ORIGINAL PAPER

F. J. Sa nchez-Va zquez á J. A. Madrid á S. ZamoraM. Tabata

Feeding entrainment of locomotor activity rhythms in the gold®shis mediated by a feeding-entrainable circadian oscillator

Accepted: 8 April 1997

Abstract Periodic food availability can act as a potentzeitgeber capable of synchronizing many biologicalrhythms in ®shes, including locomotor activity rhythms.In the present paper we investigated entrainment of lo-comotor rhythms to scheduled feeding under di�erentlight and feeding regimes. In experiment 1, ®sh wereexposed to a 12:12 h light/dark cycle and fed one singledaily meal in the middle of the light phase. In experiment2, we tested the e�ect of random versus scheduledfeeding on the daily distribution of activity. Duringrandom feeding, meals were randomly scheduled withintervals ranging from 12 to 36 h, while scheduledfeeding consisted of one single daily meal set in themiddle of the light or dark phase. Finally, in experiment3, we studied the synchronization of activity rhythms tofeeding under constant darkness (DD) and after shiftingthe feeding cycle by either advancing or delaying thefeeding cycle by 9 h. The results revealed that gold®shsynchronized to feeding, overcame light entrainmentand signi®cantly changed their daily distribution of ac-tivity according to their feeding schedule. In addition,the daily activity pattern modulated by feeding di�eredbetween layers: a peak of activity being noticeable di-rectly after feeding at the bottom, while an anticipatorybehaviour was obvious at the surface of the tank. UnderDD and no food, free-running rhythms averaging 25.5 �1.9 h (mean � SD) were detected. In conclusion, someproperties of feeding entrainment (e.g. anticipation ofthe feeding time, free-running rhythms following termi-

nation of periodic feeding, and the stability of ù aftershifting the feeding cycle) suggested that gold®sh have(a) separate but tightly coupled light- and food-en-trainable oscillators, or (b) a single oscillator that isentrainable by both light and food (one synchronizerbeing eventually stronger than the other).

Key words Locomotor activity á Feeding-entrainment á Circadian rhythms á Verticaldistribution á Gold®sh

Introduction

Food is rarely constantly available in the wild but, in-stead, shows a spatial as well as a temporal distribution.Foraging behaviour is commonly restricted to a certainperiod in which the abundance of prey is increased andthe risk of predators is reduced. Since these factors often¯uctuate periodically, the ability to predict a favourableperiod is clearly bene®cial for the animal. If the ap-proach of a meal can be anticipated, the animal canimprove food acquisition and utilization. This adaptiveresponse is thought to be mediated by an internal timingsystem or biological clock, which is synchronized to theenvironment (Ascho� 1986). Though the light/dark(LD) cycle is the most powerful environmental factor toentrain biological rhythms, periodic feeding can also actas a potent synchronizer or zeitgeber (Boulos and Ter-man 1980). Synchronization to feeding has been widelyreported in a variety of behaviours and metabolic ac-tivities of mammals and birds (review: Mistlberger1994). In ®sh, some ®eld observations as well as labo-ratory studies have also demonstrated the synchronizinge�ect of scheduled feeding (review: Spieler 1992).

The entrainment of biological rhythms to light ismediated by a light-entrainable circadian oscillator,which in rodents is localized in the suprachiasmaticnuclei (SCN) of the hypothalamus (Meijer and Rietveld1989). Ablation of the SCN leads to the abolition orserious disruption of light-entrained free-running rhythms

J Comp Physiol A (1997) 181: 121±132 Ó Springer-Verlag 1997

F.J. Sa nchez-Va zquez1 (&) á J.A. Madrid á S. ZamoraDepartment of Physiology and Pharmacology,Faculty of Biology, University of Murcia, 30100-Murcia, SpainFax: +34-68-363963; e-mail: javisan@fcu.um.es

M. TabataDepartment of Biosciences,Teikyo University of Science and Technology, Japan

1Present address:Department of Biosciences, Teikyo University of Scienceand Technology, 2525 Uenohara, Yamanashi 409-01, Japan,Fax: + 81-554 63-4431; e-mail: javisan@ntu.ac.jp

in rats (Moore and Eichler 1972; Stephan and Zucker1972) and birds (Ebihara and Kawamura 1981). Incontrast, feeding entrainment is not abolished in SCN-ablated rats, which provides strong evidence for theexistence of a feeding-entrainable circadian oscillatoroutside the SCN (Stephan et al. 1979). In ®sh, however,there is still little information on this topic and basicquestions, such as the circadian nature and the mecha-nisms of feeding-entrainment are still controversial(Spieler 1992).

Locomotor activity, among other behavioural andphysiological variables, exhibits daily rhythms that canbe entrained by a feeding cycle. In ®sh, synchronizationof locomotor activity rhythms to food has been reportedin some species, including loach, Misgurnus an-guillicaudatus (Naruse and Oishi 1994), mud-skipper,Periophtalmus cantonensis (Nishikawa and Ishibashi1975), killi®sh, Fundulus heteroclitus and bluegill, Le-pomis macrochirus (Davis and Bardach 1965), Atlanticsalmon, Salmo salar (Juell et al. 1994), and medaka,Oryzias latipes (Weber and Spieler 1987). Nevertheless,none of these studies has deeply analysed the nature ofthe endogenous timing mechanism involved in feeding-entrainment. In the case of gold®sh (Carassius auratus),although there have been some attempts to investigatethe mechanisms whereby entrainment to feeding occurs(Spieler and Noeske 1984; Spieler and Clougherty 1989),the underlying system remains unresolved.

