Journal of Neurochemistry

Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry

Presynaptic Glutamate Receptors Regulate Noradrenaline Release from Isolated Nerve Terminals *?James K. T. Wang, *Helene Andrews, and *Vijay Thukral *Program in Neurosciences and TDepartment of Physiology, Tufts University School of Medicine, Boston. Massachusetts, U.S.A.

Abstract: The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes)prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non-NMDA agonists, in a concentration-dependent manner, resulted in Caz+dependentrelease of noradrenaline from vesicular transmitter stores. The NMDA-stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non-NMDA agonists was blocked by 6-cyano-

7-nitroquinoxaline-2,3-dione, but not by MgZ+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non-NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate. Key Words: Excitatory amino acids-Neurotransmitter release-Presynaptic N-methyl-Daspartate receptors-Presynaptic non-N-methyl-D-asparkate receptors-Synaptosomes. Wang J. K. T. et al. Presynaptic glutamate receptors regulate noradrenaline release from isolated nerve terminals. J. Neurochem. 58, 204-2 1 1 (1 992).

Glutamate plays an important role in a variety of normal and abnormal neuronal processes. Its diverse actions are mediated by an array of receptors that can be broadly classified as the N-methyl-D-aspartate (NMDA) and the non-NMDA types (for review, see Monaghan et al., 1989). Non-NMDA receptors can be further distinguished pharmacologically as a-amino2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) and kainate subtypes. The vast majority of studies on glutamate actions in mammalian systems to date have focused on the postsynaptic side of the synapse, where glutamate is thought to exert its physiological actions. Consequently, the pharmacology of the postsynaptic receptors has been characterized extensively. In contrast, little is known about the localization of glutamate receptors to the presynaptic nerve terminal. The existence of presynaptic glutamate receptors has only been inferred from studies of transmitter release from brain slices. It has been shown, for example, that glutamate and some of its analogues have

complex effects on amino acid release (McBean and Roberts, 1981; Ferkany et al., 1982; Collins et al., 1983). Glutamate also stimulates the release of dopamine from striatal slices (Giorgueff et al., 1977; Roberts and Sharif, 1978; Roberts and Anderson, 1979; Snell and Johnson, 1986) and of noradrenaline (NA) from hippocampal and cortical slices (Jones et al., 1987; Vezzani et al., 1987; Fink et al., 1989). Some of these effects persist in the presence of tetrodotoxin, suggesting that they may contain a presynaptic component. However, several attempts utilizing the synaptosome preparation, which provides the simplest and most direct means of studying presynaptic receptors and transmitter release, failed to identify presynaptic glutamate receptors (Schmidt and Taylor, 1988; Fink et al., 1989), although recently a brief report of NMDA enhancing release of NA from cerebral cortical synaptosomes has appeared (Fink et al., 1990). We examined the release of NA in hippocampus, olfactory bulb, and cerebral cortex, regions which receive their major noradrenergic

Received January 9, 1991; revised manuscript received May 10, 1991; accepted May 24, 1991. Address correspondence and reprint requests to Dr. J. K. T. Wang at Program in Neurosciences, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 021 1 I , U S A . Abbreviations used: AMPA, a-amino-2,3dIhydro-5-methyl-3-0~0-

4-isoxazolepropanoic acid; ANOVA. analysis of variance; APV, 2amino-5-phosphonovaleric acid 7-Cl-KYN, 7-chlorokynurenic acid CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CPP, 3-(2-carboxypiperazin-4-y1)propyl-1 -phosphonic acid MK-80 1 , (+)-5-methyl10, I ldihydro-5Hdibenzo[a,d]cyclohepten-5,I0-imine maleate; NA, noradrenaline; NMDA, N-methyl-D-aspartate.

