Role of GABA-ergic and serotonergic systems in the anxiolytic-like mechanism of action of a 5-HT-moduline antagonist in the mouse elevated plus maze

Behavioural Brain Research 158 (2005) 339–348

Research report

Role of GABA-ergic and serotonergic systems in the anxiolytic-like mechanism of action of a 5-HT-moduline antagonist in the mouse elevated plus maze Florence Cl´eneta , Martine Hasco¨eta , Gilles Filliona , Herv´e Galonsb , Michel Bourina,∗ a

EA 3256, Neurobiologie de l’anxi´et´e et de la d´epression, Facult´e de M´edecine, BP 53508, 1 rue Gaston Veil, 44035 Nantes Cedex 01, France b Facult´ e des Sciences Pharmaceutiques et Biologiques, 4 avenue de l’Observatoire, 75006 Paris, France Received 21 June 2004; accepted 13 September 2004

Abstract 5-HT-moduline is an endogenous tetrapeptide, which acts specifically as an antagonist of 5-HT1B auto- and heteroreceptors. HG1 is an ethyl arylmethyloxypiperidine acetate and an antagonist of 5-HT-moduline, which has no 5-HT-moduline agonist effect. In a pilot study, HG1 has demonstrated an anxiolytic-like profile in three mouse models of anxiety (elevated plus maze, light/dark, four plates). The aim of our study was to examine the mechanism of the anxiolytic-like effects of HG1 in the mouse elevated plus maze. Male Swiss mice were acutely administered HG1 at active doses in association with GABA antagonists such as flumazenil, bicuculline and picrotoxine, then, with 5-HT1A (NAN 190, WAY 100635) and 5-HT1B receptor antagonist (methiothepine). Finally, we tried to potentiate non-active doses of HG1 with 5-HT1A (8-OHDPAT) and 5-HT1B receptor agonists (anpirtoline) in the mouse elevated plus maze. Regarding GABA antagonists, only flumazenil antagonised active doses of HG1 in an incomplete manner. Moreover, non-active doses of HG1 were potentiated by low doses of WAY 100635 and by anpirtoline but not by 8-OHDPAT. Finally, the anxiolytic-like effects of HG1 at active doses were antagonised by all serotonergic antagonists (WAY 100635 at higher dose, NAN 190 and methiothepin). HG1 mechanism of action in the mouse elevated plus maze seems to associate a GABA-ergic component exerting a limited regulation of 5-HT neuronal activity and a major serotonergic component, which seems to implicate presynaptic 5-HT1A and 5-HT1B receptors. © 2004 Elsevier B.V. All rights reserved. Keywords: Serotonin (5-HT); 5-HT-moduline; GABAA receptor; 5-HT1A receptor; 5-HT1B receptor; Mouse elevated plus maze (EPM)

1. Introduction 5-HT1A somatodendritic autoreceptors inhibit the electric activity of serotonergic neurons in the dorsal raphe and their activation decreases 5-HT release in brain areas such as the striatum, amygdala and hippocampus [2,20,35,30]. 5-HT1B presynaptic receptors decrease 5-HT release in the cortex, hippocampus, cerebellum and hypothalamus in vitro and in vivo in the rat. These receptors have also a function of het∗

Corresponding author. Tel.: +33 2 40 41 28 53; fax: +33 2 40 41 28 56. E-mail address: [email protected] (M. Bourin).

0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.09.015

eroreceptor whose activation, in the rat cerebellum, decreases GABA release [21]. Moreover, they modulate the release of acetylcholine in the hippocampus [28], glutamate in the cerebellum [32] and dopamine in the striatum [34]. 5-HTmoduline, an endogenous ligand has been found to modulate 5-HT1B receptors. In fact, 5-HT-moduline is an endogenous tetrapeptide (Leu-Ser-Ala-Leu), isolated and characterised by Fillion and Fillion [15], which specifically interacts in a non competitive manner with the binding of [3 H]5-HT to 5-HT1B/1D . Biochemical studies have suggested that the 5-HT-moduline binding site is different from that of 5-HT [27,33]. An

