Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2007 Jun 25;151(7):1087–1094. doi: 10.1038/sj.bjp.0707280

Functional interactions between presynaptic NMDA receptors and metabotropic glutamate receptors co-expressed on rat and human noradrenergic terminals

E Luccini 1, V Musante 1, E Neri 1, M Brambilla Bas 2, P Severi 2, M Raiteri 1,3, A Pittaluga 1,3,*
PMCID: PMC2042940  PMID: 17592518

Abstract

Background and purpose:

Electrophysiological studies described potentiation of NMDA receptor function by metabotropic glutamate receptors (mGluRs) of group I occurring postsynaptically. Since release-enhancing NMDA receptors exist on noradrenergic terminals and group I mGluRs have recently been identified on these nerve endings, we have investigated if NMDA receptor-mGluR interactions also can occur at the presynaptic level.

Experimental approach:

Rat hippocampus and human neocortex synaptosomes were labelled with [3H]noradrenaline and superfused with mGluR agonists and antagonists. NMDA-evoked [3H]noradrenaline release was produced by removal of external Mg2+ or by simultaneous application of NMDA and AMPA in Mg2+-containing solutions.

Key results:

The mGluR1/5 agonist 3,5-DHPG, inactive on its own, potentiated both the release of [3H]noradrenaline elicited by AMPA/NMDA/glycine and that evoked by NMDA/glycine following Mg2+ removal. The effect of 3,5-DHPG on the AMPA/NMDA/glycine-induced release was insensitive to the mGluR1 antagonist CPCCOEt, but it was abolished by the mGluR5 antagonist MPEP; moreover, it was potentiated by the mGluR5 positive allosteric modulator DFB. When NMDA receptors were activated by Mg2+ removal, both mGluR5 and mGluR1 contributed to the evoked release, the mGluR-mediated release being blocked only by CPCCOEt and MPEP in combination. Experiments with human neocortex synaptosomes show NMDA receptor-mGluR interactions qualitatively similar to those observed in rodents.

Conclusions and implications:

Group I mGluRs, both of the mGluR1 and mGluR5 subtypes, co-localize with NMDA receptors on noradrenergic terminals of rat hippocampus and human neocortex. Depending on the mode of activation, NMDA receptors exert differential permissive roles on the activation of presynaptic mGluR1 and mGluR5.

Keywords: mGluR1, mGluR5, NMDA receptor, AMPA receptor, noradrenaline release, synaptosomes, rat brain, human brain

Introduction

Glutamate, the principal excitatory neurotransmitter in the central nervous system (CNS), exerts its actions through activation of a receptor family that includes ionotropic and metabotropic members. Ionotropic receptors are divided into N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors, which exist as multiple heteromers formed by co-assembly of different subunits (Dingledine et al., 1999). Metabotropic glutamate receptors (mGluRs) also are heterogeneous and constitute a subfamily of eight subtypes (mGluR1–mGluR8) subdivided into three groups (I, II and III) on the basis of structural homology, pharmacological profile and associated transduction pathways (Conn and Pin, 1997). There is ample evidence that glutamate receptors exhibit both post- and presynaptic localization; at the presynaptic level, they generally mediate control of neurotransmitter release (see, for reviews, MacDermott et al., 1999; Cartmell and Schoepp, 2000; Raiteri, 2006). The most extensively studied of the presynaptic mGluRs are those of group I that exist as release-enhancing autoreceptors on glutamatergic axon terminals (Herrero et al., 1992; Rodriguez-Moreno et al., 1998; Reid et al., 1999; Thomas et al., 2000; Fazal et al., 2003; Rodriguez et al., 2005; see for a review, Cartmell and Schoepp, 2000), where they mediate enhancement of glutamate exocytosis. Only recently, release-enhancing group I mGluRs were identified on isolated cholinergic (Marti et al., 2001; Feligioni et al., 2003) and noradrenergic (Longordo et al., 2006; Parodi et al., 2006) nerve endings. In all these types of nerve endings mGluR-mediated release could be observed only when depolarizing stimuli including 4-aminopyridine (Herrero et al., 1992; Reid et al., 1999), veratrine or (−) nicotine (Parodi et al., 2006) were co-applied. However, whether or not presynaptic group I mGluRs can be made functional under more physiological conditions by glutamate acting concomitantly at ionotropic receptors coexisting on the same nerve ending, has not been investigated.

Reciprocal interactions between mGluRs of group I and NMDA receptors have been the object of several investigations carried out in recombinant and in native systems, the latter involving receptors localized postsynaptically (Bleakman et al., 1992; Bruno et al., 1995; Doherty et al., 1997; Pisani et al., 1997; Alagarsamy et al., 1999, 2005; Skeberdis et al., 2001; Benquet et al., 2002; Heidinger et al., 2002). In contrast, very little is known about functional interactions between presynaptic ionotropic and metabotropic glutamate autoreceptors coexpressed on glutamatergic terminals and, particularly, on the possible interactions between presynaptic ionotropic and metabotropic glutamate heteroreceptors that may coexist on non-glutamatergic nerve endings.

Since presynaptic NMDA receptors were found on rat hippocampal (Pittaluga and Raiteri, 1990; Raiteri et al., 1992) and rat and human neocortical (Fink et al., 1990, 1992, Pittaluga et al., 1996) noradrenergic nerve terminals, we have investigated if presynaptic NMDA receptors and group I mGluRs can interact to modulate noradrenaline release from rat and human noradrenergic nerve endings. Our results show that both mGluR1 and mGluR5 can mediate enhancement of noradrenaline release provided that coexisting NMDA receptors are activated. Moreover, depending on the mode of NMDA receptor activation, only mGluR5 or both mGluR5 and mGluR1 can exhibit releasing activity.