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input from the locus ceruleus. We found that NA release from synaptosomes prepared from the three brain regions was increased by the activation of presynaptic glutamate receptors. These receptors appear pharmacologically to be similar to both postsynaptic NMDA and non-NMDA receptors. Some of these results have been published in abstract form (Wang and Thukral, 1990).

of [3H]NA (7 nM final concentration, 13.8 Ci/mmol) or [3H]dopamine (2.4 nM final concentration, 41 Ci/mmol). The incubations were continued for 5 rnin and were terminated by 20-fold dilution with ice-cold incubation buffer and filtration over Whatman GF/B filters, followed by three 2ml washes with ice-cold buffer. Radioactivity remaining on the filters was quantitated by liquid scintillation spectrometry.

EXPERIMENTAL PROCEDURES

Specificity of transmitter uptake Olfactory bulb contains both noradrenergic innervation and dopaminergic neurons (see Halasz and Shepherd, 1983); it is, therefore, possible that part of the uptake of [3H]NAin olfactory bulb synaptosomes was actually into dopaminergic terminals. This possibility was assessed by examining the sensitivity of the uptake to blockade by the specific noradrenergic uptake inhibitor desipramine. As expected, desipramine potently inhibited the uptake of [3H]NAinto hippocampal synaptosomes, with an IC50of 6.3 nM, but did not inhibit the uptake of ['Hldopamine into striatal synaptosomes (uptake in the presence of 10 pM desipramine was 93% of uptake in the absence of desipramine; n = 2). In olfactory bulb synaptosomes, up to 90% of the uptake of either [3H]NAor [3H]dopaminewas potently inhibited by desipramine, with ICs0values of 3.5 nM and 4.9 nM, respectively. Thus, the uptake of ['HINA in this preparation was mainly into noradrenergic terminals.

Materials AMPA and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were obtained from Tocris Neuramin (Bristol, U.K.). 2-Amino-5-phosphonovaleric acid (APV), 3-(2-carboxypiperazin-4-y1)propyl-1-phosphonic acid (CPP), 7-chlorokynurenic acid (7-Cl-KYN), glutamate, kainate, (+)-5methyl- I0,l l-dihydro-5H-dibenzo[u,d]cyclohepten-5,10imine maleate (MK-801) and NMDA were obtained from RBI (Bethesda, MD, U.S.A.). [3H]NA and [3H]dopamine were obtained from Dupont-NEN (Boston, MA, U.S.A.). All other chemicals used were of reagent grade.

Preparation of synaptosomes Male Sprague-Dawley rats ( 1 50-200 g; Taconic) were killed, and the hippocampus, olfactory bulb, and cerebral cortex were rapidly dissected. Crude synaptosome fractions (P2) from these brain regions were prepared as described (Nichols et al., 1987), and purified synaptosomes using Percoll gradients were prepared as described (Dunkley et al., 1986). The results obtained from either preparation were similar, and the Pz fraction was used for the majority of the experiments, except that synaptosomes from cerebral cortex were always purified. The synaptosomes were resuspended in incubation buffer containing the following (in &): NaCl, 140; MgClZ,1;CaClZ,1; KCl, 5; NaHC03, 5; NaHZPO4,1.2; glucose, 10; HEPES, 10, pH 7.4; ascorbic acid, 0.2; and pargyline, 0.02.

Neurotransmitter release Loading ofsynaptosomes with [3H]NA(0.07 pM, 13.8 Ci/ mmol) was for 15 min at 37OC, at a protein concentration of 0.2-1 mg/ml in a total volume of 5 ml. Labeled synaptosomes were layered onto PlOO prefilters (Nuclepore) in a modified Millipore manifold and superfused with incubation buffer at a rate of 4 ml/min. After 15 rnin of superfusion, the last 3 rnin of which were with Mg2+-freebuffer in the Mg2+-freeprotocol, the trapped synaptosomes were superfused in duplicate samples with drug-containing buffer at 4 ml/min. Radioactivity in the superfwates and in the filters was quantitated by liquid scintillation spectrometry, and the released radioactivity (counts in the superfusates) is expressed as the percentage of total remaining radioactivity (superfusate counts plus filter-trapped counts) at each time point (Nichols et al., 1987). Net release of labeled transmitter was obtained by subtracting the basal control efflux from the total release in the presence of drug. For statistical analysis, Student's t test (two-tailed) or one-way analysis of variance (ANOVA), followed by Dunnett's test, was performed as indicated. Significance is defined as p < 0.05.

