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Sleep induced by low doses of apomorphine in rats
214
Electroencephalography and Clinical Neurophysiology, 1979, 46:214--219
© Elsevier/North-Holland Scientific Publishers, Ltd.
SLEEP INDUCED BY LOW DOSES OF APOMORPHINE IN RATS I G.P. MEREU, E. SCARNATI 2, E. PAGLIETTI, B. PELLEGRINI QUARANTOTTI 3, P. CHESSA, G. DI CHIARA and G.L. GESSA Institute of Pharmacology, University of Cagliari, Cagliari (Italy) (Accepted for publication: July 4, 1978)
Apomorphine, a direct stimulant of dopamine (DA) receptors (Ernst and Smelik 1966; And~n et al. 1967; Ernst 1967), exerts two opposite effects on the behaviour of experimental animals: in low doses it decreases m o t o r activity whereas in higher doses it produces hypermotility and stereotypy (Fog 1969; Str6mbom 1976). While it has been clearly demonstrated that the stimulant effect is due to the activation of postsynaptic DA receptors in the CNS (Ungerstedt 1971) it has been recently suggested that the effect of low doses of apomorphine is mediated by preferential stimulation of a special kind of DA receptor, called by Carlsson 'autoreceptors', which results in inhibition both of DA synthesis and of electrical activity of DA neurones (Kehr et al. 1972; Aghajanian and Bunney 1973; Carlsson 1975). These receptors would be a means by which these neurones could control their own activity (firing, DA synthesis and release) via DA released by the nerve endings or by dendrites in the substantia nigra (Aghajanian and Bunney 1973; Carlsson 1975; Groves et al. 1975; Geffen et al. 1976). Direct evidence that the decreased m o t o r activity is due to the stimulation of DA receptors has been recently provided (Di Chiara et
1 This investigation was supported by Tecnofarmaci S.p.A. Pomezia (Rome), Italy. 2 Present address: Institute of Physiology, Faculty of Medicine, L'Aquila, Italy. 3 Present address: Tecnofarmaci S.p.A., Pomezia (Rome), Italy.
al., 1976). However, it is not clear whether the decreased m o t o r activity reflects a true sedative or hypnotic effect. On the basis of these considerations we studied the effect of low doses of apomorphine on the EEG patterns of freely moving rats and the influence of neuroleptics on this effect.
Materials and methods Twenty-four male Sprague-Dawley rats (300--320 g) had electrodes chronically implanted in the cortex and hippocampus, according to the usual techniques for sleep recording in the rat (Michel et al. 1963; Roldan et al. 1963). An electromyogram (EMG) was recorded by means of 2 stainless steel needles implanted into the neck muscles at the beginning of each recording session. After surgery, each rat was individually housed, for the entire study period of 3--4 months, in a single cage at 24°C, with reversed light
SLEEP INDUCED BY APOMORPHINE
215
subcutaneously in the neck area through a long Teflon tube (0.1 mm internal diameter) which did not disturb the animal's movements. In a first study the animals were injected with different doses of apomorphine (1000, 500, 100, 50, 25, 12.5 and 6.2 #g/ml/ kg, within 2 min) and the EEG was recorded for 60 min thereafter. Recording was not prolonged further because apomorphine effects are of short duration due to the short half-life of the drug. After completion of the first study, a second investigation was carried out, with the same animals, to clarify the influence of different neuroleptics on the hypnotic effect of apomorphine. Pimozide (50 pg/kg), benzperidol (50 pg/kg) and L-sulpiride (500 pg/kg) were given intraperitoneally (120, 30 and 30 min before apomorphine, respectively). Pimozide was dissolved in H20 with the addition of glacial acetic acid and the solution brought to pH 5 with NaOH • L-sulpiride and benzperidol were dissolved in H20. In a third study, the effect of a continuous apomorphine infusion on EEG was studied. Apomorphine was infused for 4 h at a dose of 80 pg/600 pl/kg/h, and recording was done for the same time, starting at 15.00, at the
beginning of the infusion. Control rats were infused with the solvent alone. Cortical and hippocampal EEG and neck EMG records were used for classifying the animal's state as either awake (W), in synchronized (slow) sleep (S) or desynchronized sleep (REM) according to classical criteria (Michel et al. 1963; Roldan et al. 1963). Records were visually scored to the nearest 3 sec interval. Levels of significance were determined b y Student's t test.
