alterations might be due to the reduced availability of the endocytic RABs RAB4 and RAB5, which are involved in slow SV recycling and in alterations of RAB3A, which is the main RAB protein mediating SV exocytosis. Nevertheless, other RAB proteins may be involved in the Gdi1-null phenotype, particularly proteins that have recently been shown to be associated with SV membranes [7]. A major limitation for the study of aGDI function in the central nervous system (CNS) and the interpretation of all results obtained from the analysis of Gdi1-null mice is the fact that the complete ablation of Gdi1 occurs in all brain regions and all brain cell types throughout development in these mice. Moreover, the previously observed phenotype of the Gdi1-null mice may be due to alteration of several RAB GTPases and subsequent compensation. To solve this problem, we used the CRE/lox P recombination system to restrict Gdi1 knockout both spatially and temporally to postmitotic neurons of the anterior forebrain. These conditional Gdi1 knockout mice were viable and developed without any obvious morphological defects. We show that the conditional Gdi1 mice phenocopy the cognitive deficit and showed greatly reduced male aggression, as was previously observed in the Gdi1-null mice, confirming the pivotal role of aGDI in hippocampal, cortical and amygdala synapses in the process of memory formation. Surprisingly, the electrophysiological comparison at cortico- amygdala synaptic connections between the Gdi1-null and conditional Gdi1 mice reveals a stronger pre-synaptic phenotype in conditional Gdi1 mice. Because significant levels of recombina- tion do not start until the end of the third postnatal week, when many of the CNS neuronal pathways have already been established, these results suggest that the regulation of RABs by aGDI directly modulates complex behavior that is important for cognitive function. Results Inactivation of aGDI occurs in specific brain regions during postnatal life To generate a conditional Gdi1 mouse model, we generated a floxed allele of Gdi1 by flanking exons 2 and 3 of the mouse Gdi1 gene with two lox P sites using homologous recombination in ES cells (Fig. 1A). One homologous recombinant clone with the highest percentage of normal karyotypes (90%) was analyzed by Southern blot (Fig. 1B and C) and was injected into blastocysts. Derived germline targeted females offspring carrying one floxed allele (Gdi1lox/X) were first crossed with transgenic male mice expressing the tgFlp recombinase to excise the neomycin cassette and subsequently backcrossed into a C57BL/6N genetic back- ground. All subsequent work was performed using neo2 mice. To inactivate Gdi1 in the postnatal forebrain, we crossed heterozygous Gdi1lox/X female mice with the transgenic line CaMKII-Cre-159 [8] (provided from Dr. R. Brambilla, HSR, Milan-Italy) to generate CaMKII-Cre+-Gdi1flox/Y and CaMKII-Cre2- Gdi1lox/Y male mice (referred to as Gdi1flox/Y for Gdi1 knockout and Gdi1lox/Y for wild-type mice) for all subsequent work. Western blot analysis was performed on several brain regions at different developmental stages to assess the regional and temporal pattern of aGDI inactivation driven by CaMKII-Cre-159 transgene activity in vivo (Fig. 2). At several postnatal days (ranging from PND 14 to 56), hippocampus (Hip), cerebral cortex (CC), olfactory bulbs (OB), cerebellum (CbC) and a mixture of the thalamus (T), hypothalamus (H) and amygdala (BLA) were dissected from Gdi1flox/Y and compared with Gdi1lox/Y littermate mice (PND 56) (Fig. 2). No difference in the aGDI protein level was observed in the OB (mean 6 SD; Gdi1flox/Y 1.4460.37; Gdi1lox/Y 1.560.38), Conditional Gdi1 Deletion on Synaptic Function CbC (mean 6 SD; Gdi1flox/Y 4.1460.97; Gdi1lox/Y 4.4861) and T- H-BLA (mean 6 SD; Gdi1flox/Y 1.260.33; Gdi1lox/Y 1.5160.05) homogenates, confirming that recombination was not active in these brain regions, as previously reported [8]. At PND 28, aGDI was still detectable but down regulated by 50% in the Hip (t-test, p = 0.05) and by 65% in CC (t-test, p = 0.02). The down- regulation reached 80% at PND 56 in the Hip (t-test, p = 0.01) and 98% in CC (t-test, p = 0.005), as shown in Fig. 2B. Additionally, double immunofluorescence for aGDI and NeuN on brain slices from Gdi1lox/Y, Gdi1flox/Y and Gdi1-null mice at PND 56 revealed that aGDI down-regulation in Gdi1flox/Y was specifically observed in the anterior forebrain, as previously reported [8] (Fig. 3B–E0). Surprisingly, double immunofluorescence for aGDI and glial markers such as S100b (Fig. 3B–E0) or GFAP (data not shown), revealed that aGDI was also down-regulated in S100b and GFAP positive glial cells in Gdi1flox/Y brain regions. Gdi1flox/Y mice recapitulate the Gdi1-null cognitive and behavioral phenotypes We previously showed that adult Gdi1-null mice did not have defects in emotional or exploratory behavior but were selectively impaired in hippocampal-dependent tasks important for acquisi- tion of memory across short time intervals [5]. To assess whether the deletion of aGDI in post-natal life and in forebrain regions plays a key role in associative memory formation, Gdi1flox/Y and Gdi1lox/Y male littermates were subjected to a battery of behavioral tests at PND 60 in order to assess exploration, learning and social recognition, as previously preformed for Gdi1-null mice [5]. Exploration tests. Locomotors activity scores did not show significant genotype-dependent differences in the light-dark box, emergence test and the novelty test (data not shown), as previously observed for Gdi1-null mice [5]. Learning and memory tests Water Maze. Gdi1flox/Y and Gdi1lox/Y male littermates behaved normally in the standard hidden-platform version of the water maze. No significant differences between genotypes were observed in all the variables analyzed, confirming that Gdi1flox/Y mice do not show alterations in spatial memory, as was previously shown for Gdi1-null mice [5]. Radial Maze. Gdi1flox/Y and Gdi1lox/Y male littermate mice showed altered spatial working memory as assessed by the radial maze test. The number of total errors declined over the ten days of training in both Gdi1flox/Y and Gdi1lox/Y mice, and no difference between genotypes was observed (ANOVA genotype by repeated exposure F[1,24] = 0.545; p = 0.467; Fig. 4A). Gdi1flox/Y and Gdi1lox/Y mice had significantly different performance in the position of the first repetition (ANOVA F[1,24] = 21.47, p = 0.0001). Although Gdi1lox/Y mice eventually reached nearly perfect performance (seven out of a maximum number of eight correct successive arm visits), Gdi1flox/Y mice barely scored above chance level (5.5 correct successive arm visits after 10 days of training) (Fig. 4B). In conclusion, the Gdi1flox/Y mice seem to have no defects in procedural learning but do have a specific and severe deficit in working memory, as was seen in Gdi1-null mice [5]. Fear Conditioning. Mice were also tested for contextual and trace fear conditioning. No differences were observed between Gdi1flox/Y and Gdi1lox/Y mice in the ability to associate the CS with the US during the training session (ANOVA genotype effect: F[1,40] = 0.208, p = 0.65) as well as 24 h later when testing the context (ANOVA genotype effect: F[1,40] = 0.008, p = 0.929) and cue memory test (ANOVA genotype effect: F[1,40] = 0.239, p = 0.628) in the contextual fear conditioning (data not shown). Instead, as previously shown for the Gdi1-null mice [5], there was a PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29763