TY - JOUR AB - Transmembrane AMPA receptor regulatory proteins (TARPs), including stargazin/γ-2, are associated with AMPA receptors and participate in their surface delivery and anchoring at the postsynaptic membrane. TARPs may also act as a positive modulator of the AMPA receptor ion channel function; however, little is known about other TARP members except for stargazin/γ-2. We examined the synaptic localization of stargazin/γ-2 and γ-8 by immunoelectron microscopy and biochemical analysis. The analysis of sodium dodecyl sulfate-digested freeze-fracture replica labeling revealed that stargazin/γ-2 was concentrated in the postsynaptic area, whereas γ-8 was distributed both in synaptic and extra-synaptic plasma membranes of the hippocampal neuron. When a synaptic plasma membrane-enriched brain fraction was treated with Triton X-100 and separated by sucrose density gradient ultracentrifugation, a large proportion of NMDA receptor and stargazin/γ-2 was accumulated in raft-enriched fractions, whereas AMPA receptor and γ-8 were distributed in both the raft-enriched fractions and other Triton-insoluble fractions. Phosphorylation of stargazin/γ-2 and γ-8 was regulated by different sets of kinases and phosphatases in cultured cortical neurons. These results suggested that stargazin/γ-2 and γ-8 have distinct roles in postsynaptic membranes under the regulation of different intracellular signaling pathways. AU - Inamura, Mihoko AU - Itakura, Makoto AU - Okamoto, Hirotsugu AU - Hoka, Sumio AU - Mizoguchi, Akira AU - Fukazawa, Yugo AU - Ryuichi Shigemoto AU - Yamamori, Saori AU - Takahashi, Masami ID - 2659 IS - 1 JF - Neuroscience Research TI - Differential localization and regulation of stargazin-like protein, γ-8 and stargazin in the plasma membrane of hippocampal and cortical neurons VL - 55 ER - TY - JOUR AB - The highest densities of the two metabotropic GABA subunits, GABA B1 and GABAB2, have been reported as occurring around the glutamatergic synapses between Purkinje cell spines and parallel fibre varicosities. In order to determine how this distribution is achieved during development, we investigated the expression pattern and the cellular and subcellular localization of the GABAB1 and GABAB2 subunits in the rat cerebellum during postnatal development. At the light microscopic level, immunoreactivity for the GABAB1 and GABAB2 subunits was very prominent in the developing molecular layer, especially in Purkinje cells. Using double immunofluorescence, we demonstrated that GABAB1 was transiently expressed in glial cells. At the electron microscopic level, immunoreactivity for GABAB receptors was always detected both pre- and postsynaptically. Presynaptically, GABAB1 and GABAB2 were localized in the extrasynaptic membrane of parallel fibres at all ages, and only rarely in GABAergic axons. Postsynaptically, GABAB receptors were localized to the extrasynaptic and perisynaptic plasma membrane of Purkinje cell dendrites and spines throughout development. Quantitative analysis and three-dimensional reconstructions further revealed a progressive developmental movement of the GABAB1 subunit on the surface of Purkinje cells from dendritic shafts to its final destination, the dendritic spines. Together, these results indicate that GABAB receptors undergo dynamic regulation during cerebellar development in association with the establishment and maturation of glutamatergic synapses to Purkinje cells. AU - Luján, Rafael AU - Ryuichi Shigemoto ID - 2657 IS - 6 JF - European Journal of Neuroscience TI - Localization of metabotropic GABA receptor subunits GABAB1 and GABAB2 relative to synaptic sites in the rat developing cerebellum VL - 23 ER - TY - JOUR AB - The rocker mice are hereditary ataxic mutants that carry a point mutation in the gene encoding the CaV2.1 (P/Q-type) Ca2+ channel α1 subunit, and show the mildest symptoms among the reported CaV2.1 mutant mice. We studied the basic characteristics of the rocker mutant Ca2+ channel and their impacts on excitatory synaptic transmission in cerebellar Purkinje cells (PCs). In acutely dissociated PC somas, the rocker mutant channel showed a moderate reduction in Ca2+ channel current density, whereas its kinetics and voltage dependency of gating remained nearly normal. Despite the small changes in channel function, synaptic transmission in the parallel fiber (PF)-PC synapses was severely impaired. The climbing fiber inputs onto PCs showed a moderate impairment but could elicit normal complex spikes. Presynaptic function of the PF-PC synapses, however, was unexpectedly