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Paradoxical sleep rebound in the rat. Effects of physical procedures involved in intracisternal injection
BRAIN RESEARCH
501
P A R A D O X I C A L SLEEP R E B O U N D IN T H E RAT. E F F E C T S OF PHYSICAL P R O C E D U R E S INVOLVED IN I N T R A C I S T E R N A L I N J E C T I O N
J. MOURET, J. F. PUJOL AND S. KIYONO* Laboratoire de Mddecine Expdrimentale, Facultd de Mddecine, Lyon, and Unitd de Neuropharmacologie Biochimique, Coll~ge de France, Paris (France)
(Accepted April 20th, 1969)
INTRODUCTION It is useful in both neurophysiology and neurochemistry to obtain, through a physiological procedure, an increase in the phenomenon to be studied. This is so with paradoxical sleep (PS) the deprivation of which is followed by an important rebound in this state of sleep. As previously described in cats and rats 1,8, this deprivation can be achieved by placing the animals upon small supports surrounded by water. This situation does not allow the total relaxation of the body musculature which is one of the main features of PS. We have recently shown that an increased turnover of central norepinephrine was selectively associated with the period of PS rebound in ratsL This last study had been performed under continuous polygraphic records and stressed the importance of such controls when a correlation between functional states of the central nervous system and central metabolism of biogenic amines is investigated. In this paper, we shall describe extensively the neurophysiological influences of the recording conditions during the deprivation period on the PS rebound, and its modifications by the physical procedures, light ether anaesthesia, incision of the neck teguments and intracisternal injection of labelled norepinephrine involved in the previously reported metabolic studies. MATERIALAND METHODS Charles River rats (250-280 g) were operated under Nembutal anaesthesia (65 mg/kg). Four cortical electrodes made of stainless steel wire were placed on the frontal and parieto-occipital cortices symmetrically along the longitudinal suture. Two flexible electrodes were placed in the neck muscles, all the electrodes being sealed to a connector placed on the head of the rats and fastened by acrylic dental cement. * Present address: Department of Neurophysiology, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka, Japan. Brain Research, 15 (1969) 501-506
J. MOURETet al.
502 TABLE I
(PS) AND SLOW WAVE SLEEP ( S W S ) D U R I N G THE DEPRIVATION PERIOD Expressed as percentage of control values 4- SE (PS/RT = 8.5 ~ 2.5; SWS/RT -- 47.8 ± 6.3). RT, recording time. PARADOXICAL SLEEP
GroupI GroupII
PS
SWS
0 25.0 ± 15.0
58.1 ± 8.1 56.1± 16.9
TABLE II P S AND S W S EXPRESSED AS PERCENTAGE OF RECORDING TIME
(RT, 285 rain) D U R I N G
THE RECUPERATION
PERIOD, IN RATS RECORDED D U R I N G BOTH DEPRIVATION AND RECUPERATION, W I T H O U T ANY MANIPULATION
Group I Group II
PS/RT
SWS/RT
24.3 ± 5.2 17.5 ± 8.0
42.6 ± 10.2 50.3 4- 14.5
After at least 3 days of recovery from the surgery, the rats were continuously recorded (24 h a day) in single cages during 3 days or more, in order to secure habituation to the recording conditions. They were then used for the deprivation experiments. This deprivation was performed in metallic tanks (50 cm × 50 cm × 50 cm), the rats being placed upon supports surrounded by water (10 cm deep) with food and water ad libitum. Two types of support were used: small supports (4.5 cm diameter), the size of which does not allow total relaxation of the body musculature (Group 1); and larger supports (11.5 cm diameter), upon which the animals were expected to perform PS when used as stress control (Group II). The deprivation started at 3 p.m. and was 91 h long. At the end of this period the rats were removed from the tanks and injected intracisternally with labelled norepinephrine (20 #1). The total duration of the different manipulations involved in this injection, including the placement of rats in single cages for recuperation, never exceeded 15 min. Since the rats were killed 5 h (300 min) after the end of the deprivation, the study of the recuperation lasted 285 min. In order to obtain baseline records after such a deprivation, some rats were allowed to recuperate without any manipulation. RESULTS
Rats recorded during both privation and recuperation periods without any manipulation Twenty rats of Group I and t2 rats of Group II were recorded continuously throughout both periods. During the 15 min usually used for the intracisternal injection, they were kept awake by moving their cages.