A further feature of locomotor activity rhythms in®sh is that the general pattern of activity may show dielchanges in the vertical distribution of activity (review:Neilson and Perry 1990), so that entrainment to feedingshould account for such behaviour. For instance, if theresponse of ®sh to a feeding cycle can di�er dependingon the layer of the tank, the behavioural parametersused to de®ne feeding entrainment (i.e. anticipation tofood) will be a�ected by this factor. Surprisingly, littleattention has been paid to this fact when consideringfeeding entrainment in ®sh.

Recent research on demand-feeding and locomotoractivity circadian rhythms in single gold®sh has revealedthe existence of a highly ¯exible circadian system, whichexhibits a dual-phasing capacity, i.e. coexistence of di-urnal and nocturnal phasing, and the capacity to changephase (Sa nchez-Va zquez et al. 1996). In that report, apatent e�ect of the feeding regime on the locomotoractivity rhythms was observed: when single gold®sh thathad been entrained to a single daily meal, and whichtherefore displayed a diurnal pattern of behaviour, weregiven free access to food through a self-feeder, some ®shchanged the phase of their rhythms and became noc-turnal. Moreover, they reverted to diurnalism when theself-feeder was removed and the diurnal feeding cyclewas re-established.

In the present paper, we further investigate feedingentrainment in single gold®sh, with special attentionpaid to the endogenous nature and timing mechanismsof feeding entrainment. To this end, the synchronizationof locomotor activity rhythms to a single daily meal was

studied under di�erent light and feeding regimes, in-cluding constant darkness, shifting feeding cycles, andfood deprivation. In addition, since the general patternof activity may exhibit a spatial structure, the verticaldistribution of feeding-entrained ®sh was also consid-ered.

Material and methods

Animals and housing

The gold®sh used in the experiments were obtained from localbreeders (Shizuoka prefecture, Japan) and held at the laboratoriesof the Teikyo University of Science and Technology, where allexperiments were conducted. A total of 23-single gold®sh were usedin the present study. They were kept individually in 35-l glass tankswith a continuous supply of ®ltered and aerated water. All aquariahad a dark vinyl sheet in the front and sides to avoid ®sh beingdisturbed by the surrounding tanks. The tanks were placed insound and light-proof chambers in which water temperature wascontrolled and kept constant at 20 °C. Each aquarium was pro-vided with an overhead ¯uorescent lamp (National, 15 W), whichsupplied on average 500 lx at the water surface.

Prior to the experiments, the ®sh were maintained under a 12-hlight: 12-h dark cycle (regular LD cycle, lights on at 0600 hours ando� at 1800 hours) and fed by hand once a day during the lightperiod at irregular time intervals. For the experiments, a com-mercially available feeder for pet ®sh (Food Timer, Seiko, Japan)was installed in each aquarium. The feeder consisted of a rotatingfood container controlled by a built-in timer so that, at the pre-programmed time, the food container rotated and delivered foodpellets. The feed control slide of the food container was adjusted tofeed ®sh at a daily feeding rate of about 1.5% of body weight.

Experimental procedure

The following experiments were designed to investigate locomotoractivity rhythms under di�erent light and feeding regimes. The ®shwere basically maintained under conditions similar to those de-scribed above for the pre-experimental period.

Experiment 1: vertical distribution of feeding-entrained ®sh

We looked at the activity pattern displayed by feeding-entrainedgold®sh, with special attention paid to the vertical distribution ofactivity in the aquarium. For that purpose, three single gold®sh of97.2 g average body weight (BW) were held in individual tanks, andlocomotor activity was recorded by means of a total of nine activitysensors (three sensors per layer) symmetrically distributed from leftto right, at the upper (5 cm from the surface), middle, and lower(7 cm from the bottom) layer. The light regime varied as follows: aregular LD cycle until day 36, and constant darkness (DD) there-after. Food was supplied once a day at 1200 hours ± the middle ofthe light period ± until day 49. Thereafter, ®sh were deprived offood and left under constant conditions.

Experiment 2: random versus scheduled feeding

We investigated the e�ect of random and scheduled feeding onlocomotor rhythms under a regular LD cycle. A total of 20 singlegold®sh of 30.3 g average BW were used. Locomotor activity at theupper and lower layers was recorded by means of two photocells.Fish were initially exposed to a regular LD cycle and submitted torandom feeding (stage A) for 3 weeks and then to scheduledfeeding (stage B) for another 3 weeks. During A, single meals wererandomly scheduled in both the light and dark periods, based on a

122

table of random times. Meal intervals ranged from 12 to 36 h, sothat the average meal interval was 24 h and the average feeding ratewas 1.5% of BW. During B, one single daily meal (1.5% of BW)was set in the middle of the light phase (ML feeding group, n � 10)or in the middle of the dark phase (MD feeding group, n � 10).

