Locomotor and feeding activity rhythms in a light-entrained diurnal rodent, Octodon degus

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1 Locomotor and feeding activity rytms in a ligt-entrained diurnal rodent, Octodon degus R. GARCÍA-ALLEGUE, 1 P. LAX, 1 A. M. MADARIAGA, 2 AND J. A. MADRID 1 1 Department of Pysiology and Parmacology (Animal Pysiology Unit), University of Murcia, Murcia; and 2 Animalario University of Alicante, Alicante, Spain García-Allegue, R., P. Lax, A. M. Madariaga, and J. A. Madrid. Locomotor and feeding activity rytms in a ligtentrained diurnal rodent, Octodon degus. Am. J. Pysiol. 277 (Regulatory Integrative Comp. Pysiol. 46): R523 R531, Te weel running (WR) and feeding activity (FA) of Octodon degus, a new laboratory rodent caracterized by its diurnal abits, were recorded under different ligting conditions. Under 12:12- ligt-dark (LD 12:12) cycles, WR activity exibited a crepuscular pattern wit two peaks, M and E, associated wit dawn and dusk, respectively. In bot cases, an anticipatory activity was patent, suggesting tat, beside te masking effect of LD transitions, bot peaks ave an endogenous origin. Tis pattern, wic was also observed under a skeleton potoperiod (LD 0.5:11.5), became unimodal after LD 0.5:23.5 and constant darkness (DD) exposure. Simultaneously, FA sowed an arrytmic pattern in most animals, especially under DD, wen none of te animals exibited a significant circadian rytm. Te existence of two groups of oscillators, or two oscillators, would explain most properties of te WR rytms noted in tis species. Our results sow tat te degu s temporal feeding strategy seems mainly arrytmic, wereas its WR pattern is driven by a strongly circadian bimodal rytm. degu; entrainment; crepuscular; skeleton potoperiod; circadian Te costs of publication of tis article were defrayed in part by te payment of page carges. Te article must terefore be ereby marked advertisement in accordance wit 18 U.S.C. Section 1734 solely to indicate tis fact. OCTODON DEGUS, COMMONLY called degu, a Sout American ystricomorp rodent, as become an increasingly popular experimental animal in recent years. Degus live in central Cile and are found from sea level to 2,000 meters (for more data, see Refs. 3 and 8). Its way of life is terrestrial and fossorial, and it displays a very elaborate social beavior (5, 6). From a cronobiological point of view, te degu is of great interest. Field and laboratory studies ave sown tat tey are active during te day trougout te year, wit teir activity pattern caracterized by two main peaks of activity in te morning and late afternoon (3, 15). Degus were more active immediately before ligts-off and te 2 before ligts-on, anticipating te illumination canges. Sleep periods were mainly confined to te nocturnal period, wit a minimal amount of sleep during ligts-on (4). In accordance wit its diurnal beavior, its temperature acropase occurred at te end of te ligt period, in close association to te second activity peak (4, 14). Most animal cronobiological studies on te cronoparmacology, beavior, and pysiology of te circadian system ave been developed in nocturnal rodents suc as rat, amster, or mouse. Te degu s diurnalism, a rare caracteristic among rodents, as well as some features of its circadian rytms, suc as te variability of individual rytms, te presence of different morning-evening cronotypes, and te dramatic bimodal pattern in its circadian locomotor rytm, make te degu a model of special interest in cronobiology (10). Altoug te multioscillatory nature of te circadian system is an old issue, te assumption tat te mammalian circadian system could be based on te degree of intercommunication of several neural oscillators wit different intrinsic frequencies and varying capacities for ligt syncronization remains open (2). Te bimodality of te degu s locomotor rytms is a useful model for obtaining furter evidence to support te abovementioned ypotesis. To date, only circadian rytms of locomotor activity, body temperature, and sleep ave been studied (4 6, 9, 10, 12, 14, 15), our study being te first attempt to study feeding and weel running activity rytms (FA and WR, respectively) simultaneously in degus kept under laboratory conditions. Te aim of te present experiment was to describe te syncronization of FA and WR rytms to different ligt-dark (LD) cycles to caracterize te endogenous nature of te two main peaks of activity, described in te above studies. For tis, te