Results
(1) Effect of apomorphine injection on EEG Apomorphine was injected after 15 min of control recording, and the record continued for 60 min thereafter. Table I shows the effect of the different subcutaneous doses of apomorphine, given acutely, on the EEG pattern of the rat. Doses of apomorphine of 1.0 or 0.5 mg/kg produced stereotyped behaviour. Moreover, in agreement with previous results (Kafi and Gaillard 1976), these doses caused a marked reduction of the total sleep. In fact, the percentage of total sleep, over the 60 min recording time, fell from 18.24%, in control rats, to 4.22% after 1.0 mg/kg, and to
TABLE I Effect of different doses of apomorphine reported number (No.) of records. Apomorphine ( p g / k g s.c. )
No. of records
No. of animals
o n s l e e p in t h e r a t . E a c h v a l u e is t h e m e a n + S.E. o b t a i n e d f r o m t h e
% o f t o t a l r e c o r d i n g t i m e (1 h ) spent in: Total sleep
Solvent 1000 500 100 50 25 12.5 6.2
10 4 4 8 8 8 8 4
8 4 4 7 8 7 6 4
18.24 4.22 10.36 48.25 53.57 54.36 31.76 20.03
-+ 2 . 2 0 + 0.85 -+ 0 . 9 0 +- 5 . 1 3 _+ 4 . 8 0 + 7.85 _+ 4 . 8 2 + 1.35
* P < 0 . 0 0 1 , ** P < 0 . 0 1 ; w i t h r e s p e c t t o s a l i n e - t r e a t e d a n i m a l s .
* ** * * * **
Slow (S) sleep
Fast (D) sleep
16.26 4.22 8.90 46.53 50.06 49.78 29.30 17.95
1 . 9 8 +_ 0 . 3 3
_+ 2 . 0 7 _+ 0 . 8 5 -+ 1 . 3 5 _+ 4 . 2 4 _+ 4 . 2 1 _+ 5 . 1 2 +_ 4 . 1 8 _+ 1 . 3 0
* ** * * * *
1.46 1.72 3.51 4.58 2.26 2.08
_+ 0 . 5 3 _+ 0 . 3 3 _+ 0 . 4 8 * _+ 0 . 5 8 * +_ 0 . 3 4 + 0.22
216
G.P. MEREU ET AL.
10.36% a f t e r 0.5 m g / k g o f a p o m o r p h i n e , respectively. A f t e r 1.0 m g / k g o f a p o m o r p h i n e , t h e residual a m o u n t o f sleep was due to a brief episode o f slow sleep w h i c h usually o c c u r r e d e i t h e r a few m i n u t e s a f t e r treatment,, or at t h e end o f t h e recording period, i.e., p r e s u m a b l y at t h e t i m e w h e n t h e a p o m o r p h i n e level in t h e CNS fell into a range o f c o n c e n t r a t i o n s low e n o u g h t o elicit centxal depression. In c o n t r a s t to the above results, doses o f a p o m o r p h i n e o f 100 pg/kg or less caused behavioural sedation and increased the a m o u n t o f t o t a l sleep. This e f f e c t was already m a x i m a l with a dose o f a p o m o r p h i n e as low as 25.0 pg/kg, which increased the a m o u n t o f t o t a l sleep to 54% o f t h e r e c o r d i n g time. T h e e n h a n c e m e n t o f t o t a l sleep c o r r e s p o n d e d m a i n l y t o S (90% o f t o t a l sleep). H o w e v e r , the small doses o f a p o m o r p h i n e also increased t h e a m o u n t o f REM sleep; usually o n e or 2 episodes o f REM sleep o c c u r r e d t o w a r d t h e end o f t h e recording period. Following small doses o f a p o m o r p h i n e , the b e h a v i o u r o f t h e animal c o r r e s p o n d e d t o t h e EEG activity. Moreover, as previously r e p o r t e d (Baraldi and Benassi-Benelli 1 9 7 5 ) , small doses o f a p o m o r p h i n e caused r e c u r r e n t episodes o f y a w n i n g and penile erection. This e f f e c t was m o r e c o n s i s t e n t l y e v o k e d b y the dose o f 50 gg/kg. Each episode had a d u r a t i o n
of a few m i n u t e s . Yawning and e r e c t i o n o c c u r r e d w h e n t h e animal was fully awake, or s o o n after an episode o f S or REM sleep. In the latter t w o instances the behavioural change was associated with cortical arousal. H o w e v e r , cortical d e s y n c h r o n i z a t i o n did n o t seem to be t h e c o n s e q u e n c e of t h e behavioural change, for these events began t o g e t h e r and, at times, cortical arousal p r e c e d e d the behavioural change.
(2) Effects o f neuroleptics on apomorphine response T o clarify the d o p a m i n e - m i m e t i c n a t u r e o f the h y p n o t i c e f f e c t o f low doses o f a p o m o r phine, we studied w h e t h e r this e f f e c t m i g h t be p r e v e n t e d b y d i f f e r e n t n e u r o l e p t i c s such as p i m o z i d e , b e n z p e r i d o l and L-sulpiride. These results are r e p o r t e d in Tables II and III. As previously r e p o r t e d {Kafi and Gaillard 1976), neuroleptics, with t h e low dose and t h e t i m e schedule used, did n o t influence the overt b e h a v i o u r o f t h e animals and did n o t m o d i f y the a m o u n t o f t o t a l sleep, o f S and o f REM sleep o f t h e animals. H o w e v e r , t h e y p r e v e n t e d the hypnotic effect of apomorphine.
