Basic Mechanisms of Epileptogenesis in Pediatric Cortical Dysplasia

Introduction

Cortical neurodevelopment in mammals is a complex, dynamic process that involves the birth of neurons, their growth and differentiation, migration to their final destination, and synapse formation. This process is longer and more complex in humans, therefore increasing susceptibility to developmental errors 1, 2. Both genetic and environmental factors cumulatively dictate and influence the fate of neuronal development 3. Aberrant cell migration and differentiation is responsible for cortical dysplasia (CD), the most common histopathological substrate in surgical cases of pediatric epilepsy 4, 5.

Focal CD associated with epilepsy, first described by Taylor and associates in 1971, is characterized by dyslamination, ectopic neurons in the white matter, and the presence of abnormal cytomegalic neurons and balloon cells that exist only in one other etiology, tuberous sclerosis complex (TSC) 6. Erroneous cortical development can occur at the level of neuronal–glial proliferation and differentiation during migration of neurons to the cortical plate and during intracortical organization 7, 8. Faulty differentiation leads to the occurrence of abnormal cells in CD, in particular dysmorphic cytomegalic neurons, balloon cells, and immature pyramidal neurons. There is still debate about the proper definition of these cells, and a general consensus has not been reached. In this review, we will adhere, as much as possible, to the most recent nomenclature 9, but we also take into account previous definitions. Dysmorphic cytomegalic pyramidal neurons, sometimes called dysplastic, are misshapen, enlarged cells with abnormal orientation, cytoskeletal structure, atypical dendritic processes, and rich in neurofilaments 6, 9, 10, 11, 12. Balloon cells have thin membrane, pale, glassy and eosinophilic cytoplasm with an eccentric nucleus, express neuronal and glial markers 13, and are morphologically similar to, but usually larger than, gemistocytic astrocytes and without proliferative potential 9, 12, 14. Immature neurons derive from neuroblasts, have a small somatic size, and do not accumulate neurofilaments 9. However, not all cases of CD present these aberrant cells and the need for a classification of different types of CD based on degree of severity soon became evident as more pathological specimens from epilepsy surgery cases became available.

Recent advances in identification of different CD types, histopathological substrates, imaging, and in vitro studies using resected tissue have allowed a better understanding of the diversity of pathologies and epileptogenic mechanisms. In this review, we examine possible mechanisms of epileptogenesis in different CD types based on histopathological substrates and aberrant membrane and synaptic properties.

Classifications of CD

Several classifications of the complex structural abnormalities of CD have been proposed that account for the degree of severity depending on either the pathological characteristics or the origin of the pathology (see Figure 1). In 1995, a grading system for CD was introduced based on histopathology of resected human brain specimens. Timing of the developmental insult in early, mid‐ and late gestational stages resulted in severe, moderate and mild CD, respectively. Mild CD was characterized by pathological features such as cortical disorganization, heterotopic white matter and molecular layer neurons, persistent subpial granular cell layer, and marginal glio‐neuronal heterotopia. In moderate CD, in addition to those features, polymicrogyri and neuronal heterotopia were present. Finally, in the severe form of CD, in addition to features present in mild and moderate CD, there were balloon cells and neuronal cytomegaly 11. This classification, although not generally used today, is the only one that explicitly suggests an inverse correlation between degree of CD severity and timing of initial insult during embryogenesis.

The Palmini classification separated CD into two main types based on histopathological findings 12. Intracortical laminar and columnar disorganization was reported as the major feature of CD. This classification separated CD type I based on cortical dyslamination as well as misorientation of cells secondary to excess neurons in the subcortical white matter and CD type II based on dyslamination and the presence of abnormal cellular elements such as dysmorphic pyramidal neurons and balloon cells. CD type II was further divided into type IIa based on the presence of dysmorphic neurons and type IIb where abnormal elements also included balloon cells.

