Massimo Avoli

Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro

Seizures in patients presenting with mesial temporal lobe epilepsy result from the interaction among neuronal networks in limbic structures such as the hippocampus, amygdala and entorhinal cortex. Mesial temporal lobe epilepsy, one of the most common forms of partial epilepsy in adulthood, is generally accompanied by a pattern of brain damage known as mesial temporal sclerosis. Limbic seizures can be mimicked in vitro using preparations of combined hippocampus-entorhinal cortex slices perfused with artificial cerebrospinal fluid containing convulsants or nominally zero Mg(2+), in order to produce epileptiform synchronization. Here, we summarize experimental evidence obtained in such slices from rodents. These data indicate that in control animals: (i) prolonged, NMDA receptor-dependent epileptiform discharges, resembling electrographic limbic seizures, originate in the entorhinal cortex from where they propagate to the hippocampus via the perforant path-dentate gyrus route; (ii) the initiation and maintenance of these ictal discharges is paradoxically contributed by GABA (mainly type A) receptor-mediated mechanisms; and (iii) CA3 outputs, which relay a continuous pattern of interictal discharge at approximately 1Hz, control rather than sustain ictal discharge generation in entorhinal cortex. Recent work indicates that such a control is weakened in the pilocarpine model of epilepsy (presumably as a result of CA3 cell damage). In addition, in these experiments electrographic seizure activity spreads directly to the CA1-subiculum regions through the temporoammonic pathway. Studies reviewed here indicate that these changes in network interactions, along with other mechanisms of synaptic plasticity (e.g. axonal sprouting, decreased activation of interneurons, upregulation of bursting neurons) can confer to the epileptic, damaged limbic system, the ability to produce recurrent limbic seizures as seen in patients with mesial temporal lobe epilepsy.

GABA and Epileptogenesis

y-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS. It exerts an inhibitory action in all forebrain structures, and it may play a role in the physiopathogenesis of certain neurological conditions, including epilepsy. Impairment of GABA functions produces seizures, whereas enhancement results in an anticonvulsant effect. Accordingly, several anticonvulsant drugs, including some antiepileptic drugs (AEDs), act by enhancing the efficacy of GABAmediated mechanisms (1,2). Numerous steps in GABA synaptic function are relevant to epileptogenesis: (a) GABA synthesis; (b) GABA release; (c) GABA transport; and (d) activation of receptors, subtypes A and B. Therefore several potential targets exist for epilepsy medications that are related to GABA. GABAA receptors apparently are particularly important in epileptogenesis and therapeutics. Several reviews have described the molecular and functional aspects of GABAA receptors (3-6). In this article, we summarize recent observations, conclusions, and hypotheses regarding the possible role of GABA in epileptogenesis. Much of the information is based on presentations made at the International Symposium “Focus on Epilepsy 111: GABA and Epileptogenesis” held in Whistler, B.C., in May 1995. A list of the faculty that participated in this conference is included in the Notes section.

Sodium channels as molecular targets for antiepileptic drugs

Voltage-gated sodium channels mediate regenerative inward currents that are responsible for the initial depolarization of action potentials in brain neurons. Many of the most widely used antiepileptic drugs, as well as a number of promising new compounds suppress the abnormal neuronal excitability associated with seizures by means of complex voltage- and frequency-dependent inhibition of ionic currents through sodium channels. Over the past decade, advances in molecular biology have led to important new insights into the molecular structure of the sodium channel and have shed light on the relationship between channel structure and channel function. In this review, we examine how our current knowledge of sodium channel structure-function relationships contributes to our understanding of the action of anticonvulsant sodium channel blockers.

