Physiology & Behavior, Vol. 49, pp. 507-511. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .00
Cerebellum and Memory: An Experimental Study in the Rat Using a Passive Avoidance Conditioning Test S. G U I L L A U M I N , M. D A H H A O U I A N D J. C A S T O N l
Laboratoire de Neurophysiologie Sensorielle, Facult( des Sciences de Rouen B.P. 118, 76134 Mont-Saint-Aignan C(dex, France R e c e i v e d 4 June 1990
GUILLAUMIN, S., M. DAHHAOUI AND J. CASTON. Cerebellum and memory: An experimental study in the rat using a passive avoidance conditioning test. PHYSIOL BEHAV 49(3) 507-511, 1991.--Adult DA/HAN swain rats were submitted to a onetrial passive avoidance conditioning procedure consisting in associating darkness with a nociceptive stimulus. Seven or fourteen days after the one-trial initial experience, they were tested again in order to know whether they had forgotten it or not. The animals were divided into two experimental groups, the rats being either conditioned (COC group) or not (NOC group) before cerebellectorny, and two control groups, the animals being either intact (C group) or sham-operated (SO group). Each group was divided into two subgroups, one being given the retrieval test 7 days and the other 14 days after the initial conditioning. The results show that retention in C, SO and NOC rats was similar whether the animals were tested 7 days or 14 days after the initial one-trial conditioning. Seven days after their initial experience and the cerebellectomy, the retention in COC rats was null while when they were tested 14 days after cerebellar removal these animals had much better scores, significantly higher than 7 days after the lesion. It is concluded that the cerebellum is involved in the consolidation processes of the memory trace but is not the site of memory storage. Conditioning
Cerebellum
Memory
Rat
THE role of the cerebellum in motor learning has been the subject of many investigations. Particularly, it is known that this structure is involved in the adaptation of the vestibulo-ocular reflex to modifications of the visual environment (5, 12, 15, 22), in the classical conditioning nictitating membrane response (2, 7, 14, 16-19, 26) and in learning of alternating arm movements in the monkey (11, 24, 25) as well as in the acquisition and retention of more complex motor behaviors such as conditioned responses including the whole body (3, 4, 13, 20, 23), and several models were built to try to explain how it can interfere in learning processes (1, 8, 10, 12, 21). In a previous paper (6), some of us have demonstrated that the cerebellum is involved in learning of an avoidance conditioning task in the rat, while it is not absolutely necessary for the avoidance conditioning achievement, and suggested that cerebellectomy partly erase memory of the preoperative conditioning. The present study was intended to test, with a different experimental protocol, the role of the cerebellum in the retention of a memory trace. To this purpose, the animals were submitted to a passive avoidance conditioning task, the cerebellum being removed before or after the one-trial initial condi-
tioning, and given a retrieval test 7 or 14 days later to test the retention of the memory trace. METHOD
Experimental Device Experiments were conducted in a shuttle-box separated in two compartments by an incomplete partition wall allowing the animals to pass from one compartment to the other. One compartment (28 x 26 x 20 cm) was lighted, the other one (40 x 26 x 20 cm) dark and equipped with a grid floor. A starting box (11 x 8 x 20 cm) joined to the light compartment was separated from it by a shash-door.
Animals The animals used in this study were D A / H A N strainedmale rats (pigmented rats),3-4 months old, weighing about 200-220 g and housed in standard conditions (12 h light-12h dark, 22°(2, food and water ad lib).Forty animals divided into4 groups of 10
1Requests for reprints should be addressed to J. Caston.