Experiment 3: shifting the feeding cycle

In this experiment, we tested the synchronization of activityrhythms to feeding under DD and after shifting the feeding cycle.Special attention was paid to the phase relationship between theactive phase of ®sh and the feeding cycle. To this end, the ®sh usedin experiment 2 were transferred to DD and subjected to the pre-vious feeding cycle for 2 weeks. The feeding cycle was then shiftedby either advancing (group I, n � 10) or delaying (group II, n � 10)the feeding cycle by 9 h. After 2 weeks under these shifted cycles,feeding was suppressed and ®sh were left under truly constantconditions (DD + no food) to study free-running rhythms.

Data analysis

Locomotor activity was measured by means of infrared photocells(OMRON, mod. E3V-DS70C43S) so that the number of lightbeam interruptions was counted and stored every minute by amicrocomputer. The Chronobiology Kit (Stanford Software Sys-tems, Stanford, Calif., USA) was used for data acquisition andanalysis. Periodically, the activity records were collected andtransferred to another computer for further analysis and plotting.Software was designed to doubleplot records at a resolution of10 min, each point representing the percentage of light beam in-terruptions that occurred in 24-h intervals. The period of free-running rhythms was determined by chi-square periodogramanalysis at a con®dence level of 95% (CronobioPC, PANLAB, SA,Barcelona). To test for signi®cant di�erences in the daily number ofactivity counts, and in the positioning (ù) and length (a) of the``activity phase'' of individual ®sh, the data were subjected toanalysis of variance (ANOVA), followed by comparison of meansusing Tukey's test (Systat 5; Systat).

Results

Vertical distribution of feeding-entrained ®sh

Under a regular LD cycle, ®sh showed a diurnal swim-ming pattern modulated by feeding, a bout of activityoccurring around mid-light (the time of the daily singlemeal). However, the activity pro®le di�ered between theupper, middle and lower layers. Figure 1 shows the av-erage waveform of activity at the three di�erent layers,left and right sides being pooled. At the upper layer (A),®sh developed an anticipatory behaviour to feeding,which consisted of an increasing activity a few hoursprior to the feeding time. As a result, the mean level ofactivity during the 4 hours prior to feeding was almosttriple (from 4.4 to 12.0 counts/10 min). Later on, asso-ciated with the delivery of food pellets, the activitypro®le dropped sharply to less than 2.5 counts/10 minand remained low for 30 min. In striking contrast, at thelower layer (C), directly after feeding the oppositeseemed to be true: a peak of activity of about the sameduration as the decrease seen at the upper layer. Here, atthe time of feeding, the mean activity level rose quicklyfrom 5.7 to 12.5 counts/10 min. Between both zones,middle layer (B), there was a less clearly de®ned pattern,

which approximated the pattern observed at the upperlayer.

Eventually, an opposite daily pattern of behaviourwas observed at di�erent layers of the aquarium: thesame ®sh being nocturnal in the upper zone, and diurnalat the bottom. An example of such a nocturnal patternat the upper layer is given in Fig. 2. In this case, lightinitially appeared to entrain activity rhythms more e�-ciently than feeding, since the latter seemed to exert littlein¯uence on the overall activity pattern; however, someactivity related to feeding progressively appeared, gain-ing strength with time. This activity was particularlyevident when the ®sh were transferred to DD on day 37and entrainment to light disappeared, while light-en-trained activity seemed to join the feeding-entrainedcomponent, thus enhancing it. In general, underDD + scheduled feeding, the daily activity pro®le re-mained mostly entrained by feeding, showing an activityaggregation centred around feeding time.

Under DD + no food, rhythmic activity persistedaround the previous feeding time for the ®rst 3±4 days.However, the rhythm later became less clearly de®nedand was ®nally abolished. Figure 2 shows an interestingexample in which, when ®sh were deprived of food, theportion of activity that occurred directly after feeding(post-feeding activity) suddenly ceased, while the pre-feeding portion (anticipatory activity) persisted and be-gan to freerun with an endogenous period of 25.1 h.

Random versus scheduled feeding

Figure 3 summarizes the distribution of activity dis-played at the upper and lower layers during daytime(Light Activity = LA) of gold®sh held under a regularLD cycle and submitted ®rst to random (A) and then tomid-light feeding (B) or mid-dark feeding (C). In A, 12®sh out of 19 failed to synchronize to the LD cycle andLA was, therefore, close to 50%. On the other hand,some ®sh exhibited a more clearly de®ned pattern ateither upper or lower layers: four ®sh being mostly di-urnal (LA over 66.6%) while three ®sh mostly nocturnal(with LA under 33.6%). Furthermore, as previouslydetected in experiment 1, in one case we observed thatthe same ®sh displayed an opposite pattern, dependingon the layer: nocturnal at the upper layer and diurnal atthe lower layer. On average, LA at the upper and lowerlayers was 49.8 � 17.2% and 58.9 � 11.5% (mean �SD), respectively. We noted that well-de®ned nocturnalpatterns appeared only at the upper layer, while themajority (four out of ®ve) of clear diurnal patterns wasfound at the lower layer.

After transferring to scheduled feeding (B), there wasa notable change in the daily pattern of behaviour sincemost ®sh, from both the ML and MD feeding groups,tended to con®ne their activity to around feeding time.In 18 out of 19 animals, there was a statistically signi-®cant (ANOVA, P < 0:05) change in their LA at eitherupper or lower layer according to the scheduled feeding.

