Psychopharmacology(1992) 108:263-270
Psychopharmacology © Springer-Verlag 1992
Effects of ritanserin and chlordiazepoxide on sleep-wakefulness alterations in rats following chronic cocaine treatment Christine Dugovic, Theo F. Meert, David Ashton, and Gilbert H.C. Clincke Department of Neuropsychopharmacology,Janssen Research Foundation, B-2340 Beerse, Belgium Received November 8, 1991 / Final version March 20, 1992
Abstract. The effects of ritanserin, a 5-hydroxytryptamine2 (5-HT2) receptor antagonist, and chlordiazepoxide, a benzodiazepine agonist, on sleep-wakefulness disturbances in rats after acute administration of cocaine and after discontinuation of chronic cocaine treatment were examined. Intraperitoneal (IP) injection of chlordiazepoxide (10 mg/kg) but not ritanserin (0.63 mg/kg) prevented the increase of wakefulness (W) and the reduction of light slow wave sleep (SWS1) and deep slow wave sleep (SWS2) induced by an acute injection of cocaine (20 mg/kg IP). Daily injection of cocaine (20 mg/kg for 5 days, then 30 mg/kg for 5 days IP) at the onset of the light phase elicited an increase of W and a concomitant decrease of SWS1, SWS2 and paradoxical sleep (PS) in the light phase, followed by a rebound in SWS2 and PS in the subsequent dark phase. Following cocaine discontinuation, the circadian distribution of sleep-wakefulness states remained disturbed in saline-treated rats for at least 5 days. Both ritanserin (0.63 mg/kg IP/day) and chlordiazepoxide (10 mg/kg IP/day) reduced the alteration in the distribution of W and SWS2 throughout the lightdark cycle from the first day of administration on, but failed to prevent PS alterations. The mechanisms by which both compounds exert their effect are probably quite different. For chlordiazepoxide sedative and sleepinducing properties probably play a major role. In contrast, for ritanserin SWS2-increasing properties and its ability to reverse preference for drugs of abuse without inducing aversion might be key factors. Key words: Sleep-wakefulness - Chronic cocaine treatment - Cocaine discontinuation - Ritanserin - Chlordiazepoxide - Rat Recent investigations have shown that ritanserin, a potent serotonin antagonist at central 5-hydroxytryptamine-2 (5HT2) receptors (Leysen et al. 1985), reduced cocaine preference and cocaine intake in rats without interfering with consumatory physiological processes (Meert et al. 1990a, Correspondence to:
C. Dugovic
1991). At doses similar to those required to produce activity in this test procedure, ritanserin induced an increase of deep slow wave sleep (SWS) and a decrease of paradoxical sleep (PS) during the physiological sleep period of the rat (Dugovic and Wauquier 1987; Davenne et al. 1989; Bjorvatn and Ursin 1990; Monti et al. 1990; Silhol et al 1991) and tended to normalize the disturbed sleep-wakefulness organization in rats exposed to continuous light (Dugovic et al. 1989a). In humans given ritanserin, polysomnographic data revealed a marked increase of SWS in healthy volunteers (Idzikowski et al. 1986), poor sleepers (Adam and Oswald 1989), insomniacs (Ruiz-Primo et al. 1989) and dysthymic patients (Paiva et al. 1988). In humans, agitation, depression, anxiety and sleep disturbances have been described as abstinence symptoms following abrupt cessation of cocaine abuse (Gawin and Kleber 1986; Gillin et al. 1989; Gawin 1991). In animal models related to drug withdrawal symptoms, cocaine has been reported to produce anxiogenic-like effects (Wood and Lal 1987; Costall et al. 1989 and Emmett-Oglesby et al. 1990 for a general review). Impairments of the sleepwakefulness patterns have been also observed in animals treated with cocaine. In acute conditions, cocaine elicited cortical EEG desynchronization in cats (Wallach and Gershon 1971) and had pronounced sleep-suppressing effects in rats (Hill et al. 1977). Repetitive injections or continuous infusion of cocaine in rats resulted in a decrease of total sleep time during the course of the treatment followed by alterations in the sleep-wakefulness rhythms on drug discontinuation (Yoshimura et al. 1980; Radulovacki et al. 1990). However, the effects of cocaine withdrawal on sleep-wakefulness patterns have not been extensively studied. The purpose of the present study was to further investigate the sleep-wakefulness impairments in rats following discontinuation of chronic cocaine treatment and to examine the effects of ritanserin on these disturbances. Chlordiazepoxide was tested for comparison because of the well-known sleep-inducing properties of benzodiazepines. In addition, the direct interaction between cocaine, ritanserin and chlordiazepoxide was studied in acute experimental conditions.
264
Materials and methods
Results
Implantation procedure. Male adult Wistar rats weighing 240~
Effects of chronic cocaine administration
260 g were chronically implanted under pentobarbital anesthesia (50 mg/kg IP) with electrodes for standard sleep monitoring. Electrodes were positioned on the frontal and occipital cortices, subcutaneously on each side of the orbit and in the neck muscles for polygraphic recordings of electrocorticogram (ECoG), electro-oculogram (EOG) and electromyogram (EMG). After surgery, the animals were individually housed in Plexyglas cages and maintained under controlled environmental conditions (12 h light-dark schedule, light on 9:00 am., 22 +_ 2°C ambient temperature, food and water ad libitum).
Recording procedure. At the end of a 8-10 day recovery period from surgery and habituation to the environment, the rats were connected by a cable to a rotating connector and ECoG, EOG and EMG activities were recorded on a polygraph. The polygraphic recordings were scored visually by 30 s epochs. These epochs were classified as being either wakefulness (W), light stow wave sleep (SWS1), deep slow wave sleep (SWS2) or paradoxical sleep (PS), using the criteria of Michel et al. (1961). The scores were entered into a computer which calculated the different sleep-wakefulness parameters (amount of time spent in these four states, number and duration of episodes for each state, SWS1, SWS2 and PS latencies). Pharmacological procedure. In the first experiment, the animals (n = 20) received a daily injection of cocaine (20 mg/kg for 5 days, then 30 mg/kg for 5 days) at the onset of the light phase (9 : 00 a.m.) for a 10-day period. The dose-range of cocaine used in the present study and the duration of drug exposure have been suggested to produce measurable behavioral changes after withdrawal (Johanson and Fischman 1989). After a 5-day treatment period, the dose of cocaine was increased to 30 mg/kg in order to compensate for the possible development of tolerance upon repeated administration. On the next day the rats were treated with saline, then for a subsequent period of 10 days with either saline (n = 8), ritanserin (0,63 mg/kg/day, n = 6) or chlordiazepoxide (10 mg/kg/day, n = 6). The dose of 0.63 mg/kg ritanserin was chosen since it was the optimal dose to induce the most pronounced increase of SWS2 in the rat (Dugovic et al. 1989b). The dose of 10 mg/kg chlordiazepoxide was chosen on the basis of preliminary experiments to obtain an increase of SWS2 in the same proportions as that observed with ritanserin. Ritanserin or chlordiazepoxide treatment was started 48 h after the last injection of cocaine in order to avoid direct drug interaction. In the second experiment, a separate group of rats received an injection of ritanserin (0.63 mg/kg, n = 5) or chlordiazepoxide (10 mg/kg, n = 6) at light onset, and 30 rain later 20 mg/kg cocaine. All drugs were injected IP in a volume of 4 ml/kg body weight. Cocaine and chlordiazepoxide were dissolved in saline and ritanserin was dissolved in 1 mM tartaric acid. Data analysis. Polygraphic recordings were performed for 24 h during chronic cocaine treatment (days 5 and 8) and every second day following cocaine discontinuation (days 2, 4, 6, 8 and 10). The sleep-wakefulness parameters were analyzed per 4-h and 12-h periods and were compared to control values (later referred to as C), £e. saline injection under the same conditions before cocaine treatment was started. For the acute pharmacological effects tested in the second experiment, polygraphic recordings were performed for 8 h following the last injection. The sleep-wakefulness parameters were analyzed per 4-h periods and were compared to control values, i.e. vehicle injections under the same conditions. The duration of the time spent in the different states of vigilance was expressed in minutes (mean _+_SEM). Statistical significance of the data was assessed by means of the paired two,tailed Student t-test for within group comparisons. In addition, the non-paired two-tailed Student t-test was used for between group comparisons following cocaine discontinuation.
