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Neuroscience Letters 432 (2008) 202–205

Effects of melanotan II, a agonist, on grooming and exploration in rats after repeated restraint/immobilization Veraˇ Klenerova,´ Ivan Krejcˇ´ı, Pavel Sˇ´ıda, Zdenekˇ Hlinˇak,´ Sixtus Hynie ∗ Laboratory of Biochemical Neuropharmacology, Medical Biochemistry, First Faculty of Medicine, Charles University in Prague, Albertov 4, 128 00 Prague 2, Czech Republic Received 30 June 2007; received in revised form 5 December 2007; accepted 11 December 2007

Abstract 2 mg/kg melanotan II (MTII, administered i.p.), a cyclic peptide analog of ␣- stimulating hormone, at a single dose increased grooming in naive rats placed in an unfamiliar open-field device without changing locomotion or rearing. Male rats exposed to restraint/immobilization stress (IS) for 1 h on three consecutive days displayed increased grooming after the second stressor exposure, compared to pre-stress levels. MTII, administered to the rats after IS, enhanced the grooming response compared both to the pre- and post-stress values. The increase was greatest after the first dose and declined over the following two applications. As to the locomotion of rats in the entire experimental space, IS reduced the distance moved only after the first two stressor exposures; MTII did not influence these alterations. Locomotion in the central part of arena was not reduced by the stressor or by MTII, on the contrary, there was an increase in both groups after the third intervention. The only observed change in rearing was an increase in the MTII group after the third restraint exposure. Thus, MTII selectively increased grooming without markedly affecting the spatio-temporal structure of locomotor behavior in the open-field. The decline of MTII enhanced grooming over the three test days may be interpreted in terms of adaptation to the stressor and of the developing tolerance to the peptide. © 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Immobilization; Grooming; Exploratory behavior of rats; Open-field testing; Melanotan II

Brain melanocortin ␣-melanocyte stimulating hormone (␣- receptors, inhibited both novelty and ␣-MSH- or MTII-induced MSH) and adrenocorticotropic hormone (ACTH) modulate grooming [1]. a variety of neurophysiological phenomena including stress- Another role play in stress-induced behavioral induced neurochemical and behavioral responses and disorders responses is the induction of anxiety [4,10]. More recently, i.c.v. in laboratory rodents [1,6,7]. The demonstration of brain spe- administration of ␣-MSH and as well as MTII dose-dependently cific subtypes expressed in rat brain reduced the number of licking periods in the rat Vogel con- indicates that central actions of ␣-MSH are mediated pri- flict test, which suggested that stimulation of melanocortin marily by melanocortin MC3 and MC4 receptors [1,2]. Rat MC4 receptor causes anxiogenic-like activity [2]. In support grooming behaviors were among the first described behaviors for this view, melanocortin MC4 selective antagonist MCL0020 elicited either by mild stress or by intracerebral injection of given i.c.v. prevented swim stress-induced reduction in the melanocortin receptor agonists ␣-MSH and ACTH [6,11,12]. mouse light–dark exploration test [2]. These findings show that Recently it has been shown that melanotan II (MTII), a more melanocortin MC4 receptor might be actually related to at least selective melanocortin MC4 receptor agonist, dose-dependently some stress-induced alterations in behavioral responses. increased grooming episodes observed in rats placed in a novel In previous studies we showed that restraint/immobilization environment [1]. The role of melanocortins in modulation of one hour in duration exerts both short- and long-term effects on grooming behavior in rats is further supported by the finding spontaneous behavior of rats in the open-field test [16,17]. The that SHU919, an analogue acting as an inhibitor on melanocortin aim of the present study was to evaluate if and to what extent MTII can modulate effects of restraint stress on spontaneous behavior of rats including grooming pattern. To explore possible ∗ Corresponding author. Tel.: +420 224 968 142. anxiogenic-like action of MTII, we recorded locomotor activity E-mail address: [email protected] (S. Hynie). of rats close to the wall and in the central part of the arena, a