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autoradiographic study of the distribution of [3 H]5-HTmoduline binding sites confirmed that 5-HT1B receptors are the preferential targets for 5-HT-moduline in vivo [12]. This peptide comes from an excitable tissue as illustrated by its Ca2+ –K+ dependent release in cortical rat synaptosomes-like classical neurotransmitters [27]. In functional studies, 5-HTmoduline antagonises the inhibitory effects of 5-HT1B receptor specific agonists such as CP 93129 on evoked synaptosomal release of radiolabelled 5-HT [27]. Moreover, an intra cerebroventricular injection of 5-HT-moduline antagonises the effects of a selective 5-HT1B receptor agonist in a mouse social interaction model [27] and provokes a rapid desensitization of 5-HT1B receptors in the substantia nigra [36]. Finally, direct administration of 5-HT-moduline in the striatum induces a local augmentation of dopamine release [1]. These studies suggest that 5-HT-moduline acts as a 5-HT1B auto- and heteroreceptor antagonist modulating allosterically 5-HT and other neurotransmitter release possibly by the induction of a transconformation of 5-HT1B/1D receptors. Anxiety is a psychic disorder linked to an over activity of the central serotonergic system [6,22]. Intra hippocampic administration of a selective 5-HT1B receptor agonist (CP 93129) in the rat decreases its exploratory activity and induces a neophobic reaction in the open field test [8]. Moreover, a restraining stress is able to induce a desensitization of 5-HT1B receptors, and consequently, increases serotonin release [4] and the increase of 5-HT-moduline seems to be implicated in this response to stress [5]. The effects of the inactivation of endogenous 5-HT-moduline by polyclonal anti-5-HT-moduline antibodies, which unbalances the 5-HT-moduline/5-HT1B receptor system, have been studied in the open field and EPM in mice [19]. Blockade of endogenous 5-HT-moduline provokes a clear anxiolysis. In fact, 5-HT-moduline, which is released after an acute stress induces a desensitization of 5-HT1B receptors in vivo [5,36], which leads to an augmentation of 5-HT release and bioavailability in the synapse. Thus, 5-HTmoduline seems to play a role in the development of anxiety disorders. In a previous study [11], we evaluated the effects of a 5HT-moduline antagonist called HG1 in mice models of anxiety. In the three behavioural tests that we performed, HG1 showed an anxiolytic-like profile similar to that of diazepam. We chose to explore its mechanism of action in the EPM, possibly involving GABA-ergic and serotonergic neurotransmission systems. There are complex-modulating relationships between GABA and serotonergic systems and the effectiveness of 5HT1A antagonists in the EPM demonstrates that these receptors participate to the anxiolytic response in this test [9]. Finally, there is also a functional interaction between 5-HT1A and 5-HT1B receptors whose balance determines the degree of this response [16]. Thus, we tried to focus on the potential participation of a GABA-ergic mediation in the mechanism of action of HG1 using GABA antagonists to try to antagonise its anxiolytic effects in the mouse EPM. Then, the implication

of the 5-HT1 system to its anxiolytic effects was investigated in the mice EPM using 5-HT receptor agonists and antagonists in order to potentiate or antagonise HG1 anxiolytic-like effects.

2. Material and methods All experimental procedures were in strict accordance with the guidelines of the French Ministry of Agriculture on the use and care of laboratory animals (decree No. 87-848 of the 19 October 1987). 2.1. Animals Male Swiss weaned mice (Centre d’´elevage R. Janvier, Le Genest, France) 4 weeks old and weighing 18–20 g at the time of testing, were housed in groups of 18 per cage (40 cm × 28 cm × 17 cm), in the standard conditions of the animal room (20 ± 1 ◦ C, standard light/dark cycle light on at 7:00 h, off at 19:00 h) with free access to food and water for a period of 1 week. Each experimental group consisted of na¨ıve randomly grouped mice of the same weight, which were used only once. 2.2. Drugs and treatments HG1, 5-HT-moduline antagonist [14]; flumazenil, benzodiazepine (BZD) receptor antagonist (Ro 15-1788) (Hoffmann-La Roche SA); bicuculline methobromide, non-competitive GABAA receptor antagonist (RBI, SIGMA, France); picrotoxine, GABAA receptor antagonist which blocks chloride channel (RBI, SIGMA, France); 8-OH-DPAT hydrobromide, 5-HT1A receptor selective agonist ((±)-8-hydroxy-2-dipropylaminotetralin) (Tocris, Fisher Bioblock Scientific, France); NAN 190 hydrobromide, 5-HT1A receptor potent antagonist (1-(2-methoxyphenyl)-4-[(2-phthalimido)butyl]piperazine) (RBI, SIGMA, France); WAY 100635, 5-HT1A receptor highly selective antagonist (N-[2-[4-(2methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexane carboxamide maleate) (RBI, SIGMA, France); anpirtoline hydrochloride, 5-HT1B receptor potent agonist (Tocris, Fisher Bioblock Scientific, France); methiothepine mesylate, 5-HT1A/1B autoreceptor antagonist (RBI, SIGMA, France). All drugs were extemporaneously dissolved in distilled water except flumazenil, which was dispersed in physiological saline, to which Tween 80 (0.5 mL/8 mg) had been added. To study their effects alone, in the actimeter and EPM tests, drugs were administered intraperitoneally (i.p.) 45 min before the test under a volume of 0.5 mL/20 g of mouse weight (except HG1 which was administered i.p. 30 min before). In association studies, HG1 was administered 30 min before the test and interacting drugs, 45 min before. Control animals received vehicle only. 2.3. Dose–response studies Dose–response studies were performed in the actimeter test to determine active and non-active doses of HG1, GABA-ergic and serotonergic ligands (i.e. which do not affect locomotor and/or exploratory activity). EPM was performed with HG1, GABA-ergic and serotonergic ligands to determine active and non-active doses to be used in association studies. All tests were blind conducted to

