Intracerebral recordings of the Bereitschaftspotential demonstrate the heterogeneity of its components.

Though consisting of early and late components, the evoked potential preceding a voluntary movement (Bereitschaftspotential - BP) is often considered as a unitary phenomenon. By analyzing intracerebrally recorded BP we attempted to demonstrate that the components are electrophysiological correlates of separate operations. The BPs recorded in 42 epilepsy surgery candidates (28 men, 14 women; aged from 18 to 49 years) during self-paced clenching movements of the hand opposite to the explored hemisphere were investigated in the study. Microdeep intracerebral 5 to 15-contact electrodes were used. The averaged curves were calculated from approximately 30 trials in each case. All the records were taken with a binaural reference. The total number of explored brain regions was 235; the event-related premovement potentials were observed in 121 of them. Three types of premovement responses were observed: (i) the BP with both components; (ii) the BP with the early component only; and (iii) the BP with the late component only. The generators of the early one-component BP were demonstrated in two frontal cortical areas (precentral and middle frontal gyri) and in the parietal area known to be involved in action planning and decision making (precuneus). Some structures generating the early one-component BP were activated during movement; the others were without motor responsiveness. The results suggest a separate elaboration of functional task items in some and their integration in other brain structures, and the existence of volitional mechanisms of different hierarchical character.

New therapeutic targets to develop molecules active in drug-resistant epilepsies.

We have shown that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase involved in the endogenous phosphorylation of the alpha1 subunit of the gamma-aminobutyric acid (GABA)(A) receptor (GABA(A)R), maintaining GABA(A)-R function. GABA(A)R endogenous phosphorylation is opposed by one or several atypical phosphatases. We have shown in addition, using cerebral tissue obtained during epilepsy surgery and control tissue from patients undergoing brain tumor surgery, that both endogenous phosphorylation and GABA(A)R function are significantly reduced in the "epileptogenic" cerebral cortex when compared to control. This dysfunction likely contributes to seizure generation and/or transition from the interictal to the ictal state. The therapeutic challenge is to alleviate the endogenous phosphorylation deficiency of GABA(A)R in the epileptogenic cortical tissue, either through activating the endogenous kinase activity, or inhibiting dephosphorylation of the alpha1 subunit. Following the first trail, we have shown that spermine (the most effective polyamine) increases the GAPDH kinase activity on GABA(A)R and that subsequently such modulation potentiates its function as assessed by rundown studies on isolated neurons. Following the second trail, we have developed methods to identify these atypical membrane-bound phosphatases. Their activities were detected using two synthetic phosphopeptides corresponding to the alpha1 regions of phosphorylation by GAPDH. After purification, the active fractions are submitted to proteomic analysis by nanoLC-Maldi-TOF/TOF for protein identification. Two candidate proteins have been identified, which will be used as targets for high-throughput screening in order to develop original antiepileptic molecules.

Lability of GABAA receptor function in human partial epilepsy: possible relationship to hypometabolism.

The function of the gamma-aminobutyric acid type A receptor (GABA(A)R) is maintained by endogenous phosphorylation. We have shown that the corresponding kinase is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using the locally produced glycolytic ATP. In addition, using cerebral tissue obtained during curative surgery for epilepsy, we showed that both the endogenous phosphorylation and the GABA(A)R function are significantly reduced in the "epileptogenic" cerebral cortex when compared to "control" tissue. This dysfunction likely contributes to seizure generation and/or transition from the interictal to the ictal state. Glucose utilization is decreased in the epileptogenic cortex of patients with partial epilepsy in the interictal state, but the relationship to the disorder remains unclear. We propose that this hypometabolism is related to the deficiency in the endogenous phosphorylation of GABA(A)R and the resulting greater lability of GABAergic inhibition. Several lines of evidences indeed suggest that GABAergic inhibition is costly in terms of metabolic consumption. The deficiency of this glycolysis-dependent mechanism may thus link epileptogenicity to glucose hypometabolism. The antiepileptic effect of ketogenic diets may be mediated by the subsequent rise in the NADH/NAD(+) index, which favors GABA(A)R endogenous phosphorylation and should contribute to restoration of GABAergic inhibition in the epileptogenic zone.

Dysfunction of GABAA receptor glycolysis-dependent modulation in human partial epilepsy.

