Naunyn-Schmiedeberg's Arch Pharmacol (1995) 352:394-401 © Springer-Verlag 1995

O R I G I N A L A R T I C L E

K. Fink - V. Schmitz - C. B6ing - M. G6thert

Stimulation of serotonin release in the rat brain cortex by activation of ionotropic glutamate receptors and its modulation via 2-heteroreceptors

Received: 30 March 1995/Accepted: 25 May 1995

A b s t r a c t Rat brain cortex slices were used to study (1) the release of 5-hydroxytryptamine (5-HT) induced by activation of N-methyl-D-aspartate (NMDA) or non-NMDA receptors and (2) the ~2-adrenoceptor- mediated modulation of NMDA-evoked 5-HT release.

Cortical slices were preincubated with [-3H]5-HT in the presence of the selective noradrenaline reuptake inhibitor, maprotiline (to avoid false labelling of norad- renergic axon terminals), and then superfused with solution containing the 5-HT reuptake inhibitor, 6-nit- roquipazine. In slices superfused with Mg2+-free me- dium, NMDA and L-glutamate, in a concentration- dependent manner, elicited an overflow of tritium. The NMDA-evoked tritium overflow was abolished by omission of Ca 2 + ions, almost completely suppressed by 1.2mM Mg 2+ and only partly (by about 60%) inhibited by tetrodotoxin. Dizocilpine (formerly MK- 801), an antagonist at the phencyclidine site within the NMDA-gated channel, also decreased the NMDA- evoked overflow. The competitive NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3- pentanoic acid (CGP 37849) caused a parallel shift of the NMDA concentration-response curve to the right. The NMDA-induced tritium overflow was not affected by addition of exogenous glycine but was inhibited by 5,7-dichlorokynurenic acid, an antagonist at the glycine site of the NMDA receptor. Spermidine slightly increased the NMDA-induced tritium overflow where- as arcaine, an antagonist at the polyamine site of the NMDA-receptor, caused a decrease. Ifenprodil and eliprodil, which exhibit different affinities for NMDA receptors composed of different subunits were highly potent (in the nanomolar range) in inhibiting the NMDA-evoked tritium overflow. Noradrenaline reduced, whereas the ~2-adrenoceptor antagonist

K. Fink ( ~ ) ' V. Schmitz - C. B6ing - M. G6thert Institut fiir Pharmakologie und Toxikologie der Universitiit Bonn, Reuterstrasse 2b, D-53113 Bonn, Germany

idazoxan facilitated, the NMDA-evoked overflow. Idazoxan shifted the concentration-response curve of noradrenaline to the right. In slices superfused with solution containing 1.2 mM Mg 2 +, kainic acid or (RS)- ~-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) also caused a concentration-dependent overflow of tritium, which again was not completely (by about 75 and 50%, respectively) inhibited by tet- rodotoxin. The kainate-evoked tritium overflow was inhibited by the non-NMDA receptor antagonist 6- cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not affected by CGP 37849 or arcaine. The AMPA-evoked tritium overflow was also decreased by CNQX.

It is concluded that activation of NMDA or non- NMDA receptors elicits a release of 5-HT in the rat brain cortex. The receptors are at least partly located on the serotoninergic nerve terminals. The results with ifenprodil and eliprodil are compatible with the view that the NMDA receptor involved contains the NR2B subunit. The NMDA-evoked 5-HT release is modulated by presynaptic ~2-adrenoceptors.

K e y w o r d s NMDA receptors • NR2B subunit •

Non-NMDA receptors • Presynaptic receptors - Cerebral cortex • 5-Hydroxytryptamine release " Serotonin • c~2-adrenoceptors

Introduction

Stimulation of excitatory amino acid receptors in the corpus striatum, hippocampus and cerebral cortex has been shown to induce release of various neurotransmit- ters such as acetylcholine (Lehmann and Scatton 1982; G6thert and Fink 1989), dopamine (Roberts and Sharif 1978) and noradrenaline (Vezzani et al. 1987; Jones et al. 1987; Fink et al. 1989, 1992). Both N-methyl-D- aspartate (NMDA) and non-NMDA receptors are in- volved in this effect. At least part of the NMDA recep- tors which stimulate noradrenaline release are located

395

on the noradrenergic nerve terminals themselves (Fink et al. 1990; Pittaluga and Raiteri 1990).

