Arf4 Regulates Dendritic Spine Development Figure 6. Expression of Arf4 in brain of 4.5-month-old mice and in primary neurons, and the effect of Arf4 overexpression on dendritic morphology. (A) Hippocampi from WT mice were prepared as described in methods, followed by immunoblotting with anti-Arf4 (n = 6). (B) Mixed hippocampal and cortical neurons were prepared from E18–E19 WT mouse embryos, and neuronal lysates were collected at DIV12 and DIV19. Samples were immunoblotted with anti-Arf4. (C) Lysates from WT Neuro-2A cells were collected 48 and 72 hours after plating, followed by immunoblotting with anti-Arf4. (D–I9) Primary neuron co-transfected with GFP-b-actin and Arf4-HA (D–F9) or Arf4-mCherry (G–I9) at DIV5 and imaged at DIV14. Higher (D–I) and lower (D9– I9) magnification images are shown. (J, K) Representative images of dendrite morphology for neurons transfected with a control vector (J) or vector plus Arf4-WT (K). (L, M) The number (L) and length (M) of primary, secondary, and tertiary dendrites were quantified. (n = 12 neurons per experimental condition). All data are mean6SEM. doi:10.1371/journal.pone.0046340.g006 hundred events were analyzed for each cell and only events recorded 10 minutes after whole cell break-in were included in the data analysis. Behavioral tests Behavioral testing was performed using male and female mice that were 4–5 months of age at the time of testing. All experiments and analyses were performed blind to genotype. The pattern separation test is used to measure an animal’s ability to distinguish between similar events [40]. Mice were habituated to the pattern separation testing room for one hour prior to training. During the training period, mice were placed in an open chamber with a specific floor pattern and two identical objects, and were allowed to explore for 10 minutes. Following a 30-minute inter-trial interval, mice were placed in a second open chamber with a different floor pattern and two identical objects unique from the objects in the first trial. After 3 hours, mice were tested for 10 minutes in a chamber consisting of a floor pattern from either trial one or trial two, one object from trial one, and one object from trial two. The time each mouse spent exploring the object in the novel context (e.g., object from trial one in context from trial two) was compared with the time spent exploring the object in the old context. Exploration of an object was defined as the length of time a mouse’s nose was 1 cm away from the object, and video recordings of the trials and test period were used to manually analyze exploration time. For the Morris water maze test, the water maze pool (122 cm, diameter) was filled with opaque water (21uC) and contained a submerged platform (10 cm, diameter) during hidden trials [22,34]. The ability of mice to locate the hidden platform was determined in two sessions (3.5 hours apart) per day for 5 days. Each session consisted of two 60 sec trials with a 15 min intertrial interval. The platform location remained constant during the hidden trials, and entry points were changed for each trial. The latency to reach the hidden platform was recorded as a measure of spatial learning. Probe trials (60 sec, platform removed) were performed 24, 72, and 120 hours after the hidden trials. Memory retention was measured by the percent time spent in the target quadrant compared to the average time spent in the other three quadrants, as well as by the number of crossings over the original position of the target platform compared to the number of crossings over the equivalent platform positions in other quadrants. Following the probe trials, the ability of mice to locate a clearly visible platform was tested in three sessions (two trials/ session) to exclude differences in vision and swim speed. Performance was monitored with an EthoVision video-tracking system (Noldus Information Technology). PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e46340