Focal reduction of neuronal glutamate transporters in human neocortical epilepsy

PURPOSE: To study the differential expression of excitatory amino acid transporters (EAATs) at localized epileptic foci compared to nonepileptic regions in human neocortical epilepsy. Decreased expression of EAATs, the predominant mechanism to remove synaptic-released glutamate, may explain mechanisms of heightened excitability at these epileptic foci. METHODS: The differential expression of EAAT1-4 at the mRNA and protein levels was measured in electrically mapped human neocortical tissues using quantitative real-time PCR and immunoblotting. This required a novel way to prevent aggregation of EAAT proteins through cold solubilization. Layer-specific neuronal densities were measured to control for potential differences in neuronal density. RESULTS: While focal epileptic brain regions show marked increases in immediate early genes, they have significant reductions in the neuronal glutamate transporter mRNAs (EAAT3 and EAAT4). These changes were not associated with changes in relative neuronal density, suggesting a reduction in EAAT mRNA per neuron. Immunohistochemical staining of epileptic human neocortex confirmed the presence of EAAT1 and EAAT2 proteins in astroglial cells and EAAT3 and EAAT4 proteins in human cortical neurons. At the protein level, western blots of the same epileptic and nonepileptic regions for a subset of these patients showed a similar decrease of EAAT3 and EAAT4. Despite no change in EAAT2 mRNA, EAAT2 protein expression was significantly reduced at epileptic foci. CONCLUSION: Regional reductions in EAAT expression at human neocortical epileptic foci could produce increased local glutamate levels that in turn may contribute to both hyperexcitability and the spontaneous generation of epileptic discharges that characterize human epileptic foci.     

Electrocorticographic and neurochemical findings after local cortical valproate application in patients with pharmacoresistant focal epilepsy

Because oral pharmacological treatment of neocortical focal epilepsy is limited due to common systemic side effects and relatively low drug concentrations reached at the epileptic foci locally, application of antiepileptic agents directly onto the neocortical focus may enhance treatment tolerability and efficacy. We describe the effects of cortically applied sodium valproate (VPA) in two patients with pharmacoresistant neocortical focal epilepsy who were selected for epilepsy surgery after a circumscribed epileptic focus had been determined by invasive presurgical evaluation using subdural electrodes. Local VPA modified epileptic activity as electrocorticographically recorded from the chronic focus in both patients. In addition, VPA induced local increase of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in cortical tissue samples, whereas the excitatory glutamate was possibly decreased. In this clinical pilot study, we could show antiepileptic effects of cortically applied VPA in humans by electrocorticographic and neurochemical parameters.     

Chronic epileptic foci in monkeys: correlation between seizure frequency and proportion of pacemaker epileptic neurons.

A total of 1,802 neurons from 15 alert, undrugged Macaca mulatta monkeys were studied. Thirteen monkeys had chronic epilepsy induced by subpial alumina injections in precentral cortex. Precentral neurons were judged epileptic by the magnitude and variability of the percentage of interspike intervals less than 5 msec during periods when the monkeys were awake. This method of quantifying epileptic single neuron activity appears highly reliable in distinguishing epileptic neurons from precentral neurons in either normal cortex, cortex contralateral to, or within the focus. For the 13 epileptic monkeys, the relative proportion of strongly epileptic neurons found within foci was logarithmically correlated with the mean number of daily seizures. Because of the similarity between the physiology of the alumina focus in monkeys and epileptic foci in humans, these data imply that the severity of focal human epilepsy is a function of epileptic neuronal mass.     

Progressive neocortical damage in epilepsy

Our objective was to determine the pattern and extent of generalized and focal neocortical atrophy that develops in patients with epilepsy and the factors associated with such changes. As part of a prospective, longitudinal follow-up study of 122 patients with chronic epilepsy, 68 newly diagnosed patients, and 90 controls, serial magnetic resonance imaging scans were obtained 3.5 years apart. Image subtraction was used to identify diffuse and focal neocortical change that was quantified with a regional brain atlas and a fully automated segmentation algorithm. New focal or generalized neocortical volume losses were identified in 54% of patients with chronic epilepsy, 39% of newly diagnosed patients and 24% of controls. Patients with chronic epilepsy were significantly more likely to develop neocortical atrophy than control subjects. The increased risk of cerebral atrophy in epilepsy was not related to a history of documented seizures. Risk factors for neocortical atrophy were age and multiple antiepileptic drug exposure. Focal and generalized neocortical atrophy commonly develops in chronic epilepsy. Neocortical changes seen in a quarter of our control group over 3.5 years were likely to reflect physiological changes. Our results show that ongoing cerebral atrophy may be widespread and remote from the putative epileptic focus, possibly reflecting extensive networks and interconnections between cortical regions.     