123

On average, in the ML feeding group, LA at the upperand lower layers was 61.4 � 25.5% and 69.7 � 12.9%,respectively, while in the MD feeding group it was27.5 � 21.7% and 39.5 � 15.0%, respectively.

Figures 4 and 5 show di�erent actograms of loco-motor activity from the ML and MD feeding groups,respectively. In many cases, we observed that periodicfeeding e�ectively synchronized arrhythmic activity and,thus, a more clearly de®ned daily rhythm emerged(Fig. 4B). In other cases, the phase of the previous dailyrhythm was inverted when feeding was scheduled to theopposite phase. Examples of nocturnal and diurnalgold®sh that changed their phasing according to feedingare given in Figs. 4A, 5A and 5B, respectively. In all®shes, synchronization to feeding occurred very quicklyand usually took no more than 4 or 5 days.

Shifting the feeding cycle

When the ®sh used in the previous experiment weretransferred to constant darkness, feeding entrainmentpersisted and the phase angle (ù) and the length of theactive phase of ®sh (a) remained stable (see Fig. 6);therefore, average a and ùdid not di�er signi®cantlywhenlights were switched o� and the feeding cycle was shifted9 h (ANOVA, P > 0:05). Under LD conditions, ®shtended to con®ne their activity to the light or dark phase,depending on the phasing of the feeding cycle, and cen-tered their activity around feeding time.On average,awas10.3 � 1.8 h and ù was )77.0 � 12.2° (mean � SD,n � 16). Under DD, the average length of a remainedclose to its previous value (9.9 � 2.7 h, n � 13) but ùvaried more widely, ranging from )130° to )30° (Fig. 6).

Fig. 1A±C Average waveformof locomotor activity at theupper (A), middle (B), andlower (C) layer from three sin-gle gold®sh submitted toscheduled feeding at mid-light.Values are the mean � SDcalculated from 15 consecutivedays. The vertical dashed linerepresents the feeding time(1200 hours). The dark phase ofthe regular LD cycle (lights o�from 1800 to 0600 hours) isindicated by a shaded area

124

After shifting the feeding cycle by 9 h, the phase re-lationships previously observed in many ®shes were re-established. In general, resynchronization to the newfeeding cycle took place, the ®sh either advancing ordelaying their activity rhythms by 9 h in response.However, before ù recovered its initial position, sometransient cycles were required. Furthermore, in some®shes, a shift in their phase positioning was detected.Figures 7 and 8, show actograms of four examples, inwhich this shift was particularly evident. In Fig. 7A,after transferring to DD, ®sh delayed their activity by3 h, so that ù, which was previously set at )90° underLD, became then )30°. In the example given in Fig. 8A,in contrast, ®sh advanced the onset of its active phase6 h and, as a result, ù became )130°.

At the end of the experiment, under DD and no food,free-running rhythms were detected in 13 out of 20 an-imals, which represented 65% of the total. Table 1

summarizes the results obtained from the periodogramanalysis of the data. The average period of circadianrhythms was 25.5 � 1.9 h (mean � SD). We observed,however, a marked in¯uence of the layer on the ap-pearance of circadian rhythmicity, since in some ®sh (6out of 13) free-running rhythms were detected only atthe lower layer, while they were absent from the upperlayer (Table 1). In addition, s varied depending on in-dividuals and showed instability in its period. Figures 7and 8 show examples of ®sh displaying long free-runningrhythms (Fig. 7A, B, 8A), and one ®sh in which s wasunstable and initially ran shorter, although it later ranlonger than 24 h (Fig. 8B).

Discussion

Results from the present study indicate that scheduledfeeding acts as a potent zeitgeber, entraining locomotoractivity rhythms in gold®sh. When submitted to periodicfeeding, gold®sh synchronized to feeding, overcominglight entrainment, most of them signi®cantly changingtheir daily distribution of activity according to thefeeding schedule. In addition, we observed some prop-erties of feeding entrainment, such as anticipation of thefeeding time, free-running rhythms following termina-tion of periodic feeding and stability of ù after shiftingthe feeding cycle, that suggested the participation of aself-sustained food-entrainable oscillator (FEO).

Fig. 2 Double-plotted actogram of a locomotor activity recordregistered at the upper layer from a single gold®sh submitted toscheduled feeding at 1200 hours and exposed ®rst to a regular LDcycle, and second to constant darkness (DD). Activity records areplotted at a resolution of 10 min, the height of each point representingthe percentage of light beam interruptions that occurred in 24-hintervals. Horizontal solid and open bars at the top of the plot indicatethe light/dark regime. To test the endogenous nature of feedingentrainment, at the end of the experiment the animal was left undertruly constant conditions (i.e. DD + no food). The chi-squareperiodogram analysis of this period is indicated on the right of the plot