Uptake studies Uptake studies were performed by incubating synaptosomes (P2 fraction, 0.5-1.5 mg/ml) in 100 p1 of incubation buffer with desipramine for 5 min, followed by the addition

RESULTS

Time course of the NMDA- and glutamatestimulated [3H]NA release NMDA, when applied in the absence of Mg2+ to superfused hippocampal synaptosomes, increased the release of [3H]NA by up to 87% over unstimulated basal levels. Because the basal release of [3H]NAconsisted largely of Ca2+-independentefflux (see below) and decreased gradually over time (see legend to Fig. l), it was subtracted from the NMDA-stimulated release at each time point. This revealed the time course of the net stimulated release of NA (Fig. 1). The maximal response to NMDA occurred within 2 min, followed by a gradual decline that reached basal control levels after 10 min of treatment (data not shown). Glutamate, in the absence of Mg2+,also stimulated the release of ['HINA with a time course similar to that with NMDA (Fig. 1). Similar time courses for NMDAand glutamate-stimulated NA release were observed in synaptosomes prepared from olfactory bulb and cerebral cortex (data not shown). Glutamate agonists stimulated I3H]NA release in a concentration-dependent manner NMDA, in the absence of Mg2+,increased NA release from hippocampal synaptosomesin a concentration-dependent manner (Fig. 2), with the maximal effect occurring at 100 pM. Likewise, in the presence of Mg2+,glutamate and the selective non-NMDA agonists kainate and AMPA stimulated NA release in a concentration-dependent manner (Fig. 2). Glycine, which J. Neurochem., Val. 58, No. I . 1992

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nme (min)

FIG. 1. Time course of NMDA- and glutamate-stimulatedrelease of [3H]NA from hippocampal synaptosomes. Synaptosomes prepared from hippocampus were loaded with r3H]NA and washed (Mg2+-freeprotocol) as described in Experimental Procedures. All subsequent buffers were also MgZf-free.At time 0, the synaptosomes were superfused with control buffer or buffer containing either NMDA or glutamate. and 4-ml fractions were collectedevery minute for 5 min. The net stimulated release was obtained by subtracting the basal release at each minute from the release in the presence of drug. The basal release at each time point, beginning with the first minute, was 2.42 ? 0.24%, 1.90 k 0.08%, 1.90 ? 0.02%, 1.74 ? 0.04%, and 1.72 ? 0.06% of total transmitter stores. Results shown are the means f SEM of three experiments; 100 LclM NMDA. 0 ; 10 & glutamate, I m.

on its own had little effect on NA release (net release of 0.30 & 0.03%of total transmitter stores in the presence of 10 pM glycine, which was subtracted from the NMDA plus glycine-stimulated release),greatly potentiated the NMDA effect (Fig. 2). Similar dose-response curves for the glutamate agonists, and of potentiation of the NMDA effect by glycine, were observed in olfactory bulb and cerebral cortical synaptosomes (data not shown). None of these agonists had any significant effect on NA release evoked by K' depolarization (data not shown). These results suggest that there may be both NMDA and non-NMDA presynaptic receptors that are capable of directly increasing NA release. Presynaptic NMDA receptor To determine if the effect of NMDA on NA release is receptor-mediated,its sensitivity to selective NMDA receptor antagonists and modulators was examined. MK-801, a potent and selective noncompetitive antagonist of NMDA (see Kemp et al., 1987), abolished the NMDA-stimulated [3H]NA release from synaptosomes prepared from hippocampus, olfactorybulb, and cerebral cortex (Fig. 3). APV, another selective, but competitive, NMDA antagonist (see Watkins and 01verman, 1987), reduced the NMDA effect (Fig. 3; release of [3H]NAin the presence of NMDA plus antagonist, expressed as percentage of release in the presence of NMDA alone, was 47% for hippocampus, 56% for olfactory bulb, and 63%for cerebral cortex). Similarly, 10 pM CPP, a potent competitive NMDA antagonist, also abolished the NMDA effect in all three brain reJ. Neurochem., Vol. 58, No.

