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Increased paradoxical sleep in mice during acquisition of a shock avoidance task
Brain Research, 77 (1974) 221-230
221
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
INCREASED PARADOXICAL SLEEP IN MICE DURING ACQUISITION OF A SHOCK AVOIDANCE TASK
CARLYLE SMITH, K U N I O K I T A H A M A , JEAN-LOUIS VALATX AND MICHEL JOUVET
Department of Psychology, Trent University, Peterborough, Ontario (Canada) and Laboratoire de M~decine Expdrimentale, Universit~ Claude Bernard, Domaine Rockefeller, 69008 Lyons (France) (Accepted April 17th, 1974)
SUMMARY
Two strains of mice were subjected to a complex shock avoidance task. The C57BR (brown) strain were superior to the C57BL (black) strain in their learning ability. Both strains showed a long term increase in paradoxical sleep (PS) prior to the maximum increase in learning performance (MIP). For the brown strain, this increase was apparent in the 24 h before the MIP. For the black strain, the increase began 48 h prior to the MIP. In the brown strain the day after the MIP, a second effect appeared. PS was higher in the first half hour after sleep onset, following the training session. This increase was due to the larger numbers of slow wave sleep-paradoxical sleep (SWS-PS) cycles. The effect was not present in the black strain. It was concluded that two mechanisms are probably at work during the learning process, one during the earlier stages and one during the later stages as learning reaches criterion.
INTRODUCTION
There are now several studies which support the hypothesis that paradoxical sleep (PS) is intimately involved with the mechanisms of memory consolidation. It has been reported that selective PS deprivation prior to task acquisition results in retention deficits in both rats and micea,l% Interpolation of the PS deprivation between acquisition and retest conditions also produces retention deficits on a variety of tasks in both rats and mice4,7,s,14. In contrast, Miller et al. lz found no retention differences on a one trial avoidance task in rats when PS deprivation was imposed either before or after the acquisition condition. Negative results were also found by Albert e t al. 1 and ChernikL The combination of PS deprivation and electroconvulsive shock (ECS) has been
222
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shown to retard the acquisition of a learned task even more severely than PS deprivation alone in both rats 17 and miceL Lucero lI reported an increase in PS in the 3-h period after food-deprived rats were trained in a complex maze situation, keconte and Hennevin 0 demonstrated increases in PS in rats after a single 90-rain training session in a shuttle box. In a second study, with training sessions over a 4-day period, PS was found to be greater in test animals for days 2 and 3. This increase was confined to the first half hour of sleep after the training session and was due to an increase in the number of SWS-PS cycles 6. In another study keconte and Hennevin ~0 found that sleep deprivation in rats for 3 h after the training session drastically impaired task acquisition. In a preliminary study with two inbred strains of mice, Smith et al. 15 observed an increase in PS in the first half hour of sleep after the daily training session in a complex Y-maze avoidance situation. This increase was due to an increase in the number of SWS-PS cycles, it was also found that a long term increase in PS appeared in the 24-48-h period preceding the largest daily increase in learning performance of each individual test animal. The present study was carried out to further elucidate these findings. MATERIALS A N D M E T H O D S
The subjects were two inbred strains of mice, C57BR/cd/ORL (N = 14) and C57BL/6/ORL (N ~ 16), 50-60 days of age, weighing 22-30 g at the time of operation. All animals were chronically implanted with cortical (EEG) and electromyographic (EMG) electrodes. After 5-6 days of recovery from the operation, animals were hooked up to individual recording cables for 10 days. Following this habituation period, continuous E E G and E M G recordings were begun. This procedure continued for 9 full days and nights. The night began at 7.00 p.m. (19.00) with an artificially imposed period of darkness and ended at 7.00 a.m. with the switching on of lights. The lights remained on for 12 h. The only interruption in recording occurred on test days during the 15-min training session. The learning task The apparatus was a specially constructed multiple Y-maze. There were 5 separate Y-maze sections which fitted together as a single unit. The mazes were 8 cm wide. Each arm was l0 cm long and the sides were 12 cm high. The goal arms of each section became the start box for the next trial. This design minimized the handling of the subjects. On each day of the 5-day training period, the configuration of the maze was changed. There was a standard left-right configuration for each of the 5 days established by means of a random numbers table. The shock was delivered by a shock scrambler and the current was 0.25 mA. Groups Test animals from both strains (C57BR, N = 4; C57BL, N = 6) were given 15 1-min trials per day in the multiple Y-maze (the animals ran through the 5 sections
P S AND SHOCK AVOIDANCE
223
3 times). On each trial, an animal was required to run to the illuminated arm of the Y and avoid the non-illuminated arm. Failure to do so within the 5-sec period after the door of the start box was raised, resulted in foot shock. Shock was given if the animal did not run or if it ran to the darkened arm of the Y. Running control (RC) groups for both strains (C57BR, N = 4; C57BL, N ---- 5) were tested under the same conditions, except that no illumination cue for the Y-maze arms was provided. Subjects found the shock free arm by chance. Foot shock was begun with the raising of the start box door on each trial, since otherwise, the animals would have had the opportunity to learn shock avoidance on certain trials by reaching the shock-free arm during the 5-sec delay. To control exactly for the amount of shock received by the test group, a second special shock control (SSC) group was added (C57BR, N ---- 4; C57BL, N = 3). Mice were placed in a start box and were given shock equal in duration and in interval to the daily averages of the test animals in each strain. These shocks were administered (or omitted) at l-min intervals during each 15-min session to match the 1-min trial interval. To control for possible effects on sleep due to the shock itself, a third group was added. Animals in this non-shock control (NSC) group (C57BR, N = 2; C57BL, N ---- 2) were placed in the start box of the maze for 15 min each day for 5 days. They received no shock whatsoever. All subjects were allowed two complete baseline days with no interruptions. At the end of the third day, testing began. Animals in all groups and in both strains, were tested between 4.00 p.m. and 5.00 p.m. each day. A given animal was always run at exactly the same time. This procedure lasted for 5 days, followed by 2 more days of recording.
