402
Eleclroencephalography and Clinical Neurophysiology, 1978, 4 5 : 4 0 2 - - 4 0 8 © Elsevier/North-Holland Scientific Publishers, Ltd..
SUPRACHIASMATIC NUCLEI LESIONS IN THE RAT: ALTERATIONS IN SLEEP CIRCADIAN RHYTHMS J. MOURET, J. COINDET, G. DEBILLY and G. CHOUVET Ddpartement de Mddecine Expdrimentale, Universitd Claude-Bernard, 8, avenue Rockefeller, 693 73 Lyon Cedex 13 (France) (Accepted for publication: March 31, 1978)
Much progress has been made in recent years in understanding factors controlling sleep production, i.e., sleep amounts over a certain period of time (Jouvet 1972). In some species like the laboratory rat, the control of the circadian organization of sleep or waking is at least as important a feature as sleep production and can be affected without changes in either slow wave sleep (SWS) or paradoxical sleep (PS) amounts over successive 24 h periods; for instance pinealectomy is followed by disappearance of the circadian r h y t h m of PS whilst that of SWS is unaltered (Mouret et al. 1974). More recently Ibuka and Kawamura (1975), Ibuka et al. {1977) and our group (Coindet et al. 1975) have reported the loss of sleep-waking circadian rhythms after lesions of the suprachiasmatic nuclei (SCN) in the rat. These nuclei, which receive direct input from the retina (Moore and Lenn 1972; Moore and Klein 1974) and dense projections from the raphe nuclei (Fuxe 1965; Aghajanian et al. 1969; Saavedra et al. 1974), play an important role in the control of the r h y t h m of the pineal enzyme serotonin-N-acetyltransferase {Moore and Klein 1974) as well as in the genesis of the adrenal corticosteroid r h y t h m (Moore and Eichler 1972; Hal~sz 1972). In this paper we shall describe the effects of such lesions on SWS and PS amounts and r h y t h m s as well as the possible relationships between the reduction in amplitude of the
circadian variations and the size of the lesions in the SCN. Methods Fourteen male Wistar rats were used in this study. After 15 days to habituate to the laboratory environment (light/dark 12/12 h, light on at 7.00 a.m., food and water ad libitum, temperature 23_+ I°C), cortical and neck muscle electrodes were chronically implanted as previously described (Mouret et al. 1974). At the time of surgery their weight was about 220--230 g. They were immediately placed under the recording conditions, in single glass jars for 8 days, after which continuous polygraphic recording (24 h a day) was performed during 8 consecutive days. All records (at paper speed 5 mm/sec), were scored to the nearest minute for the presence of SWS, PS and waking. The data from visual analysis were stored in a computer for the determination of the amounts of sleep on different time bases, as well as for the study of their circadian rhythms. At the end of this control recording session, stereotaxic lesions aimed at the SCN (De Groot 1959) were made. In order to spare the superficial venous sinuses the angle o f the electrode was at 20 ° backward in the horizontal plane. In 3 rats no electrocoagulation was performed (sham rats); in the remaining 11 rats a 3 mA current was delivered for 15 sec.
SUPRACHIASMATIC NUCLEI LESIONS IN THE RAT
The rats were then replaced under the previous conditions and the records resumed for 30 days (24 h a day). At the end of the experiment the animals were killed for histological control of lesion placement. In order to quantify the circadian variations of sleep, statistical analysis was performed on hourly sleep scores according to a m e t h o d described elsewhere (Anderson 1971; Roussel et al. 1976; Kan et al. 1977), which consists in computing the best fitting curve b y least square harmonic decomposition (fundamental 24 h period T, plus harmonics at 12 h and 8 h). This m e t h o d allows the determination of the amplitude and phase of each significant (P ~< 0.05) harmonic term as well as their confidence intervals.
403
Results
According to the site of the lesion the rats could have been classified in 4 groups. However, since no changes in sleep amounts or rhythms were detected in 2 of these groups as compared with the control recording session, 3 groups were finally distinguished (Fig. 1): Group A, where no changes in sleep were detected and which will be referred to as 'control group', is composed of the 3 rats with sham lesions, and 4 rats with lesions sparing the SCN b u t located rostral or caudal to these nuclei. Group B (3 rats) is composed of the rats with incomplete lesions of the SCN, with or without extensions of the lesions caudal or rostral to these nuclei.
A 6.2
As e AT.o
A7.4 A .8 As.
A
B
C
Fig. 1. Schematic representation (De Groot atlas) of lesions in the 3 groups of rats. A: lesions localized rostrally and caudally to the SCN (4 rats). B: incomplete SCN lesions with extension rostrally and caudally to these nuclei (3 rats). C: complete SCN lesions with or without extension of the lesions (4 rats).
