Physiology& Behavior,Vol. 50, pp. 79-82. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .00
Behavioural Characteristics of Sleep in Rats Under Different Light/Dark Conditions J. N. F. VAN B E T ' I ' E R A Y , J. M. H. V O S S E N A N D A. M. L. C O E N E N
Department of Psychology, University of Nijmegen, P.O. Box 9104, 6500 HE Nijmegen, The Netherlands R e c e i v e d 19 July 1990 VAr~BETI'ERAY, J. N. F., J. M. H. VOSSEN AND A. M. L. COENEN. Behavioural characteristics of sleep in rats under different light~dark conditions. PHYSIOL BEHAV 50(1) 79-82, 1991.--In the light, rats tend to sleep with curled-up body and closed eyes, while in the dark they tend to sleep more stretched out and often with open eyes. These differences in posture may be caused by the differences in light intensity or by a diurnal rhythm. To study this, rats were provided with EEG and EMG electrodes for sleep classification and two housing conditions were created. One group of rats was maintained on a light/twilight schedule, while another group lived on a twilight/dark schedule. This way the twilight situations, while having the same absolute light level, differ in meaning because of their relative light level. Several behavioural sleep characteristics of the rat were studied, such as body posture, eye closure, position of the rat in the cage and wall contact. The results show that body posture and wall contact are influenced by the absolute light intensity, while eye closure and probably cage position are related to the circadian sleep-wake cycle which itself shows a diurnal rhythm. Sleep
Sleep posture
Diurnal rhythm
Illumination level
Light/dark schedule
Rat
group of rats (group I) the normal dark phase is replaced by a twilight phase, whereas for a second group of rats (group II) the normal light phase is replaced by a similar twilight phase. This means that the same light intensity, the twilight, has the meaning of dark for the first group of rats and the meaning of light for the second group. Comparison of both groups with respect to their sleep characteristics in the twilight phase indicates whether the behavioural characteristics of sleep are influenced by the absolute light intensity or by a (diurnal) rhythm. In the first case, the sleep characteristics found in the two groups in the twilight will not differ, while in the second case differences will be found. Moreover, when light intensity controls the characteristics, in the twilight these will be intermediate between those occurring during the light and those occurring during the dark phase. If, however, a rhythm is the regulating factor, sleep characteristics during the twilight phase will tend to be equal to those of the phase they replace. Eye closure, body posture and position of the rat in the cage (henceforth referred to as cage position) can adopt different patterns in light and dark (3). Therefore, these parameters were studied. When it was found in a pilot experiment that whether or not an animal made contact with a wall of the cage might also differ with sleep phase, this was added to the behavioural parameters.
IN the study of the behavioural characteristics of sleep in the rat, attention must be paid to the influence of light intensity. Commonly, the light/dark schedule used in various laboratories does not reflect the natural day/night cycle for rats (2,8). Laboratory rats receive much more light than wild rats, which are mostly active in dim twilight and sleep in dark burrows. Fishman and Roffwarg (4,5) argue that rats, when given a free choice, spend almost all of their time in the dark. In studying REM sleep of rats under laboratory light/dark conditions, Coenen et al. (3) found that the behavioural characteristics of sleep at REM onset in the dark period of the light/ dark cycle often differed considerably from those in the light period. While in the light the rats usually sleep with curled-up body and closed eyes, sleep in the dark also occurs when rats are lying stretched out and often with open eyes (Fig. 1). After considering these findings Roffwarg (personal communication) suggested that: "the rat treats the light as noxious and wards it off with curled-up body and lid closure." This suggestion implies that sleep characteristics such as body posture and eye closure are under the influence of the intensity of the light. Furthermore, it implies that sleep characteristics in the dark must be regarded as the more natural. An alternative explanation of the differential sleep characteristics, however, is that there is a rhythm which controls sleep characteristics in relation to the time of day. Irrespective of the actual light intensity, the rat might show one kind of sleep behaviour during the rest period and another one during the active period. In the present experiment we tried to fred out whether behavloural sleep characteristics are under the influence of actual light intensity or under the influence of relative light intensity. This was investigated by manipulating the light/dark schedule of the animals. A situation was created in which for one
METHOD Male Wistar rats with an age of 6 to 8 months were provided with permanent cortical EEG and nuchal EMG electrodes. EEG electrodes were placed in area 3 and area 17 of Krieg (7) and a reference electrode was placed on the cerebellum. The animals had at least two weeks for recovery and habituation to the re-