507
508
GUILLAUMIN, DAHHAOUI AND CAST()N
r|
• Cb
cb
cocll
,1 d
I I
flOe I f--SO
rl
I
,l
,,J
I
11 ,i
g I r
7 dnys
FIG. 1. Diagram of the experimental condition. COC: conditioned-operated-conditioned rats; NOC: na~'ve-operated-conditioned rats; SO: shamoperated rats; C: control rats. cb: cerebellectomy; ic: one-trial initial conditioning; rt: retrieval test.
rats (2 experimental and 2 control groups) were studied (Fig. 1). The two experimental groups were the following: - - C O C group (conditioned-operated-conditioned): the animals were conditioned (1 trial) and cerebellectomized 8 hours later; then, after a 7-day (5 rats) or a 14-day (5 rats) delay, they were tested again in the same conditions (retrieval test). - - N O C group (naive-operated-conditioned): the animals were cerebeUectomized while naive and conditioned (1 trial) after a 7-day delay. Then, 7 days (5 rats) or 14 days (5 rats) later, they were submitted to the retrieval test. The two control groups were the following: - - C group (controls): the animals were conditioned (1 trial) and submitted to the retrieval test 7 days (5 rats) or 14 days (5 rats) later. - - S O group (sham-operated): the animals were conditioned (1 trial) and sham-operated 8 hours later. Then, after a 7-day (5 rats) or a 14-day (5 rats) delay, they were submitted to the retrieval test.
Cerebellectomy and Sham-Operation The animals were anesthetized by IP injection of Nembutal (pentobarbital sodium) 35 mg-kg-~ and secured in a stereotaxic apparatus. The skin of the skull was incised, the muscles of the neck cut, the occipital bone drilled and removed. After cutting the dura, the cerebellum was removed by suction and the cavity filled with gelfoam. Then, the skin was sutured. Sham-operated rats were submitted to the same protocol except for removing the cerebellum which was left intact.
Experimental Procedure All the animals were isolated 48 hours before the experiments so as not to be stressed by the sudden isolation at the time of the test. Then, the rat was placed in the starting box and the shashdoor pulled up. Once the rat entered the light compartment, it displayed an exploratory behavior; after a several second delay, it spontaneously entered the dark compartment where it received an electric shock which was delivered once the four feet of the animal were in contact with the grid floor, and lasted until the rat came back into the light compartment, which required a few seconds. It was allowed to stay into the light compartment for one minute before returning to its cage in order to be able to know that the dark compartment only is associated with a nociceptive
stimulus. The time (TI) which elapses from the moment when the rat entered the light compartment to the moment when it entered the dark one was measured. Seven or 14 days later, the animals were submitted to a retrieval test which was given exactly in the same way as the initial conditioning except for the fact that the rat did not get an electric shock when entering the dark compartment. The time (T2) from the moment when the rat entered the light compartment to the moment when it entered the dark one was measured. If the difference T 2 - TI (absolute memory score) and the ratio ( T 2 - T 1 ) / T 1 (relative memory score) are null or small, one can expect, at the time of the retrieval test, the animals to have forgotten the initial nociceptive experience when entering the dark compartment; conversely, if the absolute and relative memory scores are high, one can expect the animals to have remembered this nociceptive experience. We have arbitrarily fixed an upper 10 min value to T2; in other words, if the rat did not enter the dark compartment after a 10 rain delay when given the retrieval test, it was returned to its cage. The electric shock was vibrating current (10 Hz, 45 V); such a stimulus was great enough to make the animals greatly disturbed but not too high so that the animals did not experience pain as revealed by their behavior. The intensity of the stimulus was chosen during a preexperiment in animals which are not included in this study.
Treatment of the Results For each group of 5 rats the times T1 and T2 needed for each ani___malto enter the dar_._kcompartment were respectively averaged (T1 = E T1/_5) and T2 = E T2/5), the mean values being given -+ SEM (~r~/n). Comparisons between groups were made according to the Mann-Whitney test.
