Excitability changes and glucose metabolism in experimentally ...

Excitability Changes and Glucose Metabolism in Experimentally Induced Focal Cortical Dysplasias

Malformations of cortical development are increasingly recognized in association with severe epileptic syndromes, neuropsychological disorders and mental retardation. Several clinical and experimental studies suggest that functional consequences of cortical dysplasias are not restricted to the area of the dysplastic lesion but also involve remote brain regions. In the present study cortical malformations were induced in newborn rats at day of birth by intracerebral injection of the glutamatergic agonist ibotenate. The resulting cytoarchitectonic lesion associates neuronal depopulation of deep cortical layers, ectopic neurons in superficial layers and sulcus formation, mimicking human polymicrogyria and migration disorders. Electrophysiological recordings of evoked field potentials in slice preparations of adult animals reveal hyperexcitability in widespread cortical regions surrounding the dysplasia. Low-intensity stimulation induced epileptiform activity consisting of long-lasting, multiphasic and N-methyl-D-aspartate-dependent field responses. They appeared with high variability as all-or-none events. These widespread changes in excitability were not observed in sham-operated animals with small superficial ectopias but intact deep cortical layers, indicating that focal loss of these layers induces extended alterations in cortical connectivity and imbalance of excitation and inhibition. Restricted zones of increased excitability were also found in the forelimb and hindlimb representation cortex in sham-operated and control animals, demonstrating that this activity has to be considered as an intrinsic property of specific cortical areas. Deoxyglucose autoradiography showed that the widespread hyperexcitability in ibotenate-injected animals was not accompanied by alterations in glucose metabolism, although in the area of structural abnormality a typical metabolic pattern was found, revealing an increased glucose uptake in layer I. Hypometabolism as described for many types of human dysplastic lesions was not observed. This difference between the experimental and clinical data may be due to the absence of behavioral seizures in this model. However, it can be hypothesized that in patients with developmental malformations, additional pathogenic factors contribute to the manifestation of seizure disorders. Introductions Malformations of cortical development are a heterogeneous group of structural brain anomalies frequently causing severe epileptic syndromes, neuropsychological disorders and developmental delay in children and adults (Kuzniecky et al., 1989; Palmini et al., 1991a; Barkovich et al., 1992; Rosen et al., 1995). Cortical malformations, such as ectopia, heterotopia and polymicrogyria, are supposed to result from disturbances of neuronal migration and from hypoxic–ischemic insults during cortical development (Richman et al., 1974; Ferrer, 1984; Evrard et al., 1989; Rorke, 1994). For many years, most malformations of the cerebral cortex were recognized only neuropathologically during autopsy in brains from patients with intractable partial epilepsy or in surgically removed brain tissue (Taylor et al., 1971), leading to the assumption that these lesions were relatively rare disorders. With recent improvements in modern

Christoph Redecker, Michael Lutzenburg, Pierre Gressens1, Philippe Evrard1, Otto W. Witte and Georg Hagemann Neurologische Klinik, Heinrich-Heine-Universität, Moorenstrasse 5, D-40225 Düsseldorf, Germany and 1Service de Neurologie Pédiatrique and INSERM CRI 97-01, Hôpital Robert-Debré and Faculté X. Bichat-Paris 7, F-75019 Paris, France

neuroimaging techniques, and particularly the advent of highresolution magnetic resonance imaging (MRI), developmental malformations are increasingly recognized during life, indicating that these disorders are more common than previously supposed (Barkovich and Kjos, 1992; Raymond et al., 1995; Barkovich et al., 1995). Clinical and neuroimaging studies demonstrated that >50% of children referred to epilepsy centers for intractable seizures display some type of developmental abnormality, with focal cortical dysplasia diagnosed in ∼25% of those patients with partial-onset seizures (Kuzniecky et al., 1993). In adult patients with intractable focal epilepsy these lesions were found in ∼15% (Dobyns and Kuzniecky, 1996). Neuropsychological testing of patients with epilepsy and developmental cortical lesions frequently reveals cognitive impairment (Schachter et al., 1993; Calabrese et al., 1994; Guerrini et al., 1997), but cortical malformations also have been described in non-epileptic individuals who show only focal neuropsychological deficits, such as developmental dyslexia (Galaburda and Kemper, 1979; Humphreys et al., 1990; Galaburda et al., 1994). The surgical approach to intractable partial epilepsies due to focal cortical dysplasias is often difficult, since presurgical evaluation of these patients frequently demonstrated discordance between the extension of the dysplastic lesion and the epileptogenic zone (Palmini et al., 1994). Electroencephalographic activity often extends to widespread brain regions beyond the visible lesion boundaries (Palmini et al., 1995). These discrepancies warrant extensive presurgical evaluation using invasive electroencephalographic recordings, detailed neuroimaging and metabolic studies with positron emission tomography (PET). Metabolic PET studies in patients with focal dysplastic lesions have demonstrated hypometabolism in the structurally abnormal area: in some patients this extends beyond the electrophysiological abnormalities, whereas in others it is smaller (Chugani et al., 1990; Olson et al., 1990; Lee et al., 1994). In addition Palmini et al. (1991a) reported no correlation between postsurgical outcome and completeness of excision as defined by electrophysiological studies. These discrepancies indicate the distinct electrophysiological and metabolic characteristics of cortical dysplasias that need better understanding to refine surgical strategy in these patients. In order to analyze the relationship between morphological abnormalities, electrophysiological alterations and glucose metabolism, focal cortical dysplasias were induced by intracerebral injection of the glutamatergic agonist ibotenate in developing rat brain. Ibotenate, stimulating the ionotropic N-methyl-D-aspartate (NMDA) and metabotropic glutamate receptors, has previously been used to produce focal cortical malformations in different species during brain development (Innocenti and Berbel, 1991; Marret et al., 1995b, 1996; Gressens et al., 1997; Redecker et al., 1997b). In these studies, the cytoarchitectonic pattern of the cortical malformation was Cerebral Cortex Oct/Nov 1998;8:623–634; 1047–3211/98/$4.00