Scoring of the data The E E G and E M G records allowed the categorization of the behavior of the subjects into awake, SWS, and PS. Continuous recording of EEG and E M G records for each animal were scored in the following ways. Low voltage fast wave E E G activity coupled with pronounced E M G activity was classified as 'awake'. High voltage slow wave E E G coupled with reduced E M G activity was considered to be SWS. Low voltage fast wave E E G with an almost complete absence of E M G response was considered to be PS. Sleep parameter analyses of the strains were kept separate. The learning curves for the test groups were a composite of two values, the number of correct Y-maze arm choices, and the number of shock avoidances in 15 trials. Each value provided half the trial learning score. This method of scoring was chosen because experience with pilot subjects demonstrated that animals often focussed on either the shock avoidance or the Y-maze arm choice. In using the shock avoidance score alone, it was found that some animals were poor at this while at the same time doing relatively well in Y-maze arm choice. Those initially doing poorly at Y-maze arm choice often were quick to learn the 5-sec avoidance component of the task. It was clear that the most accurate measure of learning would have to include both of these components in the learning score.
224
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RESULTS
Learning performance For the C57BR test animals, all subjects performed at a level of at least 80 ~ correct responses by the fifth training session, although some animals learned faster than others. The C57BL subjects did not reach as high a level of performance as the brown strain. Five of the 6 test animals performed at a level of proficiency between 53 To and 77 ~ correct responses by the fifth training session. A single animal, which showed no improvement whatsoever was discarded from subsequent sleep analyses. On one particular day, each test animal exhibited a maximum increase in correct performance (MIP) over the immediately preceding test day. This value was simply the largest increase in composite score over the preceding test day. In the case of two or more scores the same, the value of the first day was invariably chosen as the MIP. An examination of the relation between M I P occurrence and daily level of PS for the C57BR strain revealed that the highest number of minutes of PS per 24 h for each animal during the entire 9 day experiment always appeared in the 24-h period preceding the MIP (Kendall's coefficient -- I.G0). For the C57BL strain, the maximum observed PS per day hour period did not always precede the M I P by one day. A correlation of relative M1P size with relative levels of PS for the test animals did not reach significance (Kendall's coefficient ~- 0.80). However, as with the C57BR animals, there was always a substantial increase in the amount of PS in every test animal before the M I P was observed. In view of the apparent close relation between PS levels and learning performance and because animals within each strain did not learn at the same rate, it was decided to use a 'staggered' procedure in the analysis of the sleep data. The M I P characteristic made it a simple matter to match the learning curves of the test animals within each strain. Sleep values then corresponded in a 'staggered' fashion. For the RC group, an error was counted each time the animals entered a wrong compartment. Thus, an animal could spend much time running back and forth from the start box arm to the unsafe arm of the Y-maze, receiving shock all the while. The RC subjects were observed to exhibit a peak in the number of errors on one particular day during the 5-day session. There was then a decrease in the number of errors, apparently due to a diminution in the panic behavior of the animals, although the number of these errors always remained quite high. RC animals were matched with regard to this error decrease and compared with test animals in terms of corresponding sleep parameter values. The reduction in error was considered roughly equivalent to the MIP. The SSC group sleep parameter values were compared such that the amount of shock on succeeding test days matched that of the test animals. Since the average amount of shock for SSC animals dropped off between the second and third days of the 5-day session, the corresponding sleep values of those days were considered comparable to the sleep values associated with the M I P (and thus reduction in shock) for the test animals.
P S AND SHOCK AVOIDANCE
225
The NSC group scores were not staggered since no learning or shock differential was involved.
The long term phenomenon Since change in amounts of PS and SWS compared to baseline values as well as between test and control group values were of interest, separate analyses of variance were carried out on the difference scores between baseline and each 'staggered' test day. In analyses that were significant, 3 orthogonal comparisons were carried out to ascertain which of the 4 groups were responsible for this significant difference. The test group was compared with the combined controls, the R C group was compared with the combined SSC and NSC groups and the SSC and NSC group values were compared. Because testing began on the third baseline day and because initial handling resulted in prolonged wakefulness after the test, PS and SWS were lower than for the first two baseline days. Thus, this day was not used as part of the baseline average. For the C57BR animals, analysis of the PS differences between test and control animals 24 h prior to the M I P revealed a significant difference (F ---- 4.33, df---- 3/10, P < 0.05). An orthogonal comparison of test vs. combined control values showed a significant difference (F = 11.54, d f = 1/10, P < 0.01). The orthogonal comparisons between the control groups were not significantly different. In terms of total amount of PS per 24-h period, no differences were found at any other point. Analysis of the amount of PS during the 12 h of darkness (19.00-7.00) preceding Total
n
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ne
48Hours Illforo MIP
24 Hours Iloforo M|
p
24 Hours AftlrM|P
Fig. 1. C57BR strain showing PS means in minutes for baseline and test times before and after the maximum increase in correct performance (MIP). The dotted bars indicate the test group, the clear bars indicate the combined controls. *, significant difference between the groups, P < 0.05.