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gions (data not shown). Thus, the effect of NMDA on NA release is mediated by a presynaptic NMDA-type receptor. CNQX, a relatively selective antagonist of the nonNMDA receptor (Honor6 et al., 1988), nevertheless reduced the NMDA-stimulated [3H]NA release by more than half in synaptosomes from all three brain regions (Fig. 3; release of [3H]NA in the presence of NMDA plus CNQX, expressed as percentage of release in the presence of NMDA alone, was 50% for hippocampus, 46% for olfactory bulb, and 30%for cerebral cortex). CNQX, however, has also been shown to inhibit postsynaptic NMDA responses (Birch et al., 1988; Kleckner and Dingledine, 1989) by interacting with the glycine modulatory site of the NMDA receptor (Johnson and Ascher, 1987; Thomson, 1989) and, therefore, may be acting in a similar fashion presynaptically. This hypothesis is supported by the ability of 7-Cl-KYN, an antagonist of NMDA that acts selectively at the glycine site (Kemp et al., 1988), to reduce the NMDA-stimulated NA release by almost 90%(Fig. 3). Moreover, glycine reversed the antagonism of the NMDA effect by CNQX and by 7-Cl-KYN (data not shown). Thus, similar to the postsynaptic NMDA receptor (Kleckner and Dingledine, 1988), the presynaptic NMDA receptor is also associated with a glycine modulatory site, and some minimal levels of glycine may be required for NMDA to elicit the presynaptic response. Mg2+ is known to block the postsynaptic NMDA channel in a voltage-dependent manner (Mayer et al., 1984; Nowak et al., 1984). At a concentration of 1 mM, Mg2+also eliminated the NMDA effect on NA 3

I0

FIG. 2. Concentration dependency of glutamate agonist-stimulated release of [3H]NA. Synaptosomes prepared from hippocampus were loaded with [3H]NA and washed for 15 min with Mg2+-containing buffer. For those samples to be treated with NMDA, the last 3 min of the wash period were with Mg*+-freebuffer with or without 10 f l glycine. The synaptosomes were then superfused for 1.5 min with 6 ml of Mg2+-containingbuffer with the indicated concentrationsof glutamate (+), kainate(O),or AMPA (m), or with 6 ml of Mg*+-free buffer with (X) or without (A)10 rM glycine, containing in addition the indicated concentrationsof NMDA. Results shown are the means of three to five independent experiments, with SEM values within 20% of the means.

PRESYNAPTIC GLUTAMATE RECEPTORS REGULATE RELEASE *

release (release of t3H]NA in Mg2+-containingbuffer plus 100 pM NMDA, expressed as percentage of release in Mg2+-containingcontrol buffer, was 98 f 7% for hippocampus, 98 -+ 2% for olfactory bulb, and 99 k 4% for cerebral cortex; n = 3). Interestingly, even in the presence of 1 mM Mg2+,when neither glycine ( 10 p M ) nor NMDA (100 p M ) alone had any effect on NA release, treatment with both agents together resulted in a significant net release of [3H]NA (hippocampus: 1.38 +. 0.15% of total transmitter stores, n = 7; olfactory bulb: 1.05 k 0.21% of total transmitter stores, n = 3; cerebral cortex: 0.69 +. 0.03% of total transmitter stores, n = 3). Thus, in normal polarized synaptosomesin the presence of Mg2+,the presynaptic NMDA receptor can be activated if sufficient glycine is present.