The neocortico to mesio-basal limbic propagation of focal epileptic activity during the spike-wave complex.

In order to localize epileptogenic electrophysiological sources, a multichannel MEG system was used in 3 patients with partial epilepsy during presurgical evaluation. MEG and EEG (including scalp, sphenoidal and intracranial foramen ovale electrodes) were recorded simultaneously during a period of intensive video-EEG monitoring in order to observe single spontaneous spikes. In addition to MRI, SPECT and PET investigations were performed. Electrical activity subsequent to the activity of the epileptic focus could be localized by the MEG after noise reduction using a temporal correlation technique. Simultaneous registration of the magnetic field and the electrical field showed that the source of the primary focal epileptic activity (first period during the total spike wave complex where a dipolar magnetic field pattern is found) is localized in neocortical lateral regions, whereas another focal epileptic activity in a later phase of propagation occurs in temporal mesial regions. In 1 patient (case 1) the primary focal epileptic activity was localized in the surrounding neocortical tissue of an angioma and the middle and inferior temporal gyrus. The second phase of propagation is localized in temporo-basal-mesial regions, including para- and hippocampal structures. The latest center of activity occurred in posterior parts of the gyrus cinguli. In 2 other patients, the primary focal epileptogenic activity was localized at the insula and also spread into temporal basal mesial regions. A multi-modal approach to research of focal epilepsy, combining metabolic, electrical potential, magnetoencephalographic and morphological data, recorded by non-invasive techniques, offers new perspectives for the detection of involved brain regions. The 3-D and time-resolved localization of focal epileptic activity, correlated with the individual anatomy of the human brain, may improve the determination of neuronal populations involved in the individual epileptogenic process, especially in the interaction between temporal or extratemporal neocortex and limbic system.     

Induction of c-fos mRNA and protein in neurons and glia after traumatic brain injury: Pharmacological characterization

Focal brain injury in mice induced c-fos mRNA and protein in neurons throughout the damaged neocortex, including the piriform and the entorhinal cortices, as well as in nonneural brain cells (e.g., glia, pia, ependyma). The pattern of c-fos induction after injury suggested that injury led to spreading depression which then led to c-fos induction in neurons. Human neurons in the temporal cortex and hippocampus also showed c-fos protein induction after neurosurgical trauma. The c-fos mRNA and protein induction in mouse neurons was prevented by the noncompetitive NMDA antagonist ketamine but only partially inhibited by the voltage-dependent calcium channel antagonist nifedipine and the calmodulin antagonist trifluoperazine. The c-fos protein induction in nonneural brain cells after injury was not affected by these drugs. Thus, induction of c-fos in neocortical neurons after focal brain injury is partly NMDA receptor mediated.     

Increased discharge threshold after an interictal spike in human focal epilepsy

It is commonly assumed that interictal spikes (ISs) in focal epilepsies set off a period of inhibition that transiently reduces tissue excitability. Post-spike inhibition was described in experimental models but was never demonstrated in the human epileptic cortex. In the present study post-spike excitability was retrospectively evaluated on intracerebral stereo-electroencephalographic recordings performed in the epileptogenic cortex of five patients suffering from drug-resistant focal epilepsy secondary to Taylor-type neocortical dysplasias. Patients typically presented with highly periodic interictal spiking activity at 2.33 +/- 0.87 Hz (mean +/- SD) in the dysplastic region. During the stereo-electroencephalographic procedure, low-frequency stimulation at 1 Hz was systematically performed for diagnostic purposes to identify the epileptogenic zone. The probability of evoking an IS during the interspike period in response to 1-Hz stimuli delivered close to the ictal-onset zone was examined. Stimuli that occurred early after a spontaneous IS (within 70% of the inter-IS period) had a very low probability of generating a further IS. On the contrary, stimuli delivered during the late inter-IS period had the highest probability of evoking a further IS. The generation of stimulus-evoked ISs is occluded for several hundred milliseconds after the occurrence of a preceding spike discharge. As previously shown in animal models, these findings suggest that, during focal, periodic interictal spiking, human neocortical excitability is phasically controlled by post-spike inhibition.     

