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Restraining excitatory neurotransmission within a seizure focus provides a nondestructive treatment strategy for intractable neocortical epilepsy. Clostridial toxin light chain (LC) inhibits synaptic transmission by digesting a critical vesicle-docking protein, synaptobrevin, without directly altering neuronal health. This study tests the treatment efficacy of adenoviral vector delivered LC (AdLC) on a model of seizures in rats induced by motor cortex penicillin (PCN) injection. LC expression significantly reduced electroencephalogram (EEG) frequency, amplitude, duration, and latency compared to control groups injected with either an adenoviral vector bearing green fluorescent protein (AdGFP) or phosphate buffered solution (PBS). Correspondingly, LC gene expression improved behavioral manifestations including seizure severity and latency. There was no statistical difference in motor function before and after vector administration between treatment and control groups. Histological analysis revealed spatially discrete LC expression with corresponding synaptobrevin depletion in the cortex surrounding the injection site. Thus, vector-mediated LC gene delivery is capable of improving both the EEG and behavioral manifestations of PCN-induced focal neocortical seizures through synaptobrevin depletion.

Objective: To investigate the relationship between the apolipoprotein (ApoE) epsilon4 allele and memory performance (verbal and nonverbal) in patients with medically intractable temporal lobe epilepsy (TLE) who underwent temporal lobectomy., Methods: Presurgical and postsurgical memory performance was examined in 87 adult patients with TLE (epsilon4 = 22; non-epsilon4 = 65) to determine whether the expression of ApoE-epsilon4 may be associated with memory performance in this population and to examine how this relationship may be affected by duration of epilepsy., Results: There was a significant interaction between ApoE-epsilon4 status and duration of epilepsy such that epsilon4 carriers with a long duration of epilepsy demonstrated the poorest memory performance on both verbal and nonverbal measures. This relationship was observed both before and after temporal lobectomy, with little change in test performance over time., Conclusions: The ApoE-epsilon4 allele interacts with longstanding seizures to affect memory performance, both verbal and nonverbal, in patients with medically intractable temporal lobe epilepsy.

The recent proposal by the ILAE Task Force for Epilepsy Classification is a multiaxial, syndrome-oriented approach. Epilepsy syndromes--at least as defined by the ILAE Task Force--group patients according to multiple, usually poorly defined parameters. As a result, these syndromes frequently show significant overlap and may change with patient age. We propose a five-dimensional and patient-oriented approach to epilepsy classification. This approach shifts away from syndrome orientation, using independent criteria in each of the five dimensions similarly to the diagnostic process in general neurology. The main dimensions of this new classification consist of (1) localizing the epileptogenic zone, (2) semiology of the seizure, (3) etiology, (4) seizure frequency, and (5) related medical conditions. These dimensions characterize all information necessary for patient management, are independent parameters, and include information more pertinent than the ILAE axes with regard to patient management. All cases can be classified according to this five-dimensional system, even at initial encounter when no detailed test results are available. Information from clinical tests such as MRI and EEG are translated into the best possible working hypothesis at the time of classification, allowing increased precision of the classification as additional information becomes available.

Focal cortical dysplasia (FCD) is a common cause of pharmacoresistant epilepsy that is amenable to surgical resective treatment. The identification of structural FCD by magnetic resonance imaging (MRI) can contribute to the detection of the epileptogenic zone and improve the outcome of epilepsy surgery. MR epilepsy protocols that include specific T1 and T2 weighted, and fluid-attenuated inversion recovery (FLAIR) sequences give complementary information about the characteristic imaging features of FCD; focal cortical thickening, blurring of the gray-white junction, high FLAIR signal, and gyral anatomical abnormalities. Novel imaging techniques such as magnetic resonance spectroscopy (MRS), magnetization transfer imaging (MTI), and diffusion tensor imaging (DTI) can improve the sensitivity of MR to localize the anatomical lesion. Functional/metabolic techniques such as positron emission tomography (PET), ictal subtraction single photon emission computed tomography (SPECT), functional MRI (fMRI), and magnetic source imaging (MSI) have the potential to visualize the metabolic, vascular, and epileptogenic properties of the FCD lesion, respectively. Identification of eloquent areas of cortex, to assist in the surgical resection plan, can be obtained non-invasively through the use of fMRI and MSI. Although a significant number of FCD lesions remain unidentified using current neuroimaging techniques, future advances should result in the identification of an increasing number of these cortical malformations.

Purpose: Focal cortical dysplasia (CD) is characterized by the presence of dysmorphic neurons, laminar and columnar disorganization. A few patients with CD have balloon cells intermixed with dysmorphic neurons. The cellular characteristics of balloon cells remain unknown. This study was intended to determine further the cellular characteristics of balloon cells., Methods: Neocortical tissue resected from five patients with medically intractable focal epilepsy due to CD was studied. The presence of balloon cells (large opalescent cells with eccentric nuclei) was confirmed in all five patients by using cresylecht violet staining. Immunocytochemistry used antibodies against markers of pluripotential stem cells (CD133), multipotential progenitor cells (nestin), antiapoptotic gene products (Bcl-2), immature neurons (beta-tubulin 3, TUJ1), immature glia (vimentin), mature neurons (MAP2 and NeuN), and astrocytes (glial fibrillary acidic protein; GFAP)., Results: Balloon cells (BCs) were found to be immunoreactive to Bcl-2 (46%), vimentin (41%), Nestin (28%), CD133 (28%), MAP2 (27%), GFAP (14%), and TUJ1 (10%). An extremely small number of BCs were immunopositive for NeuN. Confocal double labeling showed that balloon cells were dually immunopositive for CD133/nestin; CD133/GFAP; CD133/Bcl-2, and nestin/GFAP., Conclusions: These results show that balloon cells are heterogeneous cell populations expressing cell-surface markers for pluripotential stem cells and proteins for multipotent progenitors, or immature neurons/glia. The presence of stem cell/progenitor markers in the balloon cells could be due to a persistent postnatal neurogenesis or early embryonic insult that resulted in arrest of proliferation/differentiation at their early stages. Additionally, the coexpression of Bcl-2 in CD133-positive balloon cells suggests that a resistance to programmed cell death may be involved in the pathogenesis of cortical dysplasia.

