In contrast, neurons with a-actinin-2 knocked down, under both conditions, continued to display the increased density of thinner, filopodia-like protrusions. This demonstrates that a-actinin-2 is required for the transition to an enlarged, mushroom-shaped spine in response to NMDA receptor stimulation and corroborates our finding that a-actinin-2 is necessary for proper spine development. NMDA receptor activation triggers post-synaptic signaling cascades that impact actin Niltubacin HDAC inhibitor filament organization and spine maturation. The “hair-like” protrusions displayed on both stimulated and un-stimulated neurons lacking a-actinin-2, imply a misorganization of actin filaments in these immature spines. Using rhodamine-phalloidin, we visualized actin filaments in spines from control neurons and a-actinin-2 knock down neurons. Interestingly, we found that spines in neurons lacking aactinin-2 were mostly devoid of detectable actin filament bundles, especially at the spine tip, in contrast to abundant phalloidinbound actin filaments visible in spines of control neurons. This finding suggests that a-actinin-2, likely through its actin cross-linking activity is needed to produce detectable actin filament bundles in the spine, which in turn drives structural changes underlying spine maturation. To address whether aactinin-2 contributes to PSD organization, we immunostained for PSD-95 in DIV 21 control cells and age-matched neurons with aactinin-2 knocked down at DIV 6–9. In contrast to control neurons, in which PSD-95 was observed in most spines, the spines of neurons with diminished levels of a-actinin-2 lacked detectable, organized PSD-95. In these neurons, PSD-95 only localized to the dendrite shaft. Loss of a-actinin-2 during mid-development, DIV16-19, when many spines have established connections with a pre-synaptic bouton, induced an increased density of immature spines that also lack PSD-95 and reduced the overall size of any pre-existing PSD in spines. This suggests that a-actinin-2 is not only required for the recruitment of post-synaptic molecules, but it is also required for the maintenance of the PSD. Importantly, coexpression of a-actinin-2-SS rescues PSD-95 localization and size in dendritic spines ; corroborating our finding that a-actinin-2 is required for PSD assembly in the spine. In agreement with previous studies, overexpression of a-actinin-2SS also increases the density of immature spines that lack PSD-95, indicating a requirement for normal synaptic amounts of a-actinin-2 to mediate PSD assembly in the spine. Since PSD-95 interacts with the NR1 subunit of NMDA receptors and a-actinin-2 directly binds to NR1 in vitro, we asked whether the NMDA receptor formed discernable structures at the tips of spines of neurons lacking a-actinin-2. We co-expressed the ubiquitous NR1 subunit of the NMDA receptor fused to a super-ecliptic pHluorin, which displays GFP fluorescence at the membrane surface when SEP is exposed to a neutral environment. Rhodamine-phalloidin was used to visualize actin-rich spines.