Contextual and auditory cued fear conditioning tests in euthyroid and adult-onset hypothyroid rats.

<p>(<b>A</b>) Schematic representation of the experimental design. Shadowed areas indicate periods of animal manipulations. (<b>B</b>) Conditioning session for auditory and contextual cues. No differences were found in neither pre-shock nor post-shock periods. (<b>C</b>) Contextual fear test sessions 1 day, 1 week and 3 weeks after conditioning. H rats showed higher freezing levels than E rats, which reach statistical differences 1 day and 3 weeks after conditioning. (<b>D</b>) Auditory cued fear testing 2 days, 1 week and 3 weeks after the conditioning session. H rats showed higher freezing levels during the tone period than their E counterparts at every test session after conditioning. Plot bars show means ± SEM. *, p<0.05; **, p<0.01; NS, no significant. n = 8 per group.</p

Auditory cued fear conditioning training, memory, extinction and spontaneous recovery tests in euthyroid and adult-onset hypothyroid rats.

<p>(<b>A</b>) Scheme showing the experimental design for Experiment 2. Shadowed areas indicate periods of animal manipulations. (<b>B</b>) Conditioning session. No differences were found in neither pre-shock nor post-shock periods. (<b>C</b>) Auditory cued fear test session 2 days after conditioning. H animals showed a significant increase in conditioned behavior. (<b>D</b>) Extinction sessions for the auditory cue fear memory. The extinction of fear memory was delayed in H animals. (<b>E</b>) Re-exposition to the tone for the assessing of spontaneous recovery of fear memory. H rats showed a remarkable increase (four-fold) in fear response as compared to E rats. Bars show mean ± SEM. *, p<0.05; **, p<0.01; NS, no significant. n = 7 for E rats and n = 8 for H rats.</p

Plasma corticosterone levels and sensitivity to foot shocks in euthyroid and hypothyroid rats.

<p>(<b>A</b>) Scheme showing the experimental design for Experiment 3. Shadowed areas indicate periods of animal manipulations. (<b>B</b>) Dynamics of the plasma corticosterone response to training in the cued fear conditioning task (0, 15, 45 and 90 min after training). (<b>C</b>) Area under the curve (AUC) analysis showed significantly increased corticosterone levels in H rats as compared to E animals. (<b>D</b>) Sensitivity to foot shocks was not altered in H animals. No differences were found in either flinching or jumping behaviors. Bars show mean ± SEM. *, p<0.05; NS, no significant. n = 7 per group.</p

Expression of glucocorticoid, mineralocorticoid and thyroid hormone receptors in the lateral and basolateral nuclei of the amygdala in euthyroid and hypothyroid rats.

<p>(<b>A</b>) Amygdala volume estimation in E and H rats. No differences were found between groups. Representative photomicrographs (40×) of MR (<b>B, C</b>) and GR (<b>E, F</b>) immunohistochemistry in LA/BLA in E (Eu) and H (Hypo) animals. Scale bar, 20 µm. Histograms show the mean gray values of GR (<b>D</b>) and MR (<b>G</b>) obtained for E and H animals (n = 4 per group). GR and MR staining were increased in H rats. Panoramic views of isotopic <i>in situ</i> hybridization for TRα1 (<b>H, I</b>) and TRβ1 (<b>K, L</b>) in coronal sections of the brain of E (Eu) and H (Hypo) animals. Scale bar, 0.15 cm. Arrows point to the amygdala. Histograms show the mean gray values in arbitrary units (AU) obtained for E and H rats for TRα1 (<b>J</b>) and TRβ1 (<b>M</b>) mRNA expression (n = 3 per group). No differences were found between groups for TRα1. TRβ1 expression was reduced in H rats. Plot bars show means ± SEM. *, p<0.05; **, p<0.01; NS, no significant.</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

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 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-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 enhances long-term synaptic potentiation.

<p>(A–B) Rectification experiments similar to the ones described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001262#pbio-1001262-g005" target="_blank">Figure 5</a>, after incubation with DL-AP5 (NMDAR inhibitor), KN-93 (CaMKII inhibitor), or KN-92 (inactive analog of KN-93). Sample traces are shown above the graphs. (C) Sample traces of evoked AMPAR-mediated synaptic responses recorded from CA1 neurons at −60 mV before (thin line) and after (thick line) LTP induction. LTP was induced by pairing presynaptic 3 Hz stimulation (540 pulses) with postsynaptic depolarization (0 mV). One of the stimulating electrodes was turned off during LTP induction (“unpaired pathway”). Organotypic slice cultures were incubated with (i) normal culture medium (control), (ii) FGL, (iii) the PKC inhibitor chelerythrine (Chel), or (iv) FGL and chelerythrine (FGL+Chel), as indicated. Treatments were for 24 h and slices were transferred to fresh culture medium (without FGL or chelerythine) for an additional 24 h prior to recordings. (D) Time course of normalized AMPAR-mediated synaptic responses before and after LTP induction (black arrow), from the slices treated as in (C). For simplicity, each time point in the plot corresponds to the average of 12 consecutive stimulations (sampling rate: 0.2 Hz). (E–F) Quantification of average synaptic potentiation from paired (“LTP”) and unpaired pathways from the last 10 min of the time-course shown in (D). The <i>p</i> value was determined with the Mann-Whitney test. <i>N</i>, number of cells.</p

FGL-triggered persistent activation of signaling pathways.

<p>(A) Left: Western blot of hippocampal extracts treated with TPA (12-<i>O</i>-tetradecanoylphorbol-13-acetate; PKC activator that served as a positive control), untreated (“0”), and treated with FGL at different time-points after FGL application. The primary antibody detects phosphorylation of endogenous proteins at PKC substrate motifs (phospho-(Ser) PKC substrate). Right: Quantification of Western blots similar to the one shown on the left, by calculating the combined intensity from all bands in each lane. <i>N</i>, number of independent experiments. The <i>p</i> values were determined with the Mann-Whitney test. (B, C) Left: Western blot of hippocampal extracts treated with FGL at different time-points after FGL application and untreated (“0”). The primary antibodies detected phosphorylated CaMKII at Thr286 (p-CaMKII) and total levels of CaMKII (T-CaMKII) (B), or phospho-GluA1 (P-S831) and total GluA1 (C). Tubulin was used as a loading control. Right: Quantification of Western blots similar to the ones shown on the left. <i>N</i>, number of independent experiments. The <i>p</i> values were determined using the Mann-Whitney test.</p