Epileptogenesis in pediatric cortical dysplasia: The dysmature cerebral developmental hypothesis

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Epilepsy & Behavior 9 (2006) 219–235

Review

Epileptogenesis in pediatric cortical dysplasia: The dysmaturecerebral developmental hypothesis q

Carlos Cepeda, Veronique M. Andre, Michael S. Levine, Noriko Salamon,Hajime Miyata, Harry V. Vinters, Gary W. Mathern *

Divisions of Neurosurgery, Neuroradiology, and Neuropathology, and Department of Neurology, The Brain Research Institute and The Mental

Retardation Research Center, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA

Department of Neuropathology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Japan

Received 21 March 2006; revised 22 May 2006; accepted 26 May 2006Available online 27 July 2006

Abstract

Cortical dysplasia (CD) is the most frequent pathology found in pediatric epilepsy surgery patients with a nearly 80% incidence inchildren younger than 3 years of age. Younger cases are more likely to have multilobar and severe forms of CD compared with olderpatients with focal and mild CD. Using clinico-pathologic techniques, we have initiated studies that unravel the timing of CD pathogen-esis that in turn suggest mechanisms of epileptogenesis. Morphological comparisons provided the first clue when we observed that cyto-megalic neurons have similarities with human subplate cells, and balloon cells have features analogous to radial glia. This suggested thatfailure of prenatal cell degeneration before birth could explain the presence of postnatal dysmorphic cells in CD tissue. Neuronal densityand MRI volumes indicate that there were more neurons than expected in CD tissue, and they were probably produced in later neuro-genesis cell cycles. Together these findings imply that there is partial failure in later phases of cortical development that might explain thedistinctive histopathology of CD. If correct, epileptogenesis should be the consequence of incomplete cellular maturation in CD tissue. In

vitro electrophysiological findings are consistent with this notion. They show that balloon cells have glial features, cytomegalic neuronsand recently discovered cytomegalic interneurons reveal atypical hyperexcitable intrinsic membrane properties, there are more GABAthan glutamate spontaneous synaptic inputs onto neurons, and in a subset of cells NMDA and GABAA receptor-mediated responsesand subunit expression are similar to those of immature neurons. Our studies support the hypothesis that there are retained prenatalcells and neurons with immature cellular and synaptic properties in pediatric CD tissue. We propose that local interactions of dysmaturecells with normal postnatal neurons produce seizures. This hypothesis will drive future studies aimed at elucidating mechanisms of epi-leptogenesis in pediatric CD tissue.� 2006 Elsevier Inc. All rights reserved.

Keywords: Review; Seizures; Epilepsy; Cytomegalic neurons; Interneurons; Balloon cell; Corticoneurogenesis; Infantile spasms; Preplate; Subplate; MRI;GABA; Glutamate; NMDA

1525-5050/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.yebeh.2006.05.012

q This study was supported by the National Institutes of Health GrantsR01 NS38992 and P01 NS02808. H.M. was supported by Grants-in-aidfrom the Ministry of Education, Culture, Sports, Science, and Technologyof Japan (17689040) and the Japan Epilepsy Research Foundation (H16-009).

* Corresponding author. Present address: Reed Neurological ResearchCenter, 710 Westwood Plaza, Rm 2123, Los Angeles, CA 90095-1769,USA. Fax: +1 310 206 8461.

E-mail address: [email protected] (G.W. Mathern).

1. Introduction

Cortical dysplasia (CD) was first identified as a patho-logic substrate associated with intractable human seizuresby David Taylor and colleagues in England more than 35years ago [1]. Initially, CD was thought to be relativelyrare, and it was not until the development of modern neu-roimaging methodologies, such as MRI, that CD becamemore widely recognized, especially in the pediatric epilepsy

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Fig. 1. Bar graph illustrating the percentage of patients by age with CDundergoing resective surgery at UCLA. CD represents close to 80% ofsurgical cases in the first 3 years of life. That percentage gradually declineswith age to 7–10% from age 15 to 19 years, which is similar to most adultsurgical series.

220 C. Cepeda et al. / Epilepsy & Behavior 9 (2006) 219–235

surgery population [2]. With increased recognition of thisentity, there has been growing interest in understandingthe seizure producing mechanisms of CD tissue [3]. In theearly 1990’s, UCLA began a series of collaborative studiesintegrating basic science, neuropathology, neuroradiology,pediatric neurology, and neurosurgery to examine resectedcortical tissue from pediatric epilepsy surgery patients [4–10]. These early in vitro electrophysiological studies werenot very informative, and in hindsight this should not havebeen a surprise because we grouped neurons from CD andnon-CD tissue together, cortical sample sites were random-ly selected, and cellular recordings were done blindly. Theintroduction of infrared videomicroscopy and Nomarskioptics allowed us to visualize viable cells prior to patching,we changed our clinical protocol to select cortical tissuebased on MRI or EEG criteria to enhance sampling dys-morphic cells, and neurons from CD were compared withthose from non-CD specimens in the experimental design.These changes greatly improved our research capabilities,and the resulting studies began to unravel cellular and syn-aptic alterations in CD tissue. This review summarizes theresultant clinico-pathologic studies since the late 1990’sfocused on mechanisms of CD pathogenesis and epilepto-genesis that are pertinent to pediatric epilepsy surgerypatients.

