Effect of ovarian hormones on synchrony of hamster circadian rhythms

Physiology & Behavior, Vol. 24, pp. 741-749. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.

Effect of Ovarian Hormones on Synchrony of Hamster Circadian Rhythms I L. P. M O R I N

Dartmouth College, Hanover, N H 03755 R e c e i v e d 10 S e p t e m b e r 1979 MORIN, L. P. Effect of ovarian hormones on synchrony of hamster circadian rhythms. PHYSIOL. BEHAV. 24(4)

741-749, 1980.--Adult ovariectomized hamsters which were implanted with Silastic capsules containing estradiol benzoate (EB), progesterone (P) or nothing (Blank) were exposed to prolonged constant light (LL) with free access to running wheels. Freerunning locomotor rhythms were recorded on an event recorder. Light energy levels averaged 12.2 uwatts/cm2 or 48.3 Lux. Rhythm splitting occurred in 1/7 animals given EB, 5/7 given P and 5/11 Blank implanted animals. Altogether, 1/7 EB, 6/7 P and 8/11 Blank implanted animals showed some form of splitting or other rhythm anomaly such as desynchrony of endogenous oscillations. There was a high incidence (10/11) of animals showing an additional transient rhythmical component running across the split condition. The study supports the concept that a multi-oscillatory system normally generates a cohesive circadian behavioral rhythm. Estradiol appears to facilitate the synchrony of these oscillations. Circadian rhythms

Estradiol

Progesterone

Activity

W H E N the circadian activity rhythm of male hamsters is entrained to a Zeitgeber (light:dark schedule), two presumed components of the nocturnal activity phase (alpha) become synchronous, stably interactive and phase-locked to the Zeitgeber phase cues of dawn (lights on) and dusk (lights off). The two components have been referred to as the morning (M) and evening (E) oscillations, respectively. In constant lighting conditions, the freerunning period of the rhythm is thought to be derived from the interaction of the two oscillations [6,10]. Upon prolonged exposure of male hamsters to constant bright light (LL), the M and E oscillators will frequently split or desynchronize and assume different frequencies with no initial stable phase relationship between them [10,11]. Eventually, most split oscillations resynchronize with a new stable relationship about 180° apart. Splitting provides some of the strongest support for multioscillator theory of circadian rhythm generation. Daan et al. [7] have suggested that a steroid hormone (testosterone) may alter the circadian period (tau) of male mouse wheel running by selectively affecting one component of the multiosciUatory circadian system. Such selective action is presumed to (a) differentially alter tau of the E or M components and/or (b) directly alter the amplitude of interaction (coupling) between E and M. Either action will change the relationship between E and M, the effect of which will be a change in the overall tau of wheelrunning [6]. Female hamsters show earlier onsets of circadian wheel running on days of the estrous cycle coincident with elevated estradiol. Ovariectomized hamsters will shorten the circadian period of wheel running when administered estradiol benzoate [9,16]. If the coupling between E and M is

Hamster

enhanced by estradiol, then this hormone might be expected to prevent splitting. Therefore, the present experiment was designed to explore the effects of constant light on the synchrony of circadian rhythms among ovariectomized and hormone treated hamsters. METHOD

Adult female hamsters (Lakeview Hamster Colony, Newfield, NJ) were housed in the colony room under a 14 hr incandescent light, 10 hr dark photoperiod (LD 14:10; lights on at 0700). Food (Purina Rat Chow) and tap water were continuously available during all phases of the experiments. After at least one month in the colony room, animals that showed at least 2 consecutive estrous cycles, as determined by the abundant postovulatory discharge, were selected for further study. Two weeks before exposure to constant light (LL), cyclic hamsters were ovariectomized and divided into three groups: estradiol benzoate (EB; n=7); progesterone (P; n=7); and no hormone controls (Blank; n= 11). Prolonged hormone administration was accomplished by subcutaneous implantation of unsterilized Silastic capsules containing crystalline hormone. Functional length of an EB capsule was 5 mm; of the P implant, 2× 10 mm; control animals received an empty 10 mm capsule. The capsules were made from Dow-Corning Silastic tubing (1.56 mm i.d. and 3.15 mm o.d.) and were sealed with Silastic adhesive (Type A). This method of delivery would probably maintain serum estradiol and P levels within the range of those found at the time of sexual receptivity onset [4,5].

