The pendulum technique for paradoxical sleep deprivation in rats

Physiology&Behavior,Vol. 25, pp. 807-811. PergamonPress and BrainResearch Publ., 1980. Printedin the U.S.A.

The Pendulum Technique for Paradoxical Sleep Deprivation in Rats Z. J. M. V A N H U L Z E N A N D A. M. L. C O E N E N 1

Department of Comparative and Physiological Psychology, University of N(jmegen 1 Montessorilaan 3, 6525 GG, N(jmegen, The Netherlands R e c e i v e d 11 July 1980 VAN HULZEN, Z. J. M. AND A. M. L. COENEN. The pendulum technique lbr paradoxical sleep deprivation in rats. PHYSIOL. BEHAV. 25(6) 807-811, 1980.--A new technique for paradoxical sleep (PS) deprivation in rats is presented. Animals are prevented from entering into PS by allowing them to sleep for only brief periods of time. This is accomplished by an apparatus which moves the animals' cages backwards and forwards like a pendulum. At the extremes of the motion postural imbalance is produced in the animals forcing them to walk downwards to the other side of their cages. A minimal amount of PS and a moderate amount of slow wave sleep (SWS) were detected during a deprivation period of 72 hrs. Following the deprivation treatment the recovery of sleep was monitored for 3 hrs; at the beginning of the light period for one group and at the beginning of the dark period for a second group. The sleep-waking patterns of two baseline groups were established at the time when the recovery sleep was examined in the deprivation groups. The deprivation treatment resulted in a significant increase in the amount of PS and a significant decrease in the amount of SWS. The extent of PS increase was similar in both deprivation groups, in spite of a large difference in the amount of SWS. The decrease of SWS mainly occurred during recovery sleep in the light. It was observed that sleep in the dark differs from sleep in the light in behavioural aspects. Paradoxical sleep

Paradoxical sleep deprivation

TWO instrumental techniques are usually employed for depriving animals of paradoxical sleep (PS). The most obvious one is the "arousal" technique, an adaptation of the technique originally used by Dement [4] in human subjects. It consists of arousing animals from sleep at the onset of each PS episode, as identified from electrophysiological indices. As soon as PS emerges an external stimulus is presented to awaken the animal. Immediately after awakening the animal is allowed to resume its sleep. Non-specific concomitants of the technique are reasonably controlled by a " y o k e d " control animal, which is aroused concurrently with the experimental animal [17]. Initially this technique effectively prevents an animal from entering into PS. However, the frequency of awakening increases rapidly, making this technique difficult to perform for more than a few hours [12]. A popular technique for long-term PS deprivation is the "watertank" technique, first described by Jouvet and her colleagues [9] in cats. Animals are maintained on small platforms, typically inverted flowerpots, surrounded by water. On this artificial island they can obtain slow wave sleep (SWS) by sitting or crouching, whereas the occurrence of PS is restricted. As the animals enter into PS the muscular tone diminishes causing them to touch the water and awaken. This technique is procedurally simple and allows a large number of animals to be deprived simultaneously. Information regarding the efficacy of the technique is provided by a few validation studies in rats [7, 11, 13]. These studies indi-

cate that the degree of PS deprivation may vary considerably, for which the diameter of the platform relative to the size of the animal seems to be mainly responsible [8, 1l, 14]. A large platform enabling the animal to curl up and have both SWS and PS without contacting the water is commonly used as control for the confoundings of the technique. Although this control may be appropriate for most of the confoundings [18], the watertank technique is still controversial in regard to its behavioural consequences. Additional controls may be tried for this technique [3,15]; a second approach is to develop an alternative technique of PS deprivation. The PS deprivation technique presented here is based on the observation that the occurrence of PS is generally preceded by SWS. Animals are allowed to have brief periods of sleep entirely composed of SWS and are awakened before PS can ensue. This is accomplished by swinging their cages backwards and forwards in a way that produces postural imbalance in the animals at regular intervals.

METHOD

Animals Adult male albino rats from the WU (SPF63 Cpb) strain [10] served as subjects. The animals were housed individually in standard laboratory cages with food and water freely available. They were maintained in a temperature controlled

1The department has changed its address from Erasmuslaan 16 to Montessorilaan 3.