The NMDA receptor is one of the ionotropic glutamate receptors essential for excitatory neurotransmission and synaptic plasticity. The present study examined the ontogenies of NMDAR1 and NMDAR2B during development (embryonic days: (E) 14, 15, 16, 17 and postnatal days: (P) 0, 5, 10, 20, and 60). The immunoreactivities of both NMDAR1 and NMDAR2B were visualized and semi- quantified simultaneously using batch-matched procedures during the developmental period. In addition, the heteromeric coassembly of NMDAR1 (NR1) and NMDAR2B (NR2B) was analyzed throughout development using immunocoprecipitation blots. Differential developmental expressions were observed between the two NMDA receptor subunits. Although both NR1 and NR2B were already expressed on cortical neurons at E14, NR1 immunoreactivity was lower than NR2B but increased up to P20 then was lower at the last sampling day (P60). By comparison, the high NR2B immunoreactivity at E14 increased until P5, and until the next sample day (P60). Despite the early embryonic protein expressions in prenatal cortex both NR1 and NR2B immunocoprecipitation analyses showed that NR1-NR2B coassembly was not initially detectable until birth (P0), and increased up to P20. This suggests that the physically interactive NMDAR1-2B complex may contribute to the plasticity of early postnatal development characterized by axonal dendritic synaptogenesis.

A better understanding of the mechanisms involved in human higher cortical functions requires a detailed knowledge of neuronal connectivity between functional cortical regions. Currently no good method for tracking in vivo neuronal connectivity exists. We investigated the inter-areal connections in vivo in the human language system using a new method, which we termed 'cortico-cortical evoked potentials' (CCEPs). Eight patients with epilepsy (age 13-42 years) underwent invasive monitoring with subdural electrodes for epilepsy surgery. Six patients had language dominance on the side of grid implantation and two had bilateral language representation by the intracarotid amobarbital test. Conventional cortical electrical stimulation was performed to identify the anterior and posterior language areas. Single pulse electrical stimuli were delivered to the anterior language (eight patients), posterior language (four patients) or face motor (two patients) area, and CCEPs were obtained by averaging electrocorticograms (ECoGs) recorded from the perisylvian and extrasylvian basal temporal language areas time-locked to the stimulus. The subjects were not asked to perform any tasks during the study. Stimulation at the anterior language area elicited CCEPs in the lateral temporo-parietal area (seven of eight patients) in the middle and posterior part of the superior temporal gyrus, the adjacent part of the middle temporal gyrus and the supramarginal gyrus. CCEPs were recorded in 3-21 electrodes per patient. CCEPs occurred at or around the particular electrodes in the posterior language area which, when stimulated, produced speech arrest. Similar early and late CCEPs were obtained from the basal temporal area by stimulating the anterior language area (three of three patients). In contrast, stimulation of the adjacent face motor area did not elicit CCEPs in language areas but rather in the postcentral gyrus. Stimulation of the posterior language area produced CCEPs in the anterior language (three of four patients) as well as in the basal temporal area (one of two patients). These CCEPs were less well defined. These findings suggest that perisylvian and extrasylvian language areas participate in the language system as components of a network by means of feed-forward and feed-back projections. Different from the classical Wernicke-Geschwind model, the present study revealed a bidirectional connection between Broca's and Wernicke's areas probably through the arcuate fasciculus and/or the cortico-subcortico-cortical pathway. CCEPs were recorded from a larger area than the posterior language area identified by electrical stimulation. This suggests the existence of a rather broad neuronal network surrounding the previously recognized core region of this area.

Objective: We sought to determine the efficacy of gamma knife radiosurgery (GKRS) in controlling mesial temporal lobe epilepsy., Methods: From August 1999 to January 2001, five patients with drug-resistant mesial temporal lobe epilepsy due to hippocampal sclerosis underwent GKRS amygdalohippocampectomy. All of the patients underwent standard epilepsy preoperative evaluation at the comprehensive epilepsy center of the Cleveland Clinic Foundation. A marginal dose of 20 Gy to the 50% isodose line was delivered to the mesial temporal structures in all patients. Postoperative follow-up included serial neurological examinations, neuroimaging studies, and neuropsychological evaluations., Results: None of the patients were seizure-free after GKRS. Two patients died, 1 month and 1 year after the procedure, as a result of complications related to recurrent seizures. At 1 year, T2-weighted magnetic resonance imaging changes were noted in all three patients, which suggested radiational changes. None of the three surviving patients had any seizure reduction, so 18, 20, and 22 months after GKRS, they underwent temporal lobectomy, which resulted in complete seizure control in all patients., Conclusion: GKRS at the 20-Gy dose level did not lead to seizure control in patients with mesial temporal lobe epilepsy due to hippocampal sclerosis.