In contrast, we failed to observe a stimulatory effect of N M D A on the release of 5-hydroxytryptamine (5- HT) in rat brain cortex slices (Fink et al. 1989). How- ever, the conditions under which that investigation was carried out were not optimal. The 5-HT transporter in the cell membrane of the serotoninergic axon terminals was not blocked by an appropriate inhibitor. Hence, the major part of the 5-HT released was probably transported back into the axon terminals and was, therefore, not available for overflow into the superfu- sion fluid. Furthermore, the possibility had to be con- sidered that, due to the powerful stimulation of nora& renaline release by NMDA, the presynaptic ~2-ad- renoceptors on the serotoninergic nerve terminals (G6thert and Huth 1980, Frankhuyzen and Mulder 1982; Maura et al. 1982; Blier et al. 1990; Trendelen- burg et al. 1994) were strongly activated, leading to an inhibition of 5-HT release.

We therefore reinvestigated under more appropri- ate conditions whether receptors for excitatory amino acids, when activated, elicit release of [-3H]5-HT in rat brain cortex slices. For this purpose, the slices were preincubated with [-3H]5-HT in the presence of map- rotiline (an inhibitor of the noradrenaline transporter) and then superfused in the presence of 6-nitroquipazine (an inhibitor of the 5-HT transporter; Vaatstra et al. 1981, Classen et al. 1984) in order to avoid false labelling of noradrenergic nerve terminals and to block reuptake of [3H]5-HT, respectively. Since, under these conditions, both N M D A and non-NMDA receptors were found to mediate stimulation of 5-HT release, two further questions were addressed. Firstly, in view of the ability of the noncompetitive N M D A receptor antagonists, ifenprodil and eliprodil, to dis- criminate between NR2A and NR2B subunits of N M D A receptors (Williams 1993; Priestley et al. 1994; Nicolas and Carter 1994; Nicolas et al. 1994), these drugs were used to study the subunit composition of the N M D A receptors eliciting, when activated, release of 5-HT. Secondly, we investigated whether the NMDA-evoked [-3H]5-HT release is modulated via ~2-adrenoceptors.

Preliminary accounts of the present data have been given at the Joint Meeting of the Association Frangaise des Pharmacologistes and the Deutsche Gesellschaft ffir Pharmakologie und Toxikologie, Lille 1993 (Fink et al. 1993) and at the 24th Meeting of the Society for Neuroscience, Miami 1994 (G6thert et al. 1994).

Materials and methods

Cerebral cortical slices (0.3 mm thick, 3 mm diameter) from male Wistar rats weighing 200-300 g were incubated for 60 rain at 37°C in

modified Krebs-Henseleit solution containing 30riM [3H]5-HT (23.8 or 28.2 Ci/mmol) and 1 gM maprotiline. Subsequently, the slices were superfused for 60-75 rain at a rate of 0.6 ml/min with [3H]5-HT-free solution containing 1 laM 6-nitroquipazine. Tritium overflow was stimulated for 5 rain after 40 min of superfusion by adding one of the following drugs (1 mM unless stated otherwise): NMDA, L-glutamate, kainic acid or AMPA. In most of the experi- ments in which NMDA or L-glutamate was used for stimulation, the Krebs-Henseleit solution was composed as follows (mM): NaC1 118, KC1 4.8, NaHCO 3 25, KH2PO 4 1.2, CaC12 1.3, glucose 11.1, ascor- bic acid 0.06, disodium EDTA 0.03 (equilibrated with 95% 0 2 and 5% CO2). In some experiments, MgSO 4 (1.2 raM) was added to, or Ca 2 + was omitted from the solution throughout superfusion. When tritium overflow was stimulated by kainic acid or AMPA, the solution contained 1.2 mM MgSO 4 during both preincubation and superfusion. Other drugs were added to the superfusion fluid from 20 min before stimulation until the end of the experiments. The superfusate was collected in 5-min samples. At the end of the experi- ments, the slices were solubilized with Soluene R, and the radioactiv- ity of the slices and superfusate samples was determined by liquid scintillation counting.