Methods

Rat brain tissue samples

Adult male rats (Sprague–Dawley; 200–250 g) were housed at constant temperature (22±1°C) and relative humidity (50%) under a regular light–dark schedule (light on from 0700 to 1900 h). Food and water were freely available. The animals were killed by decapitation and hippocampi were rapidly dissected at 0–4°C. The experimental procedures were approved by the Ethical Committee of the Pharmacology and Toxicology Section, Department of Experimental Medicine, University of Genoa, in accordance with the European legislation (European Communities Council Directive of 24 November 1986, 86/609/EEC) and with the NIH Guide for the Care and Use of Laboratory Animals.

Human brain tissue samples

Samples of human cerebral cortex were obtained from informed and consenting patients undergoing neurosurgery, each on a different day, to reach deeply seated tumours. The samples represented parts of temporal (n=7) lobes obtained from two women and five men (aged 30–70 years). Immediately after removal, the tissue was placed in a physiological salt solution at 2–4°C and synaptosomes were prepared within 90 min. The experimental procedures were approved by the Ethical Committee of the Pharmacology and Toxicology Section, Department of Experimental Medicine, University of Genoa.

Preparation of synaptosomes

Crude synaptosomes were prepared according to Raiteri et al. (1984). Briefly, brain tissues were homogenized in 40 volumes of 0.32 M sucrose, buffered at pH 7.4 with phosphate (final concentration 0.01 M). The homogenate was first centrifuged at 1000 g for 5 min, to remove nuclei and cellular debris, and synaptosomes were then isolated by centrifugation at 13 000 g for 20 min. The synaptosomal pellet was resuspended in a physiological medium having the following composition: 125 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.2 mM CaCl2, 1 mM NaH2PO4, 22 mM NaHCO3 and 10 mM glucose (pH 7.2–7.4) (gassed with 95% O2 and 5% CO2).

Release experiments

Synaptosomes were selectively labelled with [3H]noradrenaline (final concentration: 30–50 nM) in the presence of the serotonin transporter blocker 6-nitroquipazine (0.1 μM) and the dopamine transporter blocker GBR 12909 (0.1 μM) to avoid false labelling of serotonergic and dopaminergic nerve terminals, respectively. Incubation was performed at 37°C, for 15 min, in a rotary water bath and in an atmosphere of 95% O2 and 5% CO2.

After the labelling period, release experiments were performed in a multichamber superfusion system (Ugo Basile, Comerio, Varese, Italy). Identical portions of the synaptosomal suspensions were layered on microporous filters at the bottom of up to 24 parallel superfusion chambers maintained at 37°C (Raiteri and Raiteri, 2000). The amount of synaptosomes retained on each filter (2.5 cm diameter) had been calculated to constitute less than a monolayer (Raiteri et al., 1986). Synaptosomes were superfused at 0.5 ml min−1 with standard physiological solution aerated with 95% O2 and 5% CO2, at 37°C. The system was first equilibrated during 38 min of superfusion; subsequently, eight consecutive 1-min fractions were collected. Synaptosomes were exposed to agonists at the end of the first fraction collected (t=39 min) until the end of the superfusion, while antagonists were added 8 min before agonists. When indicated, the medium was replaced, at t=20 min, with a medium from which Mg2+ ions were omitted, to permit NMDA receptor activation. The radioactivity in the collected fractions and in superfused synaptosomes was counted.

Calculation and statistics

The amount of radioactivity released into each superfusate fraction was expressed as a percentage of the total synaptosomal tritium content at the start of the fraction collected (fractional efflux). Drug effects were expressed as percent increase over basal release and were calculated as the ratio between the percentage of tritium released into the fraction, where the maximal releasing effect was observed and that in the first fraction collected; this ratio was compared with the corresponding ratio obtained under control conditions (no drug added). Analysis of variance was performed by ANOVA followed by the Newman Keuls multiple comparison test. Data were considered significant at P<0.05 at least. Appropriate controls with antagonists and inhibitors were always run in parallel.

Chemicals

1-[7,8–3H]noradrenaline (specific activity 39 Ci mmol−1) was from Amersham Radiochemical Centre (Amersham, UK). NMDA, (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG), AMPA, 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP), 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), 3,3′difluorobenzaldazine (DFB) were obtained from Tocris Cookson (Bristol, UK). Glycine was purchased from Sigma Chemical Co (St Louis, MO, USA). The following compounds were gifts: 6-nitroquipazine maleate from Duphar (Amsterdam, The Netherlands), GBR 12909 from Gist-Brocades (Delft, The Netherlands), clonidine from Boehringer Ingelheim (Florence, Italy).

Results

Interactions between ionotropic and mGluR1s mediating noradrenaline release in rat hippocampus

AMPA receptors and NMDA receptors mediating noradrenaline exocytosis are present on noradrenergic terminals (Fink et al., 1990; Pittaluga and Raiteri, 1990; Wang et al., 1991; Malva et al., 1994; Pittaluga et al., 2006). The possible interactions between presynaptic mGluRs of group I and presynaptic ionotropic glutamate receptors were first studied by monitoring [3H]noradrenaline release from rat hippocampal synaptosomes during exposure to the group I mGluR agonist 3,5-DHPG and to agonists at AMPA receptors and NMDA receptors.

Activation of presynaptic AMPA receptors by 100 μM AMPA elicited [3H]noradrenaline release (40.3±4.3%; n=4; expressed as percent increase over basal). When 100 μM 3,5-DHPG was coapplied with AMPA, the release of the 3H-catecholamine remained unchanged (42.3±6.4%; n=4).

