Fig 1.
The deterioration of daily and circadian wheel running activity rhythms are delayed in BACHD females.
(A-D) Representative double-plotted actograms of BACHD male and female wheel running activity during 10 days in 12:12 LD (300 lux) and DD at 3 and 6 months of age. (E-J) Box plots representing first and third quartile (box), medians (middle line), and data range (whiskers) for male (white boxes) and female (grey boxes) BACHD mouse behavioral rhythm parameters recorded at 3 and 6 months of age in LD (E-H) or DD (I+J). Individual data points (black dots), and WT control median values are superimposed for reference (red lines—statistical significance; blue lines—no statistically significant difference). Three-way ANOVA was used to detect significant effects of genotype, sex, and age on behavioral rhythm parameters (Table 1). When main or interaction effects were identified, significant sex differences within age (*), and significant age differences within sex (#), were identified post-hoc, using the Holm-Sidak method for multiple pairwise comparisons, with P < 0.05. Rev/hr refers to wheel revolutions per hour. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01; ###P < 0.001.
Table 1.
Sex differences in BACHD activity rhythm parameters.
Effects of sex and age on BACHD mouse locomotor activity rhythm parameters (DF = 63).
Table 2.
Statistical analysis of locomotor behavior.
Three-way ANOVA results testing effects of sex and age on BACHD mouse locomotor activity rhythm parameters (DF = 63).
Fig 2.
Daytime neural activity is comparably depressed in female and male BACHD SCN neurons.
(A) Representative traces of SCN neuron spontaneous electrical activity recorded during the day. (B-E) Box plots representing the first and third quartile (box), group medians (middle line) and data range (whiskers), of male (white boxes), and female (grey boxes) SCN neuron electrophysiological properties, with data points superimposed for individual WT (white dots) and BACHD (black dots) neurons. Two-Way ANOVA was used to identify possible effects of genotype and sex while post-hoc multiple pairwise comparison testing was carried out with Holm-Sidak method (P < 0.05; Table 2). (B) BACHD SCN neuron SFR was reduced during the daytime relative to WT. (C) Inter-spike membrane potential was not altered by sex or genotype. (D) No sex or genotype differences in membrane potential recorded in the presence of TTX (1 μM) and gabazine (10 μM) to silence synaptic and electrical activity. (E) Baseline subtracted voltage responses are plotted for each group as means ± 95% CI’s. Two-Way ANOVA detected significant main effects of current injection, and an interaction of sex and genotype on voltage responses, but post-hoc pairwise comparisons using Two-Tailed T-Tests (P < 0.05) failed to detect significant voltage response differences for groups at any particular current injection magnitude. ^P < 0.05.
Table 3.
Genotype and sex effects on SCN neuron electrophysiological properties.
Two-way ANOVA results testing effects of genotype and sex on daytime action potential and resting membrane properties in WT and BACHD SCN neurons (top). When main or interaction effects were identified, significant genotypic differences within sex, and significant sex differences within genotype, were identified post-hoc using the Holm-Sidak method for multiple pairwise comparisons.
Table 4.
Statistical analysis of electrophysiological data.
Two-way ANOVA results testing effects of genotype and sex on daytime action potential and resting membrane properties in WT and BACHD SCN neurons.
Fig 3.
Sex and genotypic difference in the size of the SCN of WT and BACHD mice.
Representative images of the SCN, stained with VIP (A & B) or AVP (C &D) and counterstained with Nissl. Boxed regions in 10x image (left, scale bar = 200μm) are magnified at 40x (right, scale bar = 40μm). (E) Measurements of the Nissl-defined SCN revealed a significantly smaller SCN in 3 month-old WT females compared to age-matched WT male mice. WT females displayed a smaller and elongated SCN, while WT male SCN was larger and round. A significant reduction in the area of the SCN was found in BACHD males as compared to age-matched WT males, while females BACHD did not display pathology-associated variations. Individual data points represent the average of the left and right area of the SCN of each animal (n = 6–7) measured by 2 observers masked to the sex and genotype. No sex or genotypic differences were found in the number of VIP (F) and AVP (G) positive neurons. Individual data points represent the number of positive cell counted per animal for each group (n = 4–6). Individual data points are superimposed onto box plots representing first and third quartile (box), group medians (middle line) and data range (whiskers) for each group. Main effects of sex and genotype were identified by Two-way ANOVA *P<0.05 (see also Table 6). Significant genotypic differences within sex were identified post-hoc by Two-Tailed T-Tests, with ^P < 0.05.
Table 5.
Genotype and sex differences in BACHD SCN anatomy.
Effects of sex and genotype on different histological parameters of the SCN and the number of VIP and AVP neurons. When main or interaction effects were identified, significant sex differences within genotype, and genotype differences within sex, were identified post-hoc using the Two-Tailed T-Tests, with P < 0.05.
Table 6.
Statistical analysis of anatomical data.
Two-way ANOVA results testing effects of sex and genotype on different histological parameters of the SCN and the number of VIP and AVP positive cells.
Fig 4.
BACHD mouse sex differences in motor coordination and body weight.
(A-C) Box plots represent first and third quartile (box), group medians (middle line) and data range (whiskers), of male (white boxes) and female (grey boxes) mice, with individual data points superimposed. (A) BACHD mouse step errors on progressively narrowing challenging beams (Beams 1–4) for males at 3 months (M3) and 6 months (M6), as well as females at 3 months (F3) and 6 months (F6). Two-Way ANOVA identified main effects of sex and age on step errors. Significant sex differences within age (*) and/or significant age differences within sex (#) for each beam were identified post-hoc, using Holm-Sidak method for multiple pairwise comparisons, with P < 0.05. (B) Time to traverse all 4 challenging beams. (C) Rotorod latency to fall for WT (white dots) and BACHD (black dots) mice. Three-Way ANOVA identified main effects of genotype, and interactions of sex, age, and genotype on latency to fall. Significant genotypic differences within age and sex (^), sex differences within genotype and age (*), and significant age differences within sex and genotype (#), on latency to fall were identified post-hoc, using Two-Tailed T-Tests, with P < 0.05. (D) Body weight of WT and BACHD mice. Three-Way ANOVA identified main effects of genotype, sex, and age as well as the interaction of genotype and sex as well as sex and age (Table 4) on body weight. Post-hoc pairwise comparisons using Holm-Sidak method identified that at all age points, WT females weighed less than WT males and BACHD females, as well as ages significant differences in weight were detected between BACHD male and female mice (*P < 0.05), or BACHD male and WT male mice (^P < 0.05). Points represent mean, and error bars 95% CI.
Table 7.
Sex differences in BACHD motor coordination deterioration.
Two-way ANOVA results testing effects of sex and age on challenge beam step errors. When main or interaction effects were identified, significant sex differences within age, and age differences within sex, were identified post-hoc using the Holm-Sidak method for multiple pairwise comparisons.
Table 8.
Sex differences in BACHD motor coordination deterioration.
Table shows the latencies to fall off rotarod (sec).
Table 9.
Statistical analysis of Sex differences in BACHD motor coordination deterioration and body weight.
Three-way ANOVA results testing effects of genotype, sex, and age on rotorod latency to fall and body weight. When main or interaction effects were identified, significant genotypic differences within sex, sex differences within genotype, as well as age differences within sex and genotype were identified post-hoc using the Two-Tailed T-Tests, with P < 0.05.