Bioenergetic Mechanisms of Seizure Control.

Epilepsy is characterized by the regular occurrence of seizures, which follow a stereotypical sequence of alterations in the electroencephalogram. Seizures are typically a self limiting phenomenon, concluding finally in the cessation of hypersynchronous activity and followed by a state of decreased neuronal excitability which might underlie the cognitive and psychological symptoms the patients experience in the wake of seizures. Many efforts have been devoted to understand how seizures spontaneously stop in hope to exploit this knowledge in anticonvulsant or neuroprotective therapies. Besides the alterations in ion-channels, transmitters and neuromodulators, the successive build up of disturbances in energy metabolism have been suggested as a mechanism for seizure termination. Energy metabolism and substrate supply of the brain are tightly regulated by different mechanisms called neurometabolic and neurovascular coupling. Here we summarize the current knowledge whether these mechanisms are sufficient to cover the energy demand of hypersynchronous activity and whether a mismatch between energy need and supply could contribute to seizure control.

Contribution of Intrinsic Lactate to Maintenance of Seizure Activity in Neocortical Slices from Patients with Temporal Lobe Epilepsy and in Rat Entorhinal Cortex.

Neuronal lactate uptake supports energy metabolism associated with synaptic signaling and recovery of extracellular ion gradients following neuronal activation. Altered expression of the monocarboxylate transporters (MCT) in temporal lobe epilepsy (TLE) hampers lactate removal into the bloodstream. The resulting increase in parenchymal lactate levels might exert both, anti- and pro-ictogen effects, by causing acidosis and by supplementing energy metabolism, respectively. Hence, we assessed the contribution of lactate to the maintenance of transmembrane potassium gradients, synaptic signaling and pathological network activity in chronic epileptic human tissue. Stimulus induced and spontaneous field potentials and extracellular potassium concentration changes (∆[K⁺]O) were recorded in parallel with tissue pO2 and pH in slices from TLE patients while blocking MCTs by α-cyano-4-hydroxycinnamic acid (4-CIN) or d-lactate. Intrinsic lactate contributed to the oxidative energy metabolism in chronic epileptic tissue as revealed by the changes in pO2 following blockade of lactate uptake. However, unlike the results in rat hippocampus, ∆[K⁺]O recovery kinetics and field potential amplitude did not depend on the presence of lactate. Remarkably, inhibition of lactate uptake exerted pH-independent anti-seizure effects both in healthy rat and chronic epileptic tissue and this effect was partly mediated via adenosine 1 receptor activation following decreased oxidative metabolism.

Adenosine A1 receptor-mediated suppression of carbamazepine-resistant seizure-like events in human neocortical slices.

OBJECTIVE: The need for alternative pharmacologic strategies in treatment of epilepsies is pressing for about 30% of patients with epilepsy who do not experience satisfactory seizure control with present treatments. In temporal lobe epilepsy (TLE) even up to 80% of patients are pharmacoresistant, and surgical resection of the ictogenic tissue is only possible for a minority of TLE patients. In this study we investigate purinergic modulation of drug-resistant seizure-like events (SLEs) in human temporal cortex slices. METHODS: Layer V/VI field potentials from a total of 77 neocortical slices from 17 pharmacoresistant patients were recorded to monitor SLEs induced by application of 8 mM [K(+) ] and 50 µm bicuculline. RESULTS: Activating A1 receptors with a specific agonist completely suppressed SLEs in 73% of human temporal cortex slices. In the remaining slices, incidence of SLEs was markedly reduced. Because a subportion of slices can be pharmacosensitive, we tested effects of an A1 agonist, in slices insensitive to a high dose of carbamazepine (50 µm). Also in these cases the A1 agonist was equally efficient. Moreover, ATP and adenosine blocked or modulated SLEs, an effect mediated not by P2 receptors but rather by adenosine A1 receptors. SIGNIFICANCE: Selective activation of A1 receptors mediates a strong anticonvulsant action in human neocortical slices from pharmacoresistant patients. We propose that our human slice model of seizure-like activity is a feasible option for future studies investigating new antiepileptic drug (AED) candidates.

Drug resistance in cortical and hippocampal slices from resected tissue of epilepsy patients: no significant impact of p-glycoprotein and multidrug resistance-associated proteins.

Drug resistant patients undergoing epilepsy surgery have a good chance to become sensitive to anticonvulsant medication, suggesting that the resected brain tissue is responsible for drug resistance. Here, we address the question whether P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs) expressed in the resected tissue contribute to drug resistance in vitro. Effects of anti-epileptic drugs [carbamazepine (CBZ), sodium valproate, phenytoin] and two unspecific inhibitors of Pgp and MRPs [verapamil (VPM) and probenecid (PBN)] on seizure-like events (SLEs) induced in slices from 35 hippocampal and 35 temporal cortex specimens of altogether 51 patients (161 slices) were studied. Although in slice preparations the blood brain barrier is not functional, we found that SLEs predominantly persisted in the presence of anticonvulsant drugs (90%) and also in the presence of VPM and PBN (86%). Following subsequent co-administration of anti-epileptic drugs and drug transport inhibitors, SLEs continued in 63% of 143 slices. Drug sensitivity in slices was recognized either as transition to recurrent epileptiform transients (30%) or as suppression (7%), particularly by perfusion with CBZ in PBN containing solutions (43, 9%). Summarizing responses to co-administration from more than one slice per patient revealed that suppression of seizure-like activity in all slices was only observed in 7% of patients. Patients whose tissue was completely or partially sensitive (65%) presented with higher seizure frequencies than those with resistant tissue (35%). However, corresponding subgroups of patients do not differ with respect to expression rates of drug transporters. Our results imply that parenchymal MRPs and Pgp are not responsible for drug resistance in resected tissue.

