Available antibodies for Niltubacin a-actinin-2 cross-react with other highly homologous and equal-sized a-actinin isoforms. To clarify this issue, we used an anti-sarcomeric a-actinin antibody that is specific for a-actinin-2 and does not cross-react with a-actinin isoforms 1 and 4, which are present in CHO-K1 and COS-7 cells and rat forebrain PSD fractions . Using this reagent, we find that a-actinin-2 is enriched in hippocampal neurons and is not present in the surrounding glia cells, which contain abundant levels of a-actinin4. We also observed co-localization between a-actinin-2 and the post-synaptic protein, PSD-95, partial colocalization with the NR1 subunit of the NMDA receptor, but no co-localization with the pre-synaptic molecule, synaptophysin, indicating a-actinin-2 is only enriched on the postsynaptic side of synapses. These observations extend previous findings indicating that a-actinin-2 localizes to dendritic spines of hippocampal neurons . Knockdown of a-actinin-2 with siRNA at day in vitro 17 inhibited spine maturation and increased the number of spines along the dendrites. The spines on neurons with diminished a-actinin-2 expression were significantly thinner. While control neurons exhibited many spines with a “mushroom” morphology, e.g. a large bulbous spine head on top of a short spine neck, neurons with a-actinin-2 knocked down displayed significantly fewer mushroom-shaped spines, and more headless, filopodia-like protrusions. To show that this phenotype was specific for a-actinin-2, we co-transfected an RNAi-resistant a-actinin-2-SS with the siRNA plasmid and fixed the neurons 96 hours later. Spine density, spine head width, and the classic mushroom-shaped spine morphology, at later stages, e.g., DIV 21, were rescued by exogenous expression of aactinin-2-SS. Although difficult to quantify, many of these filopodia-like protrusions on aactinin-2 knockdown neurons appeared thinner and “hair-like” in contrast to the immature, filopodia-like spines on control neurons. Additionally, irregularly shaped protrusions containing numerous filopodia appeared on some dendrites of neurons lacking a-actinin-2. Using time-lapse confocal imaging, we found no difference in dynamics between aberrant protrusions on neurons lacking a-actinin-2 and the normal spines on control neurons, suggesting that a-actinin-2 does not regulate spine motility. Taken together, these findings show that a-actinin-2 is necessary for the proper development of dendrites and spines. Since neurons lacking normal levels of a-actinin-2 showed an increased density of immature, filopodia-like protrusions that failed to develop into mushroom-shaped spines, we hypothesized that a-actinin-2 would be required for the acute, activity-induced spine morphology changes that occur in response to chemical stimulation. To test this, we selectively activated synaptic NMDA receptors with the co-agonist glycine. As expected, 20 min following brief treatment with glycine, control neurons displayed a significant increase in the fraction of spines with wider heads and mushroom-shaped spines.