We then investigated the possibility that activation of NMDA receptors affected the function of presynaptic group I mGluRs. Activation of NMDA receptors requires removal of Mg2+ ions from the receptor channel, which can be achieved by two main methods: (i) by omitting external Mg2+ from the solutions (as it is generally performed experimentally); (ii) by concomitant activation of coexisting AMPA and NMDA receptors (this probably occurs physiologically), in the presence of external Mg2+.

AMPA receptors and NMDA receptors, originally identified on noradrenergic nerve terminals, were subsequently found to coexist on the same terminals where activation of AMPA receptors permits activation of NMDA receptors in the presence of physiological concentrations of extracellular Mg2+ (Raiteri et al., 1992). Thus, in our experimental setup, functional interactions between NMDA receptors and mGluRs can be investigated by monitoring [3H]noradrenaline release from noradrenergic terminals on which NMDA receptors are activated either by AMPA/NMDA/glycine in Mg2+-containing solutions or by NMDA/glycine following Mg2+ removal.

The release of [3H]noradrenaline evoked by AMPA/NMDA/glycine in Mg2+ (1.2 mM)-containing medium was significantly potentiated by adding 100 μM 3,5-DHPG (Figure 1a; F(3, 11)=6.89; P<0.01). This effect could be observed with 0.1 μM glycine, but not with 1 μM of the NMDA receptor coagonist. As illustrated in Figure 1b, addition to the Mg2+-free medium of 3,5-DHPG (10–100 μM) concentration-dependently potentiated the release of [3H]noradrenaline provoked by NMDA/glycine. The effects observed in Mg2+-free conditions clearly differ from those in the presence of 1.2 mM Mg2+. In particular: (i) the potentiation of the NMDA/glycine-evoked release was already significant when 3,5-DHPG was added at 10 μM; moreover, the potentiation observed in Mg2+-free solution with 100 μM of the mGluR1/5 agonist was much higher than that in Mg2+-containing medium; (ii) the potentiation of the NMDA/glycine-evoked release produced by 100 μM 3,5-DHPG occurred in the presence of both 0.1 and 1 μM glycine (Figure 1b; F(5, 25)=7.17; P<0.01 and P<0.05, respectively).

Figure 1.

Figure 1

Open in a new tab

Effects of 3,5-DHPG on the NMDA-evoked release of [3H]noradrenaline from rat hippocampal nerve endings. Synaptosomes were exposed in superfusion to 3,5-DHPG, AMPA and NMDA plus glycine at the end of the first superfusion fraction collected (t=39 min). (a) Superfusion in the presence of Mg2+ ions. (b) Superfusion in the absence of Mg2+ ions. Results are expressed as percent increase over basal release; in each panel, data are means±s.e.m. of three to six experiments run in triplicate (three superfusion chambers for each experimental condition). *P<0.05 versus respective control; **P<0.01 versus respective control. Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate; 3,5-DHPG, (RS)-3,5-dihydroxyphenylglycine; NMDA, N-methyl-D-aspartate.

Pharmacology of the mGluRs involved

The effects of AMPA/NMDA/glycine plus 3,5-DHPG in Mg2+-containing solutions and of NMDA/glycine plus 3,5-DHPG in Mg2+-free solutions were tested against selective group I mGluR antagonists and the results are shown in Figure 2. In the presence of 1.2 mM of Mg2+, the release of [3H]noradrenaline provoked by AMPA/NMDA/glycine plus 3,5-DHPG was insensitive to the selective mGluR1 antagonist CPCCOEt (5 μM); in contrast, the component of the evoked release mediated by 3,5-DHPG was abolished by 1 μM of the selective mGluR5 antagonist MPEP (Figure 2a, F(3, 8)=9.42; P<0.05). In Mg2+-free medium, neither 5 μM CPCCOEt nor 1 μM MPEP was able to inhibit significantly the release evoked by NMDA/glycine plus 3,5-DHPG. However, the component of the evoked release produced by 3,5-DHPG was attenuated when the mGluR1 and the mGluR5 antagonists were added in combination (Figure 2b; F(4, 25)=10.42; P<0.001). Notably, CPCCOEt and MPEP failed to affect significantly the release of [3H]noradrenaline induced by AMPA/NMDA/glycine in presence of Mg2+ (Figure 3a; F(2, 9)=1.079; n.s.) or that evoked by NMDA/glycine in absence of Mg2+ (Figure 3b, F(3,12) =0.93; n.s.). At the concentration applied, both antagonists did not affect the spontaneous release of noradrenaline (data not shown, but see also Longordo et al., 2006; Parodi et al., 2006).

Figure 2.

Figure 2

Open in a new tab

Effects of group I mGluR antagonists on the 3,5-DHPG potentiation of the [3H]noradrenaline release evoked by NMDA receptor activation in Mg2+-containing or Mg2+-free medium. Agonists were added at t=39 min of superfusion. Antagonists were present from 8 min before agonists. (a) Superfusion in the presence of Mg2+ ions. (b) Superfusion in the absence of Mg2+ ions. Results are expressed as percent increase over basal release; data are means±s.e.m. of three to six experiments run in triplicate. *P<0.05 versus AMPA/NMDA/glycine; **P<0.001 versus NMDA/glycine; #P<0.05 versus AMPA/NMDA/glycine/3,5-DHPG; §P<0.05 versus NMDA/glycine/3,5-DHPG. Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate; 3,5-DHPG, (RS)-3,5-dihydroxyphenylglycine; mGluR, metabotropic glutamate receptor; NMDA, N-methyl-D-aspartate.

Figure 3.

