resident the behavior was tape-recorded and the cumulative duration of aggressive behavior (biting attacks, tail rattling) and social behavior (sniffing head or back, social grooming) were determined. Video tracking, data collection and statistical analysis During all the tests, animals were video-tracked using the EthoVision 2.3 system (Noldus Information Technology, Wagen- ingen, the Netherlands, http://www.noldus.com) using an image frequency of 4.2/s. Raw data were transferred to Wintrack 2.4 (http://www.dpwolfer.ch/wintrack) for off-line analysis. Statistical computations were done using Statview 5.0 (SAS Institute, Cary, NC, USA). Gene targeting The entire mouse sequence of Gdi1 was isolated from the 129/ SvEv mouse genomic library and sequenced (accession number AF441240), as previously described [5]. The targeting vector was constructed as follow: the 59 arm consists on a KpnI/NdeI 1625 bp fragment containing the 1st exon and part of the 1st intron of Gdi1 gene; lox P sites are inserted in the 1st and in the 3rd intron, in order to delete the Gdi1 gene between exon 2 and 3; the neomycin-resistance (neo) cassette, driven by promoter and 59 UTR sequence from the mouse Pgk1 gene, flanked by frt sites was inserted in opposite orientation in the 3rd intron, immediately downstream of the 39 lox P site; the 39 arm of the targeting vector consists on EcoRV/EcoRI 7 kb containing the last 8 exons of Gdi1 and part of the 59 region of the flanking gene. After linearization with unique NotI restriction site and electroporation in AB1 embryonic stem (ES) cells, G418-resistant colonies were analyzed by PCR, from primers F4 and NEO1C with XL Expand Boheringher. PCR positive clones were confirmed by Southern blot. The 59 flanking probe S1 was an RT-PCR product of 320 bp containing exons 6 and 7 of the 16A 59 flanking gene. A 590 bp probe containing the genomic region cloned between lox P sites was used to confirm the insertion of both sites. A neo probe, obtained by PCR from primers neo2 and neo6, was used to confirm the insertion of neo cassette. One of three homologous recombinant clones was injected into blastocysts by standard methods and chimera mice were generated. Chimera female mice were crossed to C57Bl/6N mice. Genotypes were determined by PCR from tail DNA biopsy using primers Lox1 and Lox2. The primer sequence is showed in table S1. Generation of Gdi1flox mice Heterozygote Gdi1lox/X female mice were crossed with trans- genic SJL-TgN(ACTFLPe)9205Dym mice (Jackson Laboratory, Bar Harbor, ME, USA) expressing a variant of the S. cerevisiae FLP1 recombinase gene under the human b-actin promoter to delete the neo cassette. Mice were analyzed by PCR analysis of DNA extracted from the tail using primers Neo4 and Neo5, FlpF and FlpR. Heterozygote Gdi1lox/X female mice, neo2, were crossed with CaMKII-CRE-159 transgenic mouse [8], expressing the CRE recombinase under the control of the mouse aCaMKII promoter. Mice genotype was analyzed by PCR analysis of DNA extracted from the tail using primers Lox1 and Lox2, and cre1 and cre2. The primer sequence is showed in table S1. Western blot analysis Brain regions were dissected from Gdi1flox/Y (n = 3) and Gdi1lox/Y (n = 3) littermates. Proteins in sample buffer were fractionated in 10% SDS-PAGE, and Western blots were done using standard methods. Conditional Gdi1 Deletion on Synaptic Function Immunofluorescence analysis The animals were anesthetized with 2-2-2 Tribromoethanol (20 ml/gr) and transcardially perfused with 20 ml eparin in PBS1X (30 mg/100 ml), and brains were fixed in 4% (w/v) paraformaldehyde (Fluka, Buchs, Switzerland). Brains were post-fixed in the same fixative overnight at 4uC. Frozen coronal sections (15 mm thick) were established for standard immunofluorescence. Briefly, sections were washed three times with PBS 1X and then incubated in blocking buffer (PBS1X, FBS 10%, BSA 1 mg/ml and Triton 0.1%). Primary antibodies were diluted in blocking buffer and applied on sections at at the following concentration: aGDI (1:100, Zymed), NeuN (1:1000, Chemicon), S100b (1:100, SIGMA), GFAP (1:1000, Dako), Primary antibodies were incubated over night at +4uC. Section were then washed three times in PBS 1X and secondary antibodies were applied accordingly manufacturer, instructions, nuclei were stained with Dapi. Images were acquired directly from slices using the Olimpus BX51 microscope and 206 objective. Electrophysiology Slice preparation. Standard procedures were used to prepare 330 mm thick coronal slices from Gdi1-null and Gdi1 WT, Gdi1lox/Y and Gdi1flox/Y male mouse brains following a protocol approved by the European and French guidelines on animal experimentation. Briefly, the brain was dissected in ice- cold artificial cerebrospinal fluid (ACSF), mounted on an agar block and sliced with a vibratome (Leica VT1200s; Germany) at 4uC. Slices were maintained for 45 min at 35uC in an interface chamber containing ACSF equilibrated with 95% O2/5% CO2 and containing (in mM): 124 NaCl, 2.7 KCl, 2 CaCl2, 1.3 MgCl2, 26 NaHCO3, 0.4 NaH2PO4, 18 glucose, 4 ascorbate, and then for at least 45 min at room temperature before being transferred to a superfusing recording chamber. The same procedure was followed for manual brain area microdissections, the samples being immediately frozen in liquid nitrogen. Recordings. Whole-cell recordings from LA principal neurons were performed at 30–32uC in a superfusing chamber as previously described [18]. Neurons were visually identified with infrared videomicroscopy using an upright microscope equipped with a 606 objective. Patch electrodes (3–5 MV) were pulled from borosilicate glass tubing and filled with a low-chloride solution containing (in mM): 140 Cs-methylsulfonate, 5 QX314-Cl, 10 HEPES, 10 phosphocreatine, 4 Mg-ATP, and 0.3 Na-GTP (pH adjusted to 7.25 with CsOH, 295 mOsm). For current-clamp experiments, Cs-methylsulfonate was replaced with equimolar K- gluconate. All experiments were performed in the presence of picrotoxin (100 mM). Monosynaptic EPSCs or EPSPs exhibiting constant 10–90% rise times and latencies were elicited by stimulation of afferent fibers with a bipolar twisted platinum/ 10% iridium wire (25 mm diameter). In all experiments, stimulation intensity was adjusted to obtain baseline EPSC amplitudes of ,100. Data acquisition and analysis. Data were recorded with a Multiclamp700B (Molecular Devices, USA), filtered at 2 kHz and digitized at 10 kHz. Data were acquired and analyzed with pClamp10.2 (Molecular Devices). In all experiments, series resistance was monitored throughout the experiment, and if it changed by more than 15%, the data were not included in the analysis. Spontaneous and miniature events were collected using a template-based detection run onto a 3–5 minute time period. For amplitude calculation, each event was then fitted using a bi- exponential equation leading to better estimation of event characteristics. For RRP and refilling rate measurements, the last 20 (on 80 in total) EPSC amplitude were used to generate the PLoS ONE | www.plosone.org 11 January 2012 | Volume 7 | Issue 1 | e29763