Postsynaptic scaffolds of excitatory and inhibitory synapses in hippocampal neurons: Maintenance of core components independent of actin filaments and microtubules

The mechanisms responsible for anchoring molecular components of postsynapt ic specializations in the mammalian brain are not well understood but are p resumed to involve associations with cytoskeletal elements. Here we build o n previous studies of neurotransmitter receptors (Allison et al., 1998) to analyze the modes of attachment of scaffolding and signal transducing prote ins of both glutamate and GABA postsynaptic sites to either the microtubule or microfilament cytoskeleton. Hippocampal pyramidal neurons in culture we re treated with latrunculin A to depolymerize actin, with vincristine to de polymerize microtubules, or with Triton X-100 to extract soluble proteins. The synaptic clustering of PSD-95, a putative NMDA receptor anchoring prote in and a core component of the postsynaptic density (PSD), was unaffected b y actin depolymerization, microtubule depolymerization, or detergent extrac tion. The same was largely true for GKAP, a PSD-95-interacting protein. In contrast, the synaptic clustering of Ca2+ calmodulin-dependent protein kina se II (CaMKII)alpha, another core component of the PSD, was completely depe ndent on an intact actin cytoskeleton and was partially disrupted by deterg ent. Drebrin and alpha-actinin-2, actin-binding proteins concentrated in sp ines, were also dependent on F-actin for synaptic localization but were una ffected by detergent extraction. Surprisingly, the subcellular distribution s of the inhibitory synaptic proteins GABA(A)R and gephyrin, which has a tu bulin-binding motif, were unaffected by depolymerization of microtubules or actin or by detergent extraction. These studies reveal an unsuspected hete rogeneity in the modes of attachment of postsynaptic proteins to the cytosk eleton and support the idea that PSD-95 and gephyrin may be core scaffoldin g components independent of the actin or tubulin cytoskeleton.