Siegrun Gabriel

A novel mechanism underlying drug resistance in chronic epilepsy.

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. Ann Neurol 2003

Stimulus and Potassium-Induced Epileptiform Activity in the Human Dentate Gyrus from Patients with and without Hippocampal Sclerosis

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+. 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+. In both groups, the ictaform activity was sensitive to ionotropic glutamate receptor antagonists and lowering of [Ca2+]o. Our results show that, in granule cell populations of hippocampal slices from TLE patients, high K+-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.

Effects of barium on stimulus-induced rises of [K+]o in human epileptic non-sclerotic and sclerotic hippocampal area CA1

In the hippocampus of patients with therapy‐refractory temporal lobe epilepsy, glial cells of area CA1 might be less able to take up potassium ions via barium‐sensitive inwardly rectifying and voltage‐independent potassium channels. Using ion‐selective microelectrodes we investigated the effects of barium on rises in [K+]o induced by repetitive alvear stimulation in slices from surgically removed hippocampi with and without Ammons horn sclerosis (AHS and non‐AHS). In non‐AHS tissue, barium augmented rises in [K+]o by 147% and prolonged the half time of recovery by 90%. The barium effect was reversible, concentration dependent, and persisted in the presence of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA), N‐methyl‐d‐aspartate (NMDA) and γ‐aminobutyric acid [GABA(A)] receptor antagonists. In AHS tissue, barium caused a decrease in the baseline level of [K+]o. In contrast to non‐AHS slices, in AHS slices with intact synaptic transmission, barium had no effect on the stimulus‐induced rises of [K+]o, and the half time of recovery from the rise was less prolonged (by 57%). Under conditions of blocked synaptic transmission, barium augmented stimulus‐induced rises in [K+]o, but only by 40%. In both tissues, barium significantly reduced negative slow‐field potentials following repetitive stimulation but did not alter the mean population spike amplitude. The findings suggest a significant contribution of glial barium‐sensitive K+‐channels to K+‐buffering in non‐AHS tissue and an impairment of glial barium‐sensitive K+‐uptake in AHS tissue.

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 hintat 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.

Fluorescent tracer in pilocarpine‐treated rats shows widespread aberrant hippocampal neuronal connectivity

Neuronal fibres of the hippocampal formation of normal and chronic epileptic rats were investigated by fluorescent tracing methods using the pilocarpine model of limbic epilepsy. Two months after onset of spontaneous limbic seizures, hippocampal slices were prepared and maintained in vitro for 10 h. Small crystals of fluorescent dye [fluorescein (fluoro‐emerald®) and tetramethylrhodamine (fluoro‐ruby®)] were applied to different hippocampal regions. The main findings were: (i) in control rats there was no supragranular labelling when the mossy fibre tract was stained in stratum radiatum of area CA3. However, in epileptic rats a fibre network in the inner molecular layer of the dentate gyrus was retrogradely labelled; (ii) a retrograde innervation of area CA3 by CA1 pyramidal cells was disclosed by labelling remote CA1 neurons after dye injection into the stratum radiatum of area CA3 in chronic epileptic rats; (iii) labelling of CA1 neurons apart from the injection site within area CA1 was observed in epileptic rats but not in control animals; and (iv), a subicular‐hippocampal projection was present in pilocarpine‐treated rats when the tracer was injected just below the stratum pyramidale of area CA1. The findings show that fibre rearrangement in distinct regions of the epileptic hippocampal formation can occur as an aftermath of pilocarpine‐induced status epilepticus.

Alterations of Neuronal Connectivity in Area CA1 of Hippocampal Slices from Temporal Lobe Epilepsy Patients and from Pilocarpine‐Treated Epileptic Rats

Summary: Purpose: Neuronal network reorganization might be involved in epileptogenesis in human and rat limbic epilepsy. Apart from aberrant mossy fiber sprouting, a more wide‐spread fiber rearrangement in the hippocampal formation might occur. Therefore, we studied sprouting in area CA1 because this region is most affected in human temporal lobe epilepsy.

