@article{1546, abstract = {Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca2+ channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local [Ca2+] imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca2+ buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca2+ sensors for vesicular release are located at the perimeter of VGCC clusters (<30nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course.}, author = {Nakamura, Yukihiro and Harada, Harumi and Kamasawa, Naomi and Matsui, Ko and Rothman, Jason and Shigemoto, Ryuichi and Silver, R Angus and Digregorio, David and Takahashi, Tomoyuki}, issn = {1097-4199}, journal = {Neuron}, number = {1}, pages = {145 -- 158}, publisher = {Elsevier}, title = {{Nanoscale distribution of presynaptic Ca2+ channels and its impact on vesicular release during development}}, doi = {10.1016/j.neuron.2014.11.019}, volume = {85}, year = {2015}, } @article{1557, abstract = {γ-Aminobutyric acid (GABA)- and glycine-mediated hyperpolarizing inhibition is associated with a chloride influx that depends on the inwardly directed chloride electrochemical gradient. In neurons, the extrusion of chloride from the cytosol primarily depends on the expression of an isoform of potassium-chloride cotransporters (KCC2s). KCC2 is crucial in the regulation of the inhibitory tone of neural circuits, including pain processing neural assemblies. Thus we investigated the cellular distribution of KCC2 in neurons underlying pain processing in the superficial spinal dorsal horn of rats by using high-resolution immunocytochemical methods. We demonstrated that perikarya and dendrites widely expressed KCC2, but axon terminals proved to be negative for KCC2. In single ultrathin sections, silver deposits labeling KCC2 molecules showed different densities on the surface of dendritic profiles, some of which were negative for KCC2. In freeze fracture replicas and tissue sections double stained for the β3-subunit of GABAA receptors and KCC2, GABAA receptors were revealed on dendritic segments with high and also with low KCC2 densities. By measuring the distances between spots immunoreactive for gephyrin (a scaffolding protein of GABAA and glycine receptors) and KCC2 on the surface of neurokinin 1 (NK1) receptor-immunoreactive dendrites, we found that gephyrin-immunoreactive spots were located at various distances from KCC2 cotransporters; 5.7 % of them were recovered in the middle of 4-10-μm-long dendritic segments that were free of KCC2 immunostaining. The variable local densities of KCC2 may result in variable postsynaptic potentials evoked by the activation of GABAA and glycine receptors along the dendrites of spinal neurons.}, author = {Javdani, Fariba and Holló, Krisztina and Hegedűs, Krisztina and Kis, Gréta and Hegyi, Zoltán and Dócs, Klaudia and Kasugai, Yu and Fukazawa, Yugo and Shigemoto, Ryuichi and Antal, Miklós}, journal = {Journal of Comparative Neurology}, number = {13}, pages = {1967 -- 1983}, publisher = {Wiley-Blackwell}, title = {{Differential expression patterns of K+Cl- cotransporter 2 in neurons within the superficial spinal dorsal horn of rats}}, doi = {10.1002/cne.23774}, volume = {523}, year = {2015}, } @article{2241, abstract = {The brain demands high-energy supply and obstruction of blood flow causes rapid deterioration of the healthiness of brain cells. Two major events occur upon ischemia: acidosis and liberation of excess glutamate, which leads to excitotoxicity. However, cellular source of glutamate and its release mechanism upon ischemia remained unknown. Here we show a causal relationship between glial acidosis and neuronal excitotoxicity. As the major cation that flows through channelrhodopsin-2 (ChR2) is proton, this could be regarded as an optogenetic tool for instant intracellular acidification. Optical activation of ChR2 expressed in glial cells led to glial acidification and to release of glutamate. On the other hand, glial alkalization via optogenetic activation of a proton pump, archaerhodopsin (ArchT), led to cessation of glutamate release and to the relief of ischemic brain damage in vivo. Our results suggest that controlling glial pH may be an effective therapeutic strategy for intervention of ischemic brain damage.