Development of Hamster Circadian Rhythms. I. Within-Litter Synchrony of Mother and Pup Activity Rhythms at Weaning1

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1 BIOLOGY OF REPRODUCTION 33, (1985) Development of Hamster Circadian Rhythms. I. Within-Litter Synchrony of Mother and Pup Activity Rhythms at Weaning1 FRED C. DAVIS2 and ROGER A. GORSKI Laboratory of Neuroendocrinology of the Brain Research Institute and Department of Anatomy UCLA School of Medicine Los Angeles, California ABSTRACT The circadian wheel-running activity rhythms of individual hamster pups raised and maintained in constant dim light were measured beginning at 18 days of age. Records of the postweaning free-running activity rhythm were used to determine the phase of a pup s rhythm on the day of weaning and its phase relationship to its mother s rhythm. Although raised in constant light, the rhythms of pups within a litter were approximately synchronous and in phase with their mother s activity rhythm. These results indicate that the circadian oscillator underlying the activity rhythm is functional prior to weaning and is entrained by some as yet unidentified aspect of maternal rhythmicity. Furthermore, the results suggest that even in the absence of external entraining cycles, behavioral rhythms, and perhaps physiologic rhythms as well, of a mother and her offspring are normally synchronized. INTRODUCTION Although it has been known for more than 30 years that animals do not need to experience 24-h cycles during development in order to show circadian rhythmicity when mature (Aschoff, 1960; Bunning, 1973), discoveries of the last decade and a half have brought renewed focus to questions about the development of circadian rhythms. The first and most important of these discoveries is the identification in animals of discrete circadian pacemaking structures or tissues (Menaker et a!., 1978). In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is such a structure; it is both rhythmic in its neural activity (Green and Gillette, 1982; Inouye and Kawamura, 1982) and important for the control of overt rhythms (Rusak and Zucker, 1979). With the identification of a specific substrate for the generation of circadian oscillations, questions about the relationship between the structural and functional development of circadian mechanisms can now be addressed. Second, the develop- Accepted February 26, Received December 12, This research was supported by NIH grant 5R01 HD to R.A.G. and by NIH National Research Service award 5F32 HD from NICHD to F.C.D. 2Correspondence to present address: Fred C. Davis, Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA ment of a few circadian rhythms has now been well enough studied to ask not only if the circadian rhythms develop but also how the phase of the emerging rhythm is determined when development occurs in the absence of the normal external entraining agent, the light-dark cycle. Understanding when and how the phase of the oscillation is set during development can provide insight into the early functional development of circadian pacemaking tissue (Minis and Pittendrigh, 1968). In addition, the analysis of phase determination during development in mammals has revealed a previously unexplored aspect of the relationship between a mother and her offspring. Circadian rhythms of developing mammals appear to be entrained by maternal rhythms, and thus have a specific temporal relationship to them, even in the absence of any other external entraining cycle. The entrainment of developing rhythms by maternal rhythmicity was first suggested by Deguchi (1975), who studied the development of rhythmicity in the rat pineal enzyme N- acetyltransferase (NAT) under constant lighting conditions. By killing 3-wk-old pups at different times of day, he observed a rhythm in a population of pups maintained in constant darkness both before and after birth. He found that the phase of the population rhythm depended on that of the maternal rhythm and that it persisted with a predictable phase in 353

