not post-synaptic Gdi1 is important in maintaining the integrity of excitatory projections to the LA. Given the strong link between the physiology of excitatory projections to the LA and emotional learning, it is likely that these synaptic defects participate in the fear-related learning deficit that is seen in aGDI mutant mice. In addition to the functional disappearance of excitatory synapses, we also observed that the ready releasable pool size at the remaining cortico-LA synapses was significantly decreased in conditional but not Gdi1-null mutants (Fig. 8). This possibly suggests that if aGDI deletion occurs during the early phases of synaptogenesis, its absence may be overcame molecularly or functionally, which would no longer be possible in the adult consolidated synapses. Physiological experiments conducted on adult cortico-LA synapses suggest some functional differences. For example, we observed that the release probability at cortico-LA synapses, which is modified by classical fear conditioning procedures [17], is significantly higher in adult animals. Interestingly, this was observed together with a reduction in pre-synaptic expression of long-term plasticity (Y. Humeau F. Gambino, personal commu- nication). Taken together, these observations suggest that during the life of the animal, experience-driven consolidation and maturation at excitatory synapses occur in the lateral amygdala, a phenomenon that depends on the presence of aGDI and possibly specific RAB GTPases involved in SV exo- and endocytosis. We previously suggested that the lack of aGDI seems to perturb the functionality of different SV pools at the pre-synaptic site and may be due to alterations of at least two different classes of RAB GTPases, the SV-associated RAB3A and the endosomal RAB proteins RAB4 and RAB5 by altering cycling between the active GTP- and inactive GDP-bound state. Despite knowledge about the mechanisms by which RABs regulate intracellular exo- and endocitic events, it is not clear how many RABs are involved in these pathways and whether all of them are susceptible to aGDI- mediated regulation. Recently, Pavlos et al. [7], use d a combination of different techniques to conclude that SVs contained a set of RABs associated with SV membranes involved both in exocytosis and endosomal recycling, including RAB3A/B/ C, RAB4B, RAB5A, RAB10, RAB11B, RAB14 and RAB27B. They also showed that RAB27B exhibits both overlapping and distinct features from RAB3. In conclusion, our results point to a defect in pre-synaptic function that affects several parameters of short-term synaptic plasticity involved in hippocampus-related short-term memory deficits as well as in amygdala-related behavior. Stronger defects in short-term synaptic plasticity are present when Gdi1 is deleted specifically in the forebrain during adult life, suggesting a predominant role of Gdi1 via a subset of specific RAB GTPases acting specifically in these brain regions at the pre-synaptic sites and leading to learning and social alterations. In this regard, our results encourage further studies to identify RAB cell specificity and function in neurons and glial cells, which will most likely shed light on the RABs that determine the Gdi1 learning phenotype. Materials and Methods Animals All animals were maintained on a 12 hrs light/darkness cycle at 22u–25uC. Food pellets and water were available ab libitum. Experiments were done according to the animal protocols approved by the Institutional Animal Care and Use Committee San Raffaele (IACUC) (San Raffaele, Milan, Italy) and were approved by the National Ministry of Health, IACUC ID 470. All experiments were carried out in accordance with the guidelines Conditional Gdi1 Deletion on Synaptic Function established by the European Community Council Directive of 24 November 1986 on the use of animals in research (86/609/EEC). All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable results. Behavioural tests Dark/light box. A 20630-cm lit chamber with transparent Perspex walls (20 cm high) and open top was connected to a 20615620-cm plastic dark box which was completely closed except for the 7.567.5-cm door connecting it to the lit chamber. Illumination was by direct room light (500 lx). Each mouse was released in the middle of the lit compartment and observed for 5 min. Novel object test. Frames of non-reflective aluminium (37 cm high) were used to partition a round open field arena (diameter of 150 cm and 35-cm high walls) into four squares 50650-cm arenas, allowing for concurrent observation of four animals. Illumination in the room was by indirect diffuse room light (4640-W bulbs, 12 lx). The novel object was a 50-mL Falcon tube positioned vertically in the center of the arena. Each animal was observed for 30 min in the empty arena as pre-exposure. The novel object was then introduced and observation continued for another 30 min. Emergence test. It is used the same arena used in the Novel object test. In the mouse cage the day before a box made of plastic (126864 cm with opening of 864 cm) is inserted as home and during the test it is placed in a corner of the arena, at 5 cm from the nearest walls, with the opening facing away from the wall. The mice were observed for 30 min. For time course analysis, the total observation time was portioned into six periods of 10 min. Water maze. The standard hidden-platform version of the water maze was done as previously described [5]. Briefly, the test included an acquisition phase (18 trials, six/day, inter-trial time 30–40 min) followed by a reversal phase during which the platform was moved to the opposite position (12 trials, six/day). For the analysis the trials were averaged in blocks of two trials. Radial Maze. The apparatus consisted of eight arms (38 cm long, 7 cm wide) extending from an octagonal centre platform (diameter 18.5 cm) with 5-cm transparent plastic walls. The distance from the platform centre to the end of each arm was 47 cm. At the end of each arm is present a cup with a food pellet. Food-deprived mice (maintained at 85% of their free-feeding weight) were placed in the center platform and allowed to collect pellets placed at the end of each arm for 10 min. The animals were adapted to the maze for 1 day and then tested for 10 days. For each trial, the total number of arm choices, number of correct choices before the first error, total number of errors was recorded. Fear conditioning test. Auditory trace fear conditioning were performed as previously described [5]. All mice were pre- exposed to the test chamber (Ugo Basile, Italy) for 10 minutes on the two days preceding conditioning. During the training session, trial started with the presentation of the CS (15 s), followed 15 s later by the presentation of the shock for 2 s. This was repeated 5 times with a 60 seconds inter-trial intervals (ITI). Twenty-four hours after fear conditioning, mice were placed in the conditioning box again, measuring their freezing behavior in the context test (2 min without CS ‘‘contextual freezing’’). During the test, animals were video-tracked using the ANY-maze system (Anymaze, Stoelting Co, Wood Dale, IL, USA, www.anymaze. com). Resident-intruder test. Resident adult male mice were kept individually for 2 weeks prior to the test and were used as residents. C57BL/6N mice, housed in groups of 5, were used as intruders. After transfer of the intruder to the home cage of the PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e29763