Conditional Gdi1 Deletion on Synaptic Function Figure 2. Brain region- and age-specific down regulation of aGDI on Gdi1flox/Y mice. Two to eight week old male mice were analysed. (A) Protein lysates were prepared from the indicated tissues (Hip: hippocampus, CC: cerebral cortex, CbC: cerebellum, OB: olfactory bulb, T: thalamus, H: hypothalamus and BLA: amygdala) at the indicated age (PND: post natal days), fractionated on 10% SDS–PAGE gels and analyzed with a commercial anti-GDI antibody. b-tubulin was used as loading control. (B) Quantitative analysis of residual aGDI normalized by b-tubulin in the hippocampus and cerebral cortex. Protein levels were quantified by measuring the intensity of the western blot signal with the Image Quant system. Values are expressed as mean 6 SD from three independent animals at each age. Grey squares are Gdi1lox/Y animals and black squares are Gdi1flox/Y. *p,0.05, **p,0.01. doi:10.1371/journal.pone.0029763.g002 time spent sniffing the head of the intruder (ANOVA genotype effect: F[1,8] = 7.1, p = 0.03) (Fig. 4E–G). In conclusion, all of these data suggest that the down-regulation of aGDI in adult forebrain regions is sufficient to recapitulate the learning deficit previously shown in Gdi1-null mice [5]. These results suggest that aGDI acts via RAB GTPase availability to play a pivotal role in Gdi1-dependent short-term memory formation and maintenance. A defect in excitatory transmission onto lateral amygdala principal cells in the absence of aGDI points to a defect in fast SV recycling Glutamatergic lateral amygdala (LA) principal cells constitute the vast majority of neuronal cells in this amygdala nucleus [9] and can be easily identified by their electrophysiological properties [10]. They receive not only excitatory synapses from incoming projections from the cortex (cortico-LA synapses) and the medial division of the medial geniculate nucleus of the thalamus (MGm, thalamo-LA synapses) [11] but also input from neighboring principal cell collaterals. To investigate whether the emotional- related brain structures such as the amygdaloid nucleus may have a role in the fear-related behavior observed in the mutant mice, synaptic plasticity was analyzed at those synaptic connections. To better define the previously described hippocampal short- term synaptic defect [6] and to provide a comparison between Gdi1flox/Y and Gdi1-null mice, the overall number of excitatory synaptic contacts impinging onto LA principal cells in juvenile (PND 28) Gdi1-null and Gdi1 WT littermates was first examined by measuring spontaneous and miniature EPSCs (in presence of 10 mM Tetrodotoxin) (Fig. 5A, B). In control conditions, we observed a strong reduction in both the frequency (Gdi1 WT: 7.7560.85 Hz; Gdi1-null: 2.9360.26 Hz, p,0.001, n = 10 cells in both conditions) and the amplitude of recorded EPSCs (Gdi1 WT: 10.3360.80 pA; Gdi1-null: 7.0260.39 pA, p,0.001, Fig. 5B, C2). Interestingly, a significant decrease in EPSC frequency persisted in presence of 10 mM TTX (mEPSCs: Gdi1 WT: 5.7160.35 Hz, Gdi1-null: 3.0860.63 Hz, p,0.001, Fig. 5B). In contrast, mean mEPSC amplitude was similar in control and mutant animals (Gdi1 WT: 7.4460.11 pA; Gdi1-null: 6.9660.13 pA, p.0.05, Fig. 5C2). Taken together, these results indicate that in Gdi1-null slices, there is little multi-vesicular release and little of spontaneous discharge activity of incoming excitatory axons. A decrease in mEPSC frequency can result directly from a decrease in the size of the receptor field of LA principal cells, either due to a diminished dendritic tree or due to a lack of dendritic spines. To address these points, we first applied depolarizing steps to assess the cellular capacitance, but we failed to detect any difference in the cell size between genotypes (Capacitance: Gdi1 WT: 25.563.4 pF; Gdi1-null: 19.261.95 pF, p.0.05, n = 24 and 35 cells, respectively), suggesting that the main morphological properties were conserved and that probably the receptor field was similar in principal cells from Gdi1-null and control mice. Then, to directly visualize dendritic spines, we filled the recorded LA principal cells with 10 mM Alexa-488 while acquiring confocal fluorescence pictures of level III/IV dendrites using 2-photon laser scanning microscopy [12] but again did not observe any differences between Gdi1 WT and Gdi1-null mice (data not shown). These data suggest the presence of a lower release probability and/or a lower number of functional excitatory synapses onto LA principal cells but a normal quantal size in Gdi1- mice. Alteration of cortico-LA synaptic weight in the absence of aGDI Cortical projections to the LA can be directly activated by positioning a stimulation electrode within the external capsule in coronal LA-containing acute slices of mouse brain (Fig. 6A) [13]. The application of a short (1 ms) current pulse elicits a PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e29763