Effect of pinealectomy and circadian rhythm on avoidance behavior in the male rat

Physiology &Behavior, Vol. 34, pp. 327-333. Copyright©PergamonPress Ltd., 1985. Printedin the U.S.A.

0031-9384/85$3.00 + .00

Effect of Pinealectomy and Circadian Rhythm on Avoidance Behavior in the Male Rat M. D. C A T A L A ,1 F. P A L L A R D O , _A. R O M A N , P. V I L L A N U E V A A N D J. M. V I N A G I N E R

Department o f Biochemistry and Physiology, Faculty o f Medicine o f The University o f Valencia, Spain R e c e i v e d 26 A p r i l 1983 CATALA, M. D., F. PALLARDO, A. ROMAN, P. VILLANUEVA AND J. M. VIlqA GINER. Effect ofpinealectomy and circadian rhythm on avoidance behavior in the male rat. PHYSIOL BEHAV 34(3) 327-333, 1985.--Male adult albino rats were divided into six groups: two pinealectomized (Px); two sham-operated (Sh) and two serving as controls (C). Half of these groups were studied in daylight and the other half at night. The animals were open-field tested and then conditioned by the avoidance behavior test in the appropriate light period. No differences were observed among the groups when they were conditioned in the dark; however, the Px were conditioned significantly more rapidly than Sh or C in daylight. Intragroup comparisons between night/day conditioningshowed them to be similar in Px but more rapid at night in both Sh and C. The Sh group is unique and not comparable to controls. Pinealectomy

Circadian rhythm

Avoidance

Male rats

LITTLE has been published over the last twenty years relating the pineal gland to behavior and exploratory activity [6, 7, 8, 14, 15, 18]. Our primary interest is in the relationship of the pineal gland and circadian rhythm with learning. We were aware of the rat's nocturnal activity [1] and also that the pinealectomized rat is better adapted to both light and dark conditions than the unoperated animals [13]. We proposed, therefore, to study conditioning by avoidance training in control and pinealectomized rats under both daylight and nighttime conditions.

solid top. The wall which divided the box into two compartments had an opening for the tats to pass through. Each compartment had a 25 watt light suspended 25 cm above the floor. The floor was composed of metal bars connected to an electric stimulator.

Surgical Procedures The rat were pinealectomized at 12 weeks of age according to the methods of Kuszak and Rodin [9] but without cutting the superior sagittal sinus (SSS) in agreement with Quay [12] and Mouret [10]. Male rats (200-+20 g) were anesthetized with sodium pentobarbital, 60/zg/g IP. The rat was placed in a 45° head up position until a double needle had been passed under the SSS after which the animal was placed in the Trendelenburg position for one minute. Thereafter the rat was returned to the horizontal position for removal of the pineal. The excised pineals were examined under the optical and electron microscope. Sham-operated animals underwent the same procedure, but the pineal was not removed, Pinealectomized and sham-operated animals underwent surgery on the same day to correlate their response to training. Operated animals were housed in the same quarters under the same conditions as previously described. Field testing commenced when the animals were 16 weeks of age. Pinealectomized and sham-operated animals were sacrificed at the conclusion of this experiment and the brains studied to confirm no damage after the surgery. (Figs. 7-10, E.M.).

METHOD Male albino rats (N=75) were used in these experiments. Commencing at four weeks of age, the rats were exposed to the natural (ambient) light/dark cycle (Greenwich Meridian Time). A constant temperature (21°-+2°C) was maintained; and food and water were available ad lib. These experiments were done during two years from January 1980 until January 1982.

Open-Field Enclosure This enclosure was 95 in in diameter and 50 cm high with a 100 watt electric light suspended thereover. The field was divided into 19 sections: a central section, 6 internal and 12 external sectors.

Two-Way Shuttle-Box This box was 67 cm long, 37 cm wide and 35 cm high with

~Requests for reprints should be addressed to Dr. M. D. CatalA, C~itedrade Bioquimica y Fisiologia, Facultad de Medicina, Universidad de Valencia, Avenida de Blasco Ib~ifiez 17, Valencia-10, Espafia.