125

Feeding-entrained rhythms display some particularcharacteristics that denote true synchronization to thefeeding zeitgeber. Anticipation to feeding is one of thesebasic attributes, meaning that the animal is able topredict the time of feeding using a biological clock(Mistlberger 1994). In our study, gold®sh developedanticipation to feeding time when they were given a dailysingle meal at mid-light (Fig. 1A). However, since it ispossible that anticipatory activity could be linked to theexternal LD cycle, and that the animal could use thetime interval between lighting and meals, entrainment tofeeding was tested under constant conditions. Whenfeeding-entrained gold®sh were transferred from LD toDD, synchronization to feeding persisted and ®sh be-came active in anticipation of feeding time, thus provingthat feeding, and not light, was the signal that syn-chronized the activity rhythms. In addition, after shift-ing the feeding cycle by 9 h, the phase relationshipspreviously observed remained stable after a few transientcycles. These results indicate that feeding does entrainlocomotor activity rhythms under constant darkness.Nevertheless, marked changes in the phase relationshipbetween the active phase of ®sh and the feeding cyclewere detected. As indicated by the behaviour of someindividuals, the LD cycle may also in¯uence the phasepositioning of the activity rhythm (Figs. 7A, 8A). Mu-tual interactions between the light and food zeitgeberssuggest the participation of both a light-entrainable os-cillator (LEO) and a FEO, which may show partial

coupling between them. Di�erent phase relations be-tween activity onset and feeding time could also be dueto masking e�ects of lights.

Evidence for the existence of such a FEO, which iscoupled to but separate from a master LEO, has beenwidely reported in mammals (Cambras et al. 1993; Ed-monds 1977; Ottenweller et al. 1990; Rosenwasser et al.1984; Stephan 1986) and birds (Phillips et al. 1993;Rashotte and Stephan 1996), and is also thought to existin ®sh (Sa nchez-Va zquez et al. 1995; Spieler 1992). Inthe present study, under DD circadian rhythms ap-peared in the absence of periodic feeding. Furthermore,although in many cases the starting point of free-run-ning rhythms could not be traced accurately owing totheir erratic nature, circadian rhythms were observed tofreerun from the previous anticipatory portion of ac-tivity (see Fig. 2), which provides support for the exis-tence of a self-sustaining clock to explain foodanticipatory activity.

In mammals, the hypothalamic SCN has been pro-posed as the site of the master clock, which governslight-entrained circadian rhythms. In ®sh, however, thelocation of the central pacemaker remains uncertain,although some characteristics of the retinorecipient re-gion of the teleost hypothalamus show similarities withits counterpart in mammals (review: Holmqvist et al.1992). In ®sh, as well as in mammals, the location of theFEO and the physiological mechanisms involved in thetransduction of feeding entrainment are unknown. To

Fig. 3A±C Daily distributionof activity, expressed as thepercentage of locomotor activi-ty displayed during daytime(LA) at the upper (Y-axis) andlower (X-axis) layer by singlegold®sh submitted ®rst to ran-dom feeding (n � 19, A) andthen to scheduled feeding atmid-light (n � 9, B) or at mid-dark (n � 10, C). One faultyrecord was excluded from theanalysis. Values are the meancalculated from 13 consecutivedays, transient cycles after theestablishment of the feedingcycle being excluded. To helpthe visual localization of``mostly diurnal'' and ``mostlynocturnal'' ®sh, a pair of verti-cal and horizontal dotted lineshave been included at LA 33.3and 66.6%

126

Fig. 4A,B Double-plotted actograms of locomotor activity recordsof single gold®sh during experiment 2. Gold®sh were maintainedunder a LD 12:12 cycle and submitted ®rst to random-feeding and,from day 18 onwards, to scheduled feeding at mid-light. Locomotoractivity was registered through both photocells. Figure descriptionand format are the same as given in Fig. 2. Examples of gold®sh thatexhibited an unde®ned (B) or a mostly nocturnal activity pattern (A)under random-feeding, but became day active when fed at mid-light

Fig. 5A,B Double-plotted actograms of locomotor activity recordsof single gold®sh during experiment 2. Experimental conditionssimilar to those described for Fig. 4, excepting from day 18 onwardsscheduled feeding was set at mid-dark. Other ®gure descriptions andformat are the same as given in Fig. 2.A, B Two examples of gold®shwith a mostly diurnal activity pattern when random-fed, whichbecame nocturnal after scheduled feeding at mid-dark

m

.

127

date, both supporting and contradicting evidence for anutritional or behavioural entraining pathway has beenput forward (Mistlberger 1994). In gold®sh, the role ofthe diet composition in feeding-entrainment has beeninvestigated by Spieler et al. (1987), who found that dietsde®cient in some amino acids equally entrained loco-motor rhythms after shifting the feeding cycle.

Since the early paper of Davis (1963), feeding en-trainment has been studied in various ®sh species(Boujard and Leatherland 1992; Naruse and Oishi 1994;Weber and Spieler 1987), including gold®sh (Spieler andNoeske 1984; Spieler and Clougherty 1989; Gee et al.1994). The results obtained in the present experimentunder constant conditions are consistent with those re-ported by Spieler and Clougherty (1989), who describedfree-running locomotor rhythms in groups of gold®shkept under conditions of constant light or darkness. Inthat report, however, locomotor activity rhythms free-ran with a periodicity of 24 h, which contrasts with thelonger s recorded in our present experiments. It should

be noted that these researchers used smaller gold®sh(6 g) and, most importantly, ®sh were kept in groups of12, while in our experiments gold®sh were maintainedindividually. In previous experiments using light-en-trained, self-fed single gold®sh, free-running rhythmshad a 25.3-h periodicity (Sa nchez-Va zquez et al. 1996),which approaches the average s recorded in this exper-iment (25.5 h). Other authors, using light-entrainedgold®sh, have also reported circadian rhythms longerthan 24 h (Kavaliers 1981; Iigo and Tabata 1996).