2. Clinical features of CD in pediatric epilepsy surgical

patients: Impact on experimental design and research

sampling

CD is the most frequently identified pathologic substratein children undergoing epilepsy neurosurgery [11]. In thecombined adult and pediatric UCLA surgical cohort from1986 to 2005, CD (excluding tuberous sclerosis) was iden-tified in 45.5% of operated patients from age 2 months to19 years. Put another way, CD is as common in the pediat-ric epilepsy surgery population as hippocampal sclerosis isin temporal lobe epilepsy patients, and together these two

Fig. 2. MRI of patients with different forms of CD treated surgically. Right/coronal view of right hemimegalencephaly. This 8 month old began with clinicatreatment with anti-epilepsy drugs (AEDs) and ACTH the seizure frequencystatus epilepticus. Notice the diffuse enlargement of the gray and white matter inarrow). Pathology of the resected brain tissue showed severe CD. This child is seof hemispheric CD (hemi CD). This 1.9 year old began with seizures at age 2 mand generalized seizures localized to the mildly malformed but not enlargabnormalities (white arrows). Pathology showed mild CD with excessive heterosurgery. (E and F) Axial and coronal view of multilobar CD. This child beganhistory of infantile spasms, but there was mild motor asymmetry with weaknessthe frontal and parietal lobes including most of the motor-sensory cortex (whPathology showed severe CD with cytomegalic neurons but no balloon cells.control. (G and H) Axial and coronal view of lobar CD. This 4.5 year old beinvolving the collateral and para-hippocampal gyrus along with the hippocampwas severe CD, and the child is seizure free more than 1 year post-temporal logyrus. This 7.5 year old began with seizures at age 4.8 years which involved focathe left inferior frontal gyrus with a ‘‘tail’’ extending from the cortex into the wcells and dysmorphic neurons but no cytomegalic neurons. This child is seizucortex.

substrates account for roughly half of all pathologic abnor-malities in epilepsy neurosurgery cases at UCLA [2,12]. Inpediatric patients, however, this finding incompletely tellsthe story. CD incidence varies with type of resection andage at surgery. In children age 19 years or less, CD wasfound in 57% of those undergoing extratemporal resec-tions, compared with 11% for temporal lobe surgeries(Chi-square, P < 0.0001). In addition, the probability offinding CD was greater in younger children undergoingextratemporal resections with a nearly 80% incidence inthose less than 3 years of age (Fig. 1). CD was less commonwith older surgical ages approaching 7–10% for those aged16–19 years, which is the typical percentage reported forsurgical populations over 20 years of age [13,14].

CD characteristics by MRI influence the age at seizureonset and surgery in pediatric epilepsy surgery patients(Figs. 2 and 3). At UCLA, we classify CD into 5 sub-groups based on MRI criteria [15–18]. ‘‘Hemimegalenceph-aly’’ describes those CD children with an enlarged cerebral

left orientation for all figures is shown in panel (A). (A and B) Axial andl seizures at 3 months that quickly developed into infantile spasms. Despiteincreased such that at the time of surgery he was in the PICU because of

the right cerebral hemisphere with thickening of the cortical ribbon (whiteizure free 1 year post-hemispherectomy. (C and D) Axial and coronal viewonths and had infantile spasms that responded to ACTH. Residual focal

ed left hemisphere with diffuse subtle areas of gray and white mattertopias in the subcortical white matter. This child is seizure free 2 years post-seizing shortly after birth and surgery was at age 6 months. There was noof the right arm and leg. MRI reveals a large area of CD involving gyri of

ite arrows). The remainder of the hemisphere was less involved by MRI.This child is seizure free but required a hemispherectomy to achieve thatgan with seizures at age 1.3 years from a left mesial temporal area of CDus (white arrows). There was no history of infantile spasms, the pathologybectomy. (I and J) Axial and coronal view of focal CD involving a singlel motor onsets of the right body. MRI reveals a focal region of dysplasia ofhite matter toward the ventricle (white arrows). Pathology found balloonre free after focal resection that spared the motor sensory and language

c

C. Cepeda et al. / Epilepsy & Behavior 9 (2006) 219–235 221

hemisphere involving at least 3 of 4 lobes (Fig. 2A and B).Hemispheric CD cases involve diffuse MRI abnormalitiesof the gray and/or white matter of at least 3 lobes withouthemispheric enlargement (Fig. 2C and D) [15]. MultilobarCD cases are those patients where 2 contiguous brain lobes

show neuroimaging abnormalities (Fig. 2E and F) [19].Lobar CD cases are patients in whom two or more contig-uous gyri within the same lobe contain CD. In our pediat-ric experience there is no preference for CD to involve thefrontal, temporal, parietal, or occipital lobes (Fig. 2G and

Fig. 3. Box plots illustrating the differences for age at seizure onset (left graph) and age at surgery (right graph) for CD patients as classified in Fig. 2. Thebox represents the 25th–75th percentile, the line within the box the median, the lines outside the box 1.5 standard deviations of the mean, and open circlesindividual patients outside that range. The means (year ± SEM) are shown above each box. Age at seizure onset and age at surgery was progressively olderfor smaller types of CD (lobar and focal) compared with hemimegalencephaly (hemimeg) and hemispheric CD (Hemi CD, P < 0.018).


H). Finally, focal CD involves one or possible two adjoin-ing gyri within the brain, often with an abnormal whitematter ‘‘tail’’ extending toward the ventricle (Fig. 2I andJ). Using our MRI classification system, in the UCLApediatric epilepsy surgery cohort 13% of cases have hemim-egalencephaly, 17% hemispheric CD, 42% multilobar CD,17% lobar cases, and 11% focal CD. Thus, CD involvesmore than 1 lobe of the brain in 72% of our pediatric cases.Children with larger areas of CD by MRI (hemimegalen-cephaly and hemispheric CD) had a younger age at seizureonset and surgery compared with lobar and focal CDpatients (Fig. 3; P < 0.018). Age at seizure onset was 1 yearor less in 86% and before 2 years in 93% of all children withCD. Similarly, the age at surgery was 2 years or less in 50%and 5 years in 76% of our CD cohort. Hence, younger CDcases at the time of surgery most often have hemispheric ormultilobar involvement of the brain.

These clinical characteristics influence research samplingin studies involving epilepsy surgery patients with CD. Inour surgical population, CD tissue samples most oftencome from a young child (age 5 years or less) in whomthe MRI shows hemispheric or multilobar CD, any lobeof the brain will be sampled, and seizure duration will gen-erally be less than 3.5 years. By comparison, older epilepsypatients with CD will usually have many years of seizuresprior to surgery, the location of the sample site will oftenbe temporal or frontal, and the MRI will likely show lobaror focal CD. These clinical differences in CD patientsshould be considered in comparing research findings frompediatric and adult surgical cohorts (see Section 6.4).