The most recent CD classification was proposed by the International League Against Epilepsy (ILAE). It is based on a three‐tiered system that distinguishes whether pathological findings of CD are isolated or associated with other epileptogenic lesions 9. CD types I and II are considered isolated findings, while CD type III is a variant as there exists a principal lesion associated with CD (dual pathology). Similar to the Palmini classification, CD type I was defined by cytoarchitectural abnormalities. Further subdivisions were proposed with CD type Ia characterized by radial dyslamination, CD type Ib characterized by tangential dyslamination, and CD type Ic where dyslamination is both radial and tangential. In CD type II, in addition to dyslamination, there are abnormal dysmorphic neurons, type IIa, or dysmorphic neurons and balloon cells, type IIb. Lastly, in CD type III, in addition to CD, there is another lesion present, likely associated with epileptogenicity. CD type III is further divided into subtypes. CD type IIIa refers to CD associated with hippocampal sclerosis in the temporal lobe, CD type IIIb is associated with a CNS tumor, CD type IIIc is associated with a vascular malformation, and CD type IIId is associated with another lesion acquired early in life either secondary to a trauma or encephalitis. It is important to classify precisely different types of CD as it can lead to the appropriate tailoring of treatment modalities based on differential epileptogenic mechanisms 9, 15.

Clinical Manifestations

The cerebral cortex plays a vital role in a number of functions including memory, attention, perceptual awareness, thought, language, and consciousness. Accordingly, individuals affected with CD exhibit a wide array of symptoms depending on the extent and region of the brain involved. Epilepsy is the main symptom of CD, and it is often refractory to antiepileptic drugs (AEDs) 16. Symptoms may appear at any age including in adulthood, although seizures mostly start in childhood. Individuals with CD may also present with behavioral disturbances, psychomotor retardation, and learning disabilities 9, 17, 18. Other manifestations that have been reported include homonymous hemianopsia 19, 20 and sleep‐related epilepsy 21.

Children with CD type I are more likely to be mentally retarded and present maladaptive behaviors more frequently compared with children with CD type II 17. In addition, surgical outcome is significantly worse in patients with CD type I 17, 22, probably because it is more difficult to delimit the structural lesion and achieve a complete surgical resection 4, 23, 24. Patients with CD type II manifest symptoms earlier than those with CD type I. Furthermore, those with larger lesions tend to present symptoms earlier than patients with smaller lesions. Evidence suggests that patients with CD type II usually have multilobar lesions with extratemporal involvement, mainly within the frontal lobe. Hence, seizures that occur early during the neonatal period or childhood are more likely to be due to CD type II with more extensive, multilobar involvement 9. With central or parietal lesions, neurological findings include motor and sensory limb deficits and facial paresis, as well as poor bilateral hand coordination. Nystagmus and hemianopsia are observed in patients with posterior lesions. The motor deficits can become permanent if seizures are medically refractory, as is the case in status epilepticus, which poses a grave prognostic factor 20. The cognitive impairment tends to be broad, ranging from mild impairment in those with limited CD to severe cognitive disorder and autism spectrum disorder in those with extensive lesions as well as earlier onset of epilepsy. The CD cases that present with psychiatric symptoms also tend to have earlier onset of epilepsy, and their lesions are located more posteriorly 20.

MRI and EEG Findings

Magnetic resonance imagining (MRI) is the most common method used to locate the lesion(s) in CD. In children with positive MRI findings, seizures tend to be earlier in onset and intractable 25, 26, 27. The MRI in CD type I includes segmental or lobar atrophy that is usually associated with reduced volume of the subcortical white matter, some blurring of the gray–white matter junction, and abnormal patterns of gyri and sulci. This type often involves the temporal lobe 4. The MRI findings in CD type II include cortical thickening, blurring of the gray–white matter junction on T1‐ and T2‐weighted images, increase in signaling of white matter on T2‐weighted images and fluid attenuated inversion recovery (FLAIR) T2‐weighted images, and decrease in the white matter signal in T1‐weighted image. The change in white matter signaling tapers toward the ventricle, resulting in the “transmantle sign” 28. The subcortical white matter may also appear hyperintense on T2‐weighted imaging and hypointense on T1‐weighted imaging 29, 30.