}, author = {Beppu, Kaoru and Sasaki, Takuya and Tanaka, Kenji and Yamanaka, Akihiro and Fukazawa, Yugo and Shigemoto, Ryuichi and Matsui, Ko}, issn = {0896-6273}, journal = {Neuron}, number = {2}, pages = {314 -- 320}, publisher = {Elsevier}, title = {{Optogenetic countering of glial acidosis suppresses glial glutamate release and ischemic brain damage}}, doi = {10.1016/j.neuron.2013.11.011}, volume = {81}, year = {2014}, } @article{1895, abstract = {Major histocompatibility complex class I (MHCI) molecules were recently identified as novel regulators of synaptic plasticity. These molecules are expressed in various brain areas, especially in regions undergoing activity-dependent synaptic plasticity, but their role in the nucleus accumbens (NAc) is unknown. In this study, we investigated the effects of genetic disruption of MHCI function, through deletion of β2-microblobulin, which causes lack of cell surface expression of MHCI. First, we confirmed that MHCI molecules are expressed in the NAc core in wild-type mice. Second, we performed electrophysiological recordings with NAc core slices from wild-type and β2-microglobulin knock-out mice lacking cell surface expression of MHCI. We found that low frequency stimulation induced long-term depression in wild-type but not knock-out mice, whereas high frequency stimulation induced long-term potentiation in both genotypes, with a larger magnitude in knock-out mice. Furthermore, we demonstrated that knock-out mice showed more persistent behavioral sensitization to cocaine, which is a NAc-related behavior. Using this model, we analyzed the density of total AMPA receptors and their subunits GluR1 and GluR2 in the NAc core, by SDS-digested freeze-fracture replica labeling. After repeated cocaine exposure, the density of GluR1 was increased, but there was no change in total AMPA receptors and GluR2 levels in wildtype mice. In contrast, following repeated cocaine exposure, increased densities of total AMPA receptors, GluR1 and GluR2 were observed in knock-out mice. These results indicate that functional deficiency of MHCI enhances synaptic potentiation, induced by electrical and pharmacological stimulation.}, author = {Edamura, Mitsuhiro and Murakami, Gen and Meng, Hongrui and Itakura, Makoto and Shigemoto, Ryuichi and Fukuda, Atsuo and Nakahara, Daiichiro}, journal = {PLoS One}, number = {9}, publisher = {Public Library of Science}, title = {{Functional deficiency of MHC class i enhances LTP and abolishes LTD in the nucleus accumbens of mice}}, doi = {10.1371/journal.pone.0107099}, volume = {9}, year = {2014}, } @article{1898, abstract = {Fast synaptic transmission is important for rapid information processing. To explore the maximal rate of neuronal signaling and to analyze the presynaptic mechanisms, we focused on the input layer of the cerebellar cortex, where exceptionally high action potential (AP) frequencies have been reported invivo. With paired recordings between presynaptic cerebellar mossy fiber boutons and postsynaptic granule cells, we demonstrate reliable neurotransmission upto ~1 kHz. Presynaptic APs are ultrafast, with ~100μs half-duration. Both Kv1 and Kv3 potassium channels mediate the fast repolarization, rapidly inactivating sodium channels ensure metabolic efficiency, and little AP broadening occurs during bursts of up to 1.5 kHz. Presynaptic Cav2.1 (P/Q-type) calcium channels open efficiently during ultrafast APs. Furthermore, a subset of synaptic vesicles is tightly coupled to Ca2+ channels, and vesicles are rapidly recruited to the release site. These data reveal mechanisms of presynaptic AP generation and transmitter release underlying neuronal kHz signaling.}, author = {Ritzau Jost, Andreas and Delvendahl, Igor and Rings, Annika and Byczkowicz, Niklas and Harada, Harumi and Shigemoto, Ryuichi and Hirrlinger, Johannes and Eilers, Jens and Hallermann, Stefan}, journal = {Neuron}, number = {1}, pages = {152 -- 163}, publisher = {Elsevier}, title = {{Ultrafast action potentials mediate kilohertz signaling at a central synapse}}, doi = {10.1016/j.neuron.2014.08.036}, volume = {84}, year = {2014}, } @article{1919, abstract = {Long-lasting memories are formed when the stimulus is temporally distributed (spacing effect). However, the synaptic mechanisms underlying this robust phenomenon and the precise time course of the synaptic modifications that occur during learning remain unclear. Here we examined the adaptation of horizontal optokinetic response in mice that underwent 1 h of massed and spaced training at varying intervals. Despite similar acquisition by all training protocols, 1 h of spacing produced the highest memory retention at 24 h, which lasted for 1 mo. The distinct kinetics of memory are strongly correlated with the reduction of floccular parallel fiber-Purkinje cell synapses but not with AMPA receptor (AMPAR) number and synapse size. After the spaced training, we observed 25%, 23%, and 12% reduction in AMPAR density, synapse size, and synapse number, respectively. Four hours after the spaced training, half of the synapses and Purkinje cell spines had been eliminated, whereas AMPAR density and synapse size were recovered in remaining synapses. Surprisingly, massed training also produced long-term memory and halving of synapses; however, this occurred slowly over days, and the memory lasted for only 1 wk. This distinct kinetics of structural plasticity may serve as a basis for unique temporal profiles in the formation and decay of memory with or without intervals.