title: Activation-Dependent Rapid Postsynaptic Clustering of Glycine Receptors in Mature Spinal Cord Neurons.
abstract: Inhibitory synapses are established during development but continue to be generated and modulated in strength in the mature nervous system. In the spinal cord and brainstem, presynaptically released inhibitory neurotransmitter dominantly switches from GABA to glycine during normal developmentin vivo. While presynaptic mechanisms of the shift of inhibitory neurotransmission are well investigated, the contribution of postsynaptic neurotransmitter receptors to this shift is not fully elucidated. Synaptic clustering of glycine receptors (GlyRs) is regulated by activation-dependent depolarization in early development. However, GlyR activation induces hyperpolarization after the first postnatal week, and little is known whether and how presynaptically released glycine regulates postsynaptic receptors in a depolarization-independent manner in mature developmental stage. Here we developed spinal cord neuronal culture of rodents using chronic strychnine application to investigate whether initial activation of GlyRs in mature stage could change postsynaptic localization of GlyRs. Immunocytochemical analyses demonstrate that chronic blockade of GlyR activation until mature developmental stage resulted in smaller clusters of postsynaptic GlyRs that could be enlarged upon receptor activation for 1 h in the mature stage. Furthermore, live cell-imaging techniques show that GlyR activation decreases its lateral diffusion at synapses, and this phenomenon is dependent on PKC, but neither Ca2+nor CaMKII activity. These results suggest that the GlyR activation can regulate receptor diffusion and cluster size at inhibitory synapses in mature stage, providing not only new insights into the postsynaptic mechanism of shifting inhibitory neurotransmission but also the inhibitory synaptic plasticity in mature nervous system.
authors.affiliation: Division of Homeostatic Development, Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan; Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan.
authors.forename: Yoshihisa
authors.identifier: 0000-0003-3403-4527
authors.initials: Y
authors.lastname: Nakahata
authors.affiliation: Division of Homeostatic Development, Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan; Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Eto
authors.affiliation: Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan; Supportive Center for Brain Research, National Institute for Physiological Science, Okazaki 444-8585, Japan; PRESTO, Japan Science and Technology Agency (JST), Kawaguchi 332-0012, Japan.
authors.forename: Hideji
authors.initials: H
authors.lastname: Murakoshi
authors.affiliation: Department of Neurophysiology, Hamamatsu University School of Medicine , Hamamatsu 431-3192, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Department of Neurophysiology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University , Tokushima 769-2193, Japan.
authors.forename: Toshihiko
authors.identifier: 0000-0001-9125-891X
authors.initials: T
authors.lastname: Kuriu
authors.affiliation: PRESTO, Japan Science and Technology Agency (JST), Kawaguchi 332-0012, Japan; Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima 411-8540, Japan; Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima 411-8540, Japan; Department of Chemistry and Biological Science, Graduate School of Science and Engineering, Aoyama Gakuin University, Sagamihara 252-5258, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Hirata
authors.affiliation: Department of Physiology, School of Medical Sciences, University of New South Wales , Sydney 2052, Australia.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Moorhouse
authors.affiliation: Division of Homeostatic Development, Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan; Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan; Department of Physiology, Kitasato University School of Allied Health Sciences, Sagamihara 252-0373, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Ishibashi
authors.affiliation: Division of Homeostatic Development, Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan; Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan; CREST, Japan Science and Technology Agency (JST), Kawaguchi 332-0012, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Nabekura
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D018684:Glycine Agents / Q000494:pharmacology; D053444:Inhibitory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D038761:Photobleaching; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D018009:Receptors, Glycine / Q000235:genetics / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology*; D013331:Strychnine / Q000494:pharmacology; D050495:Vesicular Inhibitory Amino Acid Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D018691:Excitatory Amino Acid Antagonists; D018684:Glycine Agents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D018009:Receptors, Glycine; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; C492244:Viaat protein, mouse; C073441:gephyrin; C054562:yellow fluorescent protein, Bacteria; D013331:Strychnine
keywords: gephyrin; glycine receptor (GlyR); inhibitory synaptic plasticity; mature neuron; spinal cord; synaptic clustering