Neurotransmitters In Epileptic Activity

Changes in NT levels and function have been (1) Looked for in

(a) human epileptic tissue

(b) animals in which convulsions have been induced experimentally

(c) animals with spontaneous (genetically disposed) epilepsy (2) Induced in animals to see how they modify convulsive threshold and intensity

These approaches will be considered in respect of the different NTs although most interest has centred on the amino acids not only because of their possible involvement in the pathology, as already emphasised, but because increased neuronal activity in epilepsy must reflect, even if it is not initiated by, augmented glutamate and/or reduced GABA function.

AMINO ACID MEASUREMENTS Human studies

Reduced GABA uptake during microdialysis has been mentioned and there are reports of reduced levels of GABA in the CSF of chronic epileptics and of its synthesising enzyme glutamic acid decarboxylase (GAD) in some samples of temporal lobe tissue removed during surgery to alleviate focal seizures. Other reports find no change in GAD but an increase in GABAa receptors.

Animal studies

In addition to the loss of GAD staining (i.e. GABA) neurons and inhibitory symmetrical synapses around an alumina focus in primates (see above), studies with a chronically implanted cortical cup over a cobalt lesion (focus) in rats show an increased release of glutamate that is associated with spiking (Dodd and Bradford 1976).

Numerous acute experiments with cortical cups show that systemic convulsants increase the release of ACh but rarely that of glutamate. Even the marked convulsant EEG seen after PTZ infusion in the rat (Fig. 16.4) is not accompanied by any rise in glutamate release. This may not mean that it does not occur but that the avid uptake mechanism for glutamate ensures that levels do not rise above basal, unless the stimulation is very extreme. This may explain why perfusates of the lateral ventricle, obtained during kindled seizures induced by the stimulation of the amygdala, showed elevated glutamate levels, but only after very intense neuronal disharges. Basal GABA levels are often too low to even detect in such studies.

If kindling is regarded as a model of the development of epilepsy (epileptogenesis) then following changes in NT function, after or through its development, may be of more value than merely monitoring release during convulsions. Unfortunately results have been inconclusive. Kindling induced by the intraventricular injection of folic acid in rats produced significant increases in cortical glutamate and aspartate, but only the latter correlated directly with increased spiking. With kindling induced by electrical stimulation of the frontal cortex the only change observed alongside the increase in after-discharge was a reduction in glutamine, although this could reflect its utilisation in providing the extra glutamate required for spiking and epileptic activity.

Animals with spontaneous epilepsy

These have yielded few data apart from reports of reduced GABA and taurine in the CSF of baboons with spontaneous seizures.

AMINO ACIDS, MANIPULATION

GABA

Experimentally all GABA antagonists induce convulsions. These include the genuine receptor antagonist bicuculline, which competes with GABA for its recognition site on the GABAa receptor and picrotoxin, which binds to a different site more closely related to the chloride ion channel.

Reducing the availability of GABA by blocking the synthesising enzyme GAD also promotes convulsions. This may be achieved by substrate competition (e.g. 3-mercapto propionic acid), irreversible inhibition (e.g. allylglycine) or reducing the action or availability of its co-factor pyridoxal phosphate (e.g. various hydrazides such as semi-carbazide). In fact pyridoxal phosphate deficiency has been shown to be the cause of convulsions in children.

Clearly since a reduction in GABA function causes convulsions, then augmenting its function should provide an anticonvulsant action. This may be achieved in a number of ways as listed in Table 16.2 and indicated in Fig. 16.6. For more detail see Chapter 9.

Agonists and prodrugs

GABAa receptor agonists like muscimol and (Fig. 16.7) are active against PTZ in mice and amygdala kindling in rats but ineffective in the photosensitive baboon and in fact produce rhythmic spike and wave discharges in the EEG despite poor brain penetration. These discharges have also been seen in the few humans on which the drugs have been tested unsuccessfully.

