Relleno de Datos

Even with the methodological problems o f microdialysis, large glutamate rises would have been detectable in all studies and what may have been missed are small, transient rises. That large extracellular glutamate rises were not detected in all animal seizure models implies that these rises are not necessary for seizure activity. This is perhaps not surprising as seizures are synaptically driven events, and it is thus the local concentration o f glutamate in the synapse that is relevant. That extracellular glutamate rises may in fact be epiphenomena o f seizure activity is further confirmed by the dissociation o f the size o f extracellular glutamate rises and seizure activity such that both large and imperceptible rises in glutamate observed in my experiments can be associated with indistinguishable epileptic afterdischarges.

The rises in extracellular glutamate that do occur are usually too small to activate non-NM DA receptors; non-NMDA receptors have an EC50 o f approximately SOOpM (i.e. far in excess o f extracellular glutamate rises that are observed). Since the fast decay time course o f AMP A mediated EPSPs depends upon clearance o f glutamate from the synaptic cleft, then there is a theoretical possibility that increasing the extracellular glutamate concentration could impair diffusion o f glutamate fi*om the synaptic cleft, thus prolonging the EPSP. The magnitude o f this effect, however, is likely to be small. However, the extracellular glutamate rises that are observed are o f a sufficient magnitude to activate NM DA receptors; so that when a neuron is later depolarised either through activation o f AMP A receptors or via non-synaptic mechanisms, the magnesium blockade o f the activated NMDA receptor is removed and calcium flows into the cell. Indeed, this system has positive feedback in that activation o f NMDA receptors can result in a

decrease in high affinity glutamate uptake. Since activation o f NMDA receptors is a requisite o f kindling (McNamara et al., 1993), and since rises in extracellular glutamate are associated with kindling, then it is likely that these rises in extracellular glutamate contribute to the kindling process. Indeed, kindling can be induced by the local application o f glutamate (Croucher & Bradford, 1989). A further piece o f confirmatory evidence o f glutamate's role in kindling is the diminution in glutamate rises in the above experiments with short interstimulus intervals, and identical glutamate rises with interstimulus intervals o f 1 hour; this corresponds to the greater numbers o f stimulations required to kindle if the stimulation interval is less than one hour (Racine et al., 1973).

W hat is the role o f rises in extracellular glutamate in neurotoxicity? The role o f the glutamatergic system in cell death is in little doubt. Application o f large concentrations (lOOpM and above) o f glutamate result in cell death in cell cultures (Choi et al., 1987), and in vivo (McBean & Roberts, 1984). It is incorrect, however, to assume that large extracellular glutamate rises must thus be associated with neurotoxicty or even necessarily have a significant role. Activation o f NMDA receptors in the presence o f glycine occurs at low glutamate concentrations, and it is the association o f activation with depolarisation, which relieves the magnesium block, that results in the calcium influx causing neuronal death. In order to cause neuronal death by the application o f glutamate, large concentrations are necessary to induce a significant depolarisation, and to overcome glutamate uptake. In neuronal cell culture Choi et al. concluded that glutamate uptake was not a consideration because DHK did not potentiate the glutamate-induced neuronal death (Choi et ah, 1987). However, DHK is a weak inhibitor o f glutamate uptake in neurons (see above), and it is thus possible that the administration o f DHK resulted in minimal change to glutamate dynamics in the neuronal cell culture. The extracellular glutamate accumulation that occurs during ischaemia is surplus to requirement for the activation o f NM DA receptors, and although it may play some part in depolarisation, it is the disruption o f ionic homeostasis that is the main contributor to ischaemic depolarisation (Obrenovitch & Richards, 1994). Indeed, there is conflicting evidence for the role o f these large glutamate rises in ischaemia being directly responsible for cell death at all, as cell

death appears to be due to events that occur after the ischaemic episode. The evidence that the large glutamate rises during ischaemia do not directly contribute to cell death is as follows (Obrenovitch & Richards, 1994; Szatkowski & Attwell, 1994):

1) neuronal death can occur several hours after the period o f ischaemia; glutamate, on the other hand, is cleared from the extracellular space within minutes o f reperfusion.

2) Neurons in the area surrounding the ischaemic area (the penumbra) also die, and are very receptive to protection with glutamate antagonists, yet extracellular glutamate rises in this area during ischaemia are small.

3) Treatment with glutamate antagonists is neuroprotective in a number o f models many hours after the ischaemic episode has taken place.

The explanation for these findings is not immediately obvious. It has been noted that a large calcium influx into neurons occurs during ischaemia, and a smaller, but increased, calcium influx occurs post-ischaemia (Szatkowski & Attwell,

1994). Why the latter smaller calcium influx should result in cell death, whilst the former large calcium influx does not is a matter for speculation. It has been suggested that oxygen is necessary for the formation o f free radicals that result in cell death and that ironically the period o f hypoxia could be neuroprotective (Szatkowski & Attwell, 1994). Nevertheless, the calcium influx and the activation o f NM DA receptors that occur during ischaemia lead to a widespread and large potentiation o f synaptic transmission involving both non-NMDA and NMDA receptors (Obrenovitch & Richards, 1994; Szatkowski & Attwell, 1994). Indeed, after ischaemia the NMDA receptor current shows less voltage-dependent magnesium block at negative potentials. The combination o f this synaptic and NM DA potentiation following ischaemia result in the increased post-ischaemic calcium influx into neurons, which is susceptible to NMDA antagonist blockade. That large extracellular glutamate rises alone do not necessarily lead to neuronal death has been demonstrated by experiments in which prolonged PDC (a glutamate uptake blocker) induced rises o f extracellular glutamate to "neurotoxic" levels do not result in cell death (Massieu et al., 1995). Comparable glutamate

rises due to DHK (a different glutamate blocker) result, however, in significant neuronal death (Massieu et al., 1995). In the same study, kainic acid which resulted in no detectable rises in extracellular glutamate caused considerable neuronal death. Why the two uptake inhibitors had different effects on cell survival is not immediately clear. DHK is a relatively poor synaptic uptake blocker that has some action on glutamate-gated ion channels. PDC is a stronger more selective glutamate blocker that affects synaptic glutamate uptake. It could be that sustained synaptic rises in glutamate caused by PDC act on presynaptic terminals inhibiting the synaptic release o f glutamate, and also that these sustained rises lead to non-NMDA receptor desensitisation. Both these effects would tend to prevent large post-synaptic depolarisations from occurring, thus preventing removal o f the magnesium block o f NMDA receptors and the neurotoxic influx o f calcium. There is certainly evidence that PDC depresses excitatory synaptic transmission via glutamate activation o f presynaptic metabotropic receptors that decrease synaptic glutamate release (Maki et al.,

1994). Since synaptic concentrations o f glutamate are not affected to a great degree by DHK, then these effects would not occur.

Thus, looking for sustained extracellular glutamate rises during status epilepticus is a quixotic mission. Only small rises in extracellular glutamate are probably necessary to potentiate NMDA mediated cell death; depolarisation is probably the main activator o f these receptors in pathological conditions. Furthermore large rises in extracellular glutamate are not in themselves a sufficient explanation for neurotoxicity.

5. TREATMENT OF LATE STATUS EPILEPTICUS