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Chapter 2 Presynaptic modulation of glutamate release
O.P. Ottersen, LA. Langmoen and L. Gjerstad (Eds.) Progress in Bruin Research, Vol 116 0 1998 Elsevier Science BV. All rights reserved.
CHAPTER 2
Presynaptic modulation of glutamate release David G. Nicholls Neurosciences Institute. Department of Pharmacology, University of Dundee, Dundee DDI 9SY. Scotland, UK
Introduction The arrival of an action potential at a CNS glutamatergic varicosity does not automatically result in exocytosis of the transmitter. Instead glutamatergic varicosities in brain regions capable of synaptic plasticity show a low quanta1 release probability (Hessler et al., 1993), such that only a proportion of the action potentials passing through the varicosity lead to the release of a synaptic vesicle at a given release site. Since an action potential must pass through a varicosity to continue its propagation, this implies that there is an inherent unreliability in the coupling between the action potential and exocytosis. The high power dependency upon external Ca2 concentration of exocytosis (Augustine et al., 1985) suggests that Ca2’- entry may be the prime probability-determining step in action potential/ exocytosis coupling. Presynaptic receptors may either facilitate or inhibit release by directly or indirectly modulating this Ca2+ entry and their existence enormously enhances the potential complexity of synaptic transmission by allowing release to be controlled at each individual varicosity along the axon. The exocytosis of a single synaptic vesicle can elevate the concentration of the amino acid in the synaptic cleft to around 1mM (Clements et al., 1992), sufficient to saturate the ionotropic receptors at the post-synaptic membrane. Presynaptic +
receptors could modulate the probability of vesicle release in response to an invading action potential by a number of mechanisms: firstly by altering the kinetics of K + or Na+ channels and influencing the waveform of the depolarization; secondly by activating or inhibiting the releasecoupled Ca2+ channel and directly controlling the access of Ca2+ to the exocytotic trigger, or thirdly by acting at an intracellular locus to control some aspect of the exocytotic/endocytic cycle. The evidence, obtained from studies with isolated nerve terminals (synaptosomes) will now be reviewed that the two classes of receptor may modulate exocytosis by respectively enhancing presynaptic action potentials and inhibiting Ca2+ channels. (see Turner, this volume, for a description of Ca2+ channels coupled to glutamate release.)
Synaptosomes and the study of glutamate exocytosis Synaptosomes have proved a valuable preparation for the investigation of these presynaptic regulatory pathways (reviewed in Nicholls and SanchezPrieto, 1997; Nicholls, 1995a,b). Glutamate exocytosis greatly outweighs the release of other transmitters in preparations from cerebral cortex, hippocampus or cerebellum and is the simplest preparation which retains all the pathways for the synthesis, storage, release and reuptake of trans-
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mitter glutamate, together with a range of presynaptic receptors capable of modulating the release of the transmitter. In recent years this preparation has provided much information on the signal transduction pathways present in the glutamatergic terminal, and these provide the topic of this review. The release of glutamate can be monitored continuously by including glutamate dehydrogenase and NADP+ in the incubation medium (Nicholls and Sihra, 1986; Sanchez-Prieto et al., 1987), the released glutamate being coupled to the generation of fluorescent NADPH. The Ca2+dependent component of KC1-evoked glutamate release amounts to some 15% of the total glutamate content of the preparation (McMahon and Nicholls, 1991) and is exocytotic judged by its ATP requirement (Sanchez-Prieto et al., 1987), inhibition by tetanus and botulinum toxins (McMahon et al., 1992) and by compartmental analysis (Wilkinson and Nicholls, 1989). In addition to the conventional KC1 depolarization technique for evoking transmitter release, a second and potentially more versatile approach for evoking glutamate exocytosis is by K -A type channel inhibition (Tibbs et al., 1989). A-type K + channels activate very rapidly in response to slight depolarizations and serve as negative feedback mechanisms limiting statistical fluctuations in membrane potential from triggering spontaneous action potentials. Such channels appear to exist in nerve terminals, sensitive to a-dendrotoxin or to low concentrations of 4-aminopyridine (4AP) (Tapia and Sitges, 1982; Agoston et al., 1983; Dolezal and Tucek, 1983; Tibbs et al., 1989). Either agent causes a Ca2+-dependent, tetrodotoxin-sensitive exocytosis of glutamate from the same pool as for KCl, and utilizes the same non-L non-N type Ca2+ channels (Tibbs et al., 1989), triggering release via a highly localized pool of Ca2+ in the immediate vicinity of the Ca2+ channel (Verhage et al., 1991). The selective use of these two means of synaptosomal excitation have been central to studies dissecting the presynaptic signal transduction pathways controlling glutamatergic transmission. +
PKC and the receptor-mediated facilitation of transmitter release It has been established for some time that the application of phorbol esters leads to an enhancement of glutamate release, which can be observed with preparations from the brain slice to the isolated synaptosome (Huang et al., 1989; Segal, 1989; Barrie et al., 1991; Hefrero et al., 1992; Coffey et al., 1993). In our studies, a key finding was that the ability of phorbol esters to enhance glutamate exocytosis from cerebrocortical synaptosomes was dependent on the mode by which the preparation was depolarized: exocytosis evoked by a clamped depolarization by elevated KCl was independent of PKC activity - either phorbol ester mediated activation, or inhibition by Ro 31-8220 (Barrie et al., 1991; Coffey et al., 1993). By monitoring the phosphorylation state of endogenous MARCKS during these experiments it was possible to confirm that PKC activity was being controlled in the expected manner by these agents (Coffey et al., 1993). Thus phosphorylation of a PKC substrate is not necessary for vesicle exocytosis per se. In contrast, the use of 4AP or adendrotoxin to evoke spontaneous action