Antagonists at the NMDA recognition site and Mockers of the associated ion channel induce spontaneous tail-flicks in the rat

Europeun Journal of Pharmacology, 0

203 ( 1991) 3 15-3 18

315

1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/Y1/$03.50

ADONIS

001429999100729F

EIP 20936 Short communication

Mark J. Millan Neurobiology

Dirision,

FONDRY,

Groupe de Recherche Serrier,

7 Rue Amp&e,

92800 Puteaux, Paris. France

Received 24 May 1991, revised MS received 16 August 1991, accepted 20 August 1991

The non-competitive N-methyl-D-aspartate (NMDA) antagonists (channel blockers), MK 801, phencyclidine (PCP) and ketamine induced spontaneous tail-flicks in rats. Their order of relative potency (MK 801 > PCP > ketamine) corresponds to their relative affinities for the ion channel coupled to NMDA receptors. Drugs interacting with their other potential targets (a receptors as well as dopamine, serotonin and noradrenaline uptake sitesi failed to induce spontaneous tail-flicks. In addition, the catecholamine stimulants, methylphenidate and cocaine were inactive. CPP and CGS 19755, antagonists at the NMDA recognition site, also dose dependently elicited spontaneous tail-flicks: thtir maximal effect was equal to that of the channel blockers. In contrast, HA-966 and ifenprodil, putative antagonists at the glycine and polyamine recognition sites, respectively, failed to elicit spontaneous tail-flicks. These data demonstrate that both antagonists of the NMDA recognition site and non-competitive blockers of the associated channel induce spontaneous tail-flicks in rats. NMDA (N-methyl-D-aspartate);

MK 801; Phencyclidine (PCP); CPP (3-(2-carboxypiperazin-4-ylkpropyl-l-phosphonic CGS 19755; 5HT,, receptors; Tail flicks (spontaneous)

1. Introduction

Several subtypes of receptor for excitatory amino acids are currently recognised; these have been dassified as NMIIA (N-methyl-D-aspartate), kainate, AMPA; quisqualate, L-AP4 and metabotropic (CoIlingridge and Lester, 1989; Lehmann, 1989). NMDA receptors are involved in several physiological functions, for example, memory formation and control of nociception; in addition, they are implicated in epilepsy and neurodegeneration (Collingridge and Lester, 1989; Lehmann, 1989). Concerning control of motor behaviour, their blockade is associated with stereotyped behavioural responses such as hyperlocomotion, bodyrolling and ataxia (Koek et al., 1989; Tricklebank et al., 1989). NMDA ‘antagonists’ can be divided into several types. Non-competitive antagonists (‘channels blockers’), such as MK 801, PCP (phencyclidine) and ketamine, occupy sites within the cation-permeable channel coupled to the NMDA binding site. In contrast, drugs such as CPP and CGS 19755 act competitively at the NMDA recognition site. Additional sites within the

Correspondence to: M.J. Millan. Neurobiology Division, FONDAX, Groupe de Rechelche Servier, 7 Rue Ampere, 92X00Puteaux, Paris, France. Tel. 33.1.42.04.26.91,fax 33.1.47.28.77.66.

acid);

NMDA receptor complex comprise the glycine and polyamine sites for which HA 966 and ifenprodil, respectively, are putative competitive antagonists (Collingridge and Lester, 1989; Lehman, 1989, Scatton et al., 1991). It has recently been demonstrated that high efficacy 5HT,, agonists induced spontaneous tail-flicks in the rat (Millan et al., 1991); that is, tail-flicks in the absence of extraneous stimulation. Spontaneous tail-flicks show remarkable pharmacological specificity in that rgonists and antagonists at other S-HT receptor types, as well as at (Y,, (Ye, p,, &, D,, D,, GABA, nicotinic and muscarinic acetylcholine, p- and K-opioid receptors etc, are inactive (Millan et al., 1991). The possibility that modulation of excitatory amino acid transmission may elicit this response is interesting in view of the above-mentioned stereotyped motor effects elicited by NMDA antagonists. Further, electrophysiological studies have revealed interactions between neuronal actions mediated by NMDA and 5HT,, receptors (Murase et al., 1990). Finally, several common functional properties of NMDA antagonists and 5HT,, agonists have been identified (see Discussion). In the present study, it was evaluated whether antagonists at NMDA recognition sites (CPP and CGS 19755), the coupled glycine (HA-966) and polyamine (ifenprodil) sites as well as channel blockers (MK 801,

and ketatnine) e rat.

