Mutant mice lacking the cholecystokinin2 receptor show a dopamine-dependent hyperactivity and a behavioral sensitization to morphine

Neuroscience Letters 306 (2001) 41±44

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Mutant mice lacking the cholecystokinin2 receptor show a dopamine-dependent hyperactivity and a behavioral sensitization to morphine ValeÂrie Dauge a,*, FrancËoise Beslot a, Toshimitsu Matsui b, Bernard Pierre Roques a a

DeÂpartement de Pharmacochimie MoleÂculaire et Structurale, INSERM U266-CNRS UMR 8600, UFR des Sciences Pharmaceutiques et Biologiques, 4, avenue de l'Observatoire, 75270 Paris Cedex 06, France b Third Division Department of Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku Kobe 650-0017, Japan. Received 17 January 2001; received in revised form 17 April 2001; accepted 17 April 2001

Abstract Cholecystokinin2 (CCK2) receptor-de®cient mice were used to analyze the in vivo function of CCK2 receptor and especially the incidence of this gene invalidation on enkephalinergic and dopaminergic systems. Hyperlocomotor activity of CCK2 receptor-de®cient mice was suppressed by a selective D2 antagonist but not by a D1 antagonist. Injection of amphetamine induced a hyperlocomotor activity in both groups of mice while mutant mice were less sensitive to cocaine. Administration of 6 mg/kg of morphine once every 2 days for 5 days signi®cantly (P , 0:05) enhanced motor activity the last day compared to the ®rst day, only in CCK2 receptor-de®cient mice. These results emphasize the role of CCK2 receptors in counteracting the effects of dopaminergic systems and suggest that CCK2 receptor invalidation could lead to a slight behavioral sensitization. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cholecystokinin2 receptor-de®cient mice; Morphine; Amphetamine; Cocaine; Dopamine antagonists; Sensitization

Cholecystokinin (CCK) is a gut-brain peptide which binds to CCK1 and CCK2 receptors. The CCK2 receptor and CCK8, the sulfated octapeptide form of CCK, which are very abundant in brain, have been shown to be involved in anxiety-like behavior, nociception and memory processes [2,5,16]. In the brain mesocorticolimbic system, CCK8 and its receptors can be colocalized or not with the dopaminergic (DAergic) and opioidergic systems [6,13,15]. Con¯icting results exist on the effects of CCK on the DAergic system depending on the dose used, the structure of the brain studied and the type of receptors involved [2]. Concerning the relations with the opioidergic system, a large body of evidence has been accumulated supporting a physiological role of the CCK2 receptor in counteracting the analgesic and antidepressant-like effects of endogenous and exogenous enkephalins [11]. In addition, endogenous and exogenous enkephalins increased the DAergic systems activity especially by acting in the midbrain [7]. Dysregulations in inter-relations between DA, enkephalins and CCK at the level of the mesolimbic system could play an impor* Corresponding author. Tel.: 133-1-53-73-95-75; fax: 133-143-26-69-18. E-mail address: [email protected] (V. DaugeÂ).

tant role in diseases such as addiction, depression or schizophrenia [2,11,16]. In this study, CCK2 receptor-de®cient mice were used to analyze the in vivo function of CCK2 receptor and especially the incidence of its invalidation on morphine inducing sensitization and on dopamine drug sensitivity. Investigations were performed with CCK2 receptor de®cient mice provided with the original background 129sv/ C57BL/6 [10]. Breeding and genotype analysis have been performed by Transgenic Alliance (L'Arbesle, France). Male and female mice (3 months) were used. Animals were treated in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, 1985. Locomotor activity of mice was examined for 1 h in an actimeter. Mice were individually placed in a plastic cage (225 £ 205 cm) under a light intensity of 5 lux in a soundattenuated room. Horizontal movements of animals were counted by photocells. Same number of males and females were tested in all the experiments. In all the cases, there is no signi®cant difference between sex. Wild type and mutant mice were placed for 2 h in the actimeter and then injected s.c. with morphine at a dose of 6 mg/kg once every 2 days for 5 days, while the other 2 days, animals received saline