(3) Effect of apomorphine infusion on the EEG Because o f the short half-life o f a p o m o r phine, to clarify t h e e f f e c t o f this drug o n the sleep of the rat, we studied t h e E E G changes
TABLE II Antagonism by neuroleptics of the hypnotic effect of apomorphine 25 pg/kg s.c. Each value is the mean + S.E. obtained from the reported number of records. Neuroleptic (pg/kg i.p. )
Saline Pimozide Benzperidol L-sulpiride
-50 50 500
Time before apomorphine or solvent (min)
Total sleep as % of total recording time (1 h) Solvent
No. of records
Apomorphine
No. of records
30 120 30 30
16.32 18.12 17.54 20.85
4 4 4 4
56.43 28.35 21.75 29.32
8 8 8 8
* P < 0.001,with respect to solvent t r e a t m e n t . ** P < 0.001, with respect to apomorphine alone.
+_1.58 + 3.12 +_2.84 ± 4.31
_+8.34 +_3.86 ± 4.35 ± 4.32
* ** ** **
S L E E P I N D U C E D BY A P O M O R P H I N E
217
T A B L E III A n t a g o n i s m o f t h e h y p n o t i c e f f e c t o f a p o m o r p h i n e b y p i m o z i d e . E a c h value is t h e m e a n -+ S.E. o b t a i n e d f r o m t h e n u m b e r o f r e c o r d s r e p o r t e d in T a b l e II. Treatment
Sleep d u r a t i o n (in sec) in each 10 rain interval a f t e r t r e a t m e n t
Solvent Pimozide Apomorphine Pimozide + apomorphine
0--10
10--20
20--30
30--40
40--50
50--60
Total sleep in 60 m i n
0 0 370±31" 0
0 0 380±29" 1 6 0 ± 20
61±12 1 2 0 ± 15 400_+50' 215 ± 26
169±24 200 ± 18 470±41 * 165 ± 20
280_+17 130 ± 15 315_+30 290 ± 24
50± 6 210 ± 15 165±18 220 ± 29
5 6 0 ± 48 1 6 6 0 ± 41 2100±299" 1050 ± 130
* P < 0.001 w i t h respect t o p i m o z i d e + a p o m o r p h i n e .
T A B L E IV Effect of a p o m o r p h i n e i n f u s i o n (80 p g / k g / h ) d u r i n g 4 h, o n t h e sleep p a t t e r n . Each value is t h e m e a n ± S.E. obtained f r o m 8 r e c o r d s ( e a c h o n a d i f f e r e n t animal). L a t e n c y (in r a i n ) t o first S (slow) sleep e p i s o d e : 24 + 4, 12 -+ 2 for solvent a n d a p o m o r p h i n e , respectively. L a t e n c y (in m i n ) to first D (fast) sleep episode: 96 ± 12, 31 ± 5 for solvent a n d a p o m o r p h i n e , respectively. Mean d u r a t i o n of D episodes (in sec) = 60 ± 9, 74 ± 10 for solvent a n d a p o m o r p h i n e , respectively. Treatment
Hour
% of r e c o r d i n g t i m e s p e n t in T o t a l sleep
S sleep
D sleep
Solvent
1st 2nd 3rd 4th Total
36.26 33.54 23.19 30.41 30.85
± ± ± ± ±
4.15 3.41 2.72 4.04 3.58
35.06 29.39 19.05 27.22 27.68
_+ 4.90 ± 4.15 _+ 2.84 ± 3.35 ± 3.81
1.20 4.15 4.14 3.19 3.17
+0.10 ± 0.89 ± 0.76 _+ 0.65 _+ 0.60
Apomorphine
1st 2nd 3rd 4th Total
57.44 65.70 67.26 62.12 63.13
_+ 7.32 ± 7.10 _+ 8.11 _+ 6.31 +_ 7.21 *
51.06 59.74 58.42 56.18 56.35
_+ 3.62 _+ 4.49 _+ 5.40 ± 5.01 _+ 4.63 *
6.38 5.96 8.84 5.94 6.78
+ 0.92 +_ 0.68 _+ 1.29 _+ 0.91 _+ 0.95
* P < 0.001 w i t h r e s p e c t to s o l v e n t infusion.
produced b y a continuous subcutaneous infusion of a sedative dose of the drug. As a result (Table IV and Fig. 1) the following changes occurred during the infusion of apomorphine: (1) the latencies to the first episodes of S and REM sleep were significantly
REM
"11 SOLVENT
i
U
~
LI
U,
U
,
SS R EM
o
1
2
3
4
hours
Fig. 1. Typical h y p n o g r a m s o f rats infused w i t h solv e n t a n d a p o m o r p h i n e (80 pg/kg/h) d u r i n g 4 h. W, waking; SS, slow sleep; REM, fast sleep.
218 reduced; (2) the times spent in total sleep, in S and in REM sleep were approximately doubled with respect to rats infused with the solvent; (3) the mean duration of each episode of REM sleep was slightly increased.
G.P. MEREU ET AL. It is particularly important, both for practical and theoretical reasons, that the effect of apomorphine on sleep is not restricted to rats, since low, non-emetic doses of apomorphine cause sedation and sleep in man as well (Corsini et al. 1977).
