Distinct Functions of Period2 and Period3 in the Mouse Circadian System Revealed by In Vitro Analysis

The mammalian circadian system, which is composed of a master pacemaker in the suprachiasmatic nuclei (SCN) as well as other oscillators in the brain and peripheral tissues, controls daily rhythms of behavior and physiology. Lesions of the SCN abolish circadian rhythms of locomotor activity and transplants of fetal SCN tissue restore rhythmic behavior with the periodicity of the donor's genotype, suggesting that the SCN determines the period of the circadian behavioral rhythm. According to the model of timekeeping in the SCN, the Period (Per) genes are important elements of the transcriptional/translational feedback loops that generate the endogenous circadian rhythm. Previous studies have investigated the functions of the Per genes by examining locomotor activity in mice lacking functional PERIOD proteins. Variable behavioral phenotypes were observed depending on the line and genetic background of the mice. In the current study we assessed both wheel-running activity and Per1-promoter-driven luciferase expression (Per1-luc) in cultured SCN, pituitary, and lung explants from Per2−/− and Per3−/− mice congenic with the C57BL/6J strain. We found that the Per2−/− phenotype is enhanced in vitro compared to in vivo, such that the period of Per1-luc expression in Per2−/− SCN explants is 1.5 hours shorter than in Per2+/+ SCN, while the free-running period of wheel-running activity is only 11 minutes shorter in Per2−/− compared to Per2+/+ mice. In contrast, circadian rhythms in SCN explants from Per3−/− mice do not differ from Per3+/+ mice. Instead, the period and phase of Per1-luc expression are significantly altered in Per3−/− pituitary and lung explants compared to Per3+/+ mice. Taken together these data suggest that the function of each Per gene may differ between tissues. Per2 appears to be important for period determination in the SCN, while Per3 participates in timekeeping in the pituitary and lung

Robust Food Anticipatory Activity in BMAL1-Deficient Mice

Food availability is a potent environmental cue that directs circadian locomotor activity in rodents. Even though nocturnal rodents prefer to forage at night, daytime food anticipatory activity (FAA) is observed prior to short meals presented at a scheduled time of day. Under this restricted feeding regimen, rodents exhibit two distinct bouts of activity, a nocturnal activity rhythm that is entrained to the light-dark cycle and controlled by the master clock in the suprachiasmatic nuclei (SCN) and a daytime bout of activity that is phase-locked to mealtime. FAA also occurs during food deprivation, suggesting that a food-entrainable oscillator (FEO) keeps time in the absence of scheduled feeding. Previous studies have demonstrated that the FEO is anatomically distinct from the SCN and that FAA is observed in mice lacking some circadian genes essential for timekeeping in the SCN. In the current study, we optimized the conditions for examining FAA during restricted feeding and food deprivation in mice lacking functional BMAL1, which is critical for circadian rhythm generation in the SCN. We found that BMAL1-deficient mice displayed FAA during restricted feeding in 12hr light:12hr dark (12L:12D) and 18L:6D lighting cycles, but distinct activity during food deprivation was observed only in 18L:6D. While BMAL1-deficient mice also exhibited robust FAA during restricted feeding in constant darkness, mice were hyperactive during food deprivation so it was not clear that FAA consistently occurred at the time of previously scheduled food availability. Taken together, our findings suggest that optimization of experimental conditions such as photoperiod may be necessary to visualize FAA in genetically modified mice. Furthermore, the expression of FAA may be possible without a circadian oscillator that depends on BMAL1

Study of the doubly charmed tetraquark T+cc

Quantum chromodynamics, the theory of the strong force, describes interactions of coloured quarks and gluons and the formation of hadronic matter. Conventional hadronic matter consists of baryons and mesons made of three quarks and quark-antiquark pairs, respectively. Particles with an alternative quark content are known as exotic states. Here a study is reported of an exotic narrow state in the D0D0π+ mass spectrum just below the D*+D0 mass threshold produced in proton-proton collisions collected with the LHCb detector at the Large Hadron Collider. The state is consistent with the ground isoscalar T+cc tetraquark with a quark content of ccu⎯⎯⎯d⎯⎯⎯ and spin-parity quantum numbers JP = 1+. Study of the DD mass spectra disfavours interpretation of the resonance as the isovector state. The decay structure via intermediate off-shell D*+ mesons is consistent with the observed D0π+ mass distribution. To analyse the mass of the resonance and its coupling to the D*D system, a dedicated model is developed under the assumption of an isoscalar axial-vector T+cc state decaying to the D*D channel. Using this model, resonance parameters including the pole position, scattering length, effective range and compositeness are determined to reveal important information about the nature of the T+cc state. In addition, an unexpected dependence of the production rate on track multiplicity is observed

Characterization of Circadian Behavior in C57BL/6J <i>Per2^−/−</i> and <i>Per3^−/−</i> Mice.