Demand-feeding activity, in addition to locomotoractivity, has recently been used as a behavioural variablein a number of studies in which ®sh were trained to feedduring cycles of temporaly restricted feeding (Boujardet al. 1992; Boujard 1995; Sa nchez-Va zquez et al. 1995).In gold®sh, entrainment of demand-feeding rhythms hasalso been studied by Gee et al. (1994). In this study,when food was restricted to 1 h, gold®sh showed analmost linear increase in the number of lever pressesbetween 4 and 6 h prior to feeding. Interestingly, ®shcontinued to anticipate the onset of the feeding periodunder a continuous lighting regime. In the absence offood reward, however, the response extinguished rap-idly, making it di�cult to observe free-running rhythms(Gee et al. 1994).

During our experiments, a reversed daily pattern ofbehaviour was eventually detected at di�erent layers ofthe aquarium: the same gold®sh being predominantlynight-active at the surface, but day-active at the bottom(see daily distribution of LA, Fig. 3). Spatial-timestructures of this type (ascent to the surface at night anddescent to the bottom during the day) appear to becommonly observed behaviour. Diel vertical migrationshave been extensively reported in many aquatic organ-isms and are thought to be of adaptive signi®cance(Kerfoot 1985; Lampert 1989). Many ®sh species in bothfresh water and seawater have been found to exhibit dayand night changes in their depth position (review:Neilson and Perry 1990) but the signi®cance of suchbehaviour remains controversial. The diel pattern ofvertical migration may change on a seasonal basis(Yokota and Oishi 1992). The relative contribution ofboth exogenous and endogenous factors in the migra-tion behaviour of juvenile plaice has recently been in-vestigated by Burrows and Gibson (1995). Althoughthese vertical movements can be partially explained bylight response (Huse and Holm 1993; Appenzeller and

Fig. 6A±C Computed phase relation of feeding-entrained activityrhythms and their zeitgeber for individual gold®sh under LD 12:12(A), constant darkness (B), and after shifting the feeding cycle by 9 hin constant darkness (C). Although some gold®sh were active at anytime, the onset (ù) and length (a) of an ``active phase'' could bedetermined in well-de®ned average activity/rest waveforms fromindividual gol®sh duringA (n � 16), B (n � 13) andC (n � 11); ù wasestimated as the time in which the ®rst large peak of activity with anirreversible change in slope occurred, and a was estimated as theperiod during which the activity pro®le was above a certain levelwhich accounted for the 70% of all activity counts recorded within a24-h period. Transient cycles between A±B, and B±C were excludedfrom the analysis. The phase position of the feeding cycle ± thereference point ± is represented by a vertical dotted line (0°), while ùand a are indicated by a circle and a horizontal line, respectively

Fig. 7A,B Double-plotted actograms of locomotor activity recordsof single gold®sh during experiment 3. Gold®sh, initially kept under aLD 12:12 cycle and submitted to scheduled feeding at mid-light, weretransferred to constant darkness (DD) on day 17 and submitted to thesame feeding schedule until day 31, when the feeding cycle wasadvanced by 9 h. Two weeks later, on day 45, gold®sh were deprivedof food to study free-running rhythms. Figure description and formatare the same as given in Fig. 2. Two examples in which the phaserelationship between the feeding cycle and the active phase of ®shchanged (A) or remained (B) after transferring to DD. In both cases,the period length of the free-running rhythms was longer than 24 h.

c

128

Leggett 1995), thermoselection (Carey and Robinson1981), avoidance of predators (Clark and Levy 1988)and bioenergetics (Levy 1990), it seems that foragingbehaviour plays a central role in the control of verticalmigration. Ferno et al. (1995) proposed a trade-o� be-tween light avoidance and food attraction to explain theascent to the surface of Atlantic salmon in net pens.Moreover, as suggested by Juell et al. (1994), the verticalposition of salmon may provide information about theappetite of ®sh.

In our study, gold®sh exhibited di�erential behav-iour: anticipation of feeding time at the upper layer, anda peak of activity immediately after feeding at the lowerlayer (Fig. 1). From these results, it can be inferred thatgold®sh were waiting for food pellets near the surfaceand, when food dropped, they went down to the bottomto feed. Immediately after food delivery, both decreasedand increased activities at the upper and lower layers,respectively, may be seen as a passive response of ®sh tothe feeding stimulus (i.e. ``masking e�ect''), while an-ticipatory activity during the approach of feeding time ismost likely related to the expression of an internal tim-ing mechanism.

