Circadian rhythm of female WT and IL-1R1^−/− mice.

<p>The activity of female WT (n = 15) and female IL-1R1^−/− (n = 15) mice was recorded using the Phenotyper home cage over a period of three nights and two days. Data are plotted in 4 hour blocks and are expressed as mean±SEM and were analysed by two way repeated measures ANOVA with strain as between subjects factor and time as within subjects factor. There was no effect of strain but a significant time × strain interaction, which is denoted by *p<0.0003.</p

Cytokine and chemokine transcription and synthesis.

<p>Cytokine and chemokine transcription and synthesis.</p

Ventricular volume and synaptic density in WT and IL-1R1^−/− mice.

<p>(a–c) Representative images of the hippocampus and lateral ventricle of WT (a) and IL-1R1^−/− (b) brain. Ventricular volume in WT (n = 4) and IL-1R1^−/− mice (n = 6) was assessed using Image J Software (c). Areas were calculated for sections from each animal at 2.0 and 2.3 mm posterior to Bregma and summed and multiplied by the summed section thickness (2×10 µm: 0.02 mm). Data are non-parametric and have been presented as dot plots with median value denoted by the bar. (d–i) Synaptophysin labelling in the dorsal (see inset *) and more ventral hippocampus (approximately −3.0 mm from surface of brain; see inset *) of WT (d, g) and IL-1R1^−/− mice (e, h). Synaptic density analysis of the dorsal hippocampus: ratio of stratum radiatum to dentate gyrus molecular (f) and ventral hippocampus ratio of stratum radiatum to stratum lacunosum moleculare (i) for WT (n = 4) and IL-1R1^−/− (n = 5).</p

Performance of IL-1R1^−/− mice versus WT in the contextual fear conditioning paradigm.

<p>The time spent freezing measured across 2 minutes (pre-shock) and 5 minutes, 48 hours later (post shock), following foot shock at 0.4 mA for 2 seconds for IL-1R1^−/− mice (n = 10) and WT mice (n = 10) in the fear conditioning paradigm. Data are expressed as mean±SEM and were analysed by t-test, bars representing pre-shock freezing are insufficiently large to be clearly visible. Bonferroni post-hoc tests after a significant effect of treatment in ANOVA analysis showed that both strains showed significant differences from their pre-shock freezing (***p<0.001).</p

Open field and elevated plus maze activity in IL-1R1^−/− and WT mice.

<p>(a) Distance travelled in open field in IL-1R1^−/− mice (n = 11) compared to WT (n = 19) and (b) the number of rears recorded in open field, across 3 minutes. (c) The time spent in open arms of elevated plus maze and (d) the latency to first emerge from closed arm. Data are expressed as mean ± SEM and significant differences by t -test are denoted by ***p<0.0001 and *p<0.05.</p

Spatial and non-spatial memory in the Morris water maze in IL-1R1^−/− and WT mice.

<p>(a) The latency to reach the hidden platform, and (b) the path length were assessed in IL-1R1^−/− mice (n = 11) and WT mice (n = 20) in the spatial memory water maze task across 9 trials with 1 hour inter trial interval. (c) Probe trials performed after 9 trials and 27 trials (dotted line shows 25%), and (d) non-spatial memory (flag trial) in IL-1R1^−/− mice and WT. Data are expressed as mean±SEM and were analysed by two-way repeated-measures ANOVA with strain as between subjects factor and time as within subjects factor (a, b) or by t-test (c, d). * denotes main effect of strain (p = 0.02).</p

Impact of systemic LPS, IL-1β, TNF-α and IL-6 challenge on plasma inflammatory and stress mediators.

<p>IL-1β (a), TNF-α (b), IL-6 (c), IL-1ra (d), corticosterone (e) and PGEM (f) as measured by ELISA in plasma prepared from whole blood of C57BL/6 female mice 2 hours after systemic challenge (i.p.) with saline, LPS (100 µg/kg), IL-1β (15 µg/kg or 50 µg/kg), TNF-α (50 µg/kg or 250 µg/kg) or IL-6 (50 µg/kg or 125 µg/kg). All data groups were compared by one-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between saline, LPS and each cytokine treatment at the higher dose. Endogenous versus injected levels could not be discriminated for IL-1β, TNF-α or IL-6 (denoted peripherally administered). # denotes treatment is significantly different to saline control # p<0.05. ** denotes significant difference between treatment groups indicated by line p < 0.01, ***p < 0.001. All data have been presented as mean ± SEM. n=5 for all groups except LPS and IL-1β 50 µg/kg n=4 and IL-1β 15 µg/kg n=3.</p

c-Fos expression in the central nucleus of the amygdala (CeA; A-D) and in the pial membrane (E–H) in brains of mice 2h after i.p. treatment with saline, LPS (100µg/kg), IL-1β (15µg/kg) or TNF-α (50µg/kg).

<p>cFOS-positive neurons in the CeA are quantified and shown in the integrated table. ** denotes statistically significant difference from saline by Bonferroni post-hoc test after significant one way ANOVA.</p

Impact of systemic IL-1β or TNF-α challenge on hippocampal and hypothalamic pro-inflammatory cytokine mRNA transcription at 1, 2, 4, 8 and 24 hours.

<p>Hippocampal and hypothalamic transcription of mRNA species for IL-1β, TNF-α and IL-6 was assessed using quantitative PCR after systemic challenge (i.p.) with saline, IL-1β (15 µg/kg) or TNF-α (50 µg/kg) at 1, 2, 4, 8 and 24 hours post-injection. All data groups were compared by two-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between each IL-1β group and the equivalent saline groups and comparing the cytokines directly. # denotes IL-1β/TNF-α treatment is significantly different to equivalent saline treatment at the time point indicated; * indicates significant difference between IL-1β and TNF-α. ^#/*p<0.05 # #/**p<0.01, ^{# # #/***}p<0.001. All data have been presented as mean±SEM, n=5 for all IL-1β and TNF-α treated groups except 2 h (n=4) and saline groups n≥3.</p

Performance of IL-1R1^−/− mice versus WT on a visuo-spatial reference memory task and a working memory task.

<p>(a) Visuo-spatial reference memory was assessed using the Y-maze across 20 trials (5 trials per trial block) in IL-1R1^−/− (n = 11) compared to WT mice (n = 20). (b) Working memory was assessed in 1L-1R1^−/− (n = 11) and WT (n = 15) by T-maze alternation, over 12 days, 10 trials per day with block 1 representing performance of mice on days 1–4, block 2 the performance of mice on days 5–8 and block 3 the performance of mice on days 9–12. Data are shown as mean±SEM and were analysed by two-way repeated-measures ANOVA with strain as between subjects factor and trial block as within subjects factor. Main effects of trial block for both mazes are depicted by ***p<0.001.</p