Widespread epileptic networks in focal epilepsies: EEG-fMRI study

Purpose: To assess the extent of brain involvement during focal epileptic activity, we studied patterns of cortical and subcortical metabolic changes coinciding with interictal epileptic discharges (IEDs) using group analysis of simultaneous electroencephalography and functional magnetic resonance imaging (EEG‐fMRI) scans in patients with focal epilepsy. Methods: We selected patients with temporal lobe epilepsy (TLE, n = 32), frontal lobe epilepsy (FLE, n = 14), and posterior quadrant epilepsy (PQE, n = 20) from our 3 Tesla EEG‐fMRI database. We applied group analysis upon the blood oxygen–level dependent (BOLD) response associated with focal IEDs. Key Findings: Patients with TLE and FLE showed activations and deactivations, whereas in PQE only deactivations occurred. In TLE and FLE, the largest activation was in the mid–cingulate gyri bilaterally. In FLE, activations were also found in the ipsilateral frontal operculum, thalamus, and internal capsule, and in the contralateral cerebellum, whereas in TLE, we found additional activations in the ipsilateral mesial and neocortical temporal regions, insula, and cerebellar cortex. All three groups showed deactivations in default mode network regions, the most widespread being in the TLE group, and less in PQE and FLE. Significance: These results indicate that different epileptic syndromes result in unique and widespread networks related to focal IEDs. Default mode regions are deactivated in response to focal discharges in all three groups with syndrome specific pattern. We conclude that focal IEDs are associated with specific networks of widespread metabolic changes that may cause more substantial disturbance to brain function than might be appreciated from the focal nature of the scalp EEG discharges.     

Regulated expression of VP16CREB in neuroblastoma cells: analysis of differentiation and apoptosis

Highly malignant neuroblastoma tumors generally have defects in differentiation and apoptotic pathways. For a better understanding of these events, we use a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons in response to elevated levels of cAMP. Because one of the main downstream effectors of the cAMP signaling pathway is cAMP-response element binding (CREB), we reasoned that it might affect the expression of genes associated with differentiation and apoptotic events in NBP2 cells. To investigate this, we established tetracycline-regulated expression (TetOff) of VP16CREB, which constitutively transactivates promoters containing the CRE sequence motif. Using this system, we found that inducible expression of VP16CREB in NBP2 cells results in 1) morphological differentiation that is characterized by the formation of neurites and growth cones, 2) reversible cell differentiation unlike cAMP-induced terminal differentiation, 3) cell cycle arrest at G1, 4) no apoptosis in the presence of partial inhibition of proteasome unlike an increase in cAMP levels, and 5) changes in the expression of many genes, including down-regulation of N-myc, cyclin B1, Dickkopf-1, and Mad-2 and up-regulation of tyrosine hydroxylase, c-fos, N10, and ICER genes. Although VP16CREB expression and activation of the cAMP pathway impart many similar effects in NBP2 cells, they also bear some distinct genetic and morphological differences. Our data suggest that increased levels of cAMP function through not only CREB but also other signaling pathways that account for the additional cAMP-induced effects, including irreversible differentiation and onset of apoptosis during partial inhibition of proteasome in NBP2 cells.     

Persistent sodium current in subicular neurons isolated from patients with temporal lobe epilepsy

The persistent sodium current is a common target of anti-epileptic drugs and contributes to burst firing. Intrinsically burst firing subicular neurons are involved in the generation and spread of epileptic activity. We measured whole-cell sodium currents in pyramidal neurons isolated from the subiculum resected in drug-resistant epileptic patients and in rats. In half of the cells from both patients and rats, the sodium current inactivated within 500 ms at -30 mV. Others displayed a tetrodotoxin-sensitive slowly or non-inactivating sodium current of up to 53% of the total sodium current amplitude. Compared with the transient sodium current in the same cells, this persistent sodium current activated with normal kinetics but its voltage-dependent activation occurred 7 mV more hyperpolarized. Depolarizing voltage steps that lasted 10 s completely inactivated the persistent sodium current. Its voltage dependence did not differ from that of the transient sodium current but its slope was less steep. The voltage dependence and kinetics of the persistent sodium current in cells from patients were not different from that in subicular cells from rats. The current density and the relative amplitude contribution were 3-4 times greater in neurons from drug-resistant epilepsy patients. The abundant presence of persistent sodium current in half of the subicular neurons could lead to a larger number of neurons with intrinsic burst firing. The extraordinarily large amplitude of the persistent sodium current in this subset of subicular neurons might explain why these patients are susceptible to seizures and hard to treat pharmacologically.     