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808 room (20-22°Cl on a 12:12 hr light-dark cycle: white lights (500-600 lux) on at 09:30 and dim red lights (2-3 lux) on at 21:30 hr. Rats weighing 310 to 365 g were chronically inaplanted with a tripolar electrode set (Plastic Products Company, MS333/2-A) and a bipolar electrode set (MS303/71) for recording hippocampal E E G and nuchal EMG respectively. The three EEG electrodes were positioned in a coronal plane; two were aimed at the dorsal hippocampus, the third one served as an earth electrode. The tips of the target electrodes were spaced approximately 1.2 mm apart. The stereotaxic coordinates, with the dorsal surface of the skull horizontal, were 3.5 mm posterior to the bregma, 3.1 mm lateral to the midline and 2.5 mm ventral to the dura. The two EMG electrodes were subcutaneously placed on the dorsal neck muscles. Both electrode sets were embedded in dental cement and anchored to the skull with stainless-steel screws. Animals were allowed to recover from the operation for at least one week. They were handled daily prior to the experiment, except for the week-ends and the post-operation recovery period. Twenty-four rats showing characteristic EEG and EMG patterns were selected for the experiment.

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FIG. 1. The pendulum technique for paradoxical sleep (PS) deprivation in rats.

Apparatu.s The apparatus used for PS deprivation is pictured in Fig. 1. It consists of a swing with room for three rats in individual Perspex recording cages (30x25x35 cm), which is supported by a frame. By slowly and continuously moving the swing backwards and forwards, the orientation of the cages is changed accordingly. At the extremes of the motion the cages are tilted 42 degrees from the horizontal plane. The distance from the cages' floor to the axis of rotation is 3t.5 cm. Counterweights are used to balance the cages. The pendulum is moved by a DC motor, the speed of which can be set deliberately at different rates. The motor is mounted in a sound-insulated enclosure. Postural imbalance is produced in the animals when the cages are at the extremes of the motion. In maintaining their balance rats may stay on the tilted side of their cages with some effort. However, they quickly learn to avoid the point of imbalance by walking downwards to the other side of their cages. To take care that the rats reach the other side by walking rather than by slipping, the cages are provided with a rough bottom covered with a layer of sawdust. Climbing out by the rats is precluded by a Perspex lid. The lid is equipped with a shaft to permit passage of the recording leads. An assembly of a slipring and a spring is used to suspend the recording leads, which enables the animals to move about. Food is provided from a rack within easy reach of the animal and water is continuously available from a drinking tube with a non-drip spout.

Procedure Prior to the deprivation procedure, animals were familiarized with the recording conditions. They were put into recording cages and connected to adaptation leads for at least one week. After being placed in a stationary pendulum and attached to the recording leads for one to two days, the deprivation treatment was initiated. Animals, divided into two groups (n =6), were submitted to a deprivation period of 72 hrs. For one group of rats deprivation started at the onset of the light phase (09:30 hr), and for the second group at the onset of the dark phase (21:30 hr) of the illumination cycle. This arrangement made it possible to examine the recovery

of sleep after deprivation at the beginning of the light period (Group 1) or at the beginning of the dark period (Group 2). The speed of the pendulum was adjusted to yield sufficient PS deprivation in the animals. For both groups time between extremes was set each 12 hr-period according to the following schedule: 45, 35, 25, 20, 17 and 15 sec. Following completion of both deprivation conditions two baseline conditions (n--6) were conducted. Sleep-waking patterns were established in Group 3 and 4 at the time when the recovery sleep was monitored in the first and second group respectively. Animals were weighed immediately before the deprivation period and after termination of the recovery period. In validating the technique recordings of the hippocampal EEG and nuchal EMG and behavioural observations were made during the deprivation period. Because it would require an impractical amount of labour to continuously observe the rats throughout the deprivation period, samples of 10 min per hr were taken during the light portion of the diurnal cycle. Animals' behaviour in the moving pendulum was coded into one of three categories: (1) active movement with eyes open, (2) immobility or passive movement with eyes open, and (3) immobility or passive movement with eyes closed. The third category was taken as a behavioural index of sleep. Immediately following the deprivation treatment the recovery sleep was monitored for 3 hrs. Rats' behaviour was classified into sleep or wakefulness. Onset of sleep was indicated behaviourally by differential criteria for the light and the dark period: immobility with eyes closed, and immobility with eyes not wide open, respectively. Rats were closely observed through a window from an adjacent room. The behavioural codes were recorded on a polygraph (Mingograph-800, Elema-Schrnander) along with the hippocampal E E G and nuchai EMG signals. The recordings were evaluated by visual inspection. The sleep stages quantified were PS and SWS. Resolution of finer stage distinctions on the basis of the hippocampal E E G was felt to be difficult for a visual scorer. PS and SWS were scored for hippocampal theta-rhythm and hippocampal L I A (Large amplitude Irregular Activity) respectively during behavioural sleep. Periods of PS lasting for less than 3 sec were disregarded.

