Behavioral and genetic dissection of a mouse model for advanced sleep phase syndrome

Abstract

Study objective: The adaptive value of the endogenous circadian clock arises from its ability to synchronize (i.e., entrain) to external light-dark (LD) cycles at an appropriate phase. Studies have suggested that advanced circadian phase alignment might result from shortening of the period length of the clock. Here we explore mechanisms that contribute to an early activity phase in CAST/EiJ (CAST) mice.

Methods: We investigated circadian rhythms of wheel-running activity in C57BL/6J (B6), CAST and 2 strains of B6.CAST congenic mice, which carry CAST segments introgressed in a B6 genome.

Results: When entrained, all CAST mice initiate daily activity several hours earlier than normal mice. This difference could not be explained by alterations in the endogenous period, as activity onset did not correlate with period length. However, the photic phase-shifting responses in these mice were phase-lagged by 3 hours relative to their activity. Attenuated light masking responses were also found in CAST mice, which allow for activity normally inhibited by light. A previously identified quantitative trait locus (QTL), Era1, which contributes to the early activity trait, was confirmed and refined here using two B6.CAST congenic strains. Surprisingly, these B6.CAST mice exhibited longer rather than shorter endogenous periods, further demonstrating that the advanced phase in these mice is not due to alterations in period.

Conclusions: CAST mice have an advanced activity phase similar to human advanced sleep phase syndrome. This advanced phase is not due to its shorter period length or smaller light-induced phase shifts, but appears to be related to both light masking and altered coupling of the circadian pacemaker with various outputs. Lastly, a QTL influencing this trait was confirmed and narrowed using congenic mice as a first step toward gene identification.

Keywords: Era1; Phase angle; mouse genetics; phase response curve; quantitative trait loci.

Figure 1

CAST/EiJ (CAST) mice and C57BL/6J (B6) mice differ in phase angle of entrainment. Wheel running data from one male B6 mouse (A) and one male CAST mouse (B) were plotted in percentile actogram format. The height of the vertical tick is proportional to the number of wheel revolutions in that interval, relative to the 24-h average. Data for 9 days in LD12:12 and 11 days in constant dark are shown. Time spent in darkness is indicated by gray shading. Individual values (circles for B6 and triangles for CAST) and group means (horizontal bars) are shown for activity onset derived from LD12:12 data (C) and phase angle of entrainment derived from DD data (D). Note: (1) Only one B6 mouse had a phase angle earlier than dark onset. This value is 3 SD from the group mean and was discarded as an outlier. (2) One CAST mouse died before release into DD, thus only 9 CAST values are plotted in panel D.

Figure 2

Early runner mice exhibit early daily awakening when tested on a piezoelectric system in the absence of a wheel. Data for 4 consecutive LD 12:12 cycles recorded from 4 (B6 × CAST) × CAST backcross mice are shown. Timing of light/dark is indicated both by gray shading and the white/black bar at the top of each panel. Dashed lines show the computer-defined onset for the major wake period within each LD cycle. Sleep-wake patterns and wake onset assessed by the piezoelectric system closely matched wheel-running patterns and onsets, as shown by these 4 examples: (A) A mouse with previous wheel running onset approximately 1 h before dark; (B) and (C) Mice with previous wheel running onsets 3 h prior to dark; (D) Mouse with wheel running onset 5 h before dark. In general, all mice examined had similar activity onsets whether assessed by wheel running, or, several weeks later, by piezoelectric defined sleep and wake.

Figure 3

Circadian period is shorter in CAST than in B6 mice and does not correlate with activity onset in LD12:12 or phase angle of entrainment within the CAST strain. (A) Individual values (circles for B6 and triangles for CAST) and group means (horizontal bars) are shown for circadian period in DD. Scatterplots of circadian period against activity onset derived from LD12:12 data (B) or phase angle of entrainment derived from DD data (C) show no correlation (P > 0.40) within the CAST strain.

