Circadian activity rhythms in the solitary Cape molerat (Georychus capensis: Bathyergidae) with some evidence of splitting

Physiology & Behavior, Vol. 58, No. 4, pp. 679-685, 1995 Copyright © 1995 Elsevier Science inc. Printed in the USA. All rights reserved 0031-9384/95 $9.50 + .00

Pergamon 0031-9384(95)00106-9

Circadian Activity Rhythms in the Solitary Cape Molerat (Georychus capensis: Bathyergidae) With Some Evidence of Splitting B A R R Y G. L O V E G R O V E 1 A N D M I C H A E L E. PAPENFUS

Department of Zoology and Entomology, University of Natal, Private Bag XO1 Scottsville 3209, South Africa Received 30 January 1995 LOVEGROVE, B. G. AND M. E. PAPENFUS. Circadian activity rhythms in the solitary cape molerat (Georychus capensis: Bathyergidae) with some evidence of splitting. PHYSIOL BEHAV 58(4) 679-685, 1995.--Circadian activity patterns were measured in the solitary, subterranean Cape molerat, Georychus capensis, under fixed 12:12 LD, constant dark DD, and constant light LL photoperiods for 250 consecutive days using passive infra-red activity sensors. The molerats displayed significant nocturnal activity rhythms under 12:12 LD and free-running rhythms (23.5 h < ¢ > 25.8 h) under constant conditions. In one individual evidence was found of splitting of the activity rhythm under constant (dark; the split rhythms free-ran with short- and long-periods of 23.6 h and 24.7 h, respectively. These data show that, despite degenerate retinae, G. capensis (i) are capable of perceiving light at the level of the circadian pacemaker; (ii) can entrain endogenous activity oscillators to fixed photoperiods; and (iii) display evidence of a free-rnuning activity oscillator. Circadian

rhythms

Activity

Splitting

Entrainment

Molerats

Bathyergidae

Georychus capensis in cells of the suprachiasmatic nuclei further confirming the functionality of the RHT (29). In contrast to these data, little is known about retinal-CNS neural projections in the Bathyergidae. However, preliminary investigations of the pinealocytes of the Damara molerat (Cryptorays damarensis) from the Kalahari sands of the southwestern deserts of Africa have revealed interesting patterns of immunoreactions to photoreceptor-specific proteins. Korf (8) reports that approximately 50% of the pinealocytes in the pineal organs of this molerat are rod-opsin immunoreactive, the highest percentage reported for any mammal. Even more interesting is the observation that a "considerable" number of pinealocytes also displayed immunorcactivity to a-transducin, which has never been shown with certainty in the pineal organ of any other mammalian species (8). By acting as the signal-coupling G-protein in visual excitation of rods, the existence of a-transducin in pinealocytes markedly increases the chances of light-sensitivity of the pineal organ. Lovegrove, Heldmaier, and Ruf (14) first mooted this possibility and the likely role of a patch of white hairs, known colloquially as a bles, which can be found overly-

INTRODUCTION

ALL species of molerats of the family Bathyergidae are strictly subterranean and seldom wander aboveground (2,24). They have small atrophied eyes which are apparently incapable of forming object images (4). Sew'.ral families of subterranean mammals, such as the Spalacidae and Talpidae, also possess tiny residual eyes but these are situated below the skin surface (16,17,21). For example, the molerat Spalax ehrenbergi (Spalacidae) lacks neural projections between the retina and the lateral geniculate nucleus (21) such that strong light flashes fail to elicit potentials in the visual cortex (6). Nevertheless, although the optic tract is degenerate, functional ]projections between the retina and the ventral lateral geniculate nucleus, the optic tract nucleus, the lateroposterior nucleus, and the superior colliculus allow S. ehrenbergi to discriminate between light and dark environments (21). The retinohypothalamic tract (RHT) of S. ehrenbergi is, however, well developed, accounting for the expression of certain circadian rhythms such as activity (21). In this species, it has also recently been shown that, similar to normally sighted rodents, exposure to light during the dark phase entrains c-los expression