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F'resynaptic non-NMDA receptor Glutamate, kainate, and AMPA, in contrast to NMDA, consistently increased NA release from hippocampal and olfactory bulb synaptosomes even in the presence of 1 mMMg2+and in the absence of added glycine (Figs. 2 and 4). Similar effects were observed in cerebral cortical synaptosomes (net release of t3H]NA stimulated by 10 pM glutamate was 0.60 k 0.09% of total transmitter stores, that by 50 pM AMPA was 0.8 1 f 0.15% of total transmitter stores, and that by 10 f i A 4 kainate was 0.72 +- 0.06% of total transmitter stores; n = 3). In hippocampal and olfactory bulb synaptosomes, the effects of glutamate, AMPA, and kainate were inhibited by CNQX (Fig. 4), as would be expected for a response mediated by the non-NMDA receptor. However, the interpretation of this result is complicated by our finding that CNQX at 10 pM also partially blocked the presynaptic NMDA effect (see Fig. 3). We, therefore, examined whether the potent and selectiveNMDA antagonist MK-80 1 had any effect on the release induced by glutamate, AMPA, and kainate in the presence of Mg2+.MK-801 did not inhibit the [3H]NArelease stimulated by AMPA and by kainate, but it did reduce the effect of glutamate, most strikingly in olfactory bulb synaptosomes (Fig. 4). Thus, whereas the effects of AMPA and kainate on NA release are mediated by presynaptic non-NMDA receptors, the effect of glutamate, in the presence of Mg2+,may be mediated by both the NMDA and non-NMDA presynaptic receptors. Ca2+dependency of the NMDA-stimulated [3H]NA release To determine if the release of NA induced by NMDA and by glutamate is Ca2+-dependent, synaptosomes were exposed briefly to Ca2+-freeincubation buffer that

FIG. 3. Effects of antagonists on the NMDA-stimulated release of r3H]NA.Synaptosomes prepared from hippocampus (A), olfactory bulb (B), and cerebral cortex (C) were loaded with r3H]NA and washed (Mg2+-freeprotocol) as described in Experimental Procedures. The synaptosomes were then superfused for 1.5 min with 6 ml of Mg2+-freebuffer containing the indicated antagonists, followed by another 6 ml of the same buffer containing in addition

100 pM NMDA. The drug concentrationswere as follows: NMDA, 100 N ;MK-801, 1 pM; APV, 200 p M ; CNQX, 10 pM; and 7 - 0 KYN, 10 pM. Results shown are the means _+ SEM of three to five independent experiments. *Significantlydifferent from NMDA alone, one-way ANOVA.

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dent transmitter efflux. In contrast, the NMDA-stimulated release of [3H]NA was abolished in Ca2+-free buffer (Fig. 5), suggesting that Ca2' influx was required. Further studies showed that removal of Ca2+from the buffer, without addition of EGTA, was sufficient to block the NMDA effect (data not shown). The effect of glutamate was likewise blocked in the absence of Ca2+(data not shown).

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Reserpine pretreatment reduces the NMDAstimulated 13H]NA release Ca" dependency is characteristic of vesicular transmitter release. To provide further evidence that the NMDA-stimulated transmitter release was of vesicular origin, we examined the effect of depleting vesicular catecholamine stores by reserpine pretreatment. Incubation of synaptosomes with reserpine, before loading with [3H]NA, resulted in a fivefold decrease in incorporated label (incorporation of [3H]NAinto reserpine-pretreated synaptosomes, expressed as percentage of incorporation into control synaptosomes, was 16 k 4% for hippocampus, 19 t- 1% for olfactory bulb, and 19 1% for cerebral cortex). Reserpine pretreatment also greatly decreased the NMDA-stimulated NA release for all three brain regions (Fig. 6). Glutamatestimulated release was similarly reduced (data not shown). The residual response to NMDA after reserpine pretreatment was probably a result of incomplete depletion of the transmitter pool, because release evoked by K+ depolarization was also not completely eliminated (data not shown). These results, nevertheless, provide further evidence that the released NA originated from vesicular transmitter stores.