Figure 3

Open in a new tab

Effects of group I mGluR antagonists on the [3H]noradrenaline release evoked by NMDA receptor activation in Mg2+-containing or Mg2+-free medium. Agonists were added at t=39 min of superfusion. Antagonists were present from 8 min before agonists. (a) Superfusion in the presence of Mg2+ ions. (b) Superfusion in the absence of Mg2+ ions. Results are expressed as percent increase over basal release; data are means±s.e.m. of four experiments run in triplicate. Abbreviations: mGluR, metabotropic glutamate receptor; NMDA, N-methyl-D-aspartate.

To confirm the involvement of mGluR5 in the AMPA/NMDA/glycine-evoked release of [3H]noradrenaline, the effect of DFB, a molecule reported to act as a selective positive allosteric modulator of human and rat recombinant mGluR5 (O'Brien et al., 2003), was tested. DFB was added together with a concentration of 3,5-DHPG (30 μM), that did not enhance significantly the release of [3H]noradrenaline evoked by AMPA/NMDA/glycine (F(4,10)=90.35; n.s.; n=3). Figure 4 shows that, in the presence of 10 μM DFB, 30 μM 3,5-DHPG significantly potentiated the [3H]noradrenaline release evoked by AMPA/NMDA/glycine (F(4, 10)=90.35; P<0.01; n=3). DFB (10 μM) had no effect, on its own, either on the basal or on the AMPA/NMDA/glycine-evoked release of [3H] noradrenaline (Figure 4, F(4,10)=90.35; n.s.; n=3). Concentrations of DFB higher than 10 μM could not be tested because of the low solubility of the compound.

Figure 4.

Figure 4

Open in a new tab

Effects of DFB on the release of [3H]noradrenaline induced by AMPA/NMDA in absence or in presence of 3,5-DHPG from rat hippocampal nerve endings. Superfusion was carried out in the presence of Mg2+ ions. Synaptosomes were exposed to 3,5-DHPG, AMPA and NMDA at the end of the first fraction collected. DFB was added concomitantly with agonists. Results are expressed as percent increase over basal release; data are means±s.e.m. of three experiments run in triplicate. *P<0.01 versus 100 μM AMPA+100 μM NMDA+0.1 glycine+30 μM 3,5-DHPG. Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate; DFB, 3′difluorobenzaldazine; 3,5-DHPG, (RS)-3,5-dihydroxyphenylglycine; NMDA, N-methyl-D-aspartate.

Studies with human neocortex noradrenergic nerve endings

Human neocortical noradrenergic nerve endings possess release-enhancing NMDA receptors (Fink et al., 1992; Pittaluga et al., 1996), whereas the presence of AMPA receptors has not been investigated. NMDA receptors located on human cortical noradrenergic terminals were found to be less sensitive to agonists than the rodent ones, probably owing to a better viability of rat synaptosomes when compared to human ones (see also Longordo et al., 2006). As illustrated in Figure 5a, addition of 100 μM 3,5-DHPG, inactive on its own, more than doubled the release of [3H]noradrenaline evoked by NMDA/glycine (3 μM) in Mg2+-free solution (F(7, 26)=45.9; P<0.01 and P<0.001, respectively). The figure also shows that 3,5-DHPG was still able to significantly potentiate (Figure 5a; F(7, 26)=45.9; P<0.001) the release of [3H]noradrenaline elicited by NMDA plus 10 μM glycine, which was almost threefold higher than that observed with 3 μM of the NMDA receptor coagonist. Neither the mGluR1 antagonist CPCCOEt nor the mGluR5 antagonist MPEP affected significantly the release of [3H]noradrenaline evoked by NMDA/glycine (10 μM) plus 3,5-DHPG. However, the mGluR-dependent component of the evoked [3H]noradrenaline release was attenuated when the two antagonists were added in combination (Figure 5a; F(7, 26)=45.9; P<0.001). At the concentrations used, the antagonists failed to affect either the spontaneous or the NMDA/glycine-evoked release of [3H]noradrenaline (Figure 5b; F(3, 8)=0.56; n.s.].

Figure 5.

Figure 5

Open in a new tab

Effects of 3,5-DHPG and group I mGluR antagonists on the NMDA-evoked release of [3H]noradrenaline from human neocortical nerve endings. (a) Effects of group I mGluR antagonists on the NMDA/glycine/3,5-DHPG- induced release of tritium. (b) Effects of group I mGluR antagonists on the NMDA/glycine-induced release of tritium. Superfusion was carried out in the absence of Mg2+ ions. Synaptosomes were exposed to 3,5-DHPG and to NMDA/glycine at the end of the first fraction collected. Antagonists were present from 8 min before agonists. Results are expressed as percent increase over basal release; data are means±s.e.m. of four to six experiments run in triplicate. *P<0.01 versus NMDA/glycine; **P<0.001 versus NMDA/glycine; #P<0.001 versus NMDA/glycine/3,5-DHPG. Abbreviations: 3,5-DHPG, (RS)-3,5-dihydroxyphenylglycine; mGluR, metabotropic glutamate receptor; NMDA, N-methyl-D-aspartate.

Mode of release of the AMPA/NMDA/mGluR5 receptor-evoked release of [3H] noradrenaline

To obtain information on the mode of release of [3H]noradrenaline during activation of the AMPA/NMDA/mGlu5 receptor pathway, we evaluated the sensitivity of the evoked [3H]noradrenaline release to clonidine, a drug able to activate α2-adrenoceptors present on noradrenergic terminals and to inhibit the evoked release of the catecholamine when it occurs by exocytosis (Langer, 1981; Starke, 1981). As shown in Figure 6, the release of [3H]noradrenaline provoked in rat hippocampal nerve endings by AMPA/NMDA/3,5-DHPG (about 100% increase over basal) was about 90% inhibited by 0.1 μM clonidine (df=8, t=12.59, P<0.001; n=5), consistent with a vesicular exocytotic release.

