Article Figures & Data
Figures
- Figure 1.
Experimental design. A, The shifted group was composed of 8 wild-type and 8 duper female hamsters. B, The unshifted animals also contained 8 of each genotype and served as controls. The green arrow indicates the time point of BrdU injections at the end of the second advance. We injected animals at ZT22 and ZT12 on the first day and ZT4 on the second day for both the shifted group and the control group. Four more phase shifts (2 delays and 2 advances) were administered after the BrdU injections. Hamsters were perfused 16 d after the last advance shift, after which brains were sectioned and double-label immunocytochemistry was performed to detect BrdU and NeuN, PCNA, and DCX proteins.
- Figure 2.
Duper hamsters re-entrain more rapidly than wild types to shifts of the LD cycle. A, Double-plotted actograms of representative duper (left) and wild-type (right) hamsters subjected to successive 8 h phase delays (top) and advances (bottom) of the 14:10 h L/D cycle. Yellow shading indicates light phase. Mutants re-established the stable phase relationship of locomotor activity onset to the onset of the dark phase within 4 d, but WT hamsters required ∼14 d to re-entrain. Dupers had a positive phase angle when entrained, showed signs of scalloping (Morin et al., 1977), and reduced locomotor activity. B, Latency to re-entrain locomotor activity over the course of the experiment. Successive delays (D1, D2…D4) and advances (A1, A2…A4) were alternated (**p < 0.001, ***p < 0.0001, ****p < 0.00001, t-test). C, Duper hamsters consistently re-entrained much more quickly than wild types to all phase shifts (combined data from B). D, Mean latencies to re-entrain for each shift type, shown for each genotype. Duper hamsters show no differences in latency to reentrain to delays versus advances. WT hamsters re-entrained more rapidly to delays than advances. Results are shown as the mean ± SEM. As seen in Extended Data Figure 2-1, the mean duration of the active phase (α) was greater in both shifted and unshifted wild-type hamsters than in the corresponding groups of duper mutant hamsters over the course of the experiment (p < 0.05), but the mean number of wheel revolutions did not differ between genotypes in either shifted or unshifted hamsters.
- Figure 3.
Phase shifts and the duper mutation regulate cell division and neurogenesis in the subgranular zone of the dentate gyrus. A, Representative micrograph showing BrdU (green, top), and BrdU/NeuN (bottom) stained cells in the hamster dentate gyrus. B, Total BrdU+ cells were unaffected by circadian disruption in either genotype, but dupers had more BrdU+ cells than WTs (*p < 0.05). Results are shown as the mean ± SEM. C, Phase shifts reduced the number BrdU+/NeuN– cells in the dentate gyrus in WTs (*p < 0.05) but had no effect on duper mutants. D, Duper mutants showed more newborn neurons (BrdU+/NeuN+) than WTs (*p < 0.05), but the effect of shifts was not statistically significant in either genotype. E, Pie charts representing the effects of genotype and shifts represented as percentages (%BrdU+/NeuN– cells and %BrdU+/NeuN+ cells). Repeated phase shifts changed the proportion of newborn cells that were NeuN-IR in wild-type (p < 0.03) but had no such effect in dupers. Means were compared by two-way ANOVA, with genotype and phase shifts as main factors. As seen in Extended Data Figure 3-1, phase shifts and genotype had no significant effect on BrdU or NeuN staining in the SVZ at the 64 d survival point.
- Figure 4.
Cell proliferation in the SGZ of shifted and unshifted hamsters. A, Representative images showing PCNA (red, top left) with DAPI counterstain (top right), and DCX (green, bottom left) with DAPI counterstain (bottom, right). B, Neither genotype nor repeated shifts altered the number of PCNA+ cells in the subgranular zone, although dupers tended to have more PCNA+ cells than WTs (p = 0.09). C, Phase shifts significantly increased DCX-IR cell number in SGZ of WTs but were without significant effect (ns) in dupers. *p < 0.05. D, DCX process length was not affected by genotype or phase shifts.
Extended Data
Figure 2-1
The duper mutation reduces α but does not alter the number of wheel revolutions in shifted and unshifted animals. A, The time elapsed between onset and offset of activity is significantly greater in WT than in duper hamsters under both shift conditions (*p < 0.05). B, The average number of wheel revolutions did not differ between dupers and WTs under either shift condition. Download Figure 2-1, EPS file.
Figure 3-1
Neither phase shifts nor genotype affected the number of newborn cells in the subventricular zone at the 64 d survival interval. A, Phase shifts did not affect the number of BrdU- IR cells in either genotype, and the number of BrdU+ cells was similar in WT and duper hamsters. B, Schematic illustration of coronal section of a rodent brain showing the SVZ at the origin of the rostral migratory stream. C, Representative micrograph of right ventricle of a coronal section corresponding to B, showing BrdU+ cells (green), NeuN+ cells (red), and DAPI counterstain (blue). Download Figure 3-1, EPS file.
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