social stimuli fail to act as entraining agents of circadian rhythms in the golden hamster

J Comp Physiol A (1992) 170:181-187 J o u r n a l o f C o m p a r a t i v e ~o~, Neural,

a n d

P h y s i o l o g y A .n.~.. Phyu~gy

�9 Springer-Verlag 1992

Social stimuli fail to act as entraining agents of circadian rhythms in the golden hamster Roberto Refinetti*, Dwight E. Nelson, and Michael Menaker

Department of Biology, University of Virginia, Charlottesville, Virginia 22901, USA

Accepted November 10, 1991

Summary. The ability of social stimuli to act as entraining agents of circadian rhythms was investigated in golden hamsters (Mesoericetus auratus). In a first experiment, pairs of male hamsters (one of them enucleated and the other intact) were maintained under a light-dark (LD) cycle with a period of 23.3 h. Running-wheel activity was recorded to determine the effect of social interaction on the free-running circadian rhythm of activity. In several pairs, general activity and body temperature were also recorded. In all pairs the intact animals entrained to the LD cycle, whereas the activity rhythms of the enucleated animals free-ran with periods of approximately 24 h and showed no apparent sign of synchronization or relative coordination with the other member of the pair. In a second experiment, male hamsters maintained in constant darkness received pulses of social interaction, which have been reported to induce phase shifts of the activity rhythm. Consistent phase shifts in the running-wheel activity rhythm were not induced by the social pulses in our experiment. These results suggest strongly that social stimuli are not effective entraining agents of circadian rhythms in the golden hamster.

Key words: Circadian rhythm - Entra inment- Social interaction - Locomotor activity - Body temperature

Introduction

The level of locomotor activity of an animal, as well as the levels of many other behavioral and physiological processes, is not constant throughout the day. Periods of rest and activity alternate during the 24 h day. This daily temporal organization is driven by an endogenous timing mechanism and modulated by external agents. The endogenous timing mechanism is believed to be a circadian pacemaker with a period of approximately

Abbreviations: CT circadian time; LD light-dark * To whom offprint requests should be sent

24 h located in the suprachiasmatic nuclei of the hypo- thalamus (Moore and Eichler 1972; Ralph et al. 1990; Stephan and Zucker 1972). External agents include environmental variables such as illumination and ambient temperature and can influence circadian rhythms by af- fecting either the circadian pacemaker itself (as reflected in the period and phase of the rhythms) or simply the expression of the rhythms (as reflected in their waveform) (Aschoff 1981; Pittendrigh 1981 ; Turek 1988). The most robust and best understood exogenous agent that affects the circadian pacemaker is the daily oscillation of light and darkness. A number of experimental investi- gations have shown that social interaction can affect the expression of the circadian rhythm of activity in several mammalian species (Bovet and Oertly 1974; Crow- ley and Bovet 1980; Honrado and Mrosovsky 1989; Re- gal and Connolly 1980), but it is not clear whether social interaction can also have a direct effect on the circadian pacemaker analogous to the effect of photic stimulation. While some authors have found evidence of entrainment (i.e., a steady-state modulation of the period and phase of circadian rhythms) due to social stimulation (Mari- muthu et al. 1981; Mrosovsky 1988), others have found that the apparent entrainment, if at all observed, is actu- ally a modulation of the expressed rhythms (Davis et al. 1987; Erkert etal. 1986; Kleinknecht 1985; Richter 1970). In the golden hamster, it has been reported that pulses of social interaction can shift the phase of the free-running rhythm of locomotor activity and that this effect is dependent on the phase of stimulation (Mro- sovsky 1988). If phase shifts do occur following social interaction, then the presence of a daily cycle of social interaction should produce entrainment (or modulation and relative coordination) of the activity rhythm. The experiments described here were designed to test this prediction.

Materials and methods

Experiment 1. To investigate whether circadian rhythms of the golden hamster (Mesocrieetus auratus) can be entrained by a cycle

t 82 R. Refinetti et al. : Inefficacy of social interaction for circadian entrainment

of social interaction, we housed two animals together, one of which was blinded by bilateral enucleation. Under this condition, the light-dark cycle directly entrained the circadian rhythms of the sighted animal but not of the enueleated animal. The enucleated animal was, therefore, subjected to a controlled daily cycle of social interaction with its cage mate without being directly affected by the light-dark cycle.