0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.12.020 V. Klenerov´a et al. / Neuroscience Letters 432 (2008) 202–205 203 reduction of the latter being commonly considered as an index Experiment 2: On Day 1, all animals were tested in the open- of anxiety [18,19,21]. field device without any intervention. On Day 2, half of animals Animals: Male Wistar rats (Velaz, Czech Republic), with (n = 9) received IS + saline (control group) and the other half of starting body weights of 290–305 g, were used. Daily handled animals (n = 9) received IS + MTII (experimental group). The rats (during daily weighing animals were kept in the hand for same procedure was repeated on Day 3 and Day 4. about 1 min) had free access to a standard pellet food (ST1, The results are presented in Fig. 1. A two-way ANOVAon the Velaz, Czech Republic) and water. Rats were housed in a total time spent in grooming revealed a significant effect of treat- room maintained on a 12-h light/dark cycle (lights on 6–18) ment (F(1,64) = 30.18, p < 0.0001), a significant effect of day and temperature (21 ± 1 ◦C). Four rats were housed per cage (F(3,64) = 11.68, p < 0.0001), and treatment × day interaction (42 cm × 26 cm). Treatment of animals was in accordance with (F(3,64) = 6.26, p = 0.0009). Animals given IS + MTII devoted the Declaration of Helsinki Guiding Principles on Care and Use significantly more time to the grooming on Day 2, 3 and 4 as of Animals (DHEW Publication, NHI 80-23). compared with IS + saline group. No change in grooming time Drug: Melanotan II, a cyclic peptide analog of ␣-MSH, was dissolved in saline and injected i.p. in a dose of 2 mg per 2 ml/kg b.w. Control animals received the same volume of saline. Stress procedure: Rats were exposed to 60 min restraint/ immobilization (IS) applied by fixing front and hind legs of the rat with adhesive plaster; then the animal was restrained in a snug-fitting plastic-mesh. This mesh was bent to conform to the size of the individual animal and a bandage fixed this shape of mesh. During the stress the animals were kept in a vertical position [16,17]. After the exposure to the stressor, the animals were returned to the home cage. For the stress procedure the rats were transferred to a separate room. Two dif- ferent persons performed the stress procedure and the behavioral testing. Behavioral testing: Rats were randomly assigned to two groups in both experiments. Behavior of rats was video- monitored by an automated activity monitoring system (AnyMaze, Stoelting, U.S.A.) in a circular arena with the diam- eter of 150 cm; the walls were 50 cm high. While the movement distance and the total movement distance in the inner zone (the diameter of 110 cm) were recorded automatically, the number of rearing and the total time spent in grooming were measured by an experimenter. We did not differentiate between grooming elements (face washing, licking the body fur, genital area, limbs and tail, and scratching). Behavioral testing began 60 min after the termination of stress and/or injection (saline or MTII). Data were collected for 10 min (Experiment 1) or 5 min (Experiment 2) period. Both experiments were performed from 8 a.m. to 1 p.m. in a separate room illuminated by a fluorescent light located on the ceiling. Statistics: The Systat 10 software (SPSS, Inc., Chicago, U.S.A.) was used to evaluate behavioral data. In the second experiment, a two-way analysis of variance (ANOVA) with factors of treatment and day was followed with the Bonfer- roni adjusted post hoc tests to compare individual days within both the IS + saline (control) and IS + MTII (experimental) groups. To compare the data within a particular day between both groups the t-test was used in both experiments. Data are expressed as mean ± S.E. Statistical significance was accepted when p ≤ 0.05. Fig. 1. Effects of melanotan II (MTII) or saline (SAL) on spontaneous behavior Experiment 1: MTII-treated animals spent a significantly in the open-field arena of rats exposed repeatedly to the restraint/immobilization longer time (s) in grooming than the controls (42.4 ± 10.1, n =8 stressor (IS). Day 1: no treatment, Day 2, 3 and 4: left part = IS + SAL, ± right part = IS + MTII. From the top to the bottom: grooming-total time (s), and 10.6 3.2, n = 8, respectively; t = 3.0, p = 0.01). No differ- locomotion-total distance moved (m), inner zone distance (m), rearing-total num- ence between both groups was found in the total distance moved ber (n). Statistical significance within groups (p < 0.05): *vs. Day 1, ◦vs. Day 2 and the number of rearing. and +vs. Day 3. 