327

C A T A L , ~ ET AL

328 TABLE 1 OPEN-FIELD TEST External Sections Days *Controls (20) Sham-operated (20) Pinealectomized (20)

Internal Sections

N°Bolis

1°

2°

3°

1°

2°

3°

1°

2°

3°

23.29 16.14 19.28

16.14 11.70 21.24

11.38 12.84 20.57

1.08 1.38 1.00

0.76 0.90 0.76

0.48 0.43 0.57

3.52 2.19 2.24

2.43 2.29 2.19

3.63 2.38 1.85

*The number of animals is in brackets. This table expresses the mean number of external and internal sections crossed by each animal and also the mean number of bolis. Third hour of daylight. TABLE 2 OPEN-FIELD TEST External Sections Days *Controls (10) Sham-operated (10) Pinealectomized (10)

Internal Sections

N°Bolis

1°

2°

3°

1°

2°

3°

1°

2°

3°

49.50 46.50 45.30

29.60 40.60 35.50

28.60 39.40 35.10

4.50 3.80 6.30

1.90 2.70 4.70

1.70 2.40 4.30

1.80 0.60 0.50

1.60 1.70 3.40

1.50 1.70 3.02

*The number of animals is in brackets. This table expresses the mean number of external and internal sections crossed by each animal, and also the mean number of bolis. Third hour of darkness.

Behavioral and training sessions were c o n d u c t e d three hours after the onset o f daylight (the time previously established in our laboratory) and based on this interval, the nocturnal sessions c o m m e n c e d three hours after the onset of night. Daylight open-field testing. On three c o n s e c u t i v e days, starting at the third hour of daylight, each animal (Px, Sh and Control) was placed in the central sector and the n u m b e r of internal and external sectors entered o v e r a three minute period tallied. The n u m b e r of bolluses deposited in this time period was also counted. Nighttime open-field testing. Starting at the third hour of dark, the same procedure was carried out as above. During the nocturnal trial the conditioning r o o m was illuminated only by the light in the open-field arena.

Avoidance Behavior Training in the Shuttle-Box A v o i d a n c e b e h a v i o r training was started the day after completion o f the open-field testing-day-light.The rat was placed in one c o m p a r t m e n t of the shuttle box. E a c h animal u n d e r w e n t one training session of ten trials p e r day. E a c h trial consisted o f 5 seconds of light in one c o m p a r t m e n t ; if the animal did not cross into the o t h e r c o m p a r t m e n t in this time a l m A electric shock was applied to the grid for 5 seconds. As soon as the rat crossed to the o t h e r compartment, the light and electric shock were terminated. If the animal crossed during the light phase, no electric shock was applied. This trial was repeated e v e r y 60 seconds. The rat was considered trained w h e n it a v o i d e d being shocked in at least 80% o f the trials o v e r three c o n s e c u t i v e days. Animals not considered successful were those which did not reach the 80% level after 20 days with 10 trials per day (total 200 trials). During daylight testing the r o o m was illuminated by natural

light and a 25 W red lamp was placed behind the shuttle-box to o b s e r v e inter-trial activity.

Avoidance Training During the Third Hour of Darkness The animals were subjected to the same stimuli described above. The only illumination in the r o o m was the red light which allowed observation of activity b e t w e e n trials. The animals were scored as above. All animals were housed in their normal quarters until a few m o m e n t s before their testing session was scheduled.