dependent on the stage of brain maturation at time of injection, probably due to distinct stages of NMDA receptor maturation and subunit formation. When injected during migration of supragranular layers in newborn hamsters, ibotenate produced neuronal heterotopias and molecular ectopias (Marret et al., 1996). When administered just after completion of neuronal migration in mice at day of birth, ibotenate induced neuronal loss in deep cortical layers accompanied by an overlying microsulcus, mimicking human polymicrogyria (Marret et al., 1995b; Thompson et al., 1997; Gressens et al., 1997). In newborn rats, where migration of supragranular neurons is not yet fully completed, intracerebral injection of ibotenate resulted in typical cortical dysplasias consisting of depopulation of deep cortical layers, formation of a microsulcus and ectopias in superficial layers (Redecker et al., 1997b). This dysgenetic cortical pattern mimicks polymicrogyria and neuronal ectopias, two cortical malformations frequently associated in humans with intractable epilepsy. The ibotenate-induced cortical lesions are already present 5 days after injection and persist until adulthood, providing a suitable model to study functional consequences of focal cortical dysplasias. The present study addressed the question whether ibotenateinduced cortical dysplasias display similar epileptiform activity as previously described for the freeze-lesion model of cortical malformations (Jacobs et al., 1996; Luhmann and Raabe, 1996). To this purpose, the electrophysiological excitability of ibotenate-injected animals was examined using cortical slice preparations. Furthermore, the spatial extension of these changes was analyzed and compared with alterations in glucose metabolism. To determine whether this model might help to better understand equivocal clinical data we put special emphasis on the spatial relationship of structural and functional alterations in focal cortical malformations.

Materials and Methods Ibotenate Injections Several litters of newborn Wistar rats were used for the experiments. On the day of birth (P0), ibotenate — a glutamatergic agonist that activates both ionotropic NMDA and metabotropic receptors — was injected in the neopallium of the rat pups. Ibotenate (Sigma, St Louis, MO) was diluted in 0.1 M phosphate-buffered saline (PBS, pH 7.4). The injections were performed under diethylether inhalation as previously described (Marret et al., 1995a,b, 1996; Gressens et al., 1997; Redecker et al., 1997b). A 27-gauge needle was placed on the skull between the coronal and lambdoidal sutures, ∼2 mm from the midline, and inserted 3 mm beneath the skin surface (Fig. 1). A dose of 10 µg ibotenate per rat pup was applied by successive injections of two 1 µl boluses at an interval of 45 s. The needle was then left in place for an additional 45 s. Sham-injected animals received 2 µl PBS. After the injection procedure the pups were allowed to recover from anesthesia and returned to their dam. During caring and handling of ibotenate-injected and sham-operated animals behavioral seizures have not been observed. Histology and Immunohistochemistry The rats were killed under ether anesthesia at adult age (P50–P70), and their brains fixed with 4% paraformaldehyde. Whole brain serial sections were cut coronally and every fourth section was processed for cresyl violet staining. Unstained sections between two cresyl violet-stained sections containing neocortical lesions were used for immunohistochemistry. On coronal sections, the hemisphere opposite to the site of injection was used as control for each animal. After deparafinization, these sections reacted with polyclonal rabbit antiserum to mouse monoclonal antibodies directed against microtubule-associated protein type 2 (MAP-2) (Sigma), diluted 1/500 in 1 × PBS containing 1% normal horse serum and 0.4% Triton X-100. Sections were incubated overnight. Avidin–biotin horseradish peroxidase kits (Vector, Burlingame, CA) to