Extracellular ATP differentially affects epileptiform activity via purinergic P2X7 and adenosine A1 receptors in naive and chronic epileptic rats.

PURPOSE: Adenosine is considered an endogenous anticonvulsant. However, much less is known about the putative effects of its precursor, ATP, on epilepsy. Therefore, we tested whether ATP and its receptors are able to modulate epileptiform activity in the medial entorhinal cortex of the rat. METHODS: Recurrent epileptiform discharges (REDs) were induced by elevating extracellular potassium concentration combined with application of bicuculline in brain slices from naive and pilocarpine-treated chronic epileptic rats. Field potentials were recorded from layer V/VI of the medial entorhinal cortex. KEY FINDINGS: REDs in slices from naive animals had a higher incidence and a shorter duration than in slices from chronic epileptic animals. Exogenous application of ATP reversibly reduced the incidence of REDs in naive and chronic epileptic slices via activation of adenosine A(1) receptors without discernible P2 receptor effects. This effect was stronger in slices from chronic epileptic rats. In slices from naive rats, the P2X7 receptor antagonist A 740003 slightly but significantly reduced the amplitude of slow field potentials of REDs. In slices from chronic epileptic rats, none of the P2 receptor antagonists affected the parameters of REDs. SIGNIFICANCE: Our results suggest that endogenously released ATP differentially modulates REDs by activation of A(1) and P2X7 receptors. Although it has a minor proepileptic effect by direct activation of P2X7 receptors, its metabolite adenosine reduces the epileptiform activity via activation of A(1) receptors. The exact effect of ATP on neural activity depends on the actual activity of ectonucleotidases and the expression level of the purinergic receptors, which both alter during epileptogenesis. In addition, our data suggest that P2X7 receptor antagonists have a minor antiepileptic effect.

Functional test of multidrug transporter activity in hippocampal-neocortical brain slices from epileptic patients.

About 70% of the patients suffering from temporal lobe epilepsy (TLE) are resistant to currently available antiepileptic drugs (AEDs). For them one therapeutic option to achieve seizure control is to undergo epilepsy surgery. Expression of multidrug transporters is upregulated in resected tissue specimens from TLE patients, as well as in animal models of chronic epilepsy, which might lead to altered tissue availability of AEDs and therefore contribute to drug refractoriness. Here we describe a functional test of multidrug transporter activity in brain slices from TLE patients based on intracellular accumulation of the fluorescent multidrug transporter substrate calcein and compare functional data to the expression pattern of multidrug transporters. The rate of cytosolic calcein fluorescence increase was altered by inhibitors of multidrug transport such as probenecid (400 µM) and verapamil (40 µM) in a subset of slices, indicating the presence of functional multidrug transport proteins in human epileptic tissue. Interestingly, there were differences between the expression pattern of multidrug transporters and their ability to remove calcein-AM. Consequently, in vitro studies on multidrug transporters should always include functional tests of their activity as expression alone is not necessarily conclusive.

Impact of aquaporin-4 channels on K+ buffering and gap junction coupling in the hippocampus.

Aquaporin-4 (AQP4) is the main water channel in the brain and primarily localized to astrocytes where the channels are thought to contribute to water and K(+) homeostasis. The close apposition of AQP4 and inward rectifier K(+) channels (Kir4.1) led to the hypothesis of direct functional interactions between both channels. We investigated the impact of AQP4 on stimulus-induced alterations of the extracellular K(+) concentration ([K(+)](o)) in murine hippocampal slices. Recordings with K(+)-selective microelectrodes combined with field potential analyses were compared in wild type (wt) and AQP4 knockout (AQP4(-/-)) mice. Astrocyte gap junction coupling was assessed with tracer filling during patch clamp recording. Antidromic fiber stimulation in the alveus evoked smaller increases and slower recovery of [K(+)](o) in the stratum pyramidale of AQP4(-/-) mice indicating reduced glial swelling and a larger extracellular space when compared with control tissue. Moreover, the data hint at an impairment of the glial Na(+)/K(+) ATPase in AQP4-deficient astrocytes. In a next step, we investigated the laminar profile of [K(+)](o) by moving the recording electrode from the stratum pyramidale toward the hippocampal fissure. At distances beyond 300 µm from the pyramidal layer, the stimulation-induced, normalized increases of [K(+)](o) in AQP4(-/-) mice exceeded the corresponding values of wt mice, indicating facilitated spatial buffering. Astrocytes in AQP4(-/-) mice also displayed enhanced tracer coupling, which might underlie the improved spatial re- distribution of [K(+)](o) in the hippocampus. These findings highlight the role of AQP4 channels in the regulation of K(+) homeostasis.