404
J. MOURET ET AL.
In G r o u p C are t h e 4 rats with c o m p l e t e lesions o f t h e SCN, w i t h or w i t h o u t e x t e n sions.
h a r m o n i c d e c o m p o s i t i o n (T = 24 h), t w o interesting p o i n t s e m e r g e f r o m t h e statistical analysis (Figs. 2 a n d 3): - - a s t h e size o f the SCN lesion increases t h e r e is a progressive decrease in t h e amplit u d e o f t h e circadian v a r i a t i o n s o f b o t h SWS a n d PS; t h e a c r o p h a s e o f SWS r e m a i n s u n a l t e r e d w h e r e a s t h a t o f PS shifts t o w a r d s t h e end o f t h e i l l u m i n a t i o n p e r i o d in g r o u p C ( 1 8 h). F u r t h e r m o r e , w h e n t a k i n g into a c c o u n t t h o s e o f t h e first 4 h a r m o n i c s w h i c h r e a c h a significant value (P < 0.05), the polar p l o t s clearly s h o w t h a t SCN lesions do n o t a f f e c t t h e non-sinusoidal p a t t e r n o f t h e t e m p o r a l v a r i a t i o n s o f SWS ( e x c e p t f o r t h e r e d u c t i o n o f t h e 24 h t e r m a m p l i t u d e ) , b u t s t r o n g l y dist u r b t h e t i m e c o u r s e o f PS: In t h e 3 groups, t h e t i m e c o u r s e o f SWS can be statistically fitted b y t h e linear c o m b i n a t i o n o f 2 t e r m s : t h e 24 h f u n d a m e n t a l r h y t h m plus an 8 h t e r m w i t h a p p r o x i m a t e l y t h e s a m e a m p l i t u d e and p h a s e in t h e 3 groups. This last t e r m is likely to be t h e c o n s e q u e n c e o f t h e l i g h t e n e f f e c t . It persists in t h e animals w i t h lesions and is clearly o b s e r v e d on the t i m e p l o t s o f t h e h o u r l y scores o f SWS. Whereas a p u r e sine f u n c t i o n (T = 24 h) fits t h e PS t i m e c o u r s e in g r o u p s A a n d B, an a d d i t i o n a l significant 12 h h a r m o n i c is p r e s e n t in g r o u p C, w h o s e a m p l i t u d e is as i m p o r t a n t as t h a t o f t h e 2 4 h h a r m o n i c . Since t h e s e t w o t e r m s are r o u g h l y o u t o f p h a s e t h e y result in a
Quantitative data In o r d e r to a v o i d t h e possible non-specific e f f e c t s o f t h e s u r g e r y , we shall r e p o r t here on t h e d a t a f r o m t h e 2 2 n d to t h e 2 7 t h d a y f o l l o w i n g t h e lesion, a period w h e n t h e sleep changes, if a n y , w e r e stabilized. As s h o w n in T a b l e I, no clear m o d i f i c a t i o n o f sleep a m o u n t s o v e r successive 24 h periods was o b s e r v e d in e i t h e r g r o u p . However, a preliminary estimate of the n y c t h e m e r a l variations, based o n t h e quantitative d i f f e r e n c e s b e t w e e n t h e i l l u m i n a t i o n a n d d a r k p e r i o d s , s h o w s t h a t certain f a c t o r s were clearly a f f e c t e d in g r o u p C (SWS, PS a n d n u m b e r o f PS episodes), w h e r e a s o n l y SWS was significantly a l t e r e d in g r o u p B. In b o t h g r o u p s these changes resulted in reduct i o n o f t h e ' d a y / n i g h t ' d i f f e r e n c e s . This reduct i o n c o u l d s i m p l y be t h e result o f a shift in the p h a s e o f t h e sleep r h y t h m , o r c o u l d be due to other factors.
Circadian rhythms A p r e l i m i n a r y individual s t u d y o f each rat d e m o n s t r a t e d t h a t all animals w i t h i n each g r o u p w e r e in phase, a result which a l l o w e d p o o l i n g i n t r a g r o u p d a t a f o r the analysis o f t e m p o r a l v a r i a t i o n s o f h o u r l y sleep scores. When c o n s i d e r i n g o n l y t h e first t e r m o f
TABLE I Sleep parameters in groups A, B, C (see text}, from the 22nd to the 27th day after SCN aimed lesion. PS, paradoxical sleep (in rain); N. PS, number of paradoxical sleep episodes; SWS, slow wave sleep (in rain). In each case the amounts for 24 h (24 h), for the 13 h of darkness (black circles)and the 12 h of illumination (open circles)are shown. Values represent the average of 35 measures for group A, 15 for grOuP B and 20 for group C. PS
A B C
N. PS
SWS
24 h
•
o
24 h
•
o
24 h
•
o
103 98 105
34 36 51 *
69 62 54 *
52 56 55
18 20 26 *
34 36 29 *
634 653 651
214 252 * 279 *
420 401 * 372 *
* Significant differences (P < 0.01) from the control group (t-test).
SUPRACHIASMATIC
N U C L E I L E S I O N S IN T H E R A T
405
A
19
60
30
60
7
lh
_
:.
.
:.