624 Excitability Changes and Metabolism in Cortical Dysplasias • Redecker et al.

detect mouse antibodies were used as directed. For histological studies, to avoid regional and experimental variations in the intensity of labeling, several sections of each animal were immunoreacted in successive experiments. Anatomical regions of the injected hemisphere were compared with similar areas of the contralateral hemisphere present on the same coronal sections. Electrophysiology Electrophysiological recordings were performed in brain slices from ibotenate- and sham-injected animals as well as age-matched controls at adult age (>P60). The rats were deeply anaesthetized with diethylether, decapitated and the brain was rapidly removed. Coronal neocortical slices (400 µm) were obtained with a Series 1000 Vibratome at the site of the cortical dysplasia and at corresponding levels in control animals. The slices were maintained in an interface recording chamber at 33°C and superfused with artificial cerebrospinal f luid (ACSF; composition in mM: NaCl 124, NaHCO3 26, KCl 5, CaCl2 2, MgSO4 2, NaHPO4 1.25 and glucose 10, equilibrated with carbogen to pH 7.4). Slices were left undisturbed for at least 1 h before starting any recording or stimulation. Extracellular recordings of field potentials were performed with glass micropipettes filled with ACSF. Electrical stimulation was applied with bipolar electrodes placed in deep neocortical layers. The field potentials were recorded in superficial layers, with the two electrodes situated along a line normal to the pial surface (on-column, Fig. 3A). At each recording site a series of increasingly intense stimuli was applied to evoke responses from threshold to maximal in amplitude. Pulses lasting 50 µs were delivered at a rate of 0.05 Hz. Data were digitized, stored to disk and analyzed off-line using Signal Averager (Science Products, Frankfurt). In order to relate the electrophysiological results to the underlying histology, the slices were fixed in 4% paraformaldehyde diluted in 0.1 M phosphate buffer (pH 7.4) at the end of the experiment. Slices were then cr yoprotected by immersion in 30% sucrose in PBS (7–14 days), sectioned on a cryotome (50 µm), and processed for standard cresyl violet staining. Deoxyglucose Autoradiography The regional cerebral metabolic rate of glucose (rCMRGlc) was determined according to the [14C]deoxyglucose method (Sokoloff et al., 1977). Under inhalation anesthesia with isof lurane and N2O an arterial and venous catheter was inserted in the left femoral artery and vein. One hour after this procedure a bolus of [14C]deoxyglucose (25 µCi) dissolved in 0.4 ml isotonic NaCl solution was injected. The injections were performed within a time range of 1–2 s. Thereafter blood samples were collected for the following 45 min to determine plasma glucose concentration and plasma radioactivity. Immediately after taking the last blood sample the rat was decapitated, and the brain rapidly removed and frozen in methylene butane at –40°C, and then stored at –70°C for further processing. Coronal sections of the brains were performed on a cryotome at 20 µm. Consecutive slices were processed for standard cresyl violet staining or exposed to high-resolution autoradiography films (Amersham, hyperfilm-max, RPN9), together with an isotope standard for periods of 9–14 days. The optical densities of the films were recorded with a charge-coupled device (CCD) camera, which was connected to an online videometry processor for background subtraction and offset correction. This signal was digitized and analyzed by an imaging program. For each animals the rCMRGlc was evaluated in five coronal sections through the cortical dysplasia (distance between the sections 100 µm) and mean values were computed for the different regions of interest. The resulting values were averaged for the different experimental groups and are given as mean ± SD. Statistical differences between the different regions of interest were tested using ANOVA with Bonferroni post-test (P < 0.05).

Results Histology and Immunohistochemistry A ll adult ibotenate-injected animals (n = 37) displayed typical focal cortical dysplasias at the site of injection, similar to that previously reported (Redecker et al., 1997b). These lesions were associated with three characteristic elements: neuronal

Figure 1. Cortical lesions induced by intracerebral injection of ibotenate at day of birth (P0). (A) Schematic illustration of needle placement on head surface of newborn rat pups for ibotenate injection. (B)–(D) Nissl-stained coronal sections through a typical dysplastic lesion 15 days after injection, displaying depopulation of deep cortical layers and collections of ectopic neurons and an overlying microsulcus (arrowheads). (C) Higher magnification of the cortical lesion shown in (B). (D) Adjacent section showing the bridge of neural tissue connecting the ectopia to the remaining cortex (asterisk). Scale bars represent 1000 µm in (B) and 300 µm in (C,D). Figure 2. Dendritic organization in non-treated (A,C) and ibotenate-injected (B,D) neocortex. MAP-2 immunoreactivity shows regular organization of the dendrites in radial bundles intermixed with unlabeled nuclei in non-treated cortex (A,C) and a slight deviation of the dendrites around the cortical ectopia in ibotenate-injected cortex (B,D). Scale bars represent 200 µm (A,B) and 40 µm (C,D).

depopulation of deep cortical layers, ectopic neurons in superficial layers and sulcus formation (Fig. 1). The neuronal loss predominated in layers V and VIa accompanied by a thickening of layers II and III. The ectopic neurons were localized in the overlying molecular layer and connected to the surrounding cortical layers by a bridge of neural tissue. This morphological pattern resulted in a mushroom-like appearance of the ectopias (Fig. 1D). Close to the ectopic neurons the cortex was infolded, creating a small sulcus that was surrounded by a microgyrus. In two animals a dilation of the ventricles was found on the ipsilateral hemisphere. In ibotenate-injected animals used for immunohistochemistry (n = 14) or autoradiography (n = 8) the maximal sagittal fronto-occipital diameter of the cortical dysplasias was evaluated by counting the sections that showed the lesion. In these animals the mean diameter of the cortical dysplasia was 920 ± 270 µm. In animals used for electrophysiological recordings (n = 15) an exact evaluation of the lesion size was not possible due to the thickness of the slices (400 µm). But also in these animals the lesion was found in two or three consecutive slices. There was some variability in shape and depth of the cortical dysplasias between different litters of injected rat pups as well as in animals from the same litter. For instance, occasionally an asymmetry in the cytoarchitecture of the dysplasia was observed. In most common lesions the microgyrus extended to the depth of layer IV of the adjacent cortex, but in more severe cases extended to layer VI, with only a thin layer remaining between the microsulcus and the white matter. In the contralateral hemisphere cresyl violet-stained sections showed

