Paradoxical Sleep Deprivation in Animal Studies: Some Methodological Considerations

Paradoxical Sleep Deprivation in Animal Studies: Some Methodological Considerations

Paradoxical Sleep Deprivation in Animal Studies: Some Methodological Considerations A.M.L. COENEN and Z.J.M. VAN HULZEN Department of Comparative and ...

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Paradoxical Sleep Deprivation in Animal Studies: Some Methodological Considerations A.M.L. COENEN and Z.J.M. VAN HULZEN Department of Comparative and Physiological Psychology, University of Nijmegen, 6525 GG Nqmegen (The Netherlands)

INTRODUCTION Deprivation of paradoxical sleep (PS) is used extensively in animals as a method for investigating the possible role of PS in behavioural processes. Two hypotheses, based mainly on this approach, have received much attention in the current literature. The information processing hypothesis of PS (Jouvet, 1965; Gaarder, 1966; Moruzzi, 1966) relates PS t o learning and memory processes, whereas the neural excitability hypothesis (Cohen and Dement, 1965) assigns PS a more general role of reducing brain excitability. THE INFORMATION PROCESSING HYPOTHESIS Several lines of evidence are in agreement with the idea that PS is a state conducive to information processing (Drucker-Colin and McGaugh, 1977). Unfortunately, research devoted to delineating more precisely the role of PS in learning and memory processes has provided inconsistent results. The reasons responsible for the discrepancies between studies may be at least partly methodological. A variety of PS deprivation paradigms has been employed in studying the information processing hypothesis of PS. In the first paradigm, PS deprivation is applied for a short period of time (e.g. 3 h) immediately following learning, and the retention is measured after a period of rest. According t o the consolidation hypothesis of PS (Greenberg and Pearlman, 1974; Hennevin and Leconte, 1977) a deficit in retention is produced by the absence of PS during the critical consolidation period. In the second paradigm, animals are deprived of PS for a long period of time (e.g. 72 h) following learning, and at one of several intervals after the deprivation period an electroconvulsive shock (ECS) is administered t o the animals. They are allowed t o recover from the acute effects of PS deprivation and ECS before retention testing takes place. An impairment of retention is interpreted in terms of the memory facilitation hypothesis of PS (Fishbein and Gutwein, 1977), according t o which an already established memory trace is rendered unstable by long-term PS deprivation and susceptible t o disruption. Finally, in studies using the third paradigm, long-term PS deprivation is applied prior t o learning and an ECS is administered at various intervals after training. Again, a recovery period is allowed prior t o the retention test. The memory facilitation hypothesis of PS accounts for a retention deficit by assuming that long-term PS deprivation delays the conversion of labile into stable memory, thereby prolonging the susceptibility

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period of memory. Subsequent consolidation may take place when PS is permitted to occur during the interval between training and ECS. For the purpose of depriving animals of PS two instrumental techniques are currently available. The “arousal” technique consists of waking animals each time the onset of PS is identified from electrophysiological indices. Although this technique requires much labour, it can easily be performed for a short period of time. However, after a few hours the number of awakenings needed to prevent an animal from entering PS increases rapidly (Morden et al., 1967). For long-term PS deprivation it is common t o use the “watertank” technique. Animals are placed on small platforms surrounded by water. Under these circumstances when they enter PS, muscular atonia accompanying PS results in their touchmg the water and awakening. Inevitably, any technique of PS deprivation is more or less confounded with unintended effects, for which appropriate controls have to be designed. An obvious control for the arousal technique is the “yoked” control in which arousal takes place concurrently, irrespective of the sleep-waking activity of the control animal. The control most frequently adopted for the watertank technique is the large platform control, the adequacy of which has been thoroughly discussed in recent reviews (Vogel, 1975; Ellman et al., 1978). Studies concerning the effects of short-term PS deprivation following learning on the retention have used the watertank technique for depriving animals of PS rather than the arousal technique. Most of these studies have reported an impairment of retention (e.g. Pearlman and Greenberg, 1973; Leconte et al., 1974). In order to control for non-specific effects accompanying the technique a delayed platform was chosen. T h s is understandable in view of the fact that differences in the degree of PS deprivation only gradually develop between the large and small platform conditions. However, the adequacy of the delayed platform control within this paradigm has been questioned (e.g. Vogel, 1975). What has

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Fig. 1. Mean number of avoidances in the 5 training sessions of‘ shuttle-box avoidance (Arabic numerals) and mean amount of PS during the 4 intersession intervals (Roman numerals) and the recovery sleep (R). The intersession intervals lasted 2 h and 45 min and the recovery sleep was monitored for 1 h. (From Van Hulzen and Coenen, 1979, reprinted by permission.)