Discussion Summary Our results show that apomorphine in high doses suppresses sleep. On the contrary, small doses of apomorphine produce an increase in both S and REM sleep durations in the rat. As a sleep-inducing agent apomorphine is, to our knowledge, the most potent drug known. The finding that pimozide, L-sulpiride and benzperidol, selective blockers of dopamine receptors, prevent the hypnotic effect of apomorphine, suggests that this effect is due to the stimulation of central dopamine receptors. As to the nature of these receptors, they might be identified with the DA receptors named 'autoreceptors', whose activation results in inhibition of DA synthesis and dopaminergic firing (Aghajanian and Bunney 1973; Carlsson 1975). Alternatively, they might be a special kind of postsynaptic DA receptors, different from the DA receptors responsible for stereotypy, motor stimulation and arousal, because of a greater sensitivity to the action of the transmitter. The distinction has a physiological implication: if sleep originates from the inhibition of firing of DA neurones (i.e. from stimulation of autoreceptors), this implies that DA plays an inhibitory role on sleep. Vice versa, if sleep is due to the stimulation of some kind of postsynaptic receptors, then the DA system plays a dual role on sleep-wakefulness mechanisms: promoting and antagonistic effects in low doses and high doses, respectively. In order to clarify this problem we are currently investigating the effect of low doses of apomorphine in rats in which DA neurones axe destroyed by the intranigral or intraventricular administration of 6-hydroxydopamine.
The effect of apomorphine on the EEG of freely moving rats was studied. Apomorphine at the dose of 1 mg/kg caused stereotypy and a marked reduction of total sleep. On the contrary, acute subcutaneous administration of apomorphine at the dose of 100 pg/kg, or less, markedly increased the amount of total sleep (corresponding mostly to synchronized sleep). Moreover, the infusion of apomorphine (80 pg/kg/h) for 4 h doubled the duration of slow and REM sleep. The hypnotic effect of apomorphine was prevented by neuroleptics, such as pimozide, benzperidol and L~ulpiride, at doses which, per se, did not modify the EEG of the animals. These results suggest the existence in the CNS of DA receptors mediating sleep.
R6sum6 Sommeil induit par petites doses d'apomorphine chez les rats L'effet de l'apomorphine sur I'EEG de rats libres de leurs mouvements a ~t~ ~tudi~ pendant l'heure suivant le traitement. L'apomorphine ~ la dose de 1 mg/kg cause une st~r~otypie et une r~duction marquee du sommeil total. Au contraire, l'injection sous-cutan~e aigu~ d'apomorphine ~ la dose de 100 pg/kg, ou inf~rieure, a fortement augment~ la quantit6 de sommeil (correspondant surtout au sommeil lent). Par ailleurs, l'infusion d'apomorphine (80 pg/kg/h), pendant 4 h , a doubl~ la dur6e du sommeil lent et rapide. L'effet hypnotique de l'apomorphine a ~t6
SLEEP INDUCED BY APOMORPHINE e m p 6 c h d p a r d e s n e u r o l e p t i q u e s t e l q u e le p i m o z i d e , le b e n z p d r i d o l e t le L - s u l p i r i d e d e s d o s e s qui, p a r e l l e s - m ~ m e s , n ' o n t p a s modifi~ I'EEG de l'animal. Les rdsultats indiq u e n t l ' e x i s t e n c e , d a n s le S N C , d e r ~ c e p t e u r s d o p a m i n e r g i q u e s i m p l i q u ~ s d a n s le s o m m e i l . We thank Professor Peiro Fresia, 'Ravizza S.P.A.', Muggi6 (Milan) Italy, for supplying L-sulpiride, the active form of sulpiride.
References Aghajanian, G.K. and Bunney, B.S. Central dopaminergic neurons: neurophysiological identification and responses to drugs. In: S.H. Snyder and E. Usdin (Eds.), Frontiers in Catecholamine Research. Pergamon Press, New York, 1973: 643-648. And~n, N.-E., Rubenson, A., Fuxe, K. and HSkfelt, T. Evidence for dopamine receptor stimulation by apomorphine, J. Pharm. Pharmacol., 1967, 19: 627--629. Baraldi, M. e Benassi-Benelli, A. Induzione di erezioni ripetute nel ratto adulto mediante apomorfina. Riv. Farm. Ter., 1975, VI: 147--149. Carlsson, A., Receptor-mediated control of dopamine metabolism. In: E. Usdin and W.E. Bunney, Jr. (Eds.), Pre- and Postsynaptic Receptors. Marcel Dekker, New York, 1975: 49--65. Corsini, G.U., Del Zompo, M., Manconi, S., Cianchetti, C., Mangoni, A. and Gessa, G.L. Sedative hypnotic and antipsychotic effect of low doses of apomorphine in man. In: E. Costa and G.L. Gessa (Eds.), Advances in Biochemical Psychopharmacology, Vol. 16. Raven Press, New York, 1977 : 645-648. Di Chiara, G., Porceddu, M.L., Vargiu, L., Argiolas, A. and Gessa, G.L. Evidence for dopamine recep-