The alpha1-adrenergic receptors (alpha1ARs) play an important role in mediating sympathetic neurotransmission in peripheral organ systems; however, central alpha1ARs are not well characterized. Additionally, due to the lack of sufficiently subtype-selective drugs or high avidity antibodies, the contribution of each alpha1AR subtype to various central functions is currently unclear. Transcription regulation through alpha1AR subtypes in the CNS is also unknown. Of interest, transgenic mice that systemically overexpress the alpha1BAR show central symptoms that include age-progressive impaired mobility, neurodegeneration and susceptibility to epileptic seizure. To investigate the molecular basis of this phenotype, oligonucleotide microarray studies of whole brains of various ages were carried out to compare gene expression profiles between transgenic and normal brains. The results indicated changes in expression of apoptotic, calcium regulatory, neurodegenerative and genes involved in neurotransmission. Defects in regulation of intracellular calcium are known to play a role in cell death; thus, these genes may provide clues as to the molecular basis of alpha1BAR-induced neurodegeneration. Epilepsy is a disorder that can be caused by an imbalance between excitatory (e.g. glutamate) and inhibitory (e.g. GABA) signals. Microarray analysis of transgenic brains showed increased N-methyl-d-aspartate (NMDA) receptors and decreased GABAA, which were confirmed with immunohistochemistry, western blot and radioligand binding studies. The alpha1BAR also co-localized with the glutamatergic distribution, suggesting a glutamate imbalance as a molecular rationale for the epileptic seizures.

Purpose: Medically intractable temporal lobe epilepsy (TLE) due to hippocampal sclerosis (HS), with or without cortical dysplasia (CD), is associated with atrophy of the hippocampal formation and regional fluorodeoxyglucose positron-emission tomography (FDG-PET) hypometabolism. The relation between areas of functional and structural abnormalities is not well understood. We investigate the relation between FDG-PET metabolism and temporal lobe (TL) and hippocampal atrophy in patients with histologically proven isolated HS and HS associated with CD., Methods: Twenty-three patients underwent en bloc resection of the mesial and anterolateral neocortical structures. Ten patients were diagnosed with isolated HS; 13 patients had associated microscopic CD. Temporal lobe volumes (TLVs) and hippocampal volumes were measured. Magnetic resonance imaging (MRI) and PET were co-registered, and regions of interest (ROIs) determined as gray matter of the mesial, lateral, and anterior temporal lobe., Results: All patients (HS with or without CD) had significant ipsilateral PET hypometabolism in all three regions studied (p < 0.0001). In patients with isolated HS, the most prominent hypometabolism was in the anterior and mesial temporal lobe, whereas in dual pathology, it was in the lateral temporal lobe. TLVs and hippocampal volumes were significantly smaller on the epileptogenic side (p < 0.05). The PET asymmetries ipsilateral/contralateral to the epileptogenic zone and TLV asymmetries correlated significantly for the anterior and lateral temporal lobes (p < 0.05) in the HS+CD group, but not in the isolated HS group. Mesial temporal hypometabolism was not significantly different between the two groups., Conclusions: Temporal neocortical microscopic CD with concurrent HS is associated with more prominent lateral temporal metabolic dysfunction compared with isolated HS in TL atrophy. Further studies are needed to confirm these findings and correlate the PET hypometabolic patterns with outcome data in patients operated on for HS with or without CD.

Purpose: Malformations due to abnormal cortical development (MCDs) are common pathologic substrates of medically intractable epilepsy. The in situ epileptogenicity of these lesions as well as its relation to histopathologic changes remains unknown. The purpose of this study was to correlate the cellular patterns of MCDs with the expression of focal cortical epileptogenicity as assessed by direct extraoperative electrocorticographic (ECoG) recordings by using subdural grids., Methods: Fifteen patients with drug-resistant focal epilepsy due to pathologically confirmed MCD who underwent subdural electrode placement for extraoperative seizure localization and cortical mapping between 1997 and 2000 were included in the study. Areas of interictal spiking and ictal-onset patterns were identified and separated during surgery for further pathologic characterization (cellular and architectural). Three pathologic groups were identified: type I; architectural disorganization with/without giant neurons, type IIA; architectural disorganization with dysmorphic neurons, and type IIB; architectural disorganization, dysmorphic neurons, and balloon cells (BCs). The focal histopathologic subtypes of MCDs in cortical tissue resected were then retrospectively correlated with in situ extraoperative ECoG patterns., Results: Cortical areas with histopathologic subtype IIA showed significantly higher numbers of slow repetitive spike pattern in comparison with histopathologic type I (p = 0.007) and normal pathology (p = 0.002). The ictal onset came mainly from cortical areas with histopathologic type IIA (nine of 15 patients). None of the seizures originated from neocortical areas that showed BC-containing MCD (type IIB)., Conclusions: This study shows that areas containing BCs are less epileptogenic than are closely located dysplastic regions. These results suggest a possible protective effect of BCs or a severe disruption in the neuronal networks in BCs containing dysplastic lesions. Further studies are needed to elucidate the nature and the potential role(s) of balloon cells in MCD-induced epileptogenicity.

