Overexpression of either chronophin or cofilin prevents nLTD-induced loss and shrinkage of dendritic spines.

<p>Neurons were transfected with either the cofilin phosphatase chronophin (CIN) or wildtype cofilin itself, as indicated, together with the cell filler eGFP prior to induction of nLTD. Image width = 27.3 µm; all images show eGFP). Morphological data was quantified using eGFP images and are expressed as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, compared to control alone; ^## p<0.01, ^###p<0.001 compared to nLTD alone; ^XXp<0.01 compared to CIN or cofilin (cof) alone, two-way ANOVA, followed by Bonferroni post hoc test. (<b>A</b>) CIN overexpression prevents nLTD-associated spine loss and shrinkage. Selected dendritic regions of neurons co-expressing CIN and the cell filler eGFP at 4 min post nLTD. For spine density, number of dendrites: ctrl = 38, nLTD = 40, CIN = 32, CIN+nLTD = 59); for spine length and width, number of spines: ctrl = 659, nLTD = 551, CIN = 635, CIN+nLTD = 1109). (<b>B</b>) Cofilin overexpression prevents nLTD-associated spine loss and shrinkage. Selected dendritic regions of neurons co-expressing ectopic cofilin and the cell filler eGFP at 4 min post-nLTD. For spine density, number of dendrites: ctrl = 27, nLTD = 21, cof-wt = 54, nLTD+cof-wt = 58); For spine length and width, number of spines: ctrl = 542, nLTD = 348, cof-wt = 1063, nLTD+cof-wt = 1095). (<b>C</b>) Chronophin overexpression prevents the nLTD-induced reduction in F-actin concentration in dendritic spines. F-actin concentration in spines was quantified as the ratio of the integrated intensity of Alexa 568-phalloidin divided by spine area on neurons incubated in the absence or presence of NMDA for 4 min (nLTD). number of spines: ctrl = 304, nLTD = 259, CIN = 231, CIN+nLTD = 253; (a.u. = arbitrary units). (<b>D</b>) Chronophin overexpression prevents the LTD-induced reduction in actin filament free barbed ends (FBEs) in dendritic spines. FBE concentration was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094787#s2" target="_blank">Methods</a> on neurons incubated in the absence or presence of NMDA for 4 min (LTD). Number of spines: ctrl = 236, nLTD = 217, CIN = 96, nLTD+CIN = 167 (a.u. = arbitrary units). Experiments in this figure and its corresponding supplemental material were analyzed in a blind fashion.