2 354 DAVIS AND GORSKI constant conditions even after separation from the mothers. Although Deguchi could not measure a rhythm from an individual pup, his results indicated that as circadian rhythms in rats develop they are entrained by maternal rhythms (also see Reppert et al., 1984). The synchronization of circadian rhythms among rat pups and a relationship to maternal rhythmicity has now been indicated for the rhythm in plasma corticosterone as well (Takahashi et a!., 1979, 1982; Hiroshige et al., 1982; Takahashi and Deguchi, 1983). In a preliminary report (Davis and Gorski, 1982) and in this paper we extend the documentation of maternal entrainment to another species, golden hamsters, and to a behavioral rhythm, that of wheelrunning activity. Similarly, Takahashi et a!. (1984) have recently reported evidence for maternal entrainment of locomotor activity in rats. The wheel-running activity rhythm of rodents is particularly useful for the analysis of the synchronization between a mother and her offspring because it allows long-term and continuous records to be obtained from individual animals and consequently more accurate estimates of rhythm parameters such as phase and period than can be obtained with other rhythms. In addition, the circadian regulation of wheel-running activity by the SCN has been particularly well documented (Schwartz et al., 1980; Inouye and Kawamura, 1982; Rusak and Groos, 1982). Thus the phase of the wheelrunning activity rhythm and its entrainment during development is likely to reflect entrainment of the developing SCN. In the present paper, we describe synchronization between the activity rhythms of a hamster mother and her pups under constant lighting conditions that is established at some point in development prior to weaning but by an as yet undetermined Experimental mechanism. MATERIALS AND METHODS Protocol Male and female golden hamsters (Mesocricetus auratus) were obtained from Charles River Lakeview where they were raised on a light-dark cycle of L14.5: D9.5 (lights on h EST). They were received at 6 wk of age and subsequently maintained on a cycle of L14:D10 (lights on h PST). At 12 wk of age, the estrous cycles of individually housed females were monitored, and each was housed with two males on the projected night of ovulation only. One to 4 days later each pregnant female was transferred to a running-wheel cage maintained in dim constant light (dim LL; <1 lux) with food and water continuously available. Two females (#5 and #7) entered dim LL on Day 1 of gestation and the other two (#4 and #13) entered on Day 4. Each mother and her litter remained together undisturbed until the pups were 18 days old. On postnatal Day 18 (Day 1 is the day of birth) two pups from each litter (without regard to sex) were placed in their own individual running-wheel cage, still maintained in dim LL. From 13 to 19 h later two more pups from each litter were also placed in separate running-wheel cages. Pups were assigned to cages so that members of a litter were not adjacent to each other. The remaining pups were taken from their mothers 3 days later and weighed. The wheel-running activity of the four mothers and their 16 pups was recorded for the next 4 wk. Two of the four mothers (#7 and #13) were given a 10% solution of deuterium oxide (D20; KOR Isotopes, Cambridge, MA, 99.8%) for drinking water during part of the experiment, from either Day 6 of gestation to postnatal Day 14 (#7) or from Day 10 of gestation also to postnatal Day 14 (#13). The purpose for giving these mothers D2 0 was to increase the period of their free-running activity rhythms (Richter, 1977), thereby creating greater disparity in freerunning period among the four different mothers than there was likely to have been otherwise. This in turn assured that the phase angles of the mothers activity rhythms were not the same on postnatal Day 18 when pups were taken from them. The amount of drinking water consumed by the mothers was periodically measured. Data Analysis Wheel-running activity from mothers and pups was recorded with an Esterline-Angus event recorder. The record from each hamster was cut into 24-h segments and each day was pasted below the preceding day on poster board (Fig. 1). Using activity onset as a phase reference point, phase angle of the activity rhythm on postnatal Day 18 was determined relative to clock time for both mothers and pups. For pups, the phase angle of activity onset on Day 18 was not determined from activity on that day, but was instead taken as the Day 18 intercept of a regression line through the activity onsets of subsequent days, as shown in Fig. 2. Only unambiguous onsets were used for the regression analyses, with the number of onsets used ranging from 12 to 28 per pup with a mean of 23. The regularity of onset times varied among pups; the standard deviation of onsets around the regression line ranged from 0.34 to 1.88 h with a mean of The precision with which Day 18 phase angle was determined is indicated by the 95% confidence intervals for the Day 18 intercept. These ranged from ± 0.28 h to ± 1.74 h with a mean of ± The phase angle of a mother s activity rhythm on day 18 was determined in a similar manner, using either pre- or postweaning onsets. The 95% confidence intervals for the Day 18 phase angles of the four mothers are ± 0.68, ± 0.29, ± 0.94, and ± 0.47 h. For both mothers and pups, the regression analyses were also used to estimate free-running periods. The phase angle values determined for mothers and pups have been expressed in clock hours and

3 DEVELOPMENT OF CIRCADIAN RHYTHMS p _. - -:;_ Se.. - -a a a. -- Days E- : B = W!= E #7 I D20 Days ] Time Of Day In Hours FIG. 1. The wheel-running activity records of two mothers in dim constant light. The record of each mother has been presented in duplicate (double-plotted) to aid visualization of the free-running rhythms. The days during which mother #7 was given D2 0 are indicated as are the days when litters were born (B) and when weaning began (W).