Tritium efflux was calculated as the fraction of the tritium content in the slices at the beginning of the respective collection period (fractional rate of tritium efflux). To quantify effects of drugs on basal etttux, the fractional rate was determined in the sample collected immediately before stimulation. Basal efflux was assumed to decline linearly from the sample before to that 15-20 min after onset of stimulation. Stimulation-evoked tritium overflow was calculated by subtraction of basal from total efflux during the stimulation period and the following 15 rain and was expressed as a percentage of the tritium content in the slice at the onset of stimulation. The stimulation-induced tritium overflow was taken as a measure for the release of labelled and unlabelled 5-HT from serotoninergic neurones, and the term "5-HT release" is used in the discussion.

A sigmoidal logistic curve was fitted to concentration-response data where appropriate (Fig. 1 left-hand panel, Figs. 6 8) by iterative nonlinear regression analysis (Motulsky and Ransnas 1987); this yielded the maximal effect and the ECso (concentration producing 50% of the maximum). If less than 5 data points were available no sigmoidal curve was fitted and ICso or IC2s values (concentrations producing 50% or 25% inhibition) were determined by interpola- tion fi'om the nearest points (Figs. 2 and 5). Apparent pA 2 values of antagonists were calculated according to the formula for competi- tive reversible antagonism given by Furchgott [t972; Eq. (4)]. Re- sults are given as means _+ SEM of n experiments. For multiple comparisons with one control series, one-way ANOVA followed by Dunnett's tests was applied.

Drugs used. 5-[1, 2-3H(N)~-Hydroxytryptamine creatinine suifate ([3H]5-HT; NEN, Dreieich, Germany); (+)-noradrenaline base (Hoechst, Frankfurt/Main, Germany); N-methyl-D-aspartic acid (NMDA), tetrodotoxin (TTX), glycine sodium salt, arcaine, sper- midine, L-glutamic acid monosodium salt, kainic acid (Sigma Chemicals, St. Louis, Mo., USA); dizocilpine ( = MK-801), 6-nit- roquipazine (Research Biochemicals, Natick, Mass, USA); (RS)-e- amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), 5,7-dich- lorokynurenic acid (Tocris Cookson, Bristol, UK); DL-(E)-2-amino- 4-methyl-5-phosphono-3-pentanoic acid (CGP 37849; Ciba Geigy, Basel, Switzerland); idazoxan (Reckitt & Colman, Hull, UK); ifen- prodil, eliprodil (Synth61abo Recherche, Bagneux, France); maproti- line hydrochloride (Ciba-Geigy, Wehr, Germany). Stock solutions were prepared with dimethyl sulphoxide (CNQX, 5,7-dichloro- kynurenic acid, eliprodil), HC1 0.01 M (noradrenaline), ascorbic acid 5.7 mM ([-3HI 5-HT) or demineralized water (other drugs). Although none of the solvents, applied alone, had any effect on basal or stimulation-evoked tritium overflow, controls were carried out in the presence of the highest solvent concentration used in the respect- ive experiment.

396

Results

Basal tritium efflux

In control experiments, basal tritium efflux gradually decreased with time. In representative series of control experiments in slices superfused in the absence or pres- ence of Mg 2 ÷ (controls shown in the right-hand panels of Figs. 1 and 8, respectively), the basal tritium efflux in the collection period before stimulation (35th to 40th rain of superfusion) amounted to 0.11 _+ 0.01 and 0.15 _+ 0.01 nCi • min- ~, respectively (corresponding to fractional rates of 0.0092 _+ 0.0005 and 0.0084 _+ 0.0003 min- a, respectively). Basal tritium efflux was not modi- fied by any of the ionic conditions or drug concentra- tions investigated in the present study.