1 To whom requests for reprints should be addressed. 679

680

LOVEGROVE AND PAPENFUS

ing the parietal region of the skull in most individuals of three genera of bathyergid molerats (Bathyergus, Georychus, and Cryptomys). To date, no mammalian pineal organ has been found to be directly photosensitive. The Bathyergidae are, therefore, an important group of mammals in which to pursue this likelihood. Effective blindness in bathyergid molerats raises the interesting question about whether or not molerats display endogenous biological rhythms entrained to the day-night cycle. Recent attempts to interpret circadian activity patterns in bathyergid molerats were complicated by potential problems of social entrainment (14). Using C. damarensis, a eusocial molerat, Lovegrove, Heldmaier&Ruf (14) used passive infra-red motion detectors to measure activity patterns, instead of using running-wheels, the routine method use to measure circadian activity patterns. These authors were able to conclude that, as a colony, (a) the molerats could indeed detect a light-dark cycle; and (b) that the colony displayed a free-running period of activity ( r ) not equal to 24 h under a constant dark photoperiod regime. These data could not clearly differentiate intra-specific or individual responses to photoperiod regimes, a problem exacerbated by possible social entrainment within the colony. Secondly, it was not completely clear whether the activity ( a ) and rest ( p ) phases of the molerats occurred during the light or dark phases making it difficult to decide whether these molerats were nocturnal or diurnal. The latter problem was further confused by the identification of a short, but intense activity phase entrained to the early hours of the dark phase, the so-called nocturnal activity component (NAC), which seemed to be out-of-phase with a diurnal activity pattern displayed by one colony. It was hypothesised that the NAC may represent one of two activity oscillators specific to a particular activity such as burrowing. The lack of data on circadian biological rhythms in the Bathyergidae have led several physiological studies on molerats to assume a lack of circadian metabolic rhythms (9-11,13). In view of the apparent functional retinohypothalamic tract and endogenous activity rhythms reported by Rado et al. (21) in S. ehrenbergi, such assumptions may not be valid. This is particularly true considering the association between circadian activity rhythms and metabolic rhythms in rodents (3). Moreover, although the underground realm is characterised by constant darkness, temperature and humidity on a daily basis, this is not

necessarily true on a seasonal basis at mid-to-high latitudes (1,12,16). Like aboveground mammals, molerats may therefore require thermoregulatory and reproductive preparedness to particular seasons and may therefore depend upon a functional retinohypothalamic pathway. This study addresses the topic by investigating circadian activity rhythms in the solitary Cape molerat, Georychus capensis. The principal objectives were to establish whether G. capensis display endogenous circadian activity rhythms entrained to fixed photoperiods and, if so, whether the activity rhythms show free-running rhythms under constant conditions. MATERIALAND METHODS

Study Animals and Maintenance Seven molerats (3 males, 4 females, 127-209 g) were trapped on the New Forest Farm near Nottingham Road, Natal, South Africa, between February and March, 1993. The ages of the animals could not be determined. They were housed individually in simulated burrow systems consisting of glass terraria interconnected by 110 mm diameter glass-covered split PVC tubing (Fig. 1). The two smaller nest and toilet terraria (30 × 22 × 23 cm) were covered with glass whereas the largest terrarium (45 × 22 × 23 cm) was covered with wire mesh and was intended to represent the "aboveground" or external zone of the burrow system. Each burrow system was also provided with a 1.25 m tube leading off from the large terrarium and ending in a wooden plug in an attempt to simulate a burrowing face and entice the molerats into gnawing/burrowing behaviour. The burrow systems were housed in a constant-environment room maintained at 21°C and 50% RH on a 12:12 light:dark (LD) photoperiod. The average light intensity in the room under LL was 227 Ix. During DD the room was entered for servicing using a dim red light source. Temperature fluctuations in the room were monitored and found to vary by less than 0.1°C over a 24 h period. These temperature fluctuations were not associated with lights-on and lights-off events. The room was fitted with a white-noise generator to minimise the probability of sound entrainment. The molerats were fed a diet of chopped vegetables (sweet potato, gemsquash, butternut squash and carrots) and, occasion-

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SPLITTING OF ACTIVITY OSCILLATORS IN MOLERATS

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FIG. 2. (a) Actograms and periodograms of the activity patterns of molerat GC6 under a 12:12 LD and constant dark DD photoperiod. The actograms (left) are double-plotted to facilitate easier visual representation. The solid bars indicate the dark hours. The three periodogram graphs (right) show the Chi-square statistics (Qp) as a function of ¢, the period of activity (25). The straight lines are drawn through critical values of Xp21 for a = 0.001 (approximately the 0.01 significance level) calculated from Zar (30). The vertical broken line represents ~-= 24 h. Figure continues.