0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 86 7- 5

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V. Dauge et al. / Neuroscience Letters 306 (2001) 41±44

(Fig. 1). Mice were tested during the light period for their locomotor activity every day immediately after drug or saline administration. Under these conditions, although morphine induced an increase in motor activity in both groups of mice, this behavioral response was slightly but signi®cantly (Duncan test, P , 0:05) enhanced after chronic treatment with morphine on day 5 compared to day 1 in CCK2 receptor-de®cient mice (Fig. 1). This suggests that CCK2 receptor invalidation results in an easier development of behavioral sensitization. While the neuronal mechanisms underlying drug-induced locomotor sensitization remain to be fully understood, the DAergic system in the mesencephalon appears to play a critical role [8]. We therefore analyzed the reactivity of this system in CCK2 receptor-de®cient mice and wild type animals using various compounds capable of modifying the DA systems. In the following experiments, mice were not previously habituated to actimeter. In this condition, CCK2 receptor-de®cient mice showed a basal increase in locomotor activity. Thus, i.p. administration of the D2 selective antagonist sulpiride, at the dose of 50 mg/kg, 60 min before the actimeter test, completely blocked hyperlocomotor activity of CCK2 receptor-de®cient mice measured for 1 h, while the same dose of sulpiride did not modify the activity in wild type mice (Duncan test, P , 0:001), (Fig. 2a). On the other hand, i.p. injection of the D1 selective antagonist SCH23390, 60 min before the test, at the dose of 2 mg/kg did not change the locomotor activity measured for 1 h on both groups of mice while 10, 25 or 50 mg/kg signi®cantly decreased the motor activity of both groups of mice similarly (Fig. 2b). Therefore, the spontaneous hyperactivity of CCK2 receptor-de®cient mice appears to be dependent on

Fig. 1. Effects of morphine on the locomotor activity of CCK2 receptor-de®cient (n ˆ 8) and wild type mice (n ˆ 9). Mice were placed in actimeter 2 h before s.c. injection of 6 mg/kg of morphine or saline. Mice received morphine once every 2 days for 5 days. The two other days, they received saline. Mice were tested for their motor activity every day for 5 days. ANOVA: saline groups: genotype; F …1; 64† ˆ 1:49, P ˆ 0:215, days; F …4; 64† ˆ 0:622, P ˆ 0:441, interaction; F …4; 64† ˆ 0:593, P ˆ 0:668, morphine groups: genotype; F …1; 60† ˆ 35:107, P , 0:01, days; F …4; 60† ˆ 6:581, P , 0:05, interaction; F …4; 60† ˆ 4:376, P , 0:05. wwP , 0:01 vs. wild type group, q P , 0:05 vs. ®rst day of morphine treatment, Duncan test.

Fig. 2. Effects of the selective D2 antagonist, sulpiride (a, n ˆ 10) and of the selective D1 antagonist, SCH23390 (b, n ˆ 10) on the motor activity of CCK2 receptor-de®cient and wild type mice measured in actimeter for 1 h. Sulpiride was injected i.p. 60 min before the test at the dose of 50 mg/kg. SCH23390 was administered i.p. 60 min before the test at the dose of 2, 10, 25 and 50 mg/kg. ANOVA: sulpiride experiment: genotype; F …1; 32† ˆ 4:54, P , 0:05, treatment; F …1; 32† ˆ 138:635, P , 0:01, interaction; F …1; 32† ˆ 4:63, P , 0:05, SCH23390 (2 and 10 mg/kg) experiment: genotype; F …1; 50† ˆ 4:27, P , 0:05, treatment; F …2; 50† ˆ 16:676, P , 0:01, interaction; F …2; 50† ˆ 4.32, P , 0:05, SCH23390 (25 and 50 mg/kg) experiment: genotype; F …1; 52† ˆ 11:612, P , 0:01, treatment; F …2; 52† ˆ 190:28, P , 0:01, interaction; F …2; 52† ˆ 11:362, P , 0:01. wwP , 0:01 vs. respective saline group, qP , 0:05, qqP , 0:01 vs. respective wild type group, Duncan test.