1This research was supported by Biomedical Research Funds from Dartmouth College and National Institute of Child Health and Human Development grant HD 10740. Ms. Sarah Daniels provided excellent technical assistance.

C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/040741-09502.00/0

742

MORIN

One week following surgery, the hamsters were placed individually in translucent polypropylene cages (48x27x20 cm) each containing a 17 cm diameter running wheel. The hamsters had unrecorded access to the wheels for 1 week in LD 14:10 prior to being moved to a new room illuminated by fluorescent LL. The L L intensity measured 0.5 cm inside the cage front averaged 12.2/zwatts/cm". This corresponded to an average 48.3 Lux. Wheel running activity in L L was continuously recorded by event recorders. Continuous white noise (mean intensity=76 dB) helped mask extraneous sounds. Between group comparisons were limited to the initial 135 days of running in LL. About this time three animals implanted with EB died and the group size became too small for useful statistical comparisons. Wheel running by selected animals showing unusual rhythm patterns continued to be recorded for long term assessment of individuals. Such recording was particularly maintained if the animal's rhythmicity had not become more or less stable. When activity recording was stopped, the animal was killed with anesthesia and examined for the presence of the appropriate Silastic implant. F o r every animal, each 24 hr strip of event recorder output was pasted on a chart, day below preceding day, providing a source for visual analysis. Estimates of the circadian period were made by measuring the angle of a line visually fitted through the onsets of activity and converting the results to hours [16]. Data were also transferred from the pasted charts to a computer by using a Tektronix 4954 Graphics Tablet to trace the duration of running bouts across each consecutive 24 hr period during a 15-25 day segment of data. This procedure limited the resulution of data to about 1 revolution per minute. A periodogram analysis [12] was used to aid visual interpretation of the oscillations in the running data. Valid application of the chi square statistic to the peridogram requires an initial search for rhythm peaks at hourly intervals from 14 to 30 hr, followed by a more refined examination of the obtained peaks [12]. This procedure was not employed because high amplitude peaks were often narrowly focused on rhythm periodicities falling midway between two integer hours (e.g., 24.5 fell between 24 and 25 hr). The hourly measures of the rhythm intensity as derived from the periodogram (Qp values) on either side of the peak were often marginally significant, at best, when the chi square statistic was applied. Therefore, periodogram points were generated each tenth of an hour for the interval 5-30 hr and probability based on the chi square distribution was used as a guideline in association with visual analysis of the running record. Each method for estimating periodicities served to check the reliability of the other. Statements about periodicity of any animal were derived from the application of both methods. RESULTS

Visual and periodogram analysis of the data showed that EB, P or Blank treatments differentially affected running rhythmicity of ovariectomized hamsters. Splitting of the running rhythm, defined as a rapidly occurring, transient dissociation of two oscillations leading to a new stable relationship of about 180° between similar phase points of the two oscillations was the most frequently observed form of rhythm desynchrony among all animals. In addition to splitting, other forms of rhythmicity classified as desychrony were the presence of a high or low frequency oscillation

