Periodic feeding of SCN-lesioned pregnant rats entrains the fetal biological clock

Developmental Brain Research, 46 (1989) 291-296 Elsevier

BRD 60303

291

Short Communications

Periodic feeding of SCN-lesioned pregnant rats entrains the fetal biological clock David R. Weaver and Steven M. Reppert Laboratory of Developmental Chronobiology, Children's Service, Massachusetts General Hospital and Department of Pediatrics and Program in Neuroscience, Harvard Medical School, Boston, MA 02114 (U.S.A.)

(Accepted 13 December 1988) Key words: Circadian rhythm; Suprachiasmatic nucleus; Restricted feeding; Prenatal entrainment; Drinking behavior

Destruction of the maternal suprachiasmatic nuclei (SCN) early in gestation disrupts maternal communication of time-of-day information to the rat fetus. In the present study, we demonstrated that periodic feeding (food cue) to SCN-lesioned pregnant rats entrains the fetal biological clock. The phase of the drinking behavior rhythm was examined in pups reared in constant darkness, beginning at weaning. In several control (uncued) litters, pup phases at weaning were scattered. In other control litters where within-litter coordination of phase was observed, the average litter phase was unpredictable. In contrast, drinking rhythms of pups whose dams had received food cue during gestation were synchronized within- and between-litters, suggesting that prenatal food cue entrained the fetuses. The effect of food cue occurs prenatally, as similar results were obtained when offspring of SCN-lesioned, cued dams were fostered to lesioned, uncued dams on the day of birth. The present results, along with data from this and other laboratories, suggest that redundant mechanisms communicate time-of-day information to the fetus. Circadian rhythms are endogenously generated biological rhythms with a period length around 24 h. In mammals, circadian rhythms are generated by a biological clock located within the suprachiasmatic nuclei (SCN) of the anterior hypothalamus 1°. In adult mammals, the light-dark cycle is a potent signal for synchronization of circadian rhythms to environmental conditions (entrainment); photic information reaches the SCN directly, via the retinohypothalamic tract, and through a less direct retino-geniculo-hypothalamic pathway 11. During development, the situation is different. Fetal and neonatal rats do not respond directly to the entraining effects of light 7'8'~5, but the timing of their biological clock is nevertheless coordinated with the environmental light-dark cycle ~5. This prenatal entrainment of the fetal biological clock is the result of communication of time-of-day information from the mother to her offspring 18. Maternal,fetal communication of circadian phase is disrupted by destruction of the maternal SCN 5"17,

suggesting that the maternal entraining signal is a rhythm (or rhythms) regulated by the SCN. Since SCN lesions disrupt virtually all circadian rhythms in rodents, these experiments do little to define which aspects of maternal rhythmicity are involved in communicating time-of-day information to the fetus. Attempts to elucidate the specific signal communicating circadian phase information from mother to fetus have been unsuccessful. Removal of selected endocrine organs (pineal, pituitary, ovary, adrenal, thyroid-parathyroid; in separate experiments) does not disrupt maternal-fetal communication of phase in rats, suggesting that the rhythmic hormonal outputs from these glands are not necessary 16. A n alternative approach for investigating the mechanism for communication of time-of-day information is to artificially restore rhythmicity in a dam that has no endogenous rhythmicity. Normally , there is a circadian rhythm in food consumption in rats; this rhythm is disrupted by destruction of the SCN 12. Restricted access to food in SCN-lesioned

Correspondence: D.R. Weaver, Laboratory of Developmental Chronobiology, Massachusetts General Hospital, Boston, MA 02114, U.S.A.