potential firing in the synaptosome preparation resulted in an exocytosis which was totally controlled by PKC activity: phorbol esters could cause a 500% stimulation of release while Ro 3 1-8220 could virtually abolish the ability of 4AP to evoke release (Coffey et al., 1993). Since the distinction between 4AP-evoked glutamate release and that evoked by high KC1 is the tetrodotoxin-sensitive firing of spontaneous ‘action potentials’ (Tibbs et al., 1989), the channels involved in this process provides an obvious locus at which to search for an action of PKC. The use of membrane potential-dependent dyes to monitor the time- and population average depolarization of the synaptosomal preparation reveals that phorbol esters enhance the 4AP-evoked depolarization, indicative of an effect on an ion channel implicated in action potential firing (Barrie et al., 1991; Herrero et al., 1992a; Coffey et al., 1993; Coffey et al., 1994). The effect of the phorbol ester is
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mimicked and occluded by Ba2+ or by clofilium (Nicholls and Coffey, 1994); thus the most likely locus is a 4AP-insensitive, a-dendrotoxin-insensitive, Ba2+-sensitive, clofilium-sensitive K + channel. The PKC isoform mediating the facilitated glutamate release is potentiated by arachidonic acid (AA) at concentrations of 1-2 pM (Herrero et al., 1992a,b). In the presence of AA the threshold concentration of phorbol ester required to observe facilitation is greatly decreased (Herrero et al., 1992a). The dominant physiological agonist activating presynaptic PKC appears to be glutamate itself, since in the presence of AA a facilitation of 4AP-evoked glutamate release is observed (Herrero et al., 1992b) on addition of the mGluR agonist (lS, 3R) 1-aminocyclopentane1,3-dicarboxylic acid (ACPD). ACPD activates a metabotropic glutamate receptor (mGluR) which is coupled (in the presence of AA) to enhanced phosphorylation of the established PKC substrates MARCKS and GAP-43 (Coffey et al., 1994). The two established pathways for the formation of the diacylglycerol (DAG) required for the activation of PKC are its direct generation by phospholipase C (PLC) mediated hydrolysis of phosphatidylinositol bisphosphate and the alternative activation of phospholipase D with the initial generation of phosphatidic acid followed by its hydrolysis to DAG (for review see Klein et al., 1995). While a large and transient elevation in DAG has been reported in response to ACPD (Herrero et al., 1994), recent studies in our laboratory (unpublished) indicate a more modest (5&70% elevation) but more prolonged elevation in diacylglycerol (persisting for 15 min even in the absence of AA) in response to 100 pM ACPD. Analysis of the pharmacology of the facilitated glutamate release reveals some anomalous features: firstly ACPD-mediated enhancement is inhibited by (RS)-3,5-dihydroxyphenylglycine (DHPG) which is reported to be a selective group I mGluR agonist (Schoepp et al., 1994). An antagonist activity of DHPG has previously been reported against the ACPD-mediated activation of phospholipase D (PLD) in hippocampal slices
(Pellegrini-Giampietro et al., 1996) and preliminary experiments have indicated the presence of a PLD activity in the synaptosomal preparation with this anomalous pharmacology. Activation of presynaptic PKC by 5 nM PDBu results in a rapid desensitization of the facilitatory mGluR pathway in synaptosomes, and ACPD itself, even in the absence of added AA, causes homologous desensitization of the receptor (Herrero et al., 1994). This rapid desensitization may help to explain some of the difficulties experienced in observing the facilitatory pathway in brain slices, where the presence of extracellular glutamate may be sufficient to cause desensitization. the time-course of recovery of receptor function is slow, taking some 20 min in synaptosomes following ACPD addition, and this delay is further enhanced in the presence of okadaic acid (Herrero et al., 1994).
Receptor-mediated inhibition of glutamate exocytosis In addition to the facilitatory pathway, presynaptic glutamatergic terminals possess a variety of presynaptic inhibitory receptors (also see Bruno et al., this volume). Adenosine, acting on A1 receptors located both pre- and postsynaptically, is a potent inhibitor of neurotransmission (Fredholm and Dunwiddie, 1988). Postsynaptically adenosine hyperpolarizes by activating K -channels (De Mendonqa and Ribeiro, 1994) while the presynaptic receptor acts via a pertussis toxin-sensitive Gprotein (Dolphin and Prestwich, 1985) to inhibit the release of glutamate and other neurotransmitters (Burke and Nadler, 1988; Barrie and Nicholls, 1993). The exact presynaptic mechanism is still a matter of debate: adenosine A1 inhibition of neurotransmitter release in the avian ciliary ganglion is consistent with an activation of K t channels resulting in hyperpolarization (Bennett and Ho, 1992), however adenosine is an effective inhibitor of Ca2+ elevation and glutamate release in synaptosomes subjected to clamped KCl depolarization (Barrie and Nicholls, 1993). This is in direct contrast to the conditions discussed above +
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for observing PKC-dependent facilitation and strongly suggests a direct inhibition of a releasecoupled calcium channel, consistent with hippocampal CA1 neurons where an A1 agonist reduced presynaptic Ca2+ transients (Wu and Saggau, 1994). An inhibitory presynaptic mGluR sensitive to the agonist L-AP4 can be observed in synaptosoma1 preparations from young (1-3 week PP) but not adult rats (Vazquez et al., 1995) and contrasts with the facilitatory pathway which is not seen until week 3 but remains in terminals from the adult (Vazquez et al., 1995). This is consistent with the developmentally regulated depression of synaptic transmission by L-AP4 in the hippocampus (Baskys and Malenka, 1991), whereas in the striatum L-AP4 inhibits glutamate release from synaptosomes prepared from adult rats (East et al., 1995). The mechanism of the L-APCsensitive pathway is closely parallel to that of the adenosine A1 receptor: inhibition of both Ca2+ elevation and glutamate release is observed during clamped KCl depolarization and is insensitive to protein kinase inhibitors (Vazquez et al., 1995). The presynaptic L-AP4 receptor isoform is not clearly established: mGluR 4, 7 and 8 are expressed in the cortex (Kristensen et al., 1993; Pin and Duvoisin, 1995). ACPD-sensitive, L-AP44nsensitive inhibition is seen in the striatum acting via a pharmacologically identifiable mGluR2/3 (Lovinger, 1991). However, as discussed above this receptor appears not to be subject to the same developmental control as the cortical/hippocampal isoform/s. PKC and heterologous desensitization
