α-Flupenthixol increases slow-wave sleep in rats: Effect of dopamine receptor blockade

α-Flupenthixol increases slow-wave sleep in rats: Effect of dopamine receptor blockade

002x-3’)0x’x?,0403?3-03803 00/o Pcrgamon Press Ltd a-FLUPENTHIXOL INCREASES RATS: EFFECT OF DOPAMINE SLOW-WAVE SLEEP IN RECEPTOR BLOCKADE C. FORNAL...

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002x-3’)0x’x?,0403?3-03803 00/o Pcrgamon Press Ltd

a-FLUPENTHIXOL INCREASES RATS: EFFECT OF DOPAMINE

SLOW-WAVE SLEEP IN RECEPTOR BLOCKADE

C. FORNAL, W. J. WOJCIK* and M. RaDrrLovacKtt Department

of Pharmacology,

University

of Illinois at the Medical

(Accepted

25 Septrmhrr

Center,

Chicago.

IL 60680. U.S.A.

1981)

Summary-r-Flupenthixol (0.2 mg/kg, i.p.). a dopamine receptor blocker, significantly increased slowwave sleep and decreased wakefulness in rats when administered either at the onset of a 12-hr light period or a 12-hr dark period. The same dose of the drug strongly antagonized the dopamine-mediated stereotypy produced by an injection of apomorphme (1 mg/kg, i.p.) for at least 9-hr, indicating that dopamine receptors were blocked throughout most of the recording session. The results suggest a relationship between the blockade of dopamine receptors and the increase of slow-wave sleep time.

r-Flupenthixol (4-(3-[2-(trifluoromethyl)thioxanthen-9-ylidenelpropyli-1-piperazine ethanol) is a thioxanthene derivative and also a dopamine (DA) receptor blocker (Miiller-Nielsen, Pedersen, Nymark. Frank, Boeck, Ejellard and Christensen, 1973). When this drug was given to rats deprived of rapid-eyemovement sleep (REMS), it prevented the increase in wakefulness and reduction of sleep caused by bromocriptine (Radulovacki, Wojcik and Fornal, 1979) a DA receptor stimulant (Clark, Fhickiger and Loew, 1978). However, administration of r-flupenthixol alone showed only a trend toward decreased wakefulness and increased slow-wave sleep (SWS) with no effect on REM sleep. Since REM sleep deprivation may produce a DA receptor supersensitivity (Tufik, Lindsey and Carlini, 1978) the action of dopaminergic drugs may be modified in this experimental situation. Thus, the effects of z-flupenthixol observed on sleep in REM sleep-deprived rats may be different from that seen in normal, non-REM sleep-deprived animals. Furthermore, a recent study in rats indicated that an accelerated DA utilization in the brain prevails during the dark period as compared to the light period following tyrosine hydroxylase inhibition with D,L-a-methyl-p-tyrosine (H44/68) (Lemmer and Berger. 1978). This suggested that perhaps the effects of DA receptor blockade may be different depending on the time of the day when the DA receptor antagonist is administered. In light of the above findings, an examination was made of the effects of r-flupenthixol on sleep and waking in normal rats at time intervals corresponding

* Present address: Laboratory for Preclinical Pharmacology. NIMH, St Elizabeth’s Hospital, Washington, DC 20032, U.S.A. t Send reprint requests to Dr M. Radulovacki, Department of Pharmacology, University of Illinois at the Medical Center, 835 S. Wolcott Ave., Chicago, IL 60612, U.S.A. Key words: a-flupenthixol, dopamine receptor blockade, sleep, rat. 323

to relatively

high (i.e. 12-hr dark period) or low (i.e. 12-hr light period) central dopaminergic activity. As a measure of the intensity and duration of the antidopaminergic activity of the drug, the influence of various doses of r-flupenthixol on the DA-mediated stereotypy induced by apomorphine was also examined. METHODS

