ZIAD BOULOS**, ALAN M. ROSENWASSER and MICHAEL TERMAN***. Department of Psychology, Northeastern University, Boston, Mass. 02 i 15 (U.S.A.).
Behavioural Brain Research, 1 (1980) 39-65 © Elsevier]North-Holland Biomedical Press
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FEEDING SCHEDULES AND THE CIRCADIAN ORGANIZATION OF BEHAVIOR IN THE RAT*
ZIAD BOULOS**, ALAN M. ROSENWASSER and MICHAEL TERMAN***
Department of Psychology, Northeastern University, Boston, Mass. 02 i 15 (U.S.A.) (Received June 15th, 1979) (Revised version received August 20th, 1979) (Accepted October 5th, 1979)
Key words: circadian rhythm - feeding schedules - entrainment - drinking - anticipatory behavior - suprachiasmatic nuclei lesions - self-stimulation
SUMMARY
Feeding and drinking behavior of rats maintained in constant light were recorded before, during and after feeding schedules with periods lying within or outside the range of circadian entrainment. Regardless of period, all schedules immediately resulted in the partial or complete synchronization of drinking behavior, but failed to entrain the free-running circadian feeding and drinking rhythms. This indicates that drinking can be passively driven by periodic access to food. However, other results suggested that a separate circadian system was
* These studies were submitted by Z. Boulos in partial fulfillment of the requirements for a Ph.D. degree at Northeastern University. ** Present ad~ess: Department of Psychology, Dalhousie University, Halifax, Nova Scotia, Canada: * * * T o whom all correspondence and reprint requests should be sent at: Department of Psychology, 234 Nightingale Hall, Northeastern University. Boston, Mass. 02115, U.S.A.
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entrained by feeding schedules: First, the 24-h periodicity induced by 24-h feeding schedules often continued for several days after termination of the schedules. Second, the rats showed anticipatory activity under schedules with periods within, but not outside, the circadian range of entrainment. Third, lesions of the suprachiasmatic nuclei (SCN), which resulted in the immediate elimination of free-running rhythms, did not alter the rhythmic influences of the feeding schedules. These results provide evidence for the participation of two distinct circadian systems in the control of behavior in the rat. The two systems appear to have different entrainment characteristics and separate physiological substrates.
INTRODUCTION
Schedules of restricted daily food availability can substantially modify the temporal distribution of an animal's behavioral and physiological activities. Under a 24-h light-dark (LD) cycle and free access to food, many such activities show 24-h rhythms synchronized to the LD cycle, with daily increases and decreases occurring at times determined by the scheduling of the light transitions. When access to food is limited to a few hours per day, daily maxima and minima are still evident, but now occur at times determined, at least in part. by the time of food availability. In rodents, activities thus affected include drinking I! 5. 56], locomotor activity [44], sleep states [34]. body temperature [6, 26, 36], enzyme activity [18, 21, 22. 45], brain neurotransmitter levels [4, 26]. and the levels of hormones and other blood constituents [17, 26, 31.38. 39], Daily feeding schedules can also induce exact 24-h periodicity in various activities under constant ligllting conditions where, in the absence of food restriction, these activities show free-running circadian rhythms with periods differing slightly from 24-h [13, 30, 44, 50, 52]. Feeding schedules thus share with LD cycles the ability to control the overt phase and period of daily biological rhythms. But while in the case of LD cycles this usually represents the entrainment of one or more endogenous circadian pacemakers [I], less is known about the mechanisms of synchronization by food. The present experiments were designed to further characterize the temporal control exerted by feeding schedules on behavior, and to differentiate between alternative mechanisms by which such control might be achieved. In particular, an attempt was made to distinguish between (i) the entrainment of circadian rhythms by feeding schedules, and (ii) the direct, exogenous driving of behavior by the periodic ingestion of food. To this end, we recorded feeding (lever-
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pressing with contingent food-pellet delivery) and drinking behavior of rats under free-feeding conditions, and under restricted feeding schedules with 24-h and non-24-h periods. Non-reinforced lever-presses occurring during the deprivation hours of the feeding schedules were also recorded. In addition, several rats had continuous access to electrical brain self-stimulation (EBSS). This behavior exhibits circadian rhythmicity [53, 54], and has been shown, in short testing sessions, to be affected by food deprivation [37]. Finally, in an attempt to isolate the effects of the feeding schedules, rats were studied after receiving lesions of the suprachiasmatic nuclei (SCN) of the hypothalamus. Such lesions disrupt a variety of circadian rhythms (for a recent review, see ref. 32), but do not affect the synchronization of corticosteroid output and body temperature by daily feeding schedules [28]. METHOD
Subjects and apparatus Adult male Long-Evans rats (Blue Spruce Farms, Altamont, N.Y.) were individually maintained in cylindrical compartments of clear Plexiglas (30 cm diameter), each enclosed in a light-tight sound-attenuating chamber. Temperature was maintained at 21 + 1 °C, relative humidity at 50+ 3%. The chambers were illuminated by Vita,,Lite fluorescent bulbs partially covered with electrical tape and wire-mesh screening. All experiments were carried out under constant illumination (LL), at intensities ranging between 3 and 30 lux. Illumination intensity was measured with a Gossen Luna-Pro light meter equipped with a diffusing cap. Each compartment contained a lever mounted next to a small food cup, Every press of the lever activated an automatic feeder and resulted in the delivery of a 45-mg Noyes food pellet. A second lever provided some rats with access to EBSS, delivered through a pulley-swivel mercury commutator system [3] attached to the ceiling of the chamber. Stimulation was delivered in 0.5-sec trains of 60-Hz constant-current sinusoids at intensities of 0.04-0.15 mA (peakto-peak), and was made available on a DRL-10" (differential reinforcement of low rates) schedule under which a response is reinforced only if preceded by at least 10 sec of no responding. The DRL schedule has been shown to sustain circadian oscillations which are more stable than those obtained under continuous reinforcement [55], and which persist for many months of uninterrupted testing, although small increases in current intensity are occasionally required to maintain adequate self-stimulation rates. The DRL contingency was controlled by solid-state logic ~ E C K-series Flip Chips). An opening in the side of the compartment gave access to a drinking spout. Licks at the spout interrupted an infrared photobeam and were recorded,
42 along with lever-presses, on cumulative recorders and printing counters which were reset by hourly pulses from a digital clock. Tork timers controlled the availability of food when feeding schedules were in effect. The chambers were opened for maintenance once a week, at irregular times of day.