226
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Night
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Day
Fig. 2. C57BR strain showing PS means in minutes in the 24-h period prior to the MIP. The dotted bars represent the test group values, the clear bars the combined control group values.
the M I P was not significantly different for test and control groups, and neither was a similar analysis for the 12 daylight hours (7.00-19.00) prior to the MIP. Fig. 1 shows the mean values of PS for test and control groups. Further PS analyses of the 24-h period prior to M I P failed to show any single 3-h period significantly greater than that for the combined controls. It was noted, however, that during only one 3-h period (1.t30-3.59) was the PS of the combined controls slightly higher than that for the test group. In the other 7 blocks, PS for the test group was consistently larger (Fig. 2). There were no SWS differences found between C57BR test and control animals. For the C57BL strain, analyses of PS differences per 24-h period revealed significant differences between groups. In the 48 h prior to the MIP, test and control groups were different (F = 3.93, df = 3/10, P < 0.05). An orthogonal comparison of test vs. combined controls was significant (F = 9.00, d f - - 1t10, P < 0.05). On the other hand, there were no significant differences between the control groups. The same phenomenon was observed in the period 24 h prior to the M I P (F = 7.47, df-- 3/10, P < 0.01). The orthogonal comparison between test and combined control values was significant (F = 23.06, d f = 1/10, P < 0.1305). Comparisons among control groups were not found to be different. Analyses of the data for the daylight hours (7.130-19.00) showed that contributions to the total PS per 24 h were not heavily concentrated there. No significant differences were found at any point between test and control animals. Analysis of the night time PS accumulations (19.00-7.00) during the 24-h period prior to the M1P indicated differences between the groups (F = 5.78, df = 3/10, P < 0.05). Orthogonal comparison of test vs. combined control groups revealed a significant difference between them (F = 10.32, d f = 1/10, P < 0.01). There was also a difference between the RC and SSC, NSC combined controls (F = 10.09, d f = 1/10, P < 0.01). Fig. 3 shows the test and control PS values for the C57BL animals. Analyses of the PS accumulations in 3-h blocks revealed no periods for the 48 h prior to M I P in which a particular 3-h period of PS for test animals was significantly larger than that of the controls. In the 24 h prior to M I P the test mean was slightly higher than that of the combined controls for every 3-h block. In the 48 h prior to the
PS AND SHOCK AVOIDANCE
227
Total
i m
Day
i
HH Nn
o W
Night
!
g
NN Baseline
nh 411 H o u r s
Before M I P
24 H o u r i Befo•• M IP
24 H o u r s After MI P
Fig. 3. C57BL strain showing PS means in minutes for baseline and test times before and after the MIP. The dotted bars indicate the test group, the clear bars indicate the combined controls. *, significant difference between the two groups, P < 0.05; **, significant difference between the two groups, P < 0.01.
MIP, three 3-h blocks had control means slightly larger than the test means. Two of these occurred during the night time hours, farthest from the M1P, and one during the first 3-h block following darkness (7.00-9.59). Otherwise, the test means were all slightly larger than the control means. As with the C57BR strain, there were no differences between test and control animals with regard to SWS. A comparison of pretraining baseline PS (days 1 and 2) and the post-training baseline PS (day 9), indicated no significant differences between test and control animals for either strain.
The short term effect C57BR. An analysis of the amount of PS present in the first half hour after sleep onset following the test session was performed. Because of the 'staggered' procedure, the complete analyses of all test and control animals was possible for only 3 test days. The days included are the day of the m a x i m u m increase in learning performance, the day before this increase and the day after. An analysis of each of the days for which the scores were available comparing test and control groups in analyses of variance revealed that 24 h after M I P there was a significant difference between groups on the PS measure (F = 4.10, d f = 3/10, P < 0.05). Orthogonal comparison of the test group vs. the combined control groups was
228
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1.5
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24 Hours Before MIP
Day of MIP
24 H o u r s After MIP
Fig. 4. C57BR strain showing the number of minutes of PS in the first half hour after sleep onset following the test session. The dotted bars are test group means while the clear bars are combined control group means. *, significant differences between groups, P < 0.05. significant (F ~-- 11.68, d f --~ 1/10, P < 0.01). No other comparisons were significantly different (Fig. 4). This effect appeared to be due to an increased number of PS cycles (F ---- 6.65, d f ~- 3/10, P < 0.01). A comparison of the test group vs. the combined controls was significant (F = 19.09, d f = 1/I0, P < 0.01). None of the other comparisons among the controls was significant. Similar analyses of the SWS during these same time periods did not differentiate test and control animals. C57BL. As with the brown strain, the trials of all test and control animals could only be analysed for 3 test days. These measures included the day of the maximum learning increase and the 2 days prior to that increase. The overall analysis of variance for minutes of PS in the first half hour after sleep onset indicated that test and control groups did not differ. An analysis of the SWS in this same time period gave the same result. DISCUSSION
Both strains of mice clearly demonstrated what appears to be a prolonged or long term PS increase preceding a major increase in correct maze performance. For the C57BR group, the high PS levels in the 24 h prior to M I P seemed evenly distributed between the 12 h of light and 12 h of darkness as no significant difference between test and control groups for these periods was found. N o r was there any difference between groups when analyses of 3-h blocks of PS were carried out, although it seems clear that with the exception of the 3-h period 1.00-3.50, the test group mean for PS was always slightly larger (Fig. 2).