Figure 6.

Figure 6

Open in a new tab

Effects of the selective α2-adrenoceptor agonist clonidine on the release of [3H]noradrenaline induced by AMPA, NMDA and 3,5-DHPG from rat hippocampal nerve endings. Superfusion was carried out in presence of Mg2+ ions. Synaptosomes were exposed to 3,5-DHPG, AMPA and NMDA at the end of the first fraction collected. Results are expressed as percent increase over basal release; data are means±s.e.m. of five experiments run in triplicate. #P<0.001 versus respective control. Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate; 3,5-DHPG, (RS)-3,5-dihydroxyphenylglycine; NMDA, N-methyl-D-aspartate.

Discussion

It has often been shown that, during up–down perfusion of synaptosomal monolayers, the transmitters just released are quickly removed by the superfusion solution before they can feedback on targets of the releasing terminals or act on neighbouring particles, thus minimizing or excluding indirect effects. Accordingly, NMDA, added in Mg2+-free solutions, releases [3H]noradrenaline by directly acting at presynaptic NMDA receptors localized on noradrenergic axon terminals. This agonist which, in Mg2+-containing solutions, is unable to evoke release of noradrenaline from synaptosomes, can however acquire activity if coapplied with AMPA, indicating that AMPA receptors and NMDA receptors coexist and interact on the same noradrenergic terminals (Raiteri et al., 1992). Similarly, if a group I mGluR agonist like 3,5-DHPG, inactive on its own, becomes able to release noradrenaline when added together with a mixture of AMPA and NMDA, one explanation is that three glutamate receptors, that is, mGluRs, AMPA receptors and NMDA receptors coexist and interact on the same noradrenergic terminal. Obviously, more direct (that is, morphological) evidences of receptor colocalization would be most convincing, but they are precluded by the extremely low density of noradrenergic terminals in CNS tissues.

The first finding of the present work is that presynaptic group I mGlu heteroreceptors of the mGluR5 subtype present on noradrenergic terminals are functional when terminals are exposed to AMPA plus NMDA, a condition mimicking the physiological stimulus represented by endogenously released glutamate. A second finding is that activation of NMDA receptors due to lack of external Mg2+ ions permits activation of mGluRs of both mGluR5 and mGluR1 subtypes. Thus, activation of NMDA receptors permits group I mGluRs to become functional, consistent with the view that NMDA receptors colocalize with these mGluRs on noradrenergic axon terminals and positively couple to mGluRs. Unlike NMDA receptors, activation of AMPA receptors alone does not exert a permissive role on the function of group I mGluRs, suggesting that the two receptors types do not crosstalk on noradrenergic terminals. However, it has to be borne in mind that AMPA receptor activation permits NMDA receptor activation in presence of physiological Mg2+ ions (Raiteri et al., 1992) and that co-activation of AMPA and NMDA receptors enables mGluR5 to augment the release elicited by AMPA/NMDA/glycine (Figure 1a). Thus, activation of AMPA receptors by endogenous glutamate might represent a prerequisite for the NMDA receptor-mediated activation of mGluR5 under physiological conditions.

The mGluR1/5 agonist 3,5-DHPG was unable, on its own, to elicit release. Since transmitter release mediated by group I presynaptic mGluRs requires a concomitant depolarization of nerve terminals (Herrero et al., 1992; Parodi et al., 2006; see Cartmell and Schoepp, 2000, for a review), one might reasonably assume that presynaptic mGluRs of group I need more cytosolic Ca2+ in addition to that mobilized through the phosphatidylinositol pathway. However, our results suggest that the situation is more complex: in fact, although both presynaptic NMDA and AMPA receptors can stimulate Ca2+ influx into nerve terminals (Pittaluga and Raiteri, 1990; Raiteri et al., 1992; Malva et al., 1994), only NMDA receptors, and not AMPA receptors, mediate activation of mGluRs, implying that the mechanism by which the level of Ca2+ in the cytosol is raised may differentially affect mGluR function (see below).

AMPA receptors exert a permissive role on NMDA → mGluR5 interactions in presence of Mg2+ as supported by the results with selective antagonists as well as with DFB. This compound exhibited no releasing activity on its own; nor did it affect the release due to AMPA/NMDA/glycine, but it did strongly potentiate the effect of a slightly effective concentration of 3,5-DHPG on the AMPA/NMDA-evoked release of noradrenaline. Based on the results on DFB reported by O'Brien et al. (2003), the compound behaved as a positive allosteric modulator of mGluR5.

If activation of ‘permissive,' AMPA receptors appears to be a prerequisite to the NMDA → mGluR5 interaction in the presence of Mg2+, mGluR1 can be recruited and contribute to noradrenaline release when NMDA receptors are activated by Mg2+ removal, independently of AMPA receptors. The concomitant activation of mGluR1 and mGluR5 observed under these conditions possibly occurs because Mg2+ deficiency allows activation of the majority of NMDA receptors present on the terminals, including those that are not adjacent to permissive AMPA receptors. The finding that the mGluR-mediated component of the release evoked in Mg2+-free conditions can only be blocked by a combination of mGluR1 and mGluR5 antagonists, tends to exclude the possibility that the two receptors are on different noradrenergic terminals. However, they could coexist on the same nerve terminal on distinct regions of the plasma membrane as NMDA receptors/AMPA receptors/mGluR5 and NMDA receptors/mGluR1 combinations, respectively, but share the same transducing mechanisms.