In CD types I and II, if the lesions are very small, the MRI study may be negative regardless of the severity of the symptoms posing a challenge for surgical treatment 31, 32, 33. For example, CD type II associated with intractable partial epilepsy with early onset produces neurological deficits as well as cognitive impairment and is missed on MRI studies in one‐third of affected subjects 34. Nevertheless, detection of the “transmantle” sign in CD type II can be greatly improved using 3 Tesla MRI, which has better spatial resolution 35.

Precise localization of epileptic foci and improved outcome can benefit from the use of multimodality imaging 36. In the case where MRI is negative in individuals with intractable epilepsy, fluoro‐deoxyglucose positron emission tomography (FDG‐PET) coregistration and ictal single photon emission computed tomography (SPECT) may help to identify structural lesions 37, 38, 39, 40. In this process, FDG‐PET images are superimposed on the MRI while color‐coding the area as well as degree of hypometabolism to identify subtle CD foci. Use of FDG‐PET/MRI coregistration may be superior to MRI studies alone, as borders of the lesions are identified more clearly, thus promoting success in complete resection of these lesions 38, 41.

Electroencephalography (EEG) and intraoperative electrocorticography (ECoG) are other important tools in the localization of the epileptogenic substrate. The intrinsic epileptogenicity of CD tissue, manifested by continuous or rhythmic epileptogenic discharges (CEDs or REDs) recorded directly from the structural lesion, has been demonstrated using ECoG recordings 42. These types of discharges could be used as a diagnostic tool for CD 43. Stereo EEG confirmed a high correlation between REDs and an existing dysplastic lesion 20. Other EEG findings in CD include absence of background activity and repetitive, high‐amplitude fast spikes followed by high‐amplitude slow waves interspersed with flat periods 9. Abnormal EEG background is a common finding in CD that is multilobar or affects the posterior rather than the anterior brain in both children and adults 20. The predominant seizures observed during chronic EEG monitoring in CD are nocturnal as well as sleep‐related seizures 34. More recently, EEG high‐frequency oscillations (80–450 Hz) and fast ripples (250–500 Hz) have been used as localizing tools for CD 44, 45, 46 and also can be used as biomarkers for epileptogenic areas.

Architectural and Cellular Abnormalities in CD

Neuronal cortical growth takes place in a central to peripheral direction from the subventricular zone, and new neurons are sequentially added as new layers 47, 48, 49. This pattern continues until the formation of the six layers is completed, with each layer containing neurons of various shapes, sizes and density with different nerve fiber organization 1, 50, 51. The Reelin signaling pathway plays a crucial role in the laminar organization and orientation of radially migrating neurons 52. Reelin is a large extracellular matrix (ECM) protein 53, 54 produced by the transient Cajal–Retzius (C‐R) cells localized in the marginal zone of the developing cortex 50 and also later by some interneurons 55. Deficient Reelin signaling is the main cause of architectural malformation and polarity defects of cortical neurons 52. Interestingly, in CD types I and II, the density of C‐R cells is increased and can be used as a marker of these types of dysplasia 56. Furthermore, any disturbance in the ECM of the developing cortex can potentially interfere with the appropriate distribution of migrating neurons leading to supernumerary, misplaced neurons (Kostovic et al., 2014, this issue). Indeed, it has been demonstrated that the composition of the ECM of CD differs from that of nonmalformed tissue, mainly by the altered expression of certain ECM molecules and by the presence of an increased number of astrocytic processes 57.

In Vitro Electrophysiology of CD tissue

Pioneer work using slices in vitro demonstrated that CD tissue is highly sensitive to some proconvulsant drugs 78, 79. Treatment with 4‐aminopyridine (4‐AP), a K⁺ channel blocker that augments neurotransmitter release, leads to the generation of N‐methyl‐D‐aspartate (NMDA) receptor‐mediated ictal discharges as well as GABA and glutamate receptor‐mediated interictal discharges 80. GABA can increase the K⁺ concentration leading to initiation of seizure activity 81. In nondysplastic tissue treated with 4‐AP, ictal activity is not observed, and there is only periodic generation of interictal‐like events that are GABA mediated.