Anatomical Controls After completion of the experiments, the animals were sacrificed. They were overdosed with Nembutal, their brain was removed and placed in 10% formalin for about two weeks. Then. the brains were examined under an operation microscope. RESULTS
T1 Values of Rats During the One-Trial Initial Conditioning When the animals enter the light compartment of the shuttlebox, they display an exploratory behavior which lasts for several seconds before entering the dark compartment (T1 value). In our experiments, this T1 value in C, SO and COC rats varied from 5.4 s to 81.0 s. It was, in most cases, lower than 30 s and exceptionally higher than 1 min (3 cases out of 30). The mean T1 values calculated in C, SO and COC rats, which are 36.1 --- 7.6 s, 22.4---6.0 s and 3 9 . 7 ± 14.2 s, respectively, are not significantly different (Fig. 2). This is not surprising since the animals of these three groups were identical: their cerebellum was intact and they were submitted to the same experimental protocol. In NOC rats, the T1 values varied from 3.2 s to 296.0 s and were in most cases (7 out of 10) higher than 1 min. Such rather high values were not due to an increased exploratory behavior but to the fact that the cerebeUectomy alters the motor coordination and, consequently, the animals were clumsy and reached the dark compartment later than the nonoperated ones. However, due to the great interindividual variability, the T1 mean value of NOC rats (136.1 - 33.8 s) is not significantly different from the ones of C, SO and COC animals (Fig. 2).
T2 Values of Rats During the Retrieval Test In C, SO and NOC animals, the T2 values being not significantly different when tested 7 or 14 days after the one-trial con-
CEREBELLUM AND MEMORY
509
X,
180-
)F
600- A
150-
-
qlO S ml
~
• -
90-
B
500-
500-
120-
600"
406-
400"
300-
300-
200-
206-
1O0-~
100-
6030-
0 li C
C SO C0Cll0C FIG. 2. Initial conditioning. Time, in seconds (-SEM), elapsing from the moment when the rat entered the light compartment to the moment when it entered the dark one (T1 value) in C, SO, COC and NOC rats. No significant difference between the different groups are observed.
ditioning, they will be gathered. All the animals of the C group but one (i.e., 9 rats out of 10) stayed in the light compartment for at least 10 min and did not enter the dark one. The only exception was a rat which entered the dark compartment after a 186 s exploratory behavior in light (in this animal, the TI value was 60.0 s). Then, the mean T2 value is highly significantly higher (p-
0031-9384/91 $3.00 + .00
Cerebellum and Memory: An Experimental Study in the Rat Using a Passive Avoidance Conditioning Test S. G U I L L A U M I N , M. D A H H A O U I A N D J. C A S T O N l
Laboratoire de Neurophysiologie Sensorielle, Facult( des Sciences de Rouen B.P. 118, 76134 Mont-Saint-Aignan C(dex, France R e c e i v e d 4 June 1990
GUILLAUMIN, S., M. DAHHAOUI AND J. CASTON. Cerebellum and memory: An experimental study in the rat using a passive avoidance conditioning test. PHYSIOL BEHAV 49(3) 507-511, 1991.--Adult DA/HAN swain rats were submitted to a onetrial passive avoidance conditioning procedure consisting in associating darkness with a nociceptive stimulus. Seven or fourteen days after the one-trial initial experience, they were tested again in order to know whether they had forgotten it or not. The animals were divided into two experimental groups, the rats being either conditioned (COC group) or not (NOC group) before cerebellectorny, and two control groups, the animals being either intact (C group) or sham-operated (SO group). Each group was divided into two subgroups, one being given the retrieval test 7 days and the other 14 days after the initial conditioning. The results show that retention in C, SO and NOC rats was similar whether the animals were tested 7 days or 14 days after the initial one-trial conditioning. Seven days after their initial experience and the cerebellectomy, the retention in COC rats was null while when they were tested 14 days after cerebellar removal these animals had much better scores, significantly higher than 7 days after the lesion. It is concluded that the cerebellum is involved in the consolidation processes of the memory trace but is not the site of memory storage. Conditioning