}, author = {Aziz, Wajeeha and Wang, Wen and Kesaf, Sebnem and Mohamed, Alsayed and Fukazawa, Yugo and Shigemoto, Ryuichi}, journal = {PNAS}, number = {1}, pages = {E194 -- E202}, publisher = {National Academy of Sciences}, title = {{Distinct kinetics of synaptic structural plasticity, memory formation, and memory decay in massed and spaced learning}}, doi = {10.1073/pnas.1303317110}, volume = {111}, year = {2014}, } @article{1920, abstract = {Cerebellar motor learning is suggested to be caused by long-term plasticity of excitatory parallel fiber-Purkinje cell (PF-PC) synapses associated with changes in the number of synaptic AMPA-type glutamate receptors (AMPARs). However, whether the AMPARs decrease or increase in individual PF-PC synapses occurs in physiological motor learning and accounts for memory that lasts over days remains elusive. We combined quantitative SDS-digested freeze-fracture replica labeling for AMPAR and physical dissector electron microscopy with a simple model of cerebellar motor learning, adaptation of horizontal optokinetic response (HOKR) in mouse. After 1-h training of HOKR, short-term adaptation (STA) was accompanied with transient decrease in AMPARs by 28% in target PF-PC synapses. STA was well correlated with AMPAR decrease in individual animals and both STA and AMPAR decrease recovered to basal levels within 24 h. Surprisingly, long-termadaptation (LTA) after five consecutive daily trainings of 1-h HOKR did not alter the number of AMPARs in PF-PC synapses but caused gradual and persistent synapse elimination by 45%, with corresponding PC spine loss by the fifth training day. Furthermore, recovery of LTA after 2 wk was well correlated with increase of PF-PC synapses to the control level. Our findings indicate that the AMPARs decrease in PF-PC synapses and the elimination of these synapses are in vivo engrams in short- and long-term motor learning, respectively, showing a unique type of synaptic plasticity that may contribute to memory consolidation.}, author = {Wang, Wen and Nakadate, Kazuhiko and Masugi Tokita, Miwako and Shutoh, Fumihiro and Aziz, Wajeeha and Tarusawa, Etsuko and Lörincz, Andrea and Molnár, Elek and Kesaf, Sebnem and Li, Yunqing and Fukazawa, Yugo and Nagao, Soichi and Shigemoto, Ryuichi}, journal = {PNAS}, number = {1}, pages = {E188 -- E193}, publisher = {National Academy of Sciences}, title = {{Distinct cerebellar engrams in short-term and long-term motor learning}}, doi = {10.1073/pnas.1315541111}, volume = {111}, year = {2014}, } @article{1933, abstract = {The development of the vertebrate brain requires an exquisite balance between proliferation and differentiation of neural progenitors. Notch signaling plays a pivotal role in regulating this balance, yet the interaction between signaling and receiving cells remains poorly understood. We have found that numerous nascent neurons and/or intermediate neurogenic progenitors expressing the ligand of Notch retain apical endfeet transiently at the ventricular lumen that form adherens junctions (AJs) with the endfeet of progenitors. Forced detachment of the apical endfeet of those differentiating cells by disrupting AJs resulted in precocious neurogenesis that was preceded by the downregulation of Notch signaling. Both Notch1 and its ligand Dll1 are distributed around AJs in the apical endfeet, and these proteins physically interact with ZO-1, a constituent of the AJ. Furthermore, live imaging of a fluorescently tagged Notch1 demonstrated its trafficking from the apical endfoot to the nucleus upon cleavage. Our results identified the apical endfoot as the central site of active Notch signaling to securely prohibit inappropriate differentiation of neural progenitors.