Pairs of male hamsters, 2-3 months old at the beginning of the experiment, were formed from animals previously kept in groups of 4. One member of each pair was blinded by bilateral enucleation under sodium pentobarbital anesthesia (90 mg/kg i.p.). A programmable timer (Chrontrol model DL, Lindburg Enter- prises, San Diego, Calif.) produced a light-dark cycle (300:0 lux) with one h of light per cycle with a period of 23.3 h. Under these conditions, the circadian rhythms of the sighted animal were ex- pected to entrain to the LD cycle and have a period of 23.3 h, whereas the rhythms of the enucleated animal would retain their endogenous periods of approximately 24.0 h unless social entrainment occurred.

Eleven pairs of animals were housed in plastic cages (25 x 46 x 20 cm) lined with wood shavings and maintained inside ventilated, light-tight boxes. Purina lab chow and water were available ad libitum. Ambient temperature was 21 ~ As a control procedure, 6 sighted and 6 enucleated hamsters were housed individually under conditions otherwise identical to those described for the pairs.

For 5 of the pairs, the cages were fitted with two 17 cm diameter running wheels; for the remaining pairs and the individual animals, only one wheel was installed per cage. Microswitches attached to the wheels allowed recording of running-wheel activity by micro- computers fitted with data acquisition hardware and software (Da- taquest III, Data Sciences Inc., St. Paul, MN). In the cages with two wheels, the two microswitches were connected in parallel so that the activity from both wheels was recorded in a single channel. In addition to running-wheel records, body temperature and general activity of the enucleated animal were recorded by telemetry in the 5 two-wheel setups. Radio transmitters equipped with ther- mal sensors (Model VM-FH, Mini-Mitter Co., Sunriver, OR) were implanted intraperitoneally at the time of the enucleation surgery. The radio signals were captured by a receiver (Model RA-1010, Mini-Mitter Co., Sunriver, OR) connected to the data acquisition apparatus. Running wheel activity, telemetered body temperature, and telemetered general activity were recorded at 6-min bins and saved on disk for later analysis.

Determination of the circadian period in individual records was obtained by the method of the chi square periodogram (Sokolove and Bushell 1978). Values of the statistic Qp were calculated in the range of 21.0 to 26.0 h in 0.1 h intervals, and the highest value was considered the true period. To allow the examination of intra- subject variability in period, periodograms were calculated for con- secutive blocks of 10 days for each animal. The periods of the several blocks were then averaged for each subject to allow compar- isons between subjects.

Experiment 2. Even if social entrainment could not be demon- strated in experiment 1, it would still be possible that pulses of social interaction can have small but detectable effects on the circadian pacemaker. Therefore, this second experiment was designed to replicate partially the findings of Mrosovsky (1988). We kept male hamsters under constant darkness and 'pulsed' them with episodes of social interaction. The animals were housed individually in plastic tubs with one running wheel, as described in experiment 1. Procedures of data collection were also as described earlier.

In a first section of the experiment, we used 11 male hamsters. The animals were tested 4 times, each test consisting of one week of entrainment to a 14:10 LD cycle (300:0 lux) followed by two weeks of constant darkness. Pulses were delivered under dim red light (5 lux) at the end of the first week in constant darkness and consisted of: 1) no pulse (the animals were left undisturbed and were not exposed to the dim red light), 2) control (the animals were removed from their cages, briefly handled, and returned to the cage), 3) male pulse (a male hamster was introduced in the

cage and removed after 5 min or after fighting erupted), and 4) female pulse (a receptive female was introduced in the cage and removed after 5 min or after the first mounting by the male). The first test for all animals consisted of the no-pulse condition, whereas the order of the other 3 tests was randomized for each animal. Preliminary results from our laboratory had suggested that a phase advance of the circadian rhythm of activity could be obtained by pulses delivered 8 h before activity onset (or 'circadian time four' [CT 4], as the onset of activity is designated CT 12 [Pittendrigh 1981]). Therefore, all pulses were delivered at CT 4.

In a second section of the experiment, we tried to replicate Mrosovsky's (1988) experimental conditions more closely. Eleven male hamsters were kept under a 14:10 LD cycle for two weeks and then released into constant darkness for 5 weeks. On the 8th day of constant darkness, half the animals received a control pulse and half a social pulse at CT 2 (actual mean: 1.9, range: 0.7-2.6) to produce a predicted mean phase delay of 48 min (the largest mean phase shift observed by Mrosovsky 1988). On the 22nd day of constant darkness, animals that had previously received social pulses received control pulses, and vice versa. A social pulse consisted of moving the animal to a clean test chamber (16x24• 14 cm) and leaving it there for 30 min in the presence of another male. If intense fighting erupted, the animals were separated earlier (this happened in 18% of the encounters and resulted in social pulses of 10-15 min rather than 30 min). The control pulse was identical to the social pulse except that the second male was not introduced. All pulses were administered in total darkness with the help of infrared viewers (FJW Optical Industries Inc., Palatine, IL).

