Behavioral Profile of L 741,741, a Selective D4 Dopamine Receptor Antagonist, in Social Encounters Between Male Mice

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1 AGGRESSIVE BEHAVIOR Volume 29, pages (2003) Behavioral Profile of L 741,741, a Selective D4 Dopamine Receptor Antagonist, in Social Encounters Between Male Mice J.F. Navarro, n G. Luna and C. Pedraza Department of Psychobiology. Faculty of Psychology, University of Málaga, Spain : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : Although the role of dopamine D1 D2 D3 receptors in the modulation of aggression has been extensively documented, there is not information with respect to the implication of D4 receptor. The aim of this study was to examine the acute effects of L 741,741 (0.75, 1.5 and 3 mg/kg, i.p), a selective D4 receptor antagonist, on social encounters between male mice using an ethopharmacological approach. Ten min of diadic interactions were staged between a singly housed and an anosmic mouse in a neutral area. These encounters were videotaped and the accumulated time allocated by subjects to ten broad behavioral categories was estimated. Besides other behaviors, the aggressive and motor behaviors were evaluated 30 min after injection using an ethologically based analysis. L 741,741 did not affect significantly offensive behaviors (threat and attack), as compared with the control group. Likewise, motor and anxiety-related behaviors (such as social investigation, avoidance/flee or defense submission) were not altered after drug administration. These results suggest that dopamine D4 receptor is not involved in the modulation of aggressive behavior. Aggr. Behav. 29: , r 2003 Wiley-Liss, Inc. : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : Key Words: aggression; dopamine; D4 receptor; mice INTRODUCTION Dopamine belongs to a group of neurotransmitters called catecholamines and exerts its action by binding to specific membrane receptors, included in the family of seven transmembrane domain (7TM) G-protein coupled receptors [Vallone et al., 2000]. Dopaminergic transmission seems to be essential for the expression of aggressive behavior. In fact, most dopamine antagonists are effective antiaggressive agents. In this sense, the effects of D1 receptor selective antagonists (such as SCH 23390) on aggression have been analyzed. Overall, the results of these studies indicate that SCH has antiaggressive effects but only at doses that markedly depress motor activity [Arregui et al., 1993; Rodrı guez-arias et al., 1998], whereas at low doses it is clearly ineffective [Pedraza and n Correspondence to: José Francisco Navarro. Department of Psychobiology. Faculty of Psychology, University of Málaga, Campus de Teatinos s/n, Málaga, Spain. navahuma@uma.es Received 16 April 2002; amended version accepted 13 May Published online in Wiley Interscience ( DOI: /ab r 2003 Wiley-Liss, Inc.

2 Effects of L 741,741 on Aggression 553 Navarro, 1999]. Likewise, it has been reported that all examined dopaminergic antagonists with an anti-d2 profile (such as haloperidol, chlorpromazine, pimozide, spiperone or tiapride) show strong antiaggresive properties, although they differ by the amount of motor impairment produced [Arregui et al., 1993; Navarro et al., 1993, 2000; Manzaneque and Navarro, 1997; Navarro and Manzaneque, 1997]. Moreover, neuroleptic drugs with a mixed profile anti-d1/d2 (e.g., zuclopenthixol) and anti-d2/d3 (e.g, sulpiride or amisulpride) also exhibit an antiaggressive activity in laboratory animal models [Redolat et al., 1991; Martı n- Lo pez et al., 1993; Manzaneque and Navarro, 1999a, 1999b]. Furthermore, the new dopamine D3 antagonist U A maleate has proved to present an antiaggressive action in mice [Rodrı guez-arias et al., 1999]. On the other hand, the presynaptic dopamine function also appears to be necessary for the normal expression of aggressive behaviors [Felip et al., 2001]. Although the role of D1 D2 D3 receptors has been extensively examined, there is no information in relation to the implication of D4 dopamine receptors in aggression. Recently, significant progress has been made in the development of selective D4 