Topiramate attenuates voltage-gated sodium currents in rat cerebellar granule cells

Whole-cell, voltage-clamp recordings were made from rat cerebellar granule cells in culture under experimental conditions designed to study voltage-gated Na+ currents that were elicited by depolarizing commands from a holding potential of -60 mV up to +20 mV. These tetrodotoxin-sensitive inward currents were reduced in a dose-related manner by bath application of the structurally novel, anticonvulsant drug topiramate (10-1000 microM; n = 16). Dose-response analysis of this effect revealed an IC50 of 48.9 microM. Topiramate also made the steady-state inactivation curve of this current shift toward more negative values (midpoint of the inactivation curve -46.9 mV under control conditions and -56.5 mV during topiramate application; n = 5). We propose that these effects may contribute to control the sustained depolarizations with repetitive firing of action potentials that occur within neuronal networks during seizure activity. Therefore they may represent a mechanism of action for this novel anticonvulsant drug.

Downregulation of Tonic GABAergic Inhibition in a Mouse Model of Fragile X Syndrome

The absence of fragile X mental retardation protein results in the fragile X syndrome (FXS), a common form of mental retardation associated with attention deficit, autistic behavior, and epileptic seizures. The phenotype of FXS is reproduced in fragile X mental retardation 1 (fmr1) knockout (KO) mice that have region-specific altered expression of some gamma-aminobutyric acid (GABA(A)) receptor subunits. However, little is known about the characteristics of GABAergic inhibition in the subiculum of these animals. We employed patch-clamp recordings from subicular pyramidal cells in an in vitro slice preparation. In addition, semiquantitative polymerase chain reaction and western blot experiments were performed on subiculum obtained from wild-type (WT) and KO mice. We found that tonic GABA(A) currents were downregulated in fmr1 KO compared with WT neurons, whereas no significant differences were observed in phasic GABA(A) currents. Molecular biology analysis revealed that the tonic GABA(A) receptor subunits alpha5 and delta were underexpressed in the fmr1 KO mouse subiculum compared with WT. Because the subiculum plays a role in both cognitive functions and epileptic disorders, we propose that altered tonic inhibition in this structure contributes to the behavioral deficits and epileptic activity seen in FXS patients. This conclusion is in line with evidence implicating tonic GABA(A) inhibition in learning and memory.

Seizure‐like discharges recorded in human dysplastic neocortex maintained in vitro

Application of the convulsant drug 4-aminopyridine (50 to 100 μM) induced spontaneous seizure-like discharges (duration = 76.3 ± 46.8 sec, mean ± SD; interval of occurrence = 225.2 ± 87.9 sec) in slices of neocortex obtained from patients with a diagnosis of focal neuronal migration disorders during neurosurgical procedures for relief of drug-resistant seizures. Similar epileptiform discharges could also be elicited in these slices by single-shock stimuli delivered in the underlying white matter or within the gray matter. By contrast, neocortical slices obtained from patients suffering from temporal lobe epilepsy (which is characterized by Ammons horn sclerosis but relatively normal neocortex) did not generate any epileptiform activity during 4-aminopyridine application. Thus, our study is the first to provide experimental evidence for the intrinsic epileptogenicity that characterizes neuronal migration disorders.

Interaction of cortex and thalamus in spike and wave discharges of feline generalized penicillin epilepsy

Abstract The transition from spindles to spike and wave (SW) discharges of feline generalized penicillin epilepsy was studied using simultaneous EEG recordings from mutually related cortical and thalamic sites after i.m. injection (350,000 IU/kg) or diffuse cortical application of a weak solution (100–300 IU/hemisphere) of penicillin. Both procedures induced similar changes at cortical and thalamic levels, those in the thalamus developing at the same time or slightly later but nerver earlier than in the cortex. These changes consisted of: (i) amplitude increase of spindles, development of positive phases, and decrease in amplitude, followed by disappearance of every second spindle wave as SW discharges developed; (ii) facilitation, progressive amplitude increase, and increase or development of positive phases of recruiting responses to midline thalamic stimulation. Once SW had developed, a decrease in cortical excitability by cortical application of 15% KCl caused the cortical and thalamic SW discharges to disappear and to be replaced by spindles. These results demonstrate that important changes in thalamic activity occur during the development of cortical SW discharge whether induced by i.m. penicillin or by diffuse cortical application of a weak penicillin solution. Changes in thalamic activity appear to be secondary to changes in cortical activity. Thus, although cortical SWs are triggered by thalamocortical inputs which originally were spindle-inducing, these inputs change after penicillin, and reflect an alteration in thalamic activity imposed by the cortex through corticothalamic volleys. In their turn, they modify the cortical response.