Data analysis and presentation Hourly response totals were used for computer-assisted spectral analysis and for graphical display in the form of ranked quartile plots. The spectral analysis program first transforms the hourly data into relative deviations from a lcast-squares regression line fitted to the whole data sample. A sine and cosine function are then generated with a period T = 2.00 h, and correlated with the normalized data to yield r~inand r,o~, respectively. The peak phase of the best-fitting sine function of period T is given by, ~ = tan -m(r~Jr¢~), and its magnitude by, M = V r~u + r~s. T is then incremented in steps of 0.05 h, and the process repeated until T = 30.00 h. The resulting magnitude spectrum is expressed as proportions of the dominant spectral peak. Statistical significance of spectral components was evaluated by one-tailed t-tests. Unless otherwise specified, all effects reported here are significant at P < 0.001. The quartile plots are produced by assigning to each hour of the day a quartile rank and corresponding symbol (full box, half box, quarter box, or blank) representing the number of licks or lever-presses which occurred in that hour. Hours with no behavior are assigned to the lowest quartile and left blank. The data are double-plotted to facilitate inspection of free-running rhythms.
Surgery and histology Rats were anesthetized with intraperitoneal injections of sodium pentobarbital (50 mg/kg of body weight, Pentosol) and occasional supplementary doses of chloral hydrate solution, and surgically implanted with one or two bipolar electrodes at the start of the experiments. The rat was positioned in a Kopf stereotaxic instrument, with the skull level between bregn~..a and lambda. One electrode ~Plastic Products MS 303) was aimed at the lateral hypothalamic region and used to provide reinforcing electrical stimulation. The other, aimed at the SCN, consisted of two stainless steel wires insulated with Formvar except for the cross-section at the tips. The wires were separated by 0.5-1.0 mm to allow bilateral placement of lesions. The lesion electrode was inserted at the midfine, straddling the superior sagittal sinus 0.0-0.3 mm posterior to bregma, and was lowered 9.0mm from the surface of the skull. The lesions were made at a later date under light ether anesthesia, by passing radiofrequency current from a Grass LM-3 lesion maker through each of the two wires separately, a rectal electrode completing the circuit. The procedure for making the lesions
43 was completed in less than 2 min and the rat immediately returned to its chamber. At the end of the experiments, the rats were deeply anesthetized and perfused intracardially with 0.9% saline followed by 10% formalin solution. Brains were stored in 10% formalin for at least 10 days followed by at least 2 days in sucrose-formalin. Frozen coronal sections, 0.04 mm thick, were saved throughout the extent of the lesion and the suprachiasmatic region, and around the location of the stimulation electrode tips. The sections were mounted and stained with cresyl violet, Lesion location and extent of damage to the SCN were assessed by comparison with corresponding sections from two intact brains. A stereotaxic rat brain atlas [25] was used for verification of stimulation electrode placements. Stimulation electrode tips were located in the lateral hypothalamic region extending from the mammillary bodies to the posterior border of the anterior hypothalamic nucleus. TWENTY-FOUR-HOUR FEEDING SCHEDULES
A circadian rhythm entrained by an environmental cycle is expected, upon termination of the cycle, to start to free-run from the phase it held during entrainment. This characteristic of circadian entrainment provided the first means for distinguishing between driving anO entrainment of behavioral rhythms by daily feeding schedules. Procedure Eleven rats were used. Four of these had continuous access to EBSS. The experiments, carried out in LL, began with a baseline condition of unrestricted access to food and water. A 24-h feeding schedule (FS-24) was next imposed, under which lever-presses produced food only during an unsignalled 4-h segment which occurred at a regular time each day. In two cases, the duration of food availability was gradually reduced from 12 h to 4 h, in one case from 8 h to 4 h. The schedule was maintained for 24 to 84 days, and was followed by a return to free-feeding conditions. Results and Discussion Under the initial baseline of unrestricted feeding, all rats exhibited freerunning circadian rhythms of feeding and drinking, as seen in quartile plots of the data (Figs, l A D and 10). Spectral analysis showed the periods of these rhythms to range between 24.35 and 24.90 h. In two cases, however, circadian rhythmicity in both behaviors was lost after 2 to 4 weeks of exposure to LL (Fig. IC). Rats with access to EBSS also showed free-running rhythms in this
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