At the presynaptic terminal inhibitory receptor signal transduction pathways appear to involve a direct coupling of G-protein to the release-coupled Ca2+ channel (Barrie and Nicholls, 1993) without generation of a detectable second messenger. While the signal transduction pathways do not involve PKC they are very sensitive to PKC activation in situ and a rapid loss of inhibition is observed when PKC is activated by phorbol esters (Barrie and Nicholls, 1993; Budd and Nicholls,
1995). It is important in this context to distinguish between an in vitro suppression of a receptormediated pathway due to non-physiological activation of PKC by phorbol esters and one which is agonist evoked and might possibly have a physiological function. In contrast to the facilitatory pathway, the presynaptic inhibitory receptors do not undergo homologous desensitization. However, the adenosine and L-AP4 mediated inhibitory pathways are each suppressed when PKC is activated either by phorbol esters (Barrie and Nicholls, 1993) or by activation of the facilitatory pathway (Vazquez et al., 1995; Budd and Nicholls, 1995). This indicates incidentally that the facilitatory and inhibitory receptors are present on the same nerve terminals: indeed since no additivity is seen with the two inhibitory agonists in three week rats (Vazquez et al., 1995) the three receptors must coexist on a high proportion of cortical terminals. The site at which PKC may act to suppress the inhibitory responses has recently been clarified by expression of chimeric Ca” channel C ~ I Aand N I B subunits in HEK cells, subunits with a2 and where they could be inhibited by an endogenous somatostatin receptor. PKC-dependent phosphorylation of residues within the calcium channel’s binding site for the modulatory GPy disrupted the interaction of the G-protein subunits with the channel (Zamponi et al., 1997). AA is not required for the ACPD-mediated suppression of the inhibitory pathways (Budd and Nicholls, 1995) but greatly prolongs the duration of the suppression. The rapid reversal of the suppression in the absence of AA indicates that an active phosphatase is present to reverse the PKCmediated suppression, in contrast to the slow phosphatase activity discussed above for the homologous desensitization of the facilitatory receptor. AA is known to facilitate insertion of PKC into the membrane (Lester and Bramham, 1993), while synergistic activation of protein kinase C by arachidonic acid and diacylglycerol lead to the generation of a stable membrane-bound, cofactorindependent state of protein kinase C activity (Schachter et al., 1996). In the terminal this might
19
be predicted to result in a constitutively active PKC that remains active after the facilitatory mGluR has desensitized. However, to our surprise we find that ACPD in the absence of AA results in a translocation of several PKC isoforms to the synaptosomal membrane fraction, and that this translocation persists for several minutes (unpublished). This together with the evidence that the anomalous pharmacology of the facilitatory receptor is associated with PLD activation implies that the signal transduction pathways in the glutamatergic terminal responsible for PKC activation may be more complex than was originally proposed.
-
NMDA-R 1
-I
Physiological correlates The model system of the synaptosome discussed above shows an extensive but transient PKCdependent facilitation of glutamate exocytosis in the simultaneous presence of a glutamate agonist and a low concentration of AA. This, together with the parallel suppression of presynaptic inhibitory receptor pathways and the apparently ubiquitous presence of these pathways in cortical and hippocampal glutamatergic terminals is suggestive that these pathways might play a role in synaptic plasticity. An attractive possibility is that the AA
AMPA-R
-
Fig. 1. Schematic representation of the interactions between facilitatory and inhibitory presynaptic receptors controlling glutamate exocytosis. The scheme depicts the pre- and post-synaptic membranes of a hypothetical plastic glutamatergic synapse. A condition of maximal glutamate exocytosis is shown, such as that during the induction of long-term potentiation: post-synaptic NMDA receptor (NMDA-R) activation results in the generation of arachidonic acid (Miller et al., 1992). Overflow of glutamate in the synaptic cleft activates the facilitatory glutamate receptor (mGluR (facil); Herrero et al., 1992b), resulting in the activation of PKC. PKC rapidly desensitizes mGluR (facil), however in the presence of arachidonate as a putative retrograde messenger, PKC continues to be active even after mGluR desensitization. PKC activation results in an enhanced phosphorylation of the presynaptic phosphoproteins GAP-43 and MARCKS, an enhanced presynaptic depolarization (detected in synaptosomal preparations during 4-AP induced excitation (Coffey et al., 1993) and heterologous desensitization or suppression of inhibitory presynaptic receptors, including the adenosine A1 receptor (Barrie and Nicholls, 1993) and the inhibitory mGluR present on terminals from immature rats (VLquez et al., 1995). This retrograde messenger-dependent facilitation of release and suppression of inhibition could ensure that glutamate exocytosis is maintained at maximal probability to ensure the completion of LTP induction.