Focal clonic seizures are a frequent epileptic phenomenon. However, there are little data about their pathomechanism. In four patients with focal epilepsy and subdural electrodes, focal clonus was elicited by electrical stimulation of the motor cortex. Three additional patients underwent intraoperative stimulation of the spinal cord. Rhythmic clonic muscle responses were elicited by cortical stimulation with 20-50 Hz. The clonus consisted of simultaneous trains of compound muscle action potentials (CMAP) in agonistic and antagonistic muscles alternating with periods of muscular silence despite continuous stimulation. Clonus frequency decreased from 4.0-8.0 Hz at 50 Hz stimulation to 3.0-3.5 Hz at 20 Hz paralleled by a prolongation of the trains of CMAP. The stimulation frequency correlated with the number of stimuli blocked during relaxation. During the stable stimulation periods, the clonus frequency decreased over time. The number of stimuli which formed a train of CMAP and which were blocked during relaxation increased towards the end of the stimulation periods. Increasing intensity of stimulation at the same frequency converted a clonic to a tonic response. There was always an 1:1 relationship between stimulus and CMAP during spinal cord stimulation. We hypothesize that during cortical stimulation, clonus is elicited by synchronous activation of pyramidal tract (PT) neurons which results in excitation of intracortical GABA(B)ergic interneurons by recurrent axon-collaterals. This leads to stepwise hyperpolarization of PT neurons intermittently suppressing the output of PT neurons despite continuous stimulation. This mechanism can explain our finding that temporal and spatial summation of the stimuli were needed for clonus generation., (Copyright 2002 Elsevier Science B.V.)

The EEG characteristics of isolated hippocampal sclerosis (HS) and HS associated with other types of temporal lobe pathology are not well defined. The pathologic substrate may be an important variable in determining seizure-free outcome. The objective of this study was to define the distribution of epileptiform discharges in patients with HS and HS associated with microscopic dysplasia, and to examine their relationship with hippocampal atrophy and cell loss. Thirty-four patients (15 women and 19 men; mean age, 30.6 +/- 11.2 years), all with good outcomes after temporal lobectomy (Engel classes I and II), were included. The characteristics studied were frequency and distribution of spikes, MRI-based hippocampal volume ratios, and quantitative hippocampal cell density in various subregions. The isolated HS group showed a trend to a higher percentage of epileptiform discharges maximal at the anterior temporal electrodes (89.87 +/- 17.0%; 79.5 +/- 28.2% in the dual-pathology group). The isolated HS group had, on average, significantly more cell loss (P < 0.001). There was a significant negative correlation between the amount of cell loss in the CA1 area and both anterior temporal spikes and hippocampal ratios (P < 0.05). Isolated HS and dual pathology show minimal differences in interictal spike distribution and frequency. More widespread spike distributions in severe isolated HS compared with patients with less cell loss is probably the result of less organized limbic circuitry.

Purpose: Cortical dysplasia (CD) is a frequent cause of medically intractable focal epilepsy. The mechanisms of CD-induced epileptogenicity remain unknown. The difficulty in obtaining and testing human tissue warrants the identification and characterization of animal model(s) of CD that share most of the clinical, electroencephalographic (EEG), and histopathologic characteristics of human CD. In this study, we report on the in vivo EEG characterization of the radiation-induced model of CD., Methods: Timed-pregnant Sprague-Dawley rats were irradiated on E17 using a single dose of 145 cGy or left untreated. Their litters were identified and implanted with bifrontal epidural and hippocampal depth electrodes for prolonged continuous EEG recordings. After prolonged EEG monitoring, animals were killed and their brains sectioned and stained for histologic studies., Results: In utero-irradiated rats showed frequent spontaneous interictal epileptiform spikes and spontaneous seizures arising independently from the hippocampal or the frontal neocortical structures. No epileptiform or seizure activities were recorded from age-matched control rats. Histologic studies showed the presence of multiple cortical areas of neuronal clustering and disorganization. Moreover, pyramidal cell dispersion was seen in the CA1>CA3 areas of the hippocampal formations., Conclusions: Our results further characterize the in vivo EEG characteristics of the in utero radiation model of CD using long-term EEG monitoring. This model may be used to study the molecular and cellular changes in epileptogenic CD and to test the efficacy of newer antiepileptic medications.

The cellular mechanisms of epileptogenesis are reviewed as related to their role(s) in the expression of hyperexcitability and hypersynchrony. The data on the roles of the glutamate, GABA, acetylcholine, and adenosine receptors is discussed. The recent information on the role of glial cells in the expression of epileptogenicity is reviewed.

The advent of MR imaging techniques has immensely improved the ability to evaluate and manage patients with epilepsy. The principles of various MR techniques, the pertinent MRI neuro-anatomic features, and computer based 3D reconstruction post processing methods are discussed. Newer MR-based techniques such as magnetic resonance spectroscopy (MRS) and diffusion weighted imaging (DWI) and their applications and potential usefulness in the evaluation of patients with medically intractable epilepsy are also reviewed.