D a i l y a d m i n i s t r a t i o n of c o c a i n e (20 m g / k g for 5 days, t h e n 3 0 m g / k g for 5 days) at the o n s e t of the light p h a s e d i s r u p t e d the s l e e p - w a k e f u l n e s s d i s t r i b u t i o n t h r o u g h o u t the l i g h t - d a r k cycle. C o c a i n e a d m i n i s t r a t i o n r e s u l t e d in an i n c r e a s e of W a n d a c o n c o m i t a n t d e c r e a s e of sleep d u r a t i o n in the light p h a s e f o l l o w e d by a sleep r e b o u n d in t h e s u b s e q u e n t d a r k phase. H o w e v e r , in the t o t a l 24-h p e r i o d sleep a n d w a k e f u l n e s s a m o u n t s w e r e n o t significantly different f r o m the c o n t r o l values (C). T h e s e c h a n g e s were o b s e r v e d o n d a y 5 a n d 8, b u t w e r e m o r e p r o n o u n c e d o n d a y 8. F i g u r e 1 illustrates the s l e e p - w a k e f u l n e s s a l t e r a t i o n s a n a l y z e d per 4-h p e r i o d s o n d a y 8 of c h r o n i c c o c a i n e
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Fig. 1. Distribution of the sleep-wakefulness states per 4 h periods on day 8 of chronic cocaine treatment. Cocaine (20 mg/kg IP for 5 days, then 30 mg/kg IP for 5 days) was administered daily at the onset of the light phase. Mean values + SEM. of 20 animals were expressed in minutes. W, wakefulness; SWS1, light slow wave sleep; SWS2, deep slow wave sleep; PS, paradoxical sleep. *P < 0.05, **P < 0.01, * ** P < 0.001 (paired two-tailed Student t-test) as compared to control values (saline injection under the same conditions before chronic cocaine treatment was started). (Z~) Vehicle; ( I ) cocaine day 8
265 treatment. The administration of cocaine (30 mg/kg) at light onset was followed by a marked increase of W and a suppression of SWS1, SWS2 and PS during the first 4-h period consistent with a decrease in the number of episodes for all sleep states. The latency for the first episode of sleep (SWSt latency) was delayed by about 2-h following cocaine injection. In the second and third 4-h periods of the light phase, the amounts of the different states of vigilance returned to control values, except for PS which remained significantly reduced. During the three consecutive 4-h periods of the dark phase, SWS2 and PS rebounded while W was significantly decreased. This was due mainly to an increase in the number of SWS2 and PS episodes and to a reduction in the mean duration of W episodes.
Effects of ritanserin and chlordiazepoxide following chronic cocaine treatment The distribution of the sleep-wakefulness states during the light and dark phases in rats daily treated with either W (rnin) 0 - 12h
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Fig. 2. Amounts of wakefulness (W) expressed in minutes during the light (0-12 h) and dark (12-24 h) phases in rats given a daily IP injection of either saline, ritanserin (0.63 mg/kg) or chlordiazepoxide (10 mg/kg) at the onset of the light phase from the second day of cocaine discontinuation on. Dots represent individual results for 20 animals, and the lines connect the mean values, ap < 0.05,bp < 0.0t, cp < 0.001 (paired two-tailed Student t-test) for within group comparisons with control values (saline injection under the same conditions before chronic cocaine treatment was started). Ap < 0.05, Bp < 0.01 (non-paired two-tailed Student t-test) for between group comparisons with saline treatment
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Fig. 4. Amounts of deep slow wave sleep (SWS2) expressed in minutes during the light (0-12 h) and dark (12-24 h) phases in rats given a daily IP injection of either saline, ritanserin (0.63 mg/kg) or chlordiazepoxide (10 mg/kg) at the onset of the light phase from the second day of discontinuation on. "P < 0.05, bp < 0.01, ~P < 0.001 (paired two-tailed Student t-test) for within group comparisons with control values (saline injection under the same conditions before chronic cocaine treatment was started). Ap < 0.05, Bp < 0.01 (nonpaired two-tailed Student t-test) for between group comparisons with saline treatment
rebound was suppressed in the dark phase. Subsequently, an increase in SWS2 duration was observed from the fifth day of ritanserin or chlordiazepoxide administration on (e.g. day 6) as compared to C values. In contrast, PS alterations were not reduced in both ritanserin- and chlordiazepoxide-treated rats as compared to saline-treated rats (Fig. 5). During the 10-day period after cocaine discontinuation, PS amounts remained lower than the C values in the light phase and were slightly increased in the dark phase. As compared to C, SWS1 amounts were significantly decreased in the light phase during ritanserin treatment and were enhanced in the dark phase during chlordiazepoxide treatment (Fig. 3). Sleep latencies were modified as shown in Table 1. In saline-treated rats on the second day after cocaine discontinuation, the SWS1, SWS2 and PS latencies were significantly prolonged when compared to C values. Similar but less pronounced effects were observed on the fourth day after cocaine discontinuation. The SWS1 and SWS2 latencies were normalized in ritanserin-treated rats following the first and the second injection, respectively. In rats given chlordiazepoxide, the SWS 1, SWS2 and PS latencies
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Fig. 5. Amounts of paradoxical sleep (PS) expressed in minutes during the light (0-12 h) and dark (12 24 h) phases in rats given a daily IP injection of either saline, ritanserin (0.63 mg/kg) or chlordiazepoxide (10 mg/kg) at the onset of the light phase from the second day of cocaine discontinuation on. ap < 0.05, bp < 0.01, cp < 0.001 (paired two-tailed Student t-test) for within group comparisons with control values (saline injection under the same conditions before chronic cocaine treatment was started) returned to control values from the first day of administration on.