receptor antagonists [Oak et al., 2000]. However, published behavioral research on these compounds is as yet very limited and, to our knowledge, the effects of these substances on aggression have not been investigated. Therefore, the present study was designed to analyze the effects of L 741,741 (a selective D4 receptor antagonist) on agonistic interactions between male mice, using an ethopharmacological approach. This compund is a potent and highly selective D4 dopamine receptor antagonist, with Ki values of41700, 480 and 2.5 nm at cloned human D2, D3 and D4 receptors, respectively [Rowley et al., 1996, 1997]. MATERIAL AND METHODS Ninety albino male mice of the OF.1 strain (provided by CRIFFA, Barcelona) weighing g were used. Animals were housed under standardized lighting conditions (using a 12 h reversed cycle; white lights on: 20:00 8:00), at a constant temperature (211C) and food and tap water available ad lib, except during behavioral trials. Upon arrival in the laboratory, the subjects were allocated to two different categories. Half were housed individually in transparent plastic cages ( cm) as experimental animals. The remainder were housed in groups of five (in cages of identical dimensions) to be used as standard opponents and were rendered temporally anosmic by intranasal lavage with 4% zinc sulfate solution (Sigma Laboratories) on both 1 and 3 days before testing. Fighting in mice, as in most rodents, is closely related to olfaction. We used this type of opponent because it elicits attack but never initiates such behavior [Brain et al., 1981]. Consequently, fighting is always unidirectional, being easily quantified. All the experimental animals underwent an isolation period of 30 days before the behavioral test (isolation-induced aggression model). This experiment was carried out in accordance with the guiding principles for care and use of Laboratory Animals approved by the European Communities Council Directive of November 24, 1986 (86/609/EEC). L 741,741 (Tocris Laboratories) was diluted in physiological saline and DMSO to provide appropriate doses for injections. Four groups of mice were used. Individually housed animals were allocated randomly to one control group (n = 12) receiving vehicle (90% physiological saline and 10% DMSO) and three experimental groups (n = 11 each) receiving acute L 741,741 injections. Drug was administered acutely in three doses: 0.75, 1.5 and 3 mg/kg.

3 554 Navarro et al. Drug and vehicle were injected intraperitoneally in a volume of 10 ml/kg, being prepared freshly on test days. Thirty minutes after injection, an isolated animal and a standard opponent (marked with fur dye for identification) were confronted in a neutral area for ten minutes. This neutral cage consisted of an all glass area, measuring cm with a fresh sawdust substrate. Before the encounter, the animals were allowed 1 minute of adaptation to the neutral cage, remaining separated by means of a plastic barrier throughout this time. The social encounters were videotaped using a Sony-V8 camera. All tests were conducted under dim red light between the second and seventh hours of the dark phase of the artificial cycle of the animals. After each encounter, the neutral cage was washed out and the sawdust bedding was replaced. The tapes were analyzed using a microprocessor and a custom-developed programme [Brain et al., 1989] which facilitated estimation of time allocated to ten broad behavioral categories. The names of the categories and their constituent elements are as follows: 1. Body care (abbreviated groom, self groom, wash, shake, scratch); 2. Digging (dig, kick dig, push dig); 3. Non-social exploration (explore, rear, supported rear, scan); 4. Exploration from a distance (approach, attend, circle, head orient, stretched attention); 5. Social investigation (crawl over, crawl under, follow, groom, head groom, investigate, nose sniff, sniff, push past, walk around); 6. Threat (aggressive groom, sideways offensive, upright offensive, tail rattle); 7. Attack (charge, lunge, attack, chase); 8. Avoidance/flee (evade, flinch, retreat, ricochet, wheel, startle, jump, leave, wall, clutch); 9. Defense/submission (upright defensive, upright submissive, sideways defensive), and 10. Immobility (squat, cringe). A detailed description of all elements can be found in Brain et al. [1989]. This ethoexperimental procedure allows a complete quantification of the behavioral elements shown by the subject during the agonistic encounters. Only the behavior of the isolated animal was assessed. The analysis was carried out by a trained experimenter, unaware of the treatment of the groups. Nonparametric Kruskal-Wallis tests were initially used to assess the variance of the behavioral measures over different treatment groups. Subsequently, appropriate paired comparisons were carried out using Mann-Whitney U-tests. The analyses were performed using nonparametric statistics since the criteria for parametric statistics were not met by the data. RESULTS Table I illustrates the medians (with ranges) of the accumulated times allocated to the broad categories described above. Kruskal-Wallis analysis showed that there were no significant differences between control and experimental groups in any of the behavioral categories examined. DISCUSSION The D4 R gene is composed of five coding exons and generates a 387aa protein. This gene appears to be highly expressed in the frontal cortex, amygdala, olfactory bulb, hippocampus, and hypothalamus [Oak et al., 2000]. Several selective dopamine D4 receptor antagonists have been recently characterized. Among these novel agents is L 741,741, a potent and highly selective D4 dopamine receptor antagonist. Functional studies have demonstrated that this ligand is a full antagonist which, in absence of intrinsic activity, potently attenuates

4 Effects of L 741,741 on Aggression 555 TABLE I. Median Values (with Ranges) for Times (in Seconds) Allocated to Broad Behavioral Categories in Animals Receiving Acute Treatment with L 741,741 Doses of L 741,741 (mg/kg) Behavioral categories vehicle Body care ( ) ( ) (0 24.4) ( ) Digging ( ) (0 36.6) (0 44.2) (0 17.9) Non-social exploration ( ) ( ) ( ) ( ) Explore from a distance ( ) ( ) ( ) ( ) Social investigation ( ) ( ) ( ) ( ) Threat (0 198) (0 183) (0 170) (0 135) Attack (0 95) (0 98) (0 86) (0 319) Avoidance/flee (0 3.8) (0 2.8) (0 4.7) (0 6.5) Defence/submission (0 0) (0 0.77) (0 3.4) (0 0) Immobility (0 0) (0 0) (0 0) (0 0) dopamine-induced inhibition of forskolin-stimulated camp levels in cells stably expressing human dopamine D4 receptors [Rowley et al., 1996, 1997]. As Table I shows, no significant differences were found between control and experimental groups in the time spent in offensive behaviors (threat and attack) after L 741,741 administration. In a previous study, Garmendia et al. [1992] observed that clozapine showed antiaggressive effects with a minimal motor impairment in isolated mice. However, although clozapine has potent dopamine D4 receptor antagonist properties, it also displays high affinities for other dopaminergic receptors as well as a number of serotonergic (5 HT2, 5 HT6, 5 HT7) and a variety of other receptors (muscarinic, adrenergic, and histaminergic) [Manzaneque et al., 2002]. Our results using a selective antagonist compound of D4 receptors suggest that this receptor might not to be involved in the modulation of aggression. Motor behaviors were not significantly affected by the drug. The lack of motor effects found after L 741,741 administration is in aggreement with several behavioral studies showing selective D4 receptor antagonists do not alter spontaneus locomotion and other motor parameters [Merchant et al., 1996; Clifford and Waddington, 2000]. Furthermore, some of the behavioral categories analyzed in our study (v.g., social investigation, avoidance/ flee, defense/submission) have been frequently used to assess the anxiety-changing properties of drugs [Brain et al., 1991; Navarro and Maldonado, 1999; Maldonado and Navarro, 2001]. L 741,741 did not affect significantly any of the behavioral categories which are generally sensitive to anxiolytic or anxiogenic drugs, supporting the suggestion that selective dopamine D4 receptor antagonists are ineffective in the modulation of anxiety-related behaviors [Cao and Rodgers, 1997].