}, author = {Hatakeyama, Jun and Wakamatsu, Yoshio and Nagafuchi, Akira and Kageyama, Ryoichiro and Shigemoto, Ryuichi and Shimamura, Kenji}, journal = {Development}, number = {8}, pages = {1671 -- 1682}, publisher = {Company of Biologists}, title = {{Cadherin-based adhesions in the apical endfoot are required for active Notch signaling to control neurogenesis in vertebrates}}, doi = {10.1242/dev.102988}, volume = {141}, year = {2014}, } @article{2018, abstract = {Synaptic cell adhesion molecules are increasingly gaining attention for conferring specific properties to individual synapses. Netrin-G1 and netrin-G2 are trans-synaptic adhesion molecules that distribute on distinct axons, and their presence restricts the expression of their cognate receptors, NGL1 and NGL2, respectively, to specific subdendritic segments of target neurons. However, the neural circuits and functional roles of netrin-G isoform complexes remain unclear. Here, we use netrin-G-KO and NGL-KO mice to reveal that netrin-G1/NGL1 and netrin-G2/NGL2 interactions specify excitatory synapses in independent hippocampal pathways. In the hippocampal CA1 area, netrin-G1/NGL1 and netrin-G2/NGL2 were expressed in the temporoammonic and Schaffer collateral pathways, respectively. The lack of presynaptic netrin-Gs led to the dispersion of NGLs from postsynaptic membranes. In accord, netrin-G mutant synapses displayed opposing phenotypes in long-term and short-term plasticity through discrete biochemical pathways. The plasticity phenotypes in netrin-G-KOs were phenocopied in NGL-KOs, with a corresponding loss of netrin-Gs from presynaptic membranes. Our findings show that netrin-G/NGL interactions differentially control synaptic plasticity in distinct circuits via retrograde signaling mechanisms and explain how synaptic inputs are diversified to control neuronal activity.}, author = {Matsukawa, Hiroshi and Akiyoshi Nishimura, Sachiko and Zhang, Qi and Luján, Rafael and Yamaguchi, Kazuhiko and Goto, Hiromichi and Yaguchi, Kunio and Hashikawa, Tsutomu and Sano, Chie and Shigemoto, Ryuichi and Nakashiba, Toshiaki and Itohara, Shigeyoshi}, issn = {1529-2401}, journal = {Journal of Neuroscience}, number = {47}, pages = {15779 -- 15792}, publisher = {Society for Neuroscience}, title = {{Netrin-G/NGL complexes encode functional synaptic diversification}}, doi = {10.1523/JNEUROSCI.1141-14.2014}, volume = {34}, year = {2014}, } @article{2064, abstract = {We examined the synaptic structure, quantity, and distribution of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-type glutamate receptors (AMPARs and NMDARs, respectively) in rat cochlear nuclei by a highly sensitive freeze-fracture replica labeling technique. Four excitatory synapses formed by two distinct inputs, auditory nerve (AN) and parallel fibers (PF), on different cell types were analyzed. These excitatory synapse types included AN synapses on bushy cells (AN-BC synapses) and fusiform cells (AN-FC synapses) and PF synapses on FC (PF-FC synapses) and cartwheel cell spines (PF-CwC synapses). Immunogold labeling revealed differences in synaptic structure as well as AMPAR and NMDAR number and/or density in both AN and PF synapses, indicating a target-dependent organization. The immunogold receptor labeling also identified differences in the synaptic organization of FCs based on AN or PF connections, indicating an input-dependent organization in FCs. Among the four excitatory synapse types, the AN-BC synapses were the smallest and had the most densely packed intramembrane particles (IMPs), whereas the PF-CwC synapses were the largest and had sparsely packed IMPs. All four synapse types showed positive correlations between the IMP-cluster area and the AMPAR number, indicating a common intrasynapse-type relationship for glutamatergic synapses. Immunogold particles for AMPARs were distributed over the entire area of individual AN synapses; PF synapses often showed synaptic areas devoid of labeling. The gold-labeling for NMDARs occurred in a mosaic fashion, with less positive correlations between the IMP-cluster area and the NMDAR number. Our observations reveal target- and input-dependent features in the structure, number, and organization of AMPARs and NMDARs in AN and PF synapses.}, author = {Rubio, Maía and Fukazawa, Yugo and Kamasawa, Naomi and Clarkson, Cheryl and Molnár, Elek and Shigemoto, Ryuichi}, journal = {Journal of Comparative Neurology}, number = {18}, pages = {4023 -- 4042}, publisher = {Wiley-Blackwell}, title = {{Target- and input-dependent organization of AMPA and NMDA receptors in synaptic connections of the cochlear nucleus}}, doi = {10.1002/cne.23654}, volume = {522}, year = {2014}, }