the DAergic system via its D2 receptor type component. The DAergic system sensitivity was also studied after injection with indirect DA agonist, amphetamine and with an inhibitor of DA re-uptake, cocaine. Amphetamine, administered 30 min before the actimeter test, at the doses of 2.5 or 5 mg/ kg i.p., enhanced motor activity measured for 1 h in both groups of mice (Duncan test, P , 0:001). The effect of amphetamine was signi®cantly higher in mutant than in wild type mice (Fig. 3a). However, since the basal activity of CCK2 receptor-de®cient mice was also higher (Duncan test, P , 0:05) than the activity of wild type mice, a hypersensitivity to amphetamine in mutant mice cannot be evidenced (Fig. 3a). On the other hand, i.p. administration of cocaine immediately before the test (1 h of duration), at the doses of 10 or 20 mg/kg, induced an increase in motor activity in both groups of mice (Duncan test, P , 0:001). However, cocaine produced a higher effect in wild type

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activity of DA neurons in midbrain [14], but see also [12]. In addition, peripheral injection of a selective CCK2 agonist, BC264, increased dopamine release in the striatum [9]. Therefore, DA-dependent hyperactivity in mutant mice could be due to the consequences of opioidergic system activation since endogenous and exogenous opioids were shown to enhance dopamine release in the striatum [4,7]. In conclusion, This study shows the importance of relations existing between CCK acting on the CCK2 receptor type and enkephalinergic and DAergic systems, emphasizing the role of CCK as an antagonist system to the enkephalinergic system. In addition, they show that CCK2 receptor invalidation, could lead to a slight behavioral sensitization to morphine in mice.

Fig. 3. Effects of amphetamine (a, n ˆ 8±9) and cocaine (b, n ˆ 8±10) on the motor activity of CCK2 receptor-de®cient and wild type mice measured in actimeter for 1 h. Amphetamine was injected i.p. 30 min before the test at the doses of 0.2, 1, 2.5 or 5 mg/kg. Cocaine was injected i.p. just before the test at the doses of 5, 10 or 20 mg/kg. ANOVA: amphetamine (0.2 and 1 mg/kg) experiment: genotype; F …1; 55† ˆ 14:216, P , 0:01, treatment; F …2; 55† ˆ 253:97, P , 0:01, interaction; F …2; 55† ˆ 14:369, P , 0:01, amphetamine (2.5 and 5 mg/kg) experiment: genotype; F …1; 53† ˆ 4:25, P , 0:05, treatment; F …2; 53† ˆ 67:276, P , 0:01, interaction; F …2; 53† ˆ 4:303, P , 0:05, cocaine experiment: genotype; F …1; 59† ˆ 6:333, P , 0:01, treatment; F …3; 59† ˆ 76.788, P , 0:01, interaction; F …3; 59† ˆ 12:219, P , 0:01. w P , 0:05, wwP , 0:01 vs. respective saline group, qP , 0:05, qq P , 0:01 vs. respective wild type group, Duncan test.

compared to mutant mice, although CCK2 receptor-de®cient mice showed a basal hyperactivity compared to the wild type mice (Duncan test, P , 0:05), (Fig. 3b), showing that mutant mice were less sensitive to the cocaine effect than wild type animals. Although the mechanism of action of the antagonists are unknown, negative control of CCK2 antagonists on the cocaine effects has already been obtained. Indeed, CCK2 antagonists were effective to reduce cocaine withdrawal anxiogenesis [1] and cocaine consumption, while the inverse was obtained with CCK2 agonist [3]. The D2-dependent hyperlocomotor activity, which occurred in mutant mice, could be related to the lack of CCK2 receptor. However CCK2 antagonists were shown to decrease the

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