Figure 4

Phase response and tau response curves differ between B6 and CAST mice. Phase and tau (i.e., circadian period) responses to green light were measured in B6 (A) and CAST (B) mice exposed to 30-min green light pulses at random circadian phases at 14-day intervals when animals were housed under DD. One light pulse for B6 and two light pulses for CAST are shown as gray dots. (C) A 2-harmonic Fourier fitted phase response curve was generated from n = 65 phase shifts measured in B6 mice (circles and solid line) and n = 59 phase shifts measured in CAST mice (triangles and dashed line). The fitted curves were re-plotted to the right for clarity. (D) Adjusting the definition of the circadian phase of light exposure for each CAST mouse according to its activity onset in LD alters the timing of the phase response curve in CAST mice (triangles and dashed line). (E) The tau response curve demonstrates that light exposure does not influence circadian period in B6 (left) but has a slowing effect in CAST mice, as shown by the 2-harmonic Fourier fitted line (right).

Figure 5

Negative masking effects of light are strain and light dependent. (A) Seventy-two hours of wheel running data from B6 (top) and CAST (bottom) mice demonstrate the effects of green (left) and white (right) light exposure on wheel running activity. Animals were entrained under LD12:12 conditions using green light. The timing of the light pulse (ZT12.5-ZT13.5 on the third night shown) is illustrated by gray shading and carets at the base of each actogram. (B) The amount of wheel running during light pulse (LP) at ZT12.5-ZT13.5 and during the control dark condition is plotted individually for each B6 (upper panel) and CAST (lower panel) mouse. Baseline (BL) activity in the control dark condition is calculated as the mean wheel-running activity at the same Zeitgeber time (ZT) 2 days preceding the light pulse. P values from paired Student t-test are labeled at the top of each panel. n.s., not significant. (C) Group mean ± SEM values for wheel running during light exposure at ZT12.5-ZT13.5 are plotted as a percentage of baseline activity. ***P < 0.001, **P < 0.01, B6 vs. CAST, Student t-test. #, P < 0.05, green vs. white light within CAST mice, Student t for paired measures.

Figure 6

Activity density before dark in CAST mice housed in LD12:12 is dependent on the type of light exposure. (A) Wheel-running data are shown from one male CAST mouse housed in white light (days 1-10) or green light (days 11-20). Mean ± SEM are plotted for mathematically defined and visually adjusted activity onsets (B) and activity intensity defined by number of wheel running revolutions during the interval between activity onset and dark onset (C) for CAST mice housed in white (white bars) and green light (grey bars). *P < 0.05 green vs. white light, Student t for paired measures.

Figure 7

Congenic lines demonstrate an effect of the chromosome 18 genotype on wheel running phenotype. (A) Portions of chromosome 18 have been transferred from the CAST to the B6 background in congenic strains. The early runner phenotype was previously linked to a locus between marker D18Mit122 and D18Mit162 on chromosome 18 (Era1) by QTL analysis (upper panel reproduced from ref.15). Era1 is here aligned with the physical map of chromosome 18 and the genetic markers that have been genotyped as homozygous B6 (black in schematic representations of chromosome 18) or homozygous CAST (white in schematic representation of chromosome 18) in B6.CAST.18P and B6.CAST.18M mice. Marker and genotype information were adopted from Davis et al with modification of the B6.CAST.18M genotypes according to our more detailed genotyping results. The current proximal flanking marker is D18Mit181 and the proximal internal marker is D18Mit51. Wheel running data from one male B6.CAST.18P mouse (B) and one male B6.CAST.18M mouse (C) were double-plotted in percentile actogram format over 20 days (10 days in LD12:12 using 83 lux green light and 10 days in constant dark). Time spent in darkness is indicated by gray shading. (D) Phase angle of entrainment is altered in both congenic strains. Group mean ± SEM values of phase angle of entrainment are derived from DD data. ANOVA, F = 28.69, P < 0.001. ***P < 0.001 vs. B6, Student t. (E) Free running period length is (surprisingly) longer in congenic mice relative to B6. Group mean ± SEM values are shown. ANOVA, F = 34.47, P < 0.001. **P < 0.01; ***P < 0.001 vs. B6, Student t for unpaired measures. B6, B6.CAST.18P and B6.CAST.18M mice also differed significantly from CAST, P < 0.001, Student t for unpaired measures.