3. Histopathology of CD: Relationship to neuroimaging,

EEG, and sample site

CD is the term applied to malformations of cortical devel-opment in which the primary histopathology involves thecerebral cortex and subcortical white matter [20,21]. Whileclassification systems have been proposed based on MRI

features and genetic mutations in familial cases [14,22–24],at UCLA we classify CD from surgical specimens into mildor severe forms based on a practical grading system [25]. Forall CD cases, the minimum histopathologic criteria are cor-tical dyslamination and columnar disorganization. This isnearly always coupled with excessive heterotopic neuronsin the subcortical white matter which can (when severe)result in an indistinct gray/white matter junction (Fig. 4Aand B, black arrow). Usually associated with elemental fea-tures of CD are other histopathologic findings, including thepresence of polymicrogyria (Fig. 4C), dysmorphic (Fig. 4D)or cytomegalic neurons (Fig. 4E), balloon cells (Fig. 4F),immature-appearing neurons (Fig. 4G), and excessive het-erotopic neurons in the molecular layer (Fig. 4B, whitearrow). Dysmorphic neurons are misshapen cells in whichthe primary dendrite may be oriented away from the pialsurface, the cytoskeleton is rich in neurofilaments, and thereare aberrant basal dendrites. Cytomegalic neurons are sever-al times the diameter of adjacent normal appearing neuronsusually with preserved pyramidal or interneuron-like mor-phologies. Balloon cells are very distinctive with large somalsizes, pale glassy eosinophilic cytoplasm (resembling gemist-ocytic astrocytes), and eccentric nuclei. Immature neuronsare round or oval cells with large nuclei and a thin rim ofcytoplasm, often seen in clusters in younger CD patients.If a surgical specimen contains cortical dyslamination anda few heterotopic white matter neurons it is termed mildCD. If there is cortical dyslamination together with dysmor-phic neurons, cytomegalic neurons, and/or balloon cells thesample is classified as showing severe CD. In UCLA’s pedi-atric epilepsy surgery cohort from 1999 to 2005, the majorityof CD surgical specimens were classified as severe, 94%showing dysmorphic neurons, 60% cytomegalic neurons,and 42% balloon cells (Table 1). Thus, pediatric epilepsy sur-gical samples generally contain severe forms of CD. Thiscontrasts with specimens originating from adults where asignificant proportion of surgical cases are classified asshowing mild CD [14,26,27].

Fig. 4. Examples of the histopathology of CD using staining against NeuN (panels A–E) or H and E (panels F and G). (A and B) Compared with theautopsy case (panel A), CD is characterized by dyslamination of the cortical ribbon with increased heterotopic neurons in the white matter leading to anindistinct lower cortical to white matter junction (panel B, black arrow) and heterotopic neurons in the molecular layer (panel B, white arrow). (C) Otherfeatures of CD in surgical cases may include polymicrogyria (PMG) often consisting of undulating upper cortical neuron layers opposite a flat pial surface(panel C, arrows). (D) Dysmorphic neurons which are abnormal appearing pyramidal cells with the primary apical dendrite often oriented in directionsother than toward the pial surface is another frequent feature of CD. (E–G) Other abnormal cell types in CD can include cytomegalic neurons which aretwo to three times the size of typical pyramidal shaped and dysmorphic neurons (panel E, arrow), balloon cells with minimal aspiny processes and aneccentrically placed nucleus (panel F, arrow), and immature neurons characterized by their neuroblast-like features consisting of round to oval nuclearconfigurations with a thin rim of cytoplasm (panel G).


As might be expected from these observations, CD clas-sification by MRI and the corresponding age at surgery areassociated with finding specific histopathologic features ofsevere CD in the cortical specimen (Table 1). In our CDcohort, nearly all of the cases showed excessive heterotopicneurons in the subcortical white matter and dysmorphicneurons, and there was no preference for finding thesefeatures based on CD classification by MRI (Table 1).

However, balloon cells in the surgical specimen weremore frequently observed in older patients with focalCD compared with younger hemispheric CD cases(P = 0.017). Likewise, immature and heterotopic molecularlayer neurons along with polymicrogyria were more oftenfound in younger hemimegalencephaly and hemisphericCD cases compared with older lobar and focal CDcases (P < 0.009). At present, it is unclear if the age at

Table 1Histopathologic features of CD by MRI type at presentation

Pathologic finding (incidence) HME N = 9 Hemi CD N = 11 Multilobar N = 14 Lobar N = 12 Focal N = 9 Chi-square P-value

Heterotopic Neu WM (100%) 100% 100% 100% 100% 100% 0.99Dysmorphic neurons (94%) 89% 100% 93% 92% 100% 0.66Cytomegalic neurons (60%) 89% 36% 71% 42% 67% 0.15Polymicrogyria (53%) 78% 64% 78% 25% 11% 0.001

Heterotopic Neu Mol Lay (44%) 100% 54% 36% 25% 11% 0.001

Balloon cells (42%) 44% 18% 43% 33% 78% 0.017

Immature neurons (18%) 56% 27% 14% 0% 0% 0.009

HME, hemimegalencephaly; Hemi CD, hemispheric CD; multilobar, multilobar CD; lobar, lobar CD; and focal, focal CD (see Fig. 2).Heterotopic Neu Mol Lay, heterotopic neurons in the molecular layer and heterotopic Neu WM, heterotopic neurons in the white matter.


presentation or extent of CD based on MRI influenceswhether specific histopathologic features will be found inthe surgical specimen. However, these results suggest thatcertain features of CD, such as the presence of immatureneurons, are age specific and may evolve with time, sup-porting the notion that CD may be a dynamic changingpathological substrate (see Section 6.4). If so, this wouldhave important implications for understanding pathogene-sis and epileptogenesis of CD tissue.

Based on retrospective analyses, we discovered that find-ing dysmorphic cells in the cortical research sample sitedepended more on MRI than EEG abnormalities. Specifi-cally, we found cytomegalic neurons and balloon cells inthe research specimen more readily if that surgical samplecame from an area of pachygyria by MRI (Fig. 2F, whitearrow) with corresponding severe hypometabolism on18fluoro-2-deoxyglucose-positron emission tomography(FDG-PET) [17]. By contrast, we had difficulty finding dys-morphic cells within cortical samples with the most activeinterictal spiking or ictal onset zones by electrocorticogra-phy (ECoG). Instead, cortical regions that contained dys-morphic cells usually had severe ECoG backgroundslowing and high voltage usually rhythmic slow waves.Hence, the ability to sample dysmorphic cells for researchpurposes depended on whether we used MRI or EEG cri-teria in selecting cortical sample sites in pediatric epilepsysurgery patients with CD.

