Immunofluorescence analysis of <i>Gdi1^lox</i>, <i>Gdi1^flox/Y</i> and <i>Gdi1</i>-null mouse brain regions.

<p>Immunofluorescence analysis of 15 µm coronal sections of <i>Gdi1^lox/Y</i>. <i>Gdi1^flox/Y</i> and <i>Gdi1</i>-null. (<b>A</b>) Low magnification coronal section from <i>Gdi1^lox/Y</i> brain indicating the regions of interest reported on the right: DG (dentate gyrus, <b>B-B″</b> and <b>F-F″</b>), CA1 (hippocampal CA1 region, <b>C-C″</b> and <b>G-G″</b>), LA (lateral amygdala, <b>D-D″</b> and <b>H-H″</b>) and MGm (medial geniculate nucleus, <b>E-E″</b> and <b>I-I″</b>). (<b>A</b>) Scale bar: 1 mm. (<b>B–I″</b>) Scale bar: 0.015 mm.</p

Short-term plasticity at Cortico-LA synapses is perturbed in absence of αGDI.

<p>(<b>A</b>) Iterative stimulations (20 Hz/4 sec) were applied at Cortico-LA synapses in <i>Gdi1</i> WT and <i>Gdi1</i>-null mice. Scale bars: 50 (up) and 100 (bottom) pA and 250 msec. (<b>B</b>) The pronounced synaptic depression in <i>Gdi1</i>-null mice is well illustrated by the extraction of the last EPSCs of the train response. Scale bars: 50 (up) and 100 (bottom) pA and 10 msec. (<b>C</b>) Mean EPSC amplitude at a given stimulation for each genotype. (<b>D</b>) Cumulative plot based on the same data, allowing to visualize the deficit in refilling rate at <i>Gdi1</i>-null synapses (see material and methods section for further details). (<b>E₁</b>) Ready releasable pool size (RRP) and refilling rate (<b>E₂</b>) were calculated in <i>Gdi1</i>-null and <i>Gdi1</i> WT preparations and expressed as % of initial EPSC size. Number of recorded cells is indicated. **p<0,01.</p

Synaptic transmission at cortico-LA synapses is affected by the absence of αGDI.

<p>(<b>A</b>) Scheme of the slice preparation. (<b>B</b>) Cortico-LA synaptic currents were elicited by stimulations in the external capsule. Traces displayed typical responses for increasing strength (in mA/msec). Scale bars: 100 pA and 15 msec. (<b>C₁</b>) Recorded EPSC amplitude were averaged respective to the genotype and stimulation strength. (<b>C₂</b>) For each stimulation intensity a ∼60% decrease of EPSC size is observed in <i>Gdi1</i>-null mice. (<b>D₁</b>) 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. (<b>D₂</b>) The average time course (left) and amplitude (right) of desynchronized EPSCs were similar between <i>Gdi1</i>-null and <i>Gdi1</i> WT mice. Number of recorded cells is indicated. Scale bar: 3 pA and 10 msec.</p

Excitatory transmission onto LA principal cells of <i>Gdi1</i>-null mice.

<p>(<b>A</b>) Spontaneous (Ctrl) and miniature (TTX) EPSCs were recorded in LA principal cells from <i>Gdi1</i> WT and <i>Gdi1</i>-null littermates. Scale bars: 20 pA and 60 msec. (<b>B</b>) Mean EPSC frequency is lower in LA cells from <i>Gdi1</i>-null mice in absence or presence of TTX. The number of recorded cells is indicated. ***p<0.001. (<b>C</b>) Decrease of spontaneous – but not miniature - EPSC amplitude in absence of αGDI. (<b>C₁</b>) Spontaneous EPSC (Ctrl) and miniature EPSC (TTX) in both genotypes. Scale bars: 5 pA and 10 msec. (<b>C₂</b>) Mean EPSC values for each conditions. The number of recorded cells is indicated.</p

Brain region- and age-specific down regulation of αGDI on <i>Gdi1^flox/Y</i> mice.

<p>Two to eight week old male mice were analysed. (<b>A</b>) 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. β-tubulin was used as loading control. (<b>B</b>) Quantitative analysis of residual αGDI normalized by β-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 ± SD from three independent animals at each age. Grey squares are <i>Gdi1^lox/Y</i> animals and black squares are <i>Gdi1^flox/Y</i>. *p<0.05, **p<0.01.</p

Gene targeting and generation of <i>Gdi1^flox/Y</i> mice.

