Postsynaptic scaffolds of excitatory and inhibitory synapses in hippocampal neurons: maintenance of core components independent of actin filaments and microtubules.
(2000)
Journal - The Journal of neuroscience : the official journal of the Society for Neuroscience (UNITED STATES )
Abstract :
The mechanisms responsible for anchoring molecular components of postsynaptic specializations in the mammalian brain are not well understood but are presumed to involve associations with cytoskeletal elements. Here we build on previous studies of neurotransmitter receptors (Allison et al., 1998) to analyze the modes of attachment of scaffolding and signal transducing proteins of both glutamate and GABA postsynaptic sites to either the microtubule or microfilament cytoskeleton. Hippocampal pyramidal neurons in culture were treated with latrunculin A to depolymerize actin, with vincristine to depolymerize microtubules, or with Triton X-100 to extract soluble proteins. The synaptic clustering of PSD-95, a putative NMDA receptor anchoring protein and a core component of the postsynaptic density (PSD), was unaffected by actin depolymerization, microtubule depolymerization, or detergent extraction. The same was largely true for GKAP, a PSD-95-interacting protein. In contrast, the synaptic clustering of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)alpha, another core component of the PSD, was completely dependent on an intact actin cytoskeleton and was partially disrupted by detergent. Drebrin and alpha-actinin-2, actin-binding proteins concentrated in spines, were also dependent on F-actin for synaptic localization but were unaffected by detergent extraction. Surprisingly, the subcellular distributions of the inhibitory synaptic proteins GABA(A)R and gephyrin, which has a tubulin-binding motif, were unaffected by depolymerization of microtubules or actin or by detergent extraction. These studies reveal an unsuspected heterogeneity in the modes of attachment of postsynaptic proteins to the cytoskeleton and support the idea that PSD-95 and gephyrin may be core scaffolding components independent of the actin or tubulin cytoskeleton.
| ISSN : | 0270-6474 |
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| Mesh Heading : | Actins Animals Bicyclo Compounds, Heterocyclic Cells, Cultured Evoked Potentials Excitatory Postsynaptic Potentials Hippocampus Intracellular Signaling Peptides and Proteins Marine Toxins Membrane Proteins Microtubules Nerve Tissue Proteins Neurons Octoxynol Rats Synapses Thiazoles Thiazolidines Vincristine drug effects physiology ultrastructure pharmacology pharmacology drug effects physiology ultrastructure physiology pharmacology pharmacology pharmacology |
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| Mesh Heading Relevant : | physiology physiology physiology physiology |
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Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors.
(1998)
Journal - The Journal of neuroscience : the official journal of the Society for Neuroscience (UNITED STATES )
Abstract :
We used actin-perturbing agents and detergent extraction of primary hippocampal cultures to test directly the role of the actin cytoskeleton in localizing GABAA receptors, AMPA- and NMDA-type glutamate receptors, and potential anchoring proteins at postsynaptic sites. Excitatory postsynaptic sites on dendritic spines contained a high concentration of F-actin that was resistant to cytochalasin D but could be depolymerized using the novel compound latrunculin A. Depolymerization of F-actin led to a 40% decrease in both the number of synaptic NMDA receptor (NMDAR1) clusters and the number of AMPA receptor (GluR1)-labeled spines. The nonsynaptic NMDA receptors appeared to remain clustered and to coalesce in cell bodies. alpha-Actinin-2, which binds both actin and NMDA receptors, dissociated from the receptor clusters, but PSD-95 remained associated with both the synaptic and nonsynaptic receptor clusters, consistent with a proposed cross-linking function. AMPA receptors behaved differently; on GABAergic neurons, the clusters redistributed to nonsynaptic sites, whereas on pyramidal neurons, many of the clusters appeared to disperse. Furthermore, in control neurons, AMPA receptors were detergent extractable from pyramidal cell spines, whereas AMPA receptors on GABAergic neurons and NMDA receptors were unextractable. GABAA receptors were not dependent on F-actin for the maintenance or synaptic localization of clusters. These results indicate fundamental differences in the mechanisms of receptor anchoring at postsynaptic sites, both regarding the anchoring of a single receptor (the AMPA receptor) in pyramidal cells versus GABAergic interneurons and regarding the anchoring of different receptors (AMPA vs NMDA receptors) at a single class of postsynaptic sites on pyramidal cell dendritic spines.
| ISSN : | 0270-6474 |
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| Mesh Heading : | Actinin Actins Animals Bicyclo Compounds, Heterocyclic Cytochalasin D Cytoskeleton Dendrites Detergents Hippocampus Intracellular Signaling Peptides and Proteins Membrane Proteins Nerve Tissue Proteins Nucleic Acid Synthesis Inhibitors Pyramidal Cells Rats Receptors, AMPA Receptors, GABA-A Receptors, N-Methyl-D-Aspartate Receptors, Neurotransmitter Solubility Synapses Thiazoles Thiazolidines metabolism analysis pharmacology pharmacology chemistry drug effects metabolism chemistry metabolism ultrastructure chemistry metabolism pharmacology metabolism ultrastructure analysis metabolism analysis metabolism analysis metabolism analysis chemistry drug effects metabolism pharmacology |
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| Mesh Heading Relevant : | physiology cytology chemistry metabolism |
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