Motor ability of Q175 mutants declines with age.

<p>A. The accelerating rotarod test revealed that the age-related motor deficits in the Q175 model take up to 12 months to appear. B. 9 month old Q175 Hom mutants take longer to traverse the challenging beam test than age-matched WT and Q175 Het mice. C. The number of errors made while crossing each segment of the beam are shown, with the widest part of the beam on the left (33 mm) and narrowing towards the right (6 mm). The beam lengths are not to scale. The number of errors made while crossing the challenging beam are higher in Q175 Hom mutants. * indicates <i>P</i><0.05 in <i>post hoc</i> pairwise comparisons with WT after an effect of genotype was detected by one way ANOVA, and ^∧ indicates differences (<i>P</i><0.05) between Q175 Het and Hom mice.</p

Circadian deficits in locomotor activity in Q175 mutant mice at 12 months of age.

<p><b>A.</b> Representative double plotted actograms of wheel running activity from age-matched WT (left), Q175 Het (middle) and Q175 Hom (right) mice under 12∶12 light:dark (LD) and constant darkness (DD) are shown. The white/black bars on top indicate the LD cycle, and gray shading indicates darkness. Successive days of activity are plotted from top to bottom. <b>B.</b> Chi-square (χ²) periodograms of 10 days of activity in DD are shown from the three genotypes at 12 months, with the peak of the periodogram indicating the free-running period of each mouse. Power (%V) refers to the normalized amplitude of the periodogram. The intersecting diagonal line indicates significance to <i>P</i><0.05.</p

Circadian parameters of activity in LD and DD in WT, Q175 Het and Hom mice from 3 months to 12 months of age.

<p>Parameters were analyzed using one-way ANOVA, and the <i>F</i> and <i>P</i> statistics are reported for genotype comparisons within each age group. <i>H</i> values are reported from ANOVA on ranks comparisons in the event of failed normality or equal variance tests. * indicates that <i>post hoc</i> Bonferroni’s <i>t</i>-tests detected significant differences compared to WT, and ^∧ indicates significant differences were detected between Het and Hom groups.</p

Increased phase delay in response to a brief pulse of light at CT 16 in Q175 mutants.

<p>Data were analyzed using one way ANOVA within an age group. * indicates that <i>post hoc</i> Bonferroni’s <i>t</i>-tests detected significant differences compared to WT, and ^∧ indicates significant differences were detected between Het and Hom groups.</p

Daytime sleep is selectively affected in Q175 mutants.

<p><b>A.</b> Average waveforms of hourly sleep in WT, Q175 Het and Hom mice at 9 months (top) and 12 months of age (bottom). <b>B.</b> Amount of time spent in sleep in day is selectively decreased compared to night-time sleep in Q175 Hom mice at 9 (top) and 12 months of age (bottom). <b>C.</b> 9 (top) and 12 month (bottom) old Q175 Hom mice have more fragmented daytime sleep, with increased number of sleep episodes. <b>D.</b> Sleep episodes in Q175 Hom mice are also shorter in duration. * indicates significant difference (<i>P</i><0.05) between WT and Q175 Hom mice and ^∧ indicates differences (<i>P</i><0.05) between Q175 Het and Hom mice.</p

Neuron number and PER2 expression are unaffected in the SCN of Q175 mutants.

<p><b>A.</b> Representative images of Nissl stains at 10X magnification of coronal sections of the SCN from WT (left), Q175 Het (middle) and Hom (right) mice. 3V: 3^rd ventricle. oc: optic chiasm. <b>B.</b> Quantification of Nissl+cells in the SCN from each genotype was determined by two independent observers blind to the experimental conditions. <b>C.</b> SCN expression of the circadian protein, PER2, is unaltered in Q175 mutants. Mice at 12m of age were sacrificed at ZT 2 and ZT 14, the respective trough and peak of SCN PER2 expression. Representative images of ZT 14 SCN, 40X magnification, are shown from each genotype. <b>D.</b> The day/night difference in PER2 expression is unaltered in Q175 mutants. Quantification of PER2+cells in the SCN was determined by 2 independent observers blind to the experimental conditions.</p

Age-related decline in circadian rhythms of locomotor activity in Q175 mutants under DD conditions.

<p><b>A.</b> Free-running period (tau) is no different between the three genotypes. <b>B.</b> Power, as measured by the X² periodogram, declines dramatically in Q175 Hom mutants at 9 months. C. The cycle-to-cycle activity onset is less precise in Q175 Hom mutants from 9 months. D. The amount of activity (wheel revolutions per hour, rev/hr) plummets in Q175 Hom mutants at 9 months. * indicates significance to <i>P</i><0.05 in <i>post hoc</i> pairwise comparisons with WT within each age group after the two way ANOVA revealed an effect of genotype. Effect of age within each genotype is indicated with #. <i>F</i> and <i>P</i> statistics are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069993#pone-0069993-t002" target="_blank"><b>Table 2</b></a>.</p

Two way repeated measures ANOVA were used to determine the effects of age and genotype in the same cohort of mice from 3 to 12 months, and we report the <i>F</i> and <i>P</i> statistics for the comparisons reported in <b>Fig. 4</b> and <b>5</b> in this table.

<p>Sample sizes are as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069993#pone-0069993-t001" target="_blank"><b>Table 1</b></a>.</p

Circadian deficits in locomotor activity in Q175 mutant mice at 9 months of age.

<p><b>A.</b> Representative double plotted actograms of wheel running activity from age-matched WT (left), Q175 Het (middle) and Q175 Hom (right) mice under 12∶12 light:dark (LD) and constant darkness (DD) are shown. The white/black bars on top indicate the LD cycle, and gray shading indicates darkness. Successive days of activity are plotted from top to bottom. <b>B.</b> Chi-square (χ²) periodograms of 10 days of activity in DD are shown from the three genotypes at 9 months, with the peak of the periodogram indicating the free-running period of each mouse. Power (%V) refers to the normalized amplitude of the periodogram. The intersecting diagonal line indicates significance to <i>P</i><0.05.</p

Age-related decline in rhythms of locomotor activity in Q175 mutants in LD conditions.

<p><b>A.</b> The percentage of activity at night (nocturnality) declines at 9 months in Q175 Hom mutants. <b>B.</b> Power, as measured by the χ² periodogram, declines dramatically in Q175 Hom mutants at 9 months. <b>C.</b> The cycle-to-cycle activity onset is less precise in Q175 Hom mutants from 9 months. <b>D.</b> The amount of activity (wheel revolutions per hour, rev/hr) declines sharply in Q175 Hom mutants at 9 months. * indicates significance to <i>P</i><0.05 in <i>post hoc</i> pairwise comparisons with WT within each age group after the two way ANOVA revealed an effect of genotype. Effect of age within each genotype is indicated with #. <i>F</i> and <i>P</i> statistics are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069993#pone-0069993-t002" target="_blank"><b>Table 2</b></a>.</p