Extinction Phase To test extinction, the animals were subjected to ten trials per day o v e r twenty days. The light stimulus was given e v e r y 60 seconds, but was not followed by an electric shock [2,16]. The training was considered extinguished w h e n the animal r e s p o n d e d in only 20% of the trials o v e r three c o n s e c u t i v e days, and the e x p e r i m e n t was considered concluded. A m a x i m u m of 200 trims (10 trials per day for 20 days) was allowed. A second e x p e r i m e n t served as further validation of the results. Fifteen rats were selected at random from the controls which had c o m p l e t e d the first experiment, were pinealectomized, and the first e x p e r i m e n t was rerun. Ten animals w e r e tested and trained in daylight and 5 in the dark. (Figs. 5 and 6). RESULTS

Open-Field Test G r e a t e r exploratory activity was o b s e r v e d in all three groups at night c o m p a r e d with daytime activity and f e w e r

PINEALECTOMY AND BEHAVIOR

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4(2), 5(3), 6(2), 7, 8(2), 9, 12, 15, 16, 19 (Fig. 2). An analysis of variance (ANOVA) indicated differences existed among the three groups, F(2.42)=4.81, p<0.05. The student test showed that this effect was due to significant differences between Px and control, p<0.05, and between Px and Sh, p <0.05. There were no differences between Sh and Control.

Avoidance Behavior Training-Dark bolluses were counted (Tables 1 and 2). There were no differences among the three groups either by day or by night, nor were there any significant differences found when day/night ratios were calculated.

Avoidance Behavior Training-Daylight Only 60% (9/15) of control animals achieved the established criterion of learning (Fig. 1). The 9 successful animals fulfilled the criterion on days 5, 6, 8, 9, 10, 13, 14, 15, 17 (Fig. 2.). 66% (10/15) of the sham-operated animals were successful in learning, and reached the criterion on days 4, 5, 7 (2), 9, l l , 12, 15, 16, 19 (Fig. 2). 100% (15/15) of the pinealectomized rats achieved the learning criterion and did so on days

Control animals were 80% (8-10) successful in learning (Fig. 3). This criterion was reached on days 4(2), 7, 8, 10(2), 12, 14 (Fig. 4). The same percent of the sham-operated rats, 80% met the criterion on days 3, 4(3), 5, 6, 12, 16 (Fig. 4). 90% (9/10) of the pinealectomized rats achieved the criterion on days 4, 5(2), 7, 10, 11(2), 12, 15 (Fig. 4). The A N O V A showed no differences among the three groups, F(2,27)=0.07. When comparing the day/night results of the avoidance behavior using the Student test, significant differences were apparent between the control groups only, p<0.05. There were no significant differences between the day/night pinealectomized or sham-operated groups.

330

CATALA ET AL.

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FIG. 7. On observation, the Ductus Silvi is opening into the third ventricle, and is preserved completely of any damage. E.M.

The six groups of animals were conditioned by the usual extinction procedure and all animals were more resistant to extinction both by day and by night. DISCUSSION Our objective was to compare the behavior of control and Px rats during selected light and dark periods. The rat being a

nocturnal animal, we proposed to study its activity and training primarily during this dark period and our results indicated that avoidance behavior was learned more rapidly in the dark by all groups. The nighttime results for the three groups were similar, however, the Px rats were trained more rapidly than the other groups in daylight. The night/day learning within groups was significantly different between

PINEALECTOMY

AND BEHAVIOR

FIG. 8. The integrity of all the structures after removal of the pineal gland can be seen. E.M.

FIG. 9.The Plexus Choroideus did not suffer from the surgical procedure. The nucleus habenulae shows any damage. E.M.

331

332

CATALA E T A L .

FIG. 10, Magnification of Fig. 3 showing the integrity of the canalis centralis covering the nucleus habenulae dextro.