Effects of ritanserin and chlordiazepoxide combined with cocaine in acute conditions As shown in Tables 2 and 3, rats given an acute injection of cocaine (20 mg/kg) exhibited a long-lasting increase in the amount of time spent in W and reduced amounts of SWS1, SWS2 and PS associated with an increase in all sleep states latencies. These effects occurred in the first 4 h following the treatment and the amounts of the sleepwakefulness states returned to control values in the subsequent 4 h. Ritanserin (0.63 mg/kg) alone produced a significant increase in SWS2 duration and a significant decrease in SWS1 and PS duration during the first 4 h following the treatment (Table2), Rats given chlordiazepoxide (10 mg/kg) alone exhibited significantly increased amounts of SWS2 and reduced amounts of PS (Table 3). The SWS1 and SWS2 latencies were significantly shortened in chlordiazepoxide-treated rats whereas the PS latency was prolonged after ritanserin and chlordiazepoxide treatment. Sleep-wakefulness alterations observed after administration of cocaine were not modified by ritanserin pretreatment (Table2). In contrast, pretreatment with
267 Table 1. Sleep latencies in rats treated with either saline, ritanserin (0.63 mg/kg) or chlordiazepoxide (10 mg/kg) after cocaine discontinuation
SWS1 latency
SWS2 latency
PS latency
25.4 72.1 40.9 28.8
89.1 167.3 130.7 118.4
_+ 18.0 _+ 33.5** ,+ 34.7 ,+ 22.5
Salinetreated rats (n = 8)
Control Day 2 Day 4 Day 6
14.3 36.7 28.6 21.0
_+ 2.1 _+ 9.3* ,+ 4.3** _+ 7.2
Ritanserintreated rats (n = 6)
Control Day 2 Day 4 Day 6
13.6 _+ 2.6 19.3 ,+ 4.2 14.2 ,+ 5.1 15.3 + 4.4
17.2 _+ 2.1 39.9 + 7.0* 20.8 + 4.4 20.0 ,+ 3.8
74.3 182.4 199.8 129.6
__ 9.2 -+_30.7* ,+ 30.2* + 19.8
Chlordiazepoxidetreated rats (n = 6)
Control Day 2 Day 4 Day 6
23.7 14.0 27.7 21.7
33.0 19.1 30.2 23.0
78.2 103.9 84.1 81.9
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Values of light slow wave sleep (SWS1), deep slow wave sleep (SWS2) and paradoxical sleep (PS) latencies are means _+ SEM (min of 6-8 animals in each experimental group. Sleep latencies were evaluated on days 2, 4 and 6 after cocaine discontinuation *P < 0.05, **P < 0.01 (paired two-tailed Student t-test) as compared to control values (saline injection in the same conditions before cocaine administration was started)
Table 2. Effects of ritanserin (0.63 mg/kg) on sleep-wakefulness states and sleep latencies in cocaine-treated rats (20 mg/kg) during the first 4-h period following the treatment. Mean values + SEM of 5 animals were expressed in minutes
W SWS1 SWS2 PS SWS1 latency SWS2 latency PS latency
Vehicle + vehicle
Ritanserin + vehicle
Vehicle + cocaine
Ritanserin + cocaine
79.9 _ 9.1 15.9 _ 8.4 132.5 +_ 6.6 11.8 ,+ 3.5 13.8 ,+ 4.1 26.7 + 2.5 105.6 ,+ 25.6
71.9 + 10.1 9.3 _.+ 1.3" 152,1 + 8.2* 6.7 + 2.5* 16.3 ,+ 7.8 23.8 ,+ 9.7 177.1 ,+ 34.3*
137.0 8.9 86,5 7.6 103.2 121.4 175.6
132.7 10.0 92.7 4.6 86.9 99.0 219.0
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W, wakefulness; SWS 1, light slow wave sleep; SWS2, deep slow wave sleep; PS paradoxical sleep *P < 0.05, **P < 0.01 (paired two-tailed Student t-test) as compared to control values (vehicle injection under the same conditions)
Table 3. Effects of chlordiazepoxide (10 mg/kg) on sleep-wakefulness states and sleep latencies in cocaine-treated rats (20 mg/kg) during the first 4-h period following the treatment. Mean values _+ SEM of 6 animals were expressed in minutes
Vehicle + vehicle W SWS1 SWS2 PS SWS1 latency SWS2 latency PS latency
78.3 + 15.1 + 135.2 + 11.4 + 13.3 + 25.2 _ 96.5 --
7.6 2.1 6.0 2.9 3.4 2.6 22.8
Chlordiazepoxide + vehicle
Vehicle + cocaine
Chlordiazepoxide + cocaine
71.8 12.0 150.5 5.7 4.8 7.3 140.2
135.0 8.8 89.3 6.9 102.6 118.3 172.8
97.3 10.7 129.1 2.9 30.5 45.1 202.6
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W, wakefulness; SWS1, light slow wave sleep; SWS2, deep slow wave sleep; PS, paradoxical sleep *P < 0.05, **P < 0.01 (paired two-tailed Student t-test) as compared to control values (vehicle injection under the same conditions)
c h l o r d i a z e p o x i d e a n t a g o n i z e d the i n c r e a s e of W a n d the d e c r e a s e o f S W S 1 a n d S W S 2 i n d u c e d by c o c a i n e . T h e S W S 1 a n d S W S 2 latencies also d e c r e a s e d t o w a r d s c o n t r o l v a l u e s in c h l o r d i a z e p o x i d e - p r e t r e a t e d rats. H o w e v e r , c h l o r d i a z e p o x i d e did n o t c o u n t e r a c t the P S - s u p p r e s s i n g effects of c o c a i n e ( T a b l e 3).
Discussion I n o u r e x p e r i m e n t s , r e p e a t e d a d m i n i s t r a t i o n of c o c a i n e at the o n s e t of the light p h a s e in rats i n d u c e d m a r k e d altera t i o n s in the s l e e p - w a k e f u l n e s s r h y t h m t h r o u g h o u t the l i g h t - d a r k cycle b o t h d u r i n g the p e r i o d of c o c a i n e a d m i n -
268 istration and after discontinuation of the drug. During chronic treatment with cocaine an initial decrease of SWS1, SWS2 and PS, mainly during the first 4 h of the light phase, was followed by a rebound in SWS2 and PS during the subsequent dark phase. These initial sleep suppressing effects of cocaine are in agreement with previous data obtained in the rat during acute (Hill et al. 1977) or chronic exposure (Yoshimura et al. 1980). During continuous arterial infusion of cocaine (100 mg/kg/day) for 7 days, SWS1, SWS2 and PS were initially reduced and returned to control values by the end of the infusion (Radulovacki et al. 1990). The biphasic effect on sleep associated with repeated single injections of cocaine seen in our study has not been reported before. Hence, during chronic treatment with cocaine, sleep alterations might depend on the experimental procedure used. It is possible that the delayed sleep rebound in our experiments was observed because cocaine is a short-acting central stimulant which was administered only once a day. However, the biphasic nature of the effect illustrates that registration over the full light-dark cycle as we have done is a methodological necessity in order to guarantee complete evaluation of the drug effect. During the first days following cocaine discontinuation, the circadian distribution of the sleep-wakefulness states remained as disrupted as during chronic treatment. The amounts of SWS2 and PS were reduced in the light phase and were increased in the dark phase. Normal sleep-wakefulness rhythm recovered six days after cocaine discontinuation, except for PS which was still impaired at the end of the 10-day period. Withdrawal from chronic cocaine treatment in rats has been shown to produce sleep-wakefulness disturbances associated mainly with a SWS rebound (Yoshimura et al. 1980; Radulovacki et al. 1990) but changes in PS were not described. In humans a rebound in rapid eye movement (REM) sleep has been observed upon cocaine withdrawal (Post et al. 1974). The degree of withdrawal effects may depend upon the dose and duration of drug exposure (Johanson and Fishman 1989), but other factors may also contribute to the behavioural alterations associated with discontinuation of cocaine. For instance, conditioning effects have been discussed to play a certain role (Post and Rose 1976; Post et al. 1981; Barr et al. 1983). In the present study conditioning may have occurred since the injection with cocaine was substituted with an injection of either saline, ritanserin or chlordiazepoxide in the same conditions (same cage, same time of day). Thus, the sleep-wakefulness impairments after cocaine discontinuation in this study may be a net result of both conditioning and/or withdrawal effects. In fact conditioning is a complicating factor which can hardly be avoided in studies where animals have to be handled and injected with a drug of abuse. Hence, the ability of drugs to alter the disturbed sleep-wakefulness rhythms following cocaine discontinuation could be the result of either an antagonism of withdrawal effects, conditioning or both. Ritanserin and chlordiazepoxide when administered 48 h after the last injection of cocaine were both able to attenuate the sleep-wakefulness rhythm disturbances. Their effects following chronic cocaine treatment are presumably not due to a direct interaction since cocaine is
