Ontogeny of circadian organization in the rat

Abstract

The mammalian circadian system is orchestrated by a master pacemaker in the brain, but many peripheral tissues also contain independent or quasi-independent circadian oscillators. The adaptive significance of clocks in these structures must lie, in large part, in the phase relationships between the constituent oscillators and their micro- and macroenvironments. To examine the relationship between postnatal development, which is dependent on endogenous programs and maternal/environmental influences, and the phase of circadian oscillators, the authors assessed the circadian phase of pineal, liver, lung, adrenal, and thyroid tissues cultured from Period 1-luciferase (Per1-luc ) rat pups of various postnatal ages. The liver, thyroid, and pineal were rhythmic at birth, but the phases of their Per1-luc expression rhythms shifted remarkably during development. To determine if the timing of the phase shift in each tissue could be the result of changing environmental conditions, the behavior of pups and their mothers was monitored. The circadian phase of the liver shifted from the day to night around postnatal day (P) 22 as the pups nursed less during the light and instead ate solid food during the dark. Furthermore, the phase of Per1-luc expression in liver cultures from nursing neonates could be shifted experimentally from the day to the night by allowing pups access to the dam only during the dark. Peak Per1-luc expression also shifted from midday to early night in thyroid cultures at about P20, concurrent with the shift in eating times. The phase of Per1-luc expression in the pineal gland shifted from day to night coincident with its sympathetic innervation at around P5. Per1-luc expression was rhythmic in adrenal cultures and peaked around the time of lights-off throughout development; however, the amplitude of the rhythm increased at P25. Lung cultures were completely arrhythmic until P12 when the pups began to leave the nest. Taken together, the data suggest that the molecular machinery that generates circadian oscillations matures at different rates in different tissues and that the phase of at least some peripheral organs is malleable and may shift as the organ's function changes during development.

Figure 1. Representative bioluminescence rhythms in several tissues cultured at various postnatal days

Within one hour before lights off (arrow, light/dark cycle indicated by white and black bars, respectively), static cultures of pineal (A, B), liver (C, D), lung (E, F), adrenal (G, H), thyroid (I, J) and SCN (K, L) were prepared from Per1-luc rat pups or adults maintained in 12:12 LD. Bioluminescence measurements (in counts per second) were collected at 10 minute intervals and are plotted for 3 days.

Figure 2. Phase changes in peripheral clocks during postnatal development

Peaks of Per1-luc rhythms in pineal (A), liver (B), lung (C), adrenal (D), thyroid (E) and SCN (F) are plotted as a function of postnatal age. Black and white bars on the top indicates LD cycles in which the pups and their dams were held. The vertical axis indicates postnatal age at the time cultures were made. Average peak phase of rhythmic cultures (±SEM) from two transgenic lines of rats (L1, filled circle; L2, open circle) are plotted separately. Numbers in parentheses indicate rhythmic cultures per number of cultures tested. Note: none of lung tissue taken before P10 was rhythmic.

Figure 3. Early postnatal lung was not capable of exhibiting Per1-luc rhythmicity in culture

Neither 50 % new born calf serum (A; n=4) or 10 µM forskolin (B; n=4) stimulation could induce the rhythmicity in lung cultures made from P3 pups. Time of one hour stimulation (arrow) is indicated at the bottom of panel.

Figure 4. Representative actograms of dam’s and pups' ";nest time. "

Behavior of a mother and her pups were monitored by infra-red video camera. Events when the mother was “on” her nest are double plotted as black vertical bars (A). The number of pups that were out of the nest is indicated by the height of the black bars double plotted in (B). Black and white bars on the top of each panel indicate the LD cycles. Two other records show very similar activity patterns.

Figure 5. Effect of maternal presence/absence cycles on circadian rhythm phase in the SCN and liver

The average times (±SEM) of peaks are plotted in Figure 2. The light cycle is plotted as a black and white bar at the top of the figure. Shaded bars indicate the time when pups were with their dams (top: control, mid: light group, bottom: dark group). Both SCN and liver were cultured after either 2 or 7 days of P/A cycles as indicated. No significant differences were found in SCN (by ANOVA). The phase of liver in the dark-fed group on Day 7 was significantly different from both the control and the light-fed group (ANOVA followed by Dunnett’s test, P<0.05).