Studies have been carried out to evaluate the electrophysiological properties of normal and abnormal cells in CD. Visualization of individual brain cells is made possible by the use of infrared video microscopy in combination with differential interference contrast optics 10, 82. Use of this technology in conjunction with whole‐cell patch clamp recordings assists in more accurate characterization of different cell types and correlation with physiological function. Tissue from younger patients (<5 years of age) is optimal as there is less myelination and therefore better visualization. Biocytin is used to further characterize morphological features after electrophysiological studies.

Dysmorphic cytomegalic pyramidal neurons have abnormal passive membrane properties marked by larger cell capacitance, consistent with increased membrane area, longer time constant, and lower input resistance compared with normal‐appearing pyramidal neurons. The amplitude of macroscopic Ca²⁺ currents and Ca²⁺ influx also are larger in this type of neuron, leading to hyperexcitability 10, 73. They also have reduced sensitivity to Mg²⁺, suggesting NMDA receptors can be activated at more hyperpolarized membrane potentials 83. Changes in GABA_A receptor subunit composition have been demonstrated based on slower GABA current kinetics 15. However, the sensitivity of these cells to GABA is not changed in CD tissue compared to nondysplastic tissue 84. Similar to cytomegalic pyramidal neurons, cytomegalic GABAergic interneurons are hyperexcitable and display spontaneous membrane depolarizations and bursting activity 67.

The balloon cells, found in CD type IIb, are characterized by round cell bodies. These cells do not have voltage‐gated Na⁺ or Ca²⁺ currents. The input resistance is usually high, and there are no spontaneous synaptic currents 10, 73. These cells are not sensitive to application of excitatory amino acids, which points to lack of ability of these cells to be epileptogenic 10, 13, 85. Likewise, the “intermediate” cells observed in CD type IIb and TSC do not display active inward currents 73.

The electrophysiological properties of the misoriented pyramidal neurons and normal‐appearing pyramidal neurons with tortuous processes are normal in terms of capacitance, input resistance, and time constant 10. The neurons with immature characteristics have low membrane capacitance, high input resistance, short time constant, normal Na⁺ current but small Ca²⁺ currents, and abundance of GABA inputs 10.

Proposed Mechanisms of Epileptogenesis in CD

The exact mechanisms of epileptogenesis in pediatric CD are not yet fully understood, but they involve complex interactions among different cell types and are not simply due to an imbalance in glutamatergic/GABAergic neurotransmission 84. In addition, while some mechanisms of hyperexcitability are similar among CD types, there must be differences based on dissimilar histopathology. In CD type I, there are no dysmorphic cytomegalic neurons that could account for epileptogenic activity. Thus, other cellular types and mechanisms have to be invoked. In particular, dyslamination and the presence of immature, misoriented, and hypertrophic pyramidal neurons with or without tortuous processes could certainly lead to aberrant connectivity. In CD type II, the dysplastic neurons are postulated as essential pathogenetic elements 86 and the source of ictal discharges 20. Of the different dysplastic cells, cytomegalic pyramidal neurons are more likely to be epileptogenic, while balloon cells are not epileptogenic 85. On the other hand, the cytomegalic interneurons have been shown to be hyperexcitable and display spontaneous membrane depolarizations similar to paroxysmal depolarizing shifts, making them potential generators of epileptic activity 67.

Other Potential Mechanisms of Epileptogenesis in CD

While substantial strides have been made to understand the mechanisms of epileptogenicity in CD, many other potential factors remain underexplored. For example, upregulation of the voltage‐gated sodium channel Nav1.3 in CD type IIb could play a role in increased excitability of cytomegalic pyramidal neurons and interneurons 127. Electrotonic propagation via gap junctions also could contribute to epileptogenesis in CD tissue 128. Gap junctions play an essential role in migration, synchronization, and 4‐AP oscillatory activity 129, 130. Studies in pediatric CD tissue demonstrated a positive correlation between age and the presence of dye‐coupling 131. In addition, immature pyramidal neurons filled with biocytin frequently show coupling (personal observation).