I1k
Fig. 2. Daily variations of hourly SWS scores in groups A, B and C£ Left: t e m p o r a l representation. Ordinates: h o u r l y a m o u n t s o f SWS in min/h. Abscissae: legal t i m e in hours. F o r a b e t t e r representation, the data are duplicated over 2 successive 24 h. Individual points represent the m e a n h o u r l y SWS scores over different animals for one day. The solid line illustrates the mean time course of SWS over 5 consecutive days. The best fitting sine curve (T = 24 h) is the only term s u p e r i m p o s e d u p o n the e x p e r i m e n t a l d a t a ! R i g h t : the respective polar plots indicate the amplitude and phase of each significant h a r m o n i c (p ~ 0.05). T h e r a d i u s of the o u t e r circle corresponds to an a m o u n t of 30 rain of SWS per hour. The inner circle represents the conf!dence area of each h a r m o n i c and gives the confidence intervals (I.C.) for the corresponding a m p l i t u d e and p h ~ e . F o r c o m b i n e d r h y t h m s (two significant terms), the c i r c u m f e r e n c e o f the o u t e r circle is equivalent to the period of the considered term, and the c o n f i d e n c e area of the significant h a r m o n i c t e r m is drawn with i n t e r r u p t e d lines. The black square on top of the o u t e r circle positions local m i d n i g h t (see Roussel et al. 1976 and Kan et al. 1977 for statistical details). The corresponding quantitative values are the following: Groups
n
a
T
b
p
A
7
26.2 (0.7)
B
3
27
C
4
27.1 (1.5)
24 8 24 8 24 8
11.5 (1.5) 3.1 (1.5) 8.5(L6) 1.8(1.6) 4.7 (2.2) 2.4 (2.2)
12.0 7.7 11.9 7.9 11.3 0.3
(1)
(0.5) (1.4) (0.7) (2.8) (1.8) (2.9)
n = n u m b e r of rats; a = hourly m e a n level over 24 h in m i n / h -+ S.E.M.; T = period in h of significant h a r m o n i c terms; b = a m p l i t u d e o f t h e sine in m i n / h -+ I.C. (P = 0.95); p ffi phase (h) relatively t o local midnight ± I.C. (P = 0.95).
406
J. M O U R E T ET AL. 20
A
e14k
10
'0t
P$ o
I 60,
19...... 7
2
,lib
20
.,,.2@
10
,2".. " ' " : " . .
I'
$W$
!
19
7 Fig. 4. T h e o r e t i c a l r e c o n s t r u c t i o n of t h e t e m p o r a l v a r i a t i o n s of SWS a n d PS w i t h the significant harm'onic t e r m s p i c t u r e d with polar p l o t s (see Figs. 2 a n d 3). , control animal;------, partial lesion; . . . . . . , t o t a l lesions.
Discussion Fig. 3. Daily v a r i a t i o n s of h o u r l y PS scores. Same r e p r e s e n t a t i o n as Fig. 2. T h e radius o f the o u t e r circle c o r r e s p o n d s to 10 rain o f P S / h . T h e q u a n t i t a t i v e values are t h e following: Groups
a
T
b
A B C
4.1 ( 0 . 2 ) 4.1 ( 0 . 2 ) 4.3 ( 0 : 3 )
24 24 24 12
2.3 1.9 1 0.9
p (0.5) (0.6) (0.6) (0.6)
14.1 14:6 18.2 2.2
(0.7) (1.8) (2.2) (1.2)
drastic reduction of the light--dark differences in PS in group C. These considerations may be summarized when plotting the theoretical time course of SWS and PS, computed with the significant harmonic terms detected by the previous statistical analysis (Fig. 4). The circadian organization of PS is clearly affected by total SCN lesions, whereas that of SWS is almost unchanged under the same conditions.
Among the effects of the SCN lesions on sleep, the most important is the lack of quantitative variation in either SWS or PS on a 24 h base. This result is necessary in order to be certain that the circadian alterations observed are not simply the consequence of quantitative changes limited to one part o f the light cycle. Taken together with our previous data after pinealectomy (Mouret et al. 1974) and the results of Ibuka and Kawamura after SCN lesions (Ibuka and Kawamura 1975; Ibuka et al. 1977), this absence of quantitative alterations over the 24 h period favours a dissociation between the structures or mechanisms responsible for the production of sleep and those responsible for their circadian organization. On the one hand, due to the known control of pineal N-acetyltransferase r h y t h m s through SCN (Moore and Klein 1974), we may interpret the effects o f our lesions upon PS r h y t h m , which are similar to those observed after pinealectomy, as the result of the
SUPRACHIASMATIC NUCLEI LESIONS IN THE RAT destruction o f this control. On the other hand, after SCN lesions there is a clear-cut decrease in the amplitude of the circadian r h y t h m of SWS, which was not observed after pinealectomy. Since there is a high density of 5-HT terminals in the SCN (Fuxe 1965; Aghajanian et al. 1969; Saavedra et al. 1974) and since 5-HT is likely to be implicated in the control of SWS (Jouvet 1972}, this effect could be related to the destruction of these endings. Another possibility lies in the well known effect of SCN lesions on the circadian rhythms of drinking and activity (Stephan and Zugker 1972) which could be the cause or the consequence of the sleep alterations. Since it has been shown that f o o d may be as potent a Zeitgeber as light in the genesis of sleep rhythms (Mouret and Bobillier 1971), this possibility