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note
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Animals were housed at 21° ± 1°C with free access to food and water. Experiments were conducted in accordance with local ethical guidelines. The measurement of locomotor activity, and the open-field and elevated plus maze tests were performed as described (24). Mice were exposed to the open field for three consecutive days. The number of squares crossed was as follows: wild type: 170 ± 12, 127 ± 10, and 91 ± 11 for first, second, and third days, respectively; knockout: 256 ± 20 (P < 0.01), 158 ± 15 (NS), and 120 ± 16 (NS) for first, second, and third days, respectively (t test, n = 15 per group).
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9
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0344243655
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note
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The spontaneous alternation test was conducted as described (24). The percentage of alternation was measured as the number of times the animal visited consecutively all three arms, divided by the total number of visits during a 10-min period.
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10
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0345105910
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note
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Nociceptive thresholds were monitored by applying thermal (tail-immersion and hot-plate tests), mechanical (tail-pressure test), or chemical stimuli (writhing test) (24). For the tail-immersion test, mice were maintained in a cylinder, and their tail was immersed in water at 50°C; latency to tail withdrawal was recorded. In the hot-plate test, mice were placed on a surface heated to 50°C, and the latencies for licking their paws and jumping were recorded. For the tail-pressure test, increasing pressure (tip diameter: 1 mm) was applied to the tails of the mice until a withdrawal response was elicited. In the writhing test, mice received 0.1 ml per 10 g of body weight of a 0.6% acetic acid solution by the intraperitoneal route, and contractions of abdominal musculature (writhes) were counted between 5 and 15 min after the injection.
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Opiate tolerance was induced by daily sc injection of morphine (20 mg/kg) for 5 days followed by a sc 6 mg/kg injection of morphine.
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Opiate dependence was induced by repeated intraperitoneal injection of morphine (or saline in controls) at an interval of 12 hours, over 6 days (20 mg/kg on day 1 to 100 mg/kg on days 5 and 6). Withdrawal was precipitated by injecting naloxone (1 mg/kg, sc) 2 hours after the last morphine administration and was evaluated as reported [R. Maldonado et al., Science 273, 657 (1996)]. A global score was calculated for each animal [R. Maldonado, S. Negus, G. F. Koob, Neuropharmacology 21, 1231 (1992)].
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We thank A. Nagy for R1 embryonic stem cells, C. Dewolf and M. J. Simons for technical assistance, and D. Penninck for discussions. Supported by the Inter-university Poles of Attraction (Belgian State, Prime Minister's Office, Federal Service for Science, Technology and Culture), the Fonds de la Recherche Scientifique Médicale, the EU BIOMED II programme, and the Fondation Médicale Reine Elisabeth. The scientific responsibility is assumed by the authors. C.L. is Chercheur Qualifié of the Fonds National de la Recherche Scientifique.
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