Cerebellum
Memory
Rat
THE role of the cerebellum in motor learning has been the subject of many investigations. Particularly, it is known that this structure is involved in the adaptation of the vestibulo-ocular reflex to modifications of the visual environment (5, 12, 15, 22), in the classical conditioning nictitating membrane response (2, 7, 14, 16-19, 26) and in learning of alternating arm movements in the monkey (11, 24, 25) as well as in the acquisition and retention of more complex motor behaviors such as conditioned responses including the whole body (3, 4, 13, 20, 23), and several models were built to try to explain how it can interfere in learning processes (1, 8, 10, 12, 21). In a previous paper (6), some of us have demonstrated that the cerebellum is involved in learning of an avoidance conditioning task in the rat, while it is not absolutely necessary for the avoidance conditioning achievement, and suggested that cerebellectomy partly erase memory of the preoperative conditioning. The present study was intended to test, with a different experimental protocol, the role of the cerebellum in the retention of a memory trace. To this purpose, the animals were submitted to a passive avoidance conditioning task, the cerebellum being removed before or after the one-trial initial condi-
tioning, and given a retrieval test 7 or 14 days later to test the retention of the memory trace. METHOD
Experimental Device Experiments were conducted in a shuttle-box separated in two compartments by an incomplete partition wall allowing the animals to pass from one compartment to the other. One compartment (28 x 26 x 20 cm) was lighted, the other one (40 x 26 x 20 cm) dark and equipped with a grid floor. A starting box (11 x 8 x 20 cm) joined to the light compartment was separated from it by a shash-door.
Animals The animals used in this study were D A / H A N strainedmale rats (pigmented rats),3-4 months old, weighing about 200-220 g and housed in standard conditions (12 h light-12h dark, 22°(2, food and water ad lib).Forty animals divided into4 groups of 10
1Requests for reprints should be addressed to J. Caston.
507
508
GUILLAUMIN, DAHHAOUI AND CAST()N
r|
• Cb
cb
cocll
,1 d
I I
flOe I f--SO
rl
I
,l
,,J
I
11 ,i
g I r
7 dnys
FIG. 1. Diagram of the experimental condition. COC: conditioned-operated-conditioned rats; NOC: na~'ve-operated-conditioned rats; SO: shamoperated rats; C: control rats. cb: cerebellectomy; ic: one-trial initial conditioning; rt: retrieval test.
rats (2 experimental and 2 control groups) were studied (Fig. 1). The two experimental groups were the following: - - C O C group (conditioned-operated-conditioned): the animals were conditioned (1 trial) and cerebellectomized 8 hours later; then, after a 7-day (5 rats) or a 14-day (5 rats) delay, they were tested again in the same conditions (retrieval test). - - N O C group (naive-operated-conditioned): the animals were cerebeUectomized while naive and conditioned (1 trial) after a 7-day delay. Then, 7 days (5 rats) or 14 days (5 rats) later, they were submitted to the retrieval test. The two control groups were the following: - - C group (controls): the animals were conditioned (1 trial) and submitted to the retrieval test 7 days (5 rats) or 14 days (5 rats) later. - - S O group (sham-operated): the animals were conditioned (1 trial) and sham-operated 8 hours later. Then, after a 7-day (5 rats) or a 14-day (5 rats) delay, they were submitted to the retrieval test.
Cerebellectomy and Sham-Operation The animals were anesthetized by IP injection of Nembutal (pentobarbital sodium) 35 mg-kg-~ and secured in a stereotaxic apparatus. The skin of the skull was incised, the muscles of the neck cut, the occipital bone drilled and removed. After cutting the dura, the cerebellum was removed by suction and the cavity filled with gelfoam. Then, the skin was sutured. Sham-operated rats were submitted to the same protocol except for removing the cerebellum which was left intact.
