K-S test) or spine length (1.19 mm60.04 mm control vs. 1.2 mm60.06 mm latrunculin; n = 500 spines/5 neurons/group; p.0.05, K-S test)(Fig. 3B). Treatment with latrunculin-B also resulted in a significant decrease in overall spine density in GFP expressing neurons (n = 5 neurons/group, p,0.01, ANOVA and Tukey’s test), but not in neurons expressing the ABD of bI-spectrin (n = 5 neurons/group, p.0.05, ANOVA and Tukey’s test)(Fig. 3C). Actin in spines expressing the bI-spectrin ABD was thus resistant to the depolymerizing actions of prolonged latrunculin treatment. We next tested the effects of acute application of latrunculin-B in cells transfected with actin-GFP alone or with the bI-spectrin ABD (Fig. 4). Depolymerization of actin with latrunculin reduces the intensity of actin-GFP fluorescence in the spine head as the actin monomers that cannot polymerize diffuse out of the spine [40]. We found that acute addition of latrunculin-B (5 mm) induced a rapid (,3 min) decrease in actin-GFP intensity in control cells, as previously reported. Latrunculin application had a significantly smaller effect on the intensity of actin-GFP in spines of cells co-transfected with the ABD of bI-spectrin (n = 5 neurons/ group, p,0.001, repeated measures ANOVA). This effect was specific for the ABD of bI-spectrin, because latrunculin produced a strong decrease in the fluorescence intensity of spines co- transfected with the ABD of a-actinin-2 and actin-GFP, comparable to its effects on cells expressing actin-GFP alone. We conclude that the presence of the ABD of bI-spectrin in spines stabilizes F-actin and reduces its rate of depolymerization. The ABD of bI-spectrin decreases the morphological plasticity of dendritic spines Dynamic morphological changes in the shape of dendritic spine heads (‘‘morphing’’) occur on the order of minutes or seconds and are dependent upon actin polymerization and depolymerization in the spine head [10,40]. We therefore tested if decreases in actin depolymerization resulting from the expression of the ABD of bI- spectrin would decrease dendritic spine motility. We used live-cell imaging to compare the spontaneous, constitutive morphing of dendritic spines in neurons transfected with GFP alone or GFP together with the ABD of bI-spectrin (Fig. 5). Because the activity of both AMPARs and NMDARs may affect spine morphing [10], we also tested the effects of applying saturating concentrations of DNQX (40 mm) and AP5 (40 mm). Using GFP emission, we measured the variance of spine head diameter over a period of 10 min to quantify spine head dynamics and to control for differences in spine head size induced by the constructs. Spines expressing GFP alone exhibited significantly more spontaneous morphing than spines expressing the ABD of bI-spectrin (p,0.001, Kruskal-Wallis test), and neither were affected by DNQX and AP5 (Fig. 5C). We conclude that stabilization of actin filaments by the ABD of bI-spectrin attenuates the structural dynamics of dendritic spines. Functional consequences of expressing the ABD of bI- spectrin We next asked if there were functional correlates of the morphological changes induced by the ABD of spectrin. The postsynaptic density (PSD) is a specialized structure at the tip of the dendritic spine head comprised of densely packed glutamate receptors and signaling proteins [41,42] and its size is positively correlated with synaptic strength [2]. A principal constituent of the PSD is the scaffolding protein, PSD-95 [43]. As a first test of the possibility that the enlargement of spine heads induced by the ABD of bI-spectrin (lacking the DsRed tag) would be associated with an increase in synaptic strength, we co-expressed it with GFP- The Role of bI-Spectrin in Dendritic Spines tagged PSD-95 and cytosolic mCherry (Fig. 6A). We observed a significant increase in the area of PSD-95-GFP in dendritic spines co-expressing the ABD of bI-spectrin, as compared to cells expressing PSD-95-GFP alone (mean area = 0.20 mm260.02 mm2 in controls vs. 0.38 mm260.01 mm2 with the ABD of bI-spectrin; n = 365 spines/4 neurons/group; p,0.05, K-S test)(Fig. 6B). In agreement with previous reports [44] we observed a linear relationship between spine head size and PSD-95 area under both conditions (r = 0.83 for control and 0.79 for the ABD of bI- spectrin). We conclude that overexpression of the ABD of bI- spectrin is associated with an increase in the size of the PSD. It has been demonstrated that the number of AMPARs is proportional to PSD area [2,45,46]. We therefore asked whether the increase in PSD size produced by the ABD of bI-spectrin would be accompanied by an increase in the number of synaptic AMPARs in the spine head. To answer this question, we transfected CA1 pyramidal cells in hippocampal slice cultures with the ABD of bI-spectrin tagged with DsRed and performed whole-cell voltage-clamp recordings of spontaneous miniature excitatory postsynaptic currents (mEPSCs) on transfected and untransfected neurons in the presence of TTX (1 mm). Alexa 488 was included in the pipette solution in order to ensure that the recorded neurons were truly those expressing the spectrin construct, as apparent by green and red fluorescence. Mean mEPSC amplitudes were significantly larger in cells transfected with the ABD of bI-spectrin compared to untransfected cells in the same cultures, consistent with an increase in the number of synaptic AMPARs (mean amplitude = 20.861.4 pA for cells expressing the ABD of bI-spectrin vs. 17.161.1 pA in untrans- fected cells; n = 7 neurons/group; p,0.05, K-S test)(Fig. 7A,B). The frequency of mEPSCs was decreased ,20% after transfection with the ABD of bI-spectrin, compared to untransfected neurons recorded from the same slice (p,0.05, t-test)(Fig. 7C), consistent with the decrease in spine density described above. To test for non-specific effects of biolistic transfection on neuronal function, we also compared mEPSC amplitudes in neurons transfected with GFP alone and untransfected cells and observed no significant difference (mean amplitude = 17.160.5 pA in untransfected cells vs. 15.760.4 pA in cells expressing GFP; n = 7 neurons/group, p.0.05, unpaired t-test). The presence of the ABD of bI-spectrin in dendritic spines thus increased the sizes of the PSD and the spine head, as well as synaptic strength. The activity of the ABD of bI-spectrin is mediated by Rac3 Do the effects of the bI-spectrin ABD on spines result directly from its interaction with actin or do they result because of recruitment of some other molecule to the spine head? Our observation that only the ABD of bI-spectrin localizes to spines and caused their enlargement, although all of them bind to actin (Fig. 2), favors the later hypothesis. Small GTPases are important signaling molecules involved in the regulation of dendritic spine morphological dynamics [47]. One small GTPase, Rac1, modu- lates dendritic spine morphology [48] and function [49]. We therefore tested the role of rac proteins in the morphological changes induced by the ABD of bI-spectrin. Two Rac GTPases are expressed in hippocampal and neocortical pyramidal cells, Rac1 and Rac3 [50,51]. We first immunoprecipitated endogenous bI-spectrin from cultured corti- cal neurons and screened for the presence of Rac3 and Rac1. Surprisingly, Rac3 was enriched in immunoprecipitates of bI- spectrin, but Rac1 was absent (3 replicates)(Fig. 8A). We tested the importance of this interaction by co-expressing a dominant negative Rac3 construct together with the ABD of bI-spectrin. Under these conditions, spine head diameters were significantly PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16197