short acting. However, the mechanisms by which both compounds exert their effect are probably quite different since their own effect on disturbances after acute cocaine injections are dissimilar. Chlordiazepoxide prevented the enhancement of W and the reduction of SWS1 and SWS2 induced by an acute injection of cocaine. This could be achieved either through a direct interaction with cocaine or through the sedative and sleep-inducing properties of this drug. Benzodiazepines in general have a sleep-inducing effect in rats associated with an increase of SWS and a reduction of W and PS (Halperin et al. 1981; Mendelson et al. 1983; Depoortere et al. 1986). In addition, chlordiazepoxide has been found to reduce spontaneous locomotor activity in rats (File t984; McElroy et al. 1985) and to induce motor incoordination (Meert and Janssen 1989). In humans most benzodiazepines increase stage 2 sleep but reduce SWS (stages 3 and 4) and REM sleep (Gaillard et al. 1973; Kales et al. 1976; Feinberg et al. t977). Addicts consume benzodiazepines to compensate for acute symptoms induced by cocaine and other psychostimutants (Johanson and Fischman 1989; Goeders 1990; Hall et al. 1990). Hence, for chlordiazepoxide, sedative rather than sleep modulating effects probably account for the prevention of the acute increase of W elicited by cocaine. Chlordiazepoxide also reduced the impairment in the distribution of W and SWS2 throughout the tight-dark cycle following cocaine discontinuation but failed to restore PS alterations. Here in addition to the sleep-inducing and sedative effects, memory disruptive effects of chlordiazepoxide (Cole 1986; Meert and Janssen 1989) could have played a role through the conditioning. Ritanserin did not counteract the acute sleep-wakefulness alterations produced by cocaine, suggesting that there is no interaction between ritanserin and cocaine in acute conditions. Lack of direct interaction between ritanserin and cocaine was also reported in various other experimental models (Meert et al. 1990b). In normal rats, ritanserin has been shown to increase SWS2 without modification of the SWS1 and SWS2 latencies and to reduce PS with a prolongation of its latency (Dugovic et al. 1989b; Bjorvatn and Ursin 1990; Monti et al. 1990; Silhol et al. 1991). Ritanserin also promotes human SWS at the expense of stages 1 and 2 sleep with little effect on REM sleep (Idzikowski et al. 1986; Paiva et al. 1988; Adam and Oswald t989). Although it has pronounced effects on sleep, ritanserin did not alter locomotor activity (Awouters et al. 1988) and was completely devoid of sedative and amnesic effects (Meert and Janssen 1989). Lack of sedation might explain why ritanserin does not block acute cocaine effects. Support for the involvement of sedation comes from the fact that the decrease of PS induced by cocaine was not antagonized by chlordiazepoxide pretreatment but was even potentiated, like in ritanserinpretreated rats. When ritanserin treatment accompanied cocaine abstinence, the altered distribution of W and SWS2 was attenuated in both the light and dark phases from the first day of administration on. These data suggest that ritanserin accelerates the recovery of sleep-wakefulness rhythms in rats during early abstinence following chronic cocaine treatment. As in saline-treated rats, PS remained
269 altered which might indicate that recovery was not yet complete. This is in contrast with results obtained on disturbed sleep-wakefulness after continuous light exposure where PS also tended to recover in rats treated with ritanserin (Dugovic et al. 1989a). F r o m day 6 after cocaine discontinuation, the amounts of SWS2 were higher than the control values in rats receiving ritanserin. This effect could be attributed to the SWS2-inereasing properties of ritanserin since on this day SWS2 was normalized in saline-treated rats. Remarkably, in addition to its intrinsic effects on SWS2, ritanserin seemed to facilitate the onset of SWS1 and SWS2 after cocaine discontinuation. The latencies for the first episodes of SWS1 and SWS2 were shorter under ritanserin treatment as c o m p a r e d to saline treatment. It is unlikely that the restorative effects of ritanserin could be mediated by the induction of amnesia since in contrast to chlordiazepoxide, ritanserin is devoid of stimulus properties and does not induce state dependency (Meert and Janssen 1989). However, it has been shown that ritanserin alters the preference for drugs of abuse as shown in conditioned place preference (Nomikos and Spyraki 1988) and by the reduction of the intake of pharmacologically different drugs of abuse such as cocaine, alcohol and fentanyl in a choice drinking paradigm (Meert et al. 1990a, 1991). Hence, in animals, ritanserin seems to be able to interfere with fundamental and general mechanisms of abuse possibly related to reward a n d / o r conditioning. In preliminary clinical studies, a decrease of alcohol intake and an improvement of sleep continuity in chronic alcoholics treated with ritanserin has been reported (Monti and Alterwain 1991). In conclusion, the present data indicate that repeated treatment with cocaine disrupts the sleep-wakefulness r h y t h m of rats. The disruption lasts for at least 5 days following cocaine discontinuation. These changes could be the result of a genuine withdrawal effect, of conditioning based on the contingency of the injection and the expectation of the p r o n o u n c e d central and peripheral effects of cocaine or of a combination of these two factors. Both ritanserin and chlordiazepoxide attenuate disrupted sleep-wakefulness rhythms associated with cocaine discontinuation. However, the mechanisms by which both c o m p o u n d s exert their effect are probably quite different. Unlike ritanserin, chlordiazepoxide directly interacts with cocaine during acute administration, where its sedative properties p r o b a b l y play a major role. Chlordiazepoxide's action after cocaine discontinuation m a y depend u p o n its sedative, its sleep-inducing and its amnesic effects which could interfere with conditioning. In contrast, ritanserin had no interaction with acute cocaine and did not show sedative effects. After cocaine discontinuation it alleviated disturbed sleep-wakefulness rhythms p r o b a b l y by grace of its SWS2-increasing properties and its ability to reverse preference for drugs of abuse without inducing aversion.
Acknowledgements. The authors would like to thank Nancy Aerts, Patrick De Haes and William Melis for their skillful technical assistance.