NMDA- and L-glutamate evoked tritium overflow

Unless stated otherwise, these experiments were carried out in slices superfused with MgZ+-free medium. N M D A (Fig. 1, left-hand panel) caused an overflow of tritium in a concentration-dependent manner. The maximum response to N M D A was an overflow of 1.27 +0 .13% of tissue tritium (corresponding to

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Fig. 1 N-Methyl-D-aspartate (NMDA) -evoked tritium overflow from rat brain cortex slices and its modification by DL-(E)-2-amino- 4-methyl-5-phosphono-3-pentanoic acid (CGP 37849), tetrodotoxin (TTX), Mg 2+ ions or omission of Ca 2+. The slices were prein- cubated with [3H]5-HT (in the presence of 1 btM maprotiline) and, unless stated otherwise, superfused with Mg 2+-free medium con- taining 1 ~tM 6-nitroquipazine. N M D A was added for 5 rain after 40 rain of superfusion. Left-hand panel. Rightward shift of the con- centration-response curve for N M D A by 30 gM CGP 37849. Con- trols, • 0 ; experiments in the presence of CGP 37849 from the 20th min of superfusion onward, ©-©. Means _+ SEM of 6-18 experi- ments. • *P < 0.05 (compared to corresponding response to N M D A in the absence of CGP 37849). Right-hand panel. Effects of TTX and modification of the ionic conditions on the N M D A (1 mM)-evoked tritium overflow (CON, controls). Mg (1.2 raM) or TTX (0.3 gM) was added to, or Ca 2+ was omitted from, the medium from the 20th min of superfusion onward. Ordinate, overflow expressed as per- centage of that in controls in which superfusion medium was not changed. Means _+ SEM of 7-8 experiments. **P < 0.01 (compared to controls)

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Fig. 2 Effect of CGP 37849 and dizocilpine on the NMDA(1 mM)- evoked tritium overflow from cortex slices superfused with Mg 2+- free medium. Separate control values in the absence of drugs were determined for the two series of experiments ("ID" and "'ll"). For details, see legend to Fig. 1. CGP 37849 or dizocilpine was added to the medium from the 20th min of superfusion onward. Ordinate, overflow expressed as percentage of that in corresponding controls without drugs. Means + SEM of 5-12 experiments. *P < 0.05 (com- pared to controls)

0.18_+ 0.02 nCi) and the ECs0 was 17.4gM. The tritium overflow evoked by a standard concentration of N M D A (1 mM) was abolished by omission of Ca 2÷, almost completely suppressed by 1.2 mM Mg 2÷, but only inhibited by 57% by 0.3 gM tetrodotoxin (Fig. 1, right-hand panel).

L-glutamate at 1 and 3 mM also stimulated tritium overflow (0.53 +_ 0.04 and 0.66 _+ 0.09% of tissue tritium; n = 5-8 experiments), whereas L-glutamate concentrations of 100 gM and below did not produce a consistent stimulation. In view of the low potency of L-glutamate under the present conditions, a full con- centration-response curve was not determined.

Dizocilpine, an antagonist at the phencyclidine site of the NMDA-gated channel, and CGP 37849, a com- petitive N M D A receptor antagonist, inhibited the NMDA-evoked tritium overflow in a concentration- dependent manner. The ICs0 values were 7 nM and 1.5 gM, respectively (Fig. 2). The concentration-re- sponse curve for N M D A was shifted to the right by 30 #M CGP 37849 (apparent pAz value 5.70; Fig. 1, left-hand panel).

Glycine 10 or 100gM did not influence the NMDA-evoked tritium overflow, whereas 5,7-dich- lorokynurenic acid, an antagonist at the glycine site, caused a concentration-dependent (and at 100 ~tM al- most complete) inhibition (Fig. 3). Glycine 100 gM overcame the inhibition produced by 5,7-dich- lorokynurenic acid 100gM (Fig. 3). The NMDA- evoked overflow was increased by the polyamine spermidine (100 gM) but concentration-dependently reduced by arcaine, an antagonist at the polyamine site of the N M D A receptor (Fig. 4). Arcaine 100 gM and spermidine 100 ~M, when applied simultaneously, nei- ther decreased nor increased the NMDA-evoked tritium overflow. Ifenprodil and eliprodil (see Introduction and

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Fig. 4 Effect of spermidine, arcaine or both drugs on the N M D A (1 mM)-evoked tritium overflow from cortex slices superfused with Mg 2 +-free medium. For details, see legend to Fig. 1. Spermidine, arcaine or both were added to the medium from the 20th min of superfusion onward. Ordinate, overflow expressed as percentage of that in controls without drugs. Means -+ SEM of 7-15 experiments. *P < 0.05 (compared to controls)

Discussion) produced a concentration-dependent inhi- bition of the NMDA-induced overflow (Fig. 5). The concentration-response curve for ifenprodil appeared to level off at 1 btM. The maximum inhibition was about 50% and the IC2s value about 13 nM.

