Effects of midbrain raphe destruction on sleep and locomotor activity in rats

Physiology & Behavior, Vol. 19, pp. 535-541. Pergamon Press and Brain Research Publ., 1977. Printed in the U.S.A.

Effects of Midbrain Raphe Destruction on Sleep and Locomotor Activity in Rats A. L. BOUHUYS AND R. H. VAN DEN HOOFDAKKER

Department o f Biological Psychiatry, Psychiatric University Clinic, Oostersingel 59, Groningen, The Netherlands (Received 15 August 1975) BOUHUYS, A. L. AND R. H. VAN DEN HOOFDAKKER. Effects ofmidbrain destruction on sleep and locomotor activity in rats. PHYSIOL. BEHAV. 19(4) 535-541, 1977. - This study deals with the question whether the often reported sleep loss in animals with lesions in the rostral midbrain raphe system (the dorsal and medial raphe nuclei) might be explained by the hyperactivity shown by the animals when confronted with unfamiliar environments. Rats with electrolytic raphe lesions were compared with sham-operated rats as to both sleep and activity parameters under 3 conditions differing in familiarity. In spite of a reduction of forebrain serotonin and 5-hydroxy-indoleacetic acid of about 60-70%, raphe-lesioned rats could not be distinguished from sham-operated rats as to sleep parameters such as flow-wave sleep and REM sleep under different conditions. Thus, structural integrity of serotonin-containing neurons was not found to be a necessary condition for the normal production of sleep in rats. Locomotor activity, however, was significantly increased in raphe-lesioned rats, but this increase showed no relationship with sleep production. Raphe lesions

Sleep

Locomotor activity

Rostral midbrain Raphe system

can be expected to induce larger amounts of locomotor activity. We therefore decided to measure sleep and locomotor activity in raphe-lesioned rats under two experimental conditions. Firstly in a familiar environment, to confirm our preliminary observation that sleep loss does not occur under these circumstances. Secondly in an unfamiliar environment, to test the hypothesized relationship between locomotor activity and sleep loss in raphelesioned rats.

JOUVET [9,10] proposed a sleep hypothesis in which various biogenic amines are considered to be of critical importance in the regulation of sleep. The serotonincontaining pathways, originating from the raphe nuclei, is supposed to play a central role in starting and maintaining Slow-Wave Sleep (SWS). The hypothesis is supported by the effects of lesions in these nuclei, the dorsal and medial nuclei, (B7, B8, according to DahlstrSm et al. [4] ). In cats, the extent of the damage, the decrease in forebrain serotonin ((5-HT) 5-hydroxytryptamine) levels and the decrease in SWS are reported to show a strong correlation [9, 10, 16]. In rats, a persistent arousal pattern has been observed after lesions of these nuclei [13]. Additional evidence in favour of the 5-HT hypothesis is the insomnia resulting from PCPA (para-chlorophenyl alanine) administration in cats and rats [5, 11, 16, 17, 24], and the restoration of SWS after injection of small doses of 5-hydroxytryptophan in cats [ 11,16]. Apart from the effects on sleep, other behavioural changes following 5-HT manipulation are reported. The most prominent and consistent effect of raphe lesions observed, has been an increase in the general level of locomotor activity [7, 13, 15, 22]. Preliminary observation indicated to our surprise that the raphe-lesioned rats showed no evident sleep loss when they were adapted to the recording conditions. These rats showed more locomotor activity than controls, but the absolute level of their activity was low; at any rate so low that obvious interference with sleep production could hardly be expected. The question arose whether such interference might occur in unfamiliar circumstances, which

METHOD

Surgery In Wistar rats of 2 8 0 - 3 1 0 g, electrolytic lesions were made in the dorsal and medial raphe nuclei under ether anaesthesia. Coordinates were A 0.6, L 0.0 and V - 3 . 5 and - 1 . 5 mm [21]. The electrode, 0.2 mm in diameter, had a 1 mm uninsulated tip. An anodal direct current with a strength of 1.5 mA and a duration of 10 sec was passed through each nucleus. Control rats underwent the same l~rocedure but without current supply. Four days after the lesion, EEG electrodes were implanted under sodium pentobarbital anaesthesia (Nembutal, 50 mg/kg IP). The electrodes, of 0.5 mm diameter, were placed on the dura in the occipital area (18a, according to Krieg [ 14] ), and in the prefrontal area above the olfactory bulbs. As a reference electrode, a 7 mm long bar (0.5 mm in diameter) was fixed to the temporal muscle, contralateral to the occipital EEG electrode. The electrodes were attached to an 8-pin connector, which was then cemented to the skull. 535

