Presence of a paradoxical sleep (PS) factor in the cerebrospinal fluid of PS-deprived rats

European Journal of Pharmacology, 100 (1984) 223-226

223

Elsevier Short communication P R E S E N C E OF A P A R A D O X I C A L S L E E P (PS) F A C T O R IN T H E C E R E B R O S P I N A L F L U I D OF PS-DEPRIVED RATS JOELLE ADRIEN * and CHRISTINE DUGOVIC INSERM U3, 91 Bd de l'Hbpital, 75013 Paris, France

Received 16 January 1984, revised MS received 7 February 1984, accepted 21 February 1984

J. ADRIEN and C. DUGOVIC, Presence of a paradoxical sleep (PS) factor in the cerebrospinal fluid of PS-deprived rats, European J. Pharmacol. 100 (1984) 223-226. The lack of paradoxical sleep (PS) observed in rats after pharmacological blockade of the noradrenergic/3-receptors was reversed by intraventricular infusion of cerebrospinal fluid (CSF) from PS-deprived donor rats. The PS restoration in recipient animals was proportional to the duration of the deprivation in donors. It is concluded that some PS-inducing factor progressively accumulates in the CSF during deprivation. This factor acts beyond the noradrenergic step in the regulation of PS. Sleep factor

fl-Receptor

Cerebrospinal fluid

I. Introduction

Numerous studies have established that the noradrenergic (NA) system is involved in the regulation of paradoxical sleep (PS) (Jouvet, 1969, ref. in Lanfumey and Adrien, 1982). In particular, pharmacological blockade of adrenergic receptors has been shown to provoke sleep impairments in rats (Lanfumey and Adrien, 1982). On the other hand, several experiments have suggested that sleep inducing factors are present in the cerebrospinal fluid (CSF): the PS rebound which follows PS deprivation suggests that such factors accumulate in the CSF during the period of deprivation (Morden et al., 1967). Similarly, the transfer of CSF from a sleep-deprived donor to recipient rats increases slow waves sleep (SWS) in the recipients (Fencl et al., 1971). And finally, in the cat the para-chlorophenylalanine (PCPA)-induced insomnia can be reversed by intracerebroventricular infusion of CSF from PS-deprived donor cats (Sallanon et al., 1982). This lact experi-

* To whom all correspondence should be addressed. 0014-2999/84/'$03.00 © 1984 Elsevier Science Publishers B.V.

Sleep deprivation

ment seems to demonstrate a link between the serotoninergic system and the synthesis a n d / o r liberation of some sleep factor(s) in the CSF of cats. Thus, the question was raised if the N A system could regulate sleep by the liberation of a sleep factor. In order to test this possibility we have examined the effects of transfusing CSF from PSdeprived rats to recipient rats suffering from insomnia due to blockade of the NA system by propranolol (Lanfumey and Adrien, 1982). Our results demonstrate that the lack of PS due to propranolol blockade of the fl-receptors can indeed be reversed by intraventricular infusion of CSF from PS-deprived rats and that the amount of PS restored in the recipient animal is directly dependent on the duration of PS deprivation in the donor.

2. Materials and methods

A total of 18 rats (Sherman, 250-350 g) were used in this experiment. Under chloral hydrate anaesthesia electrodes for polygraphic sleep moni-

224 toring were i m p l a n t e d s u b c u t a n e o u s l y in the neck muscles, at the edge of the orbit a n d on the frontal a n d occipital cortices. In a d d i t i o n , a stainless steel c a n n u l a (0.9 m m external d i a m e t e r ) was stereotaxically p o s i t i o n e d in the fourth ventricle to allow infusion of CSF. In the recipient group (10 rats) the p h a r m a c o l o g i c a l p r o t o c o l was carried out after 8-10 days of h a b i t u a t i o n to the recording conditions (12 h light-dark schedule, light from 7 a.m. to 7 p.m., food a n d water ad l i b i t u m as follows: i n t r a p e r i t o n e a l injection of D L - p r o p r a n o l o l (10 m g / k g ) at 1 0 : 4 5 a.m., a n d i n t r a v e n t r i c u l a r infusion (5 min d u r a t i o n ) of 20 B1 C S F from a PS-dep r i v e d d o n o r at 1 1 : 0 0 a.m. P o l y g r a p h i c recordings were p e r f o r m e d from 9 : 30 a.m. to 7 : 00 p.m. Each recipient rat was allowed at least one d a y without p r o p r a n o l o l t r e a t m e n t between two exp e r i m e n t s in o r d e r not to create m o d i f i c a t i o n s of fl-receptor sensitivity. The C S F , p u n c t u r e d from 1 0 : 3 0 to 1 0 : 4 5 through the c a n n u l a by m e a n s of a H a m i l t o n syringe was p r o v i d e d by 8 d o n o r rats which h a d u n d e r g o n e the same surgical a n d h a b i t u a t i o n procedures, a n d which had been selectively d e p r i v e d of PS d u r i n g 1, 2, 3 or 4 d a y s using the water t a n k m e t h o d with a p e d e s t a l of 5.5 cm