without obvious splitting of the E and M oscillations or a very diffuse alpha characterized by highly erratic alpha onset and offset times associated with one or more rapid changes in tau or a phase jump of alpha. EB treatment tended to prevent all forms of rhythm desynchrony. Only 1/7 (14%) of EB implanted hamsters showed splitting; the remaining 6/7 (86%) animals maintained normal synchronous circadian rhythms with a mean tau=24.4 _+ .10 hr (Fig. 1). Splitting occurred in 5/7 P and 5/11 Blank treated animals (Figs. 2 and 3). there was no evidence that P treatment produced effects different from Blank treatment. At the onset of splitting it was impossible to determine clearly for any animal which oscillatory component of running became the leading oscillation in the split condition. However, among those animals across all groups which showed splitting into two dominant running components, wheel running records provided evidence of at least one additional component manifest at the time of splitting in 10/11 animals. This was most evident in two animals, one of which is shown in Fig. 4. During the days immediately following splitting, visual and periodogram analysis (Fig. 5) of this record suggested at least four separate oscillations. Two oscillations each with a nearly 24 hr period became phase locked about 12 hours apart and existed simultaneously with oscillations estimated by periodogram to be 21.7, 26.1 and 27.5 hr. Visual inspection of the original record supported the presence of simult.aneous 24 hr oscillations, a 21.7 hr oscillation and a 26.1 hr oscillation; the 27.5 hr oscillation was not visually evident. Among several other animals, a third oscillation assessed visually varied from being quite apparent (Fig. 2) to nearly absent. In the absence of strong supportive evidence for a third oscillation from some animals, a weakly apparent third oscillation would probably be construed as background noise. The oscillation visible at the time of splitting disappeared rapidly for all animals, but visual inspection of the running records showed that some form of high or low frequency oscillation reappeared transiently among 10/11 split animals and was particularly obvious in a few (Figs. 3 and 4). The single EB implanted animal that showed splitting also manifested the third oscillation during the split condition (Figs. 5 and 6). Periodogram analysis of this animal's record indicated a third rhythm periodicity of 23.4 hr (and its 11.7 hr harmonic), each obtaining a marginal level of statistical significance (p<0.02 and <0.01, respectively; Xe). During Days 103-118, the periodogram analysis was supported by a visual calculation [ 16] of an oscillation with a 23.4 hr period. Among Blank implanted desynchronous animals, 6/8 had continuing high or low frequency oscillations during the desynchronous condition; 5/6 P implanted animals provided similar data. Overall, 6/7 P implanted and 8/11 Blank implanted animals exhibited some form of rhythm desynchrony compared to 1/7 EB implanted animals (/9<0.025 in each case; Table 1). Statistical analysis also showed that there were no presynchrony tau differences, differences in tau during the split condition or in latency to desychrony between P and Blank groups based on animals for which such numbers could be clearly calculated (n=5 and 7, respectively). The mean predesynchrony tau for all such animals was 24.49 _+ 0.07 hr; mean split tau=23.98 _+ 0.03 hr and mean latency to desynchrony=63.7 _+ 6.7 days (range=31-114). There was no significant correlation between prior tau and latency to desychrony or tau during the split condition.

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DISCUSSION

The results demonstrate a role for estradiol in facilitating integrated circadian rhythmicity of female hamsters. EB released systemically from long term subcutaneous implants generally prevented splitting and desynchrony of wheel running rhythms generated by LL-exposed ovariectomized hamsters. Neither P nor Blank implants had such an effect. The action of estradiol to prevent dissociation of endogenous oscillations is now documented as a second known action of that steroid on a circadian rhythm system. Morin et al. [9]