degu s FA and WR, two exclusive variables, were continuously recorded under complete and skeleton potoperiods and constant darkness (DD). MATERIAL AND METHODS Animals and ousing. Ten male Octodon degus (10 mo old at te beginning of te experiments) reared in a laboratory colony at te University of Alicante were used in te study. At te beginning of te experimental period, te animals were individually oused in a modified Plexiglas cage ( cm), wic allowed te recording of FA and WR. Cages were placed in a ligt-tigt and temperature-controlled camber wit continuous ventilation ( lx during ligts-on, a temperature of 23 1 C, and 60 20% relative umidity). Dim red ligt (intensity 0.5 lx) was present all te time for nocturnal manipulations. A pelleted rat diet (Panlab) and water cups were available ad libitum. Te cages were cleaned, and te food and water were refilled every 10 days at random times of te day to prevent syncronization. Apparatus. All cages were provided wit a contact eatometer and a weel for feeding and locomotor registration. Te eatometer as been described in detail elsewere (11). Briefly, it consisted of a stainless steel grid wit a swinging grid mounted inside tat ad to be activated by te animal to eat, tus allowing te recording of FA. Te axis of te weel (9-cm wide and 25-cm diameter) was provided wit an eccentric /99 $5.00 Copyrigt 1999 te American Pysiological Society R523

2 R524 Fig. 1. Scematic representation of sequence of canges in ligting conditions during wole experimental period. LD 12:12, 12:12- ligt-dark cycle; DD, constant darkness. cylinder tat activated a microswic eac time it made a complete turn. Te WR and FA of eac animal were recorded on a 286 microprocessor wit an I/O (CIO-DIO-48, Computer Board Inc) card at 10-min intervals. FA was measured as te duration of food contacts, wereas WR was measured in revolutions. Procedure. As Fig. 1 sows, FA and WR activities were recorded for eac animal trougout 11 ligting scedules. Initially te animals were kept in continuous darkness, after wic tey were subjected to a skeleton potoperiod consisting of two pulses (30 min ligt), one at 2000 and te oter at Tirty days later, te ligt pulse at 2000 was removed, so tat te animals were exposed for anoter 30 days to only 30 min ligt per day (at 0800 local time). Te ligt was switced off (DD conditions) for 20 days, after wic te animals were kept under a 12:12- LD cycle (LD 12:12; ligts on at 0800 local time; stage LD 13 mo old) for 30 days. After anoter 80 days under DD, te animals were submitted again to an LD 12:12 cycle, ligts-on at 0800 (stage LD 17 mo old) for 50 days. After anoter 40 days under DD conditions, te Fig. 2. Tree representative actograms and mean waveforms ( SE; n 10) of weel running (WR) during exposure to 2 pulses and 1 pulse per day. Vertical dotted line in actograms and waveforms represents brigt ligt pulses (0800 and 2000). Mean waveforms were calculated from individual waveform averaged over wole experimental period after deleting first week of eac experimental pase. Data are presented as percentage of mean of overall 24, so tat tis mean equals 100%. Tis procedure normalizes ranges of diel patterns across subjects.

3 R525 animals were again submitted to LD 12:12, wic was expanded after 15 days by 3 (LD 15:9) by delaying ligts-off. Finally, a second expansion of te potoperiod was acieved 15 days later by advancing ligts-on by 3 (LD 18:6). Te animals were kept in tis condition for 15 days. Data treatment. In eac ligting condition, feeding and locomotor circadian periods were determined by 2 periodogram (17). To compare feeding and locomotor rytms te spectral power of te first 15 armonics was calculated by Fourier analysis (Cronobio-pc software, Panlab) Summary data were calculated from te means of averaged waveforms for individual animals in different experimental pases. To avoid te effects of transitional periods between experimental pases, te data referring to te first week of eac experimental pase were discounted. Te onset and te duration of te active pase ( ) were determined in well-defined averaged daily waveforms according to two different criteria related to te WR pattern recorded. 1) Wen unimodal (as occurred under DD or 1 ligt pulse), -onset was estimated as te time at wic te first large peak of activity wit an irreversible cange in slope occurred, and was estimated as te period during wic te activity profile was above te mean of daily activity counts recorded witin a 24- period. Similar procedures to estimate te onset and ave been employed by oter autors (1). 