Experimental Procedure All the animals were isolated 48 hours before the experiments so as not to be stressed by the sudden isolation at the time of the test. Then, the rat was placed in the starting box and the shashdoor pulled up. Once the rat entered the light compartment, it displayed an exploratory behavior; after a several second delay, it spontaneously entered the dark compartment where it received an electric shock which was delivered once the four feet of the animal were in contact with the grid floor, and lasted until the rat came back into the light compartment, which required a few seconds. It was allowed to stay into the light compartment for one minute before returning to its cage in order to be able to know that the dark compartment only is associated with a nociceptive
stimulus. The time (TI) which elapses from the moment when the rat entered the light compartment to the moment when it entered the dark one was measured. Seven or 14 days later, the animals were submitted to a retrieval test which was given exactly in the same way as the initial conditioning except for the fact that the rat did not get an electric shock when entering the dark compartment. The time (T2) from the moment when the rat entered the light compartment to the moment when it entered the dark one was measured. If the difference T 2 - TI (absolute memory score) and the ratio ( T 2 - T 1 ) / T 1 (relative memory score) are null or small, one can expect, at the time of the retrieval test, the animals to have forgotten the initial nociceptive experience when entering the dark compartment; conversely, if the absolute and relative memory scores are high, one can expect the animals to have remembered this nociceptive experience. We have arbitrarily fixed an upper 10 min value to T2; in other words, if the rat did not enter the dark compartment after a 10 rain delay when given the retrieval test, it was returned to its cage. The electric shock was vibrating current (10 Hz, 45 V); such a stimulus was great enough to make the animals greatly disturbed but not too high so that the animals did not experience pain as revealed by their behavior. The intensity of the stimulus was chosen during a preexperiment in animals which are not included in this study.
Treatment of the Results For each group of 5 rats the times T1 and T2 needed for each ani___malto enter the dar_._kcompartment were respectively averaged (T1 = E T1/_5) and T2 = E T2/5), the mean values being given -+ SEM (~r~/n). Comparisons between groups were made according to the Mann-Whitney test.
Anatomical Controls After completion of the experiments, the animals were sacrificed. They were overdosed with Nembutal, their brain was removed and placed in 10% formalin for about two weeks. Then. the brains were examined under an operation microscope. RESULTS
T1 Values of Rats During the One-Trial Initial Conditioning When the animals enter the light compartment of the shuttlebox, they display an exploratory behavior which lasts for several seconds before entering the dark compartment (T1 value). In our experiments, this T1 value in C, SO and COC rats varied from 5.4 s to 81.0 s. It was, in most cases, lower than 30 s and exceptionally higher than 1 min (3 cases out of 30). The mean T1 values calculated in C, SO and COC rats, which are 36.1 --- 7.6 s, 22.4---6.0 s and 3 9 . 7 ± 14.2 s, respectively, are not significantly different (Fig. 2). This is not surprising since the animals of these three groups were identical: their cerebellum was intact and they were submitted to the same experimental protocol. In NOC rats, the T1 values varied from 3.2 s to 296.0 s and were in most cases (7 out of 10) higher than 1 min. Such rather high values were not due to an increased exploratory behavior but to the fact that the cerebeUectomy alters the motor coordination and, consequently, the animals were clumsy and reached the dark compartment later than the nonoperated ones. However, due to the great interindividual variability, the T1 mean value of NOC rats (136.1 - 33.8 s) is not significantly different from the ones of C, SO and COC animals (Fig. 2).
T2 Values of Rats During the Retrieval Test In C, SO and NOC animals, the T2 values being not significantly different when tested 7 or 14 days after the one-trial con-
CEREBELLUM AND MEMORY
509
X,
180-
)F
600- A
150-
-
qlO S ml
~
• -
90-
B
500-
500-
120-
600"
406-
400"
300-
300-
200-
206-
1O0-~
100-
6030-
0 li C
C SO C0Cll0C FIG. 2. Initial conditioning. Time, in seconds (-SEM), elapsing from the moment when the rat entered the light compartment to the moment when it entered the dark one (T1 value) in C, SO, COC and NOC rats. No significant difference between the different groups are observed.
ditioning, they will be gathered. All the animals of the C group but one (i.e., 9 rats out of 10) stayed in the light compartment for at least 10 min and did not enter the dark one. The only exception was a rat which entered the dark compartment after a 186 s exploratory behavior in light (in this animal, the TI value was 60.0 s). Then, the mean T2 value is highly significantly higher (p-