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Figure 3. Characteristics of multiphasic activity. (A) Schematic illustration of the position of the stimulating and recording electrode on a coronal cortical slice that was stained with cresyl violet after electrophysiological recording. Note the cortical dysplasia. (B) Typical examples of field potentials (FP) recorded at the same position in slices of control (upper trace) or ibotenate-injected animals (lower trace). In slices of control animals a normal field response was obtained, whereas field responses recorded from an ibotenate-injected animal consisted of a normal early component (asterisks) followed by a long-lasting, multiphasic component. (C) Field responses recorded with increasing stimulation intensity from an ibotenate-injected animal. Note the occurrence of multiphasic responses at threshold stimulus intensity, the decrease in latency between 3 and 5 V, and the normal triphasic field potential with higher stimulation strength.

no alterations in cortical structure. In order to detect alterations in dendritic organization in the dysplastic lesion MAP-2 staining was performed in 14 adult ibotenate-injected animals. In the non-treated neocortex, M A P-2 labeling was regular and organized in radial bundles intermixed with unlabeled nuclei (Fig. 2). MAP-2 immunostaining was similarly distributed in the injected hemisphere, showing a normal organization of the dendrites with the exception of a slight deviation around the ectopia (Fig. 2). In accordance with previous investigations (Redecker et al., 1997b), sham-injected animals (n = 8) displayed small superficial ectopias in 50% of cases. These lesions consisted of small collections of ectopic neurons in the molecular layer without any loss of deep cortical layers or sulcus formation (see Figs 4B and 6E). The mean sagittal diameter of these lesions was 470 ± 130 µm. In one animal, only a shallow invagination of layers I and II was visible without ectopic neurons and in three animals no structural lesions could be detected in cresyl violet-stained sections.

Electrophysiology To study the electrophysiological consequences of cortical dysplasias, evoked field potentials were analyzed in slice preparations of 12 ibotenate-injected, four sham-operated and four control animals. In ibotenate-injected animals all slices containing the cortical dysplasia as well as adjacent slices were used for the experiments (n = 28). Slices from sham-operated


animals (n = 12) or controls (n = 11) came from corresponding brain regions. In all ibotenate-injected animals electrical stimulation evoked a variable, long-lasting, multiphasic field potential in widespread cortical areas (Fig. 3). This field response consisted of an early stereotyped monophasic component followed by a late variable and multiphasic activity. The early event had a latency of 3–6 ms, a duration of 10–15 ms and an amplitude that increased with stimulus intensity. The late multiphasic activity was evoked as an all-or-none response at threshold stimulus intensity with a variable latency and duration from one to the next, even for the same stimulus intensity and frequency. The early field potential most often immediately followed the multiphasic activity but latencies of 50–150 ms were also observed. The duration also varied between 45 and 570 ms (mean 148 ms). Increases in stimulus intensity frequently altered the shape of the multiphasic activity and reduced the latency as well as the duration of this field response. At high stimulus intensities the late component was completely absent and the early event became triphasic (Fig. 3). The occurrence of the late multiphasic activity was also dependent on the stimulation frequency. In the following experiments a stimulus repetition rate of 0.05 Hz was used. Higher frequencies reduced the occurrence of the multiphasic activity, whereas the early component remained unimpaired. The characteristics of this late multiphasic activity were similar to the hyperexcitability found in freeze-lesioned rats (Jacobs et al., 1996) or epileptiform events described in several chronic models of epilepsy (Prince and Tseng, 1993; Hoffman et al., 1994; Brener et al., 1997). To evaluate where this multiphasic activity can be elicited, all slices were stimulated at several positions at a distance of 2–10 mm from midline. In 86% of slices from ibotenate-injected animals this activity was found at at least two sites and in 50% of these slices at more than five sites between 2 and 10 mm from midline (Fig. 4). The distribution of sites with late multiphasic activity did not depend on the location of the dysplasia. Only in one slice could the multiphasic response not be evoked at any site, although a cortical dysplasia was present. For comparison of these electrophysiological findings with the underlying morphology, all slices were processed histologically and the cortical dysplasias were analyzed. Using cresyl violet-stained sections a strict spatial relationship between the location of the dysplasia and the occurrence of the late multiphasic activity was not obser ved. In some cases hyperexcitability was only found in the surround of the cortical dysplasia, whereas in other cases the late multiphasic activity was also evoked within the ectopia. Investigation of sham-operated and control animals revealed that this late multiphasic activity was not exclusively inducible in ibotenate-injected animals but also found as an intrinsic property of specific cortical areas (Fig. 4). Regarding the configuration of field potentials there was no difference between these groups. In 75% of slices from sham-operated and in 81% of slices from control animals a similar multiphasic activity was evoked at one position located at 3 mm from midline. In one slice from a sham-operated and two slices from two control animals this activity was additionally evoked at 5–6 mm from midline. The late multiphasic activity found in sham-operated and control animals had the same characteristics as described above, but duration and latency were shorter in both groups. In shaminjected animals the early response was immediately followed by the multiphasic component lasting 67–151 ms (mean 98 ms). In

