Physiology & Behavior, Vol. 48, pp. 523-529. Q Pergamon Press plc, 1990. Printed in the U.S.A.

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Rats Anticipate and Discriminate Between Two Daily Feeding Times ZIAD BOULOS* AND DIOMEDES E. LOGOTHETISt *Institute for Circadian Physiology, 677 Beacon Street, Boston, MA 02215 and fDepartment of Cardiology, Children’s Hospital, Harvard Medical School, Boston, MA 02115 Received 23 May 1990 BOULOS, Z. AND D. E. LOGOTHETIS. Rats anticipate and discriminate between two daily feeding times. PHYSIOL BEHAV 48(4) 523-529, 1990. -Intact rats and rats bearing lesions of the suprachiasmatic nuclei (SCNX rats) were trained to obtain food by pressing either of two levers located on opposite sides of a cylindrical cage. Intact rats were maintained in constant light (LL) and under daily light-dark (LD) cycles, SCNX rats in LL only. A restricted daily feeding schedule was next imposed, such that pressing one lever provided food for a limited duration (1 or 2 hr) at one time of day while pressing the second lever provided food for the same duration at another time of day. Most rats generally showed anticipatory lever-pressing preceding both daily feeding times, and several discriminated between the two, pressing the lever appropriate for each feeding time more than the inappropriate lever. Discrimination performance was better in intact rats in LD than in intact or SCNX rats in LL. These results indicate that rats can associate different food locations with different times of day, an ability previously known only in honeybees and birds. Food anticipation

Circadian rhythms

Suprachiasmatic nucleus

Restricted feeding schedules ‘,

ONE of the earliest examples of circadian timing in animal behavior was the demonstration that honeybees possess a memory

ously scheduled feeding time. Rats can also anticipate two daily feedings (7, 17, 18, 20), and they show increased locomotor activity at both feeding times if food is withheld entirely for one day (7). In the present study, we examined food-anticipatory lever-pressing patterns in rats that had access to two food levers in two different locations; pressing one lever produced food at one time of day, while pressing the second lever produced food at another. Our aim was to determine whether rats, like honeybees and birds, can learn to associate specific food locations with specific times of day. Both intact rats and rats bearing lesions of the suprachiasmatic nuclei (SCN) were studied. Such lesions abolish LD-entrained and free-running circadian rhythms, but they do not interfere with the development of food-anticipatory activity, nor with its persistence during food deprivation (9).

for time, or time sense (5). When allowed access to sugar water for a limited duration each day, honeybees quickly learn to arrive at the food source during or just before the time of food availability, even in the absence of external time cues, and they continue to visit the feeding place at the appropriate time, for a few days, when food is no longer available. Such training can only be achieved when food is made available at 24-hr or near-24-hr intervals, not at intervals of 19 or 48 hr, which lie outside the normal range of circadian entrainment (45). Honeybees can also be trained to two or more feeding times per day and can learn to visit one food source at one time of day and a different source at another (12,21). Thus, not only do they remember the times when food is available, but they can differentiate between one daily feeding time and another. In a natural environment, this enables honeybees to make more efficient use of their resources by limiting their visits to different flowering plants to the daily times of flower opening and of maximal nectar production (14). A capacity for associating specific food locations with specific times of day has also been demonstrated in starlings, Sturnus vulguris (lo), and in garden warblers, Sylvia borin (6). Recent studies have uncovered what appear to be similar abilities in rodents and other mammals (3,9). Rats maintained on restricted daily feeding schedules show an increase in locomotor activity or in food-motivated lever-pressing just before the scheduled feeding time. Food-anticipatory activity occurs under daily light-dark (LD) cycles as well as in constant lighting (LL) conditions, but only if food is made available at intervals that lie within the circadian range. If rats are food deprived for a few days after being exposed to restricted daily feedings, they continue to show increased locomotor or lever-pressing activity at the previ-

