Figure 5. Excitatory transmission onto LA principal cells of Gdi1-null mice. (A) Spontaneous (Ctrl) and miniature (TTX) EPSCs were recorded in LA principal cells from Gdi1 WT and Gdi1-null littermates. Scale bars: 20 pA and 60 msec. (B) Mean EPSC frequency is lower in LA cells from Gdi1-null mice in absence or presence of TTX. The number of recorded cells is indicated. ***p,0.001. (C) Decrease of spontaneous – but not miniature - EPSC amplitude in absence of aGDI. (C1) Spontaneous EPSC (Ctrl) and miniature EPSC (TTX) in both genotypes. Scale bars: 5 pA and 10 msec. (C2) Mean EPSC values for each conditions. The number of recorded cells is indicated. doi:10.1371/journal.pone.0029763.g005 The refilling rate can be assessed by high-frequency iterative stimulations: after the consumption of the pool of release-ready vesicles (RRP), neurotransmitter (NT) release usually stabilizes. The plateau level is then determined by the ‘‘refilling rate’’, which is mostly dependent on vesicular recycling at the synaptic terminal [15]. Thus, we looked at vesicular pool dynamics by applying 20 Hz/ 4 sec stimulation trains at cortico-LA synapses from Gdi1 WT and Gdi1-null animals. At a typical WT synapse, NT release first facilitates and then progressively decreases to stabilize at ,20% of the initial EPSC amplitude (Fig. 7B, C) [16]. The absence of aGDI led to major changes in the postsynaptic response to 20 Hz trains; after a first phase of facilitation due to calcium accumulation, the synaptic depression was exacerbated, and the final plateau level was significantly lower compared to Gdi1 WT littermates (Fig. 7B, C). Both RRP size and refilling rate were extracted from cumulative plots of EPSC amplitude (Fig. 7D, E1, E2) [15]. In the absence of aGDI, the refilling rate was strongly depressed (Gdi1 WT: 21.164% of initial EPSC; Gdi1-null: 9.461.7% of the initial EPSC, n = 9 and 11 cells, respectively, p,0.05, Fig. 7E2), whereas the RRP size was preserved (Gdi1 WT: 15036136% of initial EPSC; Gdi1-null: 14986257% of the initial EPSC, p.0.05, Fig. 7E1). Finally, we noticed that no post-tetanic potentiation was present at Gdi1-null synapses (data not shown), possibly because of the higher depression observed during high-frequency trains. The lack of potentiation indicated that synaptic plasticity was deeply affected in the absence of aGDI. Conditional Gdi1 mutation indicates the potential pre-synaptic role of aGDI As reported above, a progressive decrease in aGDI expression is observed in the conditional Gdi1 mouse after 4 weeks of age (PND 28) in the hippocampus, cerebral cortex and amygdala, whereas it remains elevated in the medial division of the medial geniculate nucleus (MGm, Fig. 3E-E0 and I-I0). Thus, the examination of Conditional Gdi1 Deletion on Synaptic Function Figure 6. Synaptic transmission at cortico-LA synapses is affected by the absence of aGDI. (A) Scheme of the slice preparation. (B) Cortico-LA synaptic currents were elicited by stimula- tions in the external capsule. Traces displayed typical responses for increasing strength (in mA/msec). Scale bars: 100 pA and 15 msec. (C1) Recorded EPSC amplitude were averaged respective to the genotype and stimulation strength. (C2) For each stimulation intensity a ,60% decrease of EPSC size is observed in Gdi1-null mice. (D1) Miniature EPSCs at cortico-LA synapses were obtained by perfusion of Sr2+ containing ACSF. Desynchronized EPSCs were analyzed in the tail of evoked EPSCs. Scale bars: 50 pA and 50 msec. (D2) The average time course (left) and amplitude (right) of desynchronized EPSCs were similar between Gdi1-null and Gdi1 WT mice. Number of recorded cells is indicated. Scale bar: 3 pA and 10 msec. doi:10.1371/journal.pone.0029763.g006 synaptic physiology at thalamo-LA and cortico-LA synapses in 8– 10 week-old Gdi1flox/Y animals, i.e., a time at which aGDI deletion is effective, directly examines the consequences of pre- and post- synaptic absence of aGDI at excitatory synapses. In PND 100 animals, we first compared I/O curves at the two major excitatory entries to the LA by apposing stimulation electrodes in the external and internal capsules (Fig. 8A, D) [13]. In Gdi1 WT animals, the I/O curves reach an eEPSCmax of ,2 mA at both stimulated pathways (cortico-LA: 22386272 pA, thalamo-LA: 23456279 pA, Fig. 8C, F). Similar to previous results in P100 Gdi1-null animals, both I/O curves were strongly affected (eEPSCmax: cortico-LA: 10636164 pA, thalamo-LA: 9956224 pA, both p,0.001 as compared to Gdi1 WT). A normal I/O relationship was observed in P100 Gdi1flox/Y mice in the thalamo-LA pathway (Gdi1flox/Y: 20836298 pA, p.0.05 as compared to Gdi1 WT, Fig. 8 F), indicating that postsynaptic aGDI was not crucial in determining synaptic efficacy at these synapses and that the deficit observed in the constitutive mice was due to the pre-synaptic absence of aGDI. In contrast, the delayed absence of aGDI at cortico-LA synapses was asso- ciated with a strong alteration in the I/O curve (Gdi1flox/Y: 11556103 pA, p,0.001 compared to Gdi1 WT, Fig. 8C), which PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e29763