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cording situation. One group of rats (n = 5) was maintained on a light/twilight regime with light (white light, 500-600 lux) on from 0300 till 1500 h and twilight (white light, 6-9 lux) on from 1500 till 0300 h. The second group ( n = 5 ) had twilight (white light, 6-9 lux) from 0300 h till 1500 h and dark (red light, 2-3 lux) from 1500 h till 0300 h. Habituation to the lighting schedules lasted at least six weeks. Animals were housed individually in transparent perspex cages (30 x 25 x 35). The floor of the cages was divided into squares which enabled objective position scoring. Mirrors permitted an all side view of the rats. Room temperature was 19.5 --- I°C. Based on EEG and EMG recordings and behaviour observations, a sleep classification was established, using conventional criteria (SWS has a low frequency, high amplitude EEG and a moderate/low EMG; the animals are quiet and are breathing regularly; REM sleep is characterized by a high frequency, low amplitude EEG and almost no activity in the EMG; there are often phasic phenomena like twitches of whiskers and breathing is irregular). Recordings were made for each rat in the "dark" (twilight for group I and dark for group II) as well as in the "light" period (light for group I and twilight in group II). A 90-minute period before and a similar period after the "dark" to "light" switch was covered by recordings. These 90-minute recordings were made from 1300 h till 1430 h and from 1530 h till 1600 h. In parallel, videorecordings of the rats were made. On the two audio-channels of the videorecorder, the transformed EEG and EMG signals were recorded. There was one observer. In accordance with Coenen et al. (3), observations were registered just before the onset of a REM sleep period. This provides fixed points in the sleep cycle whereby the animal has reached a certain sleep depth. At these points, eye closure, body posture, cage position and wall contact were scored. Scoring was verified by reviewing the videotapes together with the EEG's and EMG's. We determined a number of three observations per rat per period to be the minimum. The category "eye closure" had three subdivisions: closed, half open and open. In the category body posture, four subdivisions were made: "ball," when the rat is sleeping with its head tucked under the body; "curled-up," when the rat sleeps laying down nose-to-tail; "stretched-out," when the rat sleeps flat on its belly, with nose and tail as opposite poles; and " s i t , " when the rat sleeps sitting with the head lifted from the floor or only the nose touching it and the feet tucked under the body (Fig. 1). Cage position was scored in three categories: in the "comer" of the cage, along a "side," or in the "middle." In the category, wall contact, it was noted whether or not the rat was laying against one of the walls of the cage. RESULTS AND DISCUSSION
The results are presented in Fig 2. The number of observations considered are for group I: light, 22; twilight, 32; for group II: twilight, 23; dark, 20. With respect to the amount of eye closure in the twilight phases, significant differences were found (X2-test). The results show that when twilight represents the dark period, eyes are open more often than when twilight represents the light period (X2=5.76, p
0031-9384/91 $3.00 + .00
Behavioural Characteristics of Sleep in Rats Under Different Light/Dark Conditions J. N. F. VAN B E T ' I ' E R A Y , J. M. H. V O S S E N A N D A. M. L. C O E N E N
Department of Psychology, University of Nijmegen, P.O. Box 9104, 6500 HE Nijmegen, The Netherlands R e c e i v e d 19 July 1990 VAr~BETI'ERAY, J. N. F., J. M. H. VOSSEN AND A. M. L. COENEN. Behavioural characteristics of sleep in rats under different light~dark conditions. PHYSIOL BEHAV 50(1) 79-82, 1991.--In the light, rats tend to sleep with curled-up body and closed eyes, while in the dark they tend to sleep more stretched out and often with open eyes. These differences in posture may be caused by the differences in light intensity or by a diurnal rhythm. To study this, rats were provided with EEG and EMG electrodes for sleep classification and two housing conditions were created. One group of rats was maintained on a light/twilight schedule, while another group lived on a twilight/dark schedule. This way the twilight situations, while having the same absolute light level, differ in meaning because of their relative light level. Several behavioural sleep characteristics of the rat were studied, such as body posture, eye closure, position of the rat in the cage and wall contact. The results show that body posture and wall contact are influenced by the absolute light intensity, while eye closure and probably cage position are related to the circadian sleep-wake cycle which itself shows a diurnal rhythm. Sleep
Sleep posture
Diurnal rhythm
Illumination level
Light/dark schedule
Rat
group of rats (group I) the normal dark phase is replaced by a twilight phase, whereas for a second group of rats (group II) the normal light phase is replaced by a similar twilight phase. This means that the same light intensity, the twilight, has the meaning of dark for the first group of rats and the meaning of light for the second group. Comparison of both groups with respect to their sleep characteristics in the twilight phase indicates whether the behavioural characteristics of sleep are influenced by the absolute light intensity or by a (diurnal) rhythm. In the first case, the sleep characteristics found in the two groups in the twilight will not differ, while in the second case differences will be found. Moreover, when light intensity controls the characteristics, in the twilight these will be intermediate between those occurring during the light and those occurring during the dark phase. If, however, a rhythm is the regulating factor, sleep characteristics during the twilight phase will tend to be equal to those of the phase they replace. Eye closure, body posture and position of the rat in the cage (henceforth referred to as cage position) can adopt different patterns in light and dark (3). Therefore, these parameters were studied. When it was found in a pilot experiment that whether or not an animal made contact with a wall of the cage might also differ with sleep phase, this was added to the behavioural parameters.