controls and Px. The daylight results are evidently paradoxical and disappear at night. We have observed in the shuttle-box that the Px rats show less stress reaction to the electrical shock, and learn more readily than the control and sham-operated groups. As suggested by Brown [3] and Rudeen [17], it is possible that the Px rats have greater visual perception in daylight than the control groups because the pinealectomized animals do not develop the retinal lesion produced by white light in albino rats. Or, possibly; the electrical activity in the pinealectomized brain is more mature than in the controls or the sham-operated I l l ] , also taking into account that we used male rats and NIR [11] used female rats. After observing the results of utilizing a light stimulus at night, [4], it is suggested that another stimulus, e.g., sound, would be more discriminating; and, in any event, it would

appear that experiments using nocturnal animals would be more valid conducted during the dark period to observe the animals behavior. Comparing intragroup day/night effects, showed significant differences except for the sham-operated group. Thus, it appears that the sham-operated group, in agreement with other authors [5], must be considered as a special experimental group and not as a control group. ACKNOWLEDGEMENTS The author expresses her sincere appreciation to Dr. D. B. Hudson for her helpful criticism and suggestions; and also to Dr. P. S. Timiras, from Berkeley U. C. CA and to Dr. D. Castafio, Dr. V. Sim6n, Dr. V. Smith-Agreda and Dr. F. Martinez-Soriano, from Valencia, Spain.

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edited by N. Biron and W Schloot. Oxford: Pergamon Press, 1980, pp. 95-112.

4. Illnerova, H. and J. Vanececk. Effect of one-minute exposure to light and night on rat pineal serotonin N.A.T. In: The Pineal Gland o f Vertebrates Including Man, vol 52, Progress in Brain Research, edited by J. Ariens Kappers and P. Pevet. Amster-

dam: Elsevier, North Holland, 1979, pp. 241-243. 5. Kachi, T., T. K. Banerji and W. B. Quay. Circadian and ultradian changes in synaptic vesicle numbers in nerve endings on adreno medullary noradrenalin cells, and their modifications by pinealectomy and sham-operations. Neuroendocrinology 30: 291-299, 1980.

PINEALECTOMY

AND BEHAVIOR

6. Kincl, F. A., C. C. Chang and V. Zbuzkova. Observation on the influence of changing photoperiod on spontaneous wheelrunning activity of neonatally pinealectomized rats. Endocrinology 87: 38--42, 1970. 7. Kovacs, G. L., I. Gajari, G. Teledgy and K. Lissak. Effect of melatonin and pinealectomy on avoidance and exploratory activity in the rat. Physiol Behav 13: 349-355, 1974. 8. Krapp, C. H. Pineal influence on locomotion in rats. Experientia 33: 731-732, 1977. 9. Kuszak, J. and M. Rodin. A new technique of pinealectomy for adult rats. Experientia 33: 283-284, 1977. 10. Mouret, J., J. Coindet and G. Chouvet. Effect de la pin6alectomic sur les 6tats et rhythmes de sommeil du rat male. Brain Res 81: 97-105, 1974. 11. Nir, I., K. Behrooz, M. Assael, I. Ivriani and F. G. Sulman. Changes in the electrical activity of the brain following pinealectomy. Neuroendocrinology 4: 122-127, 1969. 12. Quay, W. B. Experimental evidence for pineal participation in homeostasis of brain composition. Prog Brain Res 10: 646--653, 1965.

333 13. Quay, W. B. Precocious entrainment and associated characteristics of activity patterns following pinealectomy and reversal of photoperiod. Physiol Behav 5: 1281-1290, 1970. 14. Reiss, M., R. H. Davis, M. B. Sideman and E. S. Plitcta. Pineal gland and spontaneous activity of rats. J Endocrinol 28: 127128, 1963. 15. Relkin, R. Influence of prepuberal and postpuberal pinealectomy on maze performance. Am J Physiol 218: 328--331, 1970. 16. Reynierse, J. H. and R. C. Rizley. Stimulus and response contingencies in extinction of avoidance by rats. J Comp Physiol Psychol 73: 86--92, 1970. 17. Rudeen, P. K. and W. K. O'Steen. The effects of pineal gland on light-induced retinal photoreceptor damage. Exp Eye Res 28: 37-44, 1979. 18. Wong, R. and C. B. C. Whiteside. The effect of melatonin on the wheel-running activity of rats deprived of food. J Endocrino! 40: 383-384, 1969.