0165-3806/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

292 rats artificially produces a rhythm in food consumption and results in entrainment of an oscillator distinct from the SCN capable of generating circadian rhythms in anticipatory locomotor activity2' 3,13.21 body temperature and adrenal corticosteroids 9. We report here that rhythmic ingestion of food by SCN-lesioned rats during pregnancy can entrain the fetus. Timed-pregnant, Sprague-Dawley rats (Zivic Miller Laboratories, Allison Park, PA) were housed singly in polystyrene cages within well-ventilated, light-tight environmental compartments, with lights on from 07.00 to 19.00 h daily. During the dark phase of the light-dark cycle and during periods referred to as constant darkness, dim red (>620 nm) light was provided by special fluorescent tubes within the compartment (Litho light no. 2, Chemical Products Co., N. Warren, PA). Maternal SCN lesions were performed on gestational day 7 (day 0 of gestation = day of sperm positivity) under ether anesthesia as previously described 17. Following surgery, each animal received 150,000 U penicillin G benzathine, i.m. (Bicillin, Wyeth, Philadelphia, PA) and was placed into constant darkness to prevent potential masking effects of light. After the experiment, placement and extent of lesions was determined by light microscopic examination of serial, coronal, 20-pm thick, Cresyl violet-stained sections. Except where indicated, SCN lesions resulted in complete destruction of the SCN. Maternal body temperature, activity and drinking behavior data were collected continuously and stored at 10-rain intervals by an automated data collection system (Dataquest III, Minimitter). Drinking behavior was monitored via a drinkometer relay (no. 8006; Lafayette Instruments, Lafayette, IN) wired to the water bottle's sipper spout and to the cage floor, so that each lick of the sipper spout completes the drinkometer circuit. Radiotelemetry thermistors (transmitters; model VM-FH, Minimitter, Sunriver, OR) for collection of temperature and activity data were implanted i.p. in some dams at the time of stereotaxic surgery. Pup drinking behavior was monitored in constant darkness for 2-3 weeks beginning at weaning on postnatal day 21. Computer-generated actograms of drinking activity were photographically reduced and

double-plotted. An eye-fitted line was drawn through the drinking offsets for the recording period to estimate the phase (time) of drinking offset on postnatal day 21TM.The phases of pups within a litter were analyzed using vector addition I as discussed by Davis and Gorski 4. Within-litter synchrony of pup phase was considered significant when P ~< 0.05 by the Rayleigh test, indicating that the phases of pup activity onset are not randomly (uniformly) distributed throughout the day. Experiment 1 examined the effects of food cue during gestation on circadian rhythmicity in pups from SCN-lesioned dams. Food (Purina Rat Chow no. 5012) was available ad lib to all dams until gestational day 7, when the dams received SCN lesions and were placed in constant darkness. In dams receiving periodic feeding (food cue), food access was restricted to a 4-h period (09.00-13.00 h) each day from gestational day 8 to 19. This placed the food cue opposite in phase to the normal onset of major feeding bouts in intact animals, which would occur at night. To control for the periodic entry into the environmental compartment and placing and removing food from the food bin, another group of animals was allowed constant access to food, but the food was jostled at 09.00 and 13.00 h. The food cue and food jostling procedures were discontinued on gestational day 20, when food was made available ad libitum to all animals. Dams gave birth and reared their pups in darkness. Animals were checked at random clock times throughout the remainder of the study. SCN lesions destroyed rhythmicity in drinking behavior in dams not receiving food cue (jostle control). Food cue induced generation of rhythms in drinking behavior, locomotor activity and body temperature in dams with SCN lesions (Fig. 1). During the postnatal period, uitradian rhythms in these measures predominated in both cued and uncued animals with SCN lesions. In 2 of the 6 litters in the jostle control group (J-2, J-4), the phases of pup drinking offset were scattered (Fig. 2, upper panel). In the remaining 4 litters, within-litter synchrony of pup phase was significant (Rayleigh test, P < 0.05), but the litters were not synchronized to each other (Fig. 2). This apparent synchrony of pups from SCN-lesioned dams has been observed in other studies from our laboratory;