PS AND SHOCK AVOIDANCE
229
The C57BL animals exhibited an even more prolonged long term effect than the C57BR strain. The PS of the test group was significantly greater in the 48-h period before as well as 24 h before the MIP. The actual increase in PS appeared to start about 36 h before the MIP in the daylight hours, although the statistical comparison for this 12 h of light did not reach significance. In the last 12-h period of darkness prior to the MIP, the PS for the test group was higher than for the combined controls. There was also a difference between the RC vs. SSC and NSC control groups, the PS for the RC group being higher. The reason for this difference is not clear. Despite the rather larger PS levels in the 12 h of darkness prior to the MIP, the PS levels in the daylight periods were also larger than those of the controls, although not significantly so. The more prolonged increase in PS levels prior to the MIP in the black strain is puzzling if, in fact, it is a reflection of some type of consolidation process. Perhaps PS continues to be manifest until a certain level of learning has been reached. A clear understanding of this phenomenon will require further research. It is interesting to note that the single C57BL animal which did not learn at all did not show any increase in PS. Rather, the highest recorded daily PS was observed during the second baseline day. During the following 8 days, the level of PS dropped steadily. The demonstration of increased PS in the first half hour after sleep onset for the C57BR strain on the day following the MIP was a result similar to that found by Hennevin et al. 6. No such effect was observed in the C57BL animals. This may have been due to the fact that the level of learning was not as advanced, being less than 80 9/0 in every single animal. This result appears to conflict with the preliminary report 15. However, although the PS for the test animals did increase each day by a small amount, the PS values for the combined controls were also large and differences were not significant. The data of the present experiment tend to support the hypothesis that two sleep mechanisms are involved with learning. The first appears to be connected with a long term increase in PS, while the second is a brief short term increase which is evident in the first half hour of sleep after the training session. The long term PS increase seems more important during the early phase of acquisition, while the short term effect is important as the learning curve approaches asymptote.
REFERENCES 1 ALBERT, I., CICALA, G. A., AND SIEGAL, J., The behavioral effects of REM sleep deprivation in
rats, Psychophysiology, 6 (1970) 550-560. 2 CHERNIK,D. A., Effect of REM sleep deprivation on learning and recall by humans, Percept. Motor Skills, 34 (1972) 283-294. 3 FISHBEIN,W., Interference with conversion of memory from short term to long term storage by partial sleep deprivation, Comm. Behav. Biol., 5 (1970) 171-175. 4 FISHBEIN,W., Disruptive effects of rapid eye movement sleep deprivation on long term memory, Physiol. Behav., 6 (1971) 279-282. 5 FISHBEIN,W., MCGAUGH,J. L., AND SWARZ,J. R., Retrograde amnesia: electroconvulsive shock effectsafter termination of rapid eye movement sleep deprivation, Science, 172 (1971) 80-82.
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c. SMrFH cf o/.
6 HENNEV1N, E., LECONTE, P. ET BLOCH, V., Effet du niveau d'acquisition sur l'augmentation de la dur6e du sommeil paradoxal cons6cutive b_un conditionnement d'6vitement chez le rat, C.R. Acad. Sci. (Paris), 273 (1971) 2595-2598. 7 LECONTE, P. ET BLOCH, V., Effet de la privation de sommeil paradoxal sur I'acquisition de la retention d'un conditionnement chez le rat, J. Physiol. (Paris), 62 (1970) 290-291. 8 LECONTE, P., ET BLOCH, V., D6ficit de la retention d'un conditionnement apres privation de sommeil paradoxal chez le rat, C.R. Acad. Sci. (Paris), 271 (1970) 226-229. 9 LECONTE, P., ET HENNEVIN, E., Augmentation de la dur6e de 'sommeil paradoxal' cons6cutive/t un apprentissage chez le rat, C.R. Acad. Sci. (Paris), 273 (1971) 86-88. 10 LECONTE, P., ET HENNEVIN, E., Sommeil paradoxal et memorisation: effet du d61ai d'endormissement chez le rat, J. Physiol. (Paris), 65 (1972) 255-256. ll LUCERO, M. A., Lengthening of REM sleep duration consecutive to learning in the rat, Brain Research, 20 (1970) 319-322. 12 MILLER, L., DREW, W. G., AND SCHWARTZ, I., Effect of REM sleep deprivation on retention of a one trial passive avoidance response, Percept. Motor Skills, 33 (1971) 118. 13 MYERS, J. L., Fundamentals of Experimental Design, Allyn and Bacon, Boston, Mass., 1972. 14 PEARLMAN, C. A., Latent learning impaired by R E M sleep deprivation, Psychon. Sci., 25 (1971) 135-136. 15 SMITH, C. T., K1TAHAMA, K., VALATX,J. L., ET JOUVET, M., Sommeil paradoxal et apprentissage chez deux souches consanguines de souris, C.R. Acad. Sci. (Paris), 275 (1972) 1283-1286. 16 STERN, W. C., Acquisition impairments following rapid eye movement sleep deprivation in rats, Physiol. Behav., 7 (1971) 345-352. 17 WOLFOWrrz, B. E., AND HOLDSTOCK, T. L., Paradoxical sleep deprivation and memory in rats, Comm. Behav. Biol., 6 (1971) 281-284.