In order to examine whether the NMDA-evoked tritium overflow was modulated by c~2-adrenoceptors, the influence of noradrenaline and the ct2-adrenoceptor antagonist idazoxan were examined. Noradrenaline de- creased, whereas idazoxan increased the evoked over- flow. Idazoxan 1 btM shifted the concentration-re- sponse curve of noradrenaline to the right (apparent pA2 value 7.34; Fig. 6).

Kainic acid-evoked tritium overflow

The remainder of the experiments were carried out on slices superfused with buffer containing 1.2 mM Mg a ÷.

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Fig. 5 Effect of ifenprodil or eliprodil on the NMDA (1 mM)- evoked tritium overflow from cortex slices superfused with Mg 2+- free medium. Separate control values in the absence of drugs were determined for the two series of experiments ("Q" and "(7)"). For details, see legend to Fig. 1, Ifenprodil or eliprodil was added to the medium from 20th min of superfusion onward. Ordinate, overflow expressed as percentage of that in corresponding controls without drugs. Means _+ SEM of 6-12 experiments. *P < 0.05 (compared to controls)

Under these conditions, kainic acid concentration-de- pendently stimulated tritium overflow. The maximum effect was 0.36 _+ 0.07% of tissue tritium (correspond- ing to 0.07_+ 0.01 nCi) and the ECs0 was 5.4btM (Fig. 7, left-hand panel). Tetrodotoxin reduced the overflow of tritium induced by a standard concentra- tion of 1 mM kainic acid by only 73% (Fig. 7, right- hand panel). CNQX, an antagonist of the non-NMDA type of glutamate receptor inhibited the kainic-acid evoked tritium overflow, whereas CGP 37849 or ar- caine had no effect (Fig. 7).

AMPA-evoked tritium overflow

Again in the presence of 1.2 mM Mg 2+, AMPA con- centration-dependently stimulated tritium overflow. The maximum response to AMPA was 0.41 _+ 0.06% of tissue tritium (corresponding to 0.10 + 0.02 nCi) and the ECs0 was 219 gM (Fig. 8, left-hand panel). Again 0.3 gM tetrodotoxin produced an inhibition by only 52% (Fig. 8, right-hand panel). A similar degree of inhibition was found with 30 btM CNQX (Fig. 8, right- hand panel).

Discussion

In contrast to our previous investigation (Fink et al. 1989), the present study revealed that NMDA elicits a release of 5-HT from serotoninergic neurones. A stimulatory effect was also observed with other agon- ists at excitatory amino acid receptors, namely kainic acid, AMPA and the endogenous agonist glutamate. The disclosure of an excitatory effect of NMDA on

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5-HT release is probably due to an improvement of the experimental conditions in the present study: the dura- tion of stimulation was extended from 2 to 5 min, and the reuptake of released 5-HT was blocked with 6- nitroquipazine. The possibility that the tritiated 5-HT might have been taken up and released by noradrener- gic instead of serotoninergic neurones in the slices was excluded not only by the low [-3Hl5-HT concentration used for preincubation but also by the presence of maprotiline, an inhibitor of the noradrenaline trans- porter in the cell membrane of the noradrenergic neur- ones.

The NMDA-evoked 5-HT release was caused by activation of NMDA receptors, for the following reasons.

(1) The Ca 2+-dependence of release, reflecting at least in part the influx of Ca 2 + ions via the NMDA receptor channel (see Fink and G6thert 1993), and the sensitivity to physiological Mg 2÷ concentrations (Mayer et al. 1984; Nowak et al. 1984) are characteristic features of responses evoked by NMDA receptor stimulation.

(2) CGP 37849, a competitive antagonist at the NMDA recognition site (Fagg et al. 1990), shifted the NMDA concentration-response curve to the right. The apparent pA2 value of CGP 37849 and its ICso value against a standard concentration of NMDA are com- patible with previously determined affinity or potency values of CGP 37849 at NMDA receptors in other models (Fagg et al. 1990; G6thert and Fink 1991; Fink and G6thert 1992).