In conclusion, the results presented here reveal someproperties of feeding entrainment (i.e. anticipation offeeding time, free-running rhythms under DD + nofood, and stability of ù after shifting the feeding cycle)that provide strong evidence of the existence in gold®shof a self-sustained food-entrainable circadian oscillator.However, it is still uncertain whether or not ®sh ingeneral possess a separate FEO, in addition to a LEO.Feeding-entrainment could be explained in mechanisticterms based on the properties of a complex single os-cillator, which in turn would be entrained by both lightand food. However, this is not the case for rats andbirds, since the existence of a functionally and anatom-ically separate FEO has been demonstrated. Accordingto current data, it cannot be concluded that ®sh have anindependent FEO, since it cannot be excluded that ®shmay use other mechanisms based on the information

supplied by a master LEO to anticipate food. Therefore,our data are compatible with the following two hy-potheses: gold®sh have (a) separate but tightly coupledlight- and food-entrainable oscillators, or (b) a singleoscillator that is entrainable by both light and food (onesynchronizer being eventually stronger than the other).

Acknowledgements This work was funded by the Ministry of Ed-ucation, Sports, Science and Culture, Japan (no. 07660259). Duringthis study, Dr. F.J. Sa nchez-Va zquez was supported by a postdoct-oral research fellowship from the European Commission (EU S&TFPJ, DG-XII-B-3). We would like to express our sincere gratitudeto two anonymous referees, who gave constructive and thoughtfulcomments that improved the present manuscript. Thanks are alsodue to the members of Prof. Tabata's laboratory for their assis-tance during the experiments. These experiments comply with the``Principles of animal care'' of the National Institute of Health(1985) and also with the current laws of the country in which theexperiments were conducted.

References

Appenzeller AR, Leggett WC (1995) An evaluation of light-medi-ated vertical migration of ®sh based on hydroacoustic analysisof the diel vertical movements of rainbow smolt (Osmerusmordax). Can J Fish Aquat Sci 52: 504±511

Ascho� J (1986) Anticipation of a meal: a process of ``learning''due to entrainment. Monit Zool Ital 20: 195±219

Boujard T (1995) Diel rhythms of feeding activity in the Europeancat®sh, Silurus glanis. Physiol Behav 58: 641±645

Boujard T, Leatherland JF (1992) Circadian rhythms and feedingtime in ®shes. Environ Biol Fish 35: 109±131

Boujard T, Dugy X, Genner D, Gosset C, Grig G (1992) De-scription of a modular, low cost, eater meter for the study offeeding behavior and food preferences in ®sh. Physiol Behav 52:1101±1106

Boulos Z, Terman M (1980) Food availability and daily biologicalrhythms. Neurosci Biobehav Rev 4: 119±131

Burrows MT, Gibson R (1995) The e�ect of food, predation riskand endogenous rhythmicity on the behaviour of juvenile pla-ice, Pleuronectes platessa L. Anim Behav 50: 41±52

Cambras T, Vilaplana J, DõÂ ez-Noguera A (1993) E�ects of long-term restricted feeding on motor activity rhythm in the rat. AmJ Physiol 265: R467±R473

Carey FG, Robinson BH (1981) Daily patterns in the activities ofsword®sh, Xiphias gladius, observed by acoustic telemetry. FishBull U S 79: 277±292

Clark CW, Levy DA (1988) Diel vertical migrations by juvenilesockeye salmon and the antipredation window. Am Nat 131:271±290

Davis RE, (1963) Daily ``predawn'' peak of locomotor in ®sh.Anim Behav 12: 272±283

Davis RE, Bardach E (1965) Time-co-ordinated prefeeding activityin ®sh. Anim Behav 13: 154±162

Ebihara S, Kawamura H (1981) The role of the pineal organ andthe suprachiasmatic nucleus in the control of circadian loco-motor rhythms in the Java sparrow, Padda oryzivora. J CompPhysiol A 141: 207±214

Fig. 8A,B Double-plotted actograms of locomotor activity recordsof single gold®sh during experiment 3. Experimental conditionssimilar to those described for Fig. 7, excepting on day 31 the feedingcycle was delayed by 9 h. Other ®gure descriptions and format are thesame as given in Fig. 2.A One example with a marked changed in thephase relationship between the feeding cycle and the active phase of®sh changed. B One case in which the period of the free-runningrhythm was unstable, initially running shorter but later longer than24 h

b

Table 1 Feeding entrained free-running rhythms. Chi-square periodogram analysis at a con®dence level of 95%

#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16 #17 #18 #19 #20

Upper NS ± NS ± NS 24.0 NS NS ± NS NS 28.8 NS NS 25.5 NS 25.8 NS 21.5 NSLower 23.7 26.5 28.3 23.0 NS 23.7 28.3 NS NS 24.7 NS 28.8 23.0/

25.7NS 25.0 NS 24.3 25.3 21.2/

26.7NS

131

Edmonds S (1977) Food and light as entrainers of circadian run-ning activity in the rat. Physiol Behav 18: 915±919

Ferno A, Huse I, Juell JE, Bjordal A (1995) Vertical distribution ofatlantic salmon (Salmo salar L.) in net pens: trade-o� betweensurface light avoidance and food attraction. Aquaculture 132:285±296

Gee P, Stephenson D, Wright DE (1994) Temporal discriminationlearning of operant feeding in gold®sh (Carassius auratus). J ExpAnal Behav 62: 1±13

Holmqvist BI, Ostholm T, EkstroÈ m P (1992) Retinohypothalamicprojections and the suprachiasmatic nucleus of the teleost brain.In: Ali A (ed) Rhythms in ®shes. Plenum Press, New York, pp293±318