might induce spontaneous tail-flicks

60

MU 901 PCP

3

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I

ate Wistar rats (Iffa Credo, Illskirchen, France) of 230 g were housed in groups of three in sawdustcages with free access to rat chow and water. ats were placed in horizontal, plastic cylinders of adjustable length with the tail emerging from a slit at the rear to hang over the edge of the bench. After 5 mm adaptation, the number of spontaneous tail-flicks emitted in 5 min were recorded. One spontaneous tail-flick was defined as an elevation of the tail to a level higher than that of the body axis (Millan et al., 1991). In preliminary studies, evaluation of time-course of action indicated peak effects of channel blockers at 30 min and of NMDA recognition site antagonists at 60 min post-administration: thus, dose-response curves were performed at these times, respectively. All other drugs were examined 30 min post-injection. Drugs were dissolved in sterile water and given S.C. Drug doses are in terms of the base. Drug sources and salts were as follows. DTG, R (+ )-PPP HCI. GBR 12909 HCI, (+)-MK 801 hydrogen maleate and desipramine HCI (Research Biochemicals, Natick, MA, U.S.A.); ketamine HCI (Substantia, Paris, France); CPP and HA-966 (Tocris Neuramin, Buckhurst Hill, U.K.); fhtoxetine (Lilly, Indianapolis, IN, U.S.A.); PCP HCI (Francopia. Paris, France); ifenprodil tartrate (Synthelabo. Bagneux, France); pentobarbital and cocaine (Cooperation Pharmaceutique Francaise, Paris, France). Methylphenidate was synthesized by J.L. Peglion (Servier, Paris, France). Drug structures and salts are as follows. BTCP, N-[1-(2-benzo(b)thiophenylkyclohexyllpipe~ rdine; CGS 19755, (cis4-phosphonomethyl)-2-piperi Jine carboxylic acid; CPP, 3-(2carboxypiperazin-4-yI)-propyl-1-phosphonic acid; DTG, 1,3-di-(2-tolyl)-guanidine; GBR 12909, 1-[2-[bis(4-ffuorophenyl~methoxylethyll-4-(3-phenylpropyl~piperazine-2; ( + )-MK 801, 5-methyl-lO,ll-dihydro-5H-dibenzo [a,dlcyclohepten-5,10-imine; R( + )-3-PPP, (3-hydroxyphenyl)-N-propylpiperidine; HA-966, 3-amino-l-hydroxy-pyrrolid-2-one. Data were analysed by l-way analysis of variance followed by the Newman-Keuls test, the level of significance was set at P < 0.05.

MK 801 dose dependently elicited spontaneous tailflicks over a range of doses of 0.04-0.16 mg/kg. The

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40.0

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Fig. 1. Induction of spontaneous tail-flicks by the non-competitive NMDA antagonists, MK 801, PCP and ketamine and the competitive antagonists, CPP and CGS 19755. MeanfS.E.M. depicted. N 2 6 per point. ANOVA as follows. MK 801, Ft5,50) = 15.9, P c 0.01; PCP, Ff5,43) = 6.1, P < 0.01; ketamine, F(6.46) = 5.6, P < 0.01; CPP, Ft4,38)= 27.8, P < 0.01 and CGS 19755, F(4,42)= 19.7, P < 0.01. Asterisks indicate significance of drug vs. vehicle differences. * P < 0.05, * * P < 0.01 (Newman-Keuls test). An ANOVA of the respective maximal effects yielded F(4,31) = 1.3, P > 0.05.

