Arf4 Regulates Dendritic Spine Development Figure 7. Arf4 overexpression promotes dendritic spine development in mouse primary neurons. (A–B9) Neurons transfected with GFP- b-actin alone (A–A9) or together with Arf4-HA (B–B9) at DIV5 and imaged at DIV12 (n = 9–10 neurons). (C–D9) Neurons transfected with GFP-b-actin alone (C–C9) or together with Arf4-HA (D–D9) at DIV5 and imaged at DIV14 (n = 8–10 neurons). (E–F9) Neurons transfected with GFP-b-actin alone (E– E9) or together with Arf4-HA (F–F9) at DIV5 and imaged at DIV19 (n = 8–9 neurons). (G) Averaged spine density of neurons transfected with GFP-b- actin alone or with GFP-b-actin plus Arf4-HA at several time points. All data are mean6SEM. * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0046340.g007 The open field test is a standard test for general locomotor activity, willingness to explore, and anxiety [41]. It consists of a square enclosure in which infrared detectors track animal movement. Locomotor and exploratory activity is assessed by the number of basic movements and rearings, whereas the proportion of time spent in the center of the enclosure is used as a measure of anxiety [42]. Mice were placed in the center of the chamber and were tested for 15 min. The rotarod test, using a steady-speed rotarod set at 16RPM, was performed by placing mice on rotating drums and measuring each animal’s latency to fall over a period of 300 seconds [43]. The Elevated Plus Maze is a test for rodent anxiety and is based on a rodent’s aversion to open spaces [44]. The apparatus consists of two open arms and two enclosed arms at right angles to each other. Mice were placed on the central platform and were allowed to explore the apparatus for 10 minutes. Anxiety was assessed by comparing the amount of time spent in open versus enclosed arms. Statistical analysis Unless stated otherwise, all values are expressed as mean6- SEM. Statistical analyses were performed with GraphPad Prism software. Differences between the means were assessed by t-test or one factor ANOVA followed by a Bonferroni post-hoc test. A p- value of ,0.05 was considered to be statistically significant. Statistical values are denoted as follows: * p,0.05, ** p,0.01, *** p,0.001. Results Arf4+/2 mice have impairments in a DG-dependent pattern separation task To study the roles of Arf4 in vivo, we used a gene-trapping strategy to try to generate Arf42/2 mice (Fig. 1A). Since Arf42/2 mice were embryonically lethal, we focused our in vivo studies on Arf4+/2 mice. Arf4+/2 mice were fertile, viable and showed no overt phenotype. Arf4 protein levels were reduced by 49% in the hippocampus of Arf4+/2 mice compared to wildtype (WT) littermates (Fig. 1B). X-gal staining of 4.5-month-old Arf4+/2 mice showed that Arf4 is highly expressed in the DG (Figs. 1C and D), prompting us to investigate the potential roles of Arf4 in DG- dependent memory tasks. Since the DG is known to be involved in an animal’s ability to distinguish between similar events [3], we asked whether the loss of one copy of Arf4 might affect performance in a pattern separation task. This task involved training mice to associate a certain environmental context with specific objects in two training sessions, followed by a testing session in which the rodents’ ability to recognize context-object distinctions was analyzed. Whereas WT mice spent a greater proportion of time exploring the object in the novel context than the object in the familiar context, Arf4+/ 2 mice showed no difference in the proportion of time spent with either object during the testing phase (Fig. 1E). Neither WT nor Arf4+/2 mice showed significant differences in the amount of time spent with the two identical objects in either of the training sessions PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e46340