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  • br Results br Discussion br Experimental Procedures br Autho


    Experimental Procedures
    Author Contributions
    Introduction Excitatory synaptic transmission within the brain is largely mediated by ionotropic glutamate receptors. At glutamatergic synapses, α-amino-3-hydroxy-5-methylisoxazole-4-proprionic WIN 18446 (AMPA) and N-methyl-D-aspartate (NMDA) receptors are densely clustered apposed to presynaptic terminals (Nusser, 2000), and changes in their numbers can alter postsynaptic sensitivity to glutamate release. Therefore, the maintenance of surface glutamate receptors is a critical aspect of neuronal function, and the modification of their numbers is a potential mechanism for regulating synaptic strength during plasticity (Malinow and Malenka, 2002). A fundamental mechanism of maintaining and modifying the number of synaptic glutamate receptors is their internalization from the synaptic membrane. AMPA receptors undergo rapid constitutive internalization that is regulated by synaptic activity Ehlers 2000, Lin et al. 2000. For NMDA receptors, constitutive internalization in mature neurons is slow relative to AMPA receptors and not regulated by activity but is rapid in immature neurons Ehlers 2000, Lin et al. 2000, Roche et al. 2001. Although NMDA receptors are thought to be relatively stable during synaptic plasticity, certain stimuli can induce their acute internalization Nong et al. 2003, Snyder et al. 2001. Mounting evidence suggests that the internalization of glutamate receptors is a primary mechanism of long-term depression (LTD) Beattie et al. 2000, Lin et al. 2000, Man et al. 2000, Snyder et al. 2001, Wang and Linden 2000, an electrophysiological paradigm for synaptic plasticity wherein a specific stimulus causes a decrease in synaptic strength (Bear and Malenka, 1994). Glutamate receptor internalization is thought to occur through clathrin-mediated endocytosis Beattie et al. 2000, Carroll et al. 1999, Ehlers 2000, Lin et al. 2000, Man et al. 2000, Wang and Linden 2000, a general mechanism for the internalization of proteins from the plasma membrane (Mousavi et al., 2004). However, the specific mechanisms of glutamate receptor internalization are not well understood. In cell lines, clathrin-mediated endocytosis occurs at discrete and stable “clathrin pit zones” on the membrane Gaidarov et al. 1999, Santini et al. 2002, Scott et al. 2002. A similar endocytic zone, segregated from the postsynaptic density (PSD), functions at the postsynaptic side of excitatory synapses and is presumed to be the site of the internalization of synaptic proteins, including glutamate receptors (Blanpied et al., 2002). Although electron microscopy has shown the presence of clathrin-coated pits and vesicles in dendritic spines Cooney et al. 2002, Petralia et al. 2003, Spacek and Harris 1997, Toni et al. 2001, none have been shown to traffic glutamate receptors at postsynaptic sites. Screens for plasticity-related genes have identified multiple transcripts that encode synaptic proteins, suggesting that genes induced by activity often function in normal synaptic processes (Nedivi, 1999). candidate plasticity gene 2 (cpg2) was isolated in a screen for transcripts upregulated by kainic acid-induced seizures in the rat dentate gyrus (Nedivi et al., 1993), and its expression is regulated during development and by sensory experience (Nedivi et al., 1996). cpg2 is a splice variant of the syne-1 gene, a large gene that encodes a protein with an actin binding domain at the N terminus and a nuclear transmembrane domain at the C terminus, separated by a long helical region (Starr and Han, 2003). The cpg2 transcript is derived from a portion of the separator region (Padmakumar et al., 2004), encodes a protein with homologies to dystrophin, and contains motifs predicting a structural function, including several spectrin repeats and coiled coils (Nedivi et al., 1996). Proteins with these motifs often play a central role in organizing protein complexes Burkhard et al. 2001, Djinovic-Carugo et al. 2002.