Activation of 5-HT2B Receptors in the Medial Amygdala causes Anxiolysis in the Social Interaction Test in the Rat

pp.601F608, 1997 @ 1997Elsevier ScienceLtd. All rights reserved Printed in Great Britain 0028-3908/97$17.00 + 0.00

Neuropharrnacology, Vol. 36, No. 4/5,

Pergamon PII: S002S-3908(97)00042-7

Activation of 5-HT2BReceptors in the Medial Amygdala causes Anxiolysis in the Social Interaction Test in the Rat M. S. DUXON,l G. A. KENNETT,2 S. LIGHTOWLER,2T. P. BLACKBURN2 and K. C. F. FONE1* IDepartment of Physiology and Pharmacology, University of Nottingham, Medical School, Queen’s Medical Centre, NottinghamNG72UH, U.K. and 2Department of Psychiatry Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Third Ave, Harlow, Essex CM19 SAW, U.K. (Accepted 27 January 1997) s ummary-In a recent study, we reported the presence of neuronesexpressing5-HT2Breceptor protein in the medial amygdaloid nucleus of the adult rat brain. In the present study, bilateral micro-injectionof the 5-HT2B receptor agonist l-[5-(2-thienylmethoxy)-IH-3-indolyl]propan-2-aminehydrochloride(BW 723C86,0.09 and 0.93 nmol, 5 min pretest) into the medial amygdaloid nuclei increased the total interaction time of a pair of male rats in the social interaction test, to a comparable extent to chlordiazepoxide(5 mglkg p.o., 1 hr pretest) without altering locomotor activity; indicative of anxiolytic activity. The increase in social interaction was prevented by pretreatment with the 5-HTz~/z~receptor antagonist N-(1-methyl-5-indoyl)-N’-(3-pyridyl)urea hydrochloride(SB 200646A,at 2 but not 1 mgkg p.o., 1 hr pretest), which did not alter behaviour when given alone. Intra-amygdalaBW 723C86 (0.09, 0.31 and 0.93 nmol, 5 min pretest) did not significantlyalter the number of punished responses made when the same rats were examined seven days later in a Vogel punished drinking test, although chlordiazepoxide(5 mglkg p.o., 1hr pretest) produced the expected anxiolytic profile. The results are consistent with the proposaf that activation of 5-HT2Breceptors in the medial amygdala induces anxiolysisin the social interactionmodel but has little effect on behaviourin a punishedconflict model of anxiety. These data suggest that serotonergic neurotmnsmission in this nucleus may selectively affect specific kinds of anxiety generated by different animal models. 0 1997Elsevier Science Ltd.

Keywords—5-HT2Breceptor, anxiety, medial amygdaloid nucleus, benzodiazepines, social interaction test, Vogel punished drinking test.

The most recent addition to the 5-HT2 subfamily of receptors was cloned from the rat stomach fundus (Foguet et al., 1992; Kursar et al., 1992) and is termed the 5-HT2B receptor (Hoyer et al., 1994). In smooth

muscle, the 5-HT2B receptor has been identified to mediate 5-HT-inducedcontractionof the stomachfundus (Baxter et al., 1994), endothelium-dependentrelaxation of the jugular vein (Ellis et al., 1995),contraction of the mesenteric artery of DOCA salt hypertensiverats (Watts et al., 1995, 1996)and contractionof longitudinalmuscle in the small intestine in man (Berman and Burleigh, 1995).However, the presence and any functionalrole of the this receptor subtype in the rat CNS is controversial. For example, several groups have failed to identify 5HT2Breceptor mRNA within the rat CNS (Foguet et al., 1992; Kursar et al., 1992; Pompeiano et al., 1994),

*To whom correspondence should be addressed

although it appears to be present in the spinal cord of all speciesexamined (Helton et al., 1994).This has led some workersto suggestthat the 5-HT2Breceptor may either be a peripheral counterpart of the rat 5-HT2C receptor (Schmuck et al., 1994) or that other 5-HT2 subtypes perform the CNS function of the 5-HT2Breceptor in the rat (Choi et al., 1996). In contrast, by using reverse transcription-polymerasechain reaction, Flanigan et al. (1995) identified 5-HT2B receptor transcripts in the hippocarnpus,cerebellum,cortex, mid brain and medulla of the rat. Furthermore, by using an antibody raised againstthe N-terminusof the rat 5-HT2Breceptor protein, we have recently shown the presence of 5-HT2B-like immunoreactivity (5-HT2B-LI)in discrete brain areas using immunohistochemistry (Duxon et al., 1995b, 1997).The most intenseneuronal 5-HT2B-LIwas present on cell bodies in limbic nuclei in the rat, including the lateral septum,dorsomedialhypothalamusand on a dense cluster of bipolar and multipolar cells in the medial