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been neglected is the fact that this control is not suitable for non-specific effects of the technique which potentially modulate memory storage processes, but only for proactive effects influencing subsequent retention. In a recent study (Van Hulzen and Coenen, 1979), the arousal technique was used t o deprive rats of PS during shuttle-box avoidance distributed over the light period of the diurnal cycle. Yoked control animals were taken as control for possible confoundings of the technique, whereas free sleep rats were left undisturbed during the intervals between sessions. It appeared that distributed shuttle-box avoidance proceeded normally irrespective of the almost total absence of PS during the intersession intervals (Fig. 1). Apparently, memory storage processes are not dependent on the presence of PS immediately following a period of learning, which violates the consolidation hypothesis of PS . The watertank technique of PS deprivation has also been adopted for studies on the effects on retention of long-term PS deprivation prior or subsequent to learning. Commonly, the large platform is chosen as control for non-specific concomitants of the technique (e.g. stress, sleep loss, confinement, wetness). For studies in mice this control has been abandoned, because these animals tend t o sit at the edge of the large platform and are almost as deprived of PS as the experimental animals (Fishbein and Gutwein, 1977). In order to circumvent this problem of control, Fishbein and his associates have designed experiments which are based on the combined effects of PS deprivation and ECS. The important variable t o change is the interval between termination of PS deprivation and administration of ECS. Both in the pre-learning (e.g. Linden et al., 1975) and post-learning deprivation studies (e.g. Fishbein et al., 1971) an impairment of retention was found depending on the time between PS deprivation and ECS. One-trial passive avoidance was mostly used as the learning task in t h s type of study. One post-learning deprivation experiment in rats that used one way shuttle-box avoidance yielded similar results (Wolfowitz and Holdstock, 1971). The interpretion of these findings was that the state produced by long-term deprivation is detrimental t o memory conversion and memory maintenance processes in the sense that they are rendered susceptible t o disruption. In a recent study in mice (Shiromani et al., 1979) shortterm PS deprivation, by means of the watertank technique, was applied subsequent to learning and an ECS was administered immediately thereafter. This treatment appeared ineffective in impairing retention, suggesting that short-term PS deprivation is not enough to produce a labile memory trace. The finding that memory processes are susceptible to disruption immediately after long-term PS deprivation may be alternatively interpreted by proposing that long-term PS deprivation increases central neural excitability, in which state amnesic agents are rendered more effective (Fishbein et al., 1971). T h s alternative interpretation is reminiscent of the neural excitability hypothesis of PS. Alterations in neural excitability induced by long-term PS deprivation may influence a variety of behavioural processes, in particular those with drive-motivational components (Vogel, 1975). It is in this context that one might consider possible effects of long-term PS deprivation on learning. There is some evidence that acquisition of avoidance learning is influenced by long-term PS deprivation (Albert et al., 1970; Plumer et al., 1974), although it is difficult to establish whether learning or performance variables are affected. THE NEURAL EXCITABILITY HYPOTHESIS In studying the behavioural consequences of long-term PS deprivation in the light of the neural excitability hypothesis of PS, the watertank technique has been employed for PS

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deprivation and the large platform taken as control. Several studies in rats have reported an increase in locomotor activity after long-term PS deprivation (Albert et al., 1970; Ogilvie and Broughton, 1976; Hicks and Moore, 1979), in contrast to studies in mice (Fishbein and Gutwein, 1977) in which no differences in activity level were found. The increased locomotor activity observed in the rat after long-term PS deprivation may be due to the restriction of movement imposed on the animal on the small platform rather than to PS deprivation per se. It has been observed that mice are quite active on small platforms placed in cages with wiremesh lids, whereas rats do not exlubit much activity under these circumstances (Fishbein and Gutwein, 1977). In other words, in this type of experiment restriction of movement is at least one variable for which the large platform may not be adequate as control. It seems imperative to use an alternative technique of PS deprivation in conjunction with the watertank t e c h q u e in order to be sure whether the behavioural effects of the deprivation treatment are due t o PS deprivation per se. Therefore, a PS deprivation technique was developed (Van Hulzen and Coenen, in preparation) starting from the principle that the occurrence of PS is invariably preceded by slow-wave sleep (SWS). Animals staying in their home cages are allowed t o sleep for only brief periods of time (too short to permit PS to occur) by producing postural imbalance in the animals at regular intervals. This is accomplished by an apparatus which moves the animals’ cages backwards and forwards like a pendulum, forcing the animals to regularly walk downwards t o the other side of their cages. Control rats are placed in a pendulum adjusted in a way that no imbalance is produced in the animals. They can therefore obtain sleep and PS while swinging. Using this technique it is possible to effectively deprive rats of PS for 72 h; only a few episodes of PS were detected in the animals during t h s period. The recovery sleep at the beginning of the dark phase of the illumination cycle was monitored for 3 h. Rats spent about 31% of total time in PS and 9% in SWS (Fig. 2), whereas baseline values were 6% and 11%respectively. A preliminary study of the effects of 72 h of PS deprivation on locomotor activity was carried out in Wistar rats weighing between 275 and 375 g. The number of crossings in a shuttle-box was used as index of locomotor activity. The crossings were noted during a 15 min period at the beginning of the dark period immediately following the deprivation treat-

