Histological studies.

<p>(A) NeuN immunostaining was used to evaluate cell loss in coronal slices from control (A1), resistant (A2) and epileptic (A3,A4) animals. No strain differences were apparent for control and resistant rats, while different degree of cell loss was evident in Sprague-Dawley (SD) epileptic rats as compared with Wistar (W) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048128#pone.0048128-Inostroza1" target="_blank">[13]</a>. (B) Timm staining was used to evaluate mossy fiber sprouting (MFS). Again, no apparent changes were found in control (B1) and resistant animals (B2), while MSF was obvious in the ventral hippocampus of epileptic animals (B3,B4). Scale bars are 500 µm.</p

Relationships between LTP magnitude and memory function.

<p>(A) Animals were tested in five sessions (3 min each) separated by 5 min interval and grouped in three different phases: familiarization, spatial change and novel object recognition. During the familiarization phase (two trials) five objects were simultaneously placed in the open field. In the spatial change phase (two trials) two objects were displaced (arrows). In the novel object recognition phase (1 trial), a new object was substituted for the upper-left object (arrow). (B) Discrimination ratios for the spatial change tasks for Wistar (W) and Sprague-Dawley (SD) rats. * P<0.05 for pair-wise comparisons between groups; ^## P<0.01; ^### P<0.001 for comparisons with chance level. Note poor performance of epileptic rats in Sprague-Dawley (n = 12) but not in Wistar animals (n = 13). The control grop is composed of n = 11 Wistar and n = 12 Spague-Dawley rats. Resistant rats are n = 4 Wistar and n = 5 Spraque-Dawley. (C) Discrimination ratios for the novel object recognition. Note that deficits specifically affect hippocampal-dependent spatial memory and not recognition memory. (D) Positive correlation between LTP magnitude and discrimination ratios in the spatial memory task (r² = 0.14, P = 0.017). (E) No correlation was found between LTP magnitude and discrimination ratios in the novel object recognition task.</p

Theta-burst-induced LTP was enhanced in resistant rats but reduced in epileptic animals.

<p>Upper row shows representative averaged fEPSPs recorded at the time indicated by the letters on the graph to exemplify LTP induction in different experimental groups. Graphs plot summary data of experiments where theta burst tetanization was applied (indicated by the arrow) in control (n = 16 from 11 rats), resistant (n = 7 from 7 rats) and epileptic (n = 16 from 10 rats) slices.</p

Basal synaptic transmission is altered after kainate treatment.

<p>(A) Superposition of fEPSPs (5 consecutive responses) evoked by various stimulus strengths in single representative experiments obtained from control, resistant and epileptic rat. Stimulus-response curves for fEPSP (B) and the fiber volley FV (C) obtained in slices from the control (16 slices from 11 rats), resistant (15 slices from 10 rats) and epileptic (16 slices from 13 rats) groups. (D) Input-output curves were created by comparing FV amplitudes with fEPSP slopes from data depicted in B and C. (E) FV/fEPSP ratios for different ranges of stimulus strength shown in D confirm that resistant rats present a reduction of synaptic efficacy when compared with control animals (*P<0.05).</p

Effect of systemic injections of kainate in Wistar and Sprague-Dawley rats.

<p>(A) Multiple doses of 5 mg/kg kainate were used to induce <i>status epilepticus</i> (SE) in adult male rats. The histograms show percentage of rats exhibiting SE in Wistar (n = 96) and Sprague-Dawley animals (n = 57). No differences were found between strains. (B) Progression to SE in Wistar and Sprague-Dawley rats as evaluated with the Racine scale. Data from for n = 10 rats entering SE with 2 and 3 doses in each strain. A group of rats were resistant to develop SE (n = 10 Wistar and n = 7 Sprague-Dawley). (C) Chronic electroencephalographic (EEG) recordings were obtained from the dorsal hippocampus in a group of animals. Representative examples of EEG recording during walking and awake immobility are shown for control, resistant and epileptic animals. (D,E) Epileptic rats also exhibited epileptiform events like interictal spikes (D) and spontaneous seizures (E). The discontinous line box group data obtained from the same epileptic rat.</p

Presynaptic release probability, estimated from paired-pulse facilitation ratios, was modified neither in resistant nor epileptic rats.

