S1/2^-/- mice display novelty induced hyperactivity, decreased anxiety and exploratory behavior and working memory disturbances.

<p>A–C) Open field test performed in a novel, unfamiliar test arena. WT: n = 24, S1/2^-/-: n = 26. A) Novelty-induced hyperactivity in S1/2^-/- mice as assessed by moving distance in the open field (p_MW = 0.0006). B) Analysis in 1 min bins yielded a significant E_genotype (F_(1,48) = 16.46; p = 0.0002). Moreover, Bonferroni posttest revealed the strongest difference between the genotypes in interval 3, 5 and 10 (p_Bonf<0.01, p_Bonf<0.05 and p_Bonf<0.05, respectively). C) Mutants spent more time in the center (p_MW = 0.0004) of the test arena indicating reduced anxiety when compared to controls. D-E) Hole board test performed with a subsequent modification of the open field setup by floor insert with holes. WT: n = 24, S1/2^-/-: n = 26. D) S1/2^-/- mice displayed no alterations in the overall activity measured as total distance travelled. E) S1/2^-/- mice performed less nose pokes into holes (p_MW = 0.0014) indicating decreased curiosity-related behavior compared to WT. F-G) Y-maze test. WT: n = 23, S1/2^-/-: n = 20. F) S1/2^-/- mice showed increased activity in Y-maze test (E_genotype F_(1,41) = 10.98; p = 0.0019) most evident in interval 0-5 min (p_Bonf<0.01). G) Mutant mice performed less alterations in Y-maze than control animals (E_genotype F_(1,41) = 4.86; p = 0.0331) and p_Bonf<0.05 for interval 5–10 min. H-J) S1/2^-/- mice display impairment of working memory in the radial arm water maze (RAWM). WT: n = 29, S1/2^-/-: n = 28. H) In the visible platform task, performance was similar in both genotypes (E_genotype F_(1,55) = 0.65; p = 0.4236). I-J) S1/2^-/- mice showed increased number of working errors searching for a hidden platform on the first (I) (E_genotype F_(1,55) = 3.93; p = 0.0524; I_{genotype×time} F_(3,165) = 2.68; p = 0.0486) and the second (J) day of experiment (E_genotype F_(1,55) = 9.05; p = 0.0044) and I_{genotype×time} F_(5,275) = 2.34; p = 0.0422). Bonferroni posttest revealed significant difference during the 3^rd trial of the second day (p_Bonf<0.001). WT, black bars/circles. S1/2^-/-, white bars/circles. Data were analyzed with 2-way ANOVA with Bonferroni posttest (p_Bonf) and Mann-Whitney test (p_MW) for pairwise comparisons. ***: p<0.001; **: p<0.01; *: p<0.05. E, effect; I, interaction of factors.</p

S1/2^-/- mice show alterations of prepulse inhibition (PPI) which are resistant to Clozapine treatment.

<p>A) S1/2^-/- mice display impairment of PPI (E_genotype F_(1,43) = 7.99; p = 0.0071). Bonferroni posttest revealed significant difference in prepulse 75 und 80 dB (p_Bonf<0.01 and p_Bonf<0.05, respectively). WT: n = 24, S1/2^-/-: n = 21. B) Startle response was similar in S1/2^-/- mice and WT controls (E_genotype F_(1,88) = 0.00; p = 0.9958) and not influenced significantly by vehicle injections (E_treatment F_(1,88) = 2.18; p = 0.1434). Acute clozapine treatment (3 mg/kg) reduced startle in both genotypes to similar extend (E_treatment F_(1,82) = 11.83; p = 0.0009 and E_genotype F_(1,82) = 0.01; p = 0.9030). ‘No injections’ and ‘vehicle’ groups: WT: n = 25, S1/2^-/-: n = 21; clozapine: WT: n = 20, S1/2^-/-: n = 20. C) Acute treatment with clozapine (cloz; 3 mg/kg; n = 20) reduced PPI in WT mice when compared to vehicle (veh; n = 24) treated WT animals (E_treatment F_(1,42) = 10.33; p = 0.0025). Bonferroni posttest confirmed significant difference when prepulse 70 dB was applied (p_Bonf<0.01). D) Acute treatment with clozapine (cloz; 3 mg/kg) did not influence PPI in S1/2^-/- mice (n = 20) when compared to vehicle injected mutants (n = 21) (E_treatment F_(1,39) = 0.00; p = 0.9716). Data were analyzed with 2-way ANOVA and Bonferroni posttest (p_Bonf). ***: p<0.001; **: p<0.01; *: p<0.05. E, effect.</p

Unbiased network modeling links cortical signaling with clock components via SHARP1, BMAL1 and PER1.

