Thomas-Nicolas Lehmann

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.

Differential effects of temporal pole resection with amygdalohippocampectomy versus selective amygdalohippocampectomy on material-specific memory in patients with mesial temporal lobe epilepsy.

Purpose: In the surgical treatment of mesial temporal lobe epilepsy, there is converging evidence that individually tailored or selective approaches have a favorable cognitive outcome compared to standard resections. There is, however, also evidence that due to collateral damage, selective surgery can be less selective than suggested. As part of a prospective transregional research project the present study evaluated the outcome in memory and nonmemory functions, following two selective approaches: a combined temporal pole resection with amygdalohippocampectomy (TPR+) and transsylvian selective amygdalohippocampectomy (SAH).

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.

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.

Glycinergic tonic inhibition of hippocampal neurons with depolarizing GABAergic transmission elicits histopathological signs of temporal lobe epilepsy

An increasing number of epilepsy patients are afflicted with drug‐resistant temporal lobe epilepsy (TLE) and require alternative therapeutic approaches. High‐affinity glycine receptors (haGlyRs) are functionally adapted to tonic inhibition due to their response to hippocampal ambient glycine, and their synthesis is activity‐dependent. Therefore, in our study, we scanned TLE hippocampectomies for expression of haGlyRs and characterized the effects mediated by these receptors using primary hippocampal neurons. Increased haGlyR expression occurred in TLE hippocampi obtained from patients with a severe course of disease. Furthermore, in TLE patients, haGlyR and potassium chloride cotransporter 2 (KCC2) expressions were inversely regulated. To examine this potential causal relationship with respect to TLE histopathology, we established a hippocampal cell culture system utilising tonic inhibition mediated by haGlyRs in response to hippocam‐pal ambient glycine and in the context of a high Cl equilibrium potential, as is the case in TLE hippocampal neurons. We showed that hypoactive neurons increase their ratio between glutamatergic and GABAergic synapses, reduce their dendrite length and finally undergo excitotoxicity. Pharmacological dissection of the underlying processes revealed ionotropic glutamate and TrkB receptors as critical mediators between neuronal hypoactivity and the emergence of these TLE‐characteristic histopathological signs. Moreover, our results indicate a beneficial role for KCC2, because decreasing the Cl− equilibrium potential by KCC2 expression also rescued hypoactive hippocampal neurons. Thus, our data support a causal relationship between increased haGlyR expression and the emergence of histopathological TLE‐characteristic signs, and they establish a pathophysiological role for neuronal hypoactivity in the context of a high Cl− equilibrium potential.

Nitric Oxide Modulates Spreading Depolarization Threshold in the Human and Rodent Cortex

Background and Purpose— Recent clinical data have suggested that prolonged cortical spreading depolarizations (CSDs) contribute to the pathogenesis of delayed ischemic neurologic deficits after subarachnoid hemorrhage. Elevated extracellular potassium concentrations and lowered nitric oxide (NO) levels have been detected in experimental and clinical subarachnoid hemorrhage. We investigated whether a similar extracellular composition renders the brain more susceptible to CSDs. Methods— Electrophysiologic and blood flow changes were studied in vivo in rats. Intrinsic optical signals, alterations of NO level, and electrophysiologic changes were investigated in rodent and human brain slices. Results— Elevation of subarachnoid extracellular potassium in rats in vivo triggered CSDs. Using NO-sensitive dyes, we found that CSDs induce NO synthesis in neurons and endothelial cells. When we blocked NO synthesis in vivo, CSDs occurred at a significantly lower threshold and propagated with a wave of ischemia. This increased susceptibility for CSDs by a low NO level was confirmed in rat and human neocortical slices and depended on P/Q-type calcium channels and N-methyl-d-aspartate receptors, but not on guanylate cyclase. Mice deficient in endothelial NO synthase, in contrast to mice deficient in neuronal NO synthase, had an inherently lower threshold. Conclusions— Basal NO production determined CSD threshold. The threshold effect depended predominantly on endothelial NO synthase. Reduced NO levels, as in patients with subarachnoid hemorrhage, may render the brain more susceptible to CSDs. Because CSDs have been linked to the pathogenesis of delayed ischemic neurologic deficits, raising its threshold by increasing NO availability may prove therapeutically beneficial in patients with subarachnoid hemorrhage.

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.

Splice-specific roles of glycine receptor α3 in the hippocampus

Glycine receptor (GlyR) α3 is involved in vision, and processing of acoustic and nociceptive signals, and RNA editing of GLRA3 transcripts was associated with hippocampal pathophysiology of mesial temporal lobe epilepsy (TLE). However, neither the role of GlyR α3 splicing in hippocampal neurons nor the expression of splice variants have yet been elucidated. We report here that the long (L) splice variant of GlyR α3 predominates in the brain of rodents. Cellular analysis using primary hippocampal neurons and hippocampus cryosections revealed preferential association of synaptic α3L clusters with glutamatergic nerve endings in strata granulare and pyramidale. In primary hippocampal neurons GlyR α3L clusters also preferred glutamatergic nerve endings while α3K was mainly in a diffuse state. Co‐expression of GlyR β subunit with α3L or α3K produced heteromeric receptor clusters and favoured their association with GABAergic terminals. However, heteromeric α3L was still more efficient than heteromeric α3K in associating with glutamatergic nerve endings. To give physiological relevance to these results we have finally analysed GlyR α3 splicing in human hippocampus obtained from patients with intractable TLE. As up‐regulation of α3K occurred at the expense of α3L in TLE patients with a severe course of disease and a high degree of hippocampal damage, our results again involve post‐transcriptional processing of GLRA3 transcripts in the pathophysiology of TLE.