d i a m e t e r : it was assumed that if a sleep factor was a c c u m u l a t i n g in the C S F of these d o n o r animals, its c o n c e n t r a t i o n should increase with the length o f the d e p r i v a t i o n as long as the PS r e b o u n d d e p e n d s on the d u r a t i o n of the d e p r i v a t i o n (ref. in M o r d e n et al., 1967). In the d o n o r group, sleep was m o n i t o r e d d u r i n g the d e p r i v a t i o n to check for the absence of PS, and d u r i n g the first 6-8 h of r e b o u n d . In the recipient group the restoration of sleep was a n a l y z e d as a function of the ' d o s e ' of sleep factor transferred and c o m p a r e d with baseline recordings (saline injection).

3. Results Sleep scoring in the donor group i n d i c a t e d that rats were d e p r i v e d of 93% of PS a n d only of 7% of SWS. As expressed in fig, 1A, the PS r e b o u n d s obt a i n e d d u r i n g the first 6 h of the sleep recovery p e r i o d increased with the d u r a t i o n of the d e p r i v a tion, suggesting that the a m o u n t s of sleep factor(s) in the C S F of the a n i m a l s were increasing similarly. In the p r o p r a n o l o l p r e t r e a t e d recipient rats

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after PS deprivatioon of different duration. The typical PS rebounds observed suggest a progressive accumulation of some PS factor. (B) PS restoration in propranolol-pretreated recipient rats following transfer of 20 #1 CSF from PS-deprived donors. PS amounts are calculated for the 6 h period following the transfer, and are expressed in percent of baseline recordings where the animals received the dose 0 of propranolol and 20 ,ttl of artificial CSF (the average PS amount in these baseline conditions was of 39 min per 6 h). Dots represent individual tests, and the line is the mean of all tests for a given duration of PS deprivation in donors.

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4. Discussion

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Fig. 2. Examples of hypnograms obtained from one rat during 6 h post-infusion of CSF. Top: Baseline recording (saline injection). Middle: The animal was pretreated with 10 m g / k g of DL-propranolol, and infused with CSF from a non-deprived donor. Bottom: The rat underwent the same protocol but the donor was PS-deprived during 4 days before the CSF transfer.

the results were the following (figs. 1B and 2): in agreement with a previous study (Lanfumey and Adrien, 1982), 10 m g / k g of DL-propranolol induced a PS insomnia which lasted 6 h (PS amounts reduced to 30% of baseline), with no modifications in SWS (225 _+ 22 min during the 6 h post-treatment). Infusion of artificial CSF or of CSF from non-deprived donors (dose 0) did not modify this sleep pattern, but transfer of CSF from PS deprived donors induced a progressive restoration of PS with no change in SWS. The longer the PS deprivation in the donor, the more PS was restored in the recipient rat, with amounts reaching almost normal values with 20 t~l of CSF from a donor deprived of PS for 4 days. This hypnogenic effect was highly significant (P < 0.001) as demonstrated by the regression test (correlation coefficient = 0.8). The PS restoration occurred mainly between 2 and 6 h after the CSF transfer. It was achieved by an increase in the number of episodes but without statistically significant modification of their mean duration. The latency of the first PS episode after treatment was very variable and it showed a trend, though not statistically significant, to be shorter with higher 'doses' of sleep factor (4-5 h with dose 0, versus 1-2 h after 4 days deprivation).

