Circadian clock proteins control adaptation to novel environment and memory formation

Abstract

Deficiency of the transcription factor BMAL1, a core component of the circadian clock, results in an accelerated aging phenotype in mice. The circadian clock regulates many physiological processes and was recently implicated in control of brain-based activities, such as memory formation and the regulation of emotions. Aging is accompanied by the decline in brain physiology, particularly decline in the response and adaptation to novelty. We investigated the role of the circadian clock in exploratory behavior and habituation to novelty using the open field paradigm. We found that mice with a deficiency of the circadian transcription factor BMAL1 display hyperactivity in novel environments and impaired intra- and intersession habituation, indicative of defects in short- and long-term memory formation. In contrast, mice double-deficient for the circadian proteins CRY1 and CRY2 (repressors of the BMAL1-mediated transcription) demonstrate reduced activity and accelerated habituation when compared to wild type mice. Mice with mutation in theClock gene (encoding the BMAL1 transcription partner) show normal locomotion, but increased rearing activity and impaired intersession habituation. BMAL1 is highly expressed in the neurons of the hippocampus - a brain region associated with spatial memory formation; BMAL1 deficiency disrupts circadian oscillation in gene expression and reactive oxygen species homeostasis in the brain, which may be among the possible mechanisms involved. Thus, we suggest that the BMAL1:CLOCK activity is critical for the proper exploratory and habituation behavior, and that the circadian clock prepares organism for a new round of everyday activities through optimization of behavioral learning.

Figure 1.. Open field analysis of exploratory activity and habituation of wild type and Bmal1-/- mice.

Locomotor and rearing activity were measured in 5 min increments during 1 hr. (Upper panel) locomotor and rearing activity of wt and Bmal1-/- mice on day1; (middle panel) relative locomotor activity (normalized to average distance covered on day1) and relative rearing activity (normalized to average rearing level on day1) of wt mice on days 1 and 2; (lower panel) relative locomotor and rearing activity of Bmal1-/- mice on days 1 and 2 (* P<0.05).

Figure 2.. Expression of circadian proteins in brain structures.

(a) Immunostaining of sagittal brain sections of wt and Bmal-/- mice with BMAL1 specific antibodies. Counterstaining with DAPI was used to detect nuclei. Pyramidal neurons of hippocampal areas CA1 and CA3, granular cells of the dentate gyrus and neurons of subiculum expressing BMAL1 are shown. (b) Circadian profile of Cry1 and Per2 mRNAs in the brain of wt (filled circles) and Bmal1-/- mice (open circles) as measured by real-time PCR. (* p<0.05).

Figure 3.. BMAL1 deficiency disrupts ROS homeostasis in the brain.

(a) ROS level in the brain of wt and Bmal1-/- mice was detected in the indicated time points of the circadian cycle. (b) Average ROS level in the brain of wt and Bmal1-/- mice for 24h (* P<0.05).

Figure 4.. Open field analysis of exploratory activity and habituation of Clock/Clock and Cry1,2-/- mice.

Relative locomotor and rearing activity of Clock/Clock and Cry1,2-/- mice on days 1 and 2 (normalized to the average distance/activity level on day1) (* P<0.05).

Figure 5.

Exploratory activity of wild type and circadian mutant mice. (Upper panel) Locomotor and rearing activity measured in 5 min increments during 1 hr on day1 for wt (diamonds), Bmal1-/- (squares), Clock/Clock (triangles) and Cry1,2-/- (crosses) mice. Statistically significant difference with wt activity is shown as p values. Difference between wt, Clock/Clock and Cry1,2-/- locomotor activities, and between rearing activities of Bmal1-/- vs. Clock/Clock and wt vs. Cry1,2-/- is not statistically significant. (Lower panel) Cumulative traveled distance and rearing activity on days 1 and 2; For upper panels p values of statistically significant differences between wild type and circadian mutants are indicated. For lower panels * P<0.05.

Figure 6.

Circadian mutant mice do not demonstrate anxiety phenotype. (Upper panel, left) Average horizontal velocity for all genotypes measured during 1h on days 1 and 2. Average velocity does not significantly differ between wt and Bmal1-/- animals; slight (~15%) but statistically significant decrease in average velocity is detected for Clock/Clock and Cry1,2-/- mice compared with wt and Bmal1-/-. (Upper panel, right) Cumulative time spent in the center for all genotypes measured during 1 hr on days 1 and 2. (Lower panel) Time spent in the center square of the open field arena on days 1 and 2 by wt, Clock/Clock, Bmal1-/-, and Cry1,2-/- mice. *P<0.05.