Alterations of glial cell function in temporal lobe epilepsy

Summary: Purpose: Comparison of extracellular K+ regulation in sclerotic and nonsclerotic epileptic hippocampus.

Entorhinal cortex entrains epileptiform activity in CA1 in pilocarpine-treated rats

Layer III neurons of the medial entorhinal cortex (mEC) project to CA1 via the temporoammonic pathway and exert a powerful feed-forward inhibition of CA1 pyramidal neurons. The present study evaluates the hypothesis that disrupted inhibition of CA1 pyramidal neurons causes an eased propagation of entorhinal seizures to the hippocampus via the temporoammonic pathway. Using a method to induce a confined epileptic focus in brain slices, we investigated the spread of epileptiform activity from the disinhibited mEC to CA1 in control and pilocarpine-treated rats that had displayed status epilepticus and spontaneous recurrent seizures. In pilocarpine-treated rats, the mEC showed a moderate layer III cell loss and an enhanced susceptibility to epileptiform discharges compared to control animals. Entorhinal discharges propagated to CA1 in pilocarpine-treated rats but not in controls. Disconnecting CA3 from CA1 did not affect the spread of epileptiform activity to CA1 excluding its propagation via the trisynaptic hippocampal loop. Mimicking the invasion of epileptiform discharges by repetitive stimulation of the temporoammonic pathway caused a facilitation of field potentials in CA1 that were contaminated by population spikes and afterdischarges in pilocarpine-treated but not control rats. Single cell recordings of CA1 pyramidal neurons revealed a dramatic loss of feed-forward inhibition and the occurrence of strong postsynaptic excitatory potentials in pilocarpine-treated rats. Excitatory responses in CA1 were characterized by multiple NMDA receptor-mediated afterdischarges and a strong paired-pulse facilitation in response to activation of the temporoammonic pathway. Our results suggest that, irrespective of the enhanced seizure-susceptibility of the mEC in epileptic rats, the loss of feed-forward inhibition and the enhanced NMDA receptor-mediated excitability CA1 pyramidal cells ease the spread of epileptiform activity from the mEC to CA1 via the temporoammonic pathway bypassing the classical trisynaptic hippocampal loop.

Effects of barium, furosemide, ouabaine and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on ionophoretically-induced changes in extracellular potassium concentration in hippocampal slices from rats and from patients with epilepsy.

Glial cells limit local K(+)-accumulation by K(+)-uptake through different mechanisms, sensitive to Ba(2+), ouabaine, furosemide, or DIDS. Since the relative contribution of these mechanisms has not yet been determined, we studied the effects of bath-applied barium (2 mM), ouabaine (9 microM), furosemide (2 mM), and DIDS (1 mM) on ionophoretically-induced rises in [K(+)](o) in the pyramidal layer of area CA1 from normal rat slices, in the presence of glutamate receptor (Glu-R) antagonists. We also investigated the effect of barium on ionophoretically-induced tetrapropylammonium (TPA(+))-signals in order to test for barium-induced changes of the extracellular space. Finally, we repeated the barium experiment on slices from human non-sclerotic and sclerotic hippocampal specimens to assess a reduced glial capability for barium-sensitive K(+)-uptake in sclerotic tissue from epilepsy patients. In normal rat slices barium augmented ionophoretically-induced rises in [K(+)](o) by approximately 120%, also in the presence of tetrodotoxin (TTX) (by approximately 150%), but did not significantly affect the TPA(+)-signal. Ouabaine also augmented the K(+)-signal, but only by 27%. Furosemide and DIDS had negligible effects. In slices from sclerotic human hippocampus an augmentation of the K(+)-signal by barium was absent. Thus barium augments ionophoretically-induced K(+)-signals to a similar extent as previously shown for stimulus-induced signals. We suggest that glial barium-sensitive K(+)-buffer mechanisms reduce fast local rises of [K(+)](o) by at least 50%. This capability of glial cells is extremely reduced in area CA1 of slices from human sclerotic hippocampal specimens.

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.