221
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
INCREASED PARADOXICAL SLEEP IN MICE DURING ACQUISITION OF A SHOCK AVOIDANCE TASK
CARLYLE SMITH, K U N I O K I T A H A M A , JEAN-LOUIS VALATX AND MICHEL JOUVET
Department of Psychology, Trent University, Peterborough, Ontario (Canada) and Laboratoire de M~decine Expdrimentale, Universit~ Claude Bernard, Domaine Rockefeller, 69008 Lyons (France) (Accepted April 17th, 1974)
SUMMARY
Two strains of mice were subjected to a complex shock avoidance task. The C57BR (brown) strain were superior to the C57BL (black) strain in their learning ability. Both strains showed a long term increase in paradoxical sleep (PS) prior to the maximum increase in learning performance (MIP). For the brown strain, this increase was apparent in the 24 h before the MIP. For the black strain, the increase began 48 h prior to the MIP. In the brown strain the day after the MIP, a second effect appeared. PS was higher in the first half hour after sleep onset, following the training session. This increase was due to the larger numbers of slow wave sleep-paradoxical sleep (SWS-PS) cycles. The effect was not present in the black strain. It was concluded that two mechanisms are probably at work during the learning process, one during the earlier stages and one during the later stages as learning reaches criterion.
INTRODUCTION
There are now several studies which support the hypothesis that paradoxical sleep (PS) is intimately involved with the mechanisms of memory consolidation. It has been reported that selective PS deprivation prior to task acquisition results in retention deficits in both rats and micea,l% Interpolation of the PS deprivation between acquisition and retest conditions also produces retention deficits on a variety of tasks in both rats and mice4,7,s,14. In contrast, Miller et al. lz found no retention differences on a one trial avoidance task in rats when PS deprivation was imposed either before or after the acquisition condition. Negative results were also found by Albert e t al. 1 and ChernikL The combination of PS deprivation and electroconvulsive shock (ECS) has been
222
c:. SMITH ~'l (1[.
shown to retard the acquisition of a learned task even more severely than PS deprivation alone in both rats 17 and miceL Lucero lI reported an increase in PS in the 3-h period after food-deprived rats were trained in a complex maze situation, keconte and Hennevin 0 demonstrated increases in PS in rats after a single 90-rain training session in a shuttle box. In a second study, with training sessions over a 4-day period, PS was found to be greater in test animals for days 2 and 3. This increase was confined to the first half hour of sleep after the training session and was due to an increase in the number of SWS-PS cycles 6. In another study keconte and Hennevin ~0 found that sleep deprivation in rats for 3 h after the training session drastically impaired task acquisition. In a preliminary study with two inbred strains of mice, Smith et al. 15 observed an increase in PS in the first half hour of sleep after the daily training session in a complex Y-maze avoidance situation. This increase was due to an increase in the number of SWS-PS cycles, it was also found that a long term increase in PS appeared in the 24-48-h period preceding the largest daily increase in learning performance of each individual test animal. The present study was carried out to further elucidate these findings. MATERIALS A N D M E T H O D S
The subjects were two inbred strains of mice, C57BR/cd/ORL (N = 14) and C57BL/6/ORL (N ~ 16), 50-60 days of age, weighing 22-30 g at the time of operation. All animals were chronically implanted with cortical (EEG) and electromyographic (EMG) electrodes. After 5-6 days of recovery from the operation, animals were hooked up to individual recording cables for 10 days. Following this habituation period, continuous E E G and E M G recordings were begun. This procedure continued for 9 full days and nights. The night began at 7.00 p.m. (19.00) with an artificially imposed period of darkness and ended at 7.00 a.m. with the switching on of lights. The lights remained on for 12 h. The only interruption in recording occurred on test days during the 15-min training session. The learning task The apparatus was a specially constructed multiple Y-maze. There were 5 separate Y-maze sections which fitted together as a single unit. The mazes were 8 cm wide. Each arm was l0 cm long and the sides were 12 cm high. The goal arms of each section became the start box for the next trial. This design minimized the handling of the subjects. On each day of the 5-day training period, the configuration of the maze was changed. There was a standard left-right configuration for each of the 5 days established by means of a random numbers table. The shock was delivered by a shock scrambler and the current was 0.25 mA. Groups Test animals from both strains (C57BR, N = 4; C57BL, N = 6) were given 15 1-min trials per day in the multiple Y-maze (the animals ran through the 5 sections
P S AND SHOCK AVOIDANCE
223
3 times). On each trial, an animal was required to run to the illuminated arm of the Y and avoid the non-illuminated arm. Failure to do so within the 5-sec period after the door of the start box was raised, resulted in foot shock. Shock was given if the animal did not run or if it ran to the darkened arm of the Y. Running control (RC) groups for both strains (C57BR, N = 4; C57BL, N ---- 5) were tested under the same conditions, except that no illumination cue for the Y-maze arms was provided. Subjects found the shock free arm by chance. Foot shock was begun with the raising of the start box door on each trial, since otherwise, the animals would have had the opportunity to learn shock avoidance on certain trials by reaching the shock-free arm during the 5-sec delay. To control exactly for the amount of shock received by the test group, a second special shock control (SSC) group was added (C57BR, N ---- 4; C57BL, N = 3). Mice were placed in a start box and were given shock equal in duration and in interval to the daily averages of the test animals in each strain. These shocks were administered (or omitted) at l-min intervals during each 15-min session to match the 1-min trial interval. To control for possible effects on sleep due to the shock itself, a third group was added. Animals in this non-shock control (NSC) group (C57BR, N = 2; C57BL, N ---- 2) were placed in the start box of the maze for 15 min each day for 5 days. They received no shock whatsoever. All subjects were allowed two complete baseline days with no interruptions. At the end of the third day, testing began. Animals in all groups and in both strains, were tested between 4.00 p.m. and 5.00 p.m. each day. A given animal was always run at exactly the same time. This procedure lasted for 5 days, followed by 2 more days of recording.