4 356 DAVIS AND GORSKI C. 0 u0o - V a. 0.C 41 0 ne. e U.e,., ov so C.C e u. VU C u. n V C 41 CO C Va Cl) v. C o. e n.u..e. 0 c 0 o it :. it.. i cj. a C!) 0 _;, I-v U 4. I- N. 0 N- a-v

5 DEVELOPMENT OF CIRCADIAN RHYTHMS 357 plotted on a circle representing the 24 h of postnatal Day 18 (see Fig. 3A). These values are also used to calculate phase angle differences between a pup and the time it was weaned 1pw and between a pup and its mother PM#{149} To determine if phase angles are randomly distributed in time or instead bear some relationship to each other, they are converted from hours to degrees and treated as points on a 360#{176} circle, and the null hypothesis that they are randomly distributed around the circle is tested using the Rayleigh test (Batschelet, 1972). The average phase angle for a group of values is determined by vector addition, and variation among the values is expressed by the length (r) of the mean vector. Other statistical methods it-test, analysis of variance (ANOVA)l are used where indicated. RESULTS The phase angles of activity onset on postnatal Day 18 for the four mothers and their 16 pups are graphically shown in Fig. 3A. It can be seen that the activityonsets of the pups occurred at widely different times of day and that their distribution was not significantly different from uniform. Thus the activity rhythms of the pups did not have a constant phase relationship to some factor associated with clock time. Pups were also found not to show a constant phase relationship to the time at which they were weaned (Fig. 3B). Although pups almost always show some activity upon introduction to their own running-wheel cage (see Fig. 4), the time at which this occurs does not strictly determine phase angle of the activity rhythm. If this were the case, all pups would show activity onsets near the time of weaning or at some other consistent phase relationship to it rather than the variation shown in Fig. 3B. Unlike clock time or weaning time, maternal rhythmicity appears to have a significant determing influence on the phase angle of a pup s activity rhythm. The phase relationship between the activity rhythm of each pup and its mother on the day they are separated is sufficiently similar among all pups such that the distribution of pup phase angles relative to mother time is significantly different from uniform (Fig. 3C). Pups that are beginning activity at very different clock times are clearly beginning their activity at approximately the same time relative to their mothers activity rhythms. The average phase angle difference between mothers and pups (1.6 h) indicates that, at the time of weaning, the activity portion of a pup s rhythm coincides approximately with that of its mother. The scatter in phase angles of pups relative to clock time (Fig. 3A) is apparently due to differences in average phase angle among litters. Within each litter, the mother and pups show significantly similar phase angles (r values = 0.876, 0.990, 0.866, 0.941; P<0.025 in all cases), but the average phase angles of the litters occur at very different times (r = 0.374, P>0.10). This is reflected in the distribution of maternal phase angles in Fig. 3A. The synchronization between a mother and her pups at weaning indicates that either unidirectional or mutual entrainment among A. B. C. M FIG. 3. Phase angles onset of the four mothers (M) and their 16 pups (.) on postnatal Day 18 plotted relative to clock time (A), and pup onsets plotted relative to weaning time (B) and to their mother s onset time (C). Each large circle represents the 24 h of postnatal Day 18. The average phase angle of the pups is indicated by the arrow, the length of which (r) reflects the scatter in phases. P is the probability that the distribution is not different from uniform (Rayleigh test).