Figure 4. Occurrence of multiphasic responses in ibotenate-injected (A), sham-operated (B) and control animals (C). Cresyl violet-stained sections of cortical slices previously used for the electrophysiological recordings are shown on the left side. These sections display the differential cortical patterns observed in the different experimental groups: a cortical dysplasia in an ibotenate-injected (A, arrow), a small superficial ectopia in a sham-operated (B, arrow) and no lesion in a control animal (C). Positions of stimulating and recording electrode are marked with dots and numbers. Corresponding field potentials were shown on the right side with three consecutive traces for each position. Note the widespread occurrence of multiphasic activity induced by the cortical dysplasia (A), whereas in sham-operated (B) and control animals (C) multiphasic activity is only present at position 2, which is histologically equivalent to the hindlimb and forelimb representation area HL/FL.

control animals the duration of the multiphasic response was 57–160 ms (mean 90 ms, Fig. 4). Histological evaluation of sham-operated and control animals showed that the occurrence of multiphasic activity did not depend on the presence of cortical lesions in these groups. Sham-operated animals showed a small superficial ectopia in two cases, whereas in two further sham-operated and four

control animals no structural lesions were detected with cresyl violet-stained sections. In both groups late multiphasic activity was elicited in a specific cortical area located at 3 mm from midline (Fig. 4). Cresyl violet-stained sections allowed this cortical area to be identified as hindlimb (respectively forelimb) representation cortex HL/FL using the nomenclature proposed by Zilles ( 1992).


Figure 5. Effect of the NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV, 20 µM) on field responses recorded in an ibotenate-injected animal. D-APV completely abolished the late, multiphasic component, while the early event remained unimpaired. Traces shown for each condition represent three consecutive single sweeps.

Several previous studies using other models of epileptogenesis or normal immature cortex but also specific cortical areas showed that late multiphasic activity is dependent on activation of NDM A receptors (Luhmann and Prince, 1990; NeumannHaefelin et al., 1996; Brener et al., 1997). In accordance with these studies, the effects of the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV) on late multiphasic activity was tested in six slices from different ibotenate-injected, sham-operated and control animals. In all cases, the late multiphasic activity was reversibly blocked by a concentration of 20 µM which did not affect the early field response (Fig. 5).

Deoxyglucose Autoradiography Deoxyglucose autoradiography was performed in eight ibotenate-injected, four sham-operated and four control animals at adult age. Quantitative evaluation of the mean cortical rCMRGlc did not reveal any significant differences between ibotenate-injected (0.62 ± 0.19 µmol/g/min), sham-operated (0.65 ± 0.12 µmol/g/min) and control animals (0.58 ± 0.02 µmol/ g/min). Focal changes in glucose metabolism in the area of the dysplasia were evaluated by comparing the autoradiograms with adjacent cresyl violet-stained sections (Fig. 6). Deoxyglucose autoradiograms of ibotenate-injected animals showed a typical layer-specific pattern of metabolism in the dysplastic cortex (Fig. 6). In the area of the ectopic neurons the glucose uptake (0.69 ± 0.12 µmol/g/min) was not significantly different from layers II and III of the contralateral homotopic cortex (0.69 ± 0.06 µmol/g/min). The ectopias were surrounded by a zone of higher glucose metabolism corresponding to layer I within the dysplasia (0.84 ± 0.09 µmol/g/min, Figs 6 and 7). The metabolism in this layer was significantly different from layer I in contralateral homotopic cortex (0.71 ± 0.06 µmol/g/min). In the underlying cortical layers II and III the rate of glucose metabolism (0.70 ± 0.10 µmol/g/min) was similar to layers II and III of the contralateral homotopic cortex (0.69 ± 0.06 µmol/g/ min). This pattern was observed in all ibotenate-injected animals (Fig. 6). Metabolic alterations in remote or surrounding brain

regions corresponding to the widespread hyperexcitability were not observed (Fig. 6), except for one animal (1/8) that showed an increased rCMRGlc in the adjacent lateral neo- and archicortex. In sham-injected animals a similar layer-specific pattern of glucose metabolism was observed (Fig. 6). In animals showing small superficial ectopias (3/4) these ectopic neurons displayed a rate of glucose metabolism (0.66 ± 0.10 µmol/g/min), that was not significantly different from underlying layers II and III (0.60 ± 0.14 µmol/g/min) or homotopic contralateral layers II and III (0.58 ± 0.14 µmol/g/min, Fig. 7). As described for ibotenateinjected animals, the superficial ectopias were surrounded by a zone of increased glucose consumption corresponding to layer I (0.73 ± 0.20 µmol/g/min) and showed a higher metabolism than in contralateral homotopic layer I (0.60 ± 0.16 µmol/g/min, not significant, Fig. 7). In one animal only a shallow invagination of the cortex was visible at the injection site. This animal showed a similar pattern of glucose metabolism, including activation of the molecular layer (Fig. 6). In control animals no differences in glucose uptake between ipsilateral and contralateral layer I were observed (Fig. 7).