Brain Research, 15 (1969) 501-506
PARADOXICAL SLEEP REBOUND IN THE RAT
503
TABLE III P S AND S W S EXPRESSED AS PERCENTAGES OF RECORDING TIME
(RT, 285 min) D U R I N G THE RECUPERATION
PERIOD~ IN RATS RECORDED ONLY D U R I N G THE RECUPERATION~ W I T H O U T ANY MANIPULATION
GroupI Group II
PS/RT
SWS/RT
25.6 ± 4.3 10.I ± 2.2
40.2 ± 8.7 60.2 ± 12.0
The results set out in Table I show that no rat of Group I had been able to perform PS in tanks, whereas the rats of G r o u p I I exhibited PS though in a reduced amount and with wide individual variations. In both groups there was a deprivation in slow wave sleep (SWS) which was lowered to about 50 ~ of control values, regardless to the experimental group. During the recuperation period (Table II), both groups showed a rebound in PS, far less marked in G r o u p I I than in G r o u p I, and with wide individual variations in G r o u p II.
Rats recorded only during the recuperation period without any manipulation The results in Table I I I are interesting when compared with the results of the recuperation period in Table II: while the rebound in PS of the rats of G r o u p I remain about the same, this rebound was totally absent from the rats of Group II. Evidently these rats without rebound effect were not deprived of PS while in the water tanks, whereas the rats of G r o u p I, with the same rebound, whether recorded or not during the deprivation period, were deprived of PS.
Effect of intracisternal injection of labelled norepinephrine and correlated manipulations upon PS rebound At the end of the deprivation the rats were quickly anaesthetized with ether till complete relaxation of muscular tone. The intracisternal injection was then performed through an incision of the skin of the neck. After closure of the incision with surgical clamps, the rats were placed in single cages and recorded (12 rats). As shown in Table IV, these manipulations led to a marked reduction of the amount of PS during the 285 rain of the recuperation period when the rebound was TABLE IV EFFECT OF MANIPULATIONS AND INTRACISTERNAL INJECTION ON P S AND S W S EXPRESSED AS PERCENTAGE OF RECORDING TIME I N RATS RECORDED ONLY D U R I N G THE RECUPERATION PERIOD
GroupI Group 1I
PS/RT
SWS/RT
11.7 ± 8.3 1.5 -4-2.7
45.0 ± 7.2 36.2 ± 16.9
Brain Research, 15 (1969) 501-506
504
J. MOURETet al.
TABLE V EFFECT OF EACH MANIPULATION ON THE P S TIME D U R I N G THE RECUPERATION
Results expressed as percentage of control (non-deprived animals without manipulation). Results obtained from 5 rats in each group.