In search of models of pharmacoresistant epilepsy.

Clinically available anticonvulsant drugs fail to control seizures in approximately 30% of epileptic patients. If hippocampal sclerosis is combined with focal dysplasia or similar developmental alterations, the likelihood of incomplete seizure control may reach >90%. Because only a minority of epilepsy patients benefit from epilepsy surgery, we need more research into the mechanisms of drug refractoriness. In this review we analyze different approaches to study pharmacoresistance and underlying mechanisms using in vitro models. Epileptiform discharges after prolonged application of low Mg(2+) artificial cerebrospinal fluid (ACSF) or 4-aminopyridine (4-AP), or combined application of these convulsants with bicuculline in acute hippocampal-entorhinal cortex slices reveal pharmacoresistance and point to loss of gamma-aminobutyric acid (GABA)ergic function, in part due to reduced delivery of GABA from presynaptic terminals. Interestingly, epileptiform activity in immature tissue (organotypic hippocampal slice cultures and acute intact hippocampus) is immediately resistant to available antiepileptic drugs, and preliminary evidence points to a role of alterations in Cl(-) homeostasis. Seizure-like events can also be induced in dissected tissues from human epileptic patients. Future research on human tissue may provide useful information for understanding the mechanisms underlying pharmacoresistance.

Carbamazepine-resistance in the epileptic dentate gyrus of human hippocampal slices.

Overexpression of drug efflux pumps at the blood brain barrier (BBB) has been suggested to be one important factor contributing to drug resistance in epilepsy. This would imply that resected brain tissue of drug-resistant patients is drug-sensitive in absence of the BBB. Here we studied the effects of carbamazepine (CBZ) at therapeutically relevant concentration on epileptiform activity electrophysiologically recorded in acute hippocampal slices of patients with mesial temporal lobe epilepsy (MTLE; 28 patients, 49 slices) or extra-hippocampal tumours (tumour; 6 patients, 11 slices). Epileptiform activity was induced by hilar stimulation (0.067 Hz) during elevation of extracellular potassium concentration ([K(+)](o)) and remained self-sustained in presence of 10-12 mM [K(+)](o). Quantitative analysis of data revealed that epileptiform activity in tissue of tumour-patients was predominantly suppressed by CBZ, indicating that the 'epilepsy model' used is CBZ-sensitive. In contrast, epileptiform activity in tissue of drug-resistant MTLE patients was resistant to CBZ in 82% of patients, partially suppressed in 11% and completely suppressed in 7%. The effects of CBZ in tissue of MTLE patients did not depend on the type of activity, hippocampal pathology, excitability of the tissue, or equilibration time of the drug. Considering that CBZ has direct access to all compartments of the slice, our results suggest that CBZ-resistance mechanisms are located within the parenchyma of the dentate gyrus and contribute to drug resistance in the majority of MTLE patients. BBB-located drug-resistance mechanisms per se may play a minor role in this region, because CBZ-sensitivity was only observed in 7% of CBZ-resistant patients.

Increased extracellular K+ concentration reduces the efficacy of N-methyl-D-aspartate receptor antagonists to block spreading depression-like depolarizations and spreading ischemia.

BACKGROUND AND PURPOSE: Spreading depression (SD)-like depolarizations may augment neuronal damage in neurovascular disorders such as stroke and traumatic brain injury. Spreading ischemia (SI), a particularly malignant variant of SD-like depolarization, is characterized by inverse coupling between the spreading depolarization wave and cerebral blood flow. SI has been implicated in particular in the pathophysiology of subarachnoid hemorrhage. Under physiological conditions, SD is blocked by N-methyl-D-aspartate receptor (NMDAR) antagonists. However, because both SD-like depolarizations and SI occur in presence of an increased extracellular K+ concentration ([K+]o), we tested whether this increase in baseline [K+]o would reduce the efficacy of NMDAR antagonists. METHODS: Cranial window preparations, laser Doppler flowmetry, and K+-sensitive/reference microelectrodes were used to record SD, SD-like depolarizations, and SI in rats in vivo; microelectrodes and intrinsic optical signal measurements were used to record SD and SD-like depolarizations in human and rat brain slices. RESULTS: In vivo, the noncompetitive NMDAR antagonist dizocilpine (MK-801) blocked SD propagation under physiological conditions, but did not block SD-like depolarizations or SI under high baseline [K+]o. Similar results were found in human and rat neocortical slices with both MK-801 and the competitive NMDAR antagonist D-2-amino-5-phosphonovaleric acid. CONCLUSIONS: Our data suggest that elevated baseline [K+]o reduces the efficacy of NMDAR antagonists on SD-like depolarizations and SI. In conditions of moderate energy depletion, as in the ischemic penumbra, or after subarachnoid hemorrhage, NMDAR inhibition may not be sufficient to block these depolarizations.