The development of resistance to pharmacological treatment is common to many human diseases. In chronic epilepsy, many patients develop resistance to anticonvulsant drug treatment during the course of their disease, with the underlying mechanisms remaining unclear. We have studied cellular mechanisms underlying drug resistance in resected hippocampal tissue from patients with temporal lobe epilepsy by comparing two groups of patients, the first displaying a clinical response to the anticonvulsant carbamazepine and a second group with therapy-resistant seizures. Using patch-clamp recordings, we show that the mechanism of action of carbamazepine, use-dependent block of voltage-dependent Na(+) channels, is completely lost in carbamazepine-resistant patients. Likewise, seizure activity elicited in human hippocampal slices is insensitive to carbamazepine. In marked contrast, carbamazepine-induced use-dependent block of Na(+) channels and blocked seizure activity in vitro in patients clinically responsive to this drug. Consistent with these results in human patients, we also show that use-dependent block of Na(+) channels by carbamazepine is absent in chronic experimental epilepsy. Taken together, these data suggest that a loss of Na(+) channel drug sensitivity may constitute a novel mechanism underlying the development of drug-resistant epilepsy.
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.
Generation of free radicals may have a key role in the nerve cell damage induced by prolonged or frequently recurring convulsions (status epilepticus). Mitochondrial function may also be altered due to production of free radicals during seizures. We therefore studied changes in field potentials (fp) together with measurements of extracellular, intracellular, and intramitochondrial calcium concentration ([Ca(2+)]e, [Ca(2+)]i, and [Ca(2+)]m, respectively), mitochondrial membrane potential (deltapsi), NAD(P)H auto-fluorescence, and dihydroethidium (HEt) fluorescence in hippocampal slice cultures by means of simultaneous electrophysiological and microfluorimetric measurements. As reported previously, each seizure-like event (SLE) resulted in mitochondrial depolarization associated with a delayed rise in oxidation of HEt to ethidum, presumably indicating ROS production. We show here that repeated SLEs led to a decline in intracellular and intramitochondrial Ca(2+) signals despite unaltered Ca(2+) influx. Also, mitochondrial depolarization and the NAD(P)H signal became smaller during recurring SLEs. By contrast, the ethidium fluorescence rises remained constant or even increased from SLE to SLE. After about 15 SLEs, activity changed to continuous afterdischarges with steady depolarization of mitochondrial membranes. Staining with a cell death marker, propidium iodide, indicated widespread cell damage after 2 h of recurring SLEs. The free radical scavenger, alpha-tocopherol, protected the slice cultures against this damage and also reduced the ongoing impairment of NAD(P)H production. These findings suggest involvement of reactive oxygen species (ROS) of mitochondrial origin in the epileptic cell damage and that free radical scavenging may prevent status epilepticus-induced cell loss.
Hippocampal specimens resected to cure medically intractable temporal lobe epilepsy (TLE) provide a unique possibility to study functional consequences of morphological alterations. One intriguing alteration predominantly observed in cases of hippocampal sclerosis is an uncommon network of granule cells monosynaptically interconnected via aberrant supragranular mossy fibers. We investigated whether granule cell populations in slices from sclerotic and nonsclerotic hippocampi would develop ictaform activity when challenged by low-frequency hilar stimulation in the presence of elevated extracellular potassium concentration (10 and 12 mM) and whether the experimental activity differs according to the presence of aberrant mossy fibers.We found that ictaform activity could be evoked in slices from sclerotic and nonsclerotic hippocampi (27 of 40 slices, 14 of 20 patients; and 11 of 22 slices, 6 of 12 patients, respectively). However, the two patient groups differed with respect to the pattern of ictaform discharges and the potassium concentration mandatory for its induction. Seizure-like events were already induced with 10 mM K ϩ . They exclusively occurred in slices from sclerotic hippocampi, of which 80% displayed stimulus-induced oscillatory population responses (250 -300 Hz). In slices from nonsclerotic hippocampi, atypical negative field potential shifts were predominantly evoked with 12 mM K ϩ . In both groups, the ictaform activity was sensitive to ionotropic glutamate receptor antagonists and lowering of [Ca 2ϩ ] o . Our results show that, in granule cell populations of hippocampal slices from TLE patients, high K ϩ -induced seizure-like activity and ictal spiking coincide with basic electrophysiological abnormalities, hippocampal sclerosis, and mossy fiber sprouting, suggesting that network reorganization could play a crucial role in determining type and threshold of such activity.
Metabolic dysfunction has been implicated in the pathogenesis of temporal lobe epilepsy (TLE), but its manifestation during neuronal activation in the ex vivo hippocampus from TLE patients has not been shown. We characterized metabolic and mitochondrial functions in acute hippocampal slices from pilocarpine-treated, chronic epileptic rats and from pharmaco-resistant TLE patients. Recordings of NAD(P)H fluorescence indicated the status of cellular energy metabolism, and simultaneous monitoring of extracellular potassium concentration ([K+]o) allowed us to control the induction of neuronal activation. In control rats, electrical stimulation elicited biphasic NAD(P)H fluorescence transients that were characterized by a brief initial 'drop' and a subsequent prolonged 'overshoot' correlating to enhanced NAD(P)+ reduction. In chronic epileptic rats, overshoots were significantly smaller in area CA1, but not in the subiculum as compared to controls. In TLE patients, who were histopathologically classified in groups with and without Ammon's horn sclerosis (AHS, non-AHS), large drops and very small overshoots of NAD(P)H transients were observed in dentate gyrus, CA3, CA1 and subiculum. Nevertheless, monitoring mitochondrial membrane potential (DeltaPsi(m)) by mitochondria-specific, voltage-sensitive dye (rhodamine-123) revealed similar mitochondrial responses during neuronal activation with glutamate and protonophore application in area CA1 of control and chronic-epileptic rats. Applying confocal laser scanning microscopy, these findings were confirmed in individual neurons of AHS tissue, indicating a negative DeltaPsi(m) and activation-dependent mitochondrial depolarization. Our data demonstrate severe metabolic dysfunction during neuronal activation in the hippocampus from chronic epileptic rats and humans, although mitochondria maintain negative DeltaPsi(m). Thus, our findings provide a cellular correlate for 'hypometabolism' as described for epilepsy patients and suggest mitochondrial enzyme defects in TLE.
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