has to be discussed in relation to these previous data. When the feeding schedule is restricted to the illumination period there is an important increase in PS during the dark half of the cycle, which results in a global augmentation of PS on a 24 h base. Under these conditions the rats exhibit as much SWS during the dark period as in their usual period of illumination. Thus, if the changes observed after SCN lesions were the result of such a mechanism, we should have found a global increase in PS amounts. Could all these effects be the consequence of an abolition of the adreno-cortical rhythmicity which takes place after SCN lesions {Moore and Eichler 1972; Hal~sz 1972)? This is unlikely since, in our experiments, lesions caudal to the SCN, which are followed b y the abolition of the adreno-cortical r h y t h m (Hal~sz 1972), were w i t h o u t effect on sleep. It is interesting to note that the decrease in amplitude o f the circadian variations o f PS and SWS appears to be proportional to the size of the lesion, even though we did n o t study this point specifically. This progressive decrease may represent the central counterpart of the reduction o f amplitude of activity rhythms under reduced light intensity (Aschoff 1960), and may be interpreted as a
407 quantitative transduction of light information b y the SCN. The shift of the 24 h acrophase of the PS rhythm, while that o f SWS remains as in control conditions, and the detection of an unusual 12 h harmonic term in the PS timecourse after total SCN lesion, depict in fact a desynchronization o f these 2 sleep stages which are usually synchronous. We have no interpretation of this p h e n o m e n o n b u t it is interesting to note that the effects of lesions or manipulations o f the environment on sleep rhythms have always a preferential effect u p o n PS (Mouret and Bobillier 1971; Mouret et al. 1974) which appears to be more sensitive than SWS to these procedures. All these data show that some qualitative alterations of human sleep that may appear without quantitative alterations (Mouret et al. 1972) could be related to these synchronizing structures and n o t to disturbances in the sleep centres themselves. Summary After continuous control recording (24 h/ day) during 5 days, 14 rats received sham, complete or incomplete lesions of the suprachiasmatic nuclei (SCN). Sleep records were analysed from the 22nd to the 27th day after the lesion. In rats with complete lesions of the SCN, there was a strong decrease in the amplitude of the circadian variation in slow wave (SWS) and paradoxical (PS) sleep. The acrophase of SWS remained unaltered whereas that of PS shifted towards the end of the illumination (7.00--19.00 h) period. The importance o f these changes in sleep organization seems to be correlated with the size of the SCN lesion.
R~sum~ L~sions des noyaux suprachiasmatiques chez [e rat: aItdration du rythme circadien du sommeil Des ldsions completes ou incompl~tes des
408
noyaux suprachiasmatiques (NSC) ont fit~ rfialisfies sur 14 rats d~j~ chroniquement implantfis et enregistr~s en continu pendant 5 jours. Les r~sultats des enregistrements polygraphiques montrent que du 22e a~ 27e jour apr~s la l~sion, l'amplitude des variations circadiennes du sommeil lent (SL) et du sommeil paradoxal (SP) est tr~s fortement r~duite chez les rats ayant des l~sions completes des NSC. L'acrophase du SL ne change pas tandis que celle du SP se d~cale vers la fin de la p~riode d'~clairement (7.00--19.00 h). L'importance de ces variations semble ~tre fonction de l'atteinte plus ou moins importante des NSC. This work has been supported by grants from DRME (Contract 75520) and INSERM (U 52).
References Aghajanian, G.K., Bloom, F.E. and Sheard, M.H. Electron microscopy of degeneration within the serotonin pathway of rat brain. Brain Res., 1969, 13: 266--273. Anderson, T.W. The Statistical Analysis of Time Series. Wiley, New York, 1971, 704 pp. Aschoff, J. Exogenous and endogenous components in circadian rhythms. Cold Spr. Harb. Symp. quant. Biol., 1960, 25: 11--28. Coindet, J., Chouvet, G. and Mouret, J. Effects of suprachiasmatic nuclei lesions on paradoxical sleep and slow wave sleep circadian rhythms in the rat. Neurosci. Lett., 1975, 1: 243--247. De Groot, J: The Rat Forebrain in Stereotaxic Coordinates, North-Holland, Amsterdam, 1959, 40 pp. Fuxe, K. Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system. Acta physiol, scand., 1965, Suppl. 247: a9--85. Ha!~isz, B. Hypothalamic mechanisms controlling pituitary function. Progr. Brain Res., 1972, 38: 97--119. Ibuka, N. and Kawamura, H. Loss of circadian rhythm in sleep--wakefulness cycle in the rat by