PARADOXICAL SLEEP DEPRIVATION

809 TABLE 1

MEANS AND STANDARD DEVIATIONS OF THE AMOUNTS OF PS AND SWS FOR THE FOUR TREATMENT GROUPS Recovery light

Recovery dark

Baseline light

Baseline dark

Amount of PS (sec)

3334 ± 379

3330 ± 731

1068 ± 307

640 ± 433

Amount of SWS

4546 ± 948

990 ± 663

7007 ± 140

1208 ± 704

(sec)

RESULTS AND DISCUSSION

1) and in the dark (Group 2), together with the baseline sleep in the light (Group 3) and in the dark (Group 4). The mean amounts of PS and SWS for the treatment groups are shown in Table 1; the data represent average values over the entire 3 hr-recording period. A two-way analysis of variance [19] was performed on each measure to test for the main effects of Deprivation treatment and Time-of-day. Applied to the amount of PS, the results of the analysis revealed a significant main effect for Deprivation treatment, F(1,20)= 153.60, p <0.001, no significant Time-of-day effect and no significant

In studying the behavioural consequences of long-term PS deprivation, the sleep-waking activity which forms the background of behavioural testing has to be taken into account. Therefore, in this evaluation study emphasis was placed on the changes in sleep-waking patterns occurring during the first few hrs after PS deprivation. The recovery of sleep was examined at the beginning of the light and the dark period, in reference to the characteristic sleep-waking patterns. Figure 2 depicts the recovery sleep in the light (Group

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FIG. 2. The recovery sleep after 72 hrs of PS deprivation at the beginning of the light and the dark phase of the illumination cycle, and the baseline sleep during the same portions of the illumination cycle. The mean amounts of PS (black area) and SWS (dotted area), expressed as percentage of total time, are plotted for successive 15 min periods.

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FIG. 3. A transition from wakefulness into PS without intervening SWS, sometimes occurring in the rats during their recovery sleep in the dark.

Deprivation treatment by Time-of-day interaction effect. These results indicate that the amount of PS was increased to the same extent in the recovery-light and the recovery-dark group. With respect to the amount of SWS, the analysis showed significant main effects for Deprivation treatment, F(1,20)=23.24, p<0.001 and Time-of-day, F(1,20)=283.28, p<0.001, and a significant interaction effect, F(1,20)= 16.30, p<0.001. The simple main effects of the factor Deprivation treatment were analysed by performing Scheff6 post hoc comparisons (c~=0.05). The amount of SWS was significantly decreased in the recovery-light group, and not significantly changed in the recovery-dark group, indicating that the first group was mainly responsible for the overall effect of Deprivation treatment. A similar increase in the amount of PS has occurred in the recovery-light and in the recovery-dark group in spite of a large difference in the amount of SWS between both groups. This finding suggests that the rebound of PS is only partly dependent on the presence of SWS for its expression. Studies examining the effects of short light-dark cycles on sleep-waking patterns in rats (e.g., [1]) have also reported a temporary dissociation between the states of PS and SWS. In accordance with this study, it was suggested that the mechanisms of PS are under less control of the illumination cycle than the mechanisms of SWS. The sleep data for the deprivation period were obtained from the 10 min per hr samples taken during the light portion of the illumination cycle. The mean percentages of total time spent in PS calculated for the successive deprivation days were W/b, lye, 2% for Group l, and 0%, 0%, 2% for Group 2. These negligible amounts of PS observed during the deprivation period indicate that the pendulum technique was effectively suppressing PS. This efficacy may be paralleled by the watertank technique when relatively small platforms are used [7,13]. Considerable reductions of SWS have been found under these circumstances. A high degree of PS deprivation, therefore, seems to be incompatible with the maintenance of SWS. Normal amounts of SWS only occur in association with a moderate degree of PS deprivation [11]. The pendulum technique as used in this experiment was also accompanied by a loss of SWS. The mean percentages of SWS during the successive deprivation days were 22%, 21%, 19% for Group 1 and 30%, 26%, 26% for Group 2. This loss of SWS during deprivation, however, was not compensated for by more SWS during the first part of the recovery sleep. During recovery sleep in the light a decrease rather than an increase of SWS was found. Perhaps, two hypothetical factors combined in producing this effect: a 'ceiling' effect for