Scoring of the data The E E G and E M G records allowed the categorization of the behavior of the subjects into awake, SWS, and PS. Continuous recording of EEG and E M G records for each animal were scored in the following ways. Low voltage fast wave E E G activity coupled with pronounced E M G activity was classified as 'awake'. High voltage slow wave E E G coupled with reduced E M G activity was considered to be SWS. Low voltage fast wave E E G with an almost complete absence of E M G response was considered to be PS. Sleep parameter analyses of the strains were kept separate. The learning curves for the test groups were a composite of two values, the number of correct Y-maze arm choices, and the number of shock avoidances in 15 trials. Each value provided half the trial learning score. This method of scoring was chosen because experience with pilot subjects demonstrated that animals often focussed on either the shock avoidance or the Y-maze arm choice. In using the shock avoidance score alone, it was found that some animals were poor at this while at the same time doing relatively well in Y-maze arm choice. Those initially doing poorly at Y-maze arm choice often were quick to learn the 5-sec avoidance component of the task. It was clear that the most accurate measure of learning would have to include both of these components in the learning score.
224
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RESULTS
Learning performance For the C57BR test animals, all subjects performed at a level of at least 80 ~ correct responses by the fifth training session, although some animals learned faster than others. The C57BL subjects did not reach as high a level of performance as the brown strain. Five of the 6 test animals performed at a level of proficiency between 53 To and 77 ~ correct responses by the fifth training session. A single animal, which showed no improvement whatsoever was discarded from subsequent sleep analyses. On one particular day, each test animal exhibited a maximum increase in correct performance (MIP) over the immediately preceding test day. This value was simply the largest increase in composite score over the preceding test day. In the case of two or more scores the same, the value of the first day was invariably chosen as the MIP. An examination of the relation between M I P occurrence and daily level of PS for the C57BR strain revealed that the highest number of minutes of PS per 24 h for each animal during the entire 9 day experiment always appeared in the 24-h period preceding the MIP (Kendall's coefficient -- I.G0). For the C57BL strain, the maximum observed PS per day hour period did not always precede the M I P by one day. A correlation of relative M1P size with relative levels of PS for the test animals did not reach significance (Kendall's coefficient ~- 0.80). However, as with the C57BR animals, there was always a substantial increase in the amount of PS in every test animal before the M I P was observed. In view of the apparent close relation between PS levels and learning performance and because animals within each strain did not learn at the same rate, it was decided to use a 'staggered' procedure in the analysis of the sleep data. The M I P characteristic made it a simple matter to match the learning curves of the test animals within each strain. Sleep values then corresponded in a 'staggered' fashion. For the RC group, an error was counted each time the animals entered a wrong compartment. Thus, an animal could spend much time running back and forth from the start box arm to the unsafe arm of the Y-maze, receiving shock all the while. The RC subjects were observed to exhibit a peak in the number of errors on one particular day during the 5-day session. There was then a decrease in the number of errors, apparently due to a diminution in the panic behavior of the animals, although the number of these errors always remained quite high. RC animals were matched with regard to this error decrease and compared with test animals in terms of corresponding sleep parameter values. The reduction in error was considered roughly equivalent to the MIP. The SSC group sleep parameter values were compared such that the amount of shock on succeeding test days matched that of the test animals. Since the average amount of shock for SSC animals dropped off between the second and third days of the 5-day session, the corresponding sleep values of those days were considered comparable to the sleep values associated with the M I P (and thus reduction in shock) for the test animals.
P S AND SHOCK AVOIDANCE
225
The NSC group scores were not staggered since no learning or shock differential was involved.
The long term phenomenon Since change in amounts of PS and SWS compared to baseline values as well as between test and control group values were of interest, separate analyses of variance were carried out on the difference scores between baseline and each 'staggered' test day. In analyses that were significant, 3 orthogonal comparisons were carried out to ascertain which of the 4 groups were responsible for this significant difference. The test group was compared with the combined controls, the R C group was compared with the combined SSC and NSC groups and the SSC and NSC group values were compared. Because testing began on the third baseline day and because initial handling resulted in prolonged wakefulness after the test, PS and SWS were lower than for the first two baseline days. Thus, this day was not used as part of the baseline average. For the C57BR animals, analysis of the PS differences between test and control animals 24 h prior to the M I P revealed a significant difference (F ---- 4.33, df---- 3/10, P < 0.05). An orthogonal comparison of test vs. combined control values showed a significant difference (F = 11.54, d f = 1/10, P < 0.01). The orthogonal comparisons between the control groups were not significantly different. In terms of total amount of PS per 24-h period, no differences were found at any other point. Analysis of the amount of PS during the 12 h of darkness (19.00-7.00) preceding Total
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48Hours Illforo MIP
24 Hours Iloforo M|
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24 Hours AftlrM|P
Fig. 1. C57BR strain showing PS means in minutes for baseline and test times before and after the maximum increase in correct performance (MIP). The dotted bars indicate the test group, the clear bars indicate the combined controls. *, significant difference between the groups, P < 0.05.