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CLOCK TIME (hrs) Fig. 1. Single-plotted, computer-generated records of drinking behavior, body temperature, and activity of an SCN-lesioned rat given access to food for 4 h daily (09.00-13.00 h) from gestational day 8 to 19 (shaded area). Within each panel, each horizontal line represents one 24-h period, with successive days plotted below the first. Data were collected and analyzed in 10-min bins. For drinking behavior, data were log-transformed; lower and upper limits represent 0 and 3.3, respectively. Temperature data were plotted on a linear scale with lower and upper limits set to 35.5 and 40.5 °C, respectively. Activity data were log-transformed and plotted with lower and upper limits at 0 and 3.0, respectively. The arrow indicates gestational day 7, when the dam was removed from the light-dark cycle, given a lesion aimed at the SCN, and placed into constant darkness.

importantly, when within-litter synchrony persists, the average phase of the litters is not predictable. There was no significant synchrony among the pup phases of the population taken as a whole (Fig. 2, upper right). Within each of the 4 litters from SCN-lesioned dams given food cue, pup phases were synchronized on the day of weaning (P < 0.01, Rayleigh test). Furthermore, the average phases of the litters were similar. In each case, the average phase was closely correlated with the phase (06.40 h, see below) predicted by assuming that the fetuses were entrained by the food cue (P < 0.005, Rayleigh's V-test). The population profile was also clearly synchronized (right-hand portion of Fig. 2; P < 0.05). The predicted phase was determined from assumption of pup phase at birth and observations on free-running period in rats. SCN-lesioned dams given food cue start their major eating and drinking bouts with the daily initiation of the food cue, at 09.00 h; interestingly, drinking, temperature and locomotor activity bouts initiated by the food cue persist for ca. 12 h (see Fig. 1), as they do in intact

animals. Thus, the offset of dam 'activity' occurs around 21.00 h. If we assume that the fetuses are entrained to the same circadian time as the dam, then fetal clock offset would occur at 21.00 h on the day of birth. Since the average free-running period length of the drinking rhythm in rats is 24.46 h under these conditions 17, the average phase of clock offset would free-run by about 9.7 h between birth and weaning on postnatal day 21 (21 × 0.46 h). This results in a predicted phase of clock offset of 06.40 h (21.00 plus 9.7 h) at weaning on day 2 1 . Experiment 2 examined whether the effects of food cue observed in Experiment 1 were due to effects exclusively during the prenatal period. In some studies with SCN-lesioned animals, oscillators entrained by restricted feeding can generate circadian rhythms after termination of the c u e 3"20'22. Thus, it is possible that residual, food-cue-induced rhythmicity in the dam persisted into the postnatal period and influenced developing pup rhythmicity in Experiment 1. Dams received SCN lesions on gestational day 7 and were housed in constant darkness. Food cue was provided each morning (09.00-13.00 h) from gesta-

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Fig. 2, Phases of drinking offset at weaning for pups of SCN-lesioned dams given food cue (F, middle panel) or jostle control (J, upper panel) during pregnancy (Experiment 1), or given food cue during pregnancy and fostered on the day of birth to dams not previously cued (XF, lower panel, Experiment 2). For each litter, the phases of drinking offset for individual pups are depicted (small circles or triangles) around a large circle that represents 24 h on postnatal day 21. The population profile of pup phases for each group is presented in the right-hand column. For each litter or group that was significantly synchronized (P < 0.05 by Rayleigh test), the mean phase of drinking offset is depicted by the direction of the arrow; arrow length is arbitrary.

tional day 8 through 19. On the day of birth, entire litters from food-cue dams were fostered to dams that had not received food cue. The foster dams had received SCN lesions on gestational day 7 and had given birth to litters the same day. Pup phase on postnatal day 21 was d e t e r m i n e d by monitoring drinking behavior for 2 - 3 weeks after weaning. The phase of pup drinking offsets was synchro-

nized both within and between litters in pups from dams receiving food cue during gestation and fostered to uncued dams on the day of birth (Fig. 2, lower panel). A s observed in E x p e r i m e n t 1, the average phase of the litters was close to the p r e d i c t e d phase, and analysis of the p o p u l a t i o n d a t a indicated significant synchrony (Fig. 2, lower right). All of the foster darns had incomplete lesions,