Whatever the picture, the activation of mGluR1 in Mg2+-free solutions might become relevant to some pathological conditions linked to Mg2+-deficiency. Significantly decreased concentration of Mg2+ in plasma was found in epileptic children (Benga et al., 1985). Seizures are a recognized complication of HIV-1 infection and seropositive patients with hypomagnesaemia appear to be at increased risk for convulsive status epilepticus (Van Paesschen et al., 1995). Mg2+ wasting also occurs in other segments of the population, including alcoholics and patients with acute myocardial infarction (see Tejero-Taldo et al., 2004). Of note, Figure 1 shows that the release evoked by NMDA/glycine/3,5-DHPG in Mg2+-free medium (∼210%) is much higher than that evoked by AMPA/NMDA/glycine/3,5-DHPG in presence of Mg2+ (∼120%), suggesting a relevant contribution of mGluR1. Excessive activation of mGluR1 has been related to neurotoxic events including maintenance of seizure discharges and ischaemic damage (Bruno et al., 2001; Lee et al., 2002; Pellegrini-Giampietro, 2003).

Although most of the studies on the mGluR-NMDA receptor interactions describe mGluR-induced potentiation of NMDA-mediated responses (Bleakman et al., 1992; Bruno et al., 1995; Doherty et al., 1997; Skeberdis et al., 2001), the reciprocal interaction, that is, regulation of mGluR responses by NMDA receptor activation has been the object of some reports (Luthi et al., 1994; Alagarsamy et al., 1999, 2005). Alagarsamy et al. (1999) showed that activation of NMDA receptors potentiated responses to activation of mGluR5 by reversing desensitization of this receptor possibly through activation of protein phosphatase 2B/calcineurin (Alagarsamy et al., 2005). Understanding the mechanisms by which activation of NMDA receptors permits mGluRs to contribute to noradrenaline release from noradrenergic axon terminals, including a reversal of their desensitization requires further investigations. It is important that similar interactions seem to occur in rat and in human brain, so that rat brain tissues could represent a useful model in future work.

Acknowledgments

This work was supported by grants from Istituto Superiore di Sanità (Programma Nazionale di Ricerca sull'AIDS: Progetto ‘Patologia, Clinica e Terapia dell'AIDS), from Italian Ministero dell'Istruzione, dell'Università e della Ricerca Scientifica (Projects n. 2004052809_004 (AP) and 2003053993 (MR)) and from IRCCS Istituto Neurologico Mediterraneo NEUROMED (Pozzilli, IS, Italy). We are grateful to Mrs Maura Agate for careful editorial assistance.

Abbreviations

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazole propionate

CNS

central nervous system

CPCCOEt

7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester

DFB

3,3′difluorobenzaldazine

3,5-DHPG

(RS)-3,5-dihydroxyphenylglycine

MPEP

2-methyl-6-(phenylethynyl) pyridine hydrochloride

mGluR

metabotropic glutamate receptor

NMDA

N-methyl-D-aspartate

Conflict of interest

The authors state no conflict of interest.