almost normal in terms of paired-pulse facilitation, sensitivity to extracellular Ca2+ concentration and glutamate concentration in synaptic clefts. Electron microscopic analyses including freeze-fracture replica labeling revealed that both the number and density of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors substantially decreased without gross structural changes of the PF-PC synapses. We also observed an abnormal arborization of PC dendrites in young adult rocker mice (∼ 1 month old). These lines of evidence suggest that even a moderate dysfunction of CaV2.1 Ca2+ channel can cause substantial changes in postsynaptic molecular composition of the PF-PC synapses and dendritic structure of PCs. AU - Kodama, Takashi AU - Itsukaichi-Nishida, Yuko AU - Fukazawa, Yugo AU - Wakamori, Minoru AU - Miyata, Mariko AU - Molnár, Elek AU - Mori, Yasuo AU - Ryuichi Shigemoto AU - Imoto, Keiji ID - 2663 IS - 11 JF - European Journal of Neuroscience TI - A CaV2.1 calcium channel mutation rocker reduces the number of postsynaptic AMPA receptors in parallel fiber-Purkinje cell synapses VL - 24 ER - TY - JOUR AB - GABAB receptors are the G protein-coupled receptors for the main inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA). Molecular diversity in the GABAB system arises from the GABAB1a and GABAB1b subunit isoforms that solely differ in their ectodomains by a pair of sushi repeats that is unique to GABAB1a. Using a combined genetic, physiological, and morphological approach, we now demonstrate that GABAB1 isoforms localize to distinct synaptic sites and convey separate functions in vivo. At hippocampal CA3-to-CA1 synapses, GABAB1a assembles heteroreceptors inhibiting glutamate release, while predominantly GABAB1b mediates postsynaptic inhibition. Electron microscopy reveals a synaptic distribution of GABAB1 isoforms that agrees with the observed functional differences. Transfected CA3 neurons selectively express GABAB1a in distal axons, suggesting that the sushi repeats, a conserved protein interaction motif, specify heteroreceptor localization. The constitutive absence of GABAB1a but not GABAB1b results in impaired synaptic plasticity and hippocampus-dependent memory, emphasizing molecular differences in synaptic GABAB functions. AU - Vigot, Réjan AU - Barbieri, Samuel AU - Bräuner-Osborne, Hans AU - Tureček, Rostislav AU - Ryuichi Shigemoto AU - Zhang, Yan Ping AU - Luján, Rafael AU - Jacobson, Laura H AU - Biermann, Barbara AU - Fritschy, Jean-Marc AU - Vacher, Claire-Marie AU - Müller, Matthias P AU - Sansig, Gilles AU - Guetg, Nicole AU - Cryan, John F AU - Kaupmann, Klemens AU - Gassmann, Martin AU - Oertner, Thomas G AU - Bettler, Bernhard ID - 2661 IS - 4 JF - Neuron TI - Differential Compartmentalization and Distinct Functions of GABAB Receptor Variants VL - 50 ER - TY - JOUR AB - G-protein-coupled inwardly rectifying K+ channels (Kir3 channels) coupled to metabotropic GABAB receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABAB receptors on hippocampal pyramidal cells, we used a high-resolution immunocytochemical approach. Immunoreactivity for the Kir3.2 subunit was most abundant postsynaptically and localized to the extrasynaptic plasma membrane of dendritic shafts and spines of principal cells. Quantitative analysis of immunogold particles for Kir3.2 revealed an enrichment of the protein around putative glutamatergic synapses on dendritic spines, similar to that of GABA B1. Consistent with this observation, a high degree of coclustering of Kir3.2 and GABAB1 was revealed around excitatory synapses by the highly sensitive SDS-digested freeze-fracture replica immunolabeling. In contrast, in dendritic shafts receptors and channels were found to be mainly segregated. These results suggest that Kir3.2-containing K+ channels on dendritic spines preferentially mediate the effect of GABA, whereas channels on dendritic shafts are likely to be activated by other neurotransmitters as well. Thus, Kir3 channels, localized to different subcellular compartments of hippocampal principal cells, appear to be differentially involved in synaptic integration in pyramidal cell dendrites. AU - Kulik, Ákos AU - Vida, Imre AU - Fukazawa, Yugo AU - Guetg, Nicole AU - Kasugai, Yu AU - Marker, Cheryl L AU - Rigato, Franck AU - Bettler, Bernhard AU - Wickman, Kevin D AU - Frotscher, Michael AU - Ryuichi Shigemoto ID - 2662 IS - 16 JF - Journal of Neuroscience TI - Compartment-dependent colocalization of Kir3.2-containing K+ channels and GABAB receptors in hippocampal pyramidal cells VL - 26 ER -