295 study) or a combination of rhythms could be involved. Davis and Gorski 4'5 demonstrated maternal-fetal communicaton of time-of-day information in Syrian hamsters, and have shown that maternal entrainment of the fetus is disrupted by maternal SCN lesions. More recently, Davis and Mannion 6 showed that timed daily injections of melatonin to SCNlesioned Syrian hamsters during gestation can synchronize rhythmicity in the offspring. Removal of the pineal gland (the source of circulating melatonin) does not disrupt maternal entrainment of the fetus in either rats 16 or hamsters (EC. Davis, personal communication). From the melatonin injection data 6, it appears that a rhythm that is not necessary for maternal-fetal communication of circadian phase information is nevertheless capable of synchronizing the fetal biological clock. The present study, in demonstrating that an unrelated signal can also entrain the fetal biological clock provides strong evidence for the hypothesis 6'1s that multiple, redundant signals act in concert to entrain the developing biological clock. This proposed redundancy of signals suggests that prenatal entrainment may be of considerable physiological significance.

with 30-80% of SCN tissue destroyed. Previous data show that intact SCN are necessary for maternal influences during the postnatal period ~7. While drinking behavior in the foster dams was arrhythmic, it is possible that the remnants of the SCN influenced the results observed. However, this influence would be to increase scatter within the litters, rather than to produce within-litter coordination of phase. All dams were entrained to diurnal lighting prior to surgery; if one assumes that foster dams with partial SCN lesions had a normal free-running period in constant darkness after surgery, the foster dams would be approximately 8 h out of phase from the SCN-lesioned dams given food cue in the morning during gestation. Studies with SCN-intact animals have shown that within-litter scatter is increased when the phase of the dam during the prenatal period and the phase of the foster dam are opposite at birth 19. These opposing influences on pup phase may explain the one unsynchronized litter (XF-3) in this group. The synchronization of pup phase observed in the other 3 fostered litters strongly suggests that the synchronization of pup phase observed in Experiment 1 was due to an effect of the food cue during the prenatal period. These experiments demonstrate that periodic food access (food cue) can synchronize the biological clocks of the offspring during the prenatal period. The mechanism for this prenatal effect of food cue is unclear, as food cue generates rhythms in maternal body temperature, activity and drinking behavior as well as in nutrient availability. Any of these rhythms (or another rhythm 2 not measured in the present

We thank Mark Banister, Andrew Dowd, Joseph Keohan, and Ignacio Provencio for technical assistance. This work was supported by NIH Grant HD14427 to S.M.R., and Grant 1-945 from the March of Dimes Birth Defects Foundation. D.R.W. is the recipient of NRSA fellowship HD06976. S.M.R. is an Established Investigator of the American Heart Association.

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circadian rhythms by prenatal melatonin injections, Am. J. Physiol., 255 (1988) R439-R448. 7 Duncan, M.J., Banister, M.J. and Reppert, S.M., Developmental appearance of light-dark entrainment in the rat, Brain Res., 369 (1986) 326-330. 8 Fuchs, J.L. and Moore, R.Y., Development of circadian rhythmicity and light responsiveness in the rat suprachiasmatic nucleus. A study using the 2-deoxy-[1-14C]glucose method, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 12041208. 9 Kreiger, D.T., Hause, L. and Krey, L.C., Suprachiasmatic nuclear lesions do not abolish food-shifted circadian adrenal and temperature rhythmicity, Science, 197 (1977) 398-399. 10 Moore, R.Y., Organization and function of a central nervous system oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc., 42 (1983) 2783-2789. 11 Moore, R.Y. and Card, J.P., Visual pathways and the