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the treatment in randomly designed ordered sessions. Each experiment was carried out separately in a single day.

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of other drugs were also observed and we chose ranges of doses, which did not modify the locomotor activity to be evaluated in the EPM.

2.4. Measure of locomotor activity [3] Activity was recorded using a photo-electric actimeter (OSYS, France). This apparatus consists of a stainless closed steel box containing transparent cages (270 mm × 220 mm × 110 mm) in which animal’s horizontal activity is measured in the dark by two light beams connected to a photoelectric cell. The total number of crossing beams is recorded over a period of 10 min. In order to reduce any neophobic response to the test conditions, the cages were previously dirtied by mice other than those used for the test and there was no cleaning between trials. 2.5. Elevated Plus Maze (EPM) [25] It is an ethological model of anxiety provoked in rodents by the novelty and repulsion caused by an elevated and illuminated plus maze. The apparatus consists of two open, elevated arms (16 cm × 5 cm) facing opposite to each other and separated by a central platform (5 cm × 5 cm) and two arms of the same dimension, but enclosed by walls (16 cm × 5 cm × 10 cm). A red dim light (60 W, 4 lx) focuses on the platform. The maze is raised off the ground so that open arms combine elements of unfamiliarity, openness and elevation [13]. The arms and walls are painted black. The test is carried out in a calm, tempered and dark room. Each mouse is placed on the platform, facing an open arm allowed to explore during 5 min. Time spent and number of entries onto each arm is scored by one person. The apparatus is not cleaned between each animal testing. The anxiolytic effectiveness of a drug is evaluated by a significant statistical augmentation of one of the parameters in open arms (time and/or entries). The augmentation of the percentage of entries in open arms in proportion to total entries in both arms is a good indicator of anxiolysis too, although closed arm entries and total entries reflect a motor component of the exploratory activity. 2.6. Data analysis Data were analysed by means of a global analysis of variance (ANOVA) for independent groups. For drug alone effects, if one-way ANOVA showed a significant difference between groups (p ≤ 0.05), a Dunnett a posteriori test comparing all treated groups with the control group was performed to detect which treated group was different from the control group. For association studies, if two-way ANOVA (pretreatment and treatment) showed a significant difference between groups (p ≤ 0.05), a SIDAK a posteriori test comparing all groups between each other was performed to detect which combined-treated group was different from the group treated with the corresponding dose of HG1. Analyses were performed with SPSS program for IBM compatible computer. Differences were considered statistically significant when p ≤ 0.05.

3. Results 3.1. Measure of locomotor activity The effects of HG1 (8–64 mg/kg) on the locomotor activity of mice were evaluated in our previous study [11]. The effects