Focal neocortical epilepsy affects hippocampal volume, shape, and structural integrity: a longitudinal MRI and immunohistochemistry study in a rat model

Purpose: Extratemporal epilepsy often coincides with cognitive decline, which may be associated with hippocampal dysfunction. Severe hippocampal sclerosis can be detected with conventional neuroimaging in some patients with chronic extrahippocampal epilepsy (so‐called dual pathology). However, subtle structural hippocampal changes may already develop at a much earlier phase, and in a larger number of patients. Our goal was to longitudinally characterize the development of bilateral hippocampal pathology in an experimental neocortical focal epilepsy model. Methods: Focal unilateral neocortical epilepsy was induced by microinjection of tetanus toxin in the primary motor cortex in adult male Sprague‐Dawley rats. Another group of age‐matched rats served as controls. In both groups, structural magnetic resonance imaging (MRI) was performed at 1, 3, 7, and 10 weeks of follow‐up. Bilateral hippocampi were outlined and macroscopically analyzed using a state‐of‐the‐art point‐based morphometry model. Hippocampal microstructural changes at the end of follow‐up, 10 weeks after epilepsy induction, were assessed with postmortem standard cresyl‐violet, Fluoro‐Jade, proteolipid protein 1, vimentin, glial fibrillary acidic protein, and ionized calcium binding adaptor molecule 1 stainings. Key Findings: All rats in the injected group developed seizures. The ipsilateral hippocampal volume was on average 8.76 (mean) ± 3.32% (standard deviation) smaller in the epileptic animals as compared to controls (p = 0.01) during the 10 weeks of follow‐up. The contralateral hippocampus showed a similar reduction of 8.49 (mean) ± 3.27% (standard deviation) in total volume (p = 0.02). Clear hippocampal shape differences were found between the two groups. The most affected areas after epilepsy induction were the bilateral dorso‐mediorostral, dorsolateral, and ventrolateral areas of the hippocampi. Normal developmental shape changes of the hippocampus, as detected in control rats, were largely absent in the ipsilateral hippocampus of epileptic rats. Quantitative histologic analysis revealed significant neuronal loss in the hippocampus, most pronounced in the hilar subregion, globally impaired myelination, reactive astrocytosis, and activated microglia. We found a weak but significant correlation between the number of neurons and hippocampal volume (r = 0.25, p = 0.0025). Significance: We found evidence of hippocampal pathology in both hemispheres following experimental focal neocortical epilepsy. The observed development of bilateral hippocampal pathology, with onset in the early stages of focal neocortical epilepsy, may be a significant factor in comorbidities, such as cognitive dysfunction, found in patients with extratemporal localization‐related epilepsy.     

Densities of parvalbumin-immunoreactive neurons in non-malformed hippocampal sclerosis-temporal neocortex and in cortical dysplasias

The changes in density of inhibitory parvalbumin-immunoreactive interneurons were quantitatively studied by immunohistochemistry in a series of human neocortical samples comprising the spectrum of malformations of cortical development (MCD) encountered in epilepsy surgery and the non-malformed hippocampal sclerosis-temporal neocortex in patients with refractory temporal lobe epilepsy. The highest relative density of parvalbumin-immunoreactive cells was obtained in the control samples (n = 21). The number of parvalbumin-immunoreactive neurons was significantly decreased in non-malformed hippocampal sclerosis-temporal neocortex (n = 73, 80.5% of control values). In a proportion of the latter samples as well as in two controls we observed patchy regions of absence of parvalbumin staining. The total counts of parvalbumin-immunoreactive cells in all the categories of MCD - "mild MCD" (n = 25), focal cortical dysplasia type I (n = 19) and type II (n = 15) - were decreased representing 72.4%, 55.0% and 12.2% of control values, respectively. Significantly different parvalbumin-immunoreactive cell densities were demonstrated between the focal cortical dysplasia types IIA and IIB. In "mild MCD", we observed a more pronounced decrease of parvalbumin-immunoreactive cells in the infragranular layers. No significant differences were revealed between the temporal and extratemporal examples of analogous MCD types. This study provides evidence for reduction of inhibitory parvalbumin-immunoreactive interneurons in the epileptic neocortex affected by MCD as well as in morphologically unaffected epileptic temporal neocortex, thus representing a possible mechanism for their epileptogenicity.