The reason for this disappointing response is uncertain but may be due to desensitisation of the GABAa receptors, or the actual inhibition of GABA inhibitory neurons through somatic autoreceptors which could disrupt the precise timing of physiological inhibition. Activation of the GABAB receptor with baclofen has no

Table 16.2 Drug augmentation of GABA function

A

B

1

GABA receptor, agonists

GABAa—muscimol

GABAb—baclofen

2

Gabamimetics

Progabide

Prodrugs

Gabapentin

3

GABA-t inhibitors

Ethanolamine-o-sulphate (EOS)

Na+ valproate

y vinyl-GABA (vigabatrin)

4

Uptake inhibitors

Neuronal

DABA.ACHC

Glial

Nipecotic acid-tiagabine

5

Allosteric enhancement

Benzodiazepines

6

Chloride channel openers

Mechanisms are listed under A and examples of drugs that utilise them under B. All compounds that increase the action of endogenous GABA (1-5) augment neuronal inhibition and have an anticonvulsant action. Drugs that act directly on GABA receptors have not so far proved effective. Barbiturates do not really augment GABA function; they do not act on GABA receptors or modify its destruction, but can open Cl~ channels and so increase neuronal inhibition and thus the action of GABA.

Notes:

Mechanisms are listed under A and examples of drugs that utilise them under B. All compounds that increase the action of endogenous GABA (1-5) augment neuronal inhibition and have an anticonvulsant action. Drugs that act directly on GABA receptors have not so far proved effective. Barbiturates do not really augment GABA function; they do not act on GABA receptors or modify its destruction, but can open Cl~ channels and so increase neuronal inhibition and thus the action of GABA.

Figure 16.4 Changes in the pattern of EEG activity accompanying the development of a full ictal seizure in the anaesthetised rat during the slow intravenous infusion of pentylenetetrazol. The normal control pattern (phase a) quickly takes on an arousal state (phase b, 2-5 min). This gives way to waves of steadily increasing amplitude but low frequency (2 Hz) for 8-18 min (phase c) on which a few spikes gradually appear at 20 min (phase d). Spikes gradually predominate after some 26 min (phase e) until they group to give a full ictal seizure at 30 min (phase f). Pentylenetetrazol (0.5 M) infused at 30 ^lmin-1, EEG recorded from skull screw electrodes over the parietal cortex. While this study does not mimic seizure development from a specific focus, since PTZ given systemically can act throughout the brain, it illustrates how cortical activity can become synchronised even without a primary focus. (Reproduced with permission of Macmillan Press Limited from Kent and Webster 1983)

Figure 16.4 Changes in the pattern of EEG activity accompanying the development of a full ictal seizure in the anaesthetised rat during the slow intravenous infusion of pentylenetetrazol. The normal control pattern (phase a) quickly takes on an arousal state (phase b, 2-5 min). This gives way to waves of steadily increasing amplitude but low frequency (2 Hz) for 8-18 min (phase c) on which a few spikes gradually appear at 20 min (phase d). Spikes gradually predominate after some 26 min (phase e) until they group to give a full ictal seizure at 30 min (phase f). Pentylenetetrazol (0.5 M) infused at 30 ^lmin-1, EEG recorded from skull screw electrodes over the parietal cortex. While this study does not mimic seizure development from a specific focus, since PTZ given systemically can act throughout the brain, it illustrates how cortical activity can become synchronised even without a primary focus. (Reproduced with permission of Macmillan Press Limited from Kent and Webster 1983)

general anticonvulsant effect even though it reduces reflex epilepsy in photosensitive baboons and spiking in hippocampal slices. That GABA function is important, however, in the control of epileptogenic activity is illustrated in Fig. 16.5 which shows that spiking induced in the cortex of the anaesthetised rat by leptazol occurs more readily if GABA function is reduced by the local application of its antagonist bicuculline but retarded if GABA itself is applied.