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T ~,

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FIG. 4. Non-NMDA glutamate receptors stimulate release of

[3H]NA in the presence of Mg". Synaptosomes prepared from hippocampus (A) and olfactory bulb (B) were loaded with [3H]NA and washed as described in Experimental Procedures. The synaptosomes were superfused for 1.5 min with 6 ml of Mg2+-containing buffer (open bars) or the same buffer with the addition of 1 pM MK-801 (shaded bars) or 10 pM CNQX (hatched bars), followed by another 6 ml of the respective buffer containing in addition 10 pM glutamate (Glu), 50 pM AMPA, or 10 f l kainate (KA), as indicated. Results shown are the means % SEM of three to five independent experiments. 'Significantly different from agonist alone, one-way ANOVA.

contained, in addition, 0.1 mMEGTA. This treatment did not affect the basal release of [3H]NA from hippocampal and olfactory bulb synaptosomes and had only a small effect on the basal release from cerebral cortical synaptosomes (basal release of [3H]NA during 1.5 min of superfusion, expressed as percentage of total transmitter stores, in the presence or absence of Ca", respectively, were as follows: 2.64 f 0.27% and 2.40 0.24% of total stores for hippocampus; 3.24 k 0.17% and 3.14 +- 0.32% oftotal stores for olfactory bulb; and 2.10 t- 0.12% and 1.44 -t 0.05% of total stores for cerebral cortex; means -+ SEM, n = 3-5). Thus, most of the basal release of [3H]NArepresented Ca2+-indepen+_

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FIG. 5. Effect of Ca2+depletion on the NMDA-stimulated release of r3H]NA. Synaptosomes prepared from hippocampus (HP), 01factory bulb (OB), and cerebral cortex (CX) were loaded with 13H]NA and washed (Mg*+-free protocol) as described in Experimental Procedures. The synaptosomes were then superfusedfor 1.5 min with 6 ml of Mg2+-freebuffer containing either 1 mM Ca2+(hatched bars) or no added Ca2+and 0.1 mM EGTA (open bars), followed by another 6 ml of the same buffer containing in addition 100 pM NMDA. Results shown are the means ? SEM of three to five independent experiments. 'Significantly different from Ca2+-containing controls (hatched bars) (Student's t test).

PRESYNAPTIC GLUTAMATE RECEPTORS REGULATE RELEASE

FIG. 6. Effect of reserpine pretreatment on the NMDA-stimulated release of r3H]NA. Synaptosomes prepared from hippocampus (HP), olfactory bulb (OB), and cerebral cortex (CX) were incubated with (open bars) or without (hatched bars) 1 pM reserpine at 37°C for 10 min, before being loaded with [3H]NA in the presence of reserpine for 15 min, and washed in the absence of reserpine (MgZc-freeprotocol) as described in Experimental Procedures. The synaptosomes were then superfused for 1.5 min with 6 ml of Mg2+free buffer or the same buffer containing 100 f l NMDA. Results shown are the means t SEM of three independent experiments. "Significantlydifferent from untreated controls (hatchedbars) (Student's t test).

DISCUSSION

Glutamate is the major excitatory transmitter in mammalian CNS. Many glutamate actions are thought to be mediated by a family of postsynaptic receptors. Data presented here demonstratethe existence of a class of presynaptic glutamate receptors that are present on noradrenergic terminals. In normal polarized synaptosomes, activation of these receptors by glutamate receptor agonists resulted in the release of NA from vesicular stores in a Ca*+-dependentmanner. The effect of NMDA on NA release was blocked by selective NMDA antagonists and by Mg2+,and was enhanced by glycine. Glycine appears to be an important modulator, because selective antagonists of the glycine site, such as 7-Cl-KYN, could eliminate the effect of NMDA. Moreover, the effect of NMDA without added glycine was occasionally variable, whereas addition of glycine consistently resulted in a robust increase in the response to NMDA. Indeed, glycine can partially reverse the Mg2+blockade of the NMDA action. This glycine dependence of the NMDA effect may account for past failures to detect the presynaptic NMDA receptor (Schmidt and Taylor, 1988; Fink et al., 1989). Moreover, Fink et al. ( 1990)also reported that NMDA does not affect NA release from cerebral cortical synaptosomes unless glycine has been added. Our pharmacological data indicate that the presynaptic NMDA receptor is similar to the well-studied postsynaptic NMDA receptor. It should be noted, however, that the