curve was biphasic with a further

increase in the dose leading to a loss of effect (fig. 1). The maximal number of spontaneous tail-flicks was 49.2 + 9.0 at a dose of 0.08 mg/kg. Similarly, PCP (2.5-10.0) and ketamine (10.0-20.0) dose dependently evoked spontaneous tail-flicks and yielded biphasic dose-response curves. The NMDA recognition site antagonists, CGS 19755 and CPP, each dose dependently elicted spontaneous tail-flicks. Across the dose-range evaluated, the curves were monophasic with no loss of effect at higher doses. However, owing to limited quantities of drug, it was not possible to further increase the dose of CPP and CGS 19755 to evaluate whether the dose-response curves also had a descending component. The maximum effects of MK 801, PCP, ketamine, CPP and CGS 19755 did not significantly differ from each other (see fig. 1) and were obtained at doses of 0.08, 5.0, 20.0, 20.0 and 40.0 mg/kg, respectively. Interestingly, HA 966 and ifenprodil failed to elicit spontaneous tail-flicks

317 TABLE

1

Inability of the glycine antagonist, HA-966 and the polyamine antagonist, ifenprodil as well as of drugs acting at sites other than NMDA receptors to elicit spontaneous tail-flicks in rats. Mean +S.E.M. given. N p 5 per group. ANOVA: P > 0.05.

F(9,47)= 0.59,

Drug

Dose

STFs/5 min

Vehicle HA-966 lfenprodil DTG R( + )-PPP BTCP GBR 12909 Desipramine

40.0 40.0 10.0 40.0 10.0 10.0 10.0

0.3 f 0.3 0.0 f 0.0 2.o+o.s 1.2+ 1.2 0 +o 0.5 * 0.5 1.g+ 1.1 0.2 * 0.2

Fluoxetine Methylphenidate Cocaine Pentobarbital

10.0 40.0 40.0 10.0

0 *0 1.3+ 1.1 1.4+ 1.2 0.5 f US

at doses of 2.5, 10.0 and 40.0 mg/kg. Data for the highest dose: vehicle, 0.6 + 0.3 spontaneous tailflicks/min; HA 966, 0.0 + 0.0 acd 2.0 + 0.8 spontaneous tail-flicks/min; P > 0.05 in ANOVA. As shown in table 1, the following drugs failed to elicit spontaneous tail-flicks: DTG and R-( + I-PPP (a ligands), BTCP and GBR 12909 (inhibitors of dopamine uptake), fluoxetine (inhibitor of 5-HT uptake), desipramine (inhibitor of norepinephrine uptake), methylphenidate and cocaine (catecholamine stimulants) and pentobarbital (CNS depressant). In view of the biphasic cures obtained with non-competitive NMDA antagonists, an at least l&fold range of doses of these drugs was found to be inactive (not shown).

4. Discussion This study demonstrates that both NMDA recognition site antagonists and channel blockers elicit spontaneous tail-flicks in the rat. In analogy to certain other stereotyped motor responses, the induction of spontaneous tail-flicks by NMDA channel blockers was biphasic (Koek et al., 1989; Tricklebank et al., 1989). The loss of effect upon further increasing doses probably reflects the onset of their muscle relaxant actions which interfere with the emission of coordinated motor responses (Lehmann, 1989). Over the dose-ranged tested, curves for CPP and CGS 19755 were monophasic, this difference might reflect a better separation between doses required to elicit stimulant as compared to ataxic effects of NMDA recognition site antagonists (Lehmann, 1989). Intrigingly, HA-966 (a glycine site antagonist) as well as ifenprodil (a putative polyamine