4. CD pathogenesis: A hypothesis based on clinico-pathologic

observations

While there is consensus that abnormal cortical devel-opment is responsible for CD pathogenesis [23], it isunclear when it occurs and how it produces seizures.Based on qualitative histopathologic assessments, thepresumed mechanisms of CD pathogenesis have histori-cally been defects in neuronal migration to explain sub-cortical heterotopic neurons, and altered periventricularneuroglial differentiation to account for the abnormalcytomegalic and dysmorphic neurons and balloon cells[28–31]. Thus, CD pathogenesis should be the result ofevents taking place during early phases of human corti-coneurogenesis [32]. Our investigative approach was todetermine the probable timing of CD pathogenesis by

assessing MRI and histopathologic features based onan understanding of normal human developmental neu-robiology. In other words, we asked whether MRI andhistopathologic features in postnatal CD surgical speci-mens had elements that resembled those that would beexpected in different stages of prenatal human corticaldevelopment, and if so did this tell us something aboutthe timing of CD pathogenesis.

In applying this approach it is important to under-stand what components of human cortical developmentdiffer from those in other mammals that could potential-ly explain CD histopathology [33–42]. Mammalian telen-cephalon development begins with progenitor cellproliferation in the germinal zones and migration of cellstoward the cortical surface. The earliest migrating cellsform the preplate, which is split by subsequent genera-tions of pyramidal neurons into a primodial plexiformlayer (which becomes the molecular layer or layer 1)and subplate zone. Between 17 to 32 weeks of gestation,the human cerebral cortex begins to develop identifiablegyri, and a large subplate zone exists that is several timesthe thickness of the overlying cortical ribbon, especiallyin frontal, temporal, parietal areas of eventual associative(non-primary) cortex (Figs. 5 and 6). The junctionbetween the subplate and cortical ribbon can becomevery hypercellular with an indistinct border especiallyafter 30 weeks (Fig. 6C and D) [43]. By comparison,the rodent subplate is 1–2 cells thick at the bottom oflayer 6 neurons, and the developmental gray-white mat-ter junction is fairly distinct. Cells of the human molec-ular layer and subplate zones have diversedevelopmentally mature morphologies that would beconsidered atypical and abnormal in the postnatal cere-bral cortex (Fig. 6E–H) [44,45].

We observed that prenatal human subplate cells havemorphologic features similar to those of dysmorphic neu-rons found in postnatal CD tissue (compare Figs. 4 and6). This was our first clue to the probable timing of CDpathogenesis [46]. The human subplate contains large mul-tipolar neurons with spherical or ovoid shapes similar tocytomegalic neurons, and polymorphous and fusiform neu-rons with thick primary dendrites along with inverted pyra-midal-shaped neurons or other cells in which the apicaldendrite points away from the pial surface, a feature seen

Fig. 5. Examples of human cerebral cortical development at 24 (panels A and B), 28 (panels C and D), 34 (panels E and F), and 38 (panels G and H)gestational weeks. Left column shows the lateral hemisphere surface and the right column coronal sections through the parietal-occipital cortex. At 24weeks, notice the fissure separating the forming motor sensory cortex (arrow), and superior and inferior parietal lobules (arrowhead). There is minimalgyral folding on the cut occipital surface at 24 gestational weeks. From 24 to 38 gestational weeks the lateral surface becomes increasingly convolutedand gyral folds become better defined. In addition, as the subplate degenerates the border between the cortical gray and white matter becomes betterdefined.


Fig. 6. Nissl stained sections of the developing cerebral cortex from 24 to 38 weeks gestation. From the same areas as shown in Fig. 5 right column. (A–D)At lower magnification one can see the development of the cortical mantle along with the increase in subplate cells (arrows), especially at 34 and 38 weeksof gestation. (E–H) At higher magnification, normal cells in the human subplate (arrows) can be much larger than expected or have apical dentritesoriented away from the pial surface (pial surface toward the top of each panel). Notice the similarity between normal subplate cells at 34 and 38 weeksgestation with dysmorphic and cytomegalic neurons in cortical dysplasia (compare cells in panels E–H with those of Fig. 4D and E).


in dysmorphic CD neurons [39]. Most human subplate cellsdegenerate in the 4 to 6 weeks prior to birth as thalamicaxons disengage from them and establish new connectionswith maturing cortical-plate pyramidal neurons [47,48].Relevant to MRI findings in CD patients, human subplatedegeneration coincides with increasing definition of thegray–white matter junction and secondary gyral folding(Fig. 5) [49]. Furthermore, toward the end of normal neu-rogenesis periventricular radial glial cells attach themselvesonto the tailing processes of the last produced corticalpyramidal neurons, and migrate toward the cortex andsubcortical regions, where they detach and eventuallytransform into protoplasmic astrocytes [50–52]. We andothers have noted that balloon cells in CD tissue have mor-phologic and other characteristics similar to those of radialglia [53–55].

Based on these observations, we proposed that CDpathogenesis probably involved partial failure of eventsoccurring during later phases of corticogenesis, resultingin incomplete cortical development. As a consequence, sub-plate and radial glial degeneration and transformationwould be reduced or prevented, giving the appearance ofabnormal dysmorphic cells in postnatal CD tissue. In addi-tion, failure of late cortical maturation could explain thepresence of abnormally thickened gyri with indistinct corti-cal gray-white matter junctions in MRI scans of CD

patients (compare Fig. 5F with Fig. 2A and B) [56]. Ourhypothesis would also explain why most rodent modelsof CD have failed to reproduce dysmorphic cells, becausethese neurons only appear in subplate zones of higher pri-mates and humans. We further modified our hypothesis byproposing that the timing of these events during corticaldevelopment would explain the different forms of CD byMRI and severity of CD by histopathology. Developmen-tal alterations during the late second or early third trimes-ter would account for severe CD, like hemimegalencephaly.with numerous dysmorphic cells (compare Fig. 5D withFig. 2A), while events occurring closer to birth (after thesubplate has nearly degenerated) would explain milderforms of CD (compare Fig. 5H with Fig. 2D). Likewise,events affecting one side of the brain would explain hemi-spheric CD while local effects would account for lobarand focal CD. While attractive, it is important to acknowl-edge that our hypothesis does not exclude the possibilitythat some elements of CD pathogenesis might begin muchearlier during corticoneurogenesis in surgical patients.Likewise, our hypothesis is applicable to CD associatedwith surgical cases. Other malformations of cortical devel-opment where there are known defects in neuronal migra-tion and differentiation affecting both hemispheres, such aslissencephaly, double cortex syndrome, periventricular het-erotopia with microcephaly, and others, involve different


mechanisms of pathogenesis [24]. However, our resultshave allowed us to speculate that CD histopathology forpediatric epilepsy surgery patients could be explained byfailure of processes in the later phases of cerebral develop-ment [57].