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latter receptor is subject to complex modulation and that more extensive characterization is required to ascertain that the pre- and postsynaptic NMDA receptors are indeed pharmacologically indistinguishable. In the presence of 1 mM MgZ+,glutamate and the selective non-NMDA agonists AMPA and kainate are still able to stimulate NA release despite the blockade of the NMDA channel. The effects of the three agonists are blocked by the relatively selective non-NMDA antagonist CNQX. Furthermore, the effects of AMPA and kainate are not blocked by the selective NMDA antagonist MK-80 1, whereas the effect of glutamate is partially blocked. Thus, in addition to the presynaptic NMDA receptor, there are also presynaptic nonNMDA receptors that regulate NA release. The partial inhibition of the glutamate effect by MK-80 1 suggests that, under physiological conditions, glutamate can activate both types of receptors, perhaps by first opening the non-NMDA channel to depolarize the nerve terminal, thereby relieving the Mg2+blockade of the NMDA channel. The mechanism by which activation of presynaptic glutamate receptors leads to transmitter release is presently unknown. The ability of the NMDA channel to conduct Ca" (see MacDermott and Dale, 1987) and the Ca2+dependence of the NMDA-stimulated NA release suggest that influx of this cation may be involved. Ca2+influx may directly stimulate transmitter release or may act through signal transduction cascades, such as Ca2+-dependentprotein phosphorylation pathways in the nerve terminal (Wang et al., 1988), that are known to stimulate transmitter release (Nichols et al., 1987, 1990). Similarly, activation of the non-NMDA channel, by increasing the influx of monovalent ions to depolarize the nerve terminal, could also lead to secondary Ca2+influx through the voltage-dependent channels and, hence, transmitter release. Hippocampus, olfactory bulb, and cerebral cortex are all diffusely innervated by locus ceruleus noradrenergic neurons (Pickel et al., 1974; Swanson and Hartman, 1975; Loy et al., 1980; Shipley et al., 1985). Because modulation of NA release by the presynaptic glutamate receptors occurs in synaptosomes prepared from all three brain regions, it is likely that the receptors are localized on the terminals of these widely projecting noradrenergic neurons. Thus, transmitter release from these neurons, which number only a few thousand in the rat, may be regulated not only by the activity state of the neuron, but also locally by these presynaptic receptors. Such a mechanism for discrete control of release involving presynaptic receptors has been characterized for a number of neurotransmitters (for review, see Chesselet, 1984),and may be particularly important for widely distributed transmitters, such as NA. NA modulates neuronal excitability in many brain regions (see Woodward et al., 1979; Foote et al., 1983; Halasz and Shepherd, 1983). In the hippocampus, NA increases neuronal excitability and potentiates evoked

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responses (Madison and Nicoll, 1982; Neuman and Harley, 1983; Gray and Johnston, 1987; Haas and Rose, 1987) in an NMDA-dependent fashion (Burgard et al., 1989; Stanton et al., 1989). NA has also been shown to modulate long-term potentiation (Bliss et al., 1983; Hopkins and Johnston, 1984; Stanton and Sarvey, 1985). Furthermore, NA increases glutamate release from hippocampal slices (Lynch and Bliss, 1986). This raises the possibility that the presynaptic glutamate receptors may be part of a local positive feedback loop between the two transmitters and may play a role in modulating noradrenergic functions in the hippocampus. Finally, recent evidence from our laboratory showed that dopamine release from striatal synaptosomes is also positively modulated by presynaptic glutamate receptors (Wang, 199I), suggesting that the release of a variety of transmitters may be under the regulation of presynaptic glutamate receptors. Note added in proof: Pittaluga a n d Raiteri (Eur. J . Pharmacol. 191,23 1-234, 1990) have also demonstrated presynaptic NMDA receptors that regulate NA release from hippocampal synaptosomes.

Acknowledgment: This work was supported by the PEW Charitable Trust.

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J. Neurocheni., Vol. 58, No. 1, 1992

Presynaptic glutamate receptors regulate noradrenaline release from isolated nerve terminals.

The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate...
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