Adult male Sprague-Dawley rats (35GIOOg) were used in these experiments. Each rat was anesthetized with sodium pentobarbital (40 mg/kg, i.p.), supplemented with ether as required. Atropine methyl nitrate (2 mg/kg, s.c.) was also given to prevent bronchial congestion. For recording the cortical electroencephalogram (EEG), stainless steel electrodes were screwed bilaterally into the skull over the parietal cortices, and for recording the electromyogram (EMG), stainless steel flexible wire electrodes were implanted in the dorsal neck musculature. The wires of the electrodes were soldered to the appropriate leads of a connector fixed to the skull by dental cement. Two additional stainless steel screws (size t%80 x l/S in.) were threaded into the skull, one into the occipital bone and the other into the frontal bone, to help secure the implant. After surgery, each animal was maintained in a separate cage under lighting conditions which consisted of a timer-regulated light period from 8 :00 a.m. to 8:00 p.m. All animals were housed in the same room at a constant temperature (22°C) and had free access to food and water for the duration of the study. The experiments were carried out at least 1 week after surgery and, 2 days before the recording session, all animals were habituated to a pseudo cable system. On the day of the experiment, one group of animals received either 0.2 mg/kg (i.p.) a-flupenthixol dihydrochloride (H. Lundbeck & Co.) or drug vehicle (propylene glycol) at 8:00 a.m., while a second group of animals was treated in the same manner at 8:OOp.m.

324

C. FORNAI.
I. Effect of r-llupenthixol (0.2 &kg. i.p.) administration on the sleep waking of rats during the I2-hr light and 12-hr dark periods N

Light (08.00 Dark (20.00

period 20.00 hr) period 08.00 hr)

Control r-Flupcnthi4ol Control r.-Flupenthlxol

(8) (8) (6) (5)

W 194 I45 348 277

i * i i

REMS

SWS I5 23* 26 l6*

449 501 30x 371

* * i_ *

cycle

35 30* 17 13*

77 73 64 73

i * k *

20 IO IO 5

The results arc means k SD (min). *P < 0.05 by Student’s I-test. The number of animals (N) in each group is shown in parenthesis.

Each group was polygraphically recorded for the next 12-hr during their respective light or dark periods. The dose of r-llupenthixol selected for these studies produced a strong, long-lasting blockade of DA receptors as reflected by the ability of the drug to antagonize apomorphine-induced stereotypy (see below). Evaluation of the polygraphic recordings was made using standard techniques in which each epoch of the record was dctcrmined to be either wakefulness, SW sleep or REM sleep. The epochs were 50-set long, and the speed of the paper drive was lOOsec,!page of paper. Statistics were performed on the total amount of time spent in these three states using Student’s t-test. Animals in the third group. not implanted for polygraphic recording, were injected with either various doses of rr-llupenthixol dihydrochloride (0.05. 0.2 or 0.8 “g/kg, i.p.) or drug vehicle (propylene glycol) at 8:OOa.m. One. 3 and 9 hr later, all animals received an injection of a I mg;kg (i.p.) dose of apomorphine hydrochloride (Eli Lilly & Co.) to challenge the blockade of DA receptors produced by r-Rupenthixol. The intensity of the resulting stereotypy produced by apomorphine was evaluated for 30 set every 5 min over the next IS min post-injection according to the scoring system used by Costall and Naylor (1974); (0) normal behavior; (I) exploratory activity. discontinuous sniffing; (2) continuous sniffing; (3) continuous sniffing, discontinuous licking, biting or gnawing; (4) continuous licking, biting or gnawing. The average score obtained during the l5-min observation period for each animal over the 3 time intervals was used in the final analysis. Statistics were performed using the two-way analysis of variance (ANOVA) with comparisons made to the control group by the NewmanKeuls test.

fulness by 20”,, (P < 0.05) and increased SW sleep by 209,, (P < 0.025) when compared to controls. However, REM sleep was essentially unchanged during both the light and dark periods following drug administration. Figure I shows dose and time ctTects of z-flupcnthixol on apomorphine-induced stcreotypy. It can be seen that only the 0.2 mgikg and 0.8 mg/kg doses of x-flupenthixol significantly antagonized the stereotypy induced by apomorphine for all 3 time periods. Therefore, at the 0.2 mg:kg dose of r-flupenthixol used in the sleep study, DA receptors were blocked throughout most of the polygraphic recordings.