3.2. Effects of drugs alone in the EPM (Table 1) In each association study, we could verify that HG1 was non-active at the doses of 8 and 16 mg/kg, and active at the doses of 32 and 64 mg/kg as in our previous study [11]. The effects of GABA-ergic ligands and serotonergic ligands were observed in the EPM and we chose two doses for each, which did not significantly modify the behaviour in the open arms to be associated with HG1 (Table 1). 3.3. Effects of treatments associations on the EPM (Table 2) In association with HG1 (32 mg/kg), flumazenil (2 mg/kg) decreased the number of open arm entries (F(2, 57) = 3.31, p = 0.043; SIDAK: p = 0.038) (Fig. 1a). In association with HG1 (64 mg/kg), flumazenil (2 mg/kg) increased the time spent on the open arms (F(2, 57) = 4.12; p = 0.021; SIDAK: p = 0.031) (Fig. 1b). Thus, flumazenil (2 mg/kg) antagonised significantly the anxiolytic-like effects of active doses of HG1 (32 and 64 mg/kg) (Table 2). No significant modification of the anxiolytic-like effects of HG1 (32 and 64 mg/kg) was observed when bicuculline (2 and 8 mg/kg) was associated with HG1. Thus, HG1 did not interact with GABAA receptor via its GABA site. No significant modification of the anxiolytic-like effect of HG1 (32 and 64 mg/kg) was observed when picrotoxine (2 and 8 mg/kg) was associated with HG1. Thus, HG1 did not interact with GABAA receptor via chloride-associated channel. The association of 8-OH-DPAT with HG1 at non-active doses did not modify HG1 effects on anxiety level in the test. In association with HG1 (32 mg/kg), NAN 190 (0.5 mg/kg) decreased the number of open arm entries (F(2, 27) = 3.75, p = 0.036; SIDAK: p = 0.040) (Fig. 2a). In association with HG1 (64 mg/kg), NAN 190 decreased the number of open arm entries (F(2, 27) = 7.52, p = 0.003; SIDAK: p = 0.037 (at 0.125 mg/kg), p = 0.002 (at 0.5 mg/kg)) (Fig. 2a). The time spent on the open arms was also decreased by the association of NAN 190 (0.125 and 0.5 mg/kg) (F(2, 27) = 9.86, p = 0.001; SIDAK: p = 0.002) (Fig. 2b). Percent open arm entries/total entries was decreased by the association of NAN 190 (0.5 mg/kg) (F(2, 27) = 4.21, p = 0.026; SIDAK: p = 0.023) (Fig. 2c). Thus, NAN 190 antagonised significantly the anxiolytic-like effects of active doses of HG1. In association with HG1 (16 mg/kg), WAY 100635 (0.008 mg/kg) increased the number of open arm entries (F(2, 27) = 3.65, p = 0.04; SIDAK: p = 0.05) (Fig. 3a) and the time spent on the open arms (F(2, 27) = 6.35, p = 0.005; SIDAK: p = 0.006 (at 0.008 mg/kg), p = 0.048 (at 0.002 mg/kg)) (Fig. 3b). When administered alone, WAY 100635 (2 mg/kg) displayed anxiolytic-like effects. In fact,

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Table 1 Statistical analysis of the effects of drugs alone on parameters in the open arms in the mouse EPM Drug

Entries

Time

% Entries (open arm/total)

Choice of doses for associations

HG1 8–64 mg/kg

↑ at 64 F(5, 66) = 2.89, p = 0.02; Dunnett: p = 0.017

↑ at 64 F(5, 66) = 2.9, p = 0.02; Dunnett: p = 0.002

8 and 16 (inactive); 32 and 64 (active)

Flumazenil 0.5–8 mg/kg Bicuculline 1–8 mg/kg Picrotoxine 0.03–0.5 mg/kg 8OH-DPAT 0.125–2 mg/kg WAY 100635 0.001–0.015 mg/kg

No effect F(5, 54) = 1.51, p = 0.201 No effect F(4, 45) = 1.01, p = 0.415 No effect F(5, 54) = 1.68, p = 0.155 No effect F(5, 54) = 1.52, p = 0.199 ↑ at 0.015 F(5, 54) = 4.39, p = 0.002; Dunnett: p = 0.006 No effect F(5, 54) = 1.90, p = 0.109

↑ at 32 and 64 F(5, 66) = 4.62, p = 0.001; Dunnett: p = 0.034, p = 0.004 No effect F(5, 54) = 1.80, p = 0.128 No effect F(4, 45) = 0.49, p = 0.449 No effect F(5, 54) = 1.70, p = 0.15 No effect F(5, 54) = 0.99, p = 0.433 ↑ at 0.015 F(5, 54) = 4.33, p = 0.002; Dunnett: p = 0.026 ↑ from 0.008 to 0.03 F(5, 54) = 4.06, p = 0.003; Dunnett: p = 0.008, p = 0.05, p = 0.001

WAY 100635 0.008–0.125 mg/kg

WAY 100635 0.25–4 mg/kg NAN 190 0.06–0.5 mg/kg Anpirtoline 0.125–2 mg/kg Methiothepine 0.008–0.06 mg/kg

No effect F(5, 54) = 0.72, p = 0.61 No effect F(5, 54) = 2.10, p = 0.079 ↑ at 0.5 F(5, 54) = 2.39, p = 0.05; Dunnett: p = 0.022 No effect F(4, 45) = 1.67, p = 0.173

