Canine and Feline Epilepsy. Luisa De Risio

effect of extracellular potassium is multi-faceted. Sustained potassium efflux increases extracellular potassium concentration, depolarizing the membrane and moving the intra-cellular voltage toward the threshold for sodium action potential firing. As extracellular potassium continues to accumulate, there is membrane depolarization and action potential firing increases. With further accumulation, the membrane potential becomes more depolarized than the firing threshold for sodium-action potentials, sodium channels inactivate, and neuronal firing ceases. In vitro experiments by Bikson et al. (2003) illustrate these effects of extracellular potassium accumulation. Electrographic seizure-like activity triggered in hippocampal slices by exposure to low-calcium artificial cerebro-spinal fluid (aCSF) manifested as recurrent periods of population firing followed by periods of electrographic silence lasting 12–18 s. The termination of each electrographic discharge by a period of electrographic silence resulted from transient increases in extracellular potassium to plateaus of approximately 12 mM. The depolarized state was maintained by the elevation of extracellular potassium and by the presence of persistent sodium channels that did not inactivate. Depolarization blockade-terminating seizure-like discharges have also been observed in neocortical slices in which GABA-ergic inhibition is partially blocked by picrotoxin (Pinto et al., 2005). Focal or localized increases in potassium may also trigger additional potassium release beyond the initial region of potassium accumulation. Shifts in extracellular potential, and oscillations seen at the end of hippocampal after-discharges, have been attributed to a rapid rise in extracellular potassium that triggers waves of astrocyte depolarization and a propagating rise in potassium that terminates neuronal firing (Bragin et al., 1997). In addition to its direct depolarizing effects, increased extracellular potassium may also indirectly result in membrane depolarization through the action of the potassium–chloride co-transporter KCC2. The rise in extracellular potassium can increase intracellular chloride, shifting the chloride reversal potential toward membrane depolarization. In the setting of increased intracellular chloride, the action of GABA to open chloride channels could enhance membrane depolarization to the point of becoming refractory to further firing of action potentials (Jin et al., 2005; Galanopoulou, 2007).

      Extracellular calcium levels also change markedly during paroxysmal neuronal firing and may affect the efficiency of neuron-to-neuron spread of activity. Focal seizure activity results in a decline in extracellular calcium activity of approximately 50% (Heinemann et al., 1977). This decline may inhibit synaptic transmission because synaptic vesicle fusion and neurotransmitter release are dependent on entry of extracellular calcium (King et al., 2001; Cohen and Fields, 2004). Decline in extracellular calcium also potentially affects gap junction function as hemichannel opening increases in low calcium (Thimm et al., 2005).

       Energy failure

      Sustained neuronal activation also markedly increases energy, namely ATP, utilization to restore ion gradients across the membrane. In some neurons, the presence of an ATP-gated potassium channel (KATP) reduces neuronal activity when ATP levels decline intracellularly (Yamada et al., 2001). When the ATP level falls because energy utilization outpaces energy production, potassium channels open and produce membrane hyperpolarization. Indeed, knockout mice lacking functioning KATP channels experience a myoclonic seizure on average 8.9 ± 1.1 s following onset of hypoxia, followed by generalized convulsions and death. A similar hypoxic challenge, however, does not trigger seizures in wild-type mice, indicating that KATP channels in vivo resist membrane depolarization during energy failure. Reduced levels of energy metabolites, such as glucose, may also affect seizure duration. In vitro recordings show that decreasing extracellular glucose terminates electrographic seizure-like activity in the low magnesium hippocampal slice (Kirchner et al., 2006). The effect of hypoglycaemia on seizure-like discharges in vitro was statistically significant, but not immediate. Fifty per cent fewer seizure-like discharges occurred in the 24-min period following application of low glucose artificial cerebrospinal fluid compared to the frequency of discharges in the 30 min prior to application. Low glucose also reduced the amplitude of the seizure-like discharge by 25%. These effects on the frequency and amplitude of seizure-like discharges were reversed by restoration of normal glucose levels.

       Mechanisms acting on a local network of neurons

      While seizure initiation is driven at least in part by the burst-firing properties of the individual neurons, the evolution and spread of the seizures also requires amplification and synchronization among neurons within susceptible networks. Seizure amplification occurs through the action of recurrent excitatory collaterals that form feedback loops, returning excitatory synaptic activity to the neurons within the seizure onset zone (Rutecki et al., 1989; Coulter and DeLorenzo, 1999). Seizure spread depends on the propagation and synchronization of the seizure discharge across synapses that separate neurons in the seizure onset zone from ‘normal’ neurons synaptically connected to the seizure onset zone (MacVicar and Dudek, 1980; Miles and Wong, 1983).

       Glutamate depletion

      Decrease in synaptic efficacy results in milder postsynaptic excitation, and consequently diminished amplification and spread of the seizure discharge. One mechanism limiting synaptic transmission during a sustained seizure discharge is the depletion of synaptic vesicles containing neurotransmitter. Staley et al. (1998) investigated the effects of synaptic depletion in vitro using a model of CA3 electrographic seizure discharges produced by hyperkalaemia. CA3 discharges consist of recurrent neuronal depolarizations with bursts of action-potential firing separated by period of electrographic silence. Staley et al. found that the duration of the seizure burst was proportional to the duration of the silent period preceding the burst, consistent with the hypothesis that the seizure burst duration depended on the renewed availability of immediately releasable glutamate. If glutamate-containing synaptic vesicles are replaced at a steady rate, longer inter-burst periods allow a greater resupply of immediately releasable glutamate, and an increased duration of the subsequent electro-graphic seizure discharge. Inter-burst intervals of 2–3 s or longer were necessary to achieve the longest burst durations (up to 420 ms). Thus, as the seizure discharge develops, it consumes the supply of readily releasable glutamate needed to sustain the seizure, potentially acting as a governor on excitatory drive. As the glutamate reservoir is replenished continuously, however, additional control mechanisms are necessary to prevent re-initiation of seizure activity.

       The intra- and extracellular environments

      Prolonged neuronal activity during seizure discharges may also have the effect of increasing CO2 or increasing the by-products of anaerobic metabolism, and produce extra-cellular acidosis or intracellular acidosis associated with extracellular alkalinosis (Chesler and Kaila, 1992). Glial cells may also contribute to acidification of the extracellular space in response to increases in the extracellular potassium concentration (Chesler and Kraig, 1987). In the hippocampal slice in vitro, acidification of the extracellular space to pH 6.7 terminated seizure-like burst firing facilitated by low-magnesium in the artificial CSF. The attenuation of epileptiform activity began within minutes of lowering pH (Velisek et al., 1994; Velisek, 1998). The mechanisms of action – at least in part – included decreased NMDA receptor function and loss of synaptic long-term potentiation (LTP). A milder reduction of pH to 7.1 also produced milder synaptic impairment with continued loss of LTP (Velisek, 1998). Inhibition of carbonic anhydrase, which alters extracellular pH, has some anticonvulsant benefit. In humans, the carbonic anhydrase inhibitor acetozolamide has a mild anticonvulsant effect (Thiry et al., 2007). Knockout mice deficient in carbonic anhydrase are severely acidotic and are resistant to seizures produced by flurothyl gas compared to wild-type mice (Velisek et al., 1993). Intracellular acidification may also contribute to termination of seizure discharges. Spontaneous interictal spiking following focal application of bicuculline in the piriform cortex in an in vitro whole brain preparation was associated with periodic abrupt alkanization of the extracellular space followed by a slow return to baseline pH (de Curtis et al., 1998). These observations were interpreted as evidence of intracellular acidification. Application of ammonium chloride