Figure 4. CA1 spine density is not altered in Arf4+/2 mice compared to WT mice. (A) Golgi impregnation of a WT hippocampus at 5X. (B) Representative dendrites for CA1 pyramidal neurons of WT and Arf4+/2 mice at 63X magnification. (C) Averaged total spine density in the CA1 region of WT and Arf4+/2 mice (28–30 neurons/genotype). (D) Spine densities for specific spine subtypes in the CA1 region of WT and Arf4+/2 mice. doi:10.1371/journal.pone.0046340.g004 magnification images. Z-stack sections were merged using LaserSharp2000 software. Neurons were selected randomly and 1 to 2 equivalent-length dendritic segments from each neuron were chosen for quantification of protrusions. Protrusion density and morphology were manually quantified using ImageJ software, according to criteria described [35]. cDNA and small hairpin RNA constructs HA-tagged human wildtype Arf4 was a gift from JD Lee (Scripps Research Institute, La Jolla, CA) [36] Arf4-HA point mutants (Arf4-HA-T31N and Arf4-HA-Q71L) were generated using the QuikChange II XL Site-Directed Mutagenesis kit (Strategene). mCherry-tagged human Arf4 was a gift from Paul Melancon (University of Alberta, Edmonton, Canada) [37]. The FUGW2-GFP and GFP-b-actin plasmids were gifts from Lennart Mucke and Steve Finkbeiner, respectively (Gladstone Institutes, San Francisco, CA) [13,38]. Arf4-shRNA1 and Arf4-shRNA2 constructs were expressed under the U6 promoter using the FUGW2 vector. The target sequences used for the Arf4 shRNAs are as follows: 59-TCTGGTAGATGAATTGAGA-39 (Arf4- shRNA1) and 59-AGATAGCAACGATCGTGAA-39 (Arf4- shRNA2). Flag-tagged murine ASAP1 cDNA, a gift from Paul Randazzo (National Cancer Institute, Bethesda, MD), was expressed in the pCR2.1 vector [39]. Arf4 Regulates Dendritic Spine Development Staining of mouse brains For b-galactosidase staining, brains from 4.5-month-old Arf4+/ 2 mice were removed, frozen in OCT compound, and sectioned at a thickness of 10 mm using a Leica CM1900 cryostat. Sections were washed 3 times in 0.02% Nonidet P-40/PBS, fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, and stained in PBS with 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet-P-40, and 1 mg/ml X-gal at 37uC for 16 h. After a series of ethanol washes, sections were cleared in xylene and mounted with Cytoseal. For Golgi staining, 4.5-month-old Arf4+/2 and WT littermates were stained in parallel using modified Golgi-Cox impregnation of neurons following the manufacturer’s protocol (FD Neuro- Technologies, Ellicott City, MD) (n = 4). Brains were sliced using a freezing-sliding microtome (Leica SM2000R) at a thickness of 150 mm. Images of the CA1 and DG were taken with a Leica CTR5000 brightfield 63X oil objective, coded, and analyzed in a blinded manner using ImageJ software. For hematoxylin and eosin staining, following transcardial perfusion with saline, brains from WT and Arf4+/2 mice were fixed in 4% PFA-PBS for 48 hours, transferred to 70% ethanol, and embedded in paraffin. 5 mm sagittal sections were cut for conventional hematoxylin and eosin staining. BDNF immunohistochemistry was performed on 30 mm coro- nal WT and Arf4+/2 brain sections following transcardial perfusion with saline and a 48 hour fixation in 4% PFA-PBS. Briefly, free-floating sections were treated with 3% H2O2 and 10% methanol in PBS to block endogenous peroxidase. The sections were incubated with PBS/10% normal donkey serum/1% milk/ 0.2% gelatin for 1 hour, followed by overnight incubation with primary anti-BDNF antibody (1:100, Santa Cruz Biotechnology). Sections were further processed using a biotinylated secondary donkey anti-rabbit antibody (1:1000, Jackson ImmunoResearch), avidin-peroxidase complex (ABC) and diaminobenzidine (DAB). The stained sections were mounted on slides, cleared with Xylene and coverslipped. Images were taken with a Leica CTR5000 brightfield 5X objective, and BDNF immunoreactivity was analyzed by densitometry using ImageJ software. Electrophysiology Two-month old mice were deeply anesthetized and euthanized following UCSF animal protocol guidelines. Then the brains were quickly removed and immersed in ice-cold cutting solution containing (in mM) 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 26 NaCO3, 11 glucose and 1.3 ascorbic acid, and oxygenated with 95% O2/5% CO2. Transverse slices of 325 mm were cut on a Leica VS1000 vibroslicer (Leica, Germany) and incubated at 32uC for 30 min in an interface incubation chamber (Automated Scientific, CA), after which the slices continued to be incubated in the same chamber at room temperature. For recording, slices were transferred to a submerged recording chamber and continuously perfused with oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgSO4, 26 NaCO3, 10 glucose and 2 CaCl2 at 3 mL/min (25uC). Whole-cell recordings were performed on visually identified dentate granule cells and fully matured granule cells were identified by input resistance (less than 400 MV). To isolate miniature excitatory postsynaptic currents (mEPSCs), 100 mM picrotoxin(Sigma), 5 mM bicuculline (Tocris) and 0.5 mM tetrodotoxin (Abcam) were added to the perfusate. The internal pipette solution contains (in mM) 120 CsMeSO3, 4 NaCl, 2 MgCl2, 10 HEPES, 5 EGTA, 5 MgATP, 0.3 Na3GTP and 5 QX-314. Series resistance (,30 MV) was constantly monitored and the recording was discarded if changes .15% occur. Data PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e46340