References

  1. Alagarsamy S, Marino MJ, Rouse ST, Gereau RW, IV, Heinemann SF, Conn PJ. Activation of NMDA receptors reverses desensitization of mGluR5 in native and recombinant systems. Nat Neurosci. 1999;2:234–240. doi: 10.1038/6338. [DOI] [PubMed] [Google Scholar]
  2. Alagarsamy S, Saugstad J, Warren L, Mansuy IM, Gereau IV RW, Conn PJ. NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin. Neuropharmacology. 2005;49:135–145. doi: 10.1016/j.neuropharm.2005.05.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benga I, Baltescu V, Tilinca R, Pavel O, Ghiran V, Muschevici D, et al. Plasma and cerebrospinal fluid concentrations of magnesium in epileptic children. J Neurol Sci. 1985;67:29–34. doi: 10.1016/0022-510x(85)90019-x. [DOI] [PubMed] [Google Scholar]
  4. Benquet P, Gee CE, Gerber U. Two distinct signaling pathways upregulate NMDA receptor responses via two distinct metabotropic glutamate receptor subtypes. J Neurosci. 2002;22:9679–9686. doi: 10.1523/JNEUROSCI.22-22-09679.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bleakman D, Rusin KI, Chard PS, Glaum SR, Miller RJ. Metabotropic glutamate receptors potentiate ionotropic glutamate responses in the rat dorsal horn. Mol Pharmacol. 1992;42:192–196. [PubMed] [Google Scholar]
  6. Bruno V, Battaglia G, Copani A, D'Onofrio M, Di Iorio P, De Blasi A, et al. Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs. J Cereb Blood Flow Metab. 2001;21:1013–1033. doi: 10.1097/00004647-200109000-00001. [DOI] [PubMed] [Google Scholar]
  7. Bruno V, Copani A, Knopfel T, Kuhn R, Casabona G, Dell'Albani P, et al. Activation of metabotropic glutamate receptors coupled to inositol phospholipid hydrolysis amplifies NMDA-induced neuronal regeneration in cultured cortical cells. Neuropharmacology. 1995;34:1089–1098. doi: 10.1016/0028-3908(95)00077-j. [DOI] [PubMed] [Google Scholar]
  8. Cartmell J, Schoepp DD. Regulation of neurotransmitter release by metabotropic glutamate receptors. J Neurochem. 2000;75:889–907. doi: 10.1046/j.1471-4159.2000.0750889.x. [DOI] [PubMed] [Google Scholar]
  9. Conn PJ, Pin JP. Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol. 1997;37:205–237. doi: 10.1146/annurev.pharmtox.37.1.205. [DOI] [PubMed] [Google Scholar]
  10. Dingledine R, Borges K, Bowie D, Traynelis SF. The glutamate receptor ion channels. Pharmacol Rev. 1999;51:7–61. [PubMed] [Google Scholar]
  11. Doherty AJ, Palmer MJ, Henley JM, Collingridge GL, Jane DE. RS)-2-chloro-5-hydroxyphenylglycine (CPHG) activates mGlu5, but no mGlu1, receptors expressed in CHO cells and potentiates NMDA responses in the hippocampus. Neuropharmacology. 1997;36:265–267. doi: 10.1016/s0028-3908(97)00001-4. [DOI] [PubMed] [Google Scholar]
  12. Fazal A, Parker F, Palmer AM, Croucher MJ. Characterisation of the actions of group I metabotropic glutamate receptor subtype selective ligands on excitatory amino acid release and sodium-dependent re-uptake in rat cerebrocortical minislices. J Neurochem. 2003;86:1346–1358. doi: 10.1046/j.1471-4159.2003.01932.x. [DOI] [PubMed] [Google Scholar]
  13. Feligioni M, Raiteri L, Pattarini R, Grilli M, Bruzzone S, Cavazzani P, et al. The human immunodeficiency virus-1 protein Tat and its discrete fragments evoke selective release of acetylcholine from human and rat cerebrocortical terminals through species-specific mechanisms. J Neurosci. 2003;23:6810–6818. doi: 10.1523/JNEUROSCI.23-17-06810.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fink K, Bönisch H, Göthert M. Presynaptic NMDA receptors stimulate noradrenaline release in the cerebral cortex. Eur J Pharmacol. 1990;185:115–117. doi: 10.1016/0014-2999(90)90219-v. [DOI] [PubMed] [Google Scholar]
  15. Fink K, Schultheiss R, Göthert M. Stimulation of noradrenaline release in human cerebral cortex mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Br J Pharmacol. 1992;106:67–72. doi: 10.1111/j.1476-5381.1992.tb14294.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heidinger V, Manzerra P, Wang XQ, Strasser U, Yu S-P, Choi DW, et al. Metabotropic glutamate receptor 1-induced upregulation of NMDA receptor current: mediation through the Pyk2/Src-family kinase pathway in cortical neurons. J Neurosci. 2002;22:5452–5461. doi: 10.1523/JNEUROSCI.22-13-05452.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Herrero I, Miras-Portugal MT, Sanchez-Prieto J. Positive feedback of glutamate exocytosis by metabotropic presynaptic receptor stimulation. Nature. 1992;360:163–166. doi: 10.1038/360163a0. [DOI] [PubMed] [Google Scholar]
  18. Langer SZ. Presynaptic regulation of the release of catecholamines. Pharmacol Rev. 1981;32:337–362. [PubMed] [Google Scholar]
  19. Lee AC, Wong RK, Chuang SC, Shin HS, Bianchi R. Role of synaptic metabotropic glutamate receptors in epileptiform discharges in hippocampal slices. J Neurophysiol. 2002;88:1625–1633. doi: 10.1152/jn.2002.88.4.1625. [DOI] [PubMed] [Google Scholar]
  20. Longordo F, Feligioni M, Chiaramonte G, Sbaffi P, Raiteri M, Pittaluga A. The human immunodeficiency virus-1 protein Tat upregulates NMDA receptor function by acting at mGluR1 receptors coexisting on human and rat brain noradrenergic neurons. J Pharmacol Exp Ther. 2006;317:1097–1105. doi: 10.1124/jpet.105.099630. [DOI] [PubMed] [Google Scholar]
  21. Luthi A, Gahwiler BH, Gerber U. Potentiation of a metabotropic glutamatergic response following NMDA receptor activation in rat hippocampus. Pflugers Arch. 1994;427:197–202. doi: 10.1007/BF00585965. [DOI] [PubMed] [Google Scholar]
  22. MacDermott AB, Role LW, Siegelbaum SA. Presynaptic ionotropic receptors and the control of transmitter release. Annu Rev Neurosci. 1999;22:443–485. doi: 10.1146/annurev.neuro.22.1.443. [DOI] [PubMed] [Google Scholar]