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270 Leysen JE, Gommeren W, Van Gompel P, Wynants J, Janssen P, Laduron PM (1985) Receptor binding properties in vitro and in vivo of ritanserin: a very potent and long-acting serotonin 5-HTz antagonist. Mol Pharmacol 27 : 600-611 McElroy JF, Fleming RL, Feldman RS (1985) A comparison between chlordiazepoxide and CL 218, 872 a synthetic nonbenzodiazepine ligand for benzodiazepine receptors on spontaneous locomotor activity in rats. Psychopharmacology 85:224-226 Meert TF, Janssen PAJ (1989) Psychopharmacology of ritanserin: comparison with chlordiazepoxide. Drug Dev Res I8:119-144 Meert TF, Awouters F, Janssen PAJ (1990a) The preference for alcohol and cocaine is virtually abolished by the 5-HT2 receptor antagonist ritanserin. Eur J Pharmacol 183 : 1924 Meert TF, De Haes PLAJ, Vermote PCM, Janssen PAJ (1990b) Pharmacological validation of ritanserin and risperidone in the drug discrimination test procedure in the rat. Drug Dev Res 19 : 353-373 Meert TF, Awouters F, Niemegeers CJE, Schellekens KHL, Janssen PAJ (1991) Ritanserin reduces abuse of alcohol, cocaine and fentanyl in rats. Pharmacopsychiatry 24:159 163 Mendelson WB, Cain M, Cook JM, Paul SM, Skolnick P (1983) A benzodiazepine receptor antagonist decreases sleep and reverses the hypnotic actions of flurazepam. Science 219:414-416 Michel F, Klein M, Jouvet D, Valatx JL (1961) Etude polygraphique du sommeil chez le rat. CR Soc Biol (Paris) 155:2389-2392 Monti JM, Alterwain P (1991) The effects of ritanserin on mood, sleep and alcohol intake in chronic alcoholics. Biol Psychiatry 29 : 500S Monti JM, Pineyro G, Orellana C, Boussard M, Jantos H, Labraga P, Olivera S, Alvarino F (1990) 5-HT receptor agonists 1-(2,5dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and 8-OHDPAT increase wakefulness in the rat. Biog Amines 7:145-151 Nomikos GG, Spyraki C (1988) Effects of ritanserin on the reward-
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Psychopharmacology © Springer-Verlag 1992
Effects of ritanserin and chlordiazepoxide on sleep-wakefulness alterations in rats following chronic cocaine treatment Christine Dugovic, Theo F. Meert, David Ashton, and Gilbert H.C. Clincke Department of Neuropsychopharmacology,Janssen Research Foundation, B-2340 Beerse, Belgium Received November 8, 1991 / Final version March 20, 1992
Abstract. The effects of ritanserin, a 5-hydroxytryptamine2 (5-HT2) receptor antagonist, and chlordiazepoxide, a benzodiazepine agonist, on sleep-wakefulness disturbances in rats after acute administration of cocaine and after discontinuation of chronic cocaine treatment were examined. Intraperitoneal (IP) injection of chlordiazepoxide (10 mg/kg) but not ritanserin (0.63 mg/kg) prevented the increase of wakefulness (W) and the reduction of light slow wave sleep (SWS1) and deep slow wave sleep (SWS2) induced by an acute injection of cocaine (20 mg/kg IP). Daily injection of cocaine (20 mg/kg for 5 days, then 30 mg/kg for 5 days IP) at the onset of the light phase elicited an increase of W and a concomitant decrease of SWS1, SWS2 and paradoxical sleep (PS) in the light phase, followed by a rebound in SWS2 and PS in the subsequent dark phase. Following cocaine discontinuation, the circadian distribution of sleep-wakefulness states remained disturbed in saline-treated rats for at least 5 days. Both ritanserin (0.63 mg/kg IP/day) and chlordiazepoxide (10 mg/kg IP/day) reduced the alteration in the distribution of W and SWS2 throughout the lightdark cycle from the first day of administration on, but failed to prevent PS alterations. The mechanisms by which both compounds exert their effect are probably quite different. For chlordiazepoxide sedative and sleepinducing properties probably play a major role. In contrast, for ritanserin SWS2-increasing properties and its ability to reverse preference for drugs of abuse without inducing aversion might be key factors. Key words: Sleep-wakefulness - Chronic cocaine treatment - Cocaine discontinuation - Ritanserin - Chlordiazepoxide - Rat Recent investigations have shown that ritanserin, a potent serotonin antagonist at central 5-hydroxytryptamine-2 (5HT2) receptors (Leysen et al. 1985), reduced cocaine preference and cocaine intake in rats without interfering with consumatory physiological processes (Meert et al. 1990a, Correspondence to:
C. Dugovic
1991). At doses similar to those required to produce activity in this test procedure, ritanserin induced an increase of deep slow wave sleep (SWS) and a decrease of paradoxical sleep (PS) during the physiological sleep period of the rat (Dugovic and Wauquier 1987; Davenne et al. 1989; Bjorvatn and Ursin 1990; Monti et al. 1990; Silhol et al 1991) and tended to normalize the disturbed sleep-wakefulness organization in rats exposed to continuous light (Dugovic et al. 1989a). In humans given ritanserin, polysomnographic data revealed a marked increase of SWS in healthy volunteers (Idzikowski et al. 1986), poor sleepers (Adam and Oswald 1989), insomniacs (Ruiz-Primo et al. 1989) and dysthymic patients (Paiva et al. 1988). In humans, agitation, depression, anxiety and sleep disturbances have been described as abstinence symptoms following abrupt cessation of cocaine abuse (Gawin and Kleber 1986; Gillin et al. 1989; Gawin 1991). In animal models related to drug withdrawal symptoms, cocaine has been reported to produce anxiogenic-like effects (Wood and Lal 1987; Costall et al. 1989 and Emmett-Oglesby et al. 1990 for a general review). Impairments of the sleepwakefulness patterns have been also observed in animals treated with cocaine. In acute conditions, cocaine elicited cortical EEG desynchronization in cats (Wallach and Gershon 1971) and had pronounced sleep-suppressing effects in rats (Hill et al. 1977). Repetitive injections or continuous infusion of cocaine in rats resulted in a decrease of total sleep time during the course of the treatment followed by alterations in the sleep-wakefulness rhythms on drug discontinuation (Yoshimura et al. 1980; Radulovacki et al. 1990). However, the effects of cocaine withdrawal on sleep-wakefulness patterns have not been extensively studied. The purpose of the present study was to further investigate the sleep-wakefulness impairments in rats following discontinuation of chronic cocaine treatment and to examine the effects of ritanserin on these disturbances. Chlordiazepoxide was tested for comparison because of the well-known sleep-inducing properties of benzodiazepines. In addition, the direct interaction between cocaine, ritanserin and chlordiazepoxide was studied in acute experimental conditions.
264
Materials and methods
Results
Implantation procedure. Male adult Wistar rats weighing 240~
Effects of chronic cocaine administration
260 g were chronically implanted under pentobarbital anesthesia (50 mg/kg IP) with electrodes for standard sleep monitoring. Electrodes were positioned on the frontal and occipital cortices, subcutaneously on each side of the orbit and in the neck muscles for polygraphic recordings of electrocorticogram (ECoG), electro-oculogram (EOG) and electromyogram (EMG). After surgery, the animals were individually housed in Plexyglas cages and maintained under controlled environmental conditions (12 h light-dark schedule, light on 9:00 am., 22 +_ 2°C ambient temperature, food and water ad libitum).