5 556 Navarro et al. In conclusion, despite the detailed behavioral profile obtained by ethological analysis, present results indicate that L 741,741 failed to significantly alter any aspect of behavior of the isolated male mice. Our inability to detect any behavioral effect of this compound strongly suggests that the dopamine D4 receptor is not involved in the modulation of aggression. REFERENCES Arregui A, Azpiroz A, Brain PF, Simo n VM Effects of two selective dopaminergic antagonists on ethologically-assessed encounters in male mice. Gen Pharmacol 24: Brain PF, Benton D, Childs G, Parmigiani S The effects of the opponent in tests of murine aggression. Behav Processess 6: Brain PF, Kusumorini N, Benton D Anxiety in laboratory rodents: a brief review of some recent behavioural developments. Behav Processess 25: Brain PF, McAllister KH, Walmsley S Drug effects on social behavior: methods in ethopharmacology. In: Boulton AA, Baker GB, Greenshaw AJ, editors. Neuromethods. Vol. 13. Psychopharmacology. Clifton, NJ: The Humana Press, p Cao BJ, Rodgers RJ Dopamine D4 receptor and anxiety: behavioural profiles of clozapine, L 745,870 and L 741,742 in the mouse plus-maze. Eur J Pharmacol 335: Clifford JJ, Waddington JL Topographically based search for an ethogram among a series of novel D4 dopamine receptor agonists and antagonists. Neuropsychopharmacology 22: Felip CM, Rodríguez-Arias M, Aguilar MA, Min arro J Antiaggressive and motor effects of the DA release inhibitor CGS 10746B. Aggr Behav 27: Garmendia L, Sánchez JR, Azpiroz A, Brain PF, Simo n VM Clozapine: strong antiaggressive effects with minimal motor impairment. Physiol Behav 51: Maldonado E, Navarro JF MDMA ( ecstasy ) exhibits an anxiogenic-like activity in social encounters between male mice. Pharmacol Res 44: Manzaneque JM, Navarro JF Tolerance to antiaggressive and motor activity of chlorpromazine after repeated administration to mice. Med Sci Res 25: Manzaneque JM, Navarro JF. 1999a. An ethopharmacological assessment of the effects of zuclopenthixol on agonistic interactions in male mice. Methods Find Exp Clin Pharmacol 21: Manzaneque JM, Navarro JF. 1999b. Behavioral profile of amisulpride in agonistic encounters between male mice. Aggr Behav 25: Manzaneque JM, Brain PF, Navarro JF Effect of low doses of clozapine on behaviour of isolated and group-housed male mice in the elevated plus-maze test. Prog NeuroPsychopharmacol Biol Psychiat 26: Martín-Lo pez M, Puigcerver A, Vera F, Navarro JF Sulpiride shows an antiaggressive specific effect after acute treatment in male mice. Med Sci Res 21: Merchant KM, Gill GS, Harris DW, Huff RM, Eaton MJ, Lookingland K, Lutzke BS, McCall RB, Piercy MF, Schreur PJ, Sethy VJ, Smith MW, Svenson KA, Tang AH, Vonvoigtlander PF, Tenbrink RE Pharmacological characterization of U , a dopamine D4 receptor selective antagonist. J Pharmacol Exp Ther 279: Navarro JF, Manzaneque JM Acute and subchronic effects of tiapride on isolation-induced aggression in male mice. Pharmacol Biochem Behav 58: Navarro JF, Maldonado E Behavioral profile of 3,4 methylenedioxy-methamphetamine (MDMA) in agonistic encounters between male mice. Prog NeuroPsychopharmacol Biol Psychiat 23: Navarro JF, Min arro J, Simo n VM Antiaggressive and motor effects of haloperidol show different temporal patterns in the development of tolerance. Physiol Behav 53: Navarro JF, Velasco R, Manzaneque JM Acute and subchronic effects of pimozide on isolationinduced aggression in male mice. Prog NeuroPsychopharmacol Biol Psychiat 24: Oak JN, Oldenhof J, Van Tol HHM The dopamine D4 receptor: one decade of research. Eur J Pharmacol 405: Pedraza C, Navarro JF An ethopharmacological assessment of the effects of SCH on agonistic interactions in male mice. Med Sci Res 27: Redolat R, Brain PF, Simo n VM Sulpiride has an antiaggressive effect in mice without markedly depressing motor activity. Neuropharmacology 30: Rodríguez-Arias M, Min arro J, Aguilar MA, Simo n VM Effects of risperidone and SCH on isolation-induced aggression in male mice. Eur Neuropsychopharmacol 8:

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