6 358 DAVIS AND GORSKI.01 e 0 I >1.) II) C) It) C.),B4U$Od SIBO

7 DEVELOPMENT OF CIRCADIAN RHYTHMS 359 them has occurred at some point in development prior to weaning (see Discussion). However, this entrainment need not have continued up to the time of weaning, but instead could have occurred and ended much earlier in development given two conditions: 1) the phase relationships between mothers and pups when entrainment ceased are similar to those observed at weaning, and (2) the free-running periods of the individuals within a litter are sufficiently similar that the phase relationships previously established do not change appreciably by the time of weaning. It is not known to what extent either of these conditions hold. However, estimates of postweaning free-running period for pups and mothers indicate that entrainment could have ended long before weaning. For example, assuming that entrainment occurs prenatally only and that the phase angles of a mother and her pups are identical at birth, the observed within-litter differences in free-running period are small enough that synchronization is still preserved 18 days later. In this hypothetical situation, the r values after 18 days of free running would be , 0.806, , and (P<0.05 in all cases), which are comparable to the observed values given earlier. Thus, the similarity of phase angles within a litter at weaning indicates that entrainment has occurred at some point before that time, but it cannot be specified when. Although the time of weaning was not found to determine phase angle of a pup s activity rhythm, it may nevertheless have some effect on it. The pups within each litter were weaned at two different times of day, and in three of the four litters one weaning time occurred within the 12 h before the mother s activity onset and one within the 12 h after (in the fourth litter, both weaning times occurred within the 12 h before the mother s onset). The mean phase angle difference (pm) for pups weaned before their mother s onset is 3.49 h (± SEM of 0.461) while that of those weaned after is 0.80 h (± 0.667; P<0.01, t-test), indicating an effect of weaning on phase angle. Weaning may have advanced the rhythms of one group, delayed the rhythms of the other, or had a small effect on both. Within the fourth litter, weaning time appeared to have no effect on /PM (0.10 ± 0.73 vs ± 0.48 h). The addition of D2 0 to the drinking water had the expected effect on the mothers freerunning periods. Table 1 shows that during the preweaning time of exposure to D2O, the treated mothers showed free-running periods longer than those of the untreated mothers and longer than their own free-running periods after the treatment was discontinued (postweaning). Table 1 also compares the mean postweaning free-running periods of pups from the D2 0 and H2 0 mothers, and although the pups from the D2 0 mothers were themselves undoubtedly exposed to D20, this treatment, which ended 4 days before weaning, appears to have had no long-lasting effect on period. This suggests that D2 0 does not affect circadian pacemaker development. However, although treatment with D20 began on Day 6 or 10 of gestation, as long as a week might be required for D2 0 to equilibrate with body fluids and for its full effect on period to be realized (Katz et a!., 1962; Daan and Pittendrigh, 1976). Thus, exposure of the developing hamsters to effec- TABLE 1. Free-running periods (h) of mothers before and after weaning and mean free-running periods of their litters. H20 D2O Mother number Before weaning After weaning Litter Mean SEM n Grouped pups ** *ptrnong litters, P<0.025 (ANOVA). ssbetween treatments, P>0.50 (t-test).

8 360 DAVIS AND GORSKI tive amounts of D20 may not have occurred until Day 13 of gestation in one case (mother #7) or postnatal Day 2 in the other (mother #13). These ages may be too late to affect pacemaker development. Although determined from only a small sample, the significant variation in average free-running period among the four litters of Table 1 suggests that a common developmental history or genetic relatedness affects free-running period. The amount of water consumed by the mothers was greatest during late lactation, and the variation among mothers seemed more related to the size of the litter rather than to treatment with D20 (Table 2). However, the smaller litters of the D2 0-treated mothers might be related to the treatment. In addition to the four hamsters represented in Table 2, three other hamsters became pregnant in the experiment, one of which was given D20. This mother eventually lost her litter, whereas the two receiving H20 raised litters of 9 and 11 pups. DISCUSSION We have found that, when litters of hamsters are raised under constant lighting conditions (dim LL), the phase angles of the pups circadian activity rhythms at weaning are not randomly distributed. Instead, the activity time of each pup is approximately coincident with that of its mother and with each of its siblings (Fig. 3C). Although these results demonstrate a relationship between the activity rhythms of a mother and her pups at weaning, the actual coincidence or noncoincidence of a mother s activity with that of her pups when they are together is not known. The results do not address a direct relationship between maternal and pup activity, but rather address the relationship between the circadian pacemakers underlying activity. If activity onset of the wheel-running activity rhythm of mothers and pups is assumed to represent the same phase reference point of an underlying pacemaker, then the results of Fig. 3C indicate a relationship between the pacemakers of mothers and pups established sometime prior to weaning. The postnatal activity records of the pups have been used to determine the timing of that point in the pacemaking oscillation that comes to be associated with activity onset. The synchronization within a litter strongly suggests that an entrainable circadian oscillator is functional in the pups before weaning, whether or not an overt activity rhythm is itself fully developed at that time. The relationship between mothers and pups indicates that, for any particular phase of their pacemaking oscillations, mother and pups are approximately coincident on the day of weaning. Because the time of weaning appears to have had some effect on the pups phase angles, possibly obscuring the precise phase relationship of the pups to their mother, it is unclear how much significance should be given to the specific average observed phase angle difference of 1.6 h (Fig. 3C). The within-litter synchrony among pups and between a mother and her pups could involve four different mechanisms: 1) if circadian oscillations begin at a particular point in development and at a particular phase of the oscillation, then developmental synchrony among pups could produce synchrony among the oscillations; 2) if birth occurs at a restricted phase of the mother s circadian cycle (Gattermann, 1983), then birth itself could synchronize the rhythms of pups to their mother; 3) pups could mutually entrain each other and act on the mother to entrain her rhythm; and TABLE 2. Maternal water consumption (mi/day), litter size, and mean postnatal Day 21 body weights (g) of pups not used for recording. Water consumption Mother Gestation Early lactation Body weights Late No. lactation Postweaning Pups Mean SEM n * * *10% D20.