Purpose: Diffusion-weighted MR imaging (DWI) is a novel technique to delineate focal areas of cytotoxic edema of various etiologies. We hypothesized that DWI may also detect the epileptogenic region and adjacent areas during the ictal and early postictal periods in patients with temporal lobe epilepsy (TLE)., Methods: We studied patients with intractable TLE (n = 9), due to hippocampal sclerosis (HS, n = 7), left mesial temporal lobe tumor (n = 1), and of unknown etiology (n = 1). Informed consent was obtained before inclusion in the study. All patients with single short seizures were scanned immediately after EEG-documented seizures (between 45 and 150 min); one of two patients in status was scanned 14 h after cessation of seizures. DWI results were analyzed visually and by calculating apparent diffusion coefficient (ADC) maps., Results: We found significant decreases in ADC postictally in one of six patients with TLE due to HS and single short seizures. One patient with an incompletely resected temporal lobe tumor also exhibited ADC abnormalities. One patient in focal status epilepticus revealed a decrease in ADC, and one patient with a continuous aura had no DWI abnormality., Conclusions: Postictal DWI technique may occasionally help delineate epileptic areas in some patients with TLE. Yield is low in patients with HS and single short seizures: it may be higher in patients with tumor or status epilepticus.

Purpose: Human cortical dysplasia (CD) is a frequent cause of medically intractable focal epilepsy. The neurotransmitter mechanisms of epileptogenicity in these lesions have been attributed to changes in various glutamate receptor subtypes. Increased N-methyl-D-aspartate (NMDA) receptor (NR) 2A/B coassembled with NR1 subunits has been shown in focal epileptic CD. The purpose of this study is to correlate in situ CD epileptogenicity and the expression of various glutamate receptor subtypes., Methods: The histopathological, morphological, and immunocytochemical findings in cortical tissue resected from five patients with medically intractable epilepsy and CD were correlated with electroencephalographic data recorded from subdural grids. The NMDA antibodies identified subunits NR1 (splicing variants 1a, 1b, 2a, and 2b) and NR2A/B., Results: Epileptogenic specimens displayed the following common features: (a) widespread histological abnormalities of horizontal and columnar dyslamination, neurons with inverted polarity, and more extensive dendritic changes; (b) significantly higher NR2A/B immunoreactivity in both the dysplastic somata and all their dendritic processes; and (c) no statistically significant change in NR1 subunit expression but a more pronounced staining of the apical dendrites in highly epileptogenic cortex. These abnormalities were either absent or minimal in resected specimens that did not show evidence of severe in vivo epileptogenicity., Conclusion: These studies provide direct evidence for a major contribution of the NR2A/B subunit in CD-induced epileptogenicity.

Purpose: This study was designed to quantify the relation between expressions of NMDA receptor (NMDAR) subunits (1 and 2A/B) and the epileptogenicity in human focal cortical dysplasia., Methods: Immunoblotting and immunoprecipitation were used to quantify these receptor subunits in tissue resected from EEG-verified epileptic and distal nonepileptic frontal cortical areas in each of three patients as determined by chronic subdural electrode recordings. In each patient, adjacent sections were immunostained to verify that the numbers of dysplastic neurons were greater in epileptic than in nonepileptic cortex., Results: In all patients, NMDAR2A/B expressions and their coassemblies with NMDAR1 were increased in epileptic dysplastic cortex compared with the relatively normal appearing nonepileptic cortex. For all three patients, there were no significant differences in NMDAR1 protein expressions between the two EEG groups., Conclusions: These results suggest that increased NMDAR1-NMDAR2A/B coassembly contributes to hyperexcitability in dysplastic cortical neurons and focal seizure onsets.

Cortical dysplasia (CD) is now recognized as one of the major causes of pediatric focal neocortical epilepsy, and surgical procedures have been considered early in life. However, the mechanisms involved in seizure generation and intractability in these patients are still unknown. We analyzed with immunocytochemistry for various antibodies the brain tissue from 4 children (10 months to 6 years old) with focal epilepsy due to focal CD in order to study the inhibitory and excitatory circuits in dysplastic areas. Our group had similar histopathological and clinical characteristics. In all patients we found areas of cortical disorganization with dysplastic neurons and balloon cells. We studied distributions of glial cells with glial fibrillary acidic protein (GFAP) and neurons with microtubule-associated protein 2 (MAP-2). Gliosis was present in all cases, and GFAP stained also some balloon cells. Dysplastic neurons were darkly stained by MAP-2, and we also found balloon cells weakly stained with MAP-2 in the same areas where GFAP was positive, suggesting coexpression of neuronal and glial markers in some of these cells. There was an increased expression of glutamate receptors, especially GluR2/3, but also N-methyl-D-aspartate receptors in dysplastic cortex. The inhibitory circuit does not seem to be decreased, rather we notice an increased amount of glutamate-decarboxylase-positive terminals around some of the big neurons. We discuss the possible role of these findings as mechanisms of epilepsy., (Copyright 1999 S. Karger AG, Basel)

The NMDA receptor is one of the ionotropic glutamate receptors essential for excitatory neurotransmission. The NMDAR1 subunit is inactivated by direct interaction with calmodulin. The protein levels of calmodulin, NMDAR1 and their complex were quantified in tissue resected from epileptogenic and non-epileptogenic cortical areas as determined by chronic subdural electrode recordings from three patients (aged 6, 14 and 18 years) with focal epilepsy associated with cortical dysplasia. In all patients, the co-assembly of calmodulin and NMDAR1 was decreased in epileptogenic dysplastic cortex compared with normal appearing non-epileptogenic cortex, while there was no significant difference in the total protein levels of calmodulin or NMDAR1 between the two EEG groups. These results suggest that decreased calmodulin-NMDAR1 co-assembly is a cellular mechanism that contributes to hyperexcitability in dysplastic cortical neurons and in focal seizure onsets.