Huse I, Holm JC (1993) Vertical distribution of Atlantic salmon(Salmo salar) as a function of illumination. J Fish Biol 43:147±156

Iigo M, Tabata M (1996) Circadian rhythms of locomotor activityin the gold®sh Carassius auratus. Physiol Behav 60: 775±781

Juell JE, Ferno A, Furevik DM, Huse I (1994) In¯uence of hungerlevel and food availability on the spatial distribution of Atlanticsalmon (Salmo salar L.) in sea cages. Aquacult Fish Manage 25:439±451

Kavaliers M (1981) Period lengthening and disruption of sociallyfacilitated circadian activity rhythms of gold®sh by lithium.Physiol Behav 27: 625±628

Kerfoot WC (1985) Adaptative value of vertical migration. In:Rankin MA (ed) Migration: mechanism and adaptative signif-icance. Contrib Mar Sci [Suppl] 27: 92±113

Lampert W (1989) The adaptative signi®cance of diel vertical mi-gration of zooplankton. Funct Ecol 3: 21±27

Levy DA (1990) Sensory mechanism and selective advantage fordiel vertical migration in juvenile sockeye salmon, On-corhynchus nerka. Can J Fish Aquatic Sci 47: 1796±1802

Meijer JH, Rietveld WJ (1989) Neurophysiology of the SCN cir-cadian pacemaker in rodents. Physiol Rev 69: 671±707

Mistlberger RE (1994) Circadian food-antipatory activity: formalmodels and physiological mechanisms. Neurosci Biobehav Rev18: 171±195

Moore RY, Eichler VB (1972) Loss of circadian adrenal cor-ticosterone rhythm following suprachiasmatic nucleus lesions inthe rat. Brain Res 71: 17±33

Naruse M, Oishi T (1994) E�ects of light and food as zeitgebers onlocomotor activity rhythms in the loach, Misgurnus an-guillicaudatus. Zool Sci 11: 113±119

Neilson JD, Perry RI (1990) Diel vertical migrations of marine®shes: an obligate or facultative process? Adv in Mar Biol 26:115±168

Nishikawa M, Ishibashi T (1975) Entrainment of the activityrhythm by the cycle of feeding in the mud-skipper, Per-iopthalmus cantonensis. Zool Mag 84: 184±189

Ottenweller JE, Tapp WN, Natelson BH (1990) Phase-shifting thelight-dark cycle resets the food-entrainable circadian pace-maker. Am J Physiol 258: R994±R1000

Phillips DL, Rautenberg W, Rashotte ME, Stephan FK (1993)Evidence for a separate food-entrainable circadian oscillator inthe pigeon. Physiol Behav 53: 1105±1113

Rashotte M, Stephan FK (1996) Coupling between light- and food-entrainable circadian oscillators in pigeons. Physiol Behav 59:1005±1010

Rosenwasser AM, Pelchat R, Adler NT (1984) Memory for feedingtime: possible dependence on coupled circadian oscillators.Physiol Behav 32: 25±30

Sa nchez-Va zquez FJ, Zamora S, Madrid JA (1995) Light-dark andfood restriction cycles in sea bass: e�ect of con¯icting zeitgeberson demand-feeding rhythms. Physiol Behav 58: 705±714

Sa nchez-Va zquez FJ, Madrid JA, Zamora S, Iigo M, Tabata M(1996) Demand-feeding and locomotor circadian rhythms in thegold®sh, Carassius auratus: dual and independent phasing.Physiol Behav 60: 665±674

Spieler RE (1992) Feeding-entrained circadian rhythms in ®shes.In: Ali A (ed) Rhythms in ®shes. Plenum Press, New York,pp 137±147

Spieler RE, Clougherty JJ (1989) Free-running locomotor rhythmsof feeding-entrained gold®sh. Zool Sci 6: 813±816

Spieler RE, Noeske T (1984) E�ect of photoperiod and feedingschedule on diel variations of locomotor activity, cortisol andthyroxine in gold®sh. Trans Am Fish Soc 113: 528±539

Spieler RE, Noeske-Hallin TA, DeRosier TA, Poston HA (1987)Some dietary amino acids and meal-feeding phase shifts of lo-comotor activity. Med Sci Res 15: 921±922

Stephan FK (1986) Coupling between feeding and light-entrainablecircadian pacemakers in the rat. Physiol Behav 38: 537±544

Stephan FK, Zucker I (1972) Circadian rhythms in drinkingbehaviour and locomotor acitivity of rats are eliminatedby hypothalamic lesions. Proc Natl Acad Sci USA 69: 1583±1586

Stephan FK, Swann JM, Sisk CL (1979) Entrainment of circadianrhythms by feeding schedules in rats with suprachiasmatic nu-cleus lesions. Behav Neural Biol 25: 545±554

Weber DN, Spieler RE (1987) E�ects of the light-dark cycle andscheduled feeding on behavioural and reproductive rhythms ofthe cyprinodont ®sh, medaka, Oryzias latipes. Experientia 43:621±624

Yokota T, and Oishi T (1992) Seasonal change in the locomotoractivity rhythm of the medaka, Oryzias latipes. Int J Biomete-orol 36: 39±44

132