2) Wen te pattern was bimodal, -onset was defined as te first one of tree or more consecutive 10-min intervals wit an activity iger tan te mean, wereas -offset was defined as te last of tree or more consecutive periods wit an activity iger tan te mean. was calculated as te total period between te onset and offset of measured under te ligt pase. All calculations concerning -onset and duration were performed in ours. In accordance wit te findings of Labyak et al. (10), te animals were classified in different cronotypes. For tis, te amplitude of te morning (M) or evening (E) peak was calculated as te mean of tree values: te maximum and te 10-min periods before and after tis maximum. All values were expressed as te percentage of te sum of te M and E peaks (100%). Animals wit 66% of teir activity concentrated in te M peak were considered as cronotype A, tose wit more tan 66% of teir activity related to te E peak as cronotype C, and te rest as cronotype B. A one-way ANOVA was used to determine differences in -onset, -duration, and te amplitude of te WR peaks between treatments. Individual comparisons were made by Sceffé s test. RESULTS Figure 1 sows a diagram indicating te sequence of ligting conditions and its duration trougout te experimental period. After exposure of te animals to a skeleton potoperiod consisting of two ligt pulses of 30 min separated by 12 of darkness, a bimodal pattern in WR activity emerged (Fig. 2). However, te WR activity associated wit eac pulse was different: 59% of te total daily activity occurred around te 2000 pulse and only 10% related to te 0800 pulse. As Table 1 sows, te activity related to te 2000 pulse started 3.54 before te pulse and lasted for During tis ligt pulse tere was a strong decrease in WR activity (masking effect) followed by a sarp increase, wit a maximum appearing 30 min after ligts-off. Altoug no anticipatory activity was detected in relation to te 0800 pulse, te Table 1. Analysis of bimodality under skeleton potoperiods Degu 0800 Pulse 2000 Pulse % % Skeleton potoperiod (2 pulses/day) C C C C C C C C C C Mean SE Skeleton potoperiod (1 pulse/day) C C C C C C40 C C C C Mean SE Summary data are depicted as means SE. Individual and summary data on onset, duration, and amplitude of morning and evening peaks of weel running (WR) activity under exposure to 2 and 1 ligt pulse of 30 min. Onset and duration is expressed in ours. To normalize ranges across subjects, amplitude is expressed as percentage of mean. activity started to increase after te pulse, reacing te maximum level 30 min afterward. Periodogram analysis revealed a significant circadian rytm for WR in all te animals, wereas only 30% exibited significant circadian rytmicity for FA. Moreover, FA sowed a very irregular and interindividual variable pattern. Wen te 2000 pulse was eliminated a progressive sift of te WR activity associated wit tis pulse was observed, wit most WR activity occurring around te 0800 ligt pulse after days (Fig. 2). During tis experimental pase, te animals sowing significant daily rytms for WR presented a unimodal pattern wit starting 6.39 before ligt and lasting for 8.16 (Table 1). As occurred in te previous pase wit te 2000 pulse, ligt seemed to exert a negative masking effect, te WR activity increasing after ligts-off to reac a maximum 30 min later. Also, during tis experimental pase, te FA was arrytmic for most animals (60%), wereas a significant circadian rytm appeared for WR in 80% of animals. In DD conditions (Fig. 3), WR activity started to free run from two peaks in LD, but after few days, WR exibited a unimodal pattern similar to tat observed wit te 0800 ligt pulse, but witout te reactive response to ligt. for WR was 8, and te total daily

4 Fig. 3. Representative actograms (left) and mean waveforms ( SE; n 10) of WR activity (top) and feeding activity (FA; bottom) during exposure to constant darkness. x-axis: circadian time. Mean waveforms were calculated from individual waveform averaged over wole experimental period after deleting first week of eac experimental pase. Data are presented as percentage of mean of overall circadian period, so tat tis mean equals 100%. Tis procedure normalizes ranges of circadian rytms across subjects. Fig. 4. Mean waveforms of WR (top) and FA (bottom; SE; n 10) under LD 12:12 at 2 different ages, 13 (left) and 17 mo (rigt).