(3) Dizocilpine, which blocks the NMDA-gated ca- tion channel by acting at the allosteric modulatory phencyclidine site within the channel (Wong et al. 1986), inhibited the NMDA-evoked 5-HT release with a potency similar to its antagonism against NMDA- evoked noradrenaline release (Fink et al. 1989).

(4) 5,7-Dichlorokynurenic acid, an antagonist at the modulatory glycine site of the NMDA receptor (Kemp et al. 1988; Huettner 1991), strongly inhibited the NMDA-evoked 5-HT release, whereas exogenous glycine even at high concentrations was ineffective. These findings are compatible with the presence of sufficient endogenous glycine to saturate the glycine sites. The latter have to be activated as a prerequisite for the manifestation of NMDA-evoked responses (Keith et al. 1989; Fink et al. 1990). Although exogenous glycine did not amplify the response to NMDA, it abolished the inhibitory effect of 5,7-dich- lorokynurenic acid, which is compatible with the hy- pothesis that both drugs act at the same site.

(5) Arcaine, an antagonist at the modulatory poly- amine site of the NMDA receptor (Williams et al. 1991), inhibited the NMDA-evoked 5-HT release whereas the polyamine spermidine caused an increase. No change in NMDA-evoked release was obtained when arcaine and spermidine were combined. These findings suggest that under the present conditions the polyamine sites were not fully activated by endogenous polyamines. Ifenprodil and eliprodil (former designation: SL 82.0715) have also been suggested to block the poly- amine site (Carter et al. 1990), although other mecha- nisms have also been considered for ifenprodil (Leg- endre and Westbrook 1991; Williams 1993). Irrespect- ive of the mechanism, it should be noted that both drugs inhibited NMDA-evoked 5-HT release.

The NMDA receptor probably is a pentamer com- posed of different subunits (for review see Seeburg 1993). Due to the multiple possibilities of subunit as- sembly, many NMDA receptor subtypes with different sensitivity to certain drugs have been identified. Ifen- prodil and eliprodil are capable of discriminating be- tween NMDA receptors composed of different subunits (Williams 1993; Cudennec et al. 1994). This property has mainly been studied with ifenprodil. For example,

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Fig. 7 Kainic acid - evoked tritium overflow from rat brain cortex slices and its interaction with TTX or potential antagonists. The slices were preincubated with [3H]5-HT and superfused with me- dium containing 1.2 mM Mg 2 +. Kainic acid was added for 5 rain after 40 rain of superfusion. For details, see legend to Fig. 1. Left- hand panel. Concentration-response curve for kainic acid. Means _+ SEM of 7-14 experiments. Right-hand panel. Effect of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), CGP 37849, arcaine or TTX (from the 20th min of superfusion onward) on the kainic acid (1 mM)-evoked tritium overflow (CON, controls). Ordinate, over- flow expressed as percentage of that in controls without drugs. Means _+ SEM of 7 13 experiments. *P < 0.05 (compared to con- trols)

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in studies on recombinant heteromeric NMDA rece- ptors expressed in Xenopus oocytes, ifenprodil inhib- ited NMDA-induced currents with high potency (ICso < 1 pM) at NR1A/NR2B receptors, but with about

400 times lower potency at NR1A/NR2A receptors (Williams 1993). In rat brain, populations of NMDA receptors exhibiting high and low affinities for ifen- prodil have been identified (Williams et al. 1993); in

adult animals, each population represented about 50% of NMDA receptors. High affinity of native NMDA receptors for ifenprodil has been suggested to indicate that the latter contain the NR2B subunit, whereas low affinity for ifenprodil points to the absence of this subunit but is compatible with occurrence of the NR2A subunit (Williams 1993).