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originates postsynaptically and that the PLAz is activated under conditions where the NMDA receptor is active during the establishment of synaptic plasticity. The additional presence of transmitter glutamate in the synaptic cleft would provide the synergy necessary for DAG production and PKC sensitization and could serve to reinforce the release of glutamate occurring during the period required for the establishment of synaptic plasticity. Inhibitory presynaptic receptors become operative when release is enhanced as a consequence of high-frequency stimulation (Scanziani et al., 1997; Takumi et al., this volume); the decoupling of inhibitory presynaptic receptors, would thus remove any opposing influence and further enhance the probability of release. A number of possible sources for AA in vivo can be proposed: one would be phospholipase A2 activated post-synaptically in response to NMDA receptor activation (Dumuis et al., 1990) - thus providing a mechanistic basis for proposals that AA could function as a retrograde messenger during plastic changes at the synapse (Williams et al., 1989). Weak activation of the perforant path paired with application of AA can lead to a slowonset increase in synaptic efficacy (Williams et al., 1989), while AA and diacylglycerol induce a synergistic facilitation of Ca2+-dependent glutamate release from hippocampal mossy fiber nerve endings (Zhang et al., 1996). Furthermore, the synergism between metabotropic glutamate receptor activation and arachidonic acid on glutamate release is occluded by induction of long-term potentiation in the dentate gyrus (McGahon and Lynch, 1996). While it is unreasonable that such a mechanism would chronically elevate glutamate release, a short-lasting enhancement during the induction of potentiation could act to ensure that glutamate release remained high until a synapse is securely switched to the potentiated form. Therapeutic implications
Agonists and antagonists targetting the presynaptic glutamatergic terminal greatly influence the
in vitro and in vivo release of the transmitter. The co-existence of facilitatory and inhibitory autoreceptors on the same terminal, their complex interplay and the possible influence of exogenous co-activators of PKC, such as arachidonic acid, imply that the effect of these agents on transmitter release may not always be predictable. One reason underlying the lack of consensus as to whether mGluR agonists are neuroprotective (Bruno et al., this volume) may lie in the complications resulting from these complex signal transduction pathways. References Agoston, D.V., Hargittai, P. and Nagy, A. (1983) Effects of 4AP in Ca movements and changes of membrane potential in pinched-off nerve terminals from rat cerebral cortex. J. Neurochem., 41: 745-751. Augustine, G.J., Charlton, M.P. and Smith, S.J. (1985) Calcium entry and transmitter release at voltage-clamped nerve terminals of squid. J. Physiol. Lond., 367: 163-181. Barrie, A.P. and Nicholls, D.G. (1993) Adenosine A1 receptor inhibition of glutamate exocytosis and protein kinase Cmediated decoupling. J. Neurochem., 6 0 1081-1086. Bame, A.P., Nicholls, D.G., Sanchez-Prieto, J. and Sihra, T.S. (1991) An ion channel locus for the protein kinase C potentiation of transmitter glutamate release from guinea pig cerebrocortical synaptosomes. J. Neurochem., 57: 13981404. Baskys, A. and Malenka, R.C. (1991) Agonists at metabotropic glutamate receptors presynaptically inhibit EPSCs in neonatal rat hippocampus. J. Physiol., (Lond), 444: 687701. Bennett, M.R. and Ho, S. (1992) Adenosine modulation of potassium currents in preganglionic nerve terminals of avian ciliary ganglia. Neurosci. Lett., 137: 4 1 4 . Budd, D.C. and Nicholls, D.G. (1995) Protein kinase C mediated decoupling of the presynaptic adenosine A1 receptor by a facilitatory metabotropic glutamate receptor. J. Neurochem., 65: 611621. Burke, S.P. and Nadler, J.V. (1988) Regulation of glutamate and aspartate release from slices of the hippocampal CAI area: effects of adenosine and baclofen. J . Neurochem., 51: 1541-1551. Clements, J.D., Lester, R.A.J., Tong, G., Jahr, C.E. and Westbrook, G.L. (1992) The time course of glutamate in the synaptic cleft. Science, 258: 1498-1501. Coffey, E.T., Herrero, I., Sihra, T.S., Sanchez-Prieto, J. and Nicholls, D.G. (1994) Glutamate exocytosis and MARKS phosphorylation are enhanced by a metabotropic glutamate receptor coupled to a protein kinase C synergistically
21 activated by diacylglycerol and arachidonic acid. J. Neurochem., 63: 1303-1310. Coffey, E.T., Sihra, T.S. and Nicholls, D.G. (1993) Protein kinase C and the regulation of glutamate exocytosis from cerebrocortical synaptosomes. J. Biol. Chem., 268: 2106C&21065. De MendonGa, A. and Ribeiro, J.A. (1994) Endogenous adenosine modulates long-term potentiation in the hippocampus. Neurosci., 62: 385-390. Dolezal, V. and Tucek, S. (1983) The effects of 4-aminopyridine and tetrodotoxin on the release of acetylcholine from rat striatal slices. Naunyn-Schmied. Arch. Pharmacol., 323: 9095. Dolphin, A.C. and Prestwich, S.A. (1985) Pertussis toxin reverses adenosine inhibition of neuronal glutamate release. Nature, 316: 148-150. Dumuis, A., Pin, J.P., Oomagari, K., Sebben, M. and Bockaert, J. (1990) Arachidonic acid released from striatal neurons by joint stimulation by ionotropic and metabotropic quisqualate receptors. Nature, 347: 182--184. East, S.J., Hill, M.P. and Brotchie, J.M. (1995) Metabotropic glutamate receptor agonists inhibit endogenous glutamate release from rat striatal synaptosomes. Eur. J. Pharmacol., 277: 117-121. Fredholm, B.B. and Dunwiddie, T.V. (1988) How does adenosine inhibit transmitter release? (TIPS Review). Trends Pharmacol. Sci., 9: 13G135. Herrero, I., Miras-Portugal, M.T. and Sanchez-Prieto, J. (1992a) Activation of protein kinase C by phorbol esters and arachidonic acid required for the optimal potentiation of glutamate exocytosis. J. Neurochem., 59: 157k-1577. Herrero, I., Miras-Portugal, M.T. and Sanchez-Prieto, J. (1992b) Positive feedback of glutamate exocytosis by metabotropic presynaptic receptor stimulation. Nature, 360: 163-166. Herrero, I., Mirds-Portugal. M.T. and Sanchez-Prieto, J. (1994) Rapid desensitization of the presynaptic metabotropic receptor for glutamate that facilitates glutamate release. Eur. J. Neurosci., 6: 115-120. Hessler, N.A., Shirke, A.M. and Malinow, R. (1993) The probability of transmitter release at a mammalian central synapse. Nature, 366: 569-572. Huang, H.Y., Hertting, G., Allgaier, C. and Jackisch, R. (1989) 3:4-Diaminopyridine-induced noradrenaline release from CNS tissue as a model for action potential-evoked transmitter release: effects of phorbol ester. Eur. J . Pharmacol., 169: 115-123. Klein, J., Chalifa, V., Liscovitch, M. and Loffelholz, K. (1995) Role of phospholipase D activation in nervous system physiology and pathophysiologq. J. Neurochem., 65: 1445-1455. Kristensen, P., Suzdak, P.D. and Thomsen, C. (1993) Expression pattern and pharmacology of the rat type IV metabotropic glutamate receptor. Neurosci. Lett., 155: 159-162.
