryuichi}, issn = {1529-2401}, journal = {The Journal of Neuroscience}, number = {23}, pages = {4197--4216}, publisher = {Society for Neuroscience}, title = {{Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons}}, doi = {10.1523/JNEUROSCI.1514-22.2023}, volume = {43}, year = {2023}, } @article{10890, abstract = {Upon the arrival of action potentials at nerve terminals, neurotransmitters are released from synaptic vesicles (SVs) by exocytosis. CaV2.1, 2.2, and 2.3 are the major subunits of the voltage-gated calcium channel (VGCC) responsible for increasing intraterminal calcium levels and triggering SV exocytosis in the central nervous system (CNS) synapses. The two-dimensional analysis of CaV2 distributions using sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) has revealed their numbers, densities, and nanoscale clustering patterns in individual presynaptic active zones. The variation in these properties affects the coupling of VGCCs with calcium sensors on SVs, synaptic efficacy, and temporal precision of transmission. In this study, we summarize how the morphological parameters of CaV2 distribution obtained using SDS-FRL differ depending on the different types of synapses and could correspond to functional properties in synaptic transmission.}, author = {Eguchi, Kohgaku and Montanaro-Punzengruber, Jacqueline-Claire and Le Monnier, Elodie and Shigemoto, Ryuichi}, issn = {16625129}, journal = {Frontiers in Neuroanatomy}, publisher = {Frontiers}, title = {{The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals}}, doi = {10.3389/fnana.2022.846615}, volume = {16}, year = {2022}, } @article{11419, abstract = {Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer’s disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10–20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin 1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.}, author = {Hori, Tetsuya and Eguchi, Kohgaku and Wang, Han Ying and Miyasaka, Tomohiro and Guillaud, Laurent and Taoufiq, Zacharie and Mahapatra, Satyajit and Yamada, Hiroshi and Takei, Kohji and Takahashi, Tomoyuki}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Microtubule assembly by tau impairs endocytosis and neurotransmission via dynamin sequestration in Alzheimer's disease synapse model}}, doi = {10.7554/eLife.73542}, volume = {11}, year = {2022}, } @article{7339, abstract = {Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission.}, author = {Piriya Ananda Babu, Lashmi and Wang, Han Ying and Eguchi, Kohgaku and Guillaud, Laurent and Takahashi, Tomoyuki}, issn = {15292401}, journal = {Journal of neuroscience}, number = {1}, pages = {131--142}, publisher = {Society for Neuroscience}, title = {{Microtubule and actin differentially regulate synaptic vesicle cycling to maintain high-frequency neurotransmission}}, doi = {10.1523/JNEUROSCI.1571-19.2019}, volume = {40}, year = {2020}, } @article{7665, abstract = {Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we investigated ultrastructural and electrophysiological features of synapses in mouse acute cerebellar slices prepared at ice-cold and physiological temperature (PT). In the slices prepared at CT, we found significant spine loss and reconstruction, synaptic vesicle rearrangement and decrease in synaptic proteins, all of which were not detected in slices prepared at PT. Consistent with these structural findings, slices prepared at PT showed higher release probability. Furthermore, preparation at PT allows electrophysiological recording immediately after slicing resulting in higher detectability of long-term depression (LTD) after motor learning compared with that at CT. These results indicate substantial advantages of the slice preparation at PT for investigating synaptic functions in different physiological conditions.}, author = {Eguchi, Kohgaku and Velicky, Philipp and Hollergschwandtner, Elena and Itakura, Makoto and Fukazawa, Yugo and Danzl, Johann G and Shigemoto, Ryuichi}, issn = {16625102}, journal = {Frontiers in Cellular Neuroscience}, publisher = {Frontiers Media}, title = {{Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions}}, doi = {10.3389/fncel.2020.00063}, volume = {14}, year = {2020}, } @article{7908, abstract = {Volatile anesthetics are widely used for surgery, but neuronal mechanisms of anesthesia remain unidentified. At the calyx of Held in brainstem slices from rats of either sex, isoflurane at clinical doses attenuated EPSCs by decreasing the release probability and the number of readily releasable vesicles. In presynaptic recordings of Ca2+ currents and exocytic