682

LOVEGROVE AND PAPENFUS

ally, chopped apples. The food was carefully placed in the large terrarium at random times during natural daylight hours every 2-3 days. Maintenance and cleaning of the burrow systems took place every 3 - 4 wk.

measurement. This was followed by constant dark (DD) for 129 days (days 41-170). On day 171 a 12:12 LD was once more maintained for 38 days (days 171-209) whereafter a constant light (LL) photoperiod was maintained for the remaining 40 days (days 210-250) of the study.

Protocol Statistical Analyses

Activity was measured using passive infra-red motion detectors positioned above the large terrarium (external sensor), the middle toilet terrarium (internal sensor), and at the end of the blind-ending "foraging" tunnel. Upon activation the sensors produced a 2 s 8 V output signal recorded by a micro-computer. Activity was measured by summing the number of times the sensors were activated every 6 min. Molerats were maintained on a 12:12 LD photoperiod for 3 wk prior to data measurement and for the first 40 days of

For easy visual representation, activity measures were cropped at 25 events per 6 min and double-plotted. Chi-square periodogram analyses (25) based upon Enright's (5) periodogram analysis, the most appropriate test for periodicity in circadian activity data (22), were used to calculate Chi-square statistics of the last 10 days of the uncropped data of each light regime (12:12 LD, DD, 12:12 LD, DD). These analyses tested the hypothesis that activity counts were randomly distributed among the 240

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SPLITYING OF ACTIVITY OSCILLATORS IN MOLERATS

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displayed a pattern suggesting a steady phase-delay of the onset of the morning activity bout (Fig. 3). This pattern of phase-shifting resulted in a "compression" or shortening of the duration of a (a change in the a:p ratio) into an increasingly smaller time interval as the onset of activity approached the end of the subjective night. The measured counts of activity per measurement interval increased during these "compression" periods (Fig. 3). During the initial 40-day 12:12 LD photoperiod, three molerats showed significant circadian activity rhythms on both activity sensors (r = 24.0 h, Table 1, Fig. 2a). The period of activity of two molerats (GC1 and GC5) was 24.1 h (see Fig. 3 for GC1), whereas GC4 displayed one nonsignificant period of 23.2 h on one sensor and a significant period of 24.3 h on the other (Table 1). During the second 12:12 LD regime (days 140-170) data from at least one sensor for each molerat showed significant entrainment (r = 24.0 h) to a fixed photoperiod (Fig. 2b, Table 1).

daily measurement intervals. Significant nonrandom periodicities (r) were taken as the highest Chi-square statistics (Qp values) at the p = 0.01 confidence level. RESULTS

Prior to data measurement, most of the molerats preferentially built nests under the sensor situated at the end of the blind-ending burrow resulting in ambiguous data. The recorded activity was thus not necessarily associated with gnawing or burrowing behaviour. The burrow sections were therefore removed from all systems and the activity sensors relocated to the central terrarium (see Fig. 1).

12:12 LD Photoperiods (Days 1-40 and 170-210) All molerats displayed nocturnal activity patterns. However, activity counts were nourandomly distributed (Chi-square, p < 0.01) within the subjective dark interval. The highest cumulative number of counts consistently occurred in the last few 6 rain measurement intervals prior to lights-on (Figs 2a,b) suggesting that the end of activity is entrained to the onset of light. The onset of activity varied considerably between individuals and on a day-to-day basis. For example, the activity of molerat GC1

Constant Dark (DD, Days 41-169) During the last 30 days of DD all molerats showed significant free-running (r ~ 24 h) activity rhythms (Table 1). The activity

TABLE 1 PERIODS OF ACTIVITY RHYTHMS ( r ) OF GEORYCHUS CAPENSIS UNDER FIXED AND CONSTANT PHOTOPERIODS MEASURED USING PERIODOGRAM ANALYSES AS DESCRIBED BY SOKOLOVE & BUSHELL (25) 12:12 L D Days 5 - 3 5