Lester, D.S. and Bramham, C.R. (1993) Persistent, membraneassociated protein kinase C: From model membranes to synaptic long-term potentiation. Cell Signalling, 5: 695-708. Lovinger, D.M. (1991) Trans-l-aminocyclopentane-1,3-dicarboxylic acid (tert-ACPD) decreases synaptic excitation in rat striatal slices through a presynaptic action. Neurosci. Lett., 129: 17-21. McGahon, B. and Lynch, M.A. (1996) The synergism between metabotropic glutamate receptor activation and arachidonic acid on glutamate release is occluded by induction of longterm potentiation in the dentate gyrus. Neurosci., 72: 847855. McMahon, H.T., Foran, P., Dolly, J.O., Verhage, M., Wiegant, V.M. and Nicholls, D.G. (1992) Tetanus toxin and botulinum toxins type A and B inhibit glutamate, y-aminobutyric acid, aspartate, and met-enkephalin release from synaptosomes. Clues to the locus of action. J. Biol. Chem., 267: 21338-21343. McMahon, H.T. and Nicholls, D.G. (1991) Transmitter glutamate release from isolated nerve terminals: Evidence for biphasic release and triggering by localized Ca*+. J. Neurochem., 56: 8G94. Nicholls, D.G. (1995a) The release of glutamate from synaptic terminals. In T.W. Stone (Ed.), Glutamate. (pp. 35-52). New York: CRC Press. Nicholls, D.G. (1995b) Mechanisms of glutamate release. In H. Wheal & A. Thomson (Eds.), Excitatory Amino Acids and Synaptic Transmission. (pp. 1-15). London: Academic Press. Nicholls, D.G. and Coffey, E.T. (1994) Glutamate exocytosis from isolated nerve terminals. In L. Stjame, P. Greengard, T. Hokfelt, & D. Ottoson (Eds.), Molecular and Cellular Mechanisms of Neurotransmitter Release. (pp. 189-204). New York: Raven Press. Nicholls, D.G. and Sanchez-Prieto, J. (1997) Neurotransmitter release mechanisms. In A.J. Turner & F.A. Stephenson (Eds.), Amino Acid Neurotransmission. London: Portland Press. Nicholls, D.G. and Sihra, T.S. (1986) Synaptosomes possess an exocytotic pool of glutamate. Nature, 321: 772-773. Pellegrini-Giampietro, D.E., Torregrossa, S.A. and Moroni, F. (1996) Pharmacological characterization of metabotropic glutamate receptors coupled to phospholipase D in the rat hippocampus. Br. J . Pharmacol., 118: 1035-1043. Pin, J.-P. and Duvoisin, R. (1995) The metabotropic glutamate receptors: structure and functions. Neuropharmacol. 34: 1-26. Sanchez-Prieto, J., Sihra, T.S. and Nicholls, D.G. (1987) Characterization of the exocytotic release of glutamate from guinea-pig cerebral cortical synaptosomes. J. Neurochem., 49: 58-64. Scanziani, M., Salin, P.A., Vogt, K.E., Malenka, R.C. and Nicoll, R.A. (1997) Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors. Nature, 385: 63M34.
22 Schachter, J.B., Lester, D.S. and Alkon, D.L. (1996) Synergistic activation of protein kinase C by arachidonic acid and diacylglycerols in vitro: Generation of a stable membranebound, cofactor-independent state of protein kinase C activity. Biochim. Biophys. Acta, 1291: 167-176. Schoepp, D.D., Goldsworthy, J., Johnson, B.G., Salhoff, C.R. and Baker, S.R. (1994) 3,5-dihydroxyphenylglycine is a highly selective agonist for phosphoinositide-linked metabotropic glutamate receptors in the rat hippocampus. J. Neurochem., 63: 769-772. Segal, M. (1989) Synaptic transmission between cultured rat hippocampal neurons is enhanced by activation of protein kinase-C. Neurosci. Lett., 101: 169 Tapia, R. and Sitges, M. (1982) Effect of 4-aminopyridine on transmitter release in synaptosomes. Brain Res., 250: 291299. Tibbs, G.R., Barrie, A.P., Van-Mieghem, F., McMahon, H.T. and Nicholls, D.G. (1989) Repetitive action potentials in isolated nerve terminals in the presence of 4-aminopyridine: Effects on cytosolic free Ca2' and glutamate release. J. Neurochem., 53: 1693-1699. Vazquez, E., Budd, D., Herrero, I., Nicholls, D.G. and Sanchez-Prieto, J. (1995) Co-existence and interaction between facilitatory and inhibitory metabotropic glutamate receptors and the inhibitory adenosine A1 receptor in cerebrocortical nerve terminals. Neuropharmacol., 3 4 919927. Vazquez, E., Herrero, I., Miras-Portugal, M.T. and SanchezPrieto, J. (1995) Developmental change from inhibition to
facilitation in the presynaptic control of glutamate exocytosis by metabotropic glutamate receptors. Neurosci., 68: 117124. Verhage, M., McMahon, H.T., Ghijsen, W.E.J.M., Boomsma, F., Wiegant, V. and Nicholls, D.G. (1991) Differential release of amino acids, neuropeptides and catecholamines from nerve terminals. Neuron, 6: 517-524. Wilkinson, R. and Nicholls, D.G. (1989) Compartmentation of glutamate and aspartate within cerebral cortical synaptosomes: evidence for a non-cytoplasmic origin for the Ca releasable pool of glutamate. Neurochem. In?., 15: 191-197. Williams, J.H., Errington, M.L., Lynch, M.A. and Bliss, T.V.P. (1989) Arachidonic acid induces a long-term activitydependent enhancement of synaptic transmission in the hippocampus. Nature, 341: 739-741. Wu, L. and Saggau, P. (1994) Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CAI of hippocampus. Neuron, 12: 1139-1 148. Zamponi, G.W., Bourinet, E., Nelson, D., Nargeot, J. and Snutch, T.P. (1997) Crosstalk between G proteins and protein kinase C mediated by the calcium channel a, subunit. Nature, 385: 442446. Zhang, L., Ruehr, M.L. and Dorman, R.V. (1996) Arachidonic acid and oleoylacetylglycerol induce a synergistic facilitation of Ca'+-dependent glutamate release from hippocampal mossy fiber nerve endings. J. Neurochem., 66: 177-185.
CHAPTER 2
Presynaptic modulation of glutamate release David G. Nicholls Neurosciences Institute. Department of Pharmacology, University of Dundee, Dundee DDI 9SY. Scotland, UK
Introduction The arrival of an action potential at a CNS glutamatergic varicosity does not automatically result in exocytosis of the transmitter. Instead glutamatergic varicosities in brain regions capable of synaptic plasticity show a low quanta1 release probability (Hessler et al., 1993), such that only a proportion of the action potentials passing through the varicosity lead to the release of a synaptic vesicle at a given release site. Since an action potential must pass through a varicosity to continue its propagation, this implies that there is an inherent unreliability in the coupling between the action potential and exocytosis. The high power dependency upon external Ca2 concentration of exocytosis (Augustine et al., 1985) suggests that Ca2’- entry may be the prime probability-determining step in action potential/ exocytosis coupling. Presynaptic receptors may either facilitate or inhibit release by directly or indirectly modulating this Ca2+ entry and their existence enormously enhances the potential complexity of synaptic transmission by allowing release to be controlled at each individual varicosity along the axon. The exocytosis of a single synaptic vesicle can elevate the concentration of the amino acid in the synaptic cleft to around 1mM (Clements et al., 1992), sufficient to saturate the ionotropic receptors at the post-synaptic membrane. Presynaptic +
receptors could modulate the probability of vesicle release in response to an invading action potential by a number of mechanisms: firstly by altering the kinetics of K + or Na+ channels and influencing the waveform of the depolarization; secondly by activating or inhibiting the releasecoupled Ca2+ channel and directly controlling the access of Ca2+ to the exocytotic trigger, or thirdly by acting at an intracellular locus to control some aspect of the exocytotic/endocytic cycle. The evidence, obtained from studies with isolated nerve terminals (synaptosomes) will now be reviewed that the two classes of receptor may modulate exocytosis by respectively enhancing presynaptic action potentials and inhibiting Ca2+ channels. (see Turner, this volume, for a description of Ca2+ channels coupled to glutamate release.)
Synaptosomes and the study of glutamate exocytosis Synaptosomes have proved a valuable preparation for the investigation of these presynaptic regulatory pathways (reviewed in Nicholls and SanchezPrieto, 1997; Nicholls, 1995a,b). Glutamate exocytosis greatly outweighs the release of other transmitters in preparations from cerebral cortex, hippocampus or cerebellum and is the simplest preparation which retains all the pathways for the synthesis, storage, release and reuptake of trans-
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mitter glutamate, together with a range of presynaptic receptors capable of modulating the release of the transmitter. In recent years this preparation has provided much information on the signal transduction pathways present in the glutamatergic terminal, and these provide the topic of this review. The release of glutamate can be monitored continuously by including glutamate dehydrogenase and NADP+ in the incubation medium (Nicholls and Sihra, 1986; Sanchez-Prieto et al., 1987), the released glutamate being coupled to the generation of fluorescent NADPH. The Ca2+dependent component of KC1-evoked glutamate release amounts to some 15% of the total glutamate content of the preparation (McMahon and Nicholls, 1991) and is exocytotic judged by its ATP requirement (Sanchez-Prieto et al., 1987), inhibition by tetanus and botulinum toxins (McMahon et al., 1992) and by compartmental analysis (Wilkinson and Nicholls, 1989). In addition to the conventional KC1 depolarization technique for evoking transmitter release, a second and potentially more versatile approach for evoking glutamate exocytosis is by K -A type channel inhibition (Tibbs et al., 1989). A-type K + channels activate very rapidly in response to slight depolarizations and serve as negative feedback mechanisms limiting statistical fluctuations in membrane potential from triggering spontaneous action potentials. Such channels appear to exist in nerve terminals, sensitive to a-dendrotoxin or to low concentrations of 4-aminopyridine (4AP) (Tapia and Sitges, 1982; Agoston et al., 1983; Dolezal and Tucek, 1983; Tibbs et al., 1989). Either agent causes a Ca2+-dependent, tetrodotoxin-sensitive exocytosis of glutamate from the same pool as for KCl, and utilizes the same non-L non-N type Ca2+ channels (Tibbs et al., 1989), triggering release via a highly localized pool of Ca2+ in the immediate vicinity of the Ca2+ channel (Verhage et al., 1991). The selective use of these two means of synaptosomal excitation have been central to studies dissecting the presynaptic signal transduction pathways controlling glutamatergic transmission. +
PKC and the receptor-mediated facilitation of transmitter release It has been established for some time that the application of phorbol esters leads to an enhancement of glutamate release, which can be observed with preparations from the brain slice to the isolated synaptosome (Huang et al., 1989; Segal, 1989; Barrie et al., 1991; Hefrero et al., 1992; Coffey et al., 1993). In our studies, a key finding was that the ability of phorbol esters to enhance glutamate exocytosis from cerebrocortical synaptosomes was dependent on the mode by which the preparation was depolarized: exocytosis evoked by a clamped depolarization by elevated KCl was independent of PKC activity - either phorbol ester mediated activation, or inhibition by Ro 31-8220 (Barrie et al., 1991; Coffey et al., 1993). By monitoring the phosphorylation state of endogenous MARCKS during these experiments it was possible to confirm that PKC activity was being controlled in the expected manner by these agents (Coffey et al., 1993). Thus phosphorylation of a PKC substrate is not necessary for vesicle exocytosis per se. In contrast, the use of 4AP or adendrotoxin to evoke spontaneous action potential firing in the synaptosome preparation resulted in an exocytosis which was totally controlled by PKC activity: phorbol esters could cause a 500% stimulation of release while Ro 3 1-8220 could virtually abolish the ability of 4AP to evoke release (Coffey et al., 1993). Since the distinction between 4AP-evoked glutamate release and that evoked by high KC1 is the tetrodotoxin-sensitive firing of spontaneous ‘action potentials’ (Tibbs et al., 1989), the channels involved in this process provides an obvious locus at which to search for an action of PKC. The use of membrane potential-dependent dyes to monitor the time- and population average depolarization of the synaptosomal preparation reveals that phorbol esters enhance the 4AP-evoked depolarization, indicative of an effect on an ion channel implicated in action potential firing (Barrie et al., 1991; Herrero et al., 1992a; Coffey et al., 1993; Coffey et al., 1994). The effect of the phorbol ester is
17
mimicked and occluded by Ba2+ or by clofilium (Nicholls and Coffey, 1994); thus the most likely locus is a 4AP-insensitive, a-dendrotoxin-insensitive, Ba2+-sensitive, clofilium-sensitive K + channel. The PKC isoform mediating the facilitated glutamate release is potentiated by arachidonic acid (AA) at concentrations of 1-2 pM (Herrero et al., 1992a,b). In the presence of AA the threshold concentration of phorbol ester required to observe facilitation is greatly decreased (Herrero et al., 1992a). The dominant physiological agonist activating presynaptic PKC appears to be glutamate itself, since in the presence of AA a facilitation of 4AP-evoked glutamate release is observed (Herrero et al., 1992b) on addition of the mGluR agonist (lS, 3R) 1-aminocyclopentane1,3-dicarboxylic acid (ACPD). ACPD activates a metabotropic glutamate receptor (mGluR) which is coupled (in the presence of AA) to enhanced phosphorylation of the established PKC substrates MARCKS and GAP-43 (Coffey et al., 1994). The two established pathways for the formation of the diacylglycerol (DAG) required for the activation of PKC are its direct generation by phospholipase C (PLC) mediated hydrolysis of phosphatidylinositol bisphosphate and the alternative activation of phospholipase D with the initial generation of phosphatidic acid followed by its hydrolysis to DAG (for review see Klein et al., 1995). While a large and transient elevation in DAG has been reported in response to ACPD (Herrero et al., 1994), recent studies in our laboratory (unpublished) indicate a more modest (5&70% elevation) but more prolonged elevation in diacylglycerol (persisting for 15 min even in the absence of AA) in response to 100 pM ACPD. Analysis of the pharmacology of the facilitated glutamate release reveals some anomalous features: firstly ACPD-mediated enhancement is inhibited by (RS)-3,5-dihydroxyphenylglycine (DHPG) which is reported to be a selective group I mGluR agonist (Schoepp et al., 1994). An antagonist activity of DHPG has previously been reported against the ACPD-mediated activation of phospholipase D (PLD) in hippocampal slices
(Pellegrini-Giampietro et al., 1996) and preliminary experiments have indicated the presence of a PLD activity in the synaptosomal preparation with this anomalous pharmacology. Activation of presynaptic PKC by 5 nM PDBu results in a rapid desensitization of the facilitatory mGluR pathway in synaptosomes, and ACPD itself, even in the absence of added AA, causes homologous desensitization of the receptor (Herrero et al., 1994). This rapid desensitization may help to explain some of the difficulties experienced in observing the facilitatory pathway in brain slices, where the presence of extracellular glutamate may be sufficient to cause desensitization. the time-course of recovery of receptor function is slow, taking some 20 min in synaptosomes following ACPD addition, and this delay is further enhanced in the presence of okadaic acid (Herrero et al., 1994).
Receptor-mediated inhibition of glutamate exocytosis In addition to the facilitatory pathway, presynaptic glutamatergic terminals possess a variety of presynaptic inhibitory receptors (also see Bruno et al., this volume). Adenosine, acting on A1 receptors located both pre- and postsynaptically, is a potent inhibitor of neurotransmission (Fredholm and Dunwiddie, 1988). Postsynaptically adenosine hyperpolarizes by activating K -channels (De Mendonqa and Ribeiro, 1994) while the presynaptic receptor acts via a pertussis toxin-sensitive Gprotein (Dolphin and Prestwich, 1985) to inhibit the release of glutamate and other neurotransmitters (Burke and Nadler, 1988; Barrie and Nicholls, 1993). The exact presynaptic mechanism is still a matter of debate: adenosine A1 inhibition of neurotransmitter release in the avian ciliary ganglion is consistent with an activation of K t channels resulting in hyperpolarization (Bennett and Ho, 1992), however adenosine is an effective inhibitor of Ca2+ elevation and glutamate release in synaptosomes subjected to clamped KCl depolarization (Barrie and Nicholls, 1993). This is in direct contrast to the conditions discussed above +
18
for observing PKC-dependent facilitation and strongly suggests a direct inhibition of a releasecoupled calcium channel, consistent with hippocampal CA1 neurons where an A1 agonist reduced presynaptic Ca2+ transients (Wu and Saggau, 1994). An inhibitory presynaptic mGluR sensitive to the agonist L-AP4 can be observed in synaptosoma1 preparations from young (1-3 week PP) but not adult rats (Vazquez et al., 1995) and contrasts with the facilitatory pathway which is not seen until week 3 but remains in terminals from the adult (Vazquez et al., 1995). This is consistent with the developmentally regulated depression of synaptic transmission by L-AP4 in the hippocampus (Baskys and Malenka, 1991), whereas in the striatum L-AP4 inhibits glutamate release from synaptosomes prepared from adult rats (East et al., 1995). The mechanism of the L-APCsensitive pathway is closely parallel to that of the adenosine A1 receptor: inhibition of both Ca2+ elevation and glutamate release is observed during clamped KCl depolarization and is insensitive to protein kinase inhibitors (Vazquez et al., 1995). The presynaptic L-AP4 receptor isoform is not clearly established: mGluR 4, 7 and 8 are expressed in the cortex (Kristensen et al., 1993; Pin and Duvoisin, 1995). ACPD-sensitive, L-AP44nsensitive inhibition is seen in the striatum acting via a pharmacologically identifiable mGluR2/3 (Lovinger, 1991). However, as discussed above this receptor appears not to be subject to the same developmental control as the cortical/hippocampal isoform/s. PKC and heterologous desensitization
At the presynaptic terminal inhibitory receptor signal transduction pathways appear to involve a direct coupling of G-protein to the release-coupled Ca2+ channel (Barrie and Nicholls, 1993) without generation of a detectable second messenger. While the signal transduction pathways do not involve PKC they are very sensitive to PKC activation in situ and a rapid loss of inhibition is observed when PKC is activated by phorbol esters (Barrie and Nicholls, 1993; Budd and Nicholls,
1995). It is important in this context to distinguish between an in vitro suppression of a receptormediated pathway due to non-physiological activation of PKC by phorbol esters and one which is agonist evoked and might possibly have a physiological function. In contrast to the facilitatory pathway, the presynaptic inhibitory receptors do not undergo homologous desensitization. However, the adenosine and L-AP4 mediated inhibitory pathways are each suppressed when PKC is activated either by phorbol esters (Barrie and Nicholls, 1993) or by activation of the facilitatory pathway (Vazquez et al., 1995; Budd and Nicholls, 1995). This indicates incidentally that the facilitatory and inhibitory receptors are present on the same nerve terminals: indeed since no additivity is seen with the two inhibitory agonists in three week rats (Vazquez et al., 1995) the three receptors must coexist on a high proportion of cortical terminals. The site at which PKC may act to suppress the inhibitory responses has recently been clarified by expression of chimeric Ca” channel C ~ I Aand N I B subunits in HEK cells, subunits with a2 and where they could be inhibited by an endogenous somatostatin receptor. PKC-dependent phosphorylation of residues within the calcium channel’s binding site for the modulatory GPy disrupted the interaction of the G-protein subunits with the channel (Zamponi et al., 1997). AA is not required for the ACPD-mediated suppression of the inhibitory pathways (Budd and Nicholls, 1995) but greatly prolongs the duration of the suppression. The rapid reversal of the suppression in the absence of AA indicates that an active phosphatase is present to reverse the PKCmediated suppression, in contrast to the slow phosphatase activity discussed above for the homologous desensitization of the facilitatory receptor. AA is known to facilitate insertion of PKC into the membrane (Lester and Bramham, 1993), while synergistic activation of protein kinase C by arachidonic acid and diacylglycerol lead to the generation of a stable membrane-bound, cofactorindependent state of protein kinase C activity (Schachter et al., 1996). In the terminal this might
19
be predicted to result in a constitutively active PKC that remains active after the facilitatory mGluR has desensitized. However, to our surprise we find that ACPD in the absence of AA results in a translocation of several PKC isoforms to the synaptosomal membrane fraction, and that this translocation persists for several minutes (unpublished). This together with the evidence that the anomalous pharmacology of the facilitatory receptor is associated with PLD activation implies that the signal transduction pathways in the glutamatergic terminal responsible for PKC activation may be more complex than was originally proposed.
-
NMDA-R 1
-I
Physiological correlates The model system of the synaptosome discussed above shows an extensive but transient PKCdependent facilitation of glutamate exocytosis in the simultaneous presence of a glutamate agonist and a low concentration of AA. This, together with the parallel suppression of presynaptic inhibitory receptor pathways and the apparently ubiquitous presence of these pathways in cortical and hippocampal glutamatergic terminals is suggestive that these pathways might play a role in synaptic plasticity. An attractive possibility is that the AA
AMPA-R
-
Fig. 1. Schematic representation of the interactions between facilitatory and inhibitory presynaptic receptors controlling glutamate exocytosis. The scheme depicts the pre- and post-synaptic membranes of a hypothetical plastic glutamatergic synapse. A condition of maximal glutamate exocytosis is shown, such as that during the induction of long-term potentiation: post-synaptic NMDA receptor (NMDA-R) activation results in the generation of arachidonic acid (Miller et al., 1992). Overflow of glutamate in the synaptic cleft activates the facilitatory glutamate receptor (mGluR (facil); Herrero et al., 1992b), resulting in the activation of PKC. PKC rapidly desensitizes mGluR (facil), however in the presence of arachidonate as a putative retrograde messenger, PKC continues to be active even after mGluR desensitization. PKC activation results in an enhanced phosphorylation of the presynaptic phosphoproteins GAP-43 and MARCKS, an enhanced presynaptic depolarization (detected in synaptosomal preparations during 4-AP induced excitation (Coffey et al., 1993) and heterologous desensitization or suppression of inhibitory presynaptic receptors, including the adenosine A1 receptor (Barrie and Nicholls, 1993) and the inhibitory mGluR present on terminals from immature rats (VLquez et al., 1995). This retrograde messenger-dependent facilitation of release and suppression of inhibition could ensure that glutamate exocytosis is maintained at maximal probability to ensure the completion of LTP induction.
20
originates postsynaptically and that the PLAz is activated under conditions where the NMDA receptor is active during the establishment of synaptic plasticity. The additional presence of transmitter glutamate in the synaptic cleft would provide the synergy necessary for DAG production and PKC sensitization and could serve to reinforce the release of glutamate occurring during the period required for the establishment of synaptic plasticity. Inhibitory presynaptic receptors become operative when release is enhanced as a consequence of high-frequency stimulation (Scanziani et al., 1997; Takumi et al., this volume); the decoupling of inhibitory presynaptic receptors, would thus remove any opposing influence and further enhance the probability of release. A number of possible sources for AA in vivo can be proposed: one would be phospholipase A2 activated post-synaptically in response to NMDA receptor activation (Dumuis et al., 1990) - thus providing a mechanistic basis for proposals that AA could function as a retrograde messenger during plastic changes at the synapse (Williams et al., 1989). Weak activation of the perforant path paired with application of AA can lead to a slowonset increase in synaptic efficacy (Williams et al., 1989), while AA and diacylglycerol induce a synergistic facilitation of Ca2+-dependent glutamate release from hippocampal mossy fiber nerve endings (Zhang et al., 1996). Furthermore, the synergism between metabotropic glutamate receptor activation and arachidonic acid on glutamate release is occluded by induction of long-term potentiation in the dentate gyrus (McGahon and Lynch, 1996). While it is unreasonable that such a mechanism would chronically elevate glutamate release, a short-lasting enhancement during the induction of potentiation could act to ensure that glutamate release remained high until a synapse is securely switched to the potentiated form. Therapeutic implications
Agonists and antagonists targetting the presynaptic glutamatergic terminal greatly influence the
in vitro and in vivo release of the transmitter. The co-existence of facilitatory and inhibitory autoreceptors on the same terminal, their complex interplay and the possible influence of exogenous co-activators of PKC, such as arachidonic acid, imply that the effect of these agents on transmitter release may not always be predictable. One reason underlying the lack of consensus as to whether mGluR agonists are neuroprotective (Bruno et al., this volume) may lie in the complications resulting from these complex signal transduction pathways. References Agoston, D.V., Hargittai, P. and Nagy, A. (1983) Effects of 4AP in Ca movements and changes of membrane potential in pinched-off nerve terminals from rat cerebral cortex. J. Neurochem., 41: 745-751. Augustine, G.J., Charlton, M.P. and Smith, S.J. (1985) Calcium entry and transmitter release at voltage-clamped nerve terminals of squid. J. Physiol. Lond., 367: 163-181. Barrie, A.P. and Nicholls, D.G. (1993) Adenosine A1 receptor inhibition of glutamate exocytosis and protein kinase Cmediated decoupling. J. Neurochem., 6 0 1081-1086. Bame, A.P., Nicholls, D.G., Sanchez-Prieto, J. and Sihra, T.S. (1991) An ion channel locus for the protein kinase C potentiation of transmitter glutamate release from guinea pig cerebrocortical synaptosomes. J. Neurochem., 57: 13981404. Baskys, A. and Malenka, R.C. (1991) Agonists at metabotropic glutamate receptors presynaptically inhibit EPSCs in neonatal rat hippocampus. J. Physiol., (Lond), 444: 687701. Bennett, M.R. and Ho, S. (1992) Adenosine modulation of potassium currents in preganglionic nerve terminals of avian ciliary ganglia. Neurosci. Lett., 137: 4 1 4 . Budd, D.C. and Nicholls, D.G. (1995) Protein kinase C mediated decoupling of the presynaptic adenosine A1 receptor by a facilitatory metabotropic glutamate receptor. J. Neurochem., 65: 611621. Burke, S.P. and Nadler, J.V. (1988) Regulation of glutamate and aspartate release from slices of the hippocampal CAI area: effects of adenosine and baclofen. J . Neurochem., 51: 1541-1551. Clements, J.D., Lester, R.A.J., Tong, G., Jahr, C.E. and Westbrook, G.L. (1992) The time course of glutamate in the synaptic cleft. Science, 258: 1498-1501. Coffey, E.T., Herrero, I., Sihra, T.S., Sanchez-Prieto, J. and Nicholls, D.G. (1994) Glutamate exocytosis and MARKS phosphorylation are enhanced by a metabotropic glutamate receptor coupled to a protein kinase C synergistically
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