GABA-t inhibitors

GABA transaminase is a mitochondrial enzyme which, like GAD, requires pyridoxal phosphate as co-factor. It is present in both neurons and glia and while secondary to

-i-BICUCULLINE

Figure 16.5 The importance of GABA in controlling the development of EEG epileptic spiking. The EEG records shown were taken from the anaesthetised rat during the infusion of pentylenetetrazol (PTZ). They were obtained from screw electrodes (a) in the skull over one parietal cortex and from electrodes within a cortical cup (b) on the other exposed parietal cortex which was superfused with artificial CSF to which drugs could be added. Thus while the whole cortex received PTZ only that area adjacent to the cup could be influenced by the drugs. Under control conditions the developing epileptogenic EEG was identical in both recordings. Records from the screw electrodes (a) showed the expected progressive change from wave-like (i) to spiking (ii) similar to phases c and d in Fig. 16.4. When the cortex under the cup electrodes (b) was exposed to the GABA antagonist bicuccilline the EEG had already developed spiking (bi) while that from the screw electrodes (ai) still remained wave-like. By contrast, when GABA was in the cup the EEG within it developed more slowly with wave-like activity (bii) persisting when spiking had already developed in the record from the screw electrodes (aii). Clearly GABA retards the development of spiking. (Unpublished figure but see Kent and Webster 1986 for detail and drug concentrations)

Figure 16.5 The importance of GABA in controlling the development of EEG epileptic spiking. The EEG records shown were taken from the anaesthetised rat during the infusion of pentylenetetrazol (PTZ). They were obtained from screw electrodes (a) in the skull over one parietal cortex and from electrodes within a cortical cup (b) on the other exposed parietal cortex which was superfused with artificial CSF to which drugs could be added. Thus while the whole cortex received PTZ only that area adjacent to the cup could be influenced by the drugs. Under control conditions the developing epileptogenic EEG was identical in both recordings. Records from the screw electrodes (a) showed the expected progressive change from wave-like (i) to spiking (ii) similar to phases c and d in Fig. 16.4. When the cortex under the cup electrodes (b) was exposed to the GABA antagonist bicuccilline the EEG had already developed spiking (bi) while that from the screw electrodes (ai) still remained wave-like. By contrast, when GABA was in the cup the EEG within it developed more slowly with wave-like activity (bii) persisting when spiking had already developed in the record from the screw electrodes (aii). Clearly GABA retards the development of spiking. (Unpublished figure but see Kent and Webster 1986 for detail and drug concentrations)

uptake in the degradation of GABA a number of inhibitors have proved effective experimentally and some clinically. Ethanolamine-O-sulphate was one of the first tested. It produces a large (fortyfold) and sustained increase in brain GABA accompanied by a reduction in seizures induced by maximal electroshock. Gabaculine and aminooxyacetic acid are similar but are ineffective in man whereas y-vinyl GABA (vigabatrin) has proved useful clinically. The use of this and sodium valporate is considered later.

Uptake inhibitors

GABA is removed from the synapse by a high-affinity sodium and chloride-dependent uptake into GABA neurons and surrounding glia. Blocking this process potentiates the inhibitory action of GABA applied directly to neurons in vivo and in vitro. Some inhibitors show specificity for glia and others for neuronal uptake, although since recent molecular cloning has revealed four distinct GABA transporters (Chapter 9)

this simple classification may require modification. Probably because of structural similarities to GABA, few of these compounds show brain penetration but tiagabin, a lipophilic form of nipecotic acid, has been tried successfully in refractory epilepsy.

Receptor modulators

Benzodiazepines bind to a specific site on the GABA chloride ionophore, which differs from that for GABA itself, but when occupied augments the binding and action of GABA to increase the frequency of opening of chloride ion channels. Thus they augment GABA inhibition. Many of them are potent anticonvulsants, especially when tested against PTZ and retard the development of kindling. Unfortunately their clinical value is limited by the development of tolerance.

Barbiturates also potentiate the action of GABA but as they can do this by directly increasing the duration of opening of the chloride ion channel, independently of the GABA or benzadiazepine receptor sites, they cannot strictly be considered to augment GABA. Some such as phenobarbetone are, however, of proven clinical value.