GC1 GC2 GC3 GC4 GC5 GC6

24 D D Days 1 4 0 - 1 7 0

12:12 L D Days 1 8 0 - 2 1 0

24 L L Days 2 2 0 - 2 5 0

Ext

Int

Ext

Int

Ext

Int

Ext

Int

24.1 24.0 24.0 24.3 24.1 24.0

24.1 24.0 24.0 23.2* 24.1 24.0

26.0* 23.5 25.8 23.9 24.2 24.7

24.3 23.6 25.7* 24.3 24.2 25.0

24.3* 24.0 24.0 24.0 24.0 24.0

24.0 24.1 24.0 24.0 24.0 24.0

25.5 23.2* 25.0 25.9* 24.8* 24.8

24.6 25.7* 25.2 23.9* 24.5 24.5

Ext ffi external activity sensor, Int = internal activity sensor, * ffi not significant. Each period represents the maximum Chi-square statistic (Qp) measured by each periodogram analysis.

684

LOVEGROVE AND PAPENFUS

of one molerat (GC2) consistently free-ran with a ~"< 24 h, whereas four others (GC1, GC3, GC5 and GC6) consistently free-ran with a z > 24 (Table 1). Interestingly, in the molerat GC4 system, one sensor measured significant short periods (~"< 24) whereas the other measured a significant long activity period (~-> 24; Table 1). Molerat GC6, however, displayed the most noteworthy activity patterns over the whole DD measurement period. Although, as we report above, periodicities greater than and less than 24 h were recorded on two different activity sensors in the same molerat system, similar short- and long-periods were recorded by the same sensor in both sensors in molerat GC6 (Fig. 2a). After the first 75 days of DD during which there seemed to be little free-running activity but clear "compression" of activity in this molerat, two strong, significant activity rhythms abruptly appeared between days 75-105; one was short (r < 23.6 h) and the other long (~"> 24.7 h; see periodogram in Fig. 2a). When these two opposing rhythms coincided with each other (see ca. day 95 and day 115), the free-running periodicities of each rhythm appeared to be arrested for about 10 days during which time the duration of a again became compressed into an increasing smaller time interval (Fig. 2a). After day 140 expression of the short-period rhythm tended to weaken. This is clearly evident in the reduction of the peak of activity in the periodogram between days 140-170 (Fig. 2a).

Constant Light LL (Days 211-250) All molerats displayed free-running activity rhythms (~"~: 24 h) under constant light although for two molerats (GC2 and GC5) the periodogram analyses of the rhythms were not significant (Table 1). The activity rhythms with ~"< 24 h, described earlier as one of the split rhythms in molerat GC6 above, was not visible, although the arrest of the short-period rhythm at ca. day 230 suggests the continued influence of this rhythm (Fig. 2b). DISCUSSION

Georychus capensis are clearly able to perceive light and entrain endogenous activity oscillators to 12:12 photoperiods. The endogenous nature of these oscillators is confirmed by free-running rhythms under constant conditions. It is also clear that, under the experimental conditions used, these molerats displayed nocturnal activity patterns. This contrasts with the predominantly diurnal pattern which was observed in another bathyergid molerat, the eusocial C. damarensis (14). These data suggest a functional retinohypothalamic tract and thus innervation, presumably via the retina, of the known source of endogenous oscillators, the suprachiasmatic nucleus (15,23,26). Although seasonal biological responses have not been measured in these molerats, it is likely that a functional RHT should permit seasonal thermoregulatory adjustments similar to those described by Prvet et al. (17) in S. ehrenbergi. Should seasonal biological rhythms exist, it would be of interest to determine whether pineal melatonin secretions respond to changes in the duration of the light phase.

Evidence of Splitting The data for molerat GC6 clearly indicate the phenomenon of splitting in which the nocturnal activity rhythm splits into two rhythms under constant conditions. Unfortunately these data are too limited to draw any definitive conclusions, but in several respects the patterns are unusual enough to warrant brief discussion for further research consideration. Splitting normally occurs in LL in a certain percentage of nocturnal rodents (7,19,28). Splitting has also been reported in