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Animals and Housing Adult male Long-Evans rats (200-350 g, Blue Spruce Farms, Altamont, NY) were individually maintained in cylindrical cages of clear Plexiglas (30 cm diameter), each enclosed in a light-tight, soundzattenuating, ventilated wooden chamber. Temperature was maintained at 212 1°C. relative humidity at 50 2 3%. The chambers were illuminated by Vita-Lite fluorescent bulbs partially covered with electrical tape and wire-mesh screening. Illumination intensity was measured with a Gossen Luna-Pro light meter equipped with a diffusing cap. Experiments were carried out under daily LD cycles (LD 12:12, L: 1.5-3.0 lux) and in LL (1.5-3.0 lux). One animal showed disrupted circadian rhythms of feeding and drinking in LL and was transferred to constant dim red light

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(0.7 lux). The change to dim LL resulted in the reinstatement of clear free-running rhythms in both behaviors. Each cage contained two levers, mounted on the cylindrical cage wall in diametrically opposite locations, and two small food cups, one next to each lever. Pressing the levers activated automatic feeders and resulted in the delivery of 45 mg Noyes food pellets. An opening in the cage wall next to one of the levers gave access to a drinking spout. Licks at the spout interrupted an infrared photobeam and were recorded, along with lever-presses, on cumulative recorders and printing counters which were reset by hourly pulses from a digital clock. The clock also controlled food availability during restricted feeding schedules. Maintenance activities were carried out once a week, at irregular times of day. Surgery and Histology Bilateral lesions aimed at the SCN were made under sodium pentobarbital anesthesia (50 mg/kg, IP) supplemented when necessary with chloral hydrate. The rat was placed in a Kopf stereotaxic instrument and a stainless steel wire insulated with Formvar except for the tip was lowered into the suprachiasmatic region. The lesions were produced with a Grass LM-3 radiofrequency lesion maker. At the end of the experiments, rats that had received lesions were deeply anesthetized and perfused transcardially with 0.9% saline followed by 10% formalin solution. Frozen coronal sections (40 pm) of the suprachiasmatic region were obtained and stained with cresyl violet. Procedure Five intact rats and 5 rats with SCN lesions (SCNX) were trained to obtain food by pressing either of the two food levers. All were studied in LL, but 4 of the intact rats were also studied in LD 12:12. The experiments began with a baseline condition of unlimited access to food which lasted 24-34 days. Daily feeding schedules were next imposed, such that pressing one lever produced food only during an unsignalled l- or 2-hr segment at one time of day [F(l)] , while pressing the other lever produced food for the same duration at another time of day [F(2)]. The duration of food availability had little effect on the rats’ behavior, as rats that were allowed to feed for 2 hr obtained all or most of their food in the first hour. The data from these two feeding durations were therefore combined. Interfeeding intervals (IFIs) were either 12 hr (IF’I 12-12) or 16 and 8 hr (IFI 16-8). Several animals were exposed to both IFI conditions, the change from one IFI to the other being achieved by a 4-hr shift of one of the two daily feeding times. Each condition was maintained for a minimum of 20 days. At the end of restricted feeding in LL, intact and SCNX rats were deprived of all food for 3-5 days, to determine whether food-anticipatory lever-pressing can persist in the absence of scheduled feedings, Three intact rats were also food deprived for 3 days immediately after restricted feeding in LD. Water was always available during all phases of the experiments. An additional 2 intact and 2 SCNX rats were studied under similar conditions, except that only one lever could be used to obtain food. During restricted feeding, pressing that lever produced food at two times of day (1 hr each, IFI 12-12 and/or 18-6). Intact rats were kept in LD 12:12, SCNX rats in LL. Data Analysis The hourly data (number of licks at the drinking spout and number of responses on each of the food levers) were displayed

