Masking responses to light in period mutant mice

Abstract

Masking is an acute effect of an external signal on an overt rhythm and is distinct from the process of entrainment. In the current study, we investigated the phase dependence and molecular mechanisms regulating masking effects of light pulses on spontaneous locomotor activity in mice. The circadian genes, Period1 (Per1) and Per2, are necessary components of the timekeeping machinery and entrainment by light appears to involve the induction of the expression of Per1 and Per2 mRNAs in the suprachiasmatic nuclei (SCN). We assessed the roles of the Per genes in regulating masking by assessing the effects of light pulses on nocturnal locomotor activity in C57BL/6J Per mutant mice. We found that Per1(-/-) and Per2(-/-) mice had robust negative masking responses to light. In addition, the locomotor activity of Per1(-/-)/Per2(-/-) mice appeared to be rhythmic in the light-dark (LD) cycle, and the phase of activity onset was advanced (but varied among individual mice) relative to lights off. This rhythm persisted for 1 to 2 days in constant darkness in some Per1(-/-)/Per2(-/-) mice. Furthermore, Per1(-/-)/Per2(-/-) mice exhibited robust negative masking responses to light. Negative masking was phase dependent in wild-type mice such that maximal suppression was induced by light pulses at zeitgeber time 14 (ZT14) and gradually weaker suppression occurred during light pulses at ZT16 and ZT18. By measuring the phase shifts induced by the masking protocol (light pulses were administered to mice maintained in the LD cycle), we found that the phase responsiveness of Per mutant mice was altered compared to wild-types. Together, our data suggest that negative masking responses to light are robust in Per mutant mice and that the Per1(-/-)/Per2(-/-) SCN may be a light-driven, weak/damping oscillator.

FIGURE 1

Masking responses to light are intact in Per1^−/− and Per2^−/− mice. Effect of 1-h light pulses (75–85 lux) administered at ZT14 on the wheel-running activity of wild-type (A; n = 11), Per1^−/− (B; n = 5), and Per2^−/− (C; n = 5) mice maintained in 12L:12D. Each symbol represents an individual mouse. Baseline was calculated as the total number of wheel revolutions from ZT14–15 (wheel revs/h) averaged over the 3 days prior to the light pulse. The light-pulse value represents the total number of wheel revolutions during the light pulse. Single-plotted actograms of wheel-running activity (scaled, 1-min bins) of wild-type (D), Per1^−/− (E), and Per2^−/− (F) mice. Gray shading indicates darkness and arrows indicate the day the light pulse was administered. The top panel of actograms (D, E, F) shows typical examples of the masking response of each genotype. The bottom panel of actograms (D, E, F) shows the atypical masking response of each genotype and correspond to the mice represented by the star symbol (*) in A, B, and C, respectively.

FIGURE 2

Intact masking responses to light in Per1^−/−/Per2^−/− mice. (A–E) Single-plotted actograms of wheel-running activity (scaled, 1-min bins) of Per1^−/−/Per2^−/− mice. Gray shading indicates darkness. Per1^−/−/Per2^−/− mice (n = 5) were maintained in 12L:12D and administered 1-h light pulses at ZT14 (75–85 lux). Arrows indicate the day the light pulse was administered. (F) Each symbol represents an individual mouse. Baseline was calculated as the total number of wheel revolutions from ZT14–15 (wheel revs/h) averaged over the 3 days prior to the light pulse. The light-pulse value represents the total number of wheel revolutions during the light pulse.

FIGURE 3

The phase of activity onset in some Per1^−/−/Per2^−/− mice is advanced relative to lights off. Double-plotted actograms of wheel-running activity (normalized, 6-min bins) of Per1^−/−/Per2^−/− mice maintained in 12L:12D (200–300 lux) for 5 days (in the same 12L:12D in which they were born and raised) and then released into DD. Gray shading indicates darkness.

FIGURE 4

Phase-dependent masking in wild-type mice. Effect of 1-h light pulses (75–85 lux) administered at ZT14 (A; n = 11, data from Figure 1 are replotted), ZT16 (n = 7), and ZT18 (n = 10) on wheel-running activity. Each symbol represents an individual mouse administered a single light pulse. Baseline was calculated as the total number of wheel revolutions from ZT14–15 (A), ZT16–17 (B), or ZT18–19 (C) (wheel revs/h) averaged over the 3 days prior to the light pulse. The light-pulse value represents the total number of wheel revolutions during the light pulse. The mean (± SEM) number of wheel revolutions/h during baseline (E) and light pulses (F) are shown. (G) The number of wheel revolutions during the light pulses was expressed as a percent of the number of wheel revolutions during baseline for each mouse, and then the mean (± SEM) percent of baseline values was plotted at each ZT (G). *p < .05.

FIGURE 5

Phase responses to light pulses in wild-type and Per mutant mice measured using the Type VI protocol. Mice were maintained in 12L:12D, and 1-h light pulses (75–85 lux) were administered at ZT14, ZT16, or ZT18 to wild-type, Per1^−/−, and Per2^−/− mice. Per1^−/−/Per2^−/− mice received light pulses only at ZT14. Representative single-plotted actograms of wheel-running activity (normalized, 6-min bins) of wild-type (A), Per1^−/− (B), and Per2^−/− (C) mice given light pulses at ZT14. Actograms of Per1^−/−/Per2^−/− mice administered light pulses at ZT14 are shown in Figure 2. Gray shading indicates darkness. (D) Mean phase shifts (± SEM) are shown (number of replicates reported in previous figures for wild-types; Per1^−/−: ZT14, n = 7; ZT16, n = 1; ZT18, n = 6; Per2^−/−: ZT14, n = 4; ZT16, n = 3; ZT18, n = 5; Per1^−/−/Per2^−/−: ZT14, n = 2). No error bar is shown for the ZT16 pulse of Per1^−/− mice because only one pulse was administered, or for Per1^−/−/Per2^−/− mice because the onset of activity could only be determined in two of five mice. Light pulses were not administered to Per1^−/−/Per2^−/− mice at ZT16 and ZT18 (indicated by ND; not determined).