226
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Fig. 2. C57BR strain showing PS means in minutes in the 24-h period prior to the MIP. The dotted bars represent the test group values, the clear bars the combined control group values.
the M I P was not significantly different for test and control groups, and neither was a similar analysis for the 12 daylight hours (7.00-19.00) prior to the MIP. Fig. 1 shows the mean values of PS for test and control groups. Further PS analyses of the 24-h period prior to M I P failed to show any single 3-h period significantly greater than that for the combined controls. It was noted, however, that during only one 3-h period (1.t30-3.59) was the PS of the combined controls slightly higher than that for the test group. In the other 7 blocks, PS for the test group was consistently larger (Fig. 2). There were no SWS differences found between C57BR test and control animals. For the C57BL strain, analyses of PS differences per 24-h period revealed significant differences between groups. In the 48 h prior to the MIP, test and control groups were different (F = 3.93, df = 3/10, P < 0.05). An orthogonal comparison of test vs. combined controls was significant (F = 9.00, d f - - 1t10, P < 0.05). On the other hand, there were no significant differences between the control groups. The same phenomenon was observed in the period 24 h prior to the M I P (F = 7.47, df-- 3/10, P < 0.01). The orthogonal comparison between test and combined control values was significant (F = 23.06, d f = 1/10, P < 0.1305). Comparisons among control groups were not found to be different. Analyses of the data for the daylight hours (7.130-19.00) showed that contributions to the total PS per 24 h were not heavily concentrated there. No significant differences were found at any point between test and control animals. Analysis of the night time PS accumulations (19.00-7.00) during the 24-h period prior to the M1P indicated differences between the groups (F = 5.78, df = 3/10, P < 0.05). Orthogonal comparison of test vs. combined control groups revealed a significant difference between them (F = 10.32, d f = 1/10, P < 0.01). There was also a difference between the RC and SSC, NSC combined controls (F = 10.09, d f = 1/10, P < 0.01). Fig. 3 shows the test and control PS values for the C57BL animals. Analyses of the PS accumulations in 3-h blocks revealed no periods for the 48 h prior to M I P in which a particular 3-h period of PS for test animals was significantly larger than that of the controls. In the 24 h prior to M I P the test mean was slightly higher than that of the combined controls for every 3-h block. In the 48 h prior to the
PS AND SHOCK AVOIDANCE
227
Total
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i
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o W
Night
!
g
NN Baseline
nh 411 H o u r s
Before M I P
24 H o u r i Befo•• M IP
24 H o u r s After MI P
Fig. 3. C57BL strain showing PS means in minutes for baseline and test times before and after the MIP. The dotted bars indicate the test group, the clear bars indicate the combined controls. *, significant difference between the two groups, P < 0.05; **, significant difference between the two groups, P < 0.01.
MIP, three 3-h blocks had control means slightly larger than the test means. Two of these occurred during the night time hours, farthest from the M1P, and one during the first 3-h block following darkness (7.00-9.59). Otherwise, the test means were all slightly larger than the control means. As with the C57BR strain, there were no differences between test and control animals with regard to SWS. A comparison of pretraining baseline PS (days 1 and 2) and the post-training baseline PS (day 9), indicated no significant differences between test and control animals for either strain.
The short term effect C57BR. An analysis of the amount of PS present in the first half hour after sleep onset following the test session was performed. Because of the 'staggered' procedure, the complete analyses of all test and control animals was possible for only 3 test days. The days included are the day of the m a x i m u m increase in learning performance, the day before this increase and the day after. An analysis of each of the days for which the scores were available comparing test and control groups in analyses of variance revealed that 24 h after M I P there was a significant difference between groups on the PS measure (F = 4.10, d f = 3/10, P < 0.05). Orthogonal comparison of the test group vs. the combined control groups was
228
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Fig. 4. C57BR strain showing the number of minutes of PS in the first half hour after sleep onset following the test session. The dotted bars are test group means while the clear bars are combined control group means. *, significant differences between groups, P < 0.05. significant (F ~-- 11.68, d f --~ 1/10, P < 0.01). No other comparisons were significantly different (Fig. 4). This effect appeared to be due to an increased number of PS cycles (F ---- 6.65, d f ~- 3/10, P < 0.01). A comparison of the test group vs. the combined controls was significant (F = 19.09, d f = 1/I0, P < 0.01). None of the other comparisons among the controls was significant. Similar analyses of the SWS during these same time periods did not differentiate test and control animals. C57BL. As with the brown strain, the trials of all test and control animals could only be analysed for 3 test days. These measures included the day of the maximum learning increase and the 2 days prior to that increase. The overall analysis of variance for minutes of PS in the first half hour after sleep onset indicated that test and control groups did not differ. An analysis of the SWS in this same time period gave the same result. DISCUSSION
Both strains of mice clearly demonstrated what appears to be a prolonged or long term PS increase preceding a major increase in correct maze performance. For the C57BR group, the high PS levels in the 24 h prior to M I P seemed evenly distributed between the 12 h of light and 12 h of darkness as no significant difference between test and control groups for these periods was found. N o r was there any difference between groups when analyses of 3-h blocks of PS were carried out, although it seems clear that with the exception of the 3-h period 1.00-3.50, the test group mean for PS was always slightly larger (Fig. 2).