536

BOUHUYS

Activity Recording Lo co m o t o r activity was registered automatically by means of a displacement transducer fastened to the grid floor. The data obtained were the duration of activity (in sec) over 5-rain periods. The recording device was adjusted in such a way that the data delivered showed a high correlation (r = 0.940, n = 22, Pearson) with visually observed locomotion.

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EEG Recording The rats were connected with an Elema-Sch~Snander recorder by means of a swivel. EEG's were recorded from the prefrontal and occipital electrodes against the contralateral reference electrode, with a time constant of 0.6 sec and with low-pass filtering at 30 Hz. The occipital EEG was analysed on-line by means of an analogue frequency band analyser (Ahrend van Gogh), providing the integrated amplitudes of the delta frequency band ( 0 . 9 - 2 . 0 cps) over 30-sec periods (delta output). All data were stored on magnetic tape (Honeywell), including the activity data. Finally compressed records were made (paper speed 0.05 mm/sec).

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Experimental Conditions The rats were operated on in 5 groups of 4 rats each. In 3 rats of each group lesions were made, while the remaining rat underwent a sham-operation. Of the 3 rats with lesions the most active rat was selected for further experimentation. The amount of l o c o m o t o r activity was used as an a priori criterion for the success of the lesion, because loc o m o t o r activity and forebrain 5-HT levels have been found to correlate [13]. After the experiments the locations of the lesions were biochemicaUy and histologically checked (see further). The amount of l o c o m o to r activity was assessed as the duration of visually observed locomo-

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Wakefulness, SWS and REM sleep were assessed on the basis of prefrontal olfactory bulb activity [1, 3, 6, 18], occipital EEG [23], EEG frequency analysis and activity. Figure 1 shows a compressed record (paper speed 0.05 mm/sec), together with sections of the prefrontal and occipital EEG's, recorded at a paper speed of 30 mm/sec. The prefrontal signal discriminates the abovementioned states. The voltage is highest during wakefulness, becomes lower in SWS, and nearly drops to zero in REM sleep, as also observed by others [1, 3, 6, 18]. The occipital EEG shows the characteristics in the various states described by Timo-Iaria et al. [23]. To obtain further distinction between waking and REM sleep, the activity data were used as an additional criterion. In order to achieve a high degree of objectivity in discrimination of the various states, the frequency analysis data were used. At the top of the compressed record (Fig. 1) the analyser's output (delta output) is displayed: each vertical bar represents the integrated amplitude of the delta activity in the foregoing 30 sec epoch. In the recording of each registration period, the 20 lowest delta values were measured and the mean and standard deviation served as a criterion for distinction of SWS from the other states, i.e. delta output exceeding this criterion was considered an indication of SWS in the foregoing 30 sec epoch.

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tion during the first 10 min after introduction into an unfamiliar cage. On Day 4 after the operation, the EEG electrodes were implanted in the 5 selected raphe-lesioned and in the 5 sham-operated rats. Recording took place in cages of 100 x 60 x 50 cm. The animals were supplied with water and food ad lib. Lights went on at 9:00 a.m. and off at 9:00 p.m. SWS, REM sleep and automatically determined locomotor activity were recorded under the 3 following conditions.

Condition 1. The unfamiliar environment. On Days 7 - 1 4 the raphe-lesioned and sham-operated rats were housed solitary in an experimental cage, They were connected with a swivel to habituate to the recording cable. On Day 14 after the lesioning the rats were introduced to a novel experimental cage at 10 a.m. Then the data were collected continuously during the next 5 hr. Condition 2. The familiar environment. The abovementioned rats remained in the same cage on Days 1 4 - 2 0 after the lesioning. On Days 17, 18 and 19 the rats were considered to be familiar with their cage and locomotor activity and sleep were recorded from 10 a.m. till 3 p.m. Condition 3. The social situation. For reasons to be mentioned in the discussion of the results, the rats were finally confronted with an unfamiliar male Wistar rat of the same weight. EEG recordings were made between the 4th and 8th week after the lesions and lasted from 10 a.m. until 5 p.m. Only sleep was recorded, because the technique for measuring activity did not permit of making a distinction between the activity of both animals. Three rats could not be studied because they had lost their pin-connector.