GABAergic synchronization in the limbic system and its role in the generation of epileptiform activity

GABA is the main inhibitory neurotransmitter in the adult forebrain, where it activates ionotropic type A and metabotropic type B receptors. Early studies have shown that GABA(A) receptor-mediated inhibition controls neuronal excitability and thus the occurrence of seizures. However, more complex, and at times unexpected, mechanisms of GABAergic signaling have been identified during epileptiform discharges over the last few years. Here, we will review experimental data that point at the paradoxical role played by GABA(A) receptor-mediated mechanisms in synchronizing neuronal networks, and in particular those of limbic structures such as the hippocampus, the entorhinal and perirhinal cortices, or the amygdala. After having summarized the fundamental characteristics of GABA(A) receptor-mediated mechanisms, we will analyze their role in the generation of network oscillations and their contribution to epileptiform synchronization. Whether and how GABA(A) receptors influence the interaction between limbic networks leading to ictogenesis will be also reviewed. Finally, we will consider the role of altered inhibition in the human epileptic brain along with the ability of GABA(A) receptor-mediated conductances to generate synchronous depolarizing events that may lead to ictogenesis in human epileptic disorders as well.

The kainic acid model of temporal lobe epilepsy

The kainic acid model of temporal lobe epilepsy has greatly contributed to the understanding of the molecular, cellular and pharmacological mechanisms underlying epileptogenesis and ictogenesis. This model presents with neuropathological and electroencephalographic features that are seen in patients with temporal lobe epilepsy. It is also characterized by a latent period that follows the initial precipitating injury (i.e., status epilepticus) until the appearance of recurrent seizures, as observed in the human condition. Finally, the kainic acid model can be reproduced in a variety of species using either systemic, intrahippocampal or intra-amygdaloid administrations. In this review, we describe the various methodological procedures and evaluate their differences with respect to the behavioral, electroencephalographic and neuropathological correlates. In addition, we compare the kainic acid model with other animal models of temporal lobe epilepsy such as the pilocarpine and the kindling model. We conclude that the kainic acid model is a reliable tool for understanding temporal lobe epilepsy, provided that the differences existing between methodological procedures are taken into account.

Quantitative evaluation of neuronal loss in the dorsal hippocampus in rats with long-term pilocarpine seizures

Systemic administration of the cholinergic agonist pilocarpine (350-400 mg/kg, i.p.) to rats induces acute behavioral and EEG status epilepticus followed by apparent complete neurological recovery. In rats receiving higher doses of pilocarpine (i.e., 380-400 mg/kg), recurrent seizures reappear 2-2.5 weeks later and continue to occur as long as the rats are kept alive. Stereological estimates of neurons in regions CA1, CA3 and the dentate granule cell layer in the dorsal hippocampus show a dose-dependent neuronal loss in the CA3 and CA1 subregions. The granule cell layer of the dentate gyrus is not affected. No progressive neuronal loss was observed in the regions studied after 3, 6 and 12 weeks during which the animals displayed spontaneous recurrent seizures. The temporal profile of the epileptic condition induced by pilocarpine and the resulting pattern of neuronal loss in the rat hippocampus are similar to those seen in many cases of human temporal lobe epilepsy. The neuronal loss is dose-dependent and primarily results from the acute pilocarpine-induced seizures as chronic seizures do not produce any measurable additional cell loss in the regions examined in the experimental model used in this study.