the amount of total sleep and a priority of PS rebound over a recovery of S W S The second factor may be a relative one, because total sleep deprivation studies in rats generally show a priority in the recovery of SWS [2,6] The mean percentages of passive wakefulness during deprivation were 21%, 33%, 35% for Group 1, and 24%, 23%, 26% for Group 2 It should be added that it was difficult to establish the point of transition from drowsiness into light sleep under pendulum conditions From these data it may be derived that during their stay in the moving pendulum the rats were b e h a v i o u r ally active about half of the t i m e Some remarks have to be made on the methods used in this experiment in assessing the sleep-waking p a t t e r n s E E G - E M G recordings were supplemented by behavioural o b s e r v a t i o n s In the light period the onset of sleep was indicated when the rat lay immobile (or moved passively) with his eyes closed. This could be verified electrophysiologically by the presence of hippocampal LIA. A difficulty was encountered in defining sleep onset in the dark period. On the basis of pilot work it was decided to consult the hippocampal E E G when the rat lay immobile with his eyes not wide open. The EMG record was used to detect small movements which may have remained unnoticed under observation in the dark. When full-blown hippocampal L I A was visible presence of sleep was indicated. Sleep defined in this way frequently coincided with a stretched-out posture in the rat. The curling up behaviour characterizing most of the sleep in the light seems to be less characteristic of sleep in the dark. Lying stretched-out with almost wide open eyes the rat could then enter into PS, as identified from hippocampal theta-rhythm. Eye-closure mostly occurred concomitantly with the appearance of phasic phenomena (e.g., facial muscle twitches). It was observed that brief PS episodes were not always attended by a reduction in muscular tone. Therefore, it seems hazardous to use EMG suppression as a prime index of PS. Bearing in mind a possible inaccuracy in establishing the onset of sleep, especially in the dark period, the amount of time from sleep onset to the first PS episode was determined for the four treatment groups. The latency of PS was found to be relatively short for the dark period (baseline-dark condition: 75 _+ 47 sec; recovery-dark condition: 13 _+ 11 sec), as compared to the light period (baseline-light condition: 1505 -+ 671 sec; recovery-light condition: 58 _ 30 sec). Rats of the recovery-dark group occasionally passed from wakefulness to PS without showing evidence of SWS (see Fig. 3). As a rough measure of the amount of stress accompanying the pendulum technique, the weight change occurring during the deprivation treatment was assessed. The PS de-

PARADOXICAL SLEEP DEPRIVATION

811

prived animals (n= 12) experienced a weight loss of 30.1 g

(SD: 6.5), whereas the baseline animals (n= 12) showed a weight gain of 7.9 g (SD: 4.6). In using the watertank technique weight reductions of 5 to 10 g per day have been reported in rats [11,16]. The main causes of the weight loss in the PS deprived animals may be the forced activity and the stress associated with it. Furthermore, it is uncertain whether these animals maintained their food and water intake at normal levels. A pendulum control condition was designed to control for the adverse effects of staying in a moving pendulum. After completion of the baseline-light condition rats of Group 3 were placed for 72 hrs in a pendulum moving in a way that no imbalance was produced in the animals. At one extreme of the motion their home cages were tilted 42 degrees and the other extreme only 12 degrees. These animals exhibited a minimal weight loss of 0.8 g (SD: 2.9). This treatment was completed at the onset of the light period, after which the sleep-waking patterns were established for 3 hrs. The mean amounts of PS and SWS were 1612 sec (SD: 418) and 6496 sec (SD: 829) respectively.

In comparing the pendulum and the watertank technique a number of differences are apparent. At the start of the deprivation treatment pendulum rats remain in their home cages, whereas platform rats are transferred to a new environment. PS deprivation is established in the pendulum technique by forcing the animals to walk intermittently and in the watertank technique by imposing movement restriction on the animals. The PS deprived state of the pendulum rats is taken into account by gradually increasing the speed of the pendulum. The platform situation is not modifiable according to the deprivation state of the animals. Two alternative PS deprivation techniques are now available which are based on quite different treatments. Perhaps, this provides a basis for determining whether the behavioural effects reported in the literature [5] are due to PS d e p r i v a t i o n p e r se. ACKNOWLEDGEMENT We wish to thank Arno Jansen for assistence in conducting the experiment.

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