RAPHE DESTRUCTION ON SLEEP AND LOCOMOTOR ACTIVITY

537

TABLE 1 EFFECTS OF RAPHE LESIONS ON FOREBRAIN 5-HT and 5-HIAALEVELS AND ON LOCOMOTION

Groups

% 5-HT*

% 5-HIAA*

% locomotiont

n

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100 _+ 8.7

100 -+ 13.6

19.3 -+ 6.7

5

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40.0 _ 11.4

59.5 --_ 7.5

5

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40.2 +--20.7

56.2 -+ 10.2

10

In the 5 selected, 10 non-selected raphe-lesioned and 5 sham-operated rats, the serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) values are presented, as well as the time spent o n l o c o m o t i o n . *percentage of control values. 100%, 5-HT: 461.6 ng/g tissue and 100% 5-HIAA: 410.1 ng/g tissue; - SD. tpercentage of observation time; --- SD.

Biochemistry and Histology All rats, i.e. also the raphe-lesioned rats in which no sleep recordings were made, were decapitated 5 - 8 weeks after the lesions. A transverse section of the brain was made stereotaxically, 3 mm in front of and parallel to the plane of the lesion electrode. In the anterior part, 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured by a semi-automatic fluorimetric method, described by Korf et al. [12]. In the 5 rats in which sleep recordings were made, the caudal part of the brain was fixed in 10% Formalin and 40-u sections were cut and stained (Nissl technique) for histological examination [21 ]. RESULTS

Biochemistry, Histology and Selection The activity data on the basis of which the raphelesioned rats were selected are presented in Table 1. In the same table the 5-HT and 5-HIAA levels are shown of all rats, selected, non-selected and controls. As mentioned, the highest activity was considered the best predictor of the success of the lesions. The table shows that the selected group was far more active than the controls. The activity in the selected raphe-lesioned group was only slightly higher than that in the non-selected raphe-lesioned group. Selected and non-selected rats showed the same drastic decrease of 5-HT and 5-HIAA levels as compared with the controls. These findings are in agreement with the data presented by Kostowski et al. [ 13]. Apparently 5-HT and 5-HIAA levels are closely related to locomotor activity. A further conclusion must be that our careful selection procedure was superfluous, because the lesioning technique turned out to be successful, that is to say, according to biochemical criteria. The biochemical findings are fully in line with the histological data (Fig. 2). The dorsal as well as the medial raphe nuclei in selected animals proved to have been largely destroyed. Locomotor activity and sleep data, as measured in a familiar and unfamiliar environment, are presented in the Figs. 3 and 4. The figures show the time spent (per half hour) on SWS, REM sleep and wakefulness. The time spent on locomotor activity is also presented, and the remaining time has been spent on other waking behaviour. Fig. 3

shows the means of the averaged scores per rat over 3 registration days, while in Fig. 4 the group means per registration day are presented.

Locomotor Activity To the activity data presented in the Figs. 3 and 4 a three-factor analysis of variance was applied. The factors were lesion, condition and time. A significant main effect of the lesion factor was found, F (1,8) = 12.53, p<0.01, raphe-lesioned rats were more active than sham-operated rats in both situations. There was also a significant main effect of the condition F(1,8) = 37.67, p<0.01, indicating that both groups of rats were more active in an unfamiliar environment. The lesion and the condition factor interact significantly, F(1,8) = 6.22, p<0.05. This means that raphe-lesioned rats were more active in the familiar as well as in the unfamiliar environment. Finally, the lesion, condition and time factors show significant interaction, F(9,72) = 3.36, p<0.01. As Fig. 4 shows, raphe-lesioned rats reacted differently to unfamiliar circumstances from controls: they spent more time in attaining their baseline activity.