site antagonist) failed to elicit spontaneous tail-flicks, in line with indications that they do WQ invariably mimic the behavioural effects of NMDA channel blockers or antagonists at the NMDA recognition site (Lehman, 1989; see Scatton et al., 1991). In addition to NMDA receptors, PCP is a potent l&and at u receptors (Deutsch et al., 1988). However, in contrast, DTG and R-( -t I-PPP which act as agonists at LT receptors, as well as the p:ltative u antagonists, BMY 14802 and rimcazole (Deutsch et al., 1988), failed to induce spontaneous tail-flicks (table 1 and not shown). PCP also potently interacts with neuronal sites of monoamine uptake, in particular, dopamine (Collingridge and Lester, 1989): thus, it is important that BTCP and GBR 12909, highly selective inhibitors of dopamine uptake, as well as fluoxetine and desipramine, which inhibit the uptake of serotonin and norepinephrine, respectively, fail to induce spontaneous tail-flicks (Collingridge and Lester, 1989; Koek et al., 1989). The catecholamine stimulants, methylphenidate and cocaine (Koek et al., 1989) - as well as agonists at cy,, LYE,D, and D, receptors - also do not elicit spontaneous tail-flicks (table 1; Millan et al., 1991). Further, although PCP may elicit actions via 5-HT, receptors (Nabeshima et al., 19871, 5-HT, sites do not mediate spontaneous tail-flicks (Millan et al., 1991). In addition, the anesthetic properties of PCP (Collingridge and Lester, 1989) are unlikely to be relevant since peytobarbital did not evoke spontaneous tail-flicks. These non-NMDA actions are much less pronounced with MK 801 than PCP (Lehmann, 1989) and MK 801 was more potent in inducing spontaneous tail-flicks. Indeed, the rank order of potency (MK 801 > PCP > ketamine) is identical to that for in vitro and in vivo actions at NMDA receptors (Lehmann, 1989). Importantly, NMDA recognition site antagonists, CPP and CGS 19755, which exert only minor actions at the above-mentioned sites (Koek et al., 1989; Lehmann, 19891, likewise elicited spontaneous tailflicks and pretreatment with NMDA inhibited their action (data not shown). Notably, the range of doses of channel blockers and NMDA recognition site antagonists that elicit spontaneous tail-flicks correspond to those for the inhibition of other NMDA receptormediated effects in vivo (Lehmann, 1989). Spontaneous tail-flicks is a response characteristic of a high efficacy activirtion of S-HT,, receptors. Since the drugs evaluated here possess only very low affinity (pKi < 5.0; Canton, H. and Millan, M.J., unpublished observations; Lehmann, 1989; Nabeshima et al., 1987) for 5-I-IT,, sites, a direct action at 5-HT,, receptors is unlikely to be involved in the induction of spontaneous tail-flicks. Further, the 5-HT,, antagonist, NAN 190, at a dose (2.5 mg/kg) sufficient to abolish spontaneous tail-flicks mediated by 5-HT,, receptors (Millan et al., 19911 failed to modify the action &f MK 801 (not

Evidently, NMDA antagonists mimic 5-HT,, agonists in eliciting spontaneous tail-flicks. In analogy. agonist and antagonist action at S-HT,, and NMDA eptors, respectively. is associated with, for example. a~xioIy~~c effects (Dunn et al.. 1989). Several studies ave revealed interactions between 5-HT and NMDA receptors at the neuronal level and an agonist action at 5-HT,, receptors can functionally antagonise NMDA receptormediated neuronal excitation (Murase et al., 1990). Whether the common functional properties of NMDA antagonists and 5-HT,, agonists, reflect such neuronal mechanisms will be of interest to determine. In conclusion. this study reveals that spontaneous tail-flicks are a component of the complex of stereotyped motor behaviours evoked by NMDA receptor site antagonists and channel blockers. Spontaneous tail-flicks may offer a novel, robust and quantitative parameter for the in vivo evaluation of drug actions at NMDA receptors. The further evaluation of mechanisms underlying NMDA antagonist-induced spontatail-flicks, as well as of the relationship between A and 5-HT,, receptor-mediated actions, should prove of great interest.

We thank V. Green for typing the manuscript and C. Grevoz and K. Bervoets for excellent technical assistance. BTCP and CGS 19755 were generous gifts of Drs. J.M. Kamenka aud J. Sturz. respectively. W. Koek is thanked for helpful discussions.

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Collingridge.