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amygdaloid nucleus (Duxon et al., 1995b, 1997); all areas implicated in aversion. The pharmacological characteristicsof the rat 5-HT2~ receptor (Wainscott et al., 1993; Baxter et al., 1994) are extremely homologouswith those of both the 5-HT2Aand 5-HT2C receptors, such that few ligands distinguish between these subtypes, making definitiveevaluation of the CNS functional role of each very difficult. Recent studies have, however, identified an agonist, 1-[5-(2thienylmethoxy)-lH-3-indolyl]propan-2-amine hydrochloride (BW 723C86)with a 10- and 20-fold selectivity for the rat 5-HTz~ over 5-HTz~ and 5-HT2Areceptors, respectively (Baxter et al., 1995). Following systemic administration to rats, BW 723C86 produces anxiolysis in both the social interaction and Geller–Seifter tests of anxiety, which is abolished by pretreatment with the 5HT2~,2creceptor antagonist,SB 200646A(Kennett et al., 1996).Notably, this anxiolytic-likebehaviouris opposite to the anxiogeniceffect of the 5-HT2C12B receptor agonist m-CPP, both in rats (Kennett et al., 1989; Gibson et al., 1994)and man (Mueller et al., 1985; Kahn et al., 1990), suggesting that the effect of BW 723C86 may be mediated by 5-HT2Breceptor activation. The present study attemptedto investigatethe potential functional role of neurones expressing 5-HT2~receptors in the rat medial amygdala on aversive behaviour, by examining the effect of discrete injection of the 5-HT2B agonist, BW 723C86, into this nucleus on behaviour in two models of anxiety; the social interaction (File and Hyde, 1978) and Vogel punished drinking (Vogel et al., 1971) tests. Both of these paradigms have proved sensitive to the detection of serotonergic drugs, either given systemically (Chopin and Briley, 1987), or by

intra-amygdala injection in previous studies (Higgins et al., 1991; Gonzalez et al., 1996). MATERIALS AND METHODS Animals and housing

Male Sprague–Dawley (Charles River, U.K.) rats (210-230 g) were housed in groups of six under a 12 hr light/dark cycle (lights on 0700 hr) and allowed free access to food (CRMX, Special Diet Services) and water, except where indicated in the Vogel punished drinking test. One day prior to surgery,rats were housed singly and placed in the operating suite where surgery was to be conducted. The following day rats were anaesthetized (Domitor HC1 0.4 mg/kg i.m., –30 min and Sublimaze 0.45 mg/kg i.p., –15 rein) to allow bilateral guide cannulae to be chronically implanted above the medial arnygdala (A –2.8; L t 3.8; V –6.6 mm from bregma, Paxinos and Watson (1986), Fig. l.). Anesthesia was reversed using Antiseden (0.2 mg/kg i.p.) and Nubane (0.2 mg/kg i.p.) and the rats were allowed to recover for 7 days prior to commencing behavioral tests. All behaviour was carried out between 10.00 and 17.30hr. Social interactiontest One day prior to behavioral testing (day 6), rats were weight-matched ( + 5 g) and one of each unfamiliar pair marked on the tail prior to return to the home cage. On day 7, both rats in a pair were given identical treatments (SB 200646A or vehicle p.o. 60 tin prior to, and intraarnygdala injection of BW 723C86 or saline 5 min before, or chlordiazepoxideor vehiclep.o. 60 min before, behavioral observation) and returned to their home ———— ~

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Fig. 1. Diagrammatic representation of the position of the bilateral guide cannula (left hand side) and the CNS injection sites (right hand side). Determinedby histologyfollowingbilateral injection of pontamine sky blue (0.05 (v/v), 500 nl), superimposed on a coronal section of rat brain (2.8 nun posterior to bregma) using the atlas of Paxinos and Watson (1986). The data from five rats, where injections were not located in the medial amygdala, were excluded.