Sleep The data on SWS and REM sleep, presented in the Figs. 3 and 4, were analysed in the same way as the activity data. In the unfamiliar situation smaller amounts of SWS and REM sleep were found than in the familiar situation, especially in the first hour. This was demonstrated by a significant condition (familiar, unfamiliar) effect for SWS, F(1,8) = 76.95, p<0.01, and REM sleep, F(1,8) = 44.88, p<0.01 and by a significant condition-time interaction for SWS, F(9,72) = 7.78, p<0.01, and REM sleep, F ( 9 , 7 2 ) = 2.60, p<0.01. Thus, differences in sleep amounts and in sleep distribution can be ascribed only to differences in environment. The lesion in the raphe system per se did not make any difference: both raphe-lesioned and sham-operated rats showed the same amounts and distributions of sleep, irrespective of their degree of familiarity with the environment. Thus the preliminary observations mentioned in the introduction were confirmed in familiar conditions; raphe-lesioned rats did not sleep less than controls. Although they were more active, their activity apparently did

538

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not interfere with sleep production. Consequenily iocom*.)tor activity in raphe-lesioned rals is increased H the cost,,~ other waking activities. The question whether sleep loss might occur in circumstances eliciting more activity, must be answered in the negative. Although the raphe-lesioned rats showed considerable increases of activity in an unfamiliar environment, they did not show sleep loss. Again, the locomotor activity must have been increased at the expense of other waking behaviour. Figure 4 suggests that raphe-lesioned rats in an unfamiliar environment needed more time to reach a normal sleep production than controls. This prompted a last question. Would raphe-lesioned rats have more difficulty in attaining a stable sleep level in environments with sleep-inhibiting properties? We therefore measured sleep after introduction of an unfamiliar male rat. The results are presented in Fig. 5. Again, analysis of variance reveals no differences either with respect to the amount or with respect to the distribution of SWS. In both groups the occurrence of REM sleep was too rare for statistical analysis. DISCUSSION

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The question raised in the introduction was whether the loss of sleep observed by some investigators after lesions of the midbrain raphe nuclei can be explained on the basis of increased motoractivity in lesioned animals. The results show that this is probably not so. The raphe-lesioned rats in our study produced normal amounts of sleep in spite of increased l o co m o t o r activity. Even in circumstances which elicited very large amounts of locomotor activity in raphe-lesioned rats, the sleep production remained unimpaired as compared with controls. The activity data are in agreement with most of the previously reported data [7, 13, 15, 22]. However, the activity data in a familiar environment are in contrast with those of Srebro e t al. [22], who measured no changes of activity in raphe-lesioned rats under comparable circumstances. These discrepancies may be due to the different methods employed. In our study activity was measured continuously, whereas Srebro e t al., sampled the behaviour of rats during the first 15 rain of every hour. They ranked each animal's activity 3 times (at 4 to 5 min intervals) during these 15-min periods on a 6-point scale. The sleep results are not in line with other studies [9, 10, 13, 16] showing drastic sleep loss in raphe-lesioned animals. The measurement of sleep stages and wakefulness in our study was based on prefrontal olfactory bulb activity, the occipital EEG and on l o co m o t o r activity. The automatic frequencyanalysis on the occipital EEG as well as the automatic registration of the l o co m o t o r activity garantee an identical discrimination between SWS on the one hand and the other stages on the other. Thus any unreliability connected with visual analysis was excluded, especially in the transistory phases of the records. The discrimination between REM sleep and wakefulness is less watertight. We have relied mainly on the olfactory bulbs activity. This activity has been used as one of the discriminators by a number of authors [1, 3, 6, 18]. One of the authors states that the olfactory bulb activity can be used as the sole discriminator between REM sleep and wakefulness, because this activity is always present during any kind of wakeful-