some diurnal mammals, namely in tree shrews (Tupaia belaneri) and ground squirrels (Eutamias sibiricus and Ammospermophilus leucurus) in DD (7,20,27). According to Pittendrigh and Daan (19) the unsplit activity rhythm is comprised of two coupled activity oscillators, one entrained to the start of the subjective night controlling the evening (E) component of a , and the other entrained to the end of the subjective night controlling the morning (M) component of a. Each of these oscillators has its own intrinsic period (i.e., the periods they would display if they could be uncoupled) as ~'M and '/'E for the M and E components of a, respectively. The theoretical basis for an explanation for why splitting in nocturnal rodents occurs regularly in LL but not in DD concerns the observation that z M and ~'E depend differently on light intensity: z E is a positive and ~'M a negative function of intensity (19). In DD, therefore, the intrinsic periods of the two oscillators may be very similar and the normal coupling mode between the two oscillators is easily maintained. However, during exposure to LL the difference in the intrinsic periods of the two oscillators may become so great that the normal coupling mode can no longer be maintained and splitting occurs. When splitting occurs, typically after about 50 days of DD, the most common pattern in hamsters is that the M component free-runs with a period less than the unsplit period whereas the E component continues free-running with the same period as the unsplit rhythm. The M component continues to phase-advance until it is approximately 12 h or 180 ° out-of-phase with E, after which it maintains a stable anti-phase relationship with the E component and the two components run free with the same periods [i.e., ~'M = rE, which is always less than the ~" of the previous unsplit rhythm (19,28)]. To our knowledge, it has not been observed that split rhythms fail to reach a stable antiphase relationship between the E and M components. In this study, the two oscillators split in DD and continued to free-run with opposing periods [i.e., one short ( z < 24 h) and the other long ( r > 24 h)]. The free-running oscillators periodically "collided" with each other following which they tended to become temporarily recoupled or refused for about 10 days. During this recoupling phase there was a clear shift in the a : p ratio followed by a transient jump or shift in the rhythms. Although molerat GC6 was the only molerat which displayed such a clear split rhythm, several molerats displayed evidence of activity "compression" or periodic shifting of the a:p ratio accompanied by an increase in the intensity of activity within each measurement interval. In fact, these activity compressions occurred under fixed 12:12 LD photoperiods as well, and since the onset of a tended to phase-delay toward the end of the subjective night (Fig. 3), the most significant ~" values measured by the periodogram analyses during these events were often greater than 24 h (see GC1, GC4,&GC5, Table 1) despite obvious entrainment to the end of the dark phase. Another difference displayed by the activity rhythms of G. capensis compared with other nocturnal rodents was that a was entrained to M rather than to E (see 19). As described above, in some molerats the onset of ot tended to phase-delay with respect to the onset of the subjective night. Although limited, these data may indeed suggest a) different sensitivities to light of E&M, and b) that 7 M < 24 h whereas r E > 24 h. Additional support for the latter was that the activity rhythm following the onset of DD showed "compression" before splitting indicating crossing-over of M and E (Fig. 2a). As discussed earlier, this crossing-over also occurs during gradual splitting in hamsters, but only in LL (19, 28). These observations clearly justify further research on phenomenon of splitting and the influence of light intensities on splitting patterns in these subterranean mammals, particularly in

SPLITFING OF ACTIVITY OSCILLATORS IN MOLERATS

light of the hypothesis that increasing light levels induce splitting in nocturnal animals whereas decreasing light levels induce splitring in diurnal animals (18).

Closing Remarks The importance of light or natural photoperiods to bathyergid molerats in terms of reproduction and thermoregulation remains obscure. Is there a functional significance of short- and longperiod activity oscillators in these molerats? Do free-ranging molerats manage to entrain to the Earth's natural light cycle and, if so, what role, if any, do split rhythms play? It could be argued, for example, that the opposing influence of short- and long-period activity oscillators prevems molerats from excessive free-running

685

activity patterns in the natural dark of a subterranean existence. Speculation is pointless, however, without further documentation of the extent and quantification of activity rhythms and splitting in the Bathyergidae. Moreover, it is obviously important to determine, with precision, the extent to which the pineal organ of the Bathyergidae responds, if at all, to light. ACKNOWLEDGEMENTS We thank Mr. and Mrs. N. Ross for permission to stay and trap molerats on their farm, New Forest Farm. Mr. Guy Dewar and Mr. Alan Cullis kingly assisted with computer interfacing. This research was financed by generous grants from the Foundation for Research Development Core Fund and the Natal University Research Fund to BGL.

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