We had previously demonstrated that it was possible to reverse the propranolol-induced lack of PS in rats by infusing the/~-agonist isoproterenol directly into the cerebroventricular space (Lanfumey and Adrien, 1982). In these experiments, the agonist competes with the antagonist at the receptor site, and with sufficient amounts of agonist it is possible to restore the functional state of the NA system. In the present report, it is demonstrated that the lack of PS due to the blockade of/~-receptors is reversed by the transfer of CSF from PS-deprived rats. These data support the hypothesis that some sleep factor(s) accumulate(s) progressively in the CSF during PS deprivation; factor(s) which would antagonize the adrenergic blockade of the central mechanisms leading to the appearance of PS in the recipient rat. It could be argued that the restoration of PS in recipient rats is due only to the accumulation of NA in the CSF of donors, thus acting as an agonist to stimulate the receptor sites. This is unlikely since it has been demonstrated that NA levels in the brain are not modified by PS deprivation (Stern et al., 1971). Even if the NA turnover is increased (Pujol et al., 1968; Schildkraut and Hartmann, 1972) it occurs at synaptic sites and not in CSF. It should be emphasized that if some sleep factor accumulates during PS deprivation, such a factor certainly is very potent since a transfer of just 20 ttl CSF restores normal amounts of PS. Twenty btl represent about one tenth of the total volume of CSF of the rat brain. The volume of 20 ~1 was chosen because it had previously been demonstrated to not induce sleep impairments by a simple mechanical action (Borbely and Tobler, 1979). Since infusion of a constant volume of CSF from PS-deprived donors progressively restored PS in the recipient rat as a function of the duration of deprivation the sleep modifications observed are not due to CSF volume changes per se. It remains possible that the sleep factor is liberated in the CSF through the choroid plexus under the control of the NA system. This agrees with recent data showing that the secretory function of the choroid plexus is mediated by/~-receptors (Lindvall et al., 1978).

226

To conclude, the present study suggests that some PS factor(s) accumulates in the CSF of rats during selective PS deprivation. When transferred from PS-deprived animals to rats whose NA system had been functionally blocked this factor was able to progressively restore normal amounts of PS in parallel with the duration of the deprivation in donors. This hypnogenic effect probably by-passes the noradrenergic step in the regulation of PS and it appears interesting to investigate it further in future research on insomnia.

Acknowledgement We are grateful to J.R. Theilhac for his excellent technical assistance.

References Borbely, A.A. and 1. Tobler, 1979, Cerebroventricular infusion in the rat, depression of motor activity and paradoxical sleep, Neurosci. Lett. 12, 75.

Fencl, V., G. Koski and J.R. Pappenheimer, 1971. Factors in cerebrospinal fluid from goat that affect sleep and activity in rats, J. Physiol. 216, 565. Jouvet, M., 1969, Biogenic amines and the states of sleep, Science 163, 32, Lanfumey, L. and J. Adrien, 1982, Regulation of sleep after neonatal locus coeruleus lesion: functional evidence of fladrenergic supersensitivity, European J. Pharmacol. 79, 257. Lindvall, M., E. Edvinson and C. Owman, 1978, Sympathetic nervous control of cerehrospinal fluid production from the choroid plexus, Science 201, 176. Morden, B., G. Mitchell and W. Dement, 1967, Selective REM deprivation and compensation phenomena in the rat, Brain Res. 5, 339. Pujol, J.F., J. Mouret, M. Jouvet and J. Glowinski, 1969, Increased turnover of cerebral norepinephrine during rebound of paradoxical sleep in the rat, Science 159, 112. Sallanon, M., C. Buda, M. Janin and M. Jouvet, 1982, Restoration of paradoxical sleep by cerebrospinal fluid transfer to PCPA-treated insomniac cats, Brain Res. 251, 137. Schildkraut, J.J. and E. Hartmann, 1972, Turnover and metabolism of norepinephrine in rat brain after 72 hours on a D-deprivation island, Psychopharmacol. 27, 17. Stern, W.C., F.P. Miller, R.H. Cox and R.P. Maickel, 1971, Brain norepinephrine and serotonin levels following REM sleep deprivation in the rat, Psychopharmacologia (Berlin) 22, 50.