Discussion Excitability Changes The present study clearly demonstrates that ibotenate-induced cortical dysplasias cause widespread cortical hyperexcitability in adult rats. This hyperexcitability is ref lected by long-lasting, multiphasic responses that appear with high variability as all-or-none events elicited with low-intensity stimulation applied to deep cortical layers. These multiphasic field potentials display very similar characteristics to those described in the surround of cortical freeze-lesions induced in newborn rat pups that result in cortical malformations showing a comparable cytoarchitectonic pattern, e.g. a four-layered cortex, ectopic neurons and microsulcus formation (Jacobs et al., 1996; Luhmann and Raabe, 1996). However, hyperexcitability does not only occur in the context of cortical microgyria. Several electrophysiological studies provide strong evidence that these excitability changes correspond to epileptiform activity commonly seen in chronic models of focal epilepsy (Prince and Tseng, 1993; Hoffman et al., 1994; Brener et al., 1997). Furthermore, long-lasting multiphasic activity was also obser ved when low concentrations of the GABAA receptor antagonist bicuculline or other convulsants were applied to the bathing medium (Gutnick et al., 1982; Chagnac Amitai and Connors, 1989a). A lthough there are several experimental models that mimic different aspects of human cortical malformations, electrophysiological studies focusing on the mechanisms of epileptogenesis in these disorders are relatively rare. Epileptiform activity has only been reported for the freeze-lesion model (Jacobs et al., 1996; Luhmann and Raabe, 1996) and in the present study for ibotenate-induced cortical dysplasias. Prenatal exposure of rat fetuses to irradiation or the antimitotic agent methylazoxymethanol (M A M) produces ectopias and gross

Figure 6. Pattern of glucose metabolism in ibotenate-injected and sham-operated animals at adult age. (A) Coronal [14C]deoxyglucose autoradiographic section through an ibotenate-injected brain showing a typical cortical dysplasia (frame) and in this case an ipsilateral dilatation of the ventricle (asterisk). (B) Corresponding autoradiogram of a non-injected control animal. Comparison of both autoradiograms (A,B) does not reveal any remote alterations in rCMRGlc beyond the cortical lesion. (C,D) Serial autoradiographic (C) and adjacent cresyl violet-stained sections (D) through the cortical dysplasia shown in (A) (rostro-caudal distance between the sections: 100 µm). Note the increase in rCMRGlc in layer I of the microgyrus, while glucose metabolism in the ectopia does not differ from the surrounding cortex. (E,F) Three autoradiograms and adjacent cresyl violet-stained sections through different ibotenate-induced dysplasias (E) and lesions produced by sham-injection of phosphate buffer (F). Note the similarities in the pattern of glucose metabolism in both groups. Scale bar represents 1 mm.



Figure 7. Quantitative evaluation of regional cortical glucose metabolism (rCMRGlc) in slices of ibotenate-injected (B), sham-operated (C) and control animals (D). (A) Schematic drawing of region of interest definition used for quantification. Note that the increase in rCMRGlc in layer I surrounding the ectopia is only significant in ibotenate-induced dysplasias (asterisk, ANOVA with Bonferroni post-test, P < 0.05). Data are given as mean ± SEM.

disturbances in cortical and hippocampal lamination in the offspring (Jones and Gardner, 1976; Tamaru et al., 1988). In this model an increased seizure susceptibility was reported after pharmacological reduction of seizure threshold (Roper et al., 1995, 1997) and in the MAM model a decreased threshold for hyperthermia-induced seizures was found (Germano et al., 1996). Other models, in which cortical dysgenesis is produced by injuring the neonatal rat brain (Rosen et al., 1992; Ferrer et al., 1993), have not been investigated electrophysiologically up to now. Although all these models closely display histological similarities with clinical pathologies of patients with epilepsy, behavioral seizures have not been reported. A possible explanation might be the lack of systematic observations on the animals used in these experimental studies, but it has to be considered that the association of developmental malformations and clinical epilepsy is probably rarer than suggested in clinical literature, because patients admitted for MRI are highly selected, suffering more frequently from epilepsy than the normal


