Spontaneous Recovery of Circadian Organization in Mice Lacking a Core Component of the Molecular Clockwork

Abstract

Circadian rhythms are generated by interlocked transcriptional-translational feedback loops of circadian clock genes and their protein products. Mice homozygous for a functional deletion in the Period-2 gene ( Per2m/m mice) exhibit short free-running circadian periods and eventually lose behavioral circadian rhythmicity in constant darkness (DD). We investigated Per2m/m mice in DD for several months and identified a categorical sex difference in the dependence on Per2 for maintenance of circadian rhythms. Nearly all female Per2m/m mice became circadian arrhythmic in DD, whereas free-running rhythms persisted in 37% of males. Remarkably, with extended testing, Per2m/m mice did not remain arrhythmic in DD, but after varying intervals spontaneously recovered robust, free-running circadian rhythms, with periods shorter than those expressed prior to arrhythmia. Spontaneous recovery was strikingly sex-biased, occurring in 95% of females and 33% of males. Castration in adulthood resulted in male Per2m/m mice exhibiting female-like levels of arrhythmia in DD, but did not affect spontaneous recovery. The circadian pacemaker of many gonad-intact males, but not females, can persist in DD for long intervals without a functional PER2 protein; their circadian clocks may be in an unstable equilibrium, incapable of sustaining persistent coherent circadian organization, resulting in transient cycles of circadian organization and arrhythmia.

Modified Wavelet Analyses Permit Quantification of Dynamic Interactions Between Ultradian and Circadian Rhythms

“…We next evaluated the accuracy of power structure and ridge period analyses on locomotor activity data from actual mice in which real behavioral URs were experimentally introduced. We took advantage of behavioral plasticity exhibited by Per2 m/m mice which, under prolonged exposure to constant darkness, exhibit short-period free-running CRs, followed by circadian arrhythmicity which coincides with an emergence of ultradian power (Zheng et al, 1999; Bae et al, 2001; Riggle et al, 2022). Power structure clearly documented circadian periods of 22-23 h in female Per2 m/m mice, the eventual loss of CR power, and an emergence of UR power (Figure 3B; see also Riggle et al, 2022).…”

“…Female mice ( Per2 m/m : n = 5; WT: n = 7; ~5 weeks of age) were singly housed in 12L:12D under passive infrared motion sensors. A more extensive analysis of data from these mice has been reported elsewhere (Riggle et al, 2022). The data described here (in Experiment 1.3) are a re-analysis of locomotor activity records from Per2 m/m mice that were previously reported in a study of sex differences in circadian behavior (Riggle et al, 2022); the data are used here solely for the purposes of validating the accuracy and precision of the continuous wavelet transform’s ability to measure period and power in a model of circadian arrhythmia.…”

“…A more extensive analysis of data from these mice has been reported elsewhere (Riggle et al, 2022). The data described here (in Experiment 1.3) are a re-analysis of locomotor activity records from Per2 m/m mice that were previously reported in a study of sex differences in circadian behavior (Riggle et al, 2022); the data are used here solely for the purposes of validating the accuracy and precision of the continuous wavelet transform’s ability to measure period and power in a model of circadian arrhythmia. Mice were kept in 12L:12D to establish a baseline of entrained locomotor activity and were then transferred to constant darkness (DD).…”

“…WT mice were on backgrounds of (C57BL/6 J [JAX Catalog #: 000664; black coat color] or B6(Cg)-Tyrc-2 J/J [JAX Catalog #: 000058; albino color]). Mutant mice in Experiment 1 homozygous for the Per2 Brdm1 mutation [JAX Catalog #: 003819; Per2 m/m ] were ordered directly from Jackson Labs (Riggle et al, 2022). Mice were housed in an intermediate-duration photoperiod that provided 12 h light and 12 h darkness each day (12 L:12D; ‘intermediate days’), with the exception of Experiment 3 (see Photoperiod manipulations ).…”

“…One element of this feedback loop, Period 2 (and its protein product, PER2) is critical for the integrity of the organismal circadian network: germline mutant mice with a functional deletion of the mPER2 protein dimerization PAS domain ( mPer2 Brdm1 ; Per2 m/m mice) entrain to 24 h L:D cycles, but upon transfer to constant darkness exhibit an extremely short free-running circadian period (~22-23 h), and subsequently become behaviorally circadian arrhythmic (Zheng et al, 1999). Moreover, Per2 m/m mice have been characterized as exhibiting robust increases in UR power after the loss of circadian rhythmicity in constant darkness (Zheng et al, 1999; Bae et al, 2001; Zheng et al, 2001; Abe et al, 2002; Riggle et al, 2022). We used Per2 m/m mice to evaluate the ability of the continuous wavelet transform analysis described above to detect: (1) changes in circadian period upon transfer from long day to constant darkness, (2) the loss of circadian rhythmicity following prolonged exposure to constant darkness, and (3) the emergence of power in the ultradian domain following the loss of CRs, and in doing so further characterize the ecological validity of the analytic workflow used here.…”