219 tots in the mouse brain mediating sedation. Nature (Lond.), 1976, 264: 564--567. Ernst, A.M. Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats. Psychopharmacologia (Berl.), 1967, 10: 316--323. Ernst, A.M. and Smelik, P.G. Site of action of dopamine and apomorphine on compulsive gnawing behaviour in rats. Experientia (Basel), 1966, 22: 837--838. Fog, R. Stereotyped and non-stereotyped behaviour in rats induced by various stimulant drugs. Psychopharmacologia, 1969, 14 : 299--304. Geffen, L.B., Jessell, T.M., Cuello, A.C. and Iversen L.L. Release of dopamine from dendrites in rat substantia nigra. Nature (Lond.), 1976, 260: 258-260. Groves, P.M., Wilson, C.J., Young, S.J. and Rebec, G.V. Self-inhibition by dopaminergic neurons. Science, 1975, 190: 522--529. Kafi, S. and Gaillard, J.M. Brain dopamine receptors and sleep in the rat: effects of stimulation and blockade. Europ. J. Pharmacol., 1976, 38: 357-364. Kehr, W., Carlsson, A., Lindqvist, M., Magnusson, T. and Atack, C. Evidence for a receptor-mediated feedback control of striatal tyrosine hydroxylase activity. J. Pharm. Pharmacol., 1972, 24: 744-747. Michel, F., Klein, M., Jouvet, D. e Valatx, J.L., Etude polygraphique du sommeil chez le rat. C.R. Soc. Biol. (Paris), 1963, 155: 2389--2395. Roldan, E., Weiss, T. and Fifkov~, E. Excitability changes during the sleep cycle of the rat. Electroenceph, clin. Neurophysiol., 1963, 15: 775--785. StrSmbom, U., Catecholamine receptor agonists. Effects on m o t o r activity and rate of tyrosine hydroxylation in mouse brain. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 1976, 292: 167--176. Ungerstedt, U. Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system. Acta physiol. scand., 1971, Suppl. 367: 69--93.
Electroencephalography and Clinical Neurophysiology, 1979, 46:214--219
© Elsevier/North-Holland Scientific Publishers, Ltd.
SLEEP INDUCED BY LOW DOSES OF APOMORPHINE IN RATS I G.P. MEREU, E. SCARNATI 2, E. PAGLIETTI, B. PELLEGRINI QUARANTOTTI 3, P. CHESSA, G. DI CHIARA and G.L. GESSA Institute of Pharmacology, University of Cagliari, Cagliari (Italy) (Accepted for publication: July 4, 1978)
Apomorphine, a direct stimulant of dopamine (DA) receptors (Ernst and Smelik 1966; And~n et al. 1967; Ernst 1967), exerts two opposite effects on the behaviour of experimental animals: in low doses it decreases m o t o r activity whereas in higher doses it produces hypermotility and stereotypy (Fog 1969; Str6mbom 1976). While it has been clearly demonstrated that the stimulant effect is due to the activation of postsynaptic DA receptors in the CNS (Ungerstedt 1971) it has been recently suggested that the effect of low doses of apomorphine is mediated by preferential stimulation of a special kind of DA receptor, called by Carlsson 'autoreceptors', which results in inhibition both of DA synthesis and of electrical activity of DA neurones (Kehr et al. 1972; Aghajanian and Bunney 1973; Carlsson 1975). These receptors would be a means by which these neurones could control their own activity (firing, DA synthesis and release) via DA released by the nerve endings or by dendrites in the substantia nigra (Aghajanian and Bunney 1973; Carlsson 1975; Groves et al. 1975; Geffen et al. 1976). Direct evidence that the decreased m o t o r activity is due to the stimulation of DA receptors has been recently provided (Di Chiara et
1 This investigation was supported by Tecnofarmaci S.p.A. Pomezia (Rome), Italy. 2 Present address: Institute of Physiology, Faculty of Medicine, L'Aquila, Italy. 3 Present address: Tecnofarmaci S.p.A., Pomezia (Rome), Italy.
al., 1976). However, it is not clear whether the decreased m o t o r activity reflects a true sedative or hypnotic effect. On the basis of these considerations we studied the effect of low doses of apomorphine on the EEG patterns of freely moving rats and the influence of neuroleptics on this effect.
Materials and methods Twenty-four male Sprague-Dawley rats (300--320 g) had electrodes chronically implanted in the cortex and hippocampus, according to the usual techniques for sleep recording in the rat (Michel et al. 1963; Roldan et al. 1963). An electromyogram (EMG) was recorded by means of 2 stainless steel needles implanted into the neck muscles at the beginning of each recording session. After surgery, each rat was individually housed, for the entire study period of 3--4 months, in a single cage at 24°C, with reversed light
SLEEP INDUCED BY APOMORPHINE
215
subcutaneously in the neck area through a long Teflon tube (0.1 mm internal diameter) which did not disturb the animal's movements. In a first study the animals were injected with different doses of apomorphine (1000, 500, 100, 50, 25, 12.5 and 6.2 #g/ml/ kg, within 2 min) and the EEG was recorded for 60 min thereafter. Recording was not prolonged further because apomorphine effects are of short duration due to the short half-life of the drug. After completion of the first study, a second investigation was carried out, with the same animals, to clarify the influence of different neuroleptics on the hypnotic effect of apomorphine. Pimozide (50 pg/kg), benzperidol (50 pg/kg) and L-sulpiride (500 pg/kg) were given intraperitoneally (120, 30 and 30 min before apomorphine, respectively). Pimozide was dissolved in H20 with the addition of glacial acetic acid and the solution brought to pH 5 with NaOH • L-sulpiride and benzperidol were dissolved in H20. In a third study, the effect of a continuous apomorphine infusion on EEG was studied. Apomorphine was infused for 4 h at a dose of 80 pg/600 pl/kg/h, and recording was done for the same time, starting at 15.00, at the
beginning of the infusion. Control rats were infused with the solvent alone. Cortical and hippocampal EEG and neck EMG records were used for classifying the animal's state as either awake (W), in synchronized (slow) sleep (S) or desynchronized sleep (REM) according to classical criteria (Michel et al. 1963; Roldan et al. 1963). Records were visually scored to the nearest 3 sec interval. Levels of significance were determined b y Student's t test.