<p>Representative double-plotted actograms of wheel-running activity of <i>Per2^+/+</i> (A; n = 10), <i>Per3^+/+</i> (B; n = 10), <i>Per2^−/−</i> (C; n = 12), and <i>Per3^−/−</i> (D; n = 10) mice maintained in 12L∶12D LD (lights on at 0 h and lights off at 12 h) for 7 days and then released into constant darkness (DD). The free-running period was determined by using χ² periodogram for days 1-15 in DD (E). The phase angle of entrainment (F) was determined by drawing a regression line to activity onset for days 1-5 in DD and then extending the regression line to the last day in LD. A negative phase angle was obtained when activity started before the time of lights off and a positive phase angle was obtained when activity started after the time of lights off. Data are the mean±SD; *<i>p</i><0.05.</p

<i>Per1-luc</i> Expression in Peripheral Tissues Explanted from C57BL/6J <i>Per2^−/−</i> and <i>Per3^−/−</i> Mice.

<p>Representative baseline-subtracted bioluminescence rhythms in pituitary (A) and lung (B) explants from <i>Per2^+/+</i> (black trace; top panel; n = 6) and <i>Per2^−/−</i> (red trace; top panel; n = 4-6) mice and from <i>Per3^+/+</i> (black trace; bottom panel; n = 4) and <i>Per3^−/−</i> (red trace; bottom panel; n = 5) mice. The lighting condition of the previous light/dark cycle is indicated for the first day; open bars are light and black bars are dark. The period of pituitary (C) and lung (D) explants was determined by fitting a regression line to the acrophase of the <i>Per1</i>-<i>luc</i> rhythm. The phase of pituitary (E) and lung (F) explants was designated as the first peak of <i>Per1-luc</i> expression <i>in vitro</i> and is plotted relative to the light-dark cycle before culture, where 0 h is the time of lights on and 12 h is the time of lights off (open bars are light and black bars are dark). All data are presented as the mean±SD; *<i>p</i><0.05, ***<i>p</i><0.001.</p

<i>Per1</i> Promoter Activity Is Altered in PER-deficient Tissues.

<p>Average <i>Per1-luc</i> bioluminescence (raw data) from SCN explants (A) and whole pituitary glands (B) from wildtype, heterozygous, or PER-deficient mice. Bioluminescence data from <i>Per1^−/−</i> mice were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008552#pone.0008552-Pendergast1" target="_blank">[36]</a>. The lighting condition of the previous light/dark cycle is indicated for the first day; open bars are light and black bars are dark.</p

Circadian Behavior of C57BL/6J <i>Per2^−/−</i> and <i>Per3^−/−</i> Mice.

a<p><i>Per2^+/−</i> is significantly greater than <i>Per2^+/+</i> and <i>Per2^−/−</i>.</p>*<p>The same mice were used to determine period and phase angle of entrainment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008552#pone-0008552-g001" target="_blank">Figure 1E, F</a>).</p

Food anticipatory activity in BMAL1-deficient mice in 12L:12D.

<p>Representative double-plotted actograms (A, C) and group average activity profiles (B, D) of <i>Bmal1</i>^+/+ mice (A, B; n = 3) and <i>Bmal1</i>^−/− mice (C, D; n = 7) in 12L:12D. The time when food was available is indicated by light gray shading in the activity profiles and on the left half of each actogram. The light-dark cycle is indicated by the white and black bars, respectively. On the left half of each actogram, the dark black line outlines the time of darkness. The black traces in the group average activity profiles represent the mean number of wheel revolutions (in counts/minute) plotted in 10-minute bins relative to the light-dark cycle where ZT0 is lights on and ZT12 is lights off. The SEM is shown in dark gray shading in each activity profile. AL I, FD I, RF, AL II, and FD II labels of the actograms (A, C) indicate the days used to generate the mean activity profiles <i>ad libitum</i> I, food deprivation I, restricted feeding, <i>ad libitum</i> II, and food deprivation II, respectively (B, D).</p

FAA from individual wildtype and BMAL1-deficient mice during restricted feeding and food deprivation.

<p>FAA during restricted feeding (RF; A) of individual mice in 12L:12D, 18L:6D, and DD was determined by totaling the number of wheel revolutions per minute from 4 hours before feeding time to the end of feeding time (total of 8 hours). FAA for each mouse was averaged over 9 days of restricted feeding. FAA during fasting for <i>Bmal1</i>^+/+ (B) and <i>Bmal1</i>^−/− (C) mice was defined as the total number of wheel revolutions per minute from 4 hours before feeding time to the end of previous feeding time (total of 8 hours). Wheel-running FAA for each mouse was determined separately for the first (Day 1) or second (Day 2) day of food deprivation.</p