204 V. Klenerov´a et al. / Neuroscience Letters 432 (2008) 202–205 was observed in the IS + saline-treated animals throughout the The reduction of locomotion observed after the first IS expo- experiment. In IS + MTII-treated animals the highest grooming sure only (Day 2) also points to the process of habituation to the level was found on Day 2, then it gradually decreased; never- stressor effects. The recovery of reduced locomotion after the theless, on Day 4 the level was significantly increased when second and/or third stressor exposure was also found in previ- compared with Day 1. ous study obtained under identical conditions [16]. In support of A two-way ANOVA on the total distance moved revealed the participation of adaptation process also vertical exploratory a significant effect of day (F(3,64) = 5.95, p = 0.0018), but no activity – rearing – increased on Day 4. effect of treatment (F(1,64) = 0.047, p = 0.83) and no signif- In a novel environment rodents prefer to walk close to the icant interaction between treatment and day (F(3,64) = 0.099, walls. Locomotion and time spent in the inner zone of the arena p = 0.96). In both groups there was a significant decrease of the are considered as a variable reflecting the anxiety level [8,19,20]. total movement distance on Day 2. On Day 4 the movement level In our experimental approach, i.e. using a large size open-field, returned to that exhibited on Day 1. we found no reduction in the central locomotion after IS. On A two-way ANOVA on the distance moved in the inner zone the contrary, MTII + IS-treated rats exhibited higher inner zone revealed a significant effect of day (F(3,64) = 4.42, p = 0.007), locomotor activity on Day 4. This is in contrast with findings that but no effect of treatment (F(1,64) = 0.26, p = 0.61) and no effect MTII exerts an anxiogenic-like effect after i.c.v. administration of interaction (F(3,64) = 0.29, p = 0.83). On Day 4, only animals in the test of punished drinking; a melanocortin MC4 receptor given IS + MTII exhibited significantly increased movement in antagonist prevented swim stress-induced behavioral alteration the inner zone. in the mouse light–dark exploration test [2]. Both methods may Finally, as to the number of rearing, a two-way ANOVA be considered as stronger stressogenic events than IS. We also revealed a significant effect of treatment (F(1,64) = 4.14, observed that timing of stress and testing influence the resulting p = 0.046), a significant effect of day (F(3,64) = 5.80, p < 0.01), behavioral performance in rats [16]. Lowered level of emotion- but no significant interaction between treatment and day ality induced by handling could also be involved in the observed (F(3,64) = 0.79, p = 0.50). There was no change across days in absence of movement depression in the inner zone. the control group. On the contrary, the rearing level in animals Grooming behavior induced by MTII was potentiated in IS given IS + MTII increased significantly on Day 4, but did not stressed rats. In our view, it is conceivable to assume that this reach a significant difference when compared to the controls. increase was caused by a synergic action of MTII and stress Discussion: The results revealed increased grooming after activators of MC4 brain receptors. This notion is supported by MTII given intraperitoneally. In Experiment 1, a single dose the finding of gradual decline of grooming after repeated MTII of MTII increased the amount of grooming in rats exposed to a treatment that can be explained in terms of adaptation or habit- novel environment. The main finding in Experiment 2 (Fig. 1), is uation reflected in the recovery of locomotion and rearing in IS the high enhancement of grooming activity induced by combi- stressed rats. This behavioral change indicates lesser stimulation nation of IS stressor exposure and MTII treatment. The most of hypothalamic/hypophyseal/adrenal axis. Also, in preliminary striking increase appeared on the first day of the combined experiment we observed an indication of developing tolerance treatment (Day 2), compared to grooming amount elicited by to the MTII effects. IS presentation only. Several types of stressors-like placement In conclusion, if we accept that grooming is elicited by stim- in the novel environment, handling, water immersion, noise ulation of MC4 receptors, then the present results suggest that or social interaction were found to elicit grooming [3,9,12]. MTII, given also intraperitoneally, penetrates the blood–brain Repeated restraint stress has also been reported to increase barrier to such an extent that its brain concentrations produce grooming [5], however, the restraint inducing severe stress measurable behavioral effects. reduced grooming response [12]. Stress of electrical foot-shock has been shown to suppress or to stimulate grooming depend- Acknowledgements ing on the strength or number of shocks [13–15]. In the present study IS did not increase grooming to a significant degree, how- The study was supported by grants MSM 0021620806 and ever, the effects of combination of MTII and IS suggest that GACR 309/06/0121. 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