We have generated an experimental 'double-hit' model of chronic epilepsy to recapitulate the co-existence of abnormal cortical structure and frequently recurrent seizures as observed in human focal cortical dysplasia. We induced cortical malformations by exposing rats prenatally to methylazoxymethanol acetate and triggered status epilepticus and recurrent seizures in adult methylazoxymethanol acetate rats with pilocarpine. We studied the course of epilepsy and the long-term morphologic and molecular changes induced by the occurrence of status epilepticus and subsequent chronic epilepsy in the malformed methylazoxymethanol acetate exposed brain. Behavioural and electroencephalographic analyses showed that methylazoxymethanol acetate pilocarpine rats develop more severe epilepsy than naïve rats. Morphologic and molecular analyses demonstrated that status epilepticus and subsequent seizures, but not pilocarpine treatment per se, was capable of affecting both cortical architectural and N-methyl-D-aspartate receptor abnormalities induced by methylazoxymethanol acetate. In particular, cortical thickness was further decreased and N-methyl-D-aspartate regulatory subunits were recruited at the postsynaptic membrane. In addition, methylazoxymethanol acetate pilocarpine rats showed abnormally large cortical pyramidal neurons with neurofilament over-expression. These neurons bear similarities to the hypertrophic/dysmorphic pyramidal neurons observed in acquired human focal cortical dysplasia. These data show that status epilepticus sets in motion a pathological process capable of significantly changing the cellular and molecular features of pre-existing experimental cortical malformations. They suggest that seizure recurrence in human focal cortical dysplasia might be an additional factor in establishing a pathological circuitry that favours chronic neuronal hyperexcitability.

Epilepsy is characterized by unpredictable recurrent seizures resulting from abnormal neuronal excitability. Increasing evidence indicates that aberrant astrocyte signaling to neurons plays an important role in driving the network hyperexcitability, but the underlying mechanism that alters glial signaling in epilepsy remains unknown. Increase in glutamate release by astrocytes participates in the onset and progression of seizures. Epileptic seizures are also accompanied by increase of tumor necrosis factor alpha (TNFα), a cytokine involved in the regulation of astrocyte glutamate release. Here we tested whether TNFα controls abnormal astrocyte glutamate signaling in epilepsy and through which mechanism. Combining Ca2+ imaging, optogenetics, and electrophysiology, we report that TNFα triggers a Ca2+‐dependent glutamate release from astrocytes that boosts excitatory synaptic activity in the hippocampus through a mechanism involving autocrine activation of P2Y1 receptors by astrocyte‐derived ATP/ADP. In a mouse model of temporal lobe epilepsy, such TNFα‐driven astrocytic purinergic signaling is permanently active, promotes glial glutamate release, and drives abnormal synaptic activity in the hippocampus. Blocking this pathway by inhibiting P2Y1 receptors restores normal excitatory synaptic activity in the inflamed hippocampus. Our findings indicate that targeting the coupling of TNFα with astrocyte purinergic signaling may be a therapeutic strategy for reducing glial glutamate release and normalizing synaptic activity in epilepsy.

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To investigate hypothesized effects of severe epilepsy on malformed cortex, we analyzed surgical samples from eight patients with type IIB focal cortical dysplasia (FCD) in comparison with samples from nine non-dysplastic controls. We investigated, using stereological quantification methods, where appropriate, dysplastic neurons, neuronal density, balloon cells, glia, glutamatergic synaptic input, and the expression of N-methyl-D-aspartate (NMDA) receptor subunits and associated membrane-associated guanylate kinase (MAGUK). In all FCD patients, the dysplastic areas giving rise to epileptic discharges were characterized by larger dysmorphic neurons, reduced neuronal density, and increased glutamatergic inputs, compared to adjacent areas with normal cytology. The duration of epilepsy was found to correlate directly (a) with dysmorphic neuron size, (b) reduced neuronal cell density, and (c) extent of reactive gliosis in epileptogenic/dysplastic areas. Consistent with increased glutamatergic input, western blot revealed that NMDA regulatory subunits and related MAGUK proteins were up-regulated in epileptogenic/dysplastic areas of all FCD patients examined. Taken together, these results support the hypothesis that epilepsy itself alters morphology-and probably also function-in the malformed epileptic brain. They also suggest that glutamate/NMDA/MAGUK dysregulation might be the intracellular trigger that modifies brain morphology and induces cell death.