Results

Experiment !

Examples of r u n n i n g activity of a sighted and an enucleated hamster housed individual ly are shown in Fig. 1. It can be seen that the sighted hamster en t ra ined to the LD cycle of 23.3 h with very regular daily onsets o f activity. The enucleated an imal ma in t a ined its endogenous period of 24.0 h, a l though the total a m o u n t of r u n n i n g was reduced after several weeks. Reduct ion of the a m o u n t of r u n n i n g in hamsters ma in t a ined in cons tan t darkness for 40-60 days is a c o m m o n observa t ion in our laboratory. The records shown in Fig. 1 are representative of all 12 control an imals housed individual ly. As determined by pe r iodogram analysis, the enucleated animals had a mean period (_+SD) of 23 .98+0 .07 h, whereas the sighted an imals had a mean period of 23.33 _+ 0.02 h, and this difference was statistically significant (t(10) = 13.54, P < 0.001). The periods o f indiv idual an imals ranged from 23 .88+0 .10 to 24 .07+0 .16 h (enucleated hamsters) or f rom 23 .30+0 .07 to 23.36_+0.09 (sighted animals).

Figure 2 shows the running-wheel activity records of a typical pair a long with the records of telemetered general activity and body tempera ture for the enucleated animal . As seen in the running-wheel records, and con- f irmed by the telemetered data, the c i rcadian system of the enucleated hamster was affected very little by the presence of the sighted animal . While the activity rhy thm of the sighted an imal had a period of 23.3 h, the rhy thm of the enucleated an imal remained at 24.0 h for most of the exper iment (range: 23.8 to 24.1 h) and lengthened to 24.4 h after 21/2 months . W h e n the sighted an imal

R. Refinetti et al. : Inefficacy of social interaction for circadian entrainment 183

Fig. 1. Records of running-wheel activity of a sighted and an enucleated hamster housed individually under an LD cycle (1 h of light per cycle) of 23.3 h. The data are plotted as standard acto- grams, where time in hours progresses from left to right on each day and successive lines correspond to successive days. To facilitate visualization, the data are double-plotted (i.e., each line contains

the current day plus the following day). For each 6-min bin, a dark mark is printed if one or more turns of the wheel were recorded; otherwise, the space is left blank. The sighted animal entrained to the LD cycle, whereas the enucleated animal free-ran with its endogenous period

Fig. 2. Records from a pair of hamsters housed in a single cage. Each pair consisted of an intact animal and an enucleated animal. Running-wheel activity of the pair and telemetered general activity and body temperature of the enucleated animal are shown. For each 6-rain bin, a dark mark is printed if running-wheel activity exceeds 10 revolutions, general activity exceeds 10% of peak

counts, and body temperature exceeds 36.9 ~ The heavy lines in the running wheel records indicate the duration (1 h per 23.3 h) of the light phase of the LD cycle. The impression that the activity rhythm of the enucleated hamster free-ran independently of the sighted animal is confirmed by the records of general activity and body temperature

184 R. Refinetti et al. : Inefficacy of social interaction for circadian entrainment

Fig. 3. Records of running-wheel activity of two pairs of sighted- enucleated hamsters. For each 6-min bin, a dark mark is printed if one or more turns of the wheel were recorded; otherwise the

space is left blank. The data show no indication of social synchronization of the rhythms of the two members of each pair

was removed on day 87, the rhythm of the enucleated animal was not affected (data not shown).

Although the period and phase of the enucleated animal 's rhythm were not obviously affected by the sighted animal 's activity over the 3-month period, some influence of the sighted hamster ' s activity on the enucleated hamster ' s activity was occasionally observed. In at least one record (Fig. 2), this influence can be seen as very small changes in the activity of the enucleated animal as the entrained animal ' s activity passes through it. The o telemetered general activity records of the enucleated animal also showed a minor activity component with u'~ a period of 23.3 h in several sections of the records. ~, Both of these effects may be directly due to the disruptive activity of the sighted animal in the cage (i.e., ' masking ' [Aschoff 1960]), a l though a minor degree of relative ~o m coordinat ion can not be ruled out (see Discussion).