J. MOURET ET AL. suprachiasmatic nucleus lesions. Brain Res., 1975, 96: 76--81. Ibuka, N., Inouye, S.T. and Kawamura, H. Analysis of sleep--wakefulness rhythms in male rats after suprachiasmatic nucleus lesions and ocular enucleation. Brain Res., 1977, 122: 33--47. Jouvet, M. The rote of monoamines and acetylchotine containing neurons in the regulation of the sleep-waking cycle. In: R.H. Adrian et al. (Eds.), Ergebnisse der Physiologie. Springer, Berlin, 1972: 166-307. Kan, J.P., Chouvet, G., Hdry, F., Debilly, G., Mermet, A., Glowinski, J. and Pujol, J.F. Daily variations of various parameters of serotonin metabolism in the rat brain. I. Circadian variations of tryptophan-5hydroxylase in the raphe nuclei and the striatum. Brain Res., 1977, 123: 125--136. Moore, R.Y. and Eichler, V.B. Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesion in the rat. Brain Res., 1972, 42: 201.--206. Moore, R.Y. and Klein, D.C. Visual pathways and the central neural control of a circadian rhythm in pineal serotonin-N-acetyltransferase activity. Brain Res., 1974, 71: 17--33. Moore, R.Y. and Lenn, N.J. A retino-hypothalamic projection in the rat. J. comp. Neurol., 1972, 146: 1--14 Mouret, J. and Bobillier, P., Diurnal rhythm of sleep in the rat: augmentation of paradoxical sleep following alterations of the feeding schedule. Int. J. Neurosci., 1971,2 : 265--270. Mouret, J., Renaud, B.. Quenin, P., Michel, D. et Schott, B. Monoamines et r~gulation de la vigilance. Apport et interprdtation biochimique des donndes polygraphiques. Rev. neurol., 1972, 127: 139--155. Mouret, J., Coindet, J. et Chouvet, G. Effet de la pin~alectomie sur les ~tats et rythmes de sommeil du rat m~le. Brain Res., 1974, 81 : 97--105. Roussel, B., Chouvet, G. et Debilly, G. Rythmes circadiens des temperatures internes et ambiance thermique chez le rat. Pfliigers Arch. ges. Physiol., 1976, 365: 183--189. Saavedra, J.M., Palkovits, M., Brownstein, M.J. and Axelrod, J. Serotonin distribution in the nuclei of the rat hypothalamus and preoptic region. Brain Res.,1974, 77 : 1 5 7 165. Stephan, F.K. and Zugker, I. Circadian rhythm in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc. nat. Acad. Sci. (Wash.), 1972, 69: 1583--1586.
Eleclroencephalography and Clinical Neurophysiology, 1978, 4 5 : 4 0 2 - - 4 0 8 © Elsevier/North-Holland Scientific Publishers, Ltd..
SUPRACHIASMATIC NUCLEI LESIONS IN THE RAT: ALTERATIONS IN SLEEP CIRCADIAN RHYTHMS J. MOURET, J. COINDET, G. DEBILLY and G. CHOUVET Ddpartement de Mddecine Expdrimentale, Universitd Claude-Bernard, 8, avenue Rockefeller, 693 73 Lyon Cedex 13 (France) (Accepted for publication: March 31, 1978)
Much progress has been made in recent years in understanding factors controlling sleep production, i.e., sleep amounts over a certain period of time (Jouvet 1972). In some species like the laboratory rat, the control of the circadian organization of sleep or waking is at least as important a feature as sleep production and can be affected without changes in either slow wave sleep (SWS) or paradoxical sleep (PS) amounts over successive 24 h periods; for instance pinealectomy is followed by disappearance of the circadian r h y t h m of PS whilst that of SWS is unaltered (Mouret et al. 1974). More recently Ibuka and Kawamura (1975), Ibuka et al. {1977) and our group (Coindet et al. 1975) have reported the loss of sleep-waking circadian rhythms after lesions of the suprachiasmatic nuclei (SCN) in the rat. These nuclei, which receive direct input from the retina (Moore and Lenn 1972; Moore and Klein 1974) and dense projections from the raphe nuclei (Fuxe 1965; Aghajanian et al. 1969; Saavedra et al. 1974), play an important role in the control of the r h y t h m of the pineal enzyme serotonin-N-acetyltransferase {Moore and Klein 1974) as well as in the genesis of the adrenal corticosteroid r h y t h m (Moore and Eichler 1972; Hal~sz 1972). In this paper we shall describe the effects of such lesions on SWS and PS amounts and r h y t h m s as well as the possible relationships between the reduction in amplitude of the
circadian variations and the size of the lesions in the SCN. Methods Fourteen male Wistar rats were used in this study. After 15 days to habituate to the laboratory environment (light/dark 12/12 h, light on at 7.00 a.m., food and water ad libitum, temperature 23_+ I°C), cortical and neck muscle electrodes were chronically implanted as previously described (Mouret et al. 1974). At the time of surgery their weight was about 220--230 g. They were immediately placed under the recording conditions, in single glass jars for 8 days, after which continuous polygraphic recording (24 h a day) was performed during 8 consecutive days. All records (at paper speed 5 mm/sec), were scored to the nearest minute for the presence of SWS, PS and waking. The data from visual analysis were stored in a computer for the determination of the amounts of sleep on different time bases, as well as for the study of their circadian rhythms. At the end of this control recording session, stereotaxic lesions aimed at the SCN (De Groot 1959) were made. In order to spare the superficial venous sinuses the angle o f the electrode was at 20 ° backward in the horizontal plane. In 3 rats no electrocoagulation was performed (sham rats); in the remaining 11 rats a 3 mA current was delivered for 15 sec.
