Allison D W, Gelfand V I, Spector I, Craig A M
Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois 61801, USA.
J Neurosci. 1998 Apr 1;18(7):2423-36. doi: 10.1523/JNEUROSCI.18-07-02423.1998.
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.
我们使用肌动蛋白干扰剂和去污剂提取原代海马神经元培养物，以直接测试肌动蛋白细胞骨架在将GABAA受体、AMPA和NMDA型谷氨酸受体以及潜在的锚定蛋白定位到突触后位点中的作用。树突棘上的兴奋性突触后位点含有高浓度的F-肌动蛋白，其对细胞松弛素D具有抗性，但可使用新型化合物拉春库林A使其解聚。F-肌动蛋白的解聚导致突触NMDA受体（NMDAR1）簇数量和AMPA受体（GluR1）标记的棘突数量均减少40%。非突触性NMDA受体似乎仍聚集并在细胞体中合并。既结合肌动蛋白又结合NMDA受体的α-辅肌动蛋白-2从受体簇中解离，但PSD-95仍与突触和非突触受体簇相关联，这与提出的交联功能一致。AMPA受体的行为有所不同；在GABA能神经元上，这些簇重新分布到非突触位点，而在锥体细胞上，许多簇似乎分散了。此外，在对照神经元中，AMPA受体可从锥体细胞棘突中用去污剂提取，而GABA能神经元上的AMPA受体和NMDA受体则不可提取。GABAA受体簇的维持或突触定位不依赖于F-肌动蛋白。这些结果表明，在突触后位点受体锚定机制方面存在根本差异，这既涉及锥体细胞与GABA能中间神经元中单个受体（AMPA受体）的锚定，也涉及锥体细胞树突棘上单一类突触后位点上不同受体（AMPA与NMDA受体）的锚定。
