Arf4 Regulates Dendritic Spine Development Figure 8. Overexpression of constitutively active Arf4 (Arf4-Q71L) promotes dendritic spine development to a greater extent that Arf4-WT. (A) Primary hippocampal and cortical neurons were cotransfected at DIV5 with GFP-b-actin plus Arf4-HA [wild-type (WT), T31N, or Q71L] and analyzed at DIV14 (n = 8–13 neurons). (B–C) Quantification of the effect of Arf4 and its mutants on total spine density (B) and the densities of specific spine subtypes (C) following transfection with Arf4 or its mutants. All data are mean6SEM. * p,0.05, ** p,0.01. doi:10.1371/journal.pone.0046340.g008 (Figs. 1F and G). Thus, Arf4+/2 mice were unable to effectively distinguish between two similar but unique situations. Interesting- ly, Arf4+/2 mice did not show deficits in spatial learning (Fig. 2A) or memory retention (Figs. 2B and C), suggesting that the neurological impairments are specific to DG-dependent pattern separation. Arf4+/2 mice were also not impaired in locomotor and exploratory activity (open field test) (Figs. 2D–F), motor coordi- nation (rotarod) (Fig. 2G), or anxiety-related behaviors (elevated plus maze) (Figs. 2H and I). Reduced spine density and mEPSC amplitude in Arf4+/2 DG granule cells The overall structures and morphologies of the hippocampus, cortex, and other brain regions appeared normal in Arf4+/2 brains compared to controls, as determined by hematoxylin and eosin staining (Fig. 3). We then examined the potential effects of Arf4 heterozygosity on neuronal fine structure using a modified Golgi- Cox staining protocol (Figs. 4A, 4B, and 5A). Both apical and basal dendrites of pyramidal neurons from the CA1 region (Fig. 4C), as well as dendrites from granule cells of the DG region (Fig. 5B), were analyzed (n = 4 mice/genotype). Although the CA1 region of Arf4+/2 mice did not show spine density (Figs. 4B, C) or morphology (Fig. 4D) alterations compared with controls, there was a significant decrease in total spine density in the granule cells of the DG (Figs. 5A and B), as well as a decrease in mushroom spine density (Fig. 5C), which is in line with the high level expression of Arf4 in the DG (Figs. 1C and D). Furthermore, the amplitude of miniature excitatory postsynaptic currents (mEPSCs) was 37% lower in Arf4+/2 DG granule cells than controls (Figs. 5D–F). The frequency of mEPSCs did not differ between the two genotypes (Figs. 5G and H). Thus, reducing Arf4 by 50% significantly impaired spine development and the electrophysio- logical function of granule cells in the DG. Arf4 is expressed in neurons and localizes to dendritic spines Arf4 is expressed in the hippocampi of 4.5-month-old mice, as well as in primary neurons and Neuro-2a (N2a) neuroblastoma cells (Figs. 6A–C), as determined by western blots. When a HA- (Figs. 6D–F) or mCherry-tagged (Figs. 6G–I) form of human Arf4 was expressed in mouse primary neurons, Arf4 was visible throughout the soma and dendrites, including dendritic spines. Furthermore, both Arf4-HA and Arf4-mCherry co-localized with GFP-b-actin (Figs. 6D–I), which forms networks in dendritic spines [10]. These results point to a potential role for Arf4 in modulating neuronal function at the level of dendritic spines. Arf4 promotes dendritic spine development in primary neuronal cultures To examine whether Arf4 regulates spine development, we transfected cultured primary mouse cortical and hippocampal neurons at 5 days in vitro (DIV5) with cDNA constructs encoding PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e46340