previously demonstrated that estradiol administration results in a shortened tau in female hamsters. Under the present experimental conditions, estradiol did not facilitate circadian rhythm synchrony in all animals. One animal displayed splitting and apparent additional oscillations both as the split occurred and during the stable split condition. In other instances, EB implanted in already split, ovariectomized hamsters did not restore synchrony to the unsplit state (Morin, unpub.). Hoffman [8] has presented evidence for more than two free-running oscillations in the day-active animals, Tupaia belangeri, and the complex running patterns of arctic redbacked voles [ 13] suggest more than two oscillations regulating running rhythmicity. The present data provide strong support for a multi-oscillatory system with more than two oscillations regulating female hamster circadian running rhythms. The E and M components [10] appear to be primary oscillations which separate with temporarily different frequencies during splitting. The resultant tau always become less than in the unsplit condition (present data; [11]). Nearly all hamsters with split rhythms in this experiment appeared to have at least one freerunning (secondary) oscillation traversing the coupled split rhythm. The initial manifestation of these secondary rhythmic components usually occurred during the days that the split was developing (Figs. 2, 3 and 4). This coincidence suggests the possibility that splitting may be causally related to the appearance of the secondary rhythmic components. Following divergence of the two primary components, the secondary component diminished in magnitude or disappeared altogether. Later during the stable split, at least one other secondary oscillation usually appeared; whether this was a reappearance of the earlier secondary cycle could not be determined. Nor has it been possible to determine to what extent the early or later secondary oscillations were coupled to the stable split primary oscillations. In numerous instances, the temporal patterning of running (onset, duration, termination) during the activity phase of a primary oscillation changed systematically over a period of days under the apparent influence of a secondary oscillation, but wheel running associated with a secondary oscillation generally did not occur during the inactive phase of a primary oscillation, (e.g., Figs. 3, 4 and 6). Because the running associated with a secondary oscillation tended to appear only in coincidence with an active phase of a primary oscillation, this effectively created gaps in the continuity of a secondary oscillation running record. These gaps and the general instability of the overall observed running rhythmicity detract from the periodogram's utility and reemphasizes the need for visual analysis of the records. The present results tend to mimic a set of data generated artificially. Winfree [14] used 71 slightly coupled electronic oscillators to show the existence of (a) secondary high frequency oscillations which were (b) ephemeral, but which, through an apparent process of internal phase coincidence, may have been responsible for the (c) abrupt changes in tau of the primary components. The electronic data suggest that the activity phase of a primary oscillation may be derived from coincidence of multiple high frequency oscillations each driving a discrete amount of activity. The ephemeral character of the secondary electronic or wheel running oscillations suggests that a critical phase relationship between two or more oscillations may be necessary to observe the activity of any oscillation. In this sense, a dominant oscillation could serve as a gate for the expression of a secondary oscillation, provided that the two cycles become appropri-

748

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F I G . 6. C o n t i n u o u s r e c o r d o f a n o v a r i e c t o m i z e d h a m s t e r i m p l a n t e d w i t h E B . T h i s is t h e o n l y a n i m a l i m p l a n t e d w i t h E B that s h o w e d splitting. P e r i o d o g r a m a n a l y s i s o f D a y s 100-124 is s h o w n in Fig. 5. F o r o t h e r details, see Figs. 1 a n d 2.

ESTROGEN AND RHYTHM SYNCHRONY

749

TABLE 1 EFFECTS OF OVARIANHORMONES ON CIRCADIANRHYTHMDESYNCHRONIESAMONG OVARIECTOMIZEDHAMSTERSHOUSEDIN LL Secondary oscillations: Treatment

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*EB differs from P (/9<0.025) and Blank (p<0.025); Fisher's Exact Tests.

ately phased. The concept of circadian gates have been discussed relative to Drosophila emergency where one circadian rhythm appears to regulate the expression of a second circadian rhythm [10]. A circadian gate may actually have no overt rhythmicity associated with it. As seen in Fig. 3 (around Day 125), a secondary oscillation with a period of 23.4 hr (Fig. 5) is free running through two primary oscillations. Overt rhythmic activity is dictated by the joint function of both primary and secondary oscillations. The periods of non-running during the active phase of the primary oscillations are equally rhythmic and serve to emphasize the fact that running probability during a primary oscillation is markedly affected by appropriate phase coincidence with another oscillation and not absolutely determined by any primary oscillation.* Secondary oscillations akin to those presented here have not previously been described for hamsters although dissociation of the primary oscillations in hypophysectomized hamsters has been shown [15]. The failure of other investigators to observe secondary oscillations may be related to

the sex of the animal tested or to the level of illumination. The splitting studies by Pittendrigh [10,11] used male hamsters and the light intensity was 200--400 Lux; more than two oscillations were not described. The circadian rhythm system of the hamster is sexually differentiated and males do not respond in an observable manner to exogenous estradiol (unlike the female's system; [16]). The absence of a hormone-dependent circadian rhythm response may be related to underlying sex differences in oscillatory neural structures which prevent the appearance of secondary oscillations. Alternatively, the much lower light intensity used in the present study (about 50 Lux) may have permitted the expression of rhythmicities which are normally masked [1] by higher light intensities. It is possible that the observed coincidence of oscillations is a product of a phase response curve for masking, the masking occurring differentially according to the relative phases of internal oscillations. The use of behavioral measure which is minimally masked by light could elucidate this matter [2].

*Note added in proof: an alternative view is expressed by Davis and Menaker (Am. J. Physiol., in press). REFERENCES

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