  23. Malva JO, Carvalho AP, Carvalho CM. Modulation of dopamine and noradrenaline release and of intracellular Ca2+ concentration by presynaptic glutamate receptors in hippocampus. Br J Pharmacol. 1994;113:1439–1447. doi: 10.1111/j.1476-5381.1994.tb17158.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Marti M, Paganini F, Stocchi S, Bianchi C, Beani L, Morari M. Presynaptic group I and II metabotropic glutamate receptors oppositely modulate striatal acetylcholine release. Eur J Pharmacol. 2001;14:1181–1184. doi: 10.1046/j.0953-816x.2001.01750.x. [DOI] [PubMed] [Google Scholar]
  25. O'Brien JA, Lemaire W, Chen TB, Chang RSL, Jacobson MA, Ha SN, et al. A family of highly selective allosteric modulators of the metabotropic glutamate receptor subtype 5. Mol Pharmacol. 2003;64:731–740. doi: 10.1124/mol.64.3.731. [DOI] [PubMed] [Google Scholar]
  26. Parodi M, Patti L, Grilli M, Raiteri M, Marchi M. Nicotine has a permissive role on the activation of metabotropic glutamate 5 receptors coexisting with nicotinic receptors on rat hippocampal noradrenergic nerve terminals. Neurochem Int. 2006;48:138–143. doi: 10.1016/j.neuint.2005.08.010. [DOI] [PubMed] [Google Scholar]
  27. Pellegrini-Giampietro DE. The distinct role of mGlu1 receptors in post-ischemic neuronal death. Trends Pharmacol Sci. 2003;24:461–470. doi: 10.1016/S0165-6147(03)00231-1. [DOI] [PubMed] [Google Scholar]
  28. Pisani A, Calabresi P, Centone D, Bernardi G. Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones. Br J Pharmacol. 1997;120:1007–1014. doi: 10.1038/sj.bjp.0700999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pittaluga A, Feligioni M, Longordo F, Luccini E, Raiteri M. Trafficking of presynaptic AMPA receptors mediating neurotransmitter release: neuronal selectivity and relationships with sensitivity to cyclothiazide. Neuropharmacology. 2006;50:286–296. doi: 10.1016/j.neuropharm.2005.09.004. [DOI] [PubMed] [Google Scholar]
  30. Pittaluga A, Pattarini R, Severi P, Raiteri M. Human brain N-methyl-D-aspartate receptors regulating noradrenaline release are positively modulated by HIV-1 coat protein gp120. AIDS. 1996;10:463–468. doi: 10.1097/00002030-199605000-00003. [DOI] [PubMed] [Google Scholar]
  31. Pittaluga A, Raiteri M. Release-enhancing glycine-dependent presynaptic NMDA receptors exist on noradrenergic terminals of hippocampus. Eur J Pharmacol. 1990;191:231–234. doi: 10.1016/0014-2999(90)94153-o. [DOI] [PubMed] [Google Scholar]
  32. Raiteri L, Raiteri M. Synaptosomes still viable after 25 years of superfusion. Neurochem Res. 2000;25:1265–1274. doi: 10.1023/a:1007648229795. [DOI] [PubMed] [Google Scholar]
  33. Raiteri M. Functional pharmacology in human brain. Pharmacol Rev. 2006;58:162–193. doi: 10.1124/pr.58.2.5. [DOI] [PubMed] [Google Scholar]
  34. Raiteri M, Bonanno G, Marchi M, Maura G. Is there a functional linkage between neurotransmitter uptake mechanisms and presynaptic receptors. J Pharmacol Exp Ther. 1984;231:671–677. [PubMed] [Google Scholar]
  35. Raiteri M, Garrone B, Pittaluga A. N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors regulating hippocampal norepinephrine release. II. Evidence for functional cooperation and for coexistence on the same axon terminal. J Pharmacol Exp Ther. 1992;260:238–242. [PubMed] [Google Scholar]
  36. Raiteri M, Marchi M, Caviglia AM. Studies on a possible functional coupling between presynaptic acetylcholinesterase and high-affinity choline uptake in the rat brain. J Neurochem. 1986;47:1696–1699. doi: 10.1111/j.1471-4159.1986.tb13076.x. [DOI] [PubMed] [Google Scholar]
  37. Reid ME, Toms NJ, Bedingfield JS, Roberts PJ. Group I mGlu receptors potentiate synaptosomal [3H]glutamate release independently of exogenously applied arachidonic acid. Neuropharmacology. 1999;38:477–485. doi: 10.1016/s0028-3908(98)00217-2. [DOI] [PubMed] [Google Scholar]
  38. Rodriguez-Moreno A, Sistiaga A, Lerma J, Sanchez-Prieto J. Switch from facilitation to inhibition of excitatory synaptic transmission by group I mGluR desensitization. Neuron. 1998;21:1477–1486. doi: 10.1016/s0896-6273(00)80665-0. [DOI] [PubMed] [Google Scholar]
  39. Rodriguez AL, Nong Y, Sekaran NK, Alagille D, Tamagnan GD, Conn PJ. A close structural analog of 2-methyl-6-(phenylethynyl)-pyridine acts as a neutral allosteric site ligand on metabotropic glutamate receptor subtype 5 and blocks the effects of multiple allosteric modulators. Mol Pharmacol. 2005;68:1793–1802. doi: 10.1124/mol.105.016139. [DOI] [PubMed] [Google Scholar]
  40. Skeberdis VA, Lan JY, Opitz T, Zheng X, Bennett MVL, Zukin RS. mGluR1-mediated potentiation of NMDA receptors involves a rise in intracellular calcium and activation of protein kinase C. Neuropharmacology. 2001;40:856–865. doi: 10.1016/s0028-3908(01)00005-3. [DOI] [PubMed] [Google Scholar]
  41. Starke K. Presynaptic receptors. Annu Rev Pharmacol Toxicol. 1981;21:7–30. doi: 10.1146/annurev.pa.21.040181.000255. [DOI] [PubMed] [Google Scholar]
  42. Tejero-Taldo MI, Chmielinska JJ, Gonzalez G, Mak IT, Weglicki WB. N-methyl-D-aspartate receptor blockade inhibits cardiac inflammation in the Mg2+-deficient rat. J Pharmacol Exp Ther. 2004;311:8–13. doi: 10.1124/jpet.104.070003. [DOI] [PubMed] [Google Scholar]
  43. Thomas LS, Jane DE, Croucher MJ. Metabotropic glutamate autoreceptors of the mGlu(5) subtype positively modulate neuronal glutamate release in the rat forebrain in vitro. Neuropharmacol. 2000;39:1554–1566. doi: 10.1016/s0028-3908(99)00223-3. [DOI] [PubMed] [Google Scholar]
  44. Van Paesschen W, Bodian C, Maker H. Metabolic abnormalities and new-onset seizures in human immunodeficiency virus-seropositive patients. Epilepsia. 1995;36:146–150. doi: 10.1111/j.1528-1157.1995.tb00973.x. [DOI] [PubMed] [Google Scholar]
  45. Wang JK, Andrews H, Thurkal V. Presynaptic glutamate receptors regulate noradrenaline release from isolated nerve terminals. J Neurochem. 1991;58:204–211. doi: 10.1111/j.1471-4159.1992.tb09297.x. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES