An enzyme-linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea-pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35-0.39 nmol min-1 mg of protein-1 and is not significantly affected by external Ca2+. KCl depolarization (30 mMKCl) in the absence of Ca2+ doubles this rate, whereas in the presence of Ca2+, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein-1. The final extent of Ca2+-dependent release amounts to 1.9 nmol mg of protein-1, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30 degrees C for 2 h before depolarization leads to a decrease in Ca2+-independent release and an increase in Ca2+-dependent release, consistent with an intrasynaptosomal relocation of the amino acid.

Glutamate is important in several forms of synaptic plasticity such as long-term potentiation, and in neuronal cell degeneration. Glutamate activates several types of receptors, including a metabotropic receptor that is sensitive to trans-1-amino-cyclopenthyl-1,3-dicarboxylate, coupled to G protein(s) and linked to inositol phospholipid metabolism. The activation of the metabotropic receptor in neurons generates inositol 1,4,5-trisphosphate, which causes the release of Ca2+ from intracellular stores and diacylglycerol, which activates protein kinase C. In nerve terminals, the activation of presynaptic protein kinase C with phorbol esters enhances glutamate release. But the presynaptic receptor involved in this protein kinase C-mediated increase in the release of glutamate has not yet been identified. Here we demonstrate the presence of a presynaptic glutamate receptor of the metabotropic type that mediates an enhancement of glutamate exocytosis in cerebrocortical nerve terminals. Interestingly, this potentiation of glutamate release is observed only in the presence of arachidonic acid, which may reflect that this positive feedback control of glutamate exocytosis operates in concert with other pre- or post-synaptic events of the glutamatergic neurotransmission that generate arachidonic acid. This presynaptic glutamate receptor may have a physiological role in the maintenance of long-term potentiation where there is an increase in glutamate release mediated by postsynaptically generated arachidonic acid.

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.

Technology Corporation, Japan G protein-coupled signalling is a major cellular mechanism for controlling excitability in central neurons. One of the main targets for this control at post-synaptic membranes are G protein-gated inwardly rectifying K + (GIRK or Kir3) channels, which generate slow inhibitory post-synaptic potentials following the activation of G i/o -protein-coupled receptors (North 1989 Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post-synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/ GIRK3. The post-synaptic association of GIRK subunits with GABA B receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre-synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABA B receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABA B receptors. The association of GIRK channels and GABA B receptors with excitatory synapses at both post-and pre-synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum.

Metabotropic glutamate receptors (mGluRs) from group III reduce glutamate release. Because these receptors reduce cAMP levels, we explored whether this signaling pathway contributes to release inhibition caused by mGluRs with low affinity for L-2-amino-4-phosphonobutyrate (L-AP4). In biochemical experiments with the population of cerebrocortical nerve terminals we find that L-AP4 (1 mM) inhibited the Ca 2؉ -dependent-evoked release of glutamate by 25%. This inhibitory effect was largely prevented by the pertussis toxin but was insensitive to inhibitors of protein kinase C bisindolylmaleimide and protein kinase A H-89. Furthermore, this inhibition was associated with reduction in N-type Ca 2؉ channel activity in the absence of any detectable change in cAMP levels. In the presence of forskolin, however, L-AP4 decreased the levels of cAMP. The activation of this additional signaling pathway was very efficient in counteracting the facilitation of glutamate release induced either by forskolin or the ␤-adrenergic receptor agonist isoproterenol. Imaging experiments to measure Ca 2؉ dynamics in single nerve terminals showed that L-AP4 strongly reduced the Ca 2؉ response in 28% of the nerve terminals. Moreover, immunochemical experiments showed that 25-35% of the nerve terminals that were immunopositive to synaptophysin were also immunoreactive to the low affinity L-AP4-sensitive mGluR7. Then, mGluR7 mediates the inhibition of glutamate release caused by 1 mM L-AP4, primarily by a strong inhibition of Ca 2؉ channels, although high cAMP uncovers the receptor ability to decrease cAMP. Metabotropic glutamate receptors (mGluR)1 from group III consist of four different subtypes (mGluR4, -6, -7, and -8) and are activated by the selective agonist L(ϩ)-2-amino-4-phosphonobutyrate (L-AP4) (Refs. 1-5; for review, see Refs. 6 and 7). The localization of these receptors within presynaptic active zones (8, 9) is consistent with their role as autoreceptors mediating the feedback inhibition of glutamate release (10 -13). In neuronal preparations the inhibition of glutamate release by these receptors has been considered to be mediated by the reduction of voltage-dependent Ca 2ϩ channel activity (11,14,15). However, these receptors also decrease cAMP levels both in heterologous expression systems (1-5) and in neuronal preparations (13, 16). Nevertheless, it remains unclear what influence this signaling pathway has on the inhibition of glutamate release.The activity of L-AP4-sensitive mGluRs is developmentally regulated in different brain areas (13,(17)(18)(19). In the cerebral cortex mGluRs with high affinity for L-AP4 (EC 50 2.3 M) potently reduce glutamate release in nerve terminals from young (13) but not from adult rats (18). It is therefore possible that in the cerebral cortex of adult rats, mGluRs with low affinity for L-AP4 act as presynaptic receptors that mediate synaptic inhibition. In preparations of this tissue, we have examined whether a mGluR with low affinity for L-AP4 reduces glutamate release in cerebrocortical nerve ter...