PS AND SHOCK AVOIDANCE
229
The C57BL animals exhibited an even more prolonged long term effect than the C57BR strain. The PS of the test group was significantly greater in the 48-h period before as well as 24 h before the MIP. The actual increase in PS appeared to start about 36 h before the MIP in the daylight hours, although the statistical comparison for this 12 h of light did not reach significance. In the last 12-h period of darkness prior to the MIP, the PS for the test group was higher than for the combined controls. There was also a difference between the RC vs. SSC and NSC control groups, the PS for the RC group being higher. The reason for this difference is not clear. Despite the rather larger PS levels in the 12 h of darkness prior to the MIP, the PS levels in the daylight periods were also larger than those of the controls, although not significantly so. The more prolonged increase in PS levels prior to the MIP in the black strain is puzzling if, in fact, it is a reflection of some type of consolidation process. Perhaps PS continues to be manifest until a certain level of learning has been reached. A clear understanding of this phenomenon will require further research. It is interesting to note that the single C57BL animal which did not learn at all did not show any increase in PS. Rather, the highest recorded daily PS was observed during the second baseline day. During the following 8 days, the level of PS dropped steadily. The demonstration of increased PS in the first half hour after sleep onset for the C57BR strain on the day following the MIP was a result similar to that found by Hennevin et al. 6. No such effect was observed in the C57BL animals. This may have been due to the fact that the level of learning was not as advanced, being less than 80 9/0 in every single animal. This result appears to conflict with the preliminary report 15. However, although the PS for the test animals did increase each day by a small amount, the PS values for the combined controls were also large and differences were not significant. The data of the present experiment tend to support the hypothesis that two sleep mechanisms are involved with learning. The first appears to be connected with a long term increase in PS, while the second is a brief short term increase which is evident in the first half hour of sleep after the training session. The long term PS increase seems more important during the early phase of acquisition, while the short term effect is important as the learning curve approaches asymptote.
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rats, Psychophysiology, 6 (1970) 550-560. 2 CHERNIK,D. A., Effect of REM sleep deprivation on learning and recall by humans, Percept. Motor Skills, 34 (1972) 283-294. 3 FISHBEIN,W., Interference with conversion of memory from short term to long term storage by partial sleep deprivation, Comm. Behav. Biol., 5 (1970) 171-175. 4 FISHBEIN,W., Disruptive effects of rapid eye movement sleep deprivation on long term memory, Physiol. Behav., 6 (1971) 279-282. 5 FISHBEIN,W., MCGAUGH,J. L., AND SWARZ,J. R., Retrograde amnesia: electroconvulsive shock effectsafter termination of rapid eye movement sleep deprivation, Science, 172 (1971) 80-82.
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c. SMrFH cf o/.
6 HENNEV1N, E., LECONTE, P. ET BLOCH, V., Effet du niveau d'acquisition sur l'augmentation de la dur6e du sommeil paradoxal cons6cutive b_un conditionnement d'6vitement chez le rat, C.R. Acad. Sci. (Paris), 273 (1971) 2595-2598. 7 LECONTE, P. ET BLOCH, V., Effet de la privation de sommeil paradoxal sur I'acquisition de la retention d'un conditionnement chez le rat, J. Physiol. (Paris), 62 (1970) 290-291. 8 LECONTE, P., ET BLOCH, V., D6ficit de la retention d'un conditionnement apres privation de sommeil paradoxal chez le rat, C.R. Acad. Sci. (Paris), 271 (1970) 226-229. 9 LECONTE, P., ET HENNEVIN, E., Augmentation de la dur6e de 'sommeil paradoxal' cons6cutive/t un apprentissage chez le rat, C.R. Acad. Sci. (Paris), 273 (1971) 86-88. 10 LECONTE, P., ET HENNEVIN, E., Sommeil paradoxal et memorisation: effet du d61ai d'endormissement chez le rat, J. Physiol. (Paris), 65 (1972) 255-256. ll LUCERO, M. A., Lengthening of REM sleep duration consecutive to learning in the rat, Brain Research, 20 (1970) 319-322. 12 MILLER, L., DREW, W. G., AND SCHWARTZ, I., Effect of REM sleep deprivation on retention of a one trial passive avoidance response, Percept. Motor Skills, 33 (1971) 118. 13 MYERS, J. L., Fundamentals of Experimental Design, Allyn and Bacon, Boston, Mass., 1972. 14 PEARLMAN, C. A., Latent learning impaired by R E M sleep deprivation, Psychon. Sci., 25 (1971) 135-136. 15 SMITH, C. T., K1TAHAMA, K., VALATX,J. L., ET JOUVET, M., Sommeil paradoxal et apprentissage chez deux souches consanguines de souris, C.R. Acad. Sci. (Paris), 275 (1972) 1283-1286. 16 STERN, W. C., Acquisition impairments following rapid eye movement sleep deprivation in rats, Physiol. Behav., 7 (1971) 345-352. 17 WOLFOWrrz, B. E., AND HOLDSTOCK, T. L., Paradoxical sleep deprivation and memory in rats, Comm. Behav. Biol., 6 (1971) 281-284.