Non-deprived Group i
Without manipulation
Ether
Ether ÷ incision
Ether q- incision + injection
100 263.9
69.0 208.8
26.8 147.4
1.0 120.0
TABLE VI MODIFICATION OF THE DELAY OF OCCURRENCE OF P S AFTER EACH MANIPULATION~ EXPRESSED IN MINUTES
(average values of 5 rats) Without man@ulat~n
Non-deprived Group I
32
Ether
Ether÷inc&~n
Ether+mc&~n+ inject~n
123 71
176 74
323 -E 30 138
studied. This inhibitory effect is particularly obvious on the rats of Group II with a PS even decreased as compared with control values. The considerable effect of the whole manipulation led us to try to determine the possible role of each of the manipulations involved in the overall procedures. These tests were performed on groups of 5 control rats and 5 rats of Group I. The results given in Table V show a progressive reduction in the amount of PS with each factor. This led us to wonder whether the different steps of the intracisternal injection, and the intracisternal injection itself, were able to modify the PS rebound and thus to affect its mechanisms. As shown in Table VI, the most important effect of the manipulations was to increase the delay of occurrence of the first PS phase, this effect being much more striking in the control animals. According to these results it is possible to define the 'True Recuperation Time' as the duration between the beginning of the first PS phase and the end of the 285-rain period, and then to express PS as percentage of this true recuperation time. These statistics, given in Table VII, show that this ratio in the deprived animals remains about the same after each separate or associated manipulation. This ratio remained the same in the animals allowed to recuperate without any manipulation and in the animals having received the physical procedures. Brain Research, 15 (1969) 501-506
PARADOXICAL SLEEP REBOUND IN THE RAT
505
TABLE VII RATIO OF P S TO ~TRUE RECUPERATION TIME ~
Average values for 5 rats. Without manipulation
Non-deprived Group I
29.2
Ether
Ether ÷ incision Ether + incision + injection
10.9 29.4
6.6 19.4
26.5
DISCUSSION
Effects o f the recording conditions
Since it has been shown 3 that, to some extent, the rebound is proportional to the deprivation, our experiments stress the importance of the recording conditions in the deprivation. This is fairly well exhibited by the differences in rebound in the rats of G r o u p II. The deprivation observed and proved by the rebound when the animals of this group were recorded was no longer observed when the deprivation was performed without records. Since there is no rebound effect, the deprivation in this group was due to the summation of different factors: the size of the supports and the weight of the recording cable. On the contrary, the rats of Group I showed the same rebound effect with or without recording cables. This means that the deprivation was due only to the size of the supports. That is why, in further experiments, it was possible to study the animals only during the recuperation period, without records during the deprivation, and to consider the animals of G r o u p II as stress and dampness controls. It is of interest that the relatively small size of the larger supports allowed enough space for the animals to perform PS, thus showing that one must be very careful when using supports more than 5 cm in diameter in this kind of deprivation experiment. Effects o f the intracisternal injection
According to our results, and since the ratio of PS expressed as percentage of the true recuperation time remains unmodified by the intracisternal injection, one may state that the mechanism of PS rebound is unaffected by such a non-physiological-like procedure acting only on the delay of occurrence of PS. This effect on the delay of occurrence of the rebound seems to be due, without any specific relation, to the nociceptive stimuli involved in the intracisternal injection. When the effects are compared of the manipulations on deprived and control animals the 'need' in PS becomes even more evident, since the procedures of the injection hardly allowed the control animals to perform PS within the 285-rain period. It also shows that the nociceptive effects of the manipulations are of less importance for the deprived animals. Brain Research, 15 (1969) 501-506
506
J. MOURET et al.
SUMMARY
The effects o f recording conditions during paradoxical sleep deprivation in water tanks have been studied in rats placed either on small supports or on larger areas. The PS deprivation, judged on the r e b o u n d in this stage of sleep, is a function o f the recording conditions in the group o f rats placed on the larger supports. The physical procedures involved in the intracisternal injection have been tested and shown not to affect the intensity o f PS rebound. ACKNOWLEDGEMENTS
The help o f Mrs. J. Coindet is gratefully acknowledged. The w o r k was supported partly by D R M E (Grant 143/68), I.N.S.E.R.M. and by U.S.A.F. ( E O A R ) , G r a n t 68-0039.
REFERENCES I MORDEN,B., MITCHELL,G., ANDDEMENT,W., Selective REM sleep deprivation and compensation phenomena in the rat, Brain Research, 5 (1967) 339-349. 2 PLrJOL,J.-F., MOURET,J., JOUVET, M., AND GLOWINSKI,J., Increased turnover of cerebral norepinephrine during rebound of paradoxical sleep in the rat, Science, 159 (1968) 112-114. 3 VIMONT-VICARY,P., JOUVET-MOUNIER,D., ET DELORME,F., Effets EEG et comportementaux des privations de sommeil paradoxal chez le chat, Electroenceph. clin. NeurophysioL, 20 (1966) 439-449.