The records shown in Fig. 2 are representative of the 11 pairs that were tested. Running wheel data f rom two ~"

O other pairs are shown in Fig. 3. In no case was synchro- ~| nization of the rhythms of the two animals observed, and the availability of one or two wheels had no appar- ~'~ ent influence in the results. For the 5 hamsters whose ~- activity was moni tored by telemetry, individual periods could be calculated. The shortest period was 23.90_+ ~, 0.28 h, the longest was 24.08+0.21 h, and the group ~o mean was 24.02_+0.07 h. This mean period of 24.02 h was significantly different f rom the 23.33 h period of the sighted controls ( t (9 )= 14.38, P < 0.001) but indistin- guishable f rom the 23.98 h period of the enucleated controls ( t ( 9 ) -0 .87 , P>0 .10) .

The effect o f social interaction on the expressed rhythms was particularly evident in the records of body temperature. Figure 4 shows four days of the body temperature rhythm of one of the paired animals in this

experiment and a typical temperature rhythm of a hamster housed individually. Although circadian periodicity can be observed in the records of both animals, the rhythm of the animal subjected to social interaction is characterized by marked noise that is not present in the records of undisturbed, individually housed animals.

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Fig. 4. Body temperature records of a male hamster housed with another male hamster (Top). For comparison, records of an individually housed hamster are also shown (Bottom). Over 30 individually housed hamsters have been previously studied in our laboratory and were found to have temperature rhythms similar to the one shown here. The rhythm of the group housed animal is much more irregular than that of individually housed animals


Fig. 5A-D. Running-wheel activity records of male hamsters maintained in constant darkness and subjected to environmental pulses at the time indicated by the stars. Pulses were delivered at CT 2. A and C: transfer to a small cage and 30-min exposure to a male hamster. B and D: transfer to small cage for 30 rain (control pulse). Records A and B show the typical, negligible phase shifts for both experimental (A) and control (B) pulses. Examples of the largest phase delays (up to 40 rain) for the two types of pulses are shown in records C and D

Experiment 2

Pulses delivered at CT 4 had variable results. Bouts of running for 1-2 h after the pulse were seen in animals receiving social pulses (either a female or a male intruder) but not in animals receiving control pulses or no pulses at all. Although transient phase shifts (i.e., phase advances of a few minutes on the day of the pulse) were observed in some animals, there were no steady- state phase shifts due to any of the treatments, as shown in Table 1. Steady-state phase shifts were computed by drawing separate eye-fit lines through the onsets before and after the pulse and calculating the time between these steady-state onsets on the first cycle following the pulse. Mean phase shifts varied from - 2 to +10 min and did not differ significantly from the condition of no pulse at all. However, the control and social interac-

Table 1. Phase shifts (in h) resulting from social pulses

Pulses

None Control Male Female

Mean a +0.01 +0.17 --0.04 +0.11 Variance 0.004 0.485 * 0.282 * 0.216 *

a Each value is the mean of 11 subjects. Phase advances: positive sign; phase delays : negative sign

* Significantly different from the value for no pulse (family-wise P < 0.01). Differences between means were evaluated by t tests and differences between variances by Hartley's test

tion treatments did have a disruptive effect on the activity rhythm, as the variances of all three means were significantly larger that than of the mean for the no-pulse condition.

Pulses of social interaction delivered at CT 2 caused a mean phase shift ( + SE) of - 12 + 4 min, whereas the control pulses caused a shift of - 7 + 4 min. These two mean shifts are rather small and do not differ significantly, tOO) = 0.78, P > 0.40. In either condition, little or no pulse-induced running was observed. Records of individual animals are shown in Fig. 5. Most animals had a phase delay of a few minutes (Fig. 5A" experimental, Fig. 5B: control), although one experimental and one control animal had a delay of about 40 min (Fig. 5 C: experimental, Fig. 5 D: control).

Discussion

The present results support earlier evidence that social stimulation does not act as an entraining agent of circadian rhythms in hamsters (Davis et al. 1987) or other mammals (Erkert et al. 1986; Kleinknecht 1985; Richter 1970). We found no indication of social entrainment in male hamsters paired with a conspecific of the same gender. We have not tested pairs of females, but analysis of previous data in male-female pairs (Refinetti and Menaker 1992) revealed no clear signs of social entrainment.