SUPRACHIASMATIC NUCLEI LESIONS IN THE RAT
The rats were then replaced under the previous conditions and the records resumed for 30 days (24 h a day). At the end of the experiment the animals were killed for histological control of lesion placement. In order to quantify the circadian variations of sleep, statistical analysis was performed on hourly sleep scores according to a m e t h o d described elsewhere (Anderson 1971; Roussel et al. 1976; Kan et al. 1977), which consists in computing the best fitting curve b y least square harmonic decomposition (fundamental 24 h period T, plus harmonics at 12 h and 8 h). This m e t h o d allows the determination of the amplitude and phase of each significant (P ~< 0.05) harmonic term as well as their confidence intervals.
403
Results
According to the site of the lesion the rats could have been classified in 4 groups. However, since no changes in sleep amounts or rhythms were detected in 2 of these groups as compared with the control recording session, 3 groups were finally distinguished (Fig. 1): Group A, where no changes in sleep were detected and which will be referred to as 'control group', is composed of the 3 rats with sham lesions, and 4 rats with lesions sparing the SCN b u t located rostral or caudal to these nuclei. Group B (3 rats) is composed of the rats with incomplete lesions of the SCN, with or without extensions of the lesions caudal or rostral to these nuclei.
A 6.2
As e AT.o
A7.4 A .8 As.
A
B
C
Fig. 1. Schematic representation (De Groot atlas) of lesions in the 3 groups of rats. A: lesions localized rostrally and caudally to the SCN (4 rats). B: incomplete SCN lesions with extension rostrally and caudally to these nuclei (3 rats). C: complete SCN lesions with or without extension of the lesions (4 rats).
404
J. MOURET ET AL.
In G r o u p C are t h e 4 rats with c o m p l e t e lesions o f t h e SCN, w i t h or w i t h o u t e x t e n sions.
h a r m o n i c d e c o m p o s i t i o n (T = 24 h), t w o interesting p o i n t s e m e r g e f r o m t h e statistical analysis (Figs. 2 a n d 3): - - a s t h e size o f the SCN lesion increases t h e r e is a progressive decrease in t h e amplit u d e o f t h e circadian v a r i a t i o n s o f b o t h SWS a n d PS; t h e a c r o p h a s e o f SWS r e m a i n s u n a l t e r e d w h e r e a s t h a t o f PS shifts t o w a r d s t h e end o f t h e i l l u m i n a t i o n p e r i o d in g r o u p C ( 1 8 h). F u r t h e r m o r e , w h e n t a k i n g into a c c o u n t t h o s e o f t h e first 4 h a r m o n i c s w h i c h r e a c h a significant value (P < 0.05), the polar p l o t s clearly s h o w t h a t SCN lesions do n o t a f f e c t t h e non-sinusoidal p a t t e r n o f t h e t e m p o r a l v a r i a t i o n s o f SWS ( e x c e p t f o r t h e r e d u c t i o n o f t h e 24 h t e r m a m p l i t u d e ) , b u t s t r o n g l y dist u r b t h e t i m e c o u r s e o f PS: In t h e 3 groups, t h e t i m e c o u r s e o f SWS can be statistically fitted b y t h e linear c o m b i n a t i o n o f 2 t e r m s : t h e 24 h f u n d a m e n t a l r h y t h m plus an 8 h t e r m w i t h a p p r o x i m a t e l y t h e s a m e a m p l i t u d e and p h a s e in t h e 3 groups. This last t e r m is likely to be t h e c o n s e q u e n c e o f t h e l i g h t e n e f f e c t . It persists in t h e animals w i t h lesions and is clearly o b s e r v e d on the t i m e p l o t s o f t h e h o u r l y scores o f SWS. Whereas a p u r e sine f u n c t i o n (T = 24 h) fits t h e PS t i m e c o u r s e in g r o u p s A a n d B, an a d d i t i o n a l significant 12 h h a r m o n i c is p r e s e n t in g r o u p C, w h o s e a m p l i t u d e is as i m p o r t a n t as t h a t o f t h e 2 4 h h a r m o n i c . Since t h e s e t w o t e r m s are r o u g h l y o u t o f p h a s e t h e y result in a
Quantitative data In o r d e r to a v o i d t h e possible non-specific e f f e c t s o f t h e s u r g e r y , we shall r e p o r t here on t h e d a t a f r o m t h e 2 2 n d to t h e 2 7 t h d a y f o l l o w i n g t h e lesion, a period w h e n t h e sleep changes, if a n y , w e r e stabilized. As s h o w n in T a b l e I, no clear m o d i f i c a t i o n o f sleep a m o u n t s o v e r successive 24 h periods was o b s e r v e d in e i t h e r g r o u p . However, a preliminary estimate of the n y c t h e m e r a l variations, based o n t h e quantitative d i f f e r e n c e s b e t w e e n t h e i l l u m i n a t i o n a n d d a r k p e r i o d s , s h o w s t h a t certain f a c t o r s were clearly a f f e c t e d in g r o u p C (SWS, PS a n d n u m b e r o f PS episodes), w h e r e a s o n l y SWS was significantly a l t e r e d in g r o u p B. In b o t h g r o u p s these changes resulted in reduct i o n o f t h e ' d a y / n i g h t ' d i f f e r e n c e s . This reduct i o n c o u l d s i m p l y be t h e result o f a shift in the p h a s e o f t h e sleep r h y t h m , o r c o u l d be due to other factors.