Brain Research, 15 (1969) 501-506
501
P A R A D O X I C A L SLEEP R E B O U N D IN T H E RAT. E F F E C T S OF PHYSICAL P R O C E D U R E S INVOLVED IN I N T R A C I S T E R N A L I N J E C T I O N
J. MOURET, J. F. PUJOL AND S. KIYONO* Laboratoire de Mddecine Expdrimentale, Facultd de Mddecine, Lyon, and Unitd de Neuropharmacologie Biochimique, Coll~ge de France, Paris (France)
(Accepted April 20th, 1969)
INTRODUCTION It is useful in both neurophysiology and neurochemistry to obtain, through a physiological procedure, an increase in the phenomenon to be studied. This is so with paradoxical sleep (PS) the deprivation of which is followed by an important rebound in this state of sleep. As previously described in cats and rats 1,8, this deprivation can be achieved by placing the animals upon small supports surrounded by water. This situation does not allow the total relaxation of the body musculature which is one of the main features of PS. We have recently shown that an increased turnover of central norepinephrine was selectively associated with the period of PS rebound in ratsL This last study had been performed under continuous polygraphic records and stressed the importance of such controls when a correlation between functional states of the central nervous system and central metabolism of biogenic amines is investigated. In this paper, we shall describe extensively the neurophysiological influences of the recording conditions during the deprivation period on the PS rebound, and its modifications by the physical procedures, light ether anaesthesia, incision of the neck teguments and intracisternal injection of labelled norepinephrine involved in the previously reported metabolic studies. MATERIALAND METHODS Charles River rats (250-280 g) were operated under Nembutal anaesthesia (65 mg/kg). Four cortical electrodes made of stainless steel wire were placed on the frontal and parieto-occipital cortices symmetrically along the longitudinal suture. Two flexible electrodes were placed in the neck muscles, all the electrodes being sealed to a connector placed on the head of the rats and fastened by acrylic dental cement. * Present address: Department of Neurophysiology, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka, Japan. Brain Research, 15 (1969) 501-506
J. MOURETet al.
502 TABLE I
(PS) AND SLOW WAVE SLEEP ( S W S ) D U R I N G THE DEPRIVATION PERIOD Expressed as percentage of control values 4- SE (PS/RT = 8.5 ~ 2.5; SWS/RT -- 47.8 ± 6.3). RT, recording time. PARADOXICAL SLEEP
GroupI GroupII
PS
SWS
0 25.0 ± 15.0
58.1 ± 8.1 56.1± 16.9
TABLE II P S AND S W S EXPRESSED AS PERCENTAGE OF RECORDING TIME
(RT, 285 rain) D U R I N G
THE RECUPERATION
PERIOD, IN RATS RECORDED D U R I N G BOTH DEPRIVATION AND RECUPERATION, W I T H O U T ANY MANIPULATION
Group I Group II
PS/RT
SWS/RT
24.3 ± 5.2 17.5 ± 8.0
42.6 ± 10.2 50.3 4- 14.5
After at least 3 days of recovery from the surgery, the rats were continuously recorded (24 h a day) in single cages during 3 days or more, in order to secure habituation to the recording conditions. They were then used for the deprivation experiments. This deprivation was performed in metallic tanks (50 cm × 50 cm × 50 cm), the rats being placed upon supports surrounded by water (10 cm deep) with food and water ad libitum. Two types of support were used: small supports (4.5 cm diameter), the size of which does not allow total relaxation of the body musculature (Group 1); and larger supports (11.5 cm diameter), upon which the animals were expected to perform PS when used as stress control (Group II). The deprivation started at 3 p.m. and was 91 h long. At the end of this period the rats were removed from the tanks and injected intracisternally with labelled norepinephrine (20 #1). The total duration of the different manipulations involved in this injection, including the placement of rats in single cages for recuperation, never exceeded 15 min. Since the rats were killed 5 h (300 min) after the end of the deprivation, the study of the recuperation lasted 285 min. In order to obtain baseline records after such a deprivation, some rats were allowed to recuperate without any manipulation. RESULTS
Rats recorded during both privation and recuperation periods without any manipulation Twenty rats of Group I and t2 rats of Group II were recorded continuously throughout both periods. During the 15 min usually used for the intracisternal injection, they were kept awake by moving their cages.