5-HT2~receptor mediated anxiolysis

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Fig. 2. Effect of saline (0.154 M, 500 nl, over 30 see) or BW 723C86 (BW, 0.09,0.31 or 0.9 nmol, as indicated), gi~en by bilateral injection into the medial amygdaloid nuclei 5 tin pretest following vehicle (60 rnin pretest, p.o.). Rats were used in weight-matchedpairs in the social interactiontest under bright light unfamiliar conditions on: (A) upper histograms;total social interactiontime and (B) lower histograms;correspondinglocomotoractivity (number of zone transitions), compared with that of vehicle (VEH, 1 mlkg 1% methyl cellulose) or chlordiazepoxide(CDP, 5 mgkg, p.o., 60 min pretest). All data are presented as mean+ SE, n = 9–14 per group. *P< 0.05 Duncan’sNew Multiple Range test from VEWSALfollowingone way ANOVAF(4,53) = 2.85A p = 0.03.

cage. Social interaction was assessed by placing the pair of rats into a brightly lit (150 Lux) unfamiliar white perspex box (54 x 37 x 26 cm), in which the floor was divided into 24 equal size squares (zones), for 15 min. The total time that animals spent in active social interaction (sniffing, following, biting, crawling over or under, boxing and grooming,but excludingall time spent investigatingtheir indwellingguidecannulae)was scored using a remote video and computerized score pad

(Kennett, 1992). Locomotor activity was assessed by counting the number of zone transitions made by each rat. The test arena was cleaned thoroughly with moist tissue paper between each test. Vogel punished drinking test

On day 12, individual rats were given free access to food but water was deprived for 20 hr prior to being placed in a uniformly lit operant conditioning chamber

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Fig. 3. Effect of SB 200646A(1 or 2 mgkg, p.o., 60 min pretest, hatched columns) or vehicle (VEH, 1 mlkg 1% methyl cellulose, open columns) and either saline (0.154M, 500 nl, over 30 see) or BW 723C86 (BW, 0.09 nmol) given by bilateral injection into the medial amygdaloid nuclei 5 min pretest. Rats were used in weight-matched pairs in the social interaction test under bright light unfamiliar conditions on: (A) upper histograms; total social interaction time and (B) lower histograms;correspondinglocomotoractivity (numberof zone transitions).All data are presented as mean+ SE, n = l&12 per group. *P< 0.05 from VEH/SAL and ++P <0.01 from VEH/BWO.09 Newman–Keul’stest following two way ANOVA.

5-HT2Breceptor mediated anxiolysis

(45 x 25 x 25 cm) into which a water bottle spout protruded. Rats were allowed to explore the chamber freely and drink for 3 min after the first lick of the spout, which was automatically recorded by computerized lickometer. The rat was then returned to the home cage, allowed access to water for 4 hr and then water deprived for 20 hr, before being returned to the operant chamber for 3 min on day 14. During this test day, after 30 sec continuous drinking, each subsequent 15 sec of cumulative drinkingwas punishedby an electric shock (0.25 mA for 0.2 see) delivered through the water bottle spout and the latency to the firstlick and the total number of shocks received over 3 min was recorded.

histological examination after injection of 0.05% (v/v) pontamine sky blue, as illustrated in Fig. 1. Data analysis

All results are presented as mean+ SE. Individual rat social interaction and locomotion scores and lick measurementswere analysedby Duncan’sNew Multiple Range, following one-way ANOVA (BW 723C86 dose– response studies), or two-way ANOVA followed by Newman–Keul’s test (antagonist study), P <0.05 being considered as significant. RESULTS Social interaction

Drugs

For both behavioral tests, rats were assignedto one of nine drug treatment groups. One hour prior to testing,rats were given 1% (v/v) methyl cellulose containing IOmg/ ml barium sulphate (vehicle), chlordiazepoxide (5 mg/ kg) or N-(1-methyl-5-indoyl)-N’-(3-pyridyl)urea hydrochloride (SB 200646A, 1 and 2 mg/kg) p.o. and returned to their home cage. Five minutes prior to behavioral testing, rats were bilaterally injected (500 nl, over 30 sec and the injector left in place for a further 30 see, Carnegie Micro Injector) into the medial amygdaloid nuclei with either saline (0.154 M, including all rats given chlordiazepoxide) or l-[5-(2-thienylmethoxy)-lH-3-indolyl]propan-2-amine hydrochloride (BW 723C86, 0.09, 0.31 or 0.93 nmol). All drugs were administered using a blind protocol and each rat was tested once only. Confirmation of intra-amygdala injection sites was performed by

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Pretreatment with chlordiazepoxide (5 mg/kg, p.o.) produced the expected significant increase in the total social interaction time between pairs of rats placed in an unfamiliar, brightly lit, arena (Fig. 2). A similar significant increase in social interaction was also produced following intra-amygdala injection of the 5HT2~ agonist, BW 723C86, at both 0.09 and 0.93 nmol, although an intermediate dose failed to significantly elevate interaction time (Fig. 2). Neither chlordiazepoxide nor BW 723C86 (at any dose used) produced any concomitant alteration in locomotor activity when compared with vehicle treated controls (Fig. 2), consistent with drug-induced anxiolysis. As shown in Fig. 3, pretreatment of rats with SB 200646A(1 or 2 mg/kg,p.o.) 1 hr pretest had no effect on social interaction or locomotor activity. The significant increase (F1,56= 7.303, P <0.01) in social interaction