5 R527 distance run was 1, m. Te free-running period was sorter tan 24, wit an average of In contrast, te circadian rytmicity was not significantly detected in FA in any animals. Under LD 12:12 a clear bimodal pattern of WR wit two main peaks associated wit dawn (M) and dusk (E) was recorded. As Fig. 4, top, sows, bot averaged peaks were similar in amplitude and sape, irrespective of te animal s age. In 13-mo-old animals, te M peak started 1.89 before ligts-on and lasted for Te E peak began 0.84 before ligts-off and lasted for 2.85, peaking after ligts-off (Table 2). Altoug tese crepuscular peaks cover only 274 min of te LD cycle (19%), te total activity occurring in bot peaks reaced 73% of daily WR activity. After exposure to a long period of continuous darkness and returning to LD 12:12 (LD 17 mo old) 4 mo later, te same bimodal pattern reappeared. Bot te magnitude and timing of te peaks were similar to tose during te previous exposure to LD (Fig. 4, Table 2). No differences were detected in te daily amount of WR (1, m/day during te LD 13 mo and 1, m/day during LD 17 mo). Under LD 12:12, FA sowed an irregular and igly variable daily pattern, caracterized by diurnal preferences (67% of total FA occurring during te ligt Table 2. Analysis of bimodality under LD 12:12 Degu Morning Peak % Evening Peak % LD 12:12 (13 mo) C C C C C C C C C C Mean SE LD 12:12 (17 mo) C C C C C C C C C C Mean SE Summary data are depicted as means SE. Individual and summary data on onset, duration, and amplitude of morning and evening peaks of WR activity under exposure to 12:12- ligt-dark cycle (LD 12:12). Onset and duration is expressed in ours. To normalize ranges across subjects, amplitude is expressed as percentage of mean. pase). As illustrated in Fig. 4, bottom, low levels coincided wit te M and E peaks of WR. In regard to te presence of diel rytmicity, all te records for WR sowed a significant diel rytm, wereas significant diel rytmicity for FA could only be detected in 30% of te animals in LD 13 mo and 70% in LD 17 mo. Fourier analysis confirmed te existence of differences between WR and FA. Te main differences are related to te armonics 2 and 4, corresponding to 12 and 16, respectively. Despite te ig stability of te times wen te M and E peaks occurred, teir relative amplitude differed between animals. As Fig. 5 sows, 20 30% of te animals sowed a iger M peak tan E peak (cronotype A), 50 60% exibited te same magnitude in bot peaks (cronotype B), wereas 10 20% sowed a iger E tan M(cronotype C). An individual animal s membersip of a particular cronotype did not necessarily remain te same under LD 13 mo and LD 17 mo exposure (Fig. 5, bottom). Inasmuc as before te exposure to LD 12:12 animals were exposed to DD conditions, we tested te possibility tat different cronotypes were related to ow te degu encounters te LD cycle from te free run in DD. However, only in four of ten animals was it possible to detect a close relationsip between te pase in free running and te preferential entrainment to ligt-to-dark transitions. Te influence of expanded potoperiods on WR is sown in Fig. 6. Under te tree potoperiods a bimodal pattern appeared, te second peak of activity being greater tan te first. Under LD 12:12, lasted 13.61, starting 0.35 before ligts-on and ending 1.26 after ligts-off (Table 3). Wen te potoperiod was increased to 15 of ligt by delaying te ligts-off by 3, te animals delayed te -onset by 0.93 and -offset by Te mean waveform sowed a similar pattern to tat obtained under LD 12:12, wit a burst of activity appearing 2 30 min before ligts-off (about te same local time of te previous experimental pase) and a reactive peak to darkness onset (Table 3). Under LD 18:6, no immediate response was observed in most cases, altoug some animals advanced teir -onset witout any modification of te end of (6 of 8 animals). In tese ligting conditions te waveform was similar to tat seen in LD 15:9. No statistically significant differences were detected in te onset, duration, and amplitude of peaks between te tree potoperiods, except for te duration of M peak under LD 18:6 (Table 3). During tis experimental pase te daily run was 1, m/day under LD 12:12, m/day in LD 15:9, and m/day in LD 18:6. DISCUSSION In regard to FA and WR activity, Octodon degus sould primarily be considered as a diurnal rodent species wit two crepuscular peaks in its WR activity. Te profiles of general motor activity, previously reported (3, 14), exibit some differences from te results obtained in te present experiment. Obviously, a record of general motor activity includes bot true locomotor activity and also te activity related to feeding beav-

6 R528

