p38 Mitogen-activated Protein Kinase Regulates Oscillation of Chick Pineal Circadian Clock

Extracellular signal-regulated kinase (ERK) and p38 are members of the mitogen-activated protein kinase (MAPK) family, and in some cases these kinases serve for closely related cellular functions within a cell. In a wide range of animal clock structures, ERK plays an important role in the circadian time-keeping mechanism. Here we found that immunoreactivity to p38 protein was uniformly distributed among cells in the chick pineal gland. On the other hand, a constant level of activated p38 was detected over the day, predominantly in the follicular and parafollicular pinealocytes that are potential circadian clock-containing cells. Chronic application of SB203580, a selective and reversible inhibitor of p38, to the cultured chick pineal cells markedly lengthened the period of the circadian rhythm of the melatonin release (up to 28.7 h). Noticeably, despite no significant temporal change of activated p38 level, a 4-h pulse treatment with SB203580 delayed the phase of the rhythm only when delivered during the subjective day. These results indicate a time-of-day-specific role of continuously activated p38 in the period length regulation of the chick pineal clock and suggest temporally separated regulation of the clock by two MAPKs, nighttime-activated ERK and daytime-working p38

Time-of-Day-Dependent Enhancement of Adult Neurogenesis in the Hippocampus

BACKGROUND: Adult neurogenesis occurs in specific regions of the mammalian brain such as the dentate gyrus of the hippocampus. In the neurogenic region, neural progenitor cells continuously divide and give birth to new neurons. Although biological properties of neurons and glia in the hippocampus have been demonstrated to fluctuate depending on specific times of the day, it is unclear if neural progenitors and neurogenesis in the adult brain are temporally controlled within the day. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that in the dentate gyrus of the adult mouse hippocampus, the number of M-phase cells shows a day/night variation throughout the day, with a significant increase during the nighttime. The M-phase cell number is constant throughout the day in the subventricular zone of the forebrain, another site of adult neurogenesis, indicating the daily rhythm of progenitor mitosis is region-specific. Importantly, the nighttime enhancement of hippocampal progenitor mitosis is accompanied by a nighttime increase of newborn neurons. CONCLUSIONS/SIGNIFICANCE: These results indicate that neurogenesis in the adult hippocampus occurs in a time-of-day-dependent fashion, which may dictate daily modifications of dentate gyrus physiology

Increased Anxiety in Offspring Reared by Circadian <i>Clock</i> Mutant Mice

<div><p>The maternal care that offspring receive from their mothers early in life influences the offspring’s development of emotional behavior in adulthood. Here we found that offspring reared by circadian clock-impaired mice show elevated anxiety-related behavior. <i>Clock</i> mutant mice harboring a mutation in <i>Clock</i>, a key component of the molecular circadian clock, display altered daily patterns of nursing behavior that is fragmented during the light period, instead of long bouts of nursing behavior in wild-type mice. Adult wild-type offspring fostered by <i>Clock</i> mutant mice exhibit increased anxiety-related behavior. This is coupled with reduced levels of brain serotonin at postnatal day 14, whose homeostasis during the early postnatal period is critical for normal emotional behavior in adulthood. Together, disruption of the circadian clock in mothers has an adverse impact on establishing normal anxiety levels in offspring, which may increase their risk of developing anxiety disorders.</p></div

Brain serotonin levels in offspring reared by <i>Clock</i> mutant mice.

<p>(A, B) Serotonin levels in the brain of offspring reared by wild-type mice (Wt-r) and <i>Clock</i> mutant mice (Het-r) at 14 days old (A) and 7 weeks old (B). Data are presented as mean ± SEM (n = 5–8, n.s.: not significant, *p<0.05, Student’s <i>t</i> test). t(8) = 2.45, p<0.05 in (A), t(14) = 0.530, p>0.05 in (B).</p

Elevated anxiety-related behavior in offspring reared by <i>Clock</i> mutant mice.

<p>(A–D) In the open-field test, total activity counts measured by the total number of squares crossed by the mouse (A), activity counts at the center area measured by the number of center squares crossed (B), time spent at the center area (C) and the percentage of the number of center squares crossed (100×center squares/total squares crossed) (D) are shown. Data are presented as mean ± SEM (n = 8, *p<0.05, **p<0.01, Student’s <i>t</i> test). t(14) = 3.09, p<0.01 in (A), t(7) = 2.96, p<0.05 in (B), t(7) = 3.38, p<0.05 in (C), t(7) = 3.22, p<0.05 in (D). Wt-r: wild-type mother-reared mice, Het-r: heterozygous <i>Clock</i> mutant mother-reared mice. (E–G) In the elevated plus maze test, time spent in the open arms (E), entries to the open arms (F) and the percentage of open arm entries (100×open arm/total entries) (G) are shown. Data are presented as the mean ± SEM (n = 10–14, *p<0.05, **p<0.01, Student’s <i>t</i> test). t(14) = 2.60, p<0.05 in (E), t(14) = 3.19, p<0.01 in (F), t(22) = 2.21, p<0.05 in (G). (H) Home cage activity was measured, and total activity counts over the 24-hour cycle are presented as mean ± SEM (n = 3). t(4) = −0.146, p>0.05 by Student’s <i>t</i> test. n.s.: not significant.</p

Altered diurnal pattern in locomotor activity of female heterozygous <i>Clock</i> mutant mice.

<p>(A) Activity counts over the 24-hour cycle during the light (unshaded) and dark (shaded) periods. Mean activity counts in 5-min bins of wild-type mice (blue line, n = 4) and heterozygous <i>Clock</i> mutant mice (red line, n = 4) are plotted. (B) Activity counts were accumulated over the 12-hour light and 12-hour dark periods, and total activity counts were presented as mean ± SEM (n = 4). t(6) = 0.924, p>0.05 by Student’s <i>t</i> test. n.s.: not significant. (C) Light-period activity counts were expressed as a percentage of total activity counts (n = 3). t(4) = −3.39, *p<0.05 by Student’s <i>t</i> test. Wt: wild-type mice, Het: heterozygous <i>Clock</i> mutant mice.</p

Altered diurnal pattern in nursing behavior of female heterozygous <i>Clock</i> mutant mice.

<p>(A) Representative actogram of nursing behavior in wild-type and heterozygous <i>Clock</i> mutant mice at postpartum day 2–3 under a light-dark cycle (indicated by a bar at top). Black bars represent duration of nursing bouts. (B–D) Total duration of nursing activity per day (B), duration of nursing activity during the light period (C) and mean duration of nursing bouts (D) are shown as mean ± SEM (n = 4, n.s.: not significant, *p<0.05, Student’s <i>t</i> test). t(6) = 1.82, p>0.05 in (B), t(6) = 2.93, p<0.05 in (C), t(4) = 4.00, p<0.05 and t(6) = −0.318, p>0.05 in (D). Wt: wild-type mice, Het: heterozygous <i>Clock</i> mutant mice.</p

Schematic representation of the experimental design.

<p>Wild-type male neonates (postnatal day 1, P1) were separated from their wild-type mother and fostered on postpartum wild-type mice or heterozygous <i>Clock</i> mutant mice. At P2-3, maternal behavior of mothers was video-recorded. The offspring were weaned at P28. The offspring at 7–8 weeks of age or at 14–16 weeks of age were subjected to four behavioral tests in the following order; open-field test (OFT), elevated plus maze test (EPM), forced swim test (FST) and tail suspension test (TST). Mice were given one test per day for 4 consecutive days.</p