IN the study of the behavioural characteristics of sleep in the rat, attention must be paid to the influence of light intensity. Commonly, the light/dark schedule used in various laboratories does not reflect the natural day/night cycle for rats (2,8). Laboratory rats receive much more light than wild rats, which are mostly active in dim twilight and sleep in dark burrows. Fishman and Roffwarg (4,5) argue that rats, when given a free choice, spend almost all of their time in the dark. In studying REM sleep of rats under laboratory light/dark conditions, Coenen et al. (3) found that the behavioural characteristics of sleep at REM onset in the dark period of the light/ dark cycle often differed considerably from those in the light period. While in the light the rats usually sleep with curled-up body and closed eyes, sleep in the dark also occurs when rats are lying stretched out and often with open eyes (Fig. 1). After considering these findings Roffwarg (personal communication) suggested that: "the rat treats the light as noxious and wards it off with curled-up body and lid closure." This suggestion implies that sleep characteristics such as body posture and eye closure are under the influence of the intensity of the light. Furthermore, it implies that sleep characteristics in the dark must be regarded as the more natural. An alternative explanation of the differential sleep characteristics, however, is that there is a rhythm which controls sleep characteristics in relation to the time of day. Irrespective of the actual light intensity, the rat might show one kind of sleep behaviour during the rest period and another one during the active period. In the present experiment we tried to fred out whether behavloural sleep characteristics are under the influence of actual light intensity or under the influence of relative light intensity. This was investigated by manipulating the light/dark schedule of the animals. A situation was created in which for one
METHOD Male Wistar rats with an age of 6 to 8 months were provided with permanent cortical EEG and nuchal EMG electrodes. EEG electrodes were placed in area 3 and area 17 of Krieg (7) and a reference electrode was placed on the cerebellum. The animals had at least two weeks for recovery and habituation to the re-
79
80
VAN BETrERAY, VOSSEN AND COENEN
cording situation. One group of rats (n = 5) was maintained on a light/twilight regime with light (white light, 500-600 lux) on from 0300 till 1500 h and twilight (white light, 6-9 lux) on from 1500 till 0300 h. The second group ( n = 5 ) had twilight (white light, 6-9 lux) from 0300 h till 1500 h and dark (red light, 2-3 lux) from 1500 h till 0300 h. Habituation to the lighting schedules lasted at least six weeks. Animals were housed individually in transparent perspex cages (30 x 25 x 35). The floor of the cages was divided into squares which enabled objective position scoring. Mirrors permitted an all side view of the rats. Room temperature was 19.5 --- I°C. Based on EEG and EMG recordings and behaviour observations, a sleep classification was established, using conventional criteria (SWS has a low frequency, high amplitude EEG and a moderate/low EMG; the animals are quiet and are breathing regularly; REM sleep is characterized by a high frequency, low amplitude EEG and almost no activity in the EMG; there are often phasic phenomena like twitches of whiskers and breathing is irregular). Recordings were made for each rat in the "dark" (twilight for group I and dark for group II) as well as in the "light" period (light for group I and twilight in group II). A 90-minute period before and a similar period after the "dark" to "light" switch was covered by recordings. These 90-minute recordings were made from 1300 h till 1430 h and from 1530 h till 1600 h. In parallel, videorecordings of the rats were made. On the two audio-channels of the videorecorder, the transformed EEG and EMG signals were recorded. There was one observer. In accordance with Coenen et al. (3), observations were registered just before the onset of a REM sleep period. This provides fixed points in the sleep cycle whereby the animal has reached a certain sleep depth. At these points, eye closure, body posture, cage position and wall contact were scored. Scoring was verified by reviewing the videotapes together with the EEG's and EMG's. We determined a number of three observations per rat per period to be the minimum. The category "eye closure" had three subdivisions: closed, half open and open. In the category body posture, four subdivisions were made: "ball," when the rat is sleeping with its head tucked under the body; "curled-up," when the rat sleeps laying down nose-to-tail; "stretched-out," when the rat sleeps flat on its belly, with nose and tail as opposite poles; and " s i t , " when the rat sleeps sitting with the head lifted from the floor or only the nose touching it and the feet tucked under the body (Fig. 1). Cage position was scored in three categories: in the "comer" of the cage, along a "side," or in the "middle." In the category, wall contact, it was noted whether or not the rat was laying against one of the walls of the cage. RESULTS AND DISCUSSION
The results are presented in Fig 2. The number of observations considered are for group I: light, 22; twilight, 32; for group II: twilight, 23; dark, 20. With respect to the amount of eye closure in the twilight phases, significant differences were found (X2-test). The results show that when twilight represents the dark period, eyes are open more often than when twilight represents the light period (X2=5.76, p