A reduction in GABAergic neurotransmission has been put forward as a pathophysiological mechanism for human epilepsy. However, in slices of human epileptogenic neocortex, GABAergic inhibition can be clearly demonstrated. In this article we present data showing an increase in the functional lability of GABAergic inhibition in epileptogenic tissue compared with nonepileptogenic human tissue. We have previously shown that the glycolytic enzyme GAPDH is the kinase involved in the glycolysis-dependent endogenous phosphorylation of the alpha1-subunit of GABA(A) receptor, a mechanism necessary for maintaining GABA(A) function. In human epileptogenic cortex obtained during curative surgery of patients with partial seizures, we demonstrate an intrinsic deficiency of GABA(A) receptor endogenous phosphorylation resulting in an increased lability of GABAergic currents in neurons isolated from this tissue when compared with neurons from nonepileptogenic human tissue. This feature was not related to a reduction in the number of GABA(A) receptor alpha1-subunits in the epileptogenic tissue as measured by [(3)H]flunitrazepam photoaffinity labeling. Maintaining the receptor in a phosphorylated state either by favoring the endogenous phosphorylation or by inhibiting a membrane-associated phosphatase is needed to sustain GABA(A) receptor responses in epileptogenic cortex. The increased functional lability induced by the deficiency in phosphorylation can account for transient GABAergic disinhibition favoring seizure initiation and propagation. These findings imply new therapeutic approaches and suggest a functional link to the regional cerebral glucose hypometabolism observed in patients with partial epilepsy, because the dysfunctional GABAergic mechanism depends on the locally produced glycolytic ATP.

Cellular and molecular mechanisms of epilepsy in the human brain.

Animal models have provided invaluable data for identifying the pathogenesis of epileptic disorders. Clearly, the relevance of these experimental findings would be strengthened by the demonstration that similar fundamental mechanisms are at work in the human epileptic brain. Epilepsy surgery has indeed opened the possibility to directly study the functional properties of human brain tissue in vitro, and to analyze the mechanisms underlying seizures and epileptogenesis. Here, we summarize the findings obtained over the last 40 years from electrophysiological, histochemical and molecular experiments made with the human brain tissue. In particular, this review will focus on (i) the synaptic and non-synaptic properties of neocortical neurons along with their ability to produce synchronous activity; (ii) the anatomical and functional alterations that characterize limbic structures in patients presenting with mesial temporal lobe epilepsy; (iii) the issue of antiepileptic drug action and resistance; and (iv) the pathophysiology of seizure genesis in Taylor's type focal cortical dysplasia. Finally, we will address some of the problems that are inherent to this type of experimental approach, in particular the lack of proper controls and possible strategies to obviate this limitation.

Glyceraldehyde-3-phosphate dehydrogenase is a GABAA receptor kinase linking glycolysis to neuronal inhibition.

Protein phosphorylation is crucial for regulating synaptic transmission. We describe a novel mechanism for the phosphorylation of the GABA(A) receptor, which mediates fast inhibition in the brain. A protein copurified and coimmunoprecipitated with the phosphorylated receptor alpha1 subunit; this receptor-associated protein was identified by purification and microsequencing as the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Molecular constructs demonstrated that GAPDH directly phosphorylates the long intracellular loop of GABA(A) receptor alpha1 subunit at identified serine and threonine residues. GAPDH and the alpha1 subunit were found to be colocalized at the neuronal plasma membrane. In keeping with the GAPDH/GABA(A) receptor molecular association, glycolytic ATP produced locally at plasma membranes was consumed for this alpha1 subunit phosphorylation, possibly within a single macrocomplex. The membrane-attached GAPDH is thus a dual-purpose enzyme, a glycolytic dehydrogenase, and a receptor-associated kinase. In acutely dissociated cortical neurons, the rundown of the GABA(A) responses was essentially attributable to a Mg(2+)-dependent phosphatase activity, which was sensitive to vanadate but insensitive to okadaic acid or fluoride. Rundown was significantly reduced by the addition of GAPDH or its reduced cofactor NADH and nearly abolished by the addition of its substrate glyceraldehyde-3-phosphate (G3P). The prevention of rundown by G3P was abolished by iodoacetamide, an inhibitor of the dehydrogenase activity of GAPDH, indicating that the GABA(A) responses are maintained by a glycolysis-dependent phosphorylation. Our results provide a molecular mechanism for the direct involvement of glycolysis in neurotransmission.

Epileptiform synchronization in the human dysplastic cortex.

Taylor's focal cortical dysplasia corresponds to a localized disruption of the normal cortical lamination with an excess of large, aberrant cells. Sustained epileptic discharges originate from the dysplastic neocortex and this tissue retains sufficient connectivity for expressing seizure abnormalities. In this brief review, we summarize the findings obtained by analyzing surgically-resected human tissue with focal cortical dysplasia that was maintained in vitro in a brain slice preparation. These data have been compared with those obtained from human cortex with normal structural organization; such tissue was available from patients undergoing surgery for a variety of neurological disorders, most often for mesial temporal lobe epilepsy. These studies have shown that: (i). slices obtained from focal cortical dysplastic tissue have an intrinsic ability to generate ictal-like epileptiform events when challenged with the convulsant drug 4-aminopyridine; (ii). 4-aminopyridine-induced ictal discharges are not seen in neocortical slices obtained from neocortical samples with no or minimal structural lesion; (iii). these ictal discharges are caused by the activation of excitatory amino acid receptors, and in particular those of the N-methyl-D aspartate type; (iv). focal cortical dysplastic tissue also generates synchronous potentials that are mainly contributed by GABA(A) receptor-mediated conductances.