No effect F(5, 54) = 1.27, p = 0.29 No effect F(5, 54) = 1.13, p = 0.354 No effect F(5, 54) = 2.06, p = 0.085 No effect F(4, 45) = 1.73, p = 0.159

No effect F(5, 54) = 0.81, p = 0.055 No effect F(4, 45) = 1.65, p = 0.178 No effect F(5, 54) = 1.14, p = 0.351 No effect F(5, 54) = 1.70, p = 0.15 No effect F(5, 54) = 1.70, p = 0.15

2 and 8 2 and 8 0.06 and 0.25 0.25 and 1 0.002, 0.008 and 2

↑ from 0.008 to 0.06 F(5,54) = 3.23, p = 0.013; Dunnett: p = 0.002, p = 0.041, p = 0.034, p = 0.045 No effect F(5, 54) = 1.33, p = 0.26 No effect F(5, 54) = 0.53, p = 0.755 No effect F(5, 54) = 3.45, p = 0.009; Dunnett: p > 0.05 No effect F(4, 45) = 0.97, p = 0.432

0.002, 0.008 and 2

0.002 and 0.008 and 2 0.125 and 0.5 0.25 and 1

0.015 and 0.06

Ranges of doses are of geometrical ratio of two. (↑): significant increase of the parameter vs. the control group (n = 10). F: one-way ANOVA, p: Dunnett a posteriori test.

it increased the number of open arm entries (F(3, 36) = 4.66, p = 0.007; Dunnett; p = 0.006) (Fig. 3a), the time spent on the open arms (F(3, 36) = 12.29, p < 0.001; Dunnett: p < 0.001) (Fig. 3b) and % open arm entries/total entries (F(3, 39) = 3.51, p = 0.025; Dunnett: p = 0.012) (Fig. 3c). In association with HG1 (32 mg/kg), WAY 100635 (2 mg/kg) decreased the number of open arm entries (F(3, 36) = 4.11, p = 0.013; SIDAK:

p = 0.01) (Fig. 3a). In association with HG1 (64 mg/kg); WAY 100635 (2 mg/kg) decreased the number of open arm entries (F(1, 18) = 12.92, p = 0.002) and total entries (F(1, 18) = 6.798, p = 0.018) (Fig. 3a), the time spent on the open arms (F(1, 18) = 10.480, p = 0.005) (Fig. 3b) and % open arm entries/total entries (F(1, 18) = 5.507, p = 0.031). WAY 100635, at low and sub active doses on the test (0.002 and

Table 2 Effects of the associations of GABA-ergic and serotonergic ligands with HG1 on the effects of HG1 in the mouse EPM

Antagonists of GABAA system 5-HT1A system Agonist Antagonists

HG1 sub active doses (8 and/or 16 mg/kg)

HG1 active doses (32and/or 64 mg/kg)

Flumazenil Bicuculline Picrotoxine

– – –

Incomplete Antagonism No interaction No interaction

8-OHDPAT

No interaction

–

NAN 190 WAY 100635 Low dose High dose

–

Antagonism

Potentiation –

– Antagonism

Anpirtoline Low dose High dose Methiothepine

Potentiation No interaction –

– – Antagonism

5-HT1B system Agonist Antagonist

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Fig. 1. Effects of the association flumazenil + HG1 in the mouse. (a) EPM (entries onto arms). Data are expressed as the mean number of entries onto the arms in the mouse EPM + S.E.M. (standard error of mean) (n = 20). * p ≤ 0.05, ** p ≤ 0.01 vs. appropriate parameter in the control group. + p ≤ 0.05, ++ p ≤ 0.01 vs. corresponding parameter in HG1 alone. Treatment in mg/kg. (b) EPM (time spent on the arms). Data are expressed as the mean time spent on the arms of the mouse EPM (s) + S.E.M. (n = 20). * p ≤ 0.05 vs. appropriate parameter in the control group. + p ≤ 0.05, ++ p ≤ 0.01 vs. corresponding parameter in HG1 alone. Treatment in mg/kg.