The next research question posed was whether therewere more or fewer neurons than expected in pediatricCD patients, and did the pattern of cell loss or gain provideevidence for the timing of CD pathogenesis during corticaldevelopment. Cortical pyramidal neurons migrate in suc-cessive waves from the germinal matrix, forming the corti-cal gray matter from the inside out (i.e. earliest neurons indeepest cortex). Assuming this mechanism is preserved inCD, if pathogenesis involved premature neuroglial differen-tiation, we would expect fewer neurons to be produced inlater cell cycles because of a reduced progenitor cell pool[58,59]. In addition, defects in migration would trap neu-rons in the subcortical white matter. This should result insmaller postnatal cerebral hemispheres (microcephaly),

Fig. 7. Bar graphs showing the percent change from controls (normalized for eas measured by MRI (upper left) cortical thickness (upper right), and neuronalANOVA results denoted above the bars, and significant post-hoc statistical diffthat total hemisphere volumes of both the affected and non-affected sides in CDthe affected and non-affected sides were not different between CD and controlincreased (+23%; post-hoc, P = 0.0042 compared with controls) and the non(Upper right) Mean cortical thickness was not different in CD and HME cCompared with autopsy cases, NeuN cell densities in CD and HME cases wereP < 0.0057), upper gray matter (Levels 2 and 3; ANOVA, P < 0.04; post-hoc *were also less than controls for Level 6 (P = 0.01).

reduced cortical thickness, and decreased neuronal densi-ties in the gray matter as is seen in genetic defects, like lis-sencephaly. Instead, we found in pediatric CD that MRIcerebral hemisphere volumes were normal or increased inthe case of hemimegalencephaly, and cortical thicknesswas the same or slightly increased compared with autopsycontrols [16,57]. In addition, neuronal densities wereincreased in the upper gray matter, molecular layer, andsubcortical white matter (Fig. 7). We interpreted these find-ings as evidence that there was over (not under) productionof cortical pyramidal neurons in pediatric CD tissue. Thelocation of excess neurons would be consistent with theidea that this process occurred in later periventricular cellcycles (i.e. the ones toward the end of neurogenesis). Like-wise, there were increased neurons in the subcortical whitematter and molecular layer; the expected location of resid-ual prenatal subplate and preplate cells during corticaldevelopment. Thus, heterotopic subcortical white matterneurons are likely the result of excessive late generated

xpected changes as a consequence of age) for cerebral hemisphere volumesdensities (lower) for autopsy, CD and hemimegalencephaly (HME) cases.erences compared with controls indicated (*). (Upper left) Analysis foundand HME cases were not different from controls (ANOVA, P = 0.67), and

s. However, for HME cases compared with controls, the affected side was-affected side decreased (�14%; post-hoc; P = 0.0156) in MRI volumes.ases compared with controls (right graph; ANOVA, P = 0.79). (Lower)increased in the molecular layer (Layer 1; ANOVA, P = 0.003; post-hoc *,, P < 0.0053), and superficial white matter (SWM, P = 0.01). HME cases


pyramidal neurons trying to migrate toward the corticalribbon, in combination with residual prenatal subplateneurons that failed to degenerate prior to birth.

5. Dysmature electrophysiological characteristics of

pediatric CD tissue

If CD pathogenesis involves processes that delay or pre-vent later stages of human corticogenesis, then what couldbe the consequence of these actions that explains seizuregeneration? Based on our concept of CD pathogenesis,we hypothesized that delayed cortical maturation shouldresult in CD tissue that contains prenatal-like dysmorphiccells with electrophysiological features atypical for postna-tal neurons [60]. Likewise, there should be neurons withcellular and synaptic properties similar to those in imma-ture developing cortex [61]. Furthermore, areas of severeCD (i.e. more dysplastic) should have greater signs of cel-lular dysmaturity than mild CD. Seizures would occurbecause areas of CD contain cells and synaptic propertieswith immature electrophysiological characteristics thatare hyperexcitable and ‘‘pro-epileptic’’ compared with thenormal postnatal brain.

The reader should note that at the start of our in vitro

single cell electrophysiological studies in the late 1990s,our original aim was to test whether epileptogenesisinvolved alterations in intrinsic membrane properties pro-ducing cells that spontaneously ‘‘seize’’ or alterations inexcitatory or inhibitory inputs leading to ‘‘epileptic cir-cuits’’ [62]. These concepts were generated from contempo-rary studies of human CD tissue postulating that seizuresmight arise from cytomegalic neurons and/or balloon cellsacting as ‘‘epileptic neurons’’ [30,63,64], reduced GABAmediated inhibition [65–68], or increased glutamate medi-ated excitation [64,69]. Our electrophysiological findingsturned out not to match these ideas, but instead were con-sistent with our emerging hypothesis that pediatric CD tis-sue contained atypical and dysmature cellular and synapticfeatures.

5.1. Electrophysiological properties by CD cell type: Are

there ‘‘epileptic neurons’’?

Using in vitro field potential recordings, Avoli and col-leagues previously demonstrated that 4-aminopyridine (4-AP), a K+ channel blocker that increased neurotransmitterrelease, induced epileptiform-like activity in slices frommostly adult patients with mild CD more readily than cor-tical tissue from temporal lobe epilepsy patients [70]. Otherexperiments from that laboratory further suggested thatthe 4-AP induced activity involved NMDA and GABAmediated mechanisms. However, that group did not sys-tematically identify and study dysmorphic neurons in CDtissue. Using infrared videomicroscopy, we obtainedrecordings from dysmorphic cells in pediatric CD tissueshowing that different cell types have unique electrophysio-logical characteristics [46,71].