Results
(1) Effect of apomorphine injection on EEG Apomorphine was injected after 15 min of control recording, and the record continued for 60 min thereafter. Table I shows the effect of the different subcutaneous doses of apomorphine, given acutely, on the EEG pattern of the rat. Doses of apomorphine of 1.0 or 0.5 mg/kg produced stereotyped behaviour. Moreover, in agreement with previous results (Kafi and Gaillard 1976), these doses caused a marked reduction of the total sleep. In fact, the percentage of total sleep, over the 60 min recording time, fell from 18.24%, in control rats, to 4.22% after 1.0 mg/kg, and to
TABLE I Effect of different doses of apomorphine reported number (No.) of records. Apomorphine ( p g / k g s.c. )
No. of records
No. of animals
o n s l e e p in t h e r a t . E a c h v a l u e is t h e m e a n + S.E. o b t a i n e d f r o m t h e
% o f t o t a l r e c o r d i n g t i m e (1 h ) spent in: Total sleep
Solvent 1000 500 100 50 25 12.5 6.2
10 4 4 8 8 8 8 4
8 4 4 7 8 7 6 4
18.24 4.22 10.36 48.25 53.57 54.36 31.76 20.03
-+ 2 . 2 0 + 0.85 -+ 0 . 9 0 +- 5 . 1 3 _+ 4 . 8 0 + 7.85 _+ 4 . 8 2 + 1.35
* P < 0 . 0 0 1 , ** P < 0 . 0 1 ; w i t h r e s p e c t t o s a l i n e - t r e a t e d a n i m a l s .
* ** * * * **
Slow (S) sleep
Fast (D) sleep
16.26 4.22 8.90 46.53 50.06 49.78 29.30 17.95
1 . 9 8 +_ 0 . 3 3
_+ 2 . 0 7 _+ 0 . 8 5 -+ 1 . 3 5 _+ 4 . 2 4 _+ 4 . 2 1 _+ 5 . 1 2 +_ 4 . 1 8 _+ 1 . 3 0
* ** * * * *
1.46 1.72 3.51 4.58 2.26 2.08
_+ 0 . 5 3 _+ 0 . 3 3 _+ 0 . 4 8 * _+ 0 . 5 8 * +_ 0 . 3 4 + 0.22
216
G.P. MEREU ET AL.
10.36% a f t e r 0.5 m g / k g o f a p o m o r p h i n e , respectively. A f t e r 1.0 m g / k g o f a p o m o r p h i n e , t h e residual a m o u n t o f sleep was due to a brief episode o f slow sleep w h i c h usually o c c u r r e d e i t h e r a few m i n u t e s a f t e r treatment,, or at t h e end o f t h e recording period, i.e., p r e s u m a b l y at t h e t i m e w h e n t h e a p o m o r p h i n e level in t h e CNS fell into a range o f c o n c e n t r a t i o n s low e n o u g h t o elicit centxal depression. In c o n t r a s t to the above results, doses o f a p o m o r p h i n e o f 100 pg/kg or less caused behavioural sedation and increased the a m o u n t o f t o t a l sleep. This e f f e c t was already m a x i m a l with a dose o f a p o m o r p h i n e as low as 25.0 pg/kg, which increased the a m o u n t o f t o t a l sleep to 54% o f t h e r e c o r d i n g time. T h e e n h a n c e m e n t o f t o t a l sleep c o r r e s p o n d e d m a i n l y t o S (90% o f t o t a l sleep). H o w e v e r , the small doses o f a p o m o r p h i n e also increased t h e a m o u n t o f REM sleep; usually o n e or 2 episodes o f REM sleep o c c u r r e d t o w a r d t h e end o f t h e recording period. Following small doses o f a p o m o r p h i n e , the b e h a v i o u r o f t h e animal c o r r e s p o n d e d t o t h e EEG activity. Moreover, as previously r e p o r t e d (Baraldi and Benassi-Benelli 1 9 7 5 ) , small doses o f a p o m o r p h i n e caused r e c u r r e n t episodes o f y a w n i n g and penile erection. This e f f e c t was m o r e c o n s i s t e n t l y e v o k e d b y the dose o f 50 gg/kg. Each episode had a d u r a t i o n
of a few m i n u t e s . Yawning and e r e c t i o n o c c u r r e d w h e n t h e animal was fully awake, or s o o n after an episode o f S or REM sleep. In the latter t w o instances the behavioural change was associated with cortical arousal. H o w e v e r , cortical d e s y n c h r o n i z a t i o n did n o t seem to be t h e c o n s e q u e n c e of t h e behavioural change, for these events began t o g e t h e r and, at times, cortical arousal p r e c e d e d the behavioural change.