capacitance changes, isoflurane attenuated exocytosis by inhibiting Ca2+ currents evoked by a short presynaptic depolarization, whereas it inhibited exocytosis evoked by a prolonged depolarization via directly blocking exocytic machinery downstream of Ca2+ influx. Since the length of presynaptic depolarization can simulate the frequency of synaptic inputs, isoflurane anesthesia is likely mediated by distinct dual mechanisms, depending on input frequencies. In simultaneous presynaptic and postsynaptic action potential recordings, isoflurane impaired the fidelity of repetitive spike transmission, more strongly at higher frequencies. Furthermore, in the cerebrum of adult mice, isoflurane inhibited monosynaptic corticocortical spike transmission, preferentially at a higher frequency. We conclude that dual presynaptic mechanisms operate for the anesthetic action of isoflurane, of which direct inhibition of exocytic machinery plays a low-pass filtering role in spike transmission at central excitatory synapses.}, author = {Wang, Han Ying and Eguchi, Kohgaku and Yamashita, Takayuki and Takahashi, Tomoyuki}, issn = {15292401}, journal = {Journal of Neuroscience}, number = {21}, pages = {4103--4115}, publisher = {Society for Neuroscience}, title = {{Frequency-dependent block of excitatory neurotransmission by isoflurane via dual presynaptic mechanisms}}, doi = {10.1523/JNEUROSCI.2946-19.2020}, volume = {40}, year = {2020}, } @inbook{562, abstract = {Primary neuronal cell culture preparations are widely used to investigate synaptic functions. This chapter describes a detailed protocol for the preparation of a neuronal cell culture in which giant calyx-type synaptic terminals are formed. This chapter also presents detailed protocols for utilizing the main technical advantages provided by such a preparation, namely, labeling and imaging of synaptic organelles and electrophysiological recordings directly from presynaptic terminals.}, author = {Dimitrov, Dimitar and Guillaud, Laurent and Eguchi, Kohgaku and Takahashi, Tomoyuki}, booktitle = {Neurotrophic Factors}, editor = {Skaper, Stephen D.}, pages = {201 -- 215}, publisher = {Springer}, title = {{Culture of mouse giant central nervous system synapses and application for imaging and electrophysiological analyses}}, doi = {10.1007/978-1-4939-7571-6_15}, volume = {1727}, year = {2018}, } @article{472, abstract = {α-Synuclein is a presynaptic protein the function of which has yet to be identified, but its neuronal content increases in patients of synucleinopa-thies including Parkinson’s disease. Chronic overexpression of α-synuclein reportedly expresses various phenotypes of synaptic dysfunction, but the primary target of its toxicity has not been determined. To investigate this, we acutely loaded human recombinant α-synuclein or its pathological mutants in their monomeric forms into the calyces of Held presynaptic terminals in slices from auditorily mature and immature rats of either sex. Membrane capacitance measurements revealed significant and specific inhibitory effects of WT monomeric α-synuclein on vesicle endocytosis throughout development. However, the α-synuclein A53T mutant affected vesicle endocytosis only at immature calyces, where as the A30P mutant had no effect throughout. The endocytic impairment by WTα-synuclein was rescued by intraterminal coloading of the microtubule (MT) polymerization blocker nocodazole. Furthermore, it was reversibly rescued by presynaptically loaded photostatin-1, a pho-toswitcheable inhibitor of MT polymerization, inalight-wavelength-dependent manner. Incontrast, endocyticinhibition by the A53T mutant at immature calyces was not rescued by nocodazole. Functionally, presynaptically loaded WT α-synuclein had no effect on basal synaptic transmission evoked at a low frequency, but significantly attenuated exocytosis and impaired the fidelity of neurotransmission during prolonged high-frequency stimulation. We conclude that monomeric WTα-synuclein primarily inhibits vesicle endocytosis via MT overassembly, thereby impairing high-frequency neurotransmission.}, author = {Eguchi, Kohgaku and Taoufiq, Zachari and Thorn Seshold, Oliver and Trauner, Dirk and Hasegawa, Masato and Takahashi, Tomoyuki}, issn = {02706474}, journal = {European Journal of Neuroscience}, number = {25}, pages = {6043 -- 6052}, publisher = {Wiley-Blackwell}, title = {{Wild-type monomeric α-synuclein can impair vesicle endocytosis and synaptic fidelity via tubulin polymerization at the calyx of held}}, doi = {10.1523/JNEUROSCI.0179-17.2017}, volume = {37}, year = {2017}, }