Epilepsy is characterized by unpredictable recurrent seizures resulting from hypersynchronous discharges from neuron assemblies. Increasing evidence indicates that aberrant astrocyte signalling to neurons plays an important role in driving the network hyperexcitability. Purinergic signalling is central in neuron-glia and glia-glia interactions and dysfunctions in communication pathways involving purinergic receptors have been reported in various CNS pathologies, such as Alzheimer disease, stroke, major depression, schizophrenia, and epilepsy. In the present review we will first discuss the mechanisms by which astrocytes influence neuronal activity. We will then review in more details recent evidence indicating that dysregulation of astrocyte purinergic signalling actively contributes to the appearance of abnormal neuronal activity in epilepsy.

Whether severe epilepsy could be a progressive disorder remains as yet unresolved. We previously demonstrated in a rat model of acquired focal cortical dysplasia, the methylazoxymethanol/pilocarpine - MAM/pilocarpine - rats, that the occurrence of status epilepticus (SE) and subsequent seizures fostered a pathologic process capable of modifying the morphology of cortical pyramidal neurons and NMDA receptor expression/localization. We have here extended our analysis by evaluating neocortical and hippocampal changes in MAM/pilocarpine rats at different epilepsy stages, from few days after onset up to six months of chronic epilepsy. Our findings indicate that the process triggered by SE and subsequent seizures in the malformed brain i) is steadily progressive, deeply altering neocortical and hippocampal morphology, with atrophy of neocortex and CA regions and progressive increase of granule cell layer dispersion; ii) changes dramatically the fine morphology of neurons in neocortex and hippocampus, by increasing cell size and decreasing both dendrite arborization and spine density; iii) induces reorganization of glutamatergic and GABAergic networks in both neocortex and hippocampus, favoring excitatory vs inhibitory input; iv) activates NMDA regulatory subunits. Taken together, our data indicate that, at least in experimental models of brain malformations, severe seizure activity, i.e., SE plus recurrent seizures, may lead to a widespread, steadily progressive architectural, neuronal and synaptic reorganization in the brain. They also suggest the mechanistic relevance of glutamate/NMDA hyper-activation in the seizure-related brain pathologic plasticity.

SUMMARYFocal cortical dysplasia (FCD) is a brain malformation associated with particularly severe drugresistant epilepsy that often requires surgery for seizure control. The molecular basis for such enhanced propensity to seizure generation in FCD is not as yet elucidated. To investigate cellular and molecular bases of epileptogenic mechanisms and possible effect of severe epilepsy on the malformed cortex we have here performed a parallel analysis of a rat model of acquired cortical dysplasia previously established in our laboratory, i.e., the methylazoxymethanol/pilocarpine (MAM-PILO) rats, and surgical samples from patients with type IIB FCD.Data from the MAM-PILO rat model and human FCD samples reveal in both conditions: (1) that status epilepticus (SE) and/or seizures can further modify the cellular and molecular settings of the malformed cortex; (2) excitation/inhibition imbalance, and dysregulation of the N-methyl-D-aspartate/ membrane-associated guanylate kinase (NMDA/MAGUK) expression; (3) activation of cell death in neurons and glia. The data therefore highlight the mechanistic relevance of glutamate/ NMDA hyperactivation in FCD epileptogenesis and suggest that epilepsy is a pathologic process capable of affecting structure and function of both neurons and glia.

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