<p>A) Depicted is the network model with the highest significance computed with all genes found to be differentially regulated in the cortex at ZT4 versus ZT16 (see. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110310#pone-0110310-g002" target="_blank">Figure 2A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110310#pone.0110310.s010" target="_blank">Tables S1</a>-S3 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110310#pone.0110310.s009" target="_blank">Figure S9</a> for description of symbols) including SHARP1 and -2. The network connects 14 seed nodes depicted as blue circles (higher expression levels in WT are indicated by associated small red circles) extended by 13 interactors. SHARP1 and -2 are labeled by red circles; all nodes added by the algorithm are not underlined by colored circles. The structure depicts two major clusters and places the circadian regulators SHARP1 and SHARP2 as well as PER1 at central positions. The left cluster (n = 19 objects) is mainly comprised of cellular signaling components (enkephalin A, substance P both encoded by <i>Penk</i> and the GPCRs A2A and DRD2) and downstream effectors including negative regulators (DUSP1,6 and HSPs) as well as transcriptional mediators (e.g. FOS, EGR1, JUNB). The right cluster (n = 12) comprises central components of the molecular clock (e.g. the core clock transcription factors CLOCK, NPAS2 and BMAL1 as well as clock feedback regulators and modifiers including SHARP1 and -2, PER1 and -2, CRY1, DBP and NR1D1/Rev-ERBalpha). The extended network gene list was queried against the GeneGo database for enriched correlations with diseases (B) and biological processes (C). Among the ten most significant disease associations were nine mental or mood related disease classifications (B). Among the ten highest ranked biological processes were only circadian rhythm- (rank 1 and 2) and metabolism-associated (rank 3–8) processes (C). MeSH ID, unique Medical Subject Heading disease identifier; GO ID, unique identifier of the gene ontology biological process collection; p-values determined by hypergeometric tests.</p

Attenuated sleep-wake amplitude and activity profiles in S1/2^-/- mice.

<p>A) Group means (± SEM) of the total time spend in different vigilance states over 24 h LD periods. The overall time of wake, NREM and REM sleep remained unaltered between genotypes. WT: n = 7, filled bars S1/2^-/-: n = 8, empty bars. B) Group means of light-dark or amplitude differences for wake, NREM and REM sleep. S1/2^-/- animals showed a significantly reduced light-dark amplitude for all vigilance states compared to WT animals (E_genotype F_{(1, 39)} = 19.87, p<0.0001; E_{vigilance state} F_{(2, 39)} = 19.39, p<0.0001; I_{genotype×vigilance state} F_{(2, 39)} = 1.9, p = 0.16; Post hoc two-tailed T-test: **: p<0.01 *: p<0.05). WT: n = 7, filled bars S1/2^-/-: n = 8, empty bars. C) 24 h sleep-wake distribution plotted for representative individual WT (#26) and S1/2^-/- (#828) mice with black areas given as relative amount of wakefulness obtained from 5 min bins. Note the relative difference in the amount of wakefulness during the light and dark episodes in the WT and the short periods of wakefulness in the light phase. In contrast, the S1/2^-/- mouse displayed broadened periods of sleep and wakefulness during the light and dark phases. D-F) Time course of vigilance states wakefulness (D), NREM (E) and REM sleep (F). Curves connect 2 h bin mean values (± SEM) expressed as percentage of recording time (E_time: NREM: F_(11,120) = 9.74, p<0.0001; REM F_(11,120) = 9.98, p<0.0001; E_genotype: NREM n.s.; REM F_(1,120) = 7.65, p<0.01 and I_{genotype×time}: Wakefulness F_(11,120) = 2.06, p = 0.02; NREM: F_(11,120) = 1.82, p = 0.05; REM: F_(11,120) = 1.42, p =  0.17; * =  p<0.05 in two-tailed post hoc T-test. WT: n = 7, filled circles S1/2^-/-: n = 8, empty circles. G) Diurnal wheel-running profiles depicted as accumulated activities of all recordings over a 5-day period plotted as 18 min bins. S1/2^-/- mice displayed a significantly altered activity profile in LD compared to wild-type (WT) mice (I_{genotype×time} F_(86,39040) = 1.92, p<0.0001) with reduced half maximal values of nocturnal activities at ZT 17.1 for S12^-/- mice compared to WT controls with ZT 18.3. Bonferroni posttest revealed significantly reduced activities between ZT13 and 18 (p_Bonf<0.05). n = 12 each genotype. H-I) Daytime dependent gene expression analysis of the circadian marker gene <i>Per2</i> (H) and the activity-induced gene <i>Fos</i> (I) in the cortex. Daytime dependent cortical expression of the circadian marker gene <i>Per2</i> was not substantially altered in WT and S1/2^-/- mice (H). In contrast, the mRNA expression of the activity regulated marker gene <i>Fos</i> was significantly reduced in S1/2^-/- mice at ZT16 compared to WT (I). n = 3 per timepoint and genotype. Data were analyzed with 2-way ANOVA with Bonferroni posttest (p_Bonf) and Mann-Whitney test (p_MW) for pairwise comparisons. E, effect; I, interaction of factors.</p