Uwe Heinemann

The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus.

Astrocytic gap junctions have been suggested to contribute to spatial buffering of potassium in the brain. Direct evidence has been difficult to gather because of the lack of astrocyte-specific gap junction blockers. We obtained mice with coupling-deficient astrocytes by crossing conditional connexin43-deficient mice with connexin30−/− mice. Similar to wild-type astrocytes, genetically uncoupled hippocampal astrocytes displayed negative resting membrane potentials, time- and voltage-independent whole-cell currents, and typical astrocyte morphologies. Astrocyte densities were also unchanged. Using potassium-selective microelectrodes, we assessed changes in potassium buffering in hippocampal slices of mice with coupling-deficient astrocytes. We demonstrate that astrocytic gap junctions accelerate potassium clearance, limit potassium accumulation during synchronized neuronal firing, and aid in radial potassium relocation in the stratum lacunosum moleculare. Furthermore, slices of mice with coupling-deficient astrocytes displayed a reduced threshold for the generation of epileptiform events. However, it was evident that radial relocation of potassium in the stratum radiatum was not dependent on gap junctional coupling. We suggest that the perpendicular array of individual astrocytes in the stratum radiatum makes these cells ideally suited for spatial buffering of potassium released by pyramidal cells, independent of gap junctions. In general, a surprisingly large capacity for K+ clearance was conserved in mice with coupling-deficient astrocytes, indicating that gap junction-dependent processes only partially account for K+ buffering in the hippocampus.

Lasting Blood-Brain Barrier Disruption Induces Epileptic Focus in the Rat Somatosensory Cortex

Perturbations in the integrity of the blood-brain barrier have been reported in both humans and animals under numerous pathological conditions. Although the blood-brain barrier prevents the penetration of many blood constituents into the brain extracellular space, the effect of such perturbations on the brain function and their roles in the pathogenesis of cortical diseases are unknown. In this study we established a model for focal disruption of the blood-brain barrier in the rat cortex by direct application of bile salts. Exposure of the cerebral cortex in vivo to bile salts resulted in long-lasting extravasation of serum albumin to the brain extracellular space and was associated with a prominent activation of astrocytes with no inflammatory response or marked cell loss. Using electrophysiological recordings in brain slices we found that a focus of epileptiform discharges developed within 4-7 d after treatment and could be recorded up to 49 d postoperatively in >60% of slices from treated animals but only rarely (10%) in sham-operated controls. Epileptiform activity involved both glutamatergic and GABAergic neurotransmission. Epileptiform activity was also induced by direct cortical application of native serum, denatured serum, or albumin-containing solution. In contrast, perfusion with serum-adapted electrolyte solution did not induce abnormal activity, thereby suggesting that the exposure of the serum-devoid brain environment to serum proteins underlies epileptogenesis in the blood-brain barrier-disrupted cortex. Although many neuropathologies entail a compromised blood-brain barrier, this is the first direct evidence that it may have a role in the pathogenesis of focal cortical epilepsy, a common neurological disease.

Transcriptome Profiling Reveals TGF-β Signaling Involvement in Epileptogenesis

Brain injury may result in the development of epilepsy, one of the most common neurological disorders. We previously demonstrated that albumin is critical in the generation of epilepsy after blood–brain barrier (BBB) compromise. Here, we identify TGF-β pathway activation as the underlying mechanism. We demonstrate that direct activation of the TGF-β pathway by TGF-β1 results in epileptiform activity similar to that after exposure to albumin. Coimmunoprecipitation revealed binding of albumin to TGF-β receptor II, and Smad2 phosphorylation confirmed downstream activation of this pathway. Transcriptome profiling demonstrated similar expression patterns after BBB breakdown, albumin, and TGF-β1 exposure, including modulation of genes associated with the TGF-β pathway, early astrocytic activation, inflammation, and reduced inhibitory transmission. Importantly, TGF-β pathway blockers suppressed most albumin-induced transcriptional changes and prevented the generation of epileptiform activity. Our present data identifies the TGF-β pathway as a novel putative epileptogenic signaling cascade and therapeutic target for the prevention of injury-induced epilepsy.

Induction of sharp wave-ripple complexes in vitro and reorganization of hippocampal networks

Hippocampal sharp wave–ripple complexes (SPW-Rs) occur during slow-wave sleep and behavioral immobility and are thought to represent stored information that is transferred to the neocortex during memory consolidation. Here we show that stimuli that induce long-term potentiation (LTP), a neurophysiological correlate of learning and memory, can lead to the generation of SPW-Rs in rat hippocampal slices. The induced SPW-Rs have properties that are identical to spontaneously generated SPW-Rs: they originate in CA3, propagate to CA1 and subiculum and require AMPA/kainate receptors. Their induction is dependent on NMDA receptors and involves changes in interactions between clusters of neurons in the CA3 network. Their expression is blocked by low-frequency stimulation but not by NMDA receptor antagonists. These data indicate that induction of LTP in the recurrent CA3 network may facilitate the generation of SPW-Rs.