Recording procedure. At the end of a 8-10 day recovery period from surgery and habituation to the environment, the rats were connected by a cable to a rotating connector and ECoG, EOG and EMG activities were recorded on a polygraph. The polygraphic recordings were scored visually by 30 s epochs. These epochs were classified as being either wakefulness (W), light stow wave sleep (SWS1), deep slow wave sleep (SWS2) or paradoxical sleep (PS), using the criteria of Michel et al. (1961). The scores were entered into a computer which calculated the different sleep-wakefulness parameters (amount of time spent in these four states, number and duration of episodes for each state, SWS1, SWS2 and PS latencies). Pharmacological procedure. In the first experiment, the animals (n = 20) received a daily injection of cocaine (20 mg/kg for 5 days, then 30 mg/kg for 5 days) at the onset of the light phase (9 : 00 a.m.) for a 10-day period. The dose-range of cocaine used in the present study and the duration of drug exposure have been suggested to produce measurable behavioral changes after withdrawal (Johanson and Fischman 1989). After a 5-day treatment period, the dose of cocaine was increased to 30 mg/kg in order to compensate for the possible development of tolerance upon repeated administration. On the next day the rats were treated with saline, then for a subsequent period of 10 days with either saline (n = 8), ritanserin (0,63 mg/kg/day, n = 6) or chlordiazepoxide (10 mg/kg/day, n = 6). The dose of 0.63 mg/kg ritanserin was chosen since it was the optimal dose to induce the most pronounced increase of SWS2 in the rat (Dugovic et al. 1989b). The dose of 10 mg/kg chlordiazepoxide was chosen on the basis of preliminary experiments to obtain an increase of SWS2 in the same proportions as that observed with ritanserin. Ritanserin or chlordiazepoxide treatment was started 48 h after the last injection of cocaine in order to avoid direct drug interaction. In the second experiment, a separate group of rats received an injection of ritanserin (0.63 mg/kg, n = 5) or chlordiazepoxide (10 mg/kg, n = 6) at light onset, and 30 rain later 20 mg/kg cocaine. All drugs were injected IP in a volume of 4 ml/kg body weight. Cocaine and chlordiazepoxide were dissolved in saline and ritanserin was dissolved in 1 mM tartaric acid. Data analysis. Polygraphic recordings were performed for 24 h during chronic cocaine treatment (days 5 and 8) and every second day following cocaine discontinuation (days 2, 4, 6, 8 and 10). The sleep-wakefulness parameters were analyzed per 4-h and 12-h periods and were compared to control values (later referred to as C), £e. saline injection under the same conditions before cocaine treatment was started. For the acute pharmacological effects tested in the second experiment, polygraphic recordings were performed for 8 h following the last injection. The sleep-wakefulness parameters were analyzed per 4-h periods and were compared to control values, i.e. vehicle injections under the same conditions. The duration of the time spent in the different states of vigilance was expressed in minutes (mean _+_SEM). Statistical significance of the data was assessed by means of the paired two,tailed Student t-test for within group comparisons. In addition, the non-paired two-tailed Student t-test was used for between group comparisons following cocaine discontinuation.
D a i l y a d m i n i s t r a t i o n of c o c a i n e (20 m g / k g for 5 days, t h e n 3 0 m g / k g for 5 days) at the o n s e t of the light p h a s e d i s r u p t e d the s l e e p - w a k e f u l n e s s d i s t r i b u t i o n t h r o u g h o u t the l i g h t - d a r k cycle. C o c a i n e a d m i n i s t r a t i o n r e s u l t e d in an i n c r e a s e of W a n d a c o n c o m i t a n t d e c r e a s e of sleep d u r a t i o n in the light p h a s e f o l l o w e d by a sleep r e b o u n d in t h e s u b s e q u e n t d a r k phase. H o w e v e r , in the t o t a l 24-h p e r i o d sleep a n d w a k e f u l n e s s a m o u n t s w e r e n o t significantly different f r o m the c o n t r o l values (C). T h e s e c h a n g e s were o b s e r v e d o n d a y 5 a n d 8, b u t w e r e m o r e p r o n o u n c e d o n d a y 8. F i g u r e 1 illustrates the s l e e p - w a k e f u l n e s s a l t e r a t i o n s a n a l y z e d per 4-h p e r i o d s o n d a y 8 of c h r o n i c c o c a i n e
2001 180 160q 140~
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Fig. 1. Distribution of the sleep-wakefulness states per 4 h periods on day 8 of chronic cocaine treatment. Cocaine (20 mg/kg IP for 5 days, then 30 mg/kg IP for 5 days) was administered daily at the onset of the light phase. Mean values + SEM. of 20 animals were expressed in minutes. W, wakefulness; SWS1, light slow wave sleep; SWS2, deep slow wave sleep; PS, paradoxical sleep. *P < 0.05, **P < 0.01, * ** P < 0.001 (paired two-tailed Student t-test) as compared to control values (saline injection under the same conditions before chronic cocaine treatment was started). (Z~) Vehicle; ( I ) cocaine day 8
265 treatment. The administration of cocaine (30 mg/kg) at light onset was followed by a marked increase of W and a suppression of SWS1, SWS2 and PS during the first 4-h period consistent with a decrease in the number of episodes for all sleep states. The latency for the first episode of sleep (SWSt latency) was delayed by about 2-h following cocaine injection. In the second and third 4-h periods of the light phase, the amounts of the different states of vigilance returned to control values, except for PS which remained significantly reduced. During the three consecutive 4-h periods of the dark phase, SWS2 and PS rebounded while W was significantly decreased. This was due mainly to an increase in the number of SWS2 and PS episodes and to a reduction in the mean duration of W episodes.