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Fig. 4. Effect of saline (0.154M, 500 nl, over 30 see) or BW 723C86 (BW, 0.09,0.31 or 0.9 nmol, as indicated) gi~enby bilateral injection into the medial amygdaloidnuclei 5 min pretest followingvehicle (60 tin pretest, p.o.) on the number of punished water spout licks in the Vogel conflict test, compared with that of vehicle (VEH, 1 ml/kg 1% methyl cellulose), or chlordiazepoxide(CDP, 5 mglkg, p.o., 60 min pretest). All data are presented as mean + SE, n = 10-12 per group. *P< 0.05 Duncan’s test from VEI-USALfollowing one way ANOVA F(4,49)= 2.736 P = 0.039.

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time produced by BW 723C86 (0.09 uM, intra-amygdala injection) was completely prevented by pretreatment with 2 mg/kg p.o. SB 200646 (~2,56 = 6.715, P e 0.05), once again without affecting locomotor activity (Fig. 3). Vogel punished drinking test

In the current study, the validity of performing the Vogel test 7 days after the social interaction paradigm was confirmed by the significant increase in punished responding produced by chlordiazepoxide(F4,4g= 2.736, P e 0.05, Fig. 4). In contrast, BW 723C86, also administered at the same doses used in the social interaction paradigm, failed to significantly alter the number of punishedresponsesin the Vogel test (Fig.4) in the same rats. DISCUSSION 5-HT has been implicated in anxiety and panic disordersfor over two decades, but preclinical evaluation of the functional role of each 5-HT receptor subtype in aversion remains undetermined (Graeff et al., 1993); although receptors of the 5-HT2 subtype are clearly important. Lesion studies in both man and animals show that the amygdala is pivotal to the acquisition and expression of anxiety and the action of anxiolytic drugs (Kuhar, 1986; Kahn et al., 1988; Aggleton, 1993). However, few behavioral studies have examined the effect of discrete injection of 5-HT drugs into individual amygdaloid nuclei. By using immunohistochemistryand a 5-HT2~receptor protein-directed antiserum, we recently reported that this receptor (unlike the 5-HT2Aand 5-HT2Csubtypes; Morilak et al., 1993;Abramowskiet al., 1995)has a very restricted distribution in the rat CNS, being especially abundant on neurones in the medial amygdaloid nucleus (Duxon et al., 1997).While few compoundsdiscriminate between 5-HT2Cand 5-HT2~receptors, the agonist BW 723C86 (Baxter et al., 1995) and the antagonist SB 200646A (Kennett et al., 1994), both show some selectivity for the latter. Therefore, in the current study, we examined the effect of injection of BW 723C86 into the medial amygdala on two different rat models of anxiety, to determine the specific effect of 5-HT2B receptor activationin this nucleus on aversivebehaviour. In the social interaction test, BW 723C86 significantly increased the total interaction time (at all but the 0.31 nmol dose) to a similar extent to systemic administration of the benzodiazepine, chlordiazepoxide,without affecting locomotor activity. This data suggests that intra-medial amygdaloid injection of BW 723C86 produces an anxiolytic-likeeffect in the socialinteraction test. Furthermore, the anxiolytic effect of BW 723C86 was prevented by pretreatment with 2 mg/kg p.o. of the 5-HT2B12C receptor antagonist, SB 200646A, consistent with the current proposal that this is mediated by activation of 5-HT2Breceptors in the medial amygdala. In agreement with the current data, a similar anxiolytic