0.008 mg/kg) potentiated the effects of HG1 at sub-active dose (16 mg/kg), although at these doses and at a high and anxiolytic-like one (2 mg/kg), WAY 100635 antagonised the anxiolytic-like effects of HG1 at active doses of 32 and 64 mg/kg. In association with HG1 (16 mg/kg), anpirtoline (0.25 mg/kg) increased the time spent on the open arms (F(2, 27) = 15.73, p < 0.001; SIDAK: p = 0.037) but decreased it at 1 mg/kg (SIDAK: p = 0.02) (Fig. 4). Percent open arm entries/total entries was decreased by anpirtoline (1 mg/kg) (F(2, 27) = 8.51, p = 0.001; SIDAK: p = 0.007). At low dose (0.25 mg/kg), anpirtoline potentiated the effects of HG1 at sub active dose but not at high dose (1 mg/kg), probably because of a loss of specificity for 5-HT1B receptors. In association with HG1 (32 mg/kg), methiothepine (0.06 mg/kg) decreased the number of open arm entries (F(2, 27) = 4.91, p = 0.015; SIDAK: p = 0.022) (Fig. 5a), the time spent on the open arms (F(2, 27) = 4.07, p = 0.028; SIDAK: p = 0.03) (Fig. 5b) and % open arm entries/total entries (F(2, 27) = 3.614, p = 0.041; SIDAK: p = 0.048) (Fig. 5c). In association with HG1 (64 mg/kg), methiothepine (0.06 mg/kg) decreased the number of open arm entries (F(2, 27) = 3.93,

p = 0.032; SIDAK: p = 0.044) (Fig. 5a) and the time spent on the open arms (F(2, 29) = 4.28, p = 0.024; SIDAK: p = 0.05 (at 0.06 mg/kg), p = 0.045 (at 0.015 mg/kg) (Fig. 5b). Thus, methiothepine antagonised significantly the anxiolytic-like effects of HG1 at active dose.

4. Discussion In the present study, only flumazenil interacts with HG1 but neither bicuculline nor picrotoxine, which are not active on the same site of the GABAA receptor. The GABA-ergic system does not seem to be implicated in the anxiolytic-like mechanisms of action of HG1, except a partial antagonist interaction at the BZD site of the GABAA receptor, which illustrates a moderate regulation of the GABA system on serotonergic transmission. The nature of this regulation might be explained by complex interactions between both systems in different brain areas. In fact, some GABA-ergic endings are connected with 5-HT neurons of the dorsal raphe nucleus and the application of GABA agonists, such as muscimol, in dorsal and me-

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Fig. 2. Effects of the association NAN 190 + HG1 in the mouse. (a) EPM (entries onto arms). Data are expressed as the mean number of entries onto the arms in the mouse EPM + S.E.M. (n = 10). * p ≤ 0.05 vs. appropriate parameter in the control group. + p ≤ 0.05, ++ p ≤ 0.01 vs. corresponding parameter in HG1 alone. Treatment in mg/kg. (b) (time spent on the arms). Data are expressed as the mean time spent on the arms of the mouse EPM (s) + S.E.M. (n = 10). ** p ≤ 0.01 vs. appropriate parameter in the control group. ++ p ≤ 0.01 vs. corresponding parameter in HG1 alone. Treatment in mg/kg. (c) Effects of the association NAN 190 + HG1 in the mouse EPM (% open arm entries/total entries). Data are expressed as the mean % open arm entries/total entries on the arms of the mouse EPM + S.E.M. (n = 10). ** p ≤ 0.01 vs. control group. + p ≤ 0.05 vs. HG1 alone. Treatment in mg/kg.

dial raphe nuclei (DRN and MRN) inhibit 5-HT neuronal activity, release and metabolism in forebrain. These effects are blocked by specific GABAA receptor antagonists such as bicuculline. In the DRN, GABAA receptors have a tonic influence on 5-HT neuronal activity. Finally, injection of bicuculline and picrotoxine in the DRN increases 5-HT level in the DRN, but local blockade of GABAA receptors in the MRN

and nucleus accumbens has no effects [37]. Moreover, the amygdala is the main projection site of the DRN and the inhibition of DRN 5-HT neurons decreases anxiety. Nevertheless, Gonzalez et al. [17] and Koyama et al. [24] suggest that benzodiazepine receptors and 5-HT1A receptors of the basolateral amygdala do not play any role in anxiety modelled by the EPM.

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Fig. 3. Effects of the association WAY 100635 + HG1 in the mouse. (a) EPM (entries onto arms). Data are expressed as the mean number of entries onto the arms in the mouse EPM + S.E.M. (n = 10). * p ≤ 0.05, ** p ≤ 0.01 vs. appropriate parameter in the control group, + p ≤ 0.05, ++ p ≤ 0.01 vs. corresponding parameter in HG1 alone. Treatment in mg/kg. (b) EPM (time spent on the arms). Data are expressed as the mean time spent on the arms of the mouse EPM (s) + S.E.M. (n = 10). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. control, + p ≤ 0.05, ++ p ≤ 0.01 vs. corresponding parameter in HG1 alone. Treatment in mg/kg. (c) Effects of the association WAY 100635 + HG1 in the mouse EPM (% open arm entries/total entries). Data are expressed as the mean % open arm entries/total entries on the arms of the mouse EPM + S.E.M. (n = 10). * p ≤ 0.05 vs. control group. + p ≤ 0.05 vs. HG1 alone. Treatment in mg/kg.