The exact role of balloon cells in epileptogenesis in CD type IIb also remains obscure. Several studies have found evidence for increase in mechanisms leading to clearance of glutamate in areas that contain balloon cells, hence decreasing the spread of epileptogenic activity 118, 132. In that sense, balloon cells could play a protective or antiepileptic role. As mentioned earlier, balloon cells express EAAT2/GLT1 suggesting a role in glutamate buffering 118. In addition, subsets of balloon cells and astrocytes from CD type IIb tissue also display high expression of gap junction‐forming connexin 43, allowing spatial buffering of extracellular ions and neurotransmitters 133. However, if balloon cells, which also express VGLUT2, are able to release glutamate, they could in fact contribute to epileptogenesis 134. Finally, the enlargement and tortuosity of the ECS in CD type II may alter extrasynaptic volume transmission and thus might represent another factor contributing to CD epileptogenicity 57.

Conclusions and Future Perspectives

Cortical dysplasia offers a privileged glimpse into the immature albeit pathological brain. A better definition and classification of the different CD types has helped understand basic mechanisms. But despite advances in imaging, cellular electrophysiology, and molecular biology, the exact etiological and pathophysiological mechanisms of this debilitating disorder remain unclear.

Future studies are indicated to further investigate the underlying etiology of the different types of CD. Recently, it was reported that specimens from CD type IIb cases express the human papillomavirus type 16, primarily in balloon cells, suggesting an infectious etiology, but this finding requires confirmation 135. This observation is important as this virus is a potent activator of the mTOR pathway 136. Other studies have found the presence of cytomegalovirus infection in fetal brains 137, and interestingly, the virus showed higher tropism for stem cells/radial glial cells. On the other hand, a genetic predisposition to CD also is the focus of continuous research 138.

Identifying different types of CD lesions on MRI tends to be a challenge as some are not detectable due to their subtle nature. Improvement in imaging modalities to detect CD type I and type II lesions can help achieve better outcomes 38. In addition, although there are effective neuroimaging and surgical techniques to identify and control seizures in CD 24, the diagnosis and treatment of this disorder continues to pose a challenge and many cases remain underdiagnosed and not effectively treated. The poor response to AEDs could be accounted for by a combination of mechanisms. There is altered GABA function in pediatric CD which can cause depolarization, hyperexcitability, and alterations in intracellular chloride concentration, requiring other means to renormalize the cell electrophysiology rather than the use of AEDs that increase GABA function 15. There also is altered composition of GABA_A receptor subunits as well as levels of NKCCl and KCC2 in CD type II and TSC with increase in NKCCl and decrease in KCC2. Agents such as bumetanide that inhibit NKCCl may prove effective when added to the AED therapy and are the subject of ongoing studies 102, 139, 140. Animal studies have found promising results with combination therapy with barbiturates and bumetanide in neonatal rat brain with increased efficacy of GABAergic inhibition 141, 142 (see also studies by Khazipov et al. and Dzhala and Staley, this issue). Research is currently ongoing to evaluate the safety and efficacy of this combination therapy in human neonates. The result can certainly help tailor the treatment approach for an effective seizure control in patients suffering from CD and TSC and are nonresponsive to AEDs 102.

Use of rapamycin, an inhibitor of the mTOR pathway, has proved positive in decreasing epilepsy in mice 103, 143. Although effective, this medication is accompanied by a series of adverse effects that seriously limit its use 143. It will be important to evaluate the role of this class of medications for the treatment of CD, specifically those with more desirable side effect profile than rapamycin. Electrical stimulation of thalamic nuclei also showed promise in the modulation of paroxysmal activity in CD 144. Finally, the FDA approved recently a new treatment for epilepsy using a neurostimulator implant that serves to detect the onset of seizures and deliver electrical stimulation to terminate ictal discharges 145. This can lead to future research involving more precise stimulation techniques such as the use of optogenetics 146, 147.

Conflict of interest

The authors declare no conflict of interest.