<p>(<b>A</b>) Scheme of the structural organization of the <i>Gdi1</i> gene (top), of the targeting vector (middle) and of the recombinant locus (bottom). Black boxes are coding exons or the indicated insertion cassettes; white boxes are 5′ and 3′ UTR regions. Restriction enzymes are BamHI (B), XbaI (X), NotI (NI), EcoRI (EI), NdeI (N) and KpnI (K). The position of the PCR primers for the screening of the G-418 resistant embryonic stems cell clones (F4; Neo1C) and for mice screening (Lox1; Lox2) are indicated by arrows. S1, neo and 590 bp are the probes for Southern blot analysis. (<b>B–C</b>) Southern blot analysis of the embryonic stem cell clone found positive by PCR (lane 3), compared with negative clones (lane 1–2). (<b>B</b>) Genomic DNAs were digested with BamHI, fractionated on agarose gel and hybridized with the probe 5′ S1 probe. The 5.6 Kb BamHI fragment corresponds to the wild type locus, the 7.8 Kb fragment to the recombinant locus, with neo insertion. (<b>C</b>) Genomic DNAs were digested with XbaI, fractionated on agarose gel and hybridized with the 590 bp probe, corresponding to the fragment cloned between lox <i>P</i> sites. The 6.5 kb XbaI fragment corresponds to the wild type locus and the 0.780 Kb fragment to the recombinant locus with the insertion of lox <i>P</i> sites. (<b>D</b>) PCR analysis of DNA extracted from the tails of mice, using the primers Lox1/Lox2 (330 bp correspond to the lox allele and 240 bp to the WT) and Cre1/Cre2 (750 bp correspond to the presence of the <i>CaMKII-Cre</i>-159 transgene).</p

Additional file 1: Figure S1. of IL4 induces IL6-producing M2 macrophages associated to inhibition of neuroinflammation in vitro and in vivo

Gene expression of typical M1 and M2 markers in peritoneal macrophages stimulated in vitro. (A–D) real-time RT-PCR for iNOS, Ym1, CCI17, and iL6, in non stimulated (NS), and in macrophages stimulated with different doses of iFn-γ (m1) and iL4 (M2). Gapdh has been used as a housekeeping gene. Data are shown as fold induction (Fi ± standard deviation) over NS. (TIF 42402 kb

Additional file 6: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Movie S2 Video of Catwalk gait analysis of a representative GCV-NestinTK mouse. NestinTK mice were treated with GCV, subjected to SCI and followed for locomotor recovery by BMS score when behavioral amelioration reached a plateau value, mice were subjected to Catwalk gait analysis. The video shows recording of a representative GCV-NestinTK animal (BMS score = 1). Free ambulation along an illuminated glass plate in a darkened room has been recorded for 3.22 s. Video has been captured at day 18 after SCI. (AVI 104621 kb

Additional file 2: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S2. Sorted GFP+ cells from Nestin floxGFPflox-TK mice give rise to neurospheres. Panel A and B show the gating strategy for sorting GFP+ cells from SCs bulk cultures obtained from Nestin floxGFPflox-TK mice. WT litters (A) were used to set up the gating strategy that we used to sort GFP+ cells (B). GFP+ cells were plated at the density of 8000 cells/cm2 and daily examined for the presence of neurospheres. Small spheres were observed after 3 days (C), while spheres with diameters larger than 100 μm were easily observed after 7 days (D). Scale bar 50 μm (n = 3 independent preparations). (TIFF 9717 kb

Additional file 8: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S6. Upregulation of inflammatory cues in GCV-NestinTK mice. Real-time PCR analysis of pro-inflammatory genes (A–D) in T11–T13 spinal cord tissues at different time points after the injury induction. GCV-NestinTK mice (red bars) have a increased expression of pro-inflammatory genes after injury compared with control mice (blue bars). Values indicate mean fold changes ± S.E.M (n = 3–6 for each group). Comparisons were done using the t Student test: TNFα:* p = 0.021; IL-1β:* p = 0.046; Vegfa: p = 0.008. (TIFF 7997 kb