<p>(A) Representative traces recorded at different inter-pulse intervals indicated by the numbers above the records. The first record in a row corresponds to fEPSPs evoked by the first pulses. (B) Summary data of paired-pulse facilitation ratios obtained in the same slices used to induce LTP in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048128#pone-0048128-g005" target="_blank">figure 5</a>. No significant differences were found among experimental groups (P>0.05). Data from n = 16 slices from 11 control rats; n = 7 slices from 7 resistant rats and n = 16 slices from 10 epileptic rats.</p

FGL triggers hippocampal FGFR1 phosphorylation in vitro and in vivo.

<p>(A) Cartoon structure of the double fibronectin module (FN1+FN2) of human NCAM (Protein Data Bank number 2VKW). The FGL sequence is shown in red with the two glutamine residues critical for the binding to the FGF-receptor highlighted in magenta. (B) Top: Representative immunoblot showing the in vitro phosphorylation of FGFR1 after stimulation of Trex293 cells that express Strep-tagged human FGFR1 with different concentrations of FGL and 10 ng/ml FGF1 (positive control) for 20 min. Bottom: Quantification of FGFR1 phosphorylation by FGL was performed by densitometric analysis of band intensity from four independent experiments similar to the one shown in the upper panel. (C) Phosphorylation of FGFR1 and TrkB was examined from hippocampal homogenates with an enzyme-linked immunosorbent assay (ELISA) 1 h after FGL subcutaneous injection. <i>N</i>, number of animals. Results are expressed as percentage ± SEM, with untreated controls set at 0%. (D–F) Phosphorylation of PLCγ (D), Shc (E), and FRS2 (F) in vitro was examined by Western blot, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001262#pbio-1001262-g001" target="_blank">Figure 1B</a>. Treatment with FGF1 served as the positive control. Results from four independent experiments are expressed as a percentage ± SEM, with untreated controls set at 100%. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 compared with controls. Statistics were carried out according to the <i>t</i> test.</p

FGL enhances spatial learning.

<p>(A) Mean distances swam to find the hidden platform in the Morris water maze are represented for control rats (white symbols) and FGL-treated rats (black symbols) over 2 training days (four trials each). <i>N</i>, number of animals. Statistical significance was analyzed with repeated-measures ANOVA. (B) Cumulative frequency distributions of the distances swam by individual rats. Each data point represents the distance swam by one rat in the last trial of each day.</p

FGL-induced enhanced cognition depends on PKC activity.

<p>(A, B) Mean distances traveled to find the hidden platform in the Morris water maze are represented for control rats (white circles), FGL-treated rats (black circles), and rats treated with FGL and the PKC inhibitor (grey squares; A, chelerythrine; B, GF109203X), over the 2 training days (four trials each). <i>N</i>, number of animals. Statistical significance was analyzed with repeated-measures ANOVA followed by Bonferroni's post hoc test for individual trials. A: *<i>p</i><0.05, FGL+Veh compared to FGL+Chel and Veh/Chel groups. #<i>p</i><0.05, FGL+Veh compared to FGL+Chel but not compared to Veh/Chel. B: *<i>p</i><0.05, FGL+Veh compared to FGL+GF109203X and Veh/GF109203X groups. #<i>p</i><0.05, FGL+Veh compared to Veh/GF109203X but not compared to FGL+GF109203X. (C) Probe test. Average time spent in the target quadrant of the Morris water maze (where the hidden platform had been present during training) for control rats (white column), FGL-treated rats (black column), or rats treated with FGL plus chelerythrine (grey column). Statistical significance was calculated with Bonferroni's post hoc test.</p

FGL induces AMPA receptor synaptic delivery via PKC activation.

<p>(A) Left: CA1 pyramidal neurons that express GluA1-GFP (green) on a DAPI-stained (blue) organotypic slice culture, imaged with laser-scanning confocal microscopy. Bar = 50 µm. Right: High-magnification image of GluA1-GFP-expressing neurons. Bar = 20 µm. (B) Schematic diagram that presents whole-cell recordings obtained from a neuron expressing GluA1-GFP (infected, green) and an adjacent non-fluorescent (uninfected, white) neuron. (C) AMPAR-mediated responses were recorded at −60 mV and +40 mV. The rectification index was calculated as the ratio of responses at these holding potentials. The <i>p</i> value was determined using the Mann-Whitney test. (D–H) FGL-induced rectification after incubation with inhibitors of different signal transduction pathways: MEK, PD98059 (D); PI3K, LY294002 (E); PKC, chelerythrine (F); classical PKC isoforms, GF109203X (G); atypical PKC isoforms (H). Sample traces are shown above the corresponding columns of the plot. <i>N</i>, number of cells. The <i>p</i> value was determined using the Mann-Whitney test. Scale bars = 15 pA and 10 ms.</p