Although we obtained no evidence that social interaction has a direct effect on the circadian pacemaker, as

186 R. Refinetti et al. : Inefficacy of social interaction for circadian entrainment

would be indicated by changes in the period and phase of the rhythms, our results are not in conflict with previous observations that social interaction can affect the expression of circadian rhythms (Bovet and Oertly 1974; Crowley and Bovet 1980; Honrado and Mrosovsky 1989; Regal and Connolly 1980). Specifically, the con- tention that interaction with a conspecific may disrupt the waveform of circadian rhythms is supported by the increased noise in the body temperature rhythms observed in experiment 1 as well as by the 23.3 h component in the general activity of enucleated animals.

It might be argued that the 23.3 h period of the cycle of social interaction used in experiment 1 was outside the range of entrainment for social interaction. The 23.3 h period would require at least a 0.7 h phase advance of the activity rhythm each cycle for entrainment to occur. A previous study suggested that entrainment to social interaction is possible when the period of the zeitgeber is 0.4-0.5 h shorter than the free-running period of non-entrained hamsters (Mrosovsky 1988). I f social interaction was indeed the zeitgeber in that study, we would expect at least transient modulat ion of the rhythms in our experiment. Relative coordination, for example, is known to occur for photic stimuli outside the range of entrainment (Aschoff 1981). Our experiments show very little, if any, evidence of relative coordination. Further studies could be performed to determine whether social entrainment of circadian rhythms can occur with periods of social interaction closer to 24 h. This would seem unlikely given the results from experiment 2, which indicate that pulses of social interaction do not produce phase shifts of the free-running rhythms of animals kept in constant darkness.

It is impor tan t to identify methodological differences between our experiments and the previous experiment that reported the ability of social interaction to act as an entraining agent in the golden hamster (Mrosovsky 1988). In experiment 1, our hamsters were left undisturbed in a cage to interact (or not interact) with each other without experimenter intervention. In the previous study, an intruder hamster was introduced into the cage of the experimental animal for 1 h each day to produce the reported entrainment. In addition, our animals were maintained in constant darkness (as a result of enucleation), thus avoiding complicating effects of light stimuli. In Mrosovsky ' s study, the animals were maintained in constant light o f 15-25 lux, a level o f illumination well above the threshold for the photic entrainment pathway in the golden hamster (white light threshold ~1 lux: Nelson and Takahashi, unpublished observations; see also Nelson and Takahashi 1991). It is possible that some combinat ion of social interaction and light stimulation acted as a zeitgeber in Mrosovsky ' s study. For example, phase shifts were induced by social interaction in hamsters maintained in constant light of 20-45 lux (Mro- sovsky 1988) but not in hamsters maintained in constant darkness (our experiment 2). This could be due to a socially modulated action of photic information on the circadian pacemaker. Alternatively, the phase-shifting or entraining action of social stimuli may require visual contact with the ' en t ra in ing ' conspecific.

It has also been suggested that the effects of social interaction are due to a state of arousal as determined by running-wheel activity following social interaction (Honrado and Mrosovsky 1989; Reebs and Mrosovsky 1989). In our experiments, we did not observe consistent running activity induced by the pulses of social interaction, which might explain the absence of phase shifts. However, it is not clear why social pulses would have produced arousal in Mrosovsky 's (1988) experiments but not in ours. On the other hand, we did observe some form of arousal without subsequent phase shifts. During our social pulses, the animals were clearly disturbed, as indicated by fighting, grooming, and scent marking. It is possible that these periods of arousal were not suffi- cient to induce phase shifts of the activity rhythms.

We conclude that social stimulation does not affect the circadian pacemaker of the golden hamster, even though it may disrupt the expressed rhythms. Social stimuli are quite complex and it is possible that experimental conditions that were not tested in our experiments might produce detectable results. The available evidence, however, points to the inefficacy of social stimuli as entraining agents of circadian rhythms in the golden hamster.

Acknowledgements. This work was supported by the National Sci- ence Foundation's Science and Technology Center for Biological Timing and by National Institutes of Health's NRSA Award MH- 10146 to R. Refinetti and Grant HD-13162 to M. Menaker. We thank Ms Tania Anderson (a Howard Hughes Medical Institute undergraduate fellow) for assistance in the execution of experiment 2 and Dr. Nicholas Mrosovsky for comments on an earlier version of this manuscript.

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social stimuli fail to act as entraining agents of circadian rhythms in the golden hamster

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