Purpose: We studied temporal changes of brain sodium and apparent diffusion coefficient (ADC) in a temporal lobe epilepsy (TLE) rat model using kainic acid (KA)., Methods: In situ three-dimensional 23Na magnetic resonance imaging (MRI) and proton diffusion-weighted imaging (DWI) were used. KA at a dose of 10 mg/kg body weight and 12 adult Sprague Dawley rats weighing 228-318 g (268 +/- 25 g) were used., Results: Twenty-four hours after KA injection, magnetic resonance (MR) visible sodium levels increased in both the pyriform cortex (+90%) and amygdala (+68%) and increased insignificantly in the hippocampus (+18%) and caudate-putamen (12%). The ADC in the pyriform cortex showed a -9% decrease at 5 h postictally, reaching -30% at 24 h, whereas in the amygdala decreases were -8 and -26% respectively. A significant decrease in ADC (-7%) in the hippocampus was also observed 24 h postically. Seven days later, sodium increases persisted, whereas ADC returned to normal level., Conclusions: The increase in MR visible sodium, associated with the decrease in ADC is consistent with the hypothesis that sequential seizures caused an increase in sodium influx and perturbation of membrane ion homeostasis, which eventually evolved into an irreversible phase of cellular edema, with increased MR visible intracellular sodium and decreased ADC. Return of ADC to near-control level and persistent high sodium level at 7 days may be explained by the increase in extracellular space and tissue necrosis.

Objective: Mesial temporal sclerosis (MTS) is a fairly well-recognized cause of intractable epilepsy. Its coexistence with cortical dysplasia is less commonly described. Herein, we describe the clinical and pathologic features of MTS, including cases in which cortical dysplasia was also identified., Design: Retrospective surgical series of 27 patients., Setting: Tertiary referral center with a high volume of epilepsy surgery., Patients: Patients with medically intractable epilepsy who underwent hippocampal resections and in whom an unequivocal histologic diagnosis of MTS could be made., Results: The patients studied included 18 males and 9 females ranging in age from 15 to 48 years (mean 32 years). Mesial temporal sclerosis was characterized by severe neuronal loss accompanied by gliosis occurring in the CA1/prosubiculum (27 patients, 100%), focally in the dentate gyrus (12 patients, 44%), and in the CA4 region (11 patients, 41%). Five patients (24%) had coexistent cortical dysplasia with increased molecular layer neurons (five patients), gyral fusion (two patients), diffuse architectural disorganization of the cortex (one patient), and clusters of atypical neurons and glial cells within the cortex (one patient). Twenty-five (93%) of the 27 patients had white matter neuronal heterotopia. Follow-up data were available for each patient (mean 23 months). Twenty-two of the patients are free of seizures postoperatively, including all five with coexistent cortical dysplasia; the five remaining patients have fewer seizures. There appeared to be no difference clinically between patients with MTS and no dysplasia and those with coexistent cortical dysplasia., Conclusion: We conclude that (1) MTS most severely involves the CA1/prosubiculum and CA4 regions of the hippocampus (the dentate gyrus may also be focally severely involved); (2) MTS and cortical dysplasia do occasionally exist; and (3) surgical outcome for MTS appears to be independent of coexistent cortical dysplasia.

Tropomodulin is a 40.6 kDa tropomyosin-binding protein associated with actin filaments in muscle and the membrane cytoskeleton in erythrocytes. We have detected tropomodulin mRNA and protein in brains of rats by northern and western blot analyses. In situ hybridization of rat brain and spinal cord sections shows tropomodulin expression in the cerebellum, neocortex, hippocampus, and anterior horn of the spinal cord. Tropomodulin expression is first observed around day 15 after birth and increases through day 24. The temporal and spatial changes in tropomodulin expression during cerebellar development parallel those for brain tropomyosin. Tropomodulin mRNA increases in the dentate gyrus of the hippocampus following prolonged seizure activity induced by kainic acid administration; the increase is clearly evident 8 h after initiation of seizures and is still present 1 week later. However, Western blot analysis of tropomodulin protein level in the dentate gyrus before and after seizure induction show only slight increases in tropomodulin protein concentration, suggesting tight regulation of tropomodulin expression at the translational level. The developmental expression of tropomodulin, together with the induction of tropomodulin mRNA production in the dentate gyrus after kainic acid treatment, suggests a role for tropomodulin in neuronal organization and plasticity.