BOULOS AND LOGGTHETIS graphically in double-plotted actogram format. Spectral analysis (8) was performed on baseline feeding and drinking data of all SCNX rats to verify the presence or absence of circadian periodicity. Discrimination ratios were obtained as an index of the rats’ ability to distinguish one daily feeding time from the other. The ratios were calculated for each feeding time separately and for the two feeding times combined, and were based on the last 10 days of each restricted feeding condition. They were obtained by dividing the number of correct anticipatory responses (defined as the number of times the rat pressed the lever appropriate for a given feeding time in the 3 hr immediately preceding that feeding time) by the number of incorrect anticipatory responses (i.e., the number of times the rat pressed the inappropriate lever in the same 3-hr interval). Statistical significance of the difference between the number of correct and incorrect anticipatory responses was evaluated by Wilcoxon signed-rank tests. RESULTS Intact Rats All intact rats maintained in LL with free access to food showed free-running circadian rhythms of feeding and drinking with periods longer than 24 hr. During restricted feeding in LL and LD, most of the rats’ daily water intake took place during and shortly after the daily feeding times, but the rats also showed at least some drinking associated with a free-running component in LL, or during the hours of darkness in LD (Fig. 1). Free-running components in LL were generally visible in the lever-pressing data as well. In addition, the rats showed anticipatory lever-pressing immediately preceding one or both of the daily feeding times (Fig. 1). The amount of anticipatory activity depended to some extent on the degree of overlap between the feeding times and the active phase of the animals’ circadian rhythms. Thus, in LL, the rats tended to show more anticipatory lever-pressing preceding the daily feeding time that coincided with their free-running circadian component, while in LD, anticipation of the feeding time scheduled during the dark portion of the LD cycle often exceeded that of the feeding time scheduled during the light (Fig. 1). There was also considerable day-to-day variability in the amount of anticipatory lever-pressing, both in LL and in LD (e.g., Fig. 2). This variability, however, was not specific to the dual-lever situation, as it was present in the data of the 2 rats that had access to a single lever as well (Fig. 4). Discrimination ratios for all rats with access to two levers under all conditions are given in Table 1. Two of the 4 intact rats studied in LD (rats 25 and 4L) made significantly more correct than incorrect anticipatory responses preceding each of the two daily feedings. In the case of rat 4L, this was true when the feedings were scheduled at 12-hr intervals (IF1 12- 12) as well as after a 4-hr phase delay of one of the daily feeding times (IFI 16-8). The remaining 2 animals made more correct than incorrect anticipatory responses preceding one of the daily feedings, but pressed the two levers more or less equally prior to the other daily feeding. Nevertheless, when the data from both feeding times were combined [F( 1+2)], all 4 rats had high overall discrimination ratios (range 1.50-4.77), with significantly more. correct than incorrect responses. The results in Table 1 show that all 4 rats kept on IFI 12-12 correctly anticipated F(2), which was always scheduled during the dark phase of the LD cycle, while only 2 of the 4 correctly anticipated F(l), scheduled during the light. However, discrimination ratios for both of these animals were higher for F( 1) than for

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ANTICIPATION OF TWO DAILY FEEDINGS TABLE 1

Rat 3J Intact Lever-pressing

Drinking

DISCRIMINATION RATIOS FOR 5 INTACT AND 4 SCNX RATS THAT HAD ACCESS TO 2 FOOD LEVERS

Discrimination Ratios

Intact 25

50

SCNX

75

Rat

Light

IFI

F(1)

F(2)

F(lf2)

27 35 4J 4L

LD LD LD LD

12-12 12-12 12-12 12-12

4.15** 0.93 0.92 17.42**

1.74** 3.35** 2.11** 1.75**

2.27** 2.41** 1.50* 2.32**

4L

LD

16-8

5.80**

3.46**

4.77**

25 3J 4J 3L

LL LL LL LL

12-12 12-12 12-12 12-12

1.83** 1.63* 1.67 0.96

0.83 1.04 4.38** 1.14

1.07 1.44 2.72** 1.14

3L 4L

LL LL

16-8 16-8

1.76** 2.41*

4.OQ* 1.33

1.79** 1.54

3M 4M 7M

LL LL LL

12-12 12-12 12-12

0.90 2.50** 0.47

5.50** 1.42* 3.65*

1.99** 2.07** 0.92

3M 4M 7M 8M

LL LL LL LL

16-8 16-8 16-8 16-8

0.63 1.34 0.69 1.15

4.77** 2.77** 1.61** 1.23

3.38** 2.06** 1.15 1.15

Significance levels for difference between correct and incorrect anticipatory responses: *p