The results indicate that r-flupenthixol significantly decreased wakefulness and increased SW sleep when administered to rats during their light or dark periods. The percentage reduction of wakefulness during the light period (25”,,) was not much greater than that observed during the dark period (20”~ as compared to the respective controls. However, when minute differences are compared between the two time periods. administration of r-fupenthixol appeared to have a greater effect during the dark period. A mean

31

0

Table I shows that control animals spent on the average 24 hr more time in SW sleep during the l2-hr light period as compared to those animals recorded during the 12-hr dark period. When r-flupenthixol was given to rats during the light period, wakefulness was significantly decreased by 25’!,, (P < 0.0005) and SW sleep increased by 12’>,> (P < 0.01) as compared to controls. Administration of r-flupenthixol to rats during the dark period also significantly reduced wake-

I

3

9

Time

(h)

Fig. I. Dose and time effects of r-Rupenthlxol on apomorphine-induced stereotypy. Apomorphine (I mg/kg, i.p.) was administered I, 3 or 9 hr after pretreatment with either r-flupenthixol 0.05 mg,‘kg (0). 0.2 mg; kg (0) or 0.8 mg.‘kg (w) or control vehicle (0). The results are expressed as mean behavioral scores f SD (vertical lines) for 9 animals in each group. *P < 0.01 by two-way ANOVA with comparisons made to the control group by the Newman--Keuls

test.

r-Flupenthixoi decrease of 71 min of wakefulness was observed during this period as compared to a mean decrease of 49 min during the light period. This suggests that the drug was more effective in reducing ~vakefulness during a time when DA turnover was reported to be elevated (Lemmer and Berger, 1978). These results were anticipated because when DA activity is high, more DA is released to act on post-synaptic receptors. Consequently, blockade of these receptors by r-flupenthixol would result in a larger behavioral change as compared with low DA activity. In spite of this. the differences observed between the effects of x-flupenthixol on waking during the light and dark periods were not very impressive. This could he related to a highly significant ultradian rhythm of DA-receptor binding which was reported in the rat striatum with peak-times at both 2:OOa.m. and 2:OOp.m. (Naber, Wirz-Justice, Kafka and Wehr, 1980). These identical ultradian changes in DA receptor sensitivity may cancel the differences in DA turnover during the light and dark periods. Therefore, small behavioral changes are observed between these two time periods with regard to the anti-dopaminergic action of x-flupenthixol. The enhancement of SW sleep observed in this work by g-flupenthixol is in agreement with the effects on sleep obtained with other DA receptor blockers. Thus, administration of spiroperidol or haloperidol, two potent DA receptor antagonists, to rats increased either SW sleep, or SW sleep,, respectively (Monti. 1979). Kafi and Gaillard (1976) have also demonstrated an increase in total SW sleep time after spiroperidol administration, In contrast, the antidopaminergic drug, pimozide. produced only a trend toward increased SW sleep time in rats (Hartmann, Zwilhng. Koski and List, 1973). In humans, a dose of l-4 mg of pimozide did not affect sleep while a daily administration of 4-20 mg increased total sleep time and Stage II (Gillin, Van Kammen and Bunney, 1978). Based on evidence that apomorphine induces stereotyped behavior in rodents by direct action on central dopaminergic receptors (Ernst and Smehk, 1966; Ernst, 1967) the present authors have used the blockade of this response as a behavioral index of the antidopaminergic action of a-flupenthixol. Figure I shows that the stereotypy produced by apomorphine was strongly antagonized by the selected dose of ct-flupenthixol used for the sleep study. Therefore, the observed changes in sleep-wakefulness following administration of the drug could be related to a reduction in DA receptor activity. This assumption is in accordance with reports indicating that pharmacological stimulation of DA-receptors increases wakefulness and inhibits sleep (Kafi and Gaillard, 1976; Radulovacki, Wojcik and Fornat, 1979; Cianchetti, Masala, Mangoni and Gessa, 1980). In summary, these data show that the administration of a-flupenthixol to rats significantly increased SW sleep and decreased wakefulness during a 12-hr period. Since the same dose of the drug antagonized

32s

and sleep

the DA-mediated stereotypy produced by apomorphine, the results suggest a relationship between DAreceptor blockade and the increase in SW sleep time. The obtained enhancement of SW sleep may be related to an attenuation of wakefulness. since DA neurons have been implicated in behavioral arousal (Jouvet, 1972; Jones, Bobillier, Pin and Jouvet. 1973). Acknoalrdyrmetrts--This

research was supported by ONR dihydr~chlor-

contract NO0 14-79-C-0420. ~-Flupenthixol ide was kindly supplied by H. Lundbeck hagen.

& Co., Copen-

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