Brain Research, 15 (1969) 501-506
PARADOXICAL SLEEP REBOUND IN THE RAT
503
TABLE III P S AND S W S EXPRESSED AS PERCENTAGES OF RECORDING TIME
(RT, 285 min) D U R I N G THE RECUPERATION
PERIOD~ IN RATS RECORDED ONLY D U R I N G THE RECUPERATION~ W I T H O U T ANY MANIPULATION
GroupI Group II
PS/RT
SWS/RT
25.6 ± 4.3 10.I ± 2.2
40.2 ± 8.7 60.2 ± 12.0
The results set out in Table I show that no rat of Group I had been able to perform PS in tanks, whereas the rats of G r o u p I I exhibited PS though in a reduced amount and with wide individual variations. In both groups there was a deprivation in slow wave sleep (SWS) which was lowered to about 50 ~ of control values, regardless to the experimental group. During the recuperation period (Table II), both groups showed a rebound in PS, far less marked in G r o u p I I than in G r o u p I, and with wide individual variations in G r o u p II.
Rats recorded only during the recuperation period without any manipulation The results in Table I I I are interesting when compared with the results of the recuperation period in Table II: while the rebound in PS of the rats of G r o u p I remain about the same, this rebound was totally absent from the rats of Group II. Evidently these rats without rebound effect were not deprived of PS while in the water tanks, whereas the rats of G r o u p I, with the same rebound, whether recorded or not during the deprivation period, were deprived of PS.
Effect of intracisternal injection of labelled norepinephrine and correlated manipulations upon PS rebound At the end of the deprivation the rats were quickly anaesthetized with ether till complete relaxation of muscular tone. The intracisternal injection was then performed through an incision of the skin of the neck. After closure of the incision with surgical clamps, the rats were placed in single cages and recorded (12 rats). As shown in Table IV, these manipulations led to a marked reduction of the amount of PS during the 285 rain of the recuperation period when the rebound was TABLE IV EFFECT OF MANIPULATIONS AND INTRACISTERNAL INJECTION ON P S AND S W S EXPRESSED AS PERCENTAGE OF RECORDING TIME I N RATS RECORDED ONLY D U R I N G THE RECUPERATION PERIOD
GroupI Group 1I
PS/RT
SWS/RT
11.7 ± 8.3 1.5 -4-2.7
45.0 ± 7.2 36.2 ± 16.9
Brain Research, 15 (1969) 501-506
504
J. MOURETet al.
TABLE V EFFECT OF EACH MANIPULATION ON THE P S TIME D U R I N G THE RECUPERATION
Results expressed as percentage of control (non-deprived animals without manipulation). Results obtained from 5 rats in each group.