RAPHE D E S T R U C T I O N ON SLEEP AND L O C O M O T O R A C T I V I T Y

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hess and absent during REM sleep [6]. However, Pickenhain e t al. [ 18] states that olfactory bulb activity is present during all kinds of wakefulness, except during relaxed wakefulness, i.e. during the transitions between wakefulness and sleep. Therefore the possibility that some relaxed wakefulness has been categorized as REM sleep and vice-versa cannot be fully excluded. This possibility is small in the light of the similarity of our sleepdata and those obtained by others in normal rats under comparable recording conditions [19,25]. Anyway, the conclusion must be that out methods allow a highly reliable SWS-discrimination whereas the measurement of REM sleep could have been less secure. Another fact to be taken into account when interpreting the findings is the duration of the registration. Recordings were made for 5 or 7 hr daily, during the first part of the light period. If raphe-lesioned animals exhibited sleep loss during the remaining period, it went unrecorded. Disturbances of the circadian distribution of sleep in raphe-lesioned animals have never been observed by other authors. So, if sleep loss exists, it may be expected to become most evident in that fase of the light-dark cycle during which rats produce the largest amounts of sleep, i.e. in the first part of the light period [25]. Although our data are thus highly suggestive for the absence of sleep loss in raphe-lesioned animals, they are not fully conclusive in this respect. In our study the 5-HT system was destroyed to a large extent; this observation is based on the decrease of 5-HT and 5-HIAA levels in the forebrain (Table 1), and on the histological data which indicate the destruction of 5-HTcontaining cell bodies (Fig. 2). This reduction of 5-HT and 5-HIAA found by us, is reported by others to cause a significant decrease in sleep. These discrepancies cannot be explained by differences in time interval between lesion and recording: so far as mentioned in the literature, our results were obtained in a corresponding period after the lesion. This means that possible differences in cerebral adaptation to the disturbed circuitry are not likely to be responsible for the discrepancies in outcome. We therefore doubt whether the reported sleep defects after raphe lesions can be ascribed to damage of the 5-HT system. Data supplied by Jalowiec e t aL [8] justify this doubt. They reported no effects on sleep of lesions in the dorsal and ventral

tegmentum which cause a reduction of 5-HT levels in various areas of the forebrain. This finding is in agreement with the lesion experiments carried out by Carli e t al. [21 and Zanchetti [26], but they did not determine 5-HT levels in the brain. How then to explain the reported sleep loss after midbrain lesions? Are structures other than the 5-HT neurons, but also injured by the lesioning procedure, responsible? In Jouvet's study [10] the destruction in cats transgressed the boundaries of the raphe nuclei and included parts of the medial reticular formation and the central grey matter. In the only report on rats [13], part of the forebrain reticular formation also was damaged. In our study, structures surrounding the medial and dorsal raphe nuclei were affected too. The extents and/or localizations of the lesions differ in all these studies. The available data, however, are too gross to differentiate the lesions in detail to answer this question. Further lesion studies with restricted lesions in the surroundings of the raphe nuclei, will have to be done. There is more - evidence in the field of neuropharmacology - which questions the dominant role of 5 - H T in sleep mechanisms, Apart from the studies which do report sleep loss after PCPA administration [5, 11, 16, 17, 24], there are also studies providing contradictory evidence [5, 19, 20]. It was shown after continued administration of PCPA that cats produced normal amounts of SWS and REM sleep [5]. and in rats normal amounts of sleep were measured after a single PCPA injection [19]. Furthermore, depletion of brain serotonin following intraventricular 5,7-dihydroxytryptamine fails to disrupt sleep in the rat [20]. In all cases the lack of sleep loss was assessed during periods in which the 5-HT levels in the forebrain were very low. Thus, electrolytic or pharmacological impairment of the 5-HT system apparently does not always correspond with impairment of sleep production. The 5-HT sleep hypothesis has been mainly developed in cat studies. Perhaps differences in results have to be ascribed to differences in the species studied. In our opinion, however, inter-species differences can only be taken into account if consistent relationships within each species are established. But this condition is not yet

R A P H E D E S T R U C T I O N ON S L E E P A N D L O C O M O T O R A C T I V I T Y fulfilled. N e i t h e r in cats n o r in rats are t h e e f f e c t s o f a n a t o m i c a l or p h a r m a c o l o g i c a l i m p a i r m e n t o f 5-HT m e c h a n i s m s o n sleep u n e q u i v o c a l . In brief, o u r f i n d i n g s yield a n o t h e r f r a g m e n t o f e v i d e n c e suggesting t h a t 5-HT m i g h t n o t play a crucial role in t h e r e g u l a t i o n o f sleep.

541

ACKNOWLEDGEMENTS The authors express their appreciation to Professor Dr. G. P. Baerends and Professor Dr. H. M. van Praag for their helpful suggestions and comments. We are grateful to F. Postema for the histology and J. Bakker, H. Leever, G. Haayer and K. Venema for their technical assistance.

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