In the present study, ifenprodil inhibited the NMDA-evoked 5-HT release at nanomolar concentra- tions. The concentration-response curve appeared to level off at about 1 pM ifenprodil, and a maximum of about 50% inhibition. This result is compatible with the view that about 50% of the NMDA receptors eliciting release of 5-HT contain the NR2B subunit. A potential second phase of the concentration-response curve of ifenprodil, presumably representing an interac- tion with NMDA receptors containing the NR2A subunit, could not be investigated since ifenprodil (and eliprodil) concentrations above 1 pM produced sub- stantial increases in basal tritium efflux.

The potency of NMDA in eliciting release of 5-HT was rather low, but in the same range as previously found for stimulation of acetylcholine release from stri- atal slices (Lehmann and Scatton 1982) and noradrena- line release from hippocampal (Vezzani et al. 1987) or cortical slices (Fink et al. 1989). The same holds true for the potency of glutamate in stimulating 5-HT release, which in the absence of Mg 2+ ions probably acts at least partly through NMDA receptors. The lower po- tency of glutamate than NMDA, in spite of the higher affinity of glutamate for NMDA receptors (Olvermann et al. 1986; Murphy et al. 1987), may be due to uptake into glutamatergic neurones and glial cells by the speci- fic glutamate transporters in the cell membrane.

Although under the present improved experimental conditions NMDA did induce 5-HT release, the release was still considerably lower than that of noradrenaline (compare Fink et al. 1989). This is partly due to the stimulation of inhibitory presynaptic ~2-heterorecep- tors (for references, see Introduction) on the serotonin- ergic nerve terminals by the noradrenaline released in response to NMDA: blockade of the ~a-adrenoceptors with idazoxan approximately doubled the NMDA- evoked 5-HT release. An ~2-adrenoceptor-mediated modulation of 5-HT release is supported by the ability

400

of exogenous noradrenaline to inhibit the N M D A - evoked 5-HT release, in a manner susceptible to antag- onism by idazoxan. Similarly, the release of noradrena- line evoked by N M D A has previously been shown to be inhibited by activation of presynaptic receptors, in particular ~2-autoreceptors (Fink and GiSthert 1993) and histamine U 3 heteroreceptors (Fink et al. 1994).

Tetrodotoxin only partly inhibited the N M D A - evoked 5-HT release. This finding indicates that a sub- stantial part of the N M D A receptors mediating stimu- lation of 5-HT release is located not on interneurones but on the serotoninergic nerve terminals themselves.

The ability of kainic acid and A M P A to elicit 5-HT release in cortical slices is compatible with the involve- ment of n o n - N M D A receptors. Three findings exclude that this effect is due to N M D A receptor activation. Firstly, the st imulatory effect of kainic acid and A M P A was observed in the presence of Mg 2+ ions. Secondly, C G P 37849 and arcaine, i.e. a competitive and a non- competitive N M D A receptor antagonist, failed to counteract the kainic acid-evoked 5-HT release. Third- ly, the n o n - N M D A receptor antagonist, C N Q X (Wat- kins et al. 1990), inhibited the responses to kainic acid and AMPA. The n o n - N M D A receptors also seem to be partly located on the serotoninergic nerve terminals, since the effect was partly resistant to TTX. The amount of 5-HT released in response to kainic acid and A M P A was even lower than that evoked by N M D A . Possibly the receptors eliciting 5-HT release largely are desensitized under the present conditions. Rapid desen- sitization is a typical feature of ionotropic glutamate receptors (for review see Zorumski and Thio 1992) and may also be a reason for the low quantities of 5-HT released in response to N M D A .

In conclusion, the present study showed for the first time that N M D A receptors containing the NR2B subunit as well as n o n - N M D A receptors, when ac- tivated, elicit release of 5-HT in rat brain cortex. The receptors are in part located on the serotoninergic nerve terminals themselves. The N M D A - e v o k e d 5-HT release is inhibited by endogenous noradrenaline via presynaptic c~2-adrenoceptors.

Aeknowledgements We wish to thank Mrs. H. Burisch and Mrs. I. Konrad for skilled technical assistance, Mrs. R. Milz and Mrs. S. Rudat for typing the manuscript, Dr. Waldmeier from Ciba Geigy, Basel, for the generous gift of CGP 37849 and I. Alexander from Synthelabo Recherche, Bagneux, for generous supply of ifenprodil and eliprodil. This study was supported by the Deutsche Forschungsgemeinschaft.

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