population. Furthermore, several clinical studies have reported focal minor malformations of the cortex in patients with neuropsychological deficits such as developmental dyslexia and absence of seizure disorders (Galaburda and Kemper, 1979; Kemper, 1985; Humphreys et al., 1990; Galaburda, 1993). Thus, the size of experimentally induced dysplasias could be too small for the generation of behavioral seizures, but these findings might also indicate the presence of additional factors for the manifestation of recurrent seizures in patients with clinical epilepsy. One possible additional factor might be the necessity of genetic predisposition. In this context, Lee et al. (1997) described a mutant rat that showed large subcortical heterotopias associated with spontaneous behavioral seizures. This neurological mutant rat might probably complement the models mentioned above and help to analyze the mechanisms that cause behavioral seizures. It is interesting to note that in individuals with developmental dyslexia, functional deficits in rapid processing have been described (Tallal and Piercy, 1973; Merzenich et al., 1996; Slaghuis et al., 1996), which have been assumed to be related to variable minor cortical or subcortical focal malformations (Galaburda, 1993; Galaburda et al., 1994). Similar impairment of fast auditory processing was observed in the freeze-lesion model in rats (Fitch et al., 1994; Galaburda, 1994; Herman et al., 1997), and is probably due to alterations in cortical excitability as described in the present study. Hyperexcitability in ibotenate-injected animals could be elicited not only in the surround of the structural lesion but also in remote cortical areas. Sham-operated animals, which display small superficial ectopias in 50% of cases, did not exhibit any differences in excitability to non-lesioned controls, indicating that size and depth of cortical dysgenesis might inf luence the occurrence and/or the spatial extension of the excitability changes. Superficial ectopias in sham-operated animals probably result from mechanical disruption of the pial surface and/or the underlying glia limitans caused by the needle injection (Caviness et al., 1978; Choi and Matthias, 1987; Rosen et al., 1992; Redecker et al., 1997b). Injection of ibotenate induces an excitotoxic lesion that results in the typical lesion pattern with sulcus formation and loss of deep cortical layers predominating in layer V and VI. Development of synaptic connectivity in the central nervous system has been shown to be dependent on interactions between certain targets and inputs (Goldman-Rakic, 1980; Schreyer and Jones, 1982; Easter et al., 1985). Thus, loss of deep cortical layers causes loss of specific cortico-cortical and cortico-thalamic connections or induces aberrant connectivity (Jones et al., 1981). For layer V neurons, abundance of long horizontal intracortical connections has been described (Aroniadou and Keller, 1993), indicating that excitotoxic cell death of deep cortical layers as produced by ibotenate probably induces widespread disturbances in synaptic organization beyond the boundaries of the structural lesion. These abnormalities in synaptic organization might change the balance between excitation and inhibition and cause excitability changes in widespread brain regions (Chagnac Amitai and Connors, 1989a,b). Since superficial ectopias as reported for sham-operated animals do not alter the excitability, involvement of deep cortical layers might be an important factor for the generation of widespread hyperexcitability. The finding that the depth of cortical lesions inf luences the extension of hyperexcitability has also been reported for experimentally induced infarcts in adult rats (Buchkremer-Ratzmann and Witte, 1997; Witte et al., 1997).

In accordance with clinical findings, the epileptogenic zone found in ibotenate-injected animals extends beyond the structural lesion (Palmini et al., 1991b). In order to plan a strategy in patients admitted for epilepsy surgery it has to be considered whether this extension of the epileptogenic zone is explained by propagation of epileptic activity generated in the dysplastic lesion, or whether the surrounding brain tissue is epileptogenic itself. The propagation of epileptiform activity has been described for the freeze-lesion model of microgyria (Jacobs et al., 1996; Luhmann and Raabe, 1996). In these studies multiphasic responses were evoked within a few millimeters adjacent to the microgyrus and recorded with multi-electrode arrays, and this showed that these epileptiform responses propagate across the cortex in horizontal direction. Although the propagation of the multiphasic activity was not analyzed in the present study, it clearly demonstrates that epileptiform activity can be elicited also in remote cortical areas. These results might further substantiate findings obtained in the freeze-lesion model showing that the separation of the microgyrus from the surrounding cortex with transcortical cuts did not affect the generation of epileptic activity in the disconnected surround (Prince et al., 1997), This indicates that cortical dysplastic lesions induce long-term functional alterations in structurally normal brain regions. Further studies are needed to verify these findings in the ibotenate model and to answer the question as to which mechanisms cause epileptogenesis in remote cortical areas. In sham-operated and control animals restricted zones of hyperexcitability were also obser ved in the hindlimb and forelimb representation cortex HL/FL (Zilles, 1992). The characteristics of the multiphasic responses found in these areas showed close similarities to the epileptiform activity described for ibotenate-injected animals, with the exception that the duration was commonly shorter. The occurrence of increased excitability in HL/FL of sham-operated animals was not dependent on the presence of superficial ectopias. Intrinsic hyperexcitability of specific cytoarchitectonic cortical areas has recently been described for the rostral part of the secondary occipital cortex Oc2 (Neumann-Haefelin et al., 1996) and for the auditory cortex (Metherate and Ashe, 1995), but at the present time it is still unclear which differences in cytoarchitecture, connectivity and/or receptor distribution might cause the different responsiveness to low-intensity stimulation in specific cortical areas. The secondary occipital cortex Oc2 as well as the hindlimb and forelimb representation cortex HL/FL show both a greater number of layer V neurons. For layer V, synaptic networks of intrinsically rhythmic neurons that generate synchronized oscillations have been described (Connors and Gutnick, 1990; Silva et al., 1991). Several studies further indicate that these neurons play an important role in the genesis of intracortical epileptic discharges, especially under conditions of reduced GABAergic inhibition (Connors, 1984; Hoffman et al., 1994). When the inf luence of these neuronal networks outweighs the inhibitory tone – this might be the case during low-intensity stimulation – they could generate synchronized synaptic activity. In addition, a differential regional and cellular distribution of neurotransmitter receptors might contribute to the differential excitability of various cortical areas (Fritschy and Mohler, 1995; Paysan et al., 1997). Similar, but lesion-induced, alterations in the regional distribution of neurotransmitter receptors might also occur in ibotenate-injected animals and favor the development of hyperexcitability found in widespread cortical regions.