9 DEVELOPMENT OF CIRCADIAN RHYTHMS 361 4) the mother could act on the pups to entrain each of their rhythms to her own. Although some role for each of these cannot be excluded, it is likely that entrainment by maternal rhythmicity is especially important for phase determination in the pups. The most direct evidence for this is results showing that synchrony among pups at weaning depends on normal maternal rhythmicity; litters born to mothers with lesions of their suprachiasmatic nuclei do not show the within-litter synchrony normally observed at weaning (Davis and Gorski, 1983). Neither developmental synchrony, birth, nor interactions among pups seems adequate to cause synchrony in the presence of a lesioned mother. In the present study, the phase of a mother s rhythm at weaning appeared to depend only on the free-running period of her rhythm during gestation and lactation. There was no indication that she was affected by some entraining influence that was at any time out of phase with her own rhythm (Fig. 1). Therefore, although pups could at some point have an entraining influence on their mother (mechanism 3), it is most likely that the relationship between a mother and her litter is initially established by the mother. Furthermore, there is as yet no evidence indicating that interactions among the pups within a litter play a role in the synchronization of their activity rhythms. In addition to the evidence from SCN-lesioned mothers mentioned above, rhythms in plasma corticosterone (Hiroshige et al., 1982) and in pineal NAT (Reppert et a!., 1984) appear to be free running in blind rats even when they are housed with sighted siblings exposed to a light-dark cycle. The possibility that initial synchronization among pups is at least in part caused by similar rates of development (mechanism 1) is intriguing because it requires that the initiation of oscillations be associated with a discrete and possibly identifiable point in development. Also, the conditions in the pacemaking tissue at that point would have to be related in some way to a unique phase of the oscillation. Evidence for maternal entrainment of postnatally assayed rhythms in rodent pups has now been obtained for rat pineal NAT activity (Deguchi, 1975; Reppert et al., 1984), rat plasma corticosterone (Takahashi et al., 1979, 1982; Hiroshige et al., 1982; Takahashi and Deguchi, 1983), rat locomotor activity (Takahashi et al., 1984), and hamster wheel-running activity (this paper). In addition, Reppert and Schwartz (1983) have shown that metabolic activity (2-deoxyglucose uptake) in the fetal rat suprachiasmatic nucleus varies predictably with the mother s circadian cycle. Although maternal entrainment may be a general consequence of the relationship between a mother and fetus or between a mother and newborn, the significance of such entrainment is not known. The possibilities are of two general types. First, it may be important for specific maternal and fetal rhythms in physiology or behavior to be temporally coordinated for the normal growth and development of the offspring. For example, hypothetical rhythm in the nutritional demands of the offspring may need to match a maternally derived rhythm in nutritional supply, the latter indicated by rhythms in fetal growth and in the body weights of pups during lactation (Barr, 1973; Levin and Stern, 1975). Although it is not known if such rhythms of the fetus or newborn are endogenously generated and maternally entrained or are instead only passive responses to maternal rhythmicity, they illustrate the opportunity if not the need for some mechanism of temporal coordination to evolve. Interestingly, however, weight gain of rat pups during lactation is greatest during the day when the mother spends more time with the litter, but immediately after weaning both the weight gain rhythm and a rhythm in the liver enzyme tyrosine aminotransferase reverse their phases (Honova et al., 1968; Levin and Stern, 1975). The propensity for nocturnal feeding in rats immediately after weaning and in adulthood may begin before weaning; latency to nipple attachment in rat pups is less at night than in the day (Holloway et al., 1979; Henning and Gisel, 1980). It may be that a rhythm in food intake during lactation is imposed by the mother in opposition to rather than in concert with endogenous rhythmicity of the pups. Other rhythms such as locomotor activity, pineal NAT, and plasma corticosterone do not appear to change phase at weaning and are at all times generally in phase with those same rhythms of the mother (Deguchi, 1975; Takahashi et al., 1979; Teicher and Flaum, 1979). The significance of maternal entrainment might also stem from a general need for the fetus or newborn to be entrained during development. This could be necessary for the normal development of circadian rhythms or for the eventual coordination of the newborn with external periodicities to which it could not be

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