In our study, when an inactive dose of HG1 is associated with 8-OHDPAT, no effects are observed or only a slight increase of the anxiety level reflecting only postsynaptic effects of 8-OHDPAT. Conversely, 5-HT1A receptor antagonists (WAY 100635 and NAN 190) counteract the anxiolytic-

like effects of HG1 at doses where these ligands preferentially affect presynaptic receptors. In the case of low doses of WAY 100635, the potentiation of HG1 effects could result from the combination of postsynaptic antagonism with partial presynaptic agonism. This mechanism might lie on the

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Fig. 4. Effects of the association anpirtoline + HG1 in the mouse EPM (time spent on the arms). Data are expressed as the mean time spent on the arms of the mouse EPM (s) + S.E.M. (n = 10). + p ≤ 0.05 vs. corresponding parameter in HG1 alone. Treatment in mg/kg.

Fig. 5. (a) Effects of the association methiothepine + HG1 in the mouse EPM (entries onto arms). Data are expressed as the mean number of entries onto the arms in the mouse EPM + S.E.M. (n = 10). * p ≤ 0.05 vs. appropriate parameter in control, + p ≤ 0.05 vs. corresponding parameter in HG1 alone. Treatment in mg/kg. (b) Effects of the association methiothepine + HG1 in the mouse EPM (time spent on the arms). Data are expressed as the mean time spent on the arms of the mouse EPM (s) + S.E.M. (n = 10). * p ≤ 0.05, ** p ≤ 0.01 vs. corresponding parameter in control. + p ≤ 0.05 vs. appropriate parameter in HG1 alone. Treatment in mg/kg. (c) Effects of the association methiothepine + HG1 in the mouse EPM (% open arm entries/total entries). Data are expressed as the mean % open arm entries/total entries on the arms of the mouse EPM + S.E.M. (n = 10). * p ≤ 0.05, ** p ≤ 0.01 vs. control group. + p ≤ 0.05 vs. HG1 alone. Treatment in mg/kg.

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biphasic aspect of WAY 100635 anxiolytic-like effects in the EPM. Concerning the 5-HT1B receptor system, anpirtoline is a 5HT1B receptor agonist and to a less extent, a 5-HT1D agonist and a 5-HT3 antagonist [18]. At low dose, the potentiation of HG1 effects illustrates a direct interaction with 5-HT1B receptors. Methiothepine, a nonselective presynaptic 5-HT1B receptor antagonist, limits HG1 effects in the same way as does NAN 190, but a partial agonism of 5-HT1B receptors or activation of somatodendritic 5-HT1A receptors are not excluded as methiothepin is known to increase 5-HT in vivo [31]. In fact, 5-HT1B receptors are highly present in basal ganglia (substantia nigra, globus pallidus, dorsal subiculum, cortex) and in the hypothalamus, cerebellum and DRN [7]. 5HT1B/1D receptors inhibit GABA release via heteroreceptors on striato nigral GABA-ergic afferencies. In anxiety models, 5-HT1B receptor agonists should induce or increase anxiety and antagonists, decrease it via such heteroreceptors. Numerous 5-HT1B receptor agonists are indeed anxiogenic in the mouse EPM [10]. Moreover, 5-HT1B receptor knockout (KO) mice are less anxious than wild type mice in the EPM [26,38]. Finally, flumazenil, which is antagonised by a 5-HT1B receptor agonist, also decreases anxiolytic-like effects of a 5-HT1B receptor antagonist. Moret and Briley [29] suggest that the anxiolytic-like effects of 5-HT1B receptor antagonists are due to a blockade of heteroreceptors on GABA-ergic neurons inducing an augmentation of GABA transmission. However, 5-HT1A receptors are known to interact negatively with 5-HT1B receptors. In fact, in conditions of acute stress, 5-HT-moduline increases in various brain areas, prevents 5-HT binding with 5-HT1B receptors and induces their desensitization. HG1 might restore 5-HT1B receptor participation to the limitation of 5-HT activity by blocking 5-HTmoduline, and consequently HG1 might permit the development of an anxiolytic-like response in EPM aversive conditions. Even if the interaction of HG1 with 5-HT1A receptors is only indirect, their implication in the mechanism of action is not excluded and might be predominant considering the effects of WAY 100635 associated with HG1 in our model. The nature of the interaction between 5-HT1A and 5-HT1B receptors suggested by Gardier et al. [16] and the use of more selective ligands for these receptors could help us to understand this phenomenon. In fact, functional activity of somatodendritic 5-HT1A receptors is modified in 5-HT1B receptor KO mice in comparison with wild type. In the 8-OHDPAT induced hypothermia mice model, 8-OHDPAT is more effective in KO mice than in wild type. Moreover, WAY 100635 is more effective to antagonise 8-OHDPAT induced hypothermia in mutant mice than in wild type. In addition, mutant mice are less anxious than wild type and more vulnerable to stressful conditions [38]. Moreover, systemically administered 5-HT1A and 5-HT1B receptor agonists decrease 5-HT concentration in the striatum and the effect of the 5-HT1A receptor agonist is blocked by a 5-HT1A receptor antagonist, but not by a 5-HT1B recep-