effect was observed following peripheral administration of BW 723C86 (3 mg/kg s.c.), in the same test apparatus and conditions (Kennett et al., 1996), which was also prevented by pretreatment with an equivalent dose of SB 200646A.Likewise, intra-amygdalainjection of the same doses of BW 723C86 as used in this study, produced an anxiolyticeffect in rats in the elevated plus maze (Duxon et al., 1995a). Also in accordance with the current findings,Zangrossiand Graeff (1994)showed that rnicroinjection of ketanserin (10 nmol) into the basolateral/ medial amygdala caused an anxiogenic effect in the elevated plus-maze. However, these workers did not establish that this effect was mediated by blockade of 5HT2 receptors. Although BW 723C86 has only a 10-fold selectivity for 5-HT2B over 5-HT2C receptors in ligand binding (Baxter, 1996), several factors suggest that the observed anxiolysis is likely to be mediated by activation of 5HT2Breceptors.Firstly,the dose of SB 200646Arequired to attenuatethe affect of BW 723C86,both in the current and previous systemic study (Kennett et al., 1996),is 10fold lower than that required to block the anxiogenic effect of the 5-HT2Cagonistm-CPP in the same paradigm (Kennett et al., 1994).This is also consistent with ligand binding data showing that SB 200646A has a slightly higher affinity for 5-HT2~ than 5-HT2C U3C@OrS (Kennett et al., 1994; Baxter, 1996). Secondly, m-CPP produces an anxiogenicresponse in the social interaction test, both following systemic administration (Kennett et al., 1989)and following intra-hippocampalinjection, but fails to alter socialinteractionfollowinginjection into the amygdala (Whitton and Curzon, 1990). Finally, while systemic administration of BW 723C86 induces C--OS expression(an index of neuronal activation)in the medial amygdala, m-CPP fails to induce expression of the Fos protein in this nucleus (Duxon et al., 1996). In contrast,in the currentVogel punished drinkingtest, intra-amygdala injection of BW 723C86 failed to significantlyincrease the number of punished responses at any dose used (althoughresponses tended to be higher than in vehicle treated rats), even though systemic chlordiazepoxideproduced the expected anxiolyticeffect in this paradigm. Although the Vogel punished drinking paradigmis a relatively simpletest (Vogel et al., 1971),it has proved successfulat detecting anti-conflictproperties of drugs given by systemicinjection (Chopin and Briley, 1987),making the lack of activity of BW 723C86 in this paradigm compared with the potent anxiolytic effect in the social interaction test particularly noteworthy. The precise role of 5-HT in the amygdalain anxiety is, on firstexamination,contradictory.For example, Hodges et al. (1987) found both 5-HT and 8-OH-DPAT to be anxiogenic(decreasingpunishedresponding)in a Geller– Seifter conflict paradigm following injection into the lateralhasolateral amygdala, while the non-selective 5HT2 receptor antagonist, methysergide, was anxiolytic. In contrast, injection of 8-OH-DPAT into the posteromedial cortical amygdaloid nucleus had an anxiolytic

5-HT2~receptor mediated anxiolysis

effect on the stress-induced ultrasonic vocalizations produced by rats in a Geller–Seifter conflict paradigm (Schreiber and De Vry, 1993). Furthermore, using a similar protocol, intra-amygdala injection of 5-HT3 receptor antagonists were found to be anxiolytic in the social interaction test but not in a conflict procedure (Higgins et al., 1991). Such discrepancies are, however, in accord with the suggestionthat specific5-HT receptor subtypes and particuku arnygdaloidnuclei have different roles in individual animal models of anxiety (Hodges et al., 1987;Gonzalez et al., 1996).In particular, activation of neurones in the medial amygdala is involved in affiliativebehaviourin a number of species.For example, induction of c-fos occurs in the medial amygdala following nonspecific interaction in the Syrian hamster (Kollack-Walker and Newman, 1995) and excitatory amino acid-induced lesion of this nucleus inhibits paternal behaviour in the prairie vole (Kirkpatrick et al., 1994). However, it is unlikely that intra-amygdaloid injection of BW 723C86 produces a false positive anxiolyticeffect in the socialinteractiontest by impairing the recognition of social signals, irrespective of whether they are aversive, since an identical treatment with BW 723C86 also had an anxiolytic profile in the elevated plus-maze test (Duxon et al., 1995a).The failure of BW 723C86 to elicit a clear anxiolytic profile in the current Vogel punisheddrinkingparadigmmay, therefore,reflect a greater involvement of 5-HT2Breceptors in the medial amygdala in the kind of anxiety induced by the social interactiontest, which may most closely resemblehuman generalized anxiety disorder (File, 1995). In conclusion, bilateral injection of the 5-HT2B receptor agonist, BW 723C86, into the medial amygdala produced an anxiolytic-like effect in the rat social interaction test. This was prevented by pretreatmentwith the 5-HT2B12C receptor antagonist, SB 200646A, consistent with the proposal that activation of 5-HT2B receptors in the medial amygdala may modulate forms of anxiety state most closely modeled by the social interaction paradigm. Acknowledgements—The authors are grateful to the BBSRC

and SmithKlineBeecham Pharmaceuticalsfor financialsupport of MSD and to SmithKline Beecham Pharmaceuticals for the provision of SB 200646A and BW 723C86.

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