Effects of ritanserin and chlordiazepoxide following chronic cocaine treatment The distribution of the sleep-wakefulness states during the light and dark phases in rats daily treated with either W (rnin) 0 - 12h
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rebound was suppressed in the dark phase. Subsequently, an increase in SWS2 duration was observed from the fifth day of ritanserin or chlordiazepoxide administration on (e.g. day 6) as compared to C values. In contrast, PS alterations were not reduced in both ritanserin- and chlordiazepoxide-treated rats as compared to saline-treated rats (Fig. 5). During the 10-day period after cocaine discontinuation, PS amounts remained lower than the C values in the light phase and were slightly increased in the dark phase. As compared to C, SWS1 amounts were significantly decreased in the light phase during ritanserin treatment and were enhanced in the dark phase during chlordiazepoxide treatment (Fig. 3). Sleep latencies were modified as shown in Table 1. In saline-treated rats on the second day after cocaine discontinuation, the SWS1, SWS2 and PS latencies were significantly prolonged when compared to C values. Similar but less pronounced effects were observed on the fourth day after cocaine discontinuation. The SWS1 and SWS2 latencies were normalized in ritanserin-treated rats following the first and the second injection, respectively. In rats given chlordiazepoxide, the SWS 1, SWS2 and PS latencies
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Effects of ritanserin and chlordiazepoxide combined with cocaine in acute conditions As shown in Tables 2 and 3, rats given an acute injection of cocaine (20 mg/kg) exhibited a long-lasting increase in the amount of time spent in W and reduced amounts of SWS1, SWS2 and PS associated with an increase in all sleep states latencies. These effects occurred in the first 4 h following the treatment and the amounts of the sleepwakefulness states returned to control values in the subsequent 4 h. Ritanserin (0.63 mg/kg) alone produced a significant increase in SWS2 duration and a significant decrease in SWS1 and PS duration during the first 4 h following the treatment (Table2), Rats given chlordiazepoxide (10 mg/kg) alone exhibited significantly increased amounts of SWS2 and reduced amounts of PS (Table 3). The SWS1 and SWS2 latencies were significantly shortened in chlordiazepoxide-treated rats whereas the PS latency was prolonged after ritanserin and chlordiazepoxide treatment. Sleep-wakefulness alterations observed after administration of cocaine were not modified by ritanserin pretreatment (Table2). In contrast, pretreatment with
267 Table 1. Sleep latencies in rats treated with either saline, ritanserin (0.63 mg/kg) or chlordiazepoxide (10 mg/kg) after cocaine discontinuation
SWS1 latency
SWS2 latency
PS latency
25.4 72.1 40.9 28.8
89.1 167.3 130.7 118.4
_+ 18.0 _+ 33.5** ,+ 34.7 ,+ 22.5
Salinetreated rats (n = 8)
Control Day 2 Day 4 Day 6
14.3 36.7 28.6 21.0
_+ 2.1 _+ 9.3* ,+ 4.3** _+ 7.2
Ritanserintreated rats (n = 6)
Control Day 2 Day 4 Day 6
13.6 _+ 2.6 19.3 ,+ 4.2 14.2 ,+ 5.1 15.3 + 4.4
17.2 _+ 2.1 39.9 + 7.0* 20.8 + 4.4 20.0 ,+ 3.8
74.3 182.4 199.8 129.6
__ 9.2 -+_30.7* ,+ 30.2* + 19.8
Chlordiazepoxidetreated rats (n = 6)
Control Day 2 Day 4 Day 6
23.7 14.0 27.7 21.7
33.0 19.1 30.2 23.0
78.2 103.9 84.1 81.9
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Values of light slow wave sleep (SWS1), deep slow wave sleep (SWS2) and paradoxical sleep (PS) latencies are means _+ SEM (min of 6-8 animals in each experimental group. Sleep latencies were evaluated on days 2, 4 and 6 after cocaine discontinuation *P < 0.05, **P < 0.01 (paired two-tailed Student t-test) as compared to control values (saline injection in the same conditions before cocaine administration was started)
Table 2. Effects of ritanserin (0.63 mg/kg) on sleep-wakefulness states and sleep latencies in cocaine-treated rats (20 mg/kg) during the first 4-h period following the treatment. Mean values + SEM of 5 animals were expressed in minutes
W SWS1 SWS2 PS SWS1 latency SWS2 latency PS latency
Vehicle + vehicle
Ritanserin + vehicle
Vehicle + cocaine
Ritanserin + cocaine
79.9 _ 9.1 15.9 _ 8.4 132.5 +_ 6.6 11.8 ,+ 3.5 13.8 ,+ 4.1 26.7 + 2.5 105.6 ,+ 25.6
71.9 + 10.1 9.3 _.+ 1.3" 152,1 + 8.2* 6.7 + 2.5* 16.3 ,+ 7.8 23.8 ,+ 9.7 177.1 ,+ 34.3*
137.0 8.9 86,5 7.6 103.2 121.4 175.6
132.7 10.0 92.7 4.6 86.9 99.0 219.0
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_+ 20.6* + 2.0 + 17.9' + 2.1"* _ 30.3* ,+ 32.3* ___41.9"*
W, wakefulness; SWS 1, light slow wave sleep; SWS2, deep slow wave sleep; PS paradoxical sleep *P < 0.05, **P < 0.01 (paired two-tailed Student t-test) as compared to control values (vehicle injection under the same conditions)
Table 3. Effects of chlordiazepoxide (10 mg/kg) on sleep-wakefulness states and sleep latencies in cocaine-treated rats (20 mg/kg) during the first 4-h period following the treatment. Mean values _+ SEM of 6 animals were expressed in minutes
Vehicle + vehicle W SWS1 SWS2 PS SWS1 latency SWS2 latency PS latency
78.3 + 15.1 + 135.2 + 11.4 + 13.3 + 25.2 _ 96.5 --
7.6 2.1 6.0 2.9 3.4 2.6 22.8
Chlordiazepoxide + vehicle
Vehicle + cocaine
Chlordiazepoxide + cocaine
71.8 12.0 150.5 5.7 4.8 7.3 140.2
135.0 8.8 89.3 6.9 102.6 118.3 172.8
97.3 10.7 129.1 2.9 30.5 45.1 202.6
- 7.6 + 2.1 + 8.2* _ 1.4' + 1.4' + 2.2** 4- 16.0"
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+ 14.7 + 2.4 + 12.2 + 1.0"* + 16.9 _+ 17.7 + 21.8'*
W, wakefulness; SWS1, light slow wave sleep; SWS2, deep slow wave sleep; PS, paradoxical sleep *P < 0.05, **P < 0.01 (paired two-tailed Student t-test) as compared to control values (vehicle injection under the same conditions)
c h l o r d i a z e p o x i d e a n t a g o n i z e d the i n c r e a s e of W a n d the d e c r e a s e o f S W S 1 a n d S W S 2 i n d u c e d by c o c a i n e . T h e S W S 1 a n d S W S 2 latencies also d e c r e a s e d t o w a r d s c o n t r o l v a l u e s in c h l o r d i a z e p o x i d e - p r e t r e a t e d rats. H o w e v e r , c h l o r d i a z e p o x i d e did n o t c o u n t e r a c t the P S - s u p p r e s s i n g effects of c o c a i n e ( T a b l e 3).