The present study was directed at evaluating the possible involvement of protein synthesis in excitotoxin-induced neuronal damage and prolonged expression of the proto-oncogene, c-fos. Kainic acid-induced seizure activity elicited varying degrees of neuronal damage and cell loss in selectively vulnerable regions of the adult rat limbic system. Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter behavioral seizure characteristics, but markedly attenuated damage to susceptible neuronal populations. A prolonged increase in c-fos mRNA was observed by in situ hybridization up to 16 h after the onset of seizures in regions exhibiting neuronal death. Pretreatment with cycloheximide did not affect the transient induction of c-fos observed in numerous structures, but significantly reduced the prolonged expression of c-fos mRNA in kainate-vulnerable regions. Despite producing massive seizure activity, systemic kainic acid administration during the early postnatal period did not induce any neuronal death, and did not result in prolonged c-fos expression in any brain structures. The developmental onset of selective neuronal vulnerability coincided with that of prolonged c-fos expression in susceptible neuronal populations. In adult rats, seizure activity induced by pentylenetetrazole did not produce neuronal damage nor did it produce prolonged c-fos expression. These results not only demonstrate that kainate-induced neurotoxicity and the prolonged expression of c-fos are both prevented by cycloheximide, but also strengthen idea that prolonged c-fos expression is a marker of neuronal death.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine synthesis and is regulated by both transcription-dependent and transcription-independent mechanisms. We compared the effects of asparagine, an amino acid previously shown to increase ODC activity in adult hippocampal slices, on ODC mRNA and activity in adult and neonatal hippocampal slices. In addition, we evaluated the effects of asparagine on ODC activity following seizure activity elicited by systemic administration of kainic acid (KA) in both adult and neonatal rats. Asparagine produced an increase in ODC gene expression and activity in both adult and neonatal hippocampal slices. The increase in ODC activity elicited by asparagine in hippocampal slices was the same in control animals as in animals sacrificed 16 h after KA-induced seizure activity. The asparagine-elicited increase in ODC activity in neonatal and adult hippocampal slices was blocked by the RNA synthesis inhibitor, actinomycin D. Finally, polyamines produced an inhibition of ODC activity in neonatal hippocampal slices. The results indicate that the regulation of the expression and activity of ODC is similar in neonatal and adult hippocampus.

Systemic injection of kainic acid (KA) does not cause neuronal pathology in limbic structures in rat brain prior to postnatal day (PND) 21. The present study tested if the development of the pathogenic response is associated with the maturation of a link between seizure activity and polyamine metabolism. Pathology was assessed with histological techniques and with the binding of [3H]Ro5-4864, a ligand for the peripheral type benzodiazepine binding sites (PTBBS), a marker of glial cell proliferation. In agreement with previous results, peripherally administered kainate at doses sufficient to induce intense behavioral seizures produced a loss of Nissl staining in hippocampus after PND 21 but not at earlier ages. The pattern of neuronal damage observed after PND 21 resembled that found in adult animals: extensive losses of Nissl staining in area CA3 of hippocampus and in piriform cortex, more modest effects in CA1 and sparing of the granule cells of the dentate gyrus. Similarly, no increase in [3H]Ro5-4864 binding as a result of KA administration was observed in hippocampus and piriform cortex until PND 21. Ornithine decarboxylase (ODC) activity and putrescine levels were high in the neonatal brain and decreased to reach adult values by PND 21. KA-induced seizure activity did not significantly alter both variables until PND 21. After PND 21, ODC activity and putrescine levels markedly increased 16 h after KA-induced seizure activity in hippocampus and piriform cortex. The magnitude of the effects increased between PND 21 and PND 30, at which point the changes in both parameters were comparable to those found in adults. Polyamines stimulate the activity of the calcium-dependent proteases calpain in brain fractions and may increase calpain-mediated proteolysis in situ. In accord with this, kainate-induced breakdown of spectrin, a preferred substrate of calpain, measured 16 h after KA injection followed a developmental curve parallel to that for kainate-induced increases in putrescine levels. These results indicate that the onset of vulnerability to seizure activity triggered by kainic acid is correlated with the development of an ODC/polyamine response to the seizures and further support a critical role for the ODC/polyamine pathway in neuronal pathology following a variety of insults.

Brain-derived neurotrophic factor (BDNF) mRNA expression was studied in the hippocampus at various developmental stages in normal rats and following kainic acid (KA)-induced seizure activity. Systemic administration of KA strongly elevated BDNF mRNA levels in all hippocampal subregions after postnatal day 21. In contrast, even though KA induced intense behavioral seizure activity at postnatal day 8, the seizures were not associated with elevations of BDNF mRNA levels, indicating a clear dissociation between behavioral seizures and increases in BDNF mRNA levels and contradicting the view that BDNF mRNA expression is principally regulated by neuronal activity. In the dentate gyrus at postnatal day 13, intense BDNF mRNA expression was limited to a defined area at the border between granule cell and molecular layers, suggesting the possibility that segregation of BDNF mRNA into defined subcellular compartments may play a role in establishing the well-delineated patterns of innervation in the hippocampus.

The induction of ornithine decarboxylase (ODC) in adult CNS and the resulting changes in polyamine levels are often observed under conditions associated with activation of NMDA receptors, calpain stimulation and spectrin degradation. The present study was directed at evaluating the links between these two sets of events. We measured the effects of an acute treatment of adult rats with difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, on biochemical alterations following kainate-induced seizure activity. Beside ODC activity and polyamine levels, we assayed the in situ spectrin degradation and the in vitro binding of 3H-Ro5-4864, a ligand for the peripheral benzodiazepine binding sites which is a good marker of glial proliferation, at various time intervals following systemic kainic acid (KA) injection. Kainate-induced seizure activity was followed by a transient increase in ODC activity, a long-lasting increase in putrescine levels and spectrin degradation, and a delayed increase in 3H-Ro5-4864 binding, mainly in hippocampus and piriform cortex. Treatment of the animals with DFMO markedly reduced the increase in putrescine levels up to 7 days after KA injection. It also reduced the increase in spectrin breakdown observed at 16 h but not at 4 and 7 days after KA injection. Finally, it did not modify the increase in 3H-Ro5-4864 binding measured 4 and 7 days after KA injection. The levels of putrescine were positively correlated with the extent of spectrin proteolysis in KA-treated animals whether or not they were treated with DFMO, at 16 h but not at 7 days after KA injection. The results indicate that the extent of spectrin breakdown observed shortly after KA-induced seizure activity is causally related to the changes in ODC activity and putrescine levels. Although the data are consistent with the idea that putrescine could be a marker for acute pathology, they do not support a role for polyamines in delayed neurotoxicity.