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Fig. 2. Recovery sleep after 72 h of PS deprivation at the beginning of the dark phase of the illumination cycle. Mean percentages of PS, SWS and wakefulness 0,expressed in total time, are plotted in 15 min periods.

3 29 TABLE I MEANS AND S.D. OF THE CROSSINGS IN THk SHUTTLE-BOX FOR THE 5 TREATMENT GROUPS The data were analyzed statistically using MANOVA planned comparison tests (n per group = 12). The comparisons were: (1) group A plus B plus C plus D vs group E; (2) group A plus B vs group C plus D; (3) group A vs group B; and (4) group C vs group D. The second and fourth comparisons were found to be significant: F(1,55) = 4.74,P < 0.05 and F(1,55) = 6.64,P < 0.05, respectively. Pendulum experimental (A) ~~

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ment. Two PS deprivation groups were studied: a “pendulum” experimental group and a “platform” experimental group. Animals of the latter group were placed on 6.2 cm diameter platforms. In addition, 3 control groups were run: a “pendulum” control, a “platform” control (diameter 12.8 cm) and a “free sleep” control. Table I summarizes the results. The platform experimental group showed significantly more crossings in the shuttle-box than the platform control group, whereas no significant differences were found between the pendulum experimental and the pendulum control group. These results suggest that a nonspecific effect of the small platform, probably its restriction of movement, was responsible for the increased locomotor activity in the platform experimental group. Consequently, other behavioural effects of this PS deprivation technique may be contaminated by changes in locomotor activity.

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330 Leconte, P., Hennevin, E. and Bloch, V. (1974) Duration of paradoxical sleep necessary for the acquisition of conditioned avoidance in the rat. Physiol. Behav., 13: 675-681. Linden, E.R., Bern, D. and Fishbein, W. (1975) Retrograde amnesia: prolonging the fixation phase of memory consolidation by paradoxical sleep deprivation. Physiol. Behav., 14: 409 -4 12. Morden, B., Mitchell, G. and Dement, W. (1967) Selective REM sleep deprivation and compensation phenomena in the rat. Bruin Res., 5 : 339-349. Moruzzi, G. (1966) The functional significance of sleep with particular regard to the brain mechanisms underlying consciousness. In Bruin and Conscious Experience, J.C. Eccles (Ed.), Springer-Verlag, Berlin, pp. 345-388. Ogilvie, R.D. and Broughton, R.J. (1976) Sleep deprivation and measures of emotionality in rats. Psychophysiology, 13: 249-260. Pearlman, C.A. and Greenberg, R. (1973) Posttrial REM sleep: a critical period for consolidation of shuttle-box avoidance. Apiirn. Learn. Behav., 1: 49-5 1. Plumer, S.I., Matthews, L., Tucker, M. and Cook, T.M. (1974) The water-tank technique: avoidance conditioning as a function of water level and pedestal size. Physiol. Behav., 12: 285-287. Shiromani, P., Gutwein, B.M. and Fishbein, W. (1979) Development of learning and memory in mice after brief paradoxical sleep deprivation. Physiol. Behav., 22: 971 -978. Van Hulzen, Z.J.M. and Coenen, A.M.L. (1979) Selective deprivation of paradoxical sleep and consotidation of shuttle-box avoidance. Physiol. Behav., 23: 821 -826. Vogel, G.W. (1975) A review of REM sleep deprivation. Arch. gen. Psychiat., 32: 749-761. Wolfowitz, B.E. and Holdstock, T.L. (1971) Paradoxical sleep deprivation and memory in rats. Cornrnun. Behav. Biol.. 6: 281-284.