Circadian rhythms A p r e l i m i n a r y individual s t u d y o f each rat d e m o n s t r a t e d t h a t all animals w i t h i n each g r o u p w e r e in phase, a result which a l l o w e d p o o l i n g i n t r a g r o u p d a t a f o r the analysis o f t e m p o r a l v a r i a t i o n s o f h o u r l y sleep scores. When c o n s i d e r i n g o n l y t h e first t e r m o f
TABLE I Sleep parameters in groups A, B, C (see text}, from the 22nd to the 27th day after SCN aimed lesion. PS, paradoxical sleep (in rain); N. PS, number of paradoxical sleep episodes; SWS, slow wave sleep (in rain). In each case the amounts for 24 h (24 h), for the 13 h of darkness (black circles)and the 12 h of illumination (open circles)are shown. Values represent the average of 35 measures for group A, 15 for grOuP B and 20 for group C. PS
A B C
N. PS
SWS
24 h
•
o
24 h
•
o
24 h
•
o
103 98 105
34 36 51 *
69 62 54 *
52 56 55
18 20 26 *
34 36 29 *
634 653 651
214 252 * 279 *
420 401 * 372 *
* Significant differences (P < 0.01) from the control group (t-test).
SUPRACHIASMATIC
N U C L E I L E S I O N S IN T H E R A T
405
A
19
60
30
60
7
lh
_
:.
.
:.
I1k
Fig. 2. Daily variations of hourly SWS scores in groups A, B and C£ Left: t e m p o r a l representation. Ordinates: h o u r l y a m o u n t s o f SWS in min/h. Abscissae: legal t i m e in hours. F o r a b e t t e r representation, the data are duplicated over 2 successive 24 h. Individual points represent the m e a n h o u r l y SWS scores over different animals for one day. The solid line illustrates the mean time course of SWS over 5 consecutive days. The best fitting sine curve (T = 24 h) is the only term s u p e r i m p o s e d u p o n the e x p e r i m e n t a l d a t a ! R i g h t : the respective polar plots indicate the amplitude and phase of each significant h a r m o n i c (p ~ 0.05). T h e r a d i u s of the o u t e r circle corresponds to an a m o u n t of 30 rain of SWS per hour. The inner circle represents the conf!dence area of each h a r m o n i c and gives the confidence intervals (I.C.) for the corresponding a m p l i t u d e and p h ~ e . F o r c o m b i n e d r h y t h m s (two significant terms), the c i r c u m f e r e n c e o f the o u t e r circle is equivalent to the period of the considered term, and the c o n f i d e n c e area of the significant h a r m o n i c t e r m is drawn with i n t e r r u p t e d lines. The black square on top of the o u t e r circle positions local m i d n i g h t (see Roussel et al. 1976 and Kan et al. 1977 for statistical details). The corresponding quantitative values are the following: Groups
n
a
T
b
p
A
7
26.2 (0.7)
B
3
27
C
4
27.1 (1.5)
24 8 24 8 24 8
11.5 (1.5) 3.1 (1.5) 8.5(L6) 1.8(1.6) 4.7 (2.2) 2.4 (2.2)
12.0 7.7 11.9 7.9 11.3 0.3
(1)
(0.5) (1.4) (0.7) (2.8) (1.8) (2.9)
n = n u m b e r of rats; a = hourly m e a n level over 24 h in m i n / h -+ S.E.M.; T = period in h of significant h a r m o n i c terms; b = a m p l i t u d e o f t h e sine in m i n / h -+ I.C. (P = 0.95); p ffi phase (h) relatively t o local midnight ± I.C. (P = 0.95).
406
J. M O U R E T ET AL. 20
A
e14k
10
'0t
P$ o
I 60,
19...... 7
2
,lib
20
.,,.2@
10
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19
7 Fig. 4. T h e o r e t i c a l r e c o n s t r u c t i o n of t h e t e m p o r a l v a r i a t i o n s of SWS a n d PS w i t h the significant harm'onic t e r m s p i c t u r e d with polar p l o t s (see Figs. 2 a n d 3). , control animal;------, partial lesion; . . . . . . , t o t a l lesions.
Discussion Fig. 3. Daily v a r i a t i o n s of h o u r l y PS scores. Same r e p r e s e n t a t i o n as Fig. 2. T h e radius o f the o u t e r circle c o r r e s p o n d s to 10 rain o f P S / h . T h e q u a n t i t a t i v e values are t h e following: Groups
a
T
b
A B C
4.1 ( 0 . 2 ) 4.1 ( 0 . 2 ) 4.3 ( 0 : 3 )
24 24 24 12
2.3 1.9 1 0.9
p (0.5) (0.6) (0.6) (0.6)
14.1 14:6 18.2 2.2
(0.7) (1.8) (2.2) (1.2)
drastic reduction of the light--dark differences in PS in group C. These considerations may be summarized when plotting the theoretical time course of SWS and PS, computed with the significant harmonic terms detected by the previous statistical analysis (Fig. 4). The circadian organization of PS is clearly affected by total SCN lesions, whereas that of SWS is almost unchanged under the same conditions.