(2) Effects o f neuroleptics on apomorphine response T o clarify the d o p a m i n e - m i m e t i c n a t u r e o f the h y p n o t i c e f f e c t o f low doses o f a p o m o r phine, we studied w h e t h e r this e f f e c t m i g h t be p r e v e n t e d b y d i f f e r e n t n e u r o l e p t i c s such as p i m o z i d e , b e n z p e r i d o l and L-sulpiride. These results are r e p o r t e d in Tables II and III. As previously r e p o r t e d {Kafi and Gaillard 1976), neuroleptics, with t h e low dose and t h e t i m e schedule used, did n o t influence the overt b e h a v i o u r o f t h e animals and did n o t m o d i f y the a m o u n t o f t o t a l sleep, o f S and o f REM sleep o f t h e animals. H o w e v e r , t h e y p r e v e n t e d the hypnotic effect of apomorphine.
(3) Effect of apomorphine infusion on the EEG Because o f the short half-life o f a p o m o r phine, to clarify t h e e f f e c t o f this drug o n the sleep of the rat, we studied t h e E E G changes
TABLE II Antagonism by neuroleptics of the hypnotic effect of apomorphine 25 pg/kg s.c. Each value is the mean + S.E. obtained from the reported number of records. Neuroleptic (pg/kg i.p. )
Saline Pimozide Benzperidol L-sulpiride
-50 50 500
Time before apomorphine or solvent (min)
Total sleep as % of total recording time (1 h) Solvent
No. of records
Apomorphine
No. of records
30 120 30 30
16.32 18.12 17.54 20.85
4 4 4 4
56.43 28.35 21.75 29.32
8 8 8 8
* P < 0.001,with respect to solvent t r e a t m e n t . ** P < 0.001, with respect to apomorphine alone.
+_1.58 + 3.12 +_2.84 ± 4.31
_+8.34 +_3.86 ± 4.35 ± 4.32
* ** ** **
S L E E P I N D U C E D BY A P O M O R P H I N E
217
T A B L E III A n t a g o n i s m o f t h e h y p n o t i c e f f e c t o f a p o m o r p h i n e b y p i m o z i d e . E a c h value is t h e m e a n -+ S.E. o b t a i n e d f r o m t h e n u m b e r o f r e c o r d s r e p o r t e d in T a b l e II. Treatment
Sleep d u r a t i o n (in sec) in each 10 rain interval a f t e r t r e a t m e n t
Solvent Pimozide Apomorphine Pimozide + apomorphine
0--10
10--20
20--30
30--40
40--50
50--60
Total sleep in 60 m i n
0 0 370±31" 0
0 0 380±29" 1 6 0 ± 20
61±12 1 2 0 ± 15 400_+50' 215 ± 26
169±24 200 ± 18 470±41 * 165 ± 20
280_+17 130 ± 15 315_+30 290 ± 24
50± 6 210 ± 15 165±18 220 ± 29
5 6 0 ± 48 1 6 6 0 ± 41 2100±299" 1050 ± 130
* P < 0.001 w i t h respect t o p i m o z i d e + a p o m o r p h i n e .
T A B L E IV Effect of a p o m o r p h i n e i n f u s i o n (80 p g / k g / h ) d u r i n g 4 h, o n t h e sleep p a t t e r n . Each value is t h e m e a n ± S.E. obtained f r o m 8 r e c o r d s ( e a c h o n a d i f f e r e n t animal). L a t e n c y (in r a i n ) t o first S (slow) sleep e p i s o d e : 24 + 4, 12 -+ 2 for solvent a n d a p o m o r p h i n e , respectively. L a t e n c y (in m i n ) to first D (fast) sleep episode: 96 ± 12, 31 ± 5 for solvent a n d a p o m o r p h i n e , respectively. Mean d u r a t i o n of D episodes (in sec) = 60 ± 9, 74 ± 10 for solvent a n d a p o m o r p h i n e , respectively. Treatment
Hour
% of r e c o r d i n g t i m e s p e n t in T o t a l sleep
S sleep
D sleep
Solvent
1st 2nd 3rd 4th Total
36.26 33.54 23.19 30.41 30.85
± ± ± ± ±
4.15 3.41 2.72 4.04 3.58
35.06 29.39 19.05 27.22 27.68
_+ 4.90 ± 4.15 _+ 2.84 ± 3.35 ± 3.81
1.20 4.15 4.14 3.19 3.17
+0.10 ± 0.89 ± 0.76 _+ 0.65 _+ 0.60
Apomorphine
1st 2nd 3rd 4th Total
57.44 65.70 67.26 62.12 63.13
_+ 7.32 ± 7.10 _+ 8.11 _+ 6.31 +_ 7.21 *
51.06 59.74 58.42 56.18 56.35
_+ 3.62 _+ 4.49 _+ 5.40 ± 5.01 _+ 4.63 *
6.38 5.96 8.84 5.94 6.78
+ 0.92 +_ 0.68 _+ 1.29 _+ 0.91 _+ 0.95
* P < 0.001 w i t h r e s p e c t to s o l v e n t infusion.
