Ithin 40?0 min after training, brains dissected and subjected to histological analyses. Fixed brains were embedded in paraffin and 4 mm thick coronary serial sections were 11967625 cut on a microtome and directly transferred onto SuperFrost Plus coverslips (Fisher Scientific, Schwerte, Germany). Serial sections containing basal amygdala (BA), lateral amygdala (LA) and the central amygdaloid nucleus (CE) as well as the hippocampus were taken for histological staining.Figure 1. Impaired FC in aging (Thy1)-h[A30P]aSYN mice. The indicated groups of mice were trained and after 24 h assessed for context learning (A) and after another 6 h for cued learning (B). Compared to non-shocked control mice (light bars), MedChemExpress AZP-531 age-matched old trained wild-type (wt) mice (gray bars) showed highly significant reductions of exploratory behavior in response to both context and cues. Young (Thy1)-h[A30P]aSYN transgenic (tg) mice (green bars) showed almost normal FC for context and showed significantly reduced exploratory behavior in cued FC when compared to old transgenic mice (yellow bars). The old transgenic mice showed much reduced context FC and no cued FC at all. ***p,0.0001; **p,0.01. doi:10.1371/journal.pone.0050245.gImpaired Synaptic Plasticity in Aging aSYNtg MiceFigure 2. c-Fos immunostaining in the amygdala of fear-conditioned (Thy1)-h[A30P]aSYN mice. Parallel mice as in Figure 1 were sacrificed within 40?0 min after FC training, brains dissected and amygdala sections stained with an antibody against c-Fos. Hardly any c-Fos immunoreactivity was observed in non-shocked mice under these conditions (A ), whereas shock training induced c-Fos signals even in old nontransgenic mice (J ). In contrast, young transgenic mice showed reduced c-Fos induction (M ), which in the case of old (Thy1)-h[A30P]aSYN mice was so much impaired throughout the amygdala (P ) that it failed to reach significance (S,T). ***p,0.0001; **p,0.001; *p,0.02. Size bars correspond to 20 mm. LA, lateral amygdala, BA, basal amygdala, CE, central nucleus. doi:10.1371/journal.pone.0050245.gFor immunostainings, primary antibodies were AZP-531 biological activity rabbit polyclonal antibody against c-Fos (sc-52, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100) and rabbit polyclonal antibody against Plk2 (BL1695, Bethyl Laboratories, Montgomery, TX, USA; 1:200), rat monoclonal antibody against human aSYN (15G7 [13]; 1:5) or rabbit monoclonal anti-pSer129 (cloneEP1536Y, Epitomics, Burlingame, CA, USA; 1:500). After overnight incubation at 60uC and treatment with xylene, rehydration was performed over a descending ethanol series: 100 , 95 , 75 , Tris-buffered saline, pH 7.4 (TBS). Peroxidase activity was eliminated by a 30 min treatment with 1 hydrogen peroxide followed by 3 washing steps with TBS. Antigen retrievalImpaired Synaptic Plasticity in Aging aSYNtg MiceFigure 3. Plk2 immunostaining in the amygdala of fear-conditioned (Thy1)-h[A30P]aSYN mice. Mice were processed as above and amygdala sections stained with an antibody against Plk2. Compared to non-shocked mice (A ), FC induced Plk2 signals even in old non-transgenic mice (J ). In contrast, young transgenic mice showed reduced Plk2 induction (M ), which in the case of old (Thy1)-h[A30P]aSYN mice was almost absent (P ). Size bars correspond to 20 mm. Quantifications show statistical significances (S,T). 16985061 ***p,0.001; **p,0.01; *p,0.02. LA, lateral amygdala, BA, basal amygdala, CE, central nucleus. doi:10.1371/journal.pone.0050245.gwas done by a 30 min exposure of the sectio.Ithin 40?0 min after training, brains dissected and subjected to histological analyses. Fixed brains were embedded in paraffin and 4 mm thick coronary serial sections were 11967625 cut on a microtome and directly transferred onto SuperFrost Plus coverslips (Fisher Scientific, Schwerte, Germany). Serial sections containing basal amygdala (BA), lateral amygdala (LA) and the central amygdaloid nucleus (CE) as well as the hippocampus were taken for histological staining.Figure 1. Impaired FC in aging (Thy1)-h[A30P]aSYN mice. The indicated groups of mice were trained and after 24 h assessed for context learning (A) and after another 6 h for cued learning (B). Compared to non-shocked control mice (light bars), age-matched old trained wild-type (wt) mice (gray bars) showed highly significant reductions of exploratory behavior in response to both context and cues. Young (Thy1)-h[A30P]aSYN transgenic (tg) mice (green bars) showed almost normal FC for context and showed significantly reduced exploratory behavior in cued FC when compared to old transgenic mice (yellow bars). The old transgenic mice showed much reduced context FC and no cued FC at all. ***p,0.0001; **p,0.01. doi:10.1371/journal.pone.0050245.gImpaired Synaptic Plasticity in Aging aSYNtg MiceFigure 2. c-Fos immunostaining in the amygdala of fear-conditioned (Thy1)-h[A30P]aSYN mice. Parallel mice as in Figure 1 were sacrificed within 40?0 min after FC training, brains dissected and amygdala sections stained with an antibody against c-Fos. Hardly any c-Fos immunoreactivity was observed in non-shocked mice under these conditions (A ), whereas shock training induced c-Fos signals even in old nontransgenic mice (J ). In contrast, young transgenic mice showed reduced c-Fos induction (M ), which in the case of old (Thy1)-h[A30P]aSYN mice was so much impaired throughout the amygdala (P ) that it failed to reach significance (S,T). ***p,0.0001; **p,0.001; *p,0.02. Size bars correspond to 20 mm. LA, lateral amygdala, BA, basal amygdala, CE, central nucleus. doi:10.1371/journal.pone.0050245.gFor immunostainings, primary antibodies were rabbit polyclonal antibody against c-Fos (sc-52, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100) and rabbit polyclonal antibody against Plk2 (BL1695, Bethyl Laboratories, Montgomery, TX, USA; 1:200), rat monoclonal antibody against human aSYN (15G7 [13]; 1:5) or rabbit monoclonal anti-pSer129 (cloneEP1536Y, Epitomics, Burlingame, CA, USA; 1:500). After overnight incubation at 60uC and treatment with xylene, rehydration was performed over a descending ethanol series: 100 , 95 , 75 , Tris-buffered saline, pH 7.4 (TBS). Peroxidase activity was eliminated by a 30 min treatment with 1 hydrogen peroxide followed by 3 washing steps with TBS. Antigen retrievalImpaired Synaptic Plasticity in Aging aSYNtg MiceFigure 3. Plk2 immunostaining in the amygdala of fear-conditioned (Thy1)-h[A30P]aSYN mice. Mice were processed as above and amygdala sections stained with an antibody against Plk2. Compared to non-shocked mice (A ), FC induced Plk2 signals even in old non-transgenic mice (J ). In contrast, young transgenic mice showed reduced Plk2 induction (M ), which in the case of old (Thy1)-h[A30P]aSYN mice was almost absent (P ). Size bars correspond to 20 mm. Quantifications show statistical significances (S,T). 16985061 ***p,0.001; **p,0.01; *p,0.02. LA, lateral amygdala, BA, basal amygdala, CE, central nucleus. doi:10.1371/journal.pone.0050245.gwas done by a 30 min exposure of the sectio.