7 R529 Fig. 6. Representative actogram (left) and mean waveform (rigt; SE, n 10) of WR activity during exposure to a progressively lengtening potoperiod. Saded areas in actogram indicate dark period. Dark bars in grap represent dark period of LD cycle. ior, so tat te record is less precise and more difficult to interpret. As previously reported by Labyak and Lee (9) in female degus, WR in LD conditions sowed a strong bimodal pattern caracterized by te presence of two peaks anticipating te ligt-to-dark and dark-toligt transitions, suggesting an endogenous control for tis activity. However, te precise timing and te ig amplitude of bot peaks suggest te existence of an additional masking effect induced by te ligting conditions. Similar to tat reported by Lee and Labyak (12), weel running activity of male degus sows stable free-running circadian rytms in DD caracterized by a tau 24. It is noteworty tat under DD and skeleton potoperiods, FA patterns are predominantly arrytmic in most animals on a circadian basis. Furtermore, under LD, some animals did not sow any statistically significant circadian rytm in FA. Tis uncoupling between WR and FA was also evident under different illumination conditions, especially under DD, wen all te animals displayed circadian rytms for WR and none for FA. In tose animals in wic FA exibited a significant circadian rytm, its periodicity coincided wit tat observed for WR, suggesting tat bot variables are under te control of a common pacemaker or different coupled pacemakers. However, te loss of FA rytmicity under DD supports te ypotesis tat circadian rytm in FA may be te result of internal masking elicited by WR. Obviously, wen an animal is running in a weel it cannot eat simultaneously, wic justifies te coincidence between te peak in WR and te fall in FA under LD conditions. However, under DD, te pattern of WR was unimodal and less clearly defined and so te internal masking would be weaker tan under LD, allowing tat FA was arrytmic in all animals. Examples of suc a dramatic uncoupling in oter species are rare (16). Trougout te experimental period, no important age-related differences were detected in WR, probably because tis species as a long life span and te ; Fig. 5. Mean waveforms ( SE; n 10) and superimposed actogram (all records belonging to same cronotype are represented in same actogram) of WR of 13-mo-old (left) and 17-mo-old (rigt) degus exposed to LD 12:12. Animals were classified in cronotypes according to criterion sown above. M, morning peak; E, evening peak. Eac animal is represent by a number inside te graps. A, cronotype A; B, cronotype B; C, cronotype C. Animals wit 66% of activity concentrated in M peak were considered cronotype A, tose wit 66% of activity related to E peak were considered cronotype C, and te rest were considered cronotype B.

8 R530 Table 3. Analysis of bimodality under expanded potoperiod Degu Morning Peak Evening Peak Expanded potoperiod (LD 12:12) C C C C C C C C C C Mean 7.65* 0.92* 6.58* 16.80* 4.46* 11.22* SE Expanded potoperiod C C C C C C C C C C Mean 8.58* 1.12* 4.70* 17.35* 5.38* 8.45* SE Expanded potoperiod C C C C C C C C C C Mean 8.54* * 17.96* 4.40* 11.97* SE Summary data are depicted as means SE. Individual and summary data on onset, duration, and amplitude of morning and evening peaks of WR activity under exposure to expanded potoperiod. Onset and duration is expressed in ours. To normalize ranges across subjects, amplitude is expressed as percentage of mean. Values for a given experimental period tat do not sare a symbol are significantly different (P 0.05). duration of our experiments was not sufficient to sow suc an evolution. Te crepuscular caracter of te degu as also been observed in natural environmental conditions. However, altoug it as been suggested tat tis beavior is te consequence of its low tolerance of ig daytime temperatures (3, 15), te coincidence of te records obtained under natural conditions and tose obtained under laboratory conditions involving constant temperature suggests tat te pattern of motor activity is strongly defined in tis species and is not exclusively controlled by environmental temperatures. It sould be pointed out tat te dramatic bimodal pattern in locomotor activity observed under our laboratory conditions could be enanced by te presence of weels togeter wit te ig ambient temperature. According to Jaeger (7), WR in te laboratory may be analogous to foraging beavior in te wild. In te degu, field studies sow tat tis activity is predominant at dawn and dusk and may enable te animal to accumulate food to be consumed later wen foraging subsides. Similar daily foraging strategies are employed by many species, including wild rats (18) In regard to te nature of te M and E peaks of WR, it seems clear tat bot peaks are endogenous components entrained to LD cycle. Te following remarks support tis contention. 1) Under LD 12:12, bot peaks sowed an anticipatory caracter to ligt-to-dark and dark-to-ligt transitions. 2) Wen te animals were transferred to DD, te free-running rytms started to run from te two peaks. 