Induction of the proto-oncogene c-fos is often considered to be a marker of increased neuronal activity. We have used in situ hybridization to study the pattern of c-fos expression in limbic structures following kainic acid-induced seizures during the postnatal period in the rat. Prior to postnatal day 13 (P13), seizure activity did not result in c-fos induction in any limbic structure. Between P13 and P25, a gradual increase in c-fos expression was observed in hippocampus and cortical structures. These results were corroborated by nuclear run-off transcription assay. Thus, alterations in c-fos transcription that may facilitate stimulus-transcription coupling occur during postnatal development. The possible relationship between the postnatal maturation of c-fos expression and the increase in susceptibility of specific neuronal populations to seizure-induced cell damage is discussed.

In situ hybridization techniques were used to analyse the spatiotemporal pattern of brain-derived neurotrophic factor messenger RNA elevation associated with kainic acid-induced seizure activity in the rat. Pronounced increases in hippocampal brain-derived neurotrophic factor messenger RNA levels were observed as early as 30 min following the onset of behavioral seizures. The greatest increase (10-fold) occurred in the dentate granule cell layer, while pyramidal layers CA1, CA3, and CA4 exhibited increases of two- to six-fold. Peak elevation of brain-derived neurotrophic factor messenger RNA in CA1 hippocampal region was evident at 4 h in CA3, and in the dentate granule layer at 30 min postseizure. Elevations persisted in the dentate and hilar regions to four days, while the increases in CA1 and CA3 returned to control levels by 16 h following seizure. Significant increases in brain-derived neurotrophic factor messenger RNA were also observed in the superficial layers of cortex (II and III) and in the piriform cortex which reached peak elevations by 8 h. No detectable changes were observed in the dorsomedial thalamus. Although histologically defined pyramidal and granule cell layers displayed relatively uniform increases in brain-derived neurotrophic factor messenger RNA in response to kainate, a closer examination of the labeling patterns using emulsion autoradiography revealed discrete areas of high grain densities overlapping uniform, moderate hybridization densities in the dentate granule cell layer and CA3, suggesting that the capacity to upregulate brain-derived neurotrophic factor messenger RNA in these regions may differ among individual neurons. In conclusion, our studies revealed that brain-derived neurotrophic factor messenger RNA induction in response to systemic kainate administration differs in hippocampal and cortical areas, in magnitude, time of onset and duration. The observed temperospatial pattern does not correspond in a simple way to increases in metabolic or electrical activity associated with seizures or neuronal vulnerability coincident with the seizures.

Impairment of polyamine synthesis by treatment with difluoromehtylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been shown to alter normal brain development. In the present study we determined the effect of DFMO treatment during a discrete developmental period on polyamine levels and on the in situ activity of calpain, as reflected by the level of degradation of spectrin, in various brain regions of rat pups. DFMO treatment from postnatal days 5 to 10 produced a marked decrease in putrescine levels in every brain structure and a significant decrease in spectrin breakdown levels in hippocampus and cortex but not in cerebellum. The results indicate that the ODC/polyamine pathway partly regulates the in situ activity of calpain and that polyamines may play a role in both growth and degeneration phenomena.

Polyamine synthesis is induced by various extracellular signals, and it is widely held that this biochemical response participates in cell growth and differentiation. Certain of the triggers for synthesis in brain tissues also increase the breakdown of high-molecular-weight structural proteins, apparently by activating calcium-dependent proteases (calpains). The present experiments tested the possibility that calpain activity is modulated by polyamines. Spermine, spermidine, and putrescine all increased calcium-dependent proteolysis of [14C]casein by soluble fractions of rat brain. The order of potency was spermine greater than spermidine greater than putrescine, with apparent affinities of 30, 300, and 6,000 microM, respectively. Each of the three polyamines at physiological concentrations also potentiated the calcium-dependent breakdown of two endogenous high-molecular-weight structural proteins known to be substrates of calpain, in both supernatant and membrane fractions. The thiol protease inhibitor leupeptin, a known calpain inhibitor, also inhibited calcium-dependent proteolysis in the presence and absence of polyamines. The polyamines did not increase the activity of purified calpain I or calpain II determined with either [14C]casein or purified spectrin as the substrate, nor did they interfere with the inhibitory effects of calpastatin, an endogenous inhibitor of calpain. However, polyamines potentiated the stimulation of endogenous but not purified calpain activity produced by an endogenous calpain activator. These results suggest a role for polyamines in protein degradation as well as protein synthesis.

The effects of six phenolic compounds (o-, m-, and p-cresol and 2-, 3-, and 4-ethylphenol) on the anaerobic biodegradation of phenol was examined in batch methanogenic cultures. Results showed that ethylphenols were more inhibitory of phenol degradation than were cresols. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.