Blood–brain barrier breakdown-inducing astrocytic transformation: Novel targets for the prevention of epilepsy

Epileptogenesis is common following brain insults such as trauma, ischemia and infection. However, the mechanisms underlying injury-related epileptogenesis remain unknown. Recent studies demonstrated impaired integrity of the blood-brain barrier (BBB) during epileptogenesis. Here we review accumulating experimental evidence supporting the potential involvement of primary BBB lesion in epileptogenesis. Data from animal experiments demonstrate that primary breakdown of the BBB prone animals to develop focal neocortical epilepsy that is followed by neuronal loss and impaired functions. The extravasation of albumin from the circulation into the brain neuropil was found to be sufficient for the induction of epileptogenesis. Albumin binds to transforming growth factor beta receptor 2 (TGFbetaR2) in astrocytes and induces rapid transcriptional modifications, astrocytic transformation and dysfunction. We highlight a novel cascade of events which is initiated by increased BBB permeability, eventually leading to neuronal dysfunction, epilepsy and cell loss. We review potential mechanisms and existing experimental evidence for the important role of astrocytes and the TGFbeta pathway in epileptogenesis. Finally, we review evidence from human clinical data supporting the involvement of BBB lesion in epilepsy. We propose that primary vascular injury, and specifically BBB breakdown and repair, are key elements in altered interactions within the neurovascular unit and thus may serve as new therapeutic targets.

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

Astrocytic dysfunction in epileptogenesis: consequence of altered potassium and glutamate homeostasis?

Focal epilepsy often develops following traumatic, ischemic, or infectious brain injury. While the electrical activity of the epileptic brain is well characterized, the mechanisms underlying epileptogenesis are poorly understood. We have recently shown that in the rat neocortex, long-lasting breakdown of the blood–brain barrier (BBB) or direct exposure of the neocortex to serum-derived albumin leads to rapid upregulation of the astrocytic marker GFAP (glial fibrillary acidic protein), followed by delayed (within 4–7 d) development of an epileptic focus. We investigated the role of astrocytes in epileptogenesis in the BBB-breakdown and albumin models of epileptogenesis. We found similar, robust changes in astrocytic gene expression in the neocortex within hours following treatment with deoxycholic acid (BBB breakdown) or albumin. These changes predict reduced clearance capacity for both extracellular glutamate and potassium. Electrophysiological recordings in vitro confirmed the reduced clearance of activity-dependent accumulation of both potassium and glutamate 24 h following exposure to albumin. We used a NEURON model to simulate the consequences of reduced astrocytic uptake of potassium and glutamate on EPSPs. The model predicted that the accumulation of glutamate is associated with frequency-dependent (>100 Hz) decreased facilitation of EPSPs, while potassium accumulation leads to frequency-dependent (10–50 Hz) and NMDA-dependent synaptic facilitation. In vitro electrophysiological recordings during epileptogenesis confirmed frequency-dependent synaptic facilitation leading to seizure-like activity. Our data indicate a transcription-mediated astrocytic transformation early during epileptogenesis. We suggest that the resulting reduction in the clearance of extracellular potassium underlies frequency-dependent neuronal hyperexcitability and network synchronization.

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.

Blood-brain barrier dysfunction, TGFβ signaling, and astrocyte dysfunction in epilepsy.

Brain insults, including traumatic and ischemic injuries, are frequently followed by acute seizures and delayed development of epilepsy. Dysfunction of the blood‐brain barrier (BBB) is a hallmark of brain insults and is usually surrounding the core lesion. Recent studies from several laboratories confirmed that vascular pathology is involved in the development of epilepsy and demonstrate a key role for astroglia in this process. In this review, we focus on glia‐related mechanisms linking vascular pathology, and specifically BBB dysfunction, to seizures and epilepsy. We summarize molecular and physiological experimental data demonstrating that the function of astrocytes is altered due to direct exposure to serum albumin, mediated by transforming growth factor beta signaling. We discuss the reported changes and their potential role in the observed hyperexcitability as well as potential implications of these findings for the future development of new diagnostic modalities and treatments to allow a full implementation of the gained knowledge for the benefit of patients with epilepsy.

Impairment of intracortical GABAergic inhibition in a rat model of absence epilepsy

The WAG/Rij rat strain is characterized in its EEG by the manifestation of spike-wave discharges which resemble in their spontaneous appearance and pharmacological sensitivity the absence epilepsy observed in humans. In order to test the hypothesis whether cellular intrinsic membrane and/or synaptic network properties in the neocortex are modified in this form of epilepsy, we analyzed with extra- and intracellular recording techniques the functional status of neocortical slices obtained from adult epileptic WAG/Rij rats and compared them with the data acquired from non-epileptic control Wistar rats. Intrinsic membrane properties, like resting membrane potential, neuronal input resistance and basic cellular firing characteristics, did not differ between these two strains. However, the analysis of extra- and intracellularly recorded synaptic responses revealed an intracortical hyperexcitability which was accompanied by a significant reduction in the efficiency of GABAergic inhibition. Our data indicate that the imbalance between intracortical excitatory and inhibitory mechanisms may at least contribute to the expression and augmentation of spike-wave discharges in epileptic WAG/Rij rats.