Balloon cells, for example, were cells with propertiessimilar to glia. Morphologically, they lacked dendriticspines and axons (Fig. 8). Electrophysiologically, theylacked inward currents, had very high membrane inputresistances, did not generate action potentials when mem-branes were depolarized (Fig. 9A), and displayed no spon-taneous synaptic currents or responses to exogenousapplication of excitatory amino acids [17,46]. Thus, bal-loon cells were unusual cells in that they do not appearcapable of spontaneously firing action potentials like onewould expect of neurons [72].

By contrast, cytomegalic neurons with pyramidal mor-phologies had atypical hyperexcitable characteristics. Mor-phologically, cytomegalic neurons had robust and matureappearing dendritic processes with variable spine densities,basal dendrites, and axons (Fig. 8). Functionally, they dis-played large capacitance, very low input resistance anduncharacteristic intrinsic membrane properties, such asincreased voltage-gated Ca2+ currents that allowed thesecells to fire repeated Ca2+ spikes once they reached firingthresholds (Fig. 9B) [46]. This latter property was neverobserved in normal pyramidal neurons from mild CD ornon-CD tissue. In addition, cytomegalic neurons respond-ed to exogenous application of excitatory and inhibitoryamino acids, and due to their large membrane areasligand-gated currents appeared increased compared withnormal-sized pyramidal neurons. However, other featuresof cytomegalic neurons were inconsistent with the notionthat they were ‘‘epileptic neurons’’. Their resting mem-brane potentials were relatively hyperpolarized, and cur-rent density measurements indicated that the density ofpostsynaptic receptors per area of membrane was reducedcompared with normal pyramidal neurons. In addition,using current clamp recording techniques, none of the cyto-megalic neurons showed spontaneous depolarizations lead-ing to self generated action potentials in the in vitro slice[17]. We interpreted these findings as evidence that cytome-galic neurons have hyperexcitable electrophysiologicalproperties, as might be expected of atypical cells, makingthem capable of exciting neighboring cells to amplify sei-zure activity. However, by themselves they do not havethe intrinsic membrane properties necessary to initiate ictalevents in CD tissue.

Recently, we have identified in severe CD tissue anothergroup of cytomegalic cells that have the appearance ofGABAergic interneurons. Electrophysiological recordingssuggest that these cells could spontaneously produce‘‘epileptic-like’’ activity [73]. These were very large basket,stellate, or bitufted shaped cells (Fig. 8) with firing proper-ties similar to those of normal interneurons. Cytomegalicinterneurons showed signs of hyperexcitability by produc-ing spontaneous membrane depolarizations with actionpotentials in current clamp (Fig. 9C), and rhythmic burstswhen depolarized with suprathreshold continuous positivecurrent. While only a handful of cytomegalic interneuronshave been sampled thus far, these cells could possiblysynchronize neuronal activity and be candidate ‘‘epileptic

Fig. 8. Camera lucida drawings of biocytin-filled cells from pediatric CD tissue samples. Cell types include (left to right, top to bottom): balloon cell,normal-appearing pyramidal neuron, immature neuron, cytomegalic pyramidal neuron, cytomegalic interneuron. Notice that the balloon cell shows alarge soma with thin tortuous processes practically devoid of spines. In addition, the balloon cell has no apparent axon. By comparison, cytomegalicneurons and interneurons are very large cells with axons, diffuse dendrites and spines. See Fig. 9, text and Table 2 for electrophysiological details.


neurons’’ using GABA as their primary neurotransmitter.However, several unanswered questions remain about theseatypical cells. It is unknown if they appear in all or only aproportion of CD cases. Recall that cytomegalic pyrami-dal-shaped neurons were found in about 60% of pediatricCD cases (Table 1). If cytomegalic interneurons are foundin only a few cases, then their presence cannot explain epi-leptogenesis for every CD patient. Moreover, we do notknow the origin of these atypical neurons and whether theyrelease more than one type of neurotransmitter. Based onour hypothesis of CD pathogenesis it would be worthwhileto investigate whether there are cells with similar morphol-ogies and electrophysiological characteristics in the prena-tal human subplate.

Equally intriguing was our examination of immaturepyramidal-shaped neurons. These cells appear in clustersin younger CD cases, such as those with hemimegalenceph-aly [16,74]. Immature looking neurons have underdevel-oped dendritic processes with relatively few spines(Fig. 8), very high input resistances; and are unique in thatthey universally display robust GABAergic synaptic activ-ity (Fig. 9D) that depolarizes (excite) cell membranes, andthese inputs are often very rhythmic (clock-like) [46].GABA-mediated activation of immature pyramidal neu-rons could induce rhythmic firing, thus creating islets ofhyperexcitability in severe CD tissue. However, becauseof their underdeveloped processes we do not know howwell immature neurons are connected with other neurons

Fig. 9. Electrophysiological abnormalities found in cells from severe CD tissue. Recordings obtained using the whole-cell patch clamp technique in voltage(panels A, B and D) or current clamp (Panel C) modes. The pipette solution contained K-gluconate (A and C) or Cs-methanesulfonate (B and D). (A) Aseries of depolarizing voltage commands was unable to induce active inward currents in a balloon cell recorded in an in vitro slice from the temporal regionof a patient with tuberous sclerosis complex (12 years of age). In normal pyramidal neurons the same protocol would induce inward Na+ currents. (B) In acytomegalic pyramidal neuron, a step voltage command (�70–�20 mV) induced repetitive Ca2+ mediated spikes. This is an abnormal finding not seen innormal cells and is a sign of cellular hyperexcitability. Cell recorded in the parietal region of a severe CD case (0.92 year old). (C) Spontaneous membranedepolarizations, reminiscent of paroxysmal depolarization shifts, recorded in a cytomegalic interneuron in the frontal region of a patient withhemimegalencephaly (0.75 year old). (D) A depolarizing ramp voltage command (�90–50 mV) induced a series of inward (Na+ and Ca2+) and outward(K+) currents in an immature pyramidal neuron. Interestingly, superimposed on those currents were small spontaneous synaptic currents that wereblocked by the GABAA receptor antagonist bicuculline. Cell recorded in the parietal region in a case of severe CD (0.92 year old).


in CD tissue. Likewise, their relative scarcity in mostlyyounger severe CD cases make them unlikely candidatesto be ‘‘epileptic neurons’’ capable of spontaneous seizuregeneration in all CD tissue. Other cell types sampled inCD tissue, such as misoriented pyramidal neurons and neu-rons with dysmorphic (tortuous) dendrites and axons hadelectrophysiologic properties similar to those of normal-ap-pearing pyramidal neurons, and are unlikely candidates tobe ‘‘epileptic neurons’’.