3) Te angle of pase between te M and E peaks and te ligts-on and -off canged wit different potoperiods. However, te sape of te peaks was modified by te masking effect of ligt, brigt ligt exerting a negative masking effect on WR, wereas te onset of darkness seems to stimulate WR activity. Tis seems to be te case not only under LD conditions but also under skeleton potoperiods. Wen we tried to analyze more deeply te origin of te two activity peaks of WR activity a few questions arose. Are te M and E peaks te output of a single clock-controlled peak and a second peak primarily te result of masking? Te anticipatory activity in bot peaks to ligt-to-dark transitions under LD 12:12 and te fact tat under DD, WR started to free run from two peaks in LD suggest an endogenous origin for bot peaks. Are te M and E peaks te output of two different oscillators or, on te contrary, are tey produced by one oscillator wit two separate outputs? Te appearance of a unimodal pattern of WR after a few days under DD and a ligt pulse suggests tat at least two different oscillators or groups of oscillators are involved. Are tere two single oscillators or two groups of oscillators? Altoug it is difficult to answer tis question, te presence of components associated wit one peak of activity tat free run until tey become entrained to te oter peak supports te ypotesis tat it is a multioscillatory system in wic te oscillators are associated in two groups. An oscillator seems to be composed of a functional network of supraciasmatic nuclei (SCN) neurons (2). Experimental evidences of suc a multioscillatory nature ave been recently sown in te mammalian SCN (13). A noteworty caracteristic of degu s WR rytms is te wide variation in te relative amplitudes of te M and E peaks between animals, wic permits teir classification into tree main groups: morning type (A), wit 66% of teir activity related to M peak, evening type (C), wit 33% of teir activity related to M peak, and intermediate type (B), in wic te M peak represents 33 66% of activity. Altoug te number of

9 R531 animals studied was not sufficient to allow a statistical analysis, te percentage of animals belonging to eac cronotype is similar to tat described by Labyak et al. (10) using body temperature rytms and general motor activity as variables. It is possible to ypotesize tat te circadian system of te degu tat is responsible for controlling WR is composed of multiple oscillators, eac wit its own particular period, pase angle of entrainability and pase response curve (2). Tus different oscillators would entrain preferentially to dawn or dusk, altoug some of tem sould be able to entrain indiscriminately to bot transitions. Wen animals are exposed to DD or one brigt ligt pulse, all te oscillators became coupled and generated a unimodal pattern. Te existence of different cronotypes could be te consequence of different proportions of M and E oscillators in eac individual. In conclusion, our results sow tat te degu s temporal feeding strategy seems mainly arrytmic, wereas its WR patterns appear to be driven by a strongly circadian bimodal rytm. Te bimodal nature of te WR activity of te degu can be explained by te existence of two groups of oscillators being responsible for te M and E peaks. Tese would be entrained to a complete LD cycle, altoug some of tem may be able to intercange teir pase angle of entrainment. Perspectives In recent years, because of its diurnal beavior and certain particular pysiological caracteristics, te degu as become an interesting experimental model for different researc fields, including cronobiology. Our results, obtained wit a different population of degus tan used for most oter publications on circadian rytms of te species, sow a similar pattern of circadian entrainment to tat reported by oter laboratories, suggesting tis animal model is sufficiently robust for use in future cronobiological researc. In addition, tese results suggest new and interesting fields of study, suc as 1) te different pattern of rytmicity between WR and FA (a strong circadian rytm in WR wile, simultaneously, FA is more arrytmic tan circadian); 2) a study of cronotype stability and te response of te morning and evening peaks to ligt pulses could provide important evidence to support te multioscillatory nature of circadian system; and 3) finally, a diurnal rodent caracterized by te existence of different cronotypes could provide an experimental model better tan nocturnal rodents for applied cronobiological studies in parmacology, psycology, and etology. Address for reprint requests and oter correspondence: R. García Allegue, Dpto. Fisiología Animal, Facultad de Biología, Universidad de Murcia, Campus de Espinardo, Murcia, Spain ( rallegue@fcu.um.es). Received 22 January 1998; accepted in final form 22 April REFERENCES 1. Ascoff, J. Anticipation of a meal: a process of learning due to entrainment. Monit. 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