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tor antagonist. Nevertheless, the effect of the 5-HT1B receptor agonist is blocked by a 5-HT1B receptor antagonist but not by a 5-HT1A receptor antagonist. 8-OHDPAT, systemically administered in the rat, decreases 5-HT concentration in the hippocampus and striatum via a presynaptic target and this effect also occurs after local injection in raphe nuclei. 5-HT1B/1D receptor antagonists bind to receptors located on DRN neurons and induce a local augmentation of extracellular 5-HT, which activates 5-HT1A autoreceptors leading to 5-HT decrease in the striatum [23]. Finally, we may wonder about the synaptic targets implicated in the observed effects. In fact, the attenuation of 5-HT neurotransmission is anxiolytic-like in animal models and the anxiolytic-like effects of 5-HT1 receptor ligands could result from an agonistic action at somatodendritic 5-HT1A autoreceptors and/or an antagonism of postsynaptic 5-HT1A receptors. Cao and Rodgers study [9] has shown that the mouse EPM is a model of anxiety sensitive to 5-HT1A receptor ligands. In fact, high doses of WAY 100635 increase 5-HT via the tonic inhibition of somatodendritic 5-HT1A receptors and this effect counteracts the action of agonistic compounds on postsynaptic 5-HT1A receptor sites. The mechanism of action of HG1 in the mouse EPM could associate several systems of neurotransmission but it seems that the main way is through the regulation of 5-HT1A and BZD receptors. More selective compounds would be helpful to clearly examine the role of each serotonin receptor subtype, and particularly 5-HT1B receptors, but their brain distribution after i.p. administration remains very low. References [1] Bentu´e-Ferrer D, Reymann JM, Rousselle JC, Massot O, Bourin M, Allain H, et al. 5-HT-moduline, a 5-HT1B/1D receptor endogenous modulator, interacts with dopamine release measured in vivo by microdialysis. Eur J Pharmacol 1998;358:129–37. [2] Blier P, Pineyro G, El Mansari M, Bergeron R, De Montigny C. Role of somatodendritic 5-HT autoreceptors in modulating 5-HT neurotransmission. Ann NY Acad Sci 1998;15:204–16. [3] Boissier JR, Simon P. Action de la caffeine sur la motilit´e spontan´ee de la souris. Arch Int Pharmacodyn 1965;158:212–21. [4] Bolanos-Jimenez F, Manhaes De Castro R, Seguin L, Clo¨ez-Tayarani I, Monneret V, Drieu K, et al. Effects of stress on the functional properties of pre- and post-synaptic 5-HT1B receptors in the rat brain. Eur J Pharmacol 1995;294:531–40. [5] Bonnin A, Grimaldi B, Fillion MP, Fillion G. Acute stress induces a differential increase of 5-HT-moduline (LSAL) tissue content in various rat brain areas. Brain Res 1999;825:152–60. [6] Briley M, Chopin P, Moret C. Effects of serotonin lesions on anxious behaviour measured in the elevated-plus-maze test in the rat. Psychopharmacology 1990;101:187–9. [7] Bruinvels AT, Palacios JM, Hoyer D. Autoradiographic characterization and localisation of serotonin 5-HT1B compared to 5-HT1D binding sites in rat brain. Naunyn-Schmiedeberg’s Arch Pharmacol 1993;347:569–82. [8] Buhot MC, Naili S. Changes in exploratory activity following stimulation of hippocampal 5-HT1A and 5-HT1B receptors in the rat. Hippocampus 1995;5:198–208. [9] Cao BJ, Rodgers RJ. Influence of 5-HT1A receptor antagonism on plus-maze behaviour in mice II. WAY 100635 SDZ

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