5.2. Dysmature synaptic properties of pediatric CD tissue

During normal corticogenesis, GABA synaptic signalingappears before glutamate, and involves early interneuronfunction and GABAA receptor activation [75]. By electronmicroscopy, GABA symmetric synapses comprise 50% oftotal synapses in the early cortex that decline to 15% inthe adult [76]. In addition, GABA-positive cells and aGABA-containing axonal plexus have been identified inthe prenatal marginal zone, indicating an abundant sourceof GABA during early corticoneurogenesis [77].

As might be expected based on our dysmaturity hypoth-esis, we found more GABAergic relative to glutamatergicsynaptic inputs onto normal-appearing pyramidal neuronsand cytomegalic pyramidal-shaped neurons in areas ofsevere CD tissue compared with mild CD [17]. In severe

CD, this finding supports the notion that neurotransmis-sion retains immature features with GABA as the predom-inant neurotransmitter. Initial cellular electrophysiologicalstudies also indicate that GABAA synaptic responses arealtered in pediatric CD tissue with findings similar to thoseof immature cortex [78]. Using acutely dissociated cells, wefound that GABA-induced current peak amplitudes (butnot current densities) were larger in cytomegalic neuronscompared with normal pyramidal neurons. In addition,GABAA current decay times were slower in cytomegalicneurons (like immature cells), suggesting differential recep-tor subunit composition. Our studies of GABA-mediatedneurotransmission are just beginning, but support the con-tention that because of high intracellular chloride concen-tration from developmentally regulated chloride iontransporters, seizure generation in pediatric CD could bedue in part to GABA acting as an excitatory neurotrans-mitter in selected cell types [76,79–84]. In addition, it willbe important to determine if recently discovered cytome-galic interneurons provide a source of enhanced clock-likeGABAergic inputs onto neurons in pediatric CD tissueleading to synchronized activity.

While glutamate-mediated synaptic inputs appear to berelatively decreased in severe CD, this does not rule outthe possibility that postsynaptic glutamate receptors couldcontribute to cellular hyperexcitability in pediatric CD


patients [85]. Human studies have reported increased somalimmunostaining for NMDA receptor subunits in CD tissue[63,64,86]. In addition, we reported decreased Mg2+ sensi-tivity of postsynaptic NMDA receptors in cytomegalic neu-rons and a proportion of normal appearing pyramidalneurons in regions of severe CD compared with mild andnon-CD cases [87]. Reduced Mg2+ sensitivity of the NMDAreceptor is a feature of the developing cerebral cortex [88].The functional NMDA receptor changes were associatedwith less effective blockade with ifenprodil (an NR2B selec-tive antagonist), and decreased NR2B subunit mRNA andprotein expression [87] as was similarly reported in theMAM rat model of CD [89]. Thus, in pediatric CD NMDAreceptors appear to be dysmature with reduced Mg2+ sensi-tivity and this is associated with changes in NMDA receptorsubunit composition. How glutamate receptor changes con-tribute to seizure generation in the context of GABA-med-iated neurotransmission is as yet unexplored.

6. Hypothesis critique and future research directions

Our clinico-pathologic studies of pediatric epilepsy sur-gery patients support the hypothesis that components ofCD pathogenesis involve partial failure of processes in laterphases of cortical development, and as a consequence CDtissue has signs of cellular and synaptic immaturity andarrested development (summarized in Table 2). The simi-larities between cytomegalic and dysmorphic neurons withprenatal human subplate cells, increased densities of lategenerated pyramidal neurons, predominance of GABA asa neurotransmitter, presence of immature pyramidal neu-rons with depolarizing GABA-mediated synaptic inputs,reduced Mg2+ sensitivity of NMDA and altered GABAA

receptor responses, and hyperexcitable cellular propertiesof cytomegalic neurons and cytomegalic interneurons allsupport the notion that severe CD has ‘‘pro-epileptic’’ fea-tures similar to those of immature developing cortex. Insevere CD, such findings suggest that seizure generationmight arise from unexpected mechanisms, such as depolar-izing GABA-mediated neurotransmission in one group ofcells interacting with neighboring more mature neurons[90]. While intriguing, our human studies are at a very

Table 2Summary of intrinsic membrane properties and synaptic features by cell type

Cell type Basic membrane properties Excitability

Cm (pF) Ri (MX) Tau (ms)

Balloon — › — fl No inward curCytomegalic pyramidal › fl › › Repetitive Ca2

Cytomegalic interneuron › fl › › Spontaneous PImmature fl › fl › Depolarizing G

—, ›, fl = No change, increase and decrease, respectively. Comparisons are betcells or between normal and cytomegalic interneurons.Cm, cell membrane capacitance, Ri, input resistance, Tau, membrane time coPGA, pacemaker GABA synaptic events (5–10 Hz).PDS, paroxysmal-like depolarizing shifts.

preliminary phase, and these conclusions and hypothesesshould be viewed cautiously. The limitations of our exper-imental techniques and assumptions necessary to interpretour results should be evaluated in future hypothesis-drivenstudies, as noted below.

6.1. The dysmature cerebral developmental hypothesis

While an attractive proposition that has, so far,explained the histopathology and cellular electrophysiolog-ical features of severe CD tissue, this concept requires fur-ther experimental testing and verification. The reader isreminded that the minimal histopathology of CD is corticaldyslamination with excessive heterotopic white matter neu-rons. In other words, medically refractory seizures occur inpatients with mild CD that do not contain dysmorphiccells, polymicrogyria, or other features of severe CD. Canthe dysmature cerebral developmental hypothesis explainseizure generation in mild CD cases? To address this ques-tion, future studies should focus on the morphologic andelectrophysiologic properties of subcortical and gray mat-ter neurons, and the interactions of these two cell popula-tions in CD tissue. The interactions of these cells couldproduce seizures, and it will be important to determine ifthis involves immature cellular and synaptic features.