Discussion I n o u r e x p e r i m e n t s , r e p e a t e d a d m i n i s t r a t i o n of c o c a i n e at the o n s e t of the light p h a s e in rats i n d u c e d m a r k e d altera t i o n s in the s l e e p - w a k e f u l n e s s r h y t h m t h r o u g h o u t the l i g h t - d a r k cycle b o t h d u r i n g the p e r i o d of c o c a i n e a d m i n -
268 istration and after discontinuation of the drug. During chronic treatment with cocaine an initial decrease of SWS1, SWS2 and PS, mainly during the first 4 h of the light phase, was followed by a rebound in SWS2 and PS during the subsequent dark phase. These initial sleep suppressing effects of cocaine are in agreement with previous data obtained in the rat during acute (Hill et al. 1977) or chronic exposure (Yoshimura et al. 1980). During continuous arterial infusion of cocaine (100 mg/kg/day) for 7 days, SWS1, SWS2 and PS were initially reduced and returned to control values by the end of the infusion (Radulovacki et al. 1990). The biphasic effect on sleep associated with repeated single injections of cocaine seen in our study has not been reported before. Hence, during chronic treatment with cocaine, sleep alterations might depend on the experimental procedure used. It is possible that the delayed sleep rebound in our experiments was observed because cocaine is a short-acting central stimulant which was administered only once a day. However, the biphasic nature of the effect illustrates that registration over the full light-dark cycle as we have done is a methodological necessity in order to guarantee complete evaluation of the drug effect. During the first days following cocaine discontinuation, the circadian distribution of the sleep-wakefulness states remained as disrupted as during chronic treatment. The amounts of SWS2 and PS were reduced in the light phase and were increased in the dark phase. Normal sleep-wakefulness rhythm recovered six days after cocaine discontinuation, except for PS which was still impaired at the end of the 10-day period. Withdrawal from chronic cocaine treatment in rats has been shown to produce sleep-wakefulness disturbances associated mainly with a SWS rebound (Yoshimura et al. 1980; Radulovacki et al. 1990) but changes in PS were not described. In humans a rebound in rapid eye movement (REM) sleep has been observed upon cocaine withdrawal (Post et al. 1974). The degree of withdrawal effects may depend upon the dose and duration of drug exposure (Johanson and Fishman 1989), but other factors may also contribute to the behavioural alterations associated with discontinuation of cocaine. For instance, conditioning effects have been discussed to play a certain role (Post and Rose 1976; Post et al. 1981; Barr et al. 1983). In the present study conditioning may have occurred since the injection with cocaine was substituted with an injection of either saline, ritanserin or chlordiazepoxide in the same conditions (same cage, same time of day). Thus, the sleep-wakefulness impairments after cocaine discontinuation in this study may be a net result of both conditioning and/or withdrawal effects. In fact conditioning is a complicating factor which can hardly be avoided in studies where animals have to be handled and injected with a drug of abuse. Hence, the ability of drugs to alter the disturbed sleep-wakefulness rhythms following cocaine discontinuation could be the result of either an antagonism of withdrawal effects, conditioning or both. Ritanserin and chlordiazepoxide when administered 48 h after the last injection of cocaine were both able to attenuate the sleep-wakefulness rhythm disturbances. Their effects following chronic cocaine treatment are presumably not due to a direct interaction since cocaine is
short acting. However, the mechanisms by which both compounds exert their effect are probably quite different since their own effect on disturbances after acute cocaine injections are dissimilar. Chlordiazepoxide prevented the enhancement of W and the reduction of SWS1 and SWS2 induced by an acute injection of cocaine. This could be achieved either through a direct interaction with cocaine or through the sedative and sleep-inducing properties of this drug. Benzodiazepines in general have a sleep-inducing effect in rats associated with an increase of SWS and a reduction of W and PS (Halperin et al. 1981; Mendelson et al. 1983; Depoortere et al. 1986). In addition, chlordiazepoxide has been found to reduce spontaneous locomotor activity in rats (File t984; McElroy et al. 1985) and to induce motor incoordination (Meert and Janssen 1989). In humans most benzodiazepines increase stage 2 sleep but reduce SWS (stages 3 and 4) and REM sleep (Gaillard et al. 1973; Kales et al. 1976; Feinberg et al. t977). Addicts consume benzodiazepines to compensate for acute symptoms induced by cocaine and other psychostimutants (Johanson and Fischman 1989; Goeders 1990; Hall et al. 1990). Hence, for chlordiazepoxide, sedative rather than sleep modulating effects probably account for the prevention of the acute increase of W elicited by cocaine. Chlordiazepoxide also reduced the impairment in the distribution of W and SWS2 throughout the tight-dark cycle following cocaine discontinuation but failed to restore PS alterations. Here in addition to the sleep-inducing and sedative effects, memory disruptive effects of chlordiazepoxide (Cole 1986; Meert and Janssen 1989) could have played a role through the conditioning. Ritanserin did not counteract the acute sleep-wakefulness alterations produced by cocaine, suggesting that there is no interaction between ritanserin and cocaine in acute conditions. Lack of direct interaction between ritanserin and cocaine was also reported in various other experimental models (Meert et al. 1990b). In normal rats, ritanserin has been shown to increase SWS2 without modification of the SWS1 and SWS2 latencies and to reduce PS with a prolongation of its latency (Dugovic et al. 1989b; Bjorvatn and Ursin 1990; Monti et al. 1990; Silhol et al. 1991). Ritanserin also promotes human SWS at the expense of stages 1 and 2 sleep with little effect on REM sleep (Idzikowski et al. 1986; Paiva et al. 1988; Adam and Oswald t989). Although it has pronounced effects on sleep, ritanserin did not alter locomotor activity (Awouters et al. 1988) and was completely devoid of sedative and amnesic effects (Meert and Janssen 1989). Lack of sedation might explain why ritanserin does not block acute cocaine effects. Support for the involvement of sedation comes from the fact that the decrease of PS induced by cocaine was not antagonized by chlordiazepoxide pretreatment but was even potentiated, like in ritanserinpretreated rats. When ritanserin treatment accompanied cocaine abstinence, the altered distribution of W and SWS2 was attenuated in both the light and dark phases from the first day of administration on. These data suggest that ritanserin accelerates the recovery of sleep-wakefulness rhythms in rats during early abstinence following chronic cocaine treatment. As in saline-treated rats, PS remained
269 altered which might indicate that recovery was not yet complete. This is in contrast with results obtained on disturbed sleep-wakefulness after continuous light exposure where PS also tended to recover in rats treated with ritanserin (Dugovic et al. 1989a). F r o m day 6 after cocaine discontinuation, the amounts of SWS2 were higher than the control values in rats receiving ritanserin. This effect could be attributed to the SWS2-inereasing properties of ritanserin since on this day SWS2 was normalized in saline-treated rats. Remarkably, in addition to its intrinsic effects on SWS2, ritanserin seemed to facilitate the onset of SWS1 and SWS2 after cocaine discontinuation. The latencies for the first episodes of SWS1 and SWS2 were shorter under ritanserin treatment as c o m p a r e d to saline treatment. It is unlikely that the restorative effects of ritanserin could be mediated by the induction of amnesia since in contrast to chlordiazepoxide, ritanserin is devoid of stimulus properties and does not induce state dependency (Meert and Janssen 1989). However, it has been shown that ritanserin alters the preference for drugs of abuse as shown in conditioned place preference (Nomikos and Spyraki 1988) and by the reduction of the intake of pharmacologically different drugs of abuse such as cocaine, alcohol and fentanyl in a choice drinking paradigm (Meert et al. 1990a, 1991). Hence, in animals, ritanserin seems to be able to interfere with fundamental and general mechanisms of abuse possibly related to reward a n d / o r conditioning. In preliminary clinical studies, a decrease of alcohol intake and an improvement of sleep continuity in chronic alcoholics treated with ritanserin has been reported (Monti and Alterwain 1991). In conclusion, the present data indicate that repeated treatment with cocaine disrupts the sleep-wakefulness r h y t h m of rats. The disruption lasts for at least 5 days following cocaine discontinuation. These changes could be the result of a genuine withdrawal effect, of conditioning based on the contingency of the injection and the expectation of the p r o n o u n c e d central and peripheral effects of cocaine or of a combination of these two factors. Both ritanserin and chlordiazepoxide attenuate disrupted sleep-wakefulness rhythms associated with cocaine discontinuation. However, the mechanisms by which both c o m p o u n d s exert their effect are probably quite different. Unlike ritanserin, chlordiazepoxide directly interacts with cocaine during acute administration, where its sedative properties p r o b a b l y play a major role. Chlordiazepoxide's action after cocaine discontinuation m a y depend u p o n its sedative, its sleep-inducing and its amnesic effects which could interfere with conditioning. In contrast, ritanserin had no interaction with acute cocaine and did not show sedative effects. After cocaine discontinuation it alleviated disturbed sleep-wakefulness rhythms p r o b a b l y by grace of its SWS2-increasing properties and its ability to reverse preference for drugs of abuse without inducing aversion.
Acknowledgements. The authors would like to thank Nancy Aerts, Patrick De Haes and William Melis for their skillful technical assistance.
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