Non-deprived Group i
Without manipulation
Ether
Ether ÷ incision
Ether q- incision + injection
100 263.9
69.0 208.8
26.8 147.4
1.0 120.0
TABLE VI MODIFICATION OF THE DELAY OF OCCURRENCE OF P S AFTER EACH MANIPULATION~ EXPRESSED IN MINUTES
(average values of 5 rats) Without man@ulat~n
Non-deprived Group I
32
Ether
Ether÷inc&~n
Ether+mc&~n+ inject~n
123 71
176 74
323 -E 30 138
studied. This inhibitory effect is particularly obvious on the rats of Group II with a PS even decreased as compared with control values. The considerable effect of the whole manipulation led us to try to determine the possible role of each of the manipulations involved in the overall procedures. These tests were performed on groups of 5 control rats and 5 rats of Group I. The results given in Table V show a progressive reduction in the amount of PS with each factor. This led us to wonder whether the different steps of the intracisternal injection, and the intracisternal injection itself, were able to modify the PS rebound and thus to affect its mechanisms. As shown in Table VI, the most important effect of the manipulations was to increase the delay of occurrence of the first PS phase, this effect being much more striking in the control animals. According to these results it is possible to define the 'True Recuperation Time' as the duration between the beginning of the first PS phase and the end of the 285-rain period, and then to express PS as percentage of this true recuperation time. These statistics, given in Table VII, show that this ratio in the deprived animals remains about the same after each separate or associated manipulation. This ratio remained the same in the animals allowed to recuperate without any manipulation and in the animals having received the physical procedures. Brain Research, 15 (1969) 501-506
PARADOXICAL SLEEP REBOUND IN THE RAT
505
TABLE VII RATIO OF P S TO ~TRUE RECUPERATION TIME ~
Average values for 5 rats. Without manipulation
Non-deprived Group I
29.2
Ether
Ether ÷ incision Ether + incision + injection
10.9 29.4
6.6 19.4
26.5
DISCUSSION
Effects o f the recording conditions
Since it has been shown 3 that, to some extent, the rebound is proportional to the deprivation, our experiments stress the importance of the recording conditions in the deprivation. This is fairly well exhibited by the differences in rebound in the rats of G r o u p II. The deprivation observed and proved by the rebound when the animals of this group were recorded was no longer observed when the deprivation was performed without records. Since there is no rebound effect, the deprivation in this group was due to the summation of different factors: the size of the supports and the weight of the recording cable. On the contrary, the rats of Group I showed the same rebound effect with or without recording cables. This means that the deprivation was due only to the size of the supports. That is why, in further experiments, it was possible to study the animals only during the recuperation period, without records during the deprivation, and to consider the animals of G r o u p II as stress and dampness controls. It is of interest that the relatively small size of the larger supports allowed enough space for the animals to perform PS, thus showing that one must be very careful when using supports more than 5 cm in diameter in this kind of deprivation experiment. Effects o f the intracisternal injection
According to our results, and since the ratio of PS expressed as percentage of the true recuperation time remains unmodified by the intracisternal injection, one may state that the mechanism of PS rebound is unaffected by such a non-physiological-like procedure acting only on the delay of occurrence of PS. This effect on the delay of occurrence of the rebound seems to be due, without any specific relation, to the nociceptive stimuli involved in the intracisternal injection. When the effects are compared of the manipulations on deprived and control animals the 'need' in PS becomes even more evident, since the procedures of the injection hardly allowed the control animals to perform PS within the 285-rain period. It also shows that the nociceptive effects of the manipulations are of less importance for the deprived animals. Brain Research, 15 (1969) 501-506
506
J. MOURET et al.
SUMMARY
The effects o f recording conditions during paradoxical sleep deprivation in water tanks have been studied in rats placed either on small supports or on larger areas. The PS deprivation, judged on the r e b o u n d in this stage of sleep, is a function o f the recording conditions in the group o f rats placed on the larger supports. The physical procedures involved in the intracisternal injection have been tested and shown not to affect the intensity o f PS rebound. ACKNOWLEDGEMENTS
The help o f Mrs. J. Coindet is gratefully acknowledged. The w o r k was supported partly by D R M E (Grant 143/68), I.N.S.E.R.M. and by U.S.A.F. ( E O A R ) , G r a n t 68-0039.
REFERENCES I MORDEN,B., MITCHELL,G., ANDDEMENT,W., Selective REM sleep deprivation and compensation phenomena in the rat, Brain Research, 5 (1967) 339-349. 2 PLrJOL,J.-F., MOURET,J., JOUVET, M., AND GLOWINSKI,J., Increased turnover of cerebral norepinephrine during rebound of paradoxical sleep in the rat, Science, 159 (1968) 112-114. 3 VIMONT-VICARY,P., JOUVET-MOUNIER,D., ET DELORME,F., Effets EEG et comportementaux des privations de sommeil paradoxal chez le chat, Electroenceph. clin. NeurophysioL, 20 (1966) 439-449.
Brain Research, 15 (1969) 501-506