6.2. The in vitro preparation

Currently the best experimental methods to study singlecells from human brain tissue are the in vitro slice and dis-sociated cell techniques, in which individual cells can beidentified and sampled. However, this is an artificial envi-ronment, and we do not know how CD cells behave in vivo

with a full complement of glutamate and GABA inputs.Neurons, like cytomegalic cells, might be capable of spon-taneous depolarization in situ that we cannot detect exper-imentally. Methods that are able to characterizeelectrophysiological properties of cells in a tissue block per-haps using high field strength MRI or other techniques willbe necessary in order to understand how atypical andimmature cells interact with more normal cells within alocal cortical milieu.

in severe pediatric CD tissue

Synapticinputs

NMDA responses GABA responses

rents None None None+ spikes Variable

PGAReduced Mg2+ sensitivity Slower decay time

DS ? ? ?ABA events › PGA Reduced Mg2+ sensitivity? ›

ween normal pyramidal and balloon, cytomegalic pyramidal and immature

nstant.


6.3. The supposition that subplate cells and radial glia are

cytomegalic neurons and balloon cells

It is imperative to acknowledge that our hypothesisregarding the etiology of cytomegalic neurons and ballooncells were derived from correlative observations and cir-cumstantial evidence. This emphasizes the importance ofcontinued studies of human CD tissue as this is currentlythe only source of material to study these unusual cells.Unexplored is whether cytomegalic neurons can be depo-larized by GABA, as might be expected if these are dysma-ture cells. Likewise, despite our findings to date, ballooncells should not be completely dismissed from contributingto seizure generation. With their astroglial properties, theycould be an extrasynaptic source of released glutamate thatinitiates paroxysmal depolarization shifts [91]. It will alsobe critical to compare and contrast cytomegalic neuronsand interneurons with normal subplate cells and ballooncells with radial glia. This may be difficult, as access to nor-mal developing cortex in the last trimester of pregnancy islimited, and studies of sub-human primates may be theonly ethically acceptable alternative to understand the elec-trophysiological features of normal subplate cells.

6.4. Comparisons of pediatric and adult CD

The literature already contains published reports fromadult CD cohorts that are inconsistent with our findingsin pediatric CD patients. By contrast with our pediatricdata, adult CD patients reportedly show decreased corticalneuronal densities, increased NR2B receptor subunits incytomegalic neurons, and reduced spontaneous GABAsynaptic inputs [92–95]. These discrepancies need to beresolved, and probably relate to differences in CD clinicalcharacteristics and sample sites, as outlined at the begin-ning of this review. It is imperative that research studiesfrom human CD cohorts clearly define the source andMRI characteristics of CD cases and sample sites so thatpediatric and adult studies can be compared. In addition,other potential experimental confounds should be carefullycontrolled and accounted for, such as source and age ofcomparison tissue for human studies.

Another possibility for consideration in future studies isthat the histopathology and electrophysiological character-istics of CD tissue may not be static. As we previously not-ed, most CD patients begin with seizures early in life, andthe incidence of specific cell types, like immature neuronsand balloon cells were age and MRI specific (Table 1). Thissuggests that CD may evolve with age and longer seizuredurations, and this should be tested.

6.5. Interactions of CD tissue with normal cerebral cortex

While in vitro experiments can address changes in cellsand synaptic circuits within a limited geographic region,these studies do not provide information about how areasof CD interact with distant cortical structures, like the thal-

amus, basal ganglia, brainstem, and cerebral cortex of thecontralateral hemisphere. These questions are relevant inunderstanding how localized pathologies, like CD, oftenproduce what appear to be generalized seizures in infantsand children and age-specific syndromes like infantilespasms. Likewise, we found that age at seizure onset wasolder for smaller CD pathologies suggesting that interac-tions based on size with other cortical structures influenceclinical seizure characteristics. How CD size influencesage at seizure onset is unknown, but probably involvesinteractions with other cortical and subcortical structures.Furthermore, it is unclear what seizure-related mechanismsin CD induce epileptic encephalopathy, which is a commonfeature in infants with early onset epilepsy [96]. Thus,future research studies should focus on following subcorti-cal, white matter, and cortical maturation in vivo using lon-gitudinal studies to identify how the postnatal human brainmatures with and without constant seizures, and whetherseizure-induced encephalopathy can be identified andprevented.

6.6. Therapeutic implications

Ultimately, the goal of human research should be toprovide insight into novel and rational treatment methods,and our hypothesis may have future impact on how wetreat children with seizures from CD [97]. Currently, mostfirst line drugs used to treat pediatric epilepsy act byenhancing GABA mediated neurotransmission. This maynot be appropriate in pediatric CD, especially if GABAis found to depolarize dysmorphic and immature neuronsinto firing action potentials. Instead, agents that speedthe developmental maturation, such as transition of chlo-ride transporters that would alter GABA receptors fromdepolarizing to hyperpolarizing, could be novel approachesto treat seizures in pediatric CD patients [98]. Pharmaco-logic studies of tissue resected at surgery as an adjunct intherapy discovery will be important in testing these notionsin pediatric CD.

Acknowledgments

Special thanks to Robin Fisher, Marea K. Boylan, Nan-ping Wu, Irene Yamazaki, Jorge Flores-Hernandez, Ray-mond S. Hurst, Marissa Andres, Snow T. Nguyen, JuliaW. Chang, My N. Huynh, Susan Koh, Joyce Wu, DennisChute, P. Sarat Chandra, Joao P. Leite, and Luciano Ne-der who assisted in our studies, past and present. Theauthors also thank Sergey Popov in the Pathology Depart-ment of Pediatric Hospital No1 in St. Petersburg, Russiafor supplying prenatal human brain specimens.

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Epileptogenesis in pediatric cortical dysplasia: The dysmature cerebral developmental hypothesis