The Glutamate Synapse

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Glutamate is a potent neurotoxin and there is evidence that its neurotoxicity is mediated by free oxygen radicals (20,22-24,45,61,71). The glutamate synapse (Fig. 1) possesses several features that indicate that oxidative stress mediated by free oxygen radicals, antioxidant defenses, and, in addition, nitric oxide, play a role in its function.

Figure 1 Simplified diagram of the glutamate synapse. AA, arachidonic acid; C, ascor-bate; DA, dopamine; DAQ, dopamine quinones; glu, glutamate; M glu, metabotropic glutamate receptors; NO, nitric oxide; NS, cytoskeleton; P.N., proteases and nucleases; PGH, prostaglandin H; PGH syn, prostaglandin H synthase (cyclooxygenase); ROS, reactive oxygen species. In loci that do not contain dopamine, the cofactor for PGH synthase will be some other molecule.

Figure 1 Simplified diagram of the glutamate synapse. AA, arachidonic acid; C, ascor-bate; DA, dopamine; DAQ, dopamine quinones; glu, glutamate; M glu, metabotropic glutamate receptors; NO, nitric oxide; NS, cytoskeleton; P.N., proteases and nucleases; PGH, prostaglandin H; PGH syn, prostaglandin H synthase (cyclooxygenase); ROS, reactive oxygen species. In loci that do not contain dopamine, the cofactor for PGH synthase will be some other molecule.

1. When glutamate is released into the synapse from the axon terminal its action is terminated by rapid reuptake by the glutamate transporter. The energy for this is provided by an Na+/K+-dependent ATPase. Glutamate uptake is accompanied by simultaneous release by the glutamate transporter of ascorbate into the synaptic cleft (66,68). This ascorbate/glu hetero exchange is not tightly linked but rather is buffered by a further process such as competition for a common intracyto-plasmic binding site (35). Ascorbate is the principal extracellular anti-oxidant in brain.

2. The antioxidant dipeptide carnosine is colocalized with glutamate in the synaptic vesicle and is released with glutamate into the synaptic cleft (6,74).

3. The N-methyl-d-aspartate (NMDA) receptor for glutamate possesses a redox-sensitive site containing sulfydryl groups, oxidation of which downregulates the receptor. Since, as we will see, activation of the NMDA receptor leads to the release of neurotoxic free radicals, this provides a negative feedback mechanism to protect the receptor and synapse against glutamate neurotoxicity.

The sources of free radicals at the glutamate synapse include the following:

1. Activation of the NMDA receptor opens a calcium channel. The entry of calcium into the dendritic spine activates phospholipase A2. This in turn acts on membrane phospholipids to release arachidonic acid, which leads to the activation of prostaglandin H synthase, the rate-limiting step in prostaglandin synthesis. This reaction leads to the release of large amounts of reactive oxygen species including hydrogen peroxide (H2O2), which is a freely diffusible molecule and so can enter the synaptic cleft. Here, in the presence of minute amounts of ''free'' iron, it could form the highly cytotoxic hydroxyl radical. The status of free iron in the brain is controversial. Recently, Mumby et al. (55) distinguished between free iron (iron free of high-affinity binding to transferrin), loosely bound iron (associated with proteins such as albumin), and labile iron (can be mobilized from biological ligands by oxi-dative stress). During periods of oxidative stress low molecular weight labile ferrous iron appears in the cerebrospinal fluid (CSF) (36) and so free iron could be present in the extracellular fluid of the synaptic space.

2. Entry of calcium into the postsynaptic region also activates nitric oxide synthase. This also leads to the release of reactive oxygen species. The predominant redox form of nitric oxide is the highly neurotoxic nitric oxide radical NOV Nitric oxide is also a freely diffusible molecule and can diffuse back into the synaptic cleft. Moreover, nitric oxide and H2O2 can interact independently of free iron to produce hydroxyl radicals (57).

It is therefore possible that one factor in synaptic plasticity may be the redox balance at the glutamate synapse between neurotoxic oxidants (such as H2O2 and NOO, on the one hand, and neuroprotective antioxidants (such as ascor-bate, carnosine, and possibly glutathione) on the other (88). However, in at least some parts of the brain, synaptic plasticity is a function of learning and learning depends in part on positive reinforcement. One chemical signal of reinforcement received by the organism is the widespread release of dopamine throughout the higher cortex (77), especially the prefrontal cortex. Most dopamine terminals are nonsynaptic boutons-en-passage carried on widely diffuse networks. Many of these terminals are closely attached to glutamate synapses in the cortex and stria-tum (44). This would allow dopamine to diffuse into the glutamate synapse. The neurotoxic effects of dopamine are mediated by its oxidative quinone metabolites acting not on dopamine receptors but on NMDA receptors (3,9,47,53). This implies that dopamine or its metabolites can reach NMDA receptors. Pickel et al. (64) have presented evidence that dopamine can diffuse through extracellular space in this manner. Moreover, dopamine boutons-en-passage in the prefrontal cortex have low levels of the dopamine transporter (reuptake) molecule (86), which suggests that the dopamine released may be diffusing elsewhere. It may therefore be significant that dopamine is a potent antioxidant (48). The antioxi-dant effect of dopamine is mediated by redox cycling between dopamine and dopamine o-quinone. When dopamine reduces a reactive oxygen species (ROS) molecule it is converted to dopamine o-quinone. The latter is reconverted to dopa-mine by ambient antioxidants, in particular ascorbate and glutathione. Only when these are exhausted is the dopamine o-quinone further (irreversibly) oxidized to dopaminochrome (13,15,60). Thus, the release of dopamine into the glutamate synapse following reinforcement would tilt the redox balance toward neuropro-tective reduction leading to the growth of that synapse. Lack of dopamine would tilt the redox balance in the other neurotoxic oxidant direction and so would promote deletion of that synapse. In this way glutamatergic synapses that were active during the period of dopamine release would be conserved and their growth (mediated by metabotropic glutamate receptors) would not be inhibited, whereas synapses that were active in the absence of dopamine release would tend to be pruned by unbuffered toxic free radicals. This suggested mechanism of the action of dopamine is meant to complement its other actions via its own receptors linked to postsynaptic cascades involving cyclic nucleotides that may also be involved in synaptic plasticity.

Other factors in this reaction are the metabotropic glutamate receptors which are connected with a postsynaptic cascade in which phosphatases are activated. These act on the cytoskeletal proteins and result in growth of the spine (72).

However, the action of ascorbate at synapses is more complex than simply that mediated by its antioxidant properties. It also exhibits the following properties: blockade of NMDA, adrenergic, 5-hydroxytryptamine (5-HT), and dopamine receptors (11); inhibition of glutamate binding to the NMDA receptor (35); inhibition of NMDA-evoked currents (32); inhibition of N+,K+-activated ATPase and of dopamine-sensitive adenylate cyclase; stimulation of release of acetylcholine and norepinephrine from synaptic vesicles; acting as a cofactor for dopamine P-hydroxylase (54); inhibition of dopamine uptake (4); promotion of synthesis of catecholamines by two mechanisms, i.e., reduction of pteridins (es sential cofactors for tyrosine hydroxylase) and induction of a threefold increase in mRNA production for enzymes involved in catecholamine synthesis (81). The bad news is that ascorbate interacts with the nitrosium ion to form the nitric acid radical (33) and also inhibits glutathione uptake (37). Clearly, the action of ascorbate at synapses is very complex.

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