Glutamate

NMDA receptor antagonists such as AP5 and AP7 were first shown to be anticonvulsant following introcerebroventricular injection into DBA/2 mice susceptable to audiogenic seizures. In addition, they offer protection to PTZ, reduce the afterdischarge in amygdala kindled rats and can actually retard the development of kindling. Although AP7 has some effect in photosensitive baboons, systemically active compounds have proved difficult to synthesise. Recently felbamate, an antagonist at the glycine-sensitive site on the NMDA receptor, has shown systemic anticonvulsant activity and clinical efficacy.

Inhibition of glutamate release was thought to be the mode of action of lamotrigine. It reduces MES and kindling and also glutamate (and to a lesser extent GABA) release induced in brain slices by veratridine, which opens sodium channels. But it now seems likely that the actual block of sodium channels is its primary action (see later).

The epileptic discharges induced in hippocampal slices by tetanic stimulation has been shown to be accompanied by reduced GABA-mediated IPSPs (Stelzer, Slater and Bruggencate 1987). Since AP7 not only reduced the discharges but also restored the response to GABA some linkage between NMDA and GABAA receptors seems probable. In fact the interaction between glutamate and GABA probably means that both of them and possibly their different receptors may need to be manipulated appropriately to control convulsive activity. This has been shown in fact experimentally when bicuculline was infused intravenously for short periods in the rat to give a burst of epileptic-like spiking in the EEG. Superfusion of the cortex using the cup technique with the glutamate AMPA antagonist CNQX or the GABAb agonist baclofen reduced the actual number (initiation) of spikes but not their amplitude, while NMDA antagonists (AP7) and the GABAA agonist muscimal reduced the size (development and spread of excitation) and not the number of spikes (Zia-Gharib and Webster 1991). Clearly more than one aspect of amino acid function may need to be controlled.

Other NTs have been implicated in the aetiology of epilepsy but direct evidence is lacking. They will be considered briefly.

ACETYLCHOLINE (ACh)

Cholinergic agonists, e.g. carbachol, applied to the rat cortex cause focal spiking and even seizures which can also be induced by large doses of CNS-penetrating anti-cholinesterases such as physostigmine (reversible inhibitor) or di-isopropylfluoro-phosphate (irreversible). Many studies have also shown that cortical ACh release increases in proportion to EEG activity during the administration of a wide range of convulsants. Nevertheless while cholinergic-induced seizures can be suppressed by antimuscarinic drugs they have no effect against any epilepsy in humans and ACh release presumably reflects rather than directly causes cortical activity.

MONOAMINES

The widespread and diverging nature of ascending monoamine pathways to the cortex suggest that NA and 5-HT are more likely to have a secondary modifying rather than a primary effect on the initiation of epileptic activity. In reality this is the case and their secondary role is even a minor one. Generally a reduction in monoamine function facilitates experimentally induced seizures (see Meldrum 1989) while increasing it reduces seizure susceptibility. The variability of the procedures used and results obtained do not justify more detailed analysis here.

Some mention should perhaps be made of dopamine, considering its role in the control of motor function. It is perhaps not surprising that DA agonists like apo-morphine block the myoclonus induced in photosensitive baboons and audiogenic seizures in DBA/2 mice while neuroleptics (DA antagonists) may have a weak procon-vulsant effect in humans. Also in rats with absence seizures dopa, apomorphine and Dj agonists reduce facial clonus and spike and wave discharges, while the Dj antagonist SCH 23390 increases them. Nevertheless, there is no evidence of a significant role for DA (or NA and 5-HT) in human epilepsies.

ADENOSINE

A number of studies have shown that adenosine inhibits neuronal firing both in vitro and in vivo and is itself released during intense neuronal activity. It can protect against PTZ seizures in rodents while the antagonist theophylline is proconvulsant. No clear picture of its role in human epilepsy has emerged.

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