Glucose Metabolism The present study demonstrates that ibotenate-induced cortical dysplasias show only focal alterations of cerebral glucose metabolism in the area of the structural lesion, whereas remote cortical areas that display pronounced changes in cortical excitability remain unimpaired. These focal changes in regional glucose metabolism correspond to the morphology of the malformation, revealing a layer-specific pattern with significant increase in glucose uptake in layer I and normal metabolism in the ectopia as well as in the underlying cortical layers. Also, sham-operated animals showing small superficial ectopias display a similar metabolic pattern with increased glucose uptake in layer I, although not to a significant level. Abnormal glucose metabolism in developmental malformations has been described in several clinical studies using [18F]f luorodeoxyglucose (FDG) PET (Bairamian et al., 1985; Chugani et al., 1990; Rintahaka et al., 1993; Lee et al., 1994). Most of those have demonstrated interictal hypometabolism in the structural abnormal area (Chugani et al., 1990; Olson et al., 1990). However, due to the various differences in patient populations a wide range of metabolic patterns has been described, e.g. subcortical heterotopias can be diagnosed because of displaced gray matter metabolic activity within otherwise hypometabolic white matter (Lee et al., 1994). This is the first study to combine electrophysiological and metabolic investigations in experimentally induced cortical malformations. In accordance with clinical findings, epileptiform activity was electrophysiologically detected in cortical regions that showed normal glucose metabolism, but focal hypometabolism in the area of the structural abnormality, as described in clinical PET studies, was not found in ibotenateinduced cortical dysplasias. This difference between the clinical and experimental data could be due to the absence of behavioral seizures in this model as discussed above, but it might also provide further evidence for the hypothesis that patients with cortical malformations require additional pathogenic factors to develop seizure disorders. In clinical studies it remains unclear whether hypometabolism found in patients with cortical dysplastic lesions is due to structural abnormality or functional derangement. Lee et al. (1994) reported that in a group of patients with cortical dysplasias and decreased glucose metabolism, structural alterations such as gliosis contribute to the finding of FDG PET hypometabolism. Extensive reactive gliosis has been reported to occur in the first days after ibotenate injection in newborn mice but, in adulthood, gliosis was limited to focal glial scar at the site of injection (Marret et al., 1995b). Furthermore cytological properties of neurons within the ibotenate-induced ectopia as well as the microgyrus appear normal, whereas several pathological studies and case reports on post-surgical specimens of human dysplastic cortex have been described consisting of numerous enlarged atypical neurons (Taylor et al., 1971; Ferrer et al., 1992; Battaglia et al., 1996; Preul et al., 1997). At the present time it is still unclear whether these structural alterations are the consequence of secondary damage due to recurrent seizures or whether they are an intrinsic pathogenic feature of certain types of cortical dysplasias that are primary involved in epileptogenesis. Furthermore, functional alterations contributing to the hypometabolism in clinical PET studies have to be considered. Several clinical studies have reported that epileptic foci induce widespread functional alterations in glucose metabolism beyond the structural lesion and even in remote areas (Engel et al., 1982; Theodore et al., 1988; Witte et


al., 1994; Arnold et al., 1996). Experimental investigations suggest that these widespread changes might be mediated via cortico-thalamo-cortical circuits (Bruehl and Witte, 1995; Bruehl et al., 1995; Redecker et al., 1997a). The present findings show a clear increase in glucose metabolism in layer I of the cortical lesion that showed significance in ibotenate-induced dysplasias but was also present in sham-injected animals showing small superficial ectopias. This regional increase in glucose uptake ref lects abnormal synaptic activity in layer I. It is still unclear whether this hypermetabolism is the consequence of an overactivation or increased density of structurally normal synapses or whether it is due to abnormal synaptic organization in this layer. Due to the limited resolution of clinical PET scans, layer-specific analysis of glucose metabolism in human cortical dysplasias is not possible. Conclusions The current study clearly demonstrates that ibotenate-induced cortical dysplasias cause widespread cortical hyperexcitability. These widespread changes in excitability could not be observed in sham-operated animals with intact deep cortical layers, indicating that focal loss of these layers induces extended alterations in cortical connectivity and imbalance of excitation and inhibition. Restricted zones of increased excitability were also found in the forelimb and hindlimb representation cortex FL/HL of sham and control animals, demonstrating this activity to be an intrinsic property of specific cortical areas. In contrast to the electrophysiological results, glucose metabolism was altered only in the structurally abnormal area displaying a characteristic pattern. Hypometabolism as described for many types of human dysplastic lesions has not been observed. This difference between the experimental and clinical data may be due to the absence of behavioral seizures in this model. However, the present findings add further evidence to the hypothesis that in patients with developmental malformations additional pathogenic factors contribute to the manifestation of seizure disorders.

Notes The authors wish to thank S. Hamm for excellent technical assistance, J.F. Staiger from the Institut für Hirnforschung for his help regarding the identification of the cortical areas, M. Schroeter for helpful comments on the histologies, and H. Steinmetz for continuous support and stimulating discussions. This work was supported by grants from Deutsche Forschungsgemeinschaft Re 1315/1–1 to C.R. and SFB 194. Address correspondence to Georg Hagemann, Neurologische Klinik, Heinrich-Heine-Universität, Moorenstrasse 5, D-40225 Düsseldorf, Germany. Email: [email protected].

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