produced b y a continuous subcutaneous infusion of a sedative dose of the drug. As a result (Table IV and Fig. 1) the following changes occurred during the infusion of apomorphine: (1) the latencies to the first episodes of S and REM sleep were significantly
REM
"11 SOLVENT
i
U
~
LI
U,
U
,
SS R EM
o
1
2
3
4
hours
Fig. 1. Typical h y p n o g r a m s o f rats infused w i t h solv e n t a n d a p o m o r p h i n e (80 pg/kg/h) d u r i n g 4 h. W, waking; SS, slow sleep; REM, fast sleep.
218 reduced; (2) the times spent in total sleep, in S and in REM sleep were approximately doubled with respect to rats infused with the solvent; (3) the mean duration of each episode of REM sleep was slightly increased.
G.P. MEREU ET AL. It is particularly important, both for practical and theoretical reasons, that the effect of apomorphine on sleep is not restricted to rats, since low, non-emetic doses of apomorphine cause sedation and sleep in man as well (Corsini et al. 1977).
Discussion Summary Our results show that apomorphine in high doses suppresses sleep. On the contrary, small doses of apomorphine produce an increase in both S and REM sleep durations in the rat. As a sleep-inducing agent apomorphine is, to our knowledge, the most potent drug known. The finding that pimozide, L-sulpiride and benzperidol, selective blockers of dopamine receptors, prevent the hypnotic effect of apomorphine, suggests that this effect is due to the stimulation of central dopamine receptors. As to the nature of these receptors, they might be identified with the DA receptors named 'autoreceptors', whose activation results in inhibition of DA synthesis and dopaminergic firing (Aghajanian and Bunney 1973; Carlsson 1975). Alternatively, they might be a special kind of postsynaptic DA receptors, different from the DA receptors responsible for stereotypy, motor stimulation and arousal, because of a greater sensitivity to the action of the transmitter. The distinction has a physiological implication: if sleep originates from the inhibition of firing of DA neurones (i.e. from stimulation of autoreceptors), this implies that DA plays an inhibitory role on sleep. Vice versa, if sleep is due to the stimulation of some kind of postsynaptic receptors, then the DA system plays a dual role on sleep-wakefulness mechanisms: promoting and antagonistic effects in low doses and high doses, respectively. In order to clarify this problem we are currently investigating the effect of low doses of apomorphine in rats in which DA neurones axe destroyed by the intranigral or intraventricular administration of 6-hydroxydopamine.
The effect of apomorphine on the EEG of freely moving rats was studied. Apomorphine at the dose of 1 mg/kg caused stereotypy and a marked reduction of total sleep. On the contrary, acute subcutaneous administration of apomorphine at the dose of 100 pg/kg, or less, markedly increased the amount of total sleep (corresponding mostly to synchronized sleep). Moreover, the infusion of apomorphine (80 pg/kg/h) for 4 h doubled the duration of slow and REM sleep. The hypnotic effect of apomorphine was prevented by neuroleptics, such as pimozide, benzperidol and L~ulpiride, at doses which, per se, did not modify the EEG of the animals. These results suggest the existence in the CNS of DA receptors mediating sleep.
R6sum6 Sommeil induit par petites doses d'apomorphine chez les rats L'effet de l'apomorphine sur I'EEG de rats libres de leurs mouvements a ~t~ ~tudi~ pendant l'heure suivant le traitement. L'apomorphine ~ la dose de 1 mg/kg cause une st~r~otypie et une r~duction marquee du sommeil total. Au contraire, l'injection sous-cutan~e aigu~ d'apomorphine ~ la dose de 100 pg/kg, ou inf~rieure, a fortement augment~ la quantit6 de sommeil (correspondant surtout au sommeil lent). Par ailleurs, l'infusion d'apomorphine (80 pg/kg/h), pendant 4 h , a doubl~ la dur6e du sommeil lent et rapide. L'effet hypnotique de l'apomorphine a ~t6
SLEEP INDUCED BY APOMORPHINE e m p 6 c h d p a r d e s n e u r o l e p t i q u e s t e l q u e le p i m o z i d e , le b e n z p d r i d o l e t le L - s u l p i r i d e d e s d o s e s qui, p a r e l l e s - m ~ m e s , n ' o n t p a s modifi~ I'EEG de l'animal. Les rdsultats indiq u e n t l ' e x i s t e n c e , d a n s le S N C , d e r ~ c e p t e u r s d o p a m i n e r g i q u e s i m p l i q u ~ s d a n s le s o m m e i l . We thank Professor Peiro Fresia, 'Ravizza S.P.A.', Muggi6 (Milan) Italy, for supplying L-sulpiride, the active form of sulpiride.
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