Among the effects of the SCN lesions on sleep, the most important is the lack of quantitative variation in either SWS or PS on a 24 h base. This result is necessary in order to be certain that the circadian alterations observed are not simply the consequence of quantitative changes limited to one part o f the light cycle. Taken together with our previous data after pinealectomy (Mouret et al. 1974) and the results of Ibuka and Kawamura after SCN lesions (Ibuka and Kawamura 1975; Ibuka et al. 1977), this absence of quantitative alterations over the 24 h period favours a dissociation between the structures or mechanisms responsible for the production of sleep and those responsible for their circadian organization. On the one hand, due to the known control of pineal N-acetyltransferase r h y t h m s through SCN (Moore and Klein 1974), we may interpret the effects o f our lesions upon PS r h y t h m , which are similar to those observed after pinealectomy, as the result of the
SUPRACHIASMATIC NUCLEI LESIONS IN THE RAT destruction o f this control. On the other hand, after SCN lesions there is a clear-cut decrease in the amplitude of the circadian r h y t h m of SWS, which was not observed after pinealectomy. Since there is a high density of 5-HT terminals in the SCN (Fuxe 1965; Aghajanian et al. 1969; Saavedra et al. 1974) and since 5-HT is likely to be implicated in the control of SWS (Jouvet 1972}, this effect could be related to the destruction of these endings. Another possibility lies in the well known effect of SCN lesions on the circadian rhythms of drinking and activity (Stephan and Zugker 1972) which could be the cause or the consequence of the sleep alterations. Since it has been shown that f o o d may be as potent a Zeitgeber as light in the genesis of sleep rhythms (Mouret and Bobillier 1971), this possibility has to be discussed in relation to these previous data. When the feeding schedule is restricted to the illumination period there is an important increase in PS during the dark half of the cycle, which results in a global augmentation of PS on a 24 h base. Under these conditions the rats exhibit as much SWS during the dark period as in their usual period of illumination. Thus, if the changes observed after SCN lesions were the result of such a mechanism, we should have found a global increase in PS amounts. Could all these effects be the consequence of an abolition of the adreno-cortical rhythmicity which takes place after SCN lesions {Moore and Eichler 1972; Hal~sz 1972)? This is unlikely since, in our experiments, lesions caudal to the SCN, which are followed b y the abolition of the adreno-cortical r h y t h m (Hal~sz 1972), were w i t h o u t effect on sleep. It is interesting to note that the decrease in amplitude o f the circadian variations o f PS and SWS appears to be proportional to the size of the lesion, even though we did n o t study this point specifically. This progressive decrease may represent the central counterpart of the reduction o f amplitude of activity rhythms under reduced light intensity (Aschoff 1960), and may be interpreted as a
407 quantitative transduction of light information b y the SCN. The shift of the 24 h acrophase of the PS rhythm, while that o f SWS remains as in control conditions, and the detection of an unusual 12 h harmonic term in the PS timecourse after total SCN lesion, depict in fact a desynchronization o f these 2 sleep stages which are usually synchronous. We have no interpretation of this p h e n o m e n o n b u t it is interesting to note that the effects of lesions or manipulations o f the environment on sleep rhythms have always a preferential effect u p o n PS (Mouret and Bobillier 1971; Mouret et al. 1974) which appears to be more sensitive than SWS to these procedures. All these data show that some qualitative alterations of human sleep that may appear without quantitative alterations (Mouret et al. 1972) could be related to these synchronizing structures and n o t to disturbances in the sleep centres themselves. Summary After continuous control recording (24 h/ day) during 5 days, 14 rats received sham, complete or incomplete lesions of the suprachiasmatic nuclei (SCN). Sleep records were analysed from the 22nd to the 27th day after the lesion. In rats with complete lesions of the SCN, there was a strong decrease in the amplitude of the circadian variation in slow wave (SWS) and paradoxical (PS) sleep. The acrophase of SWS remained unaltered whereas that of PS shifted towards the end of the illumination (7.00--19.00 h) period. The importance o f these changes in sleep organization seems to be correlated with the size of the SCN lesion.
R~sum~ L~sions des noyaux suprachiasmatiques chez [e rat: aItdration du rythme circadien du sommeil Des ldsions completes ou incompl~tes des
408
noyaux suprachiasmatiques (NSC) ont fit~ rfialisfies sur 14 rats d~j~ chroniquement implantfis et enregistr~s en continu pendant 5 jours. Les r~sultats des enregistrements polygraphiques montrent que du 22e a~ 27e jour apr~s la l~sion, l'amplitude des variations circadiennes du sommeil lent (SL) et du sommeil paradoxal (SP) est tr~s fortement r~duite chez les rats ayant des l~sions completes des NSC. L'acrophase du SL ne change pas tandis que celle du SP se d~cale vers la fin de la p~riode d'~clairement (7.00--19.00 h). L'importance de ces variations semble ~tre fonction de l'atteinte plus ou moins importante des NSC. This work has been supported by grants from DRME (Contract 75520) and INSERM (U 52).
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