Abnormal maturation of non-dysmorphic neurons in focal cortical dysplasia: Immunohistochemical considerations

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
  • 2.1. FCD surgical tissue preparation
  • 2.2. Normal-looking neurons
  • 2.3. Immunohistochemistry
  • 2.4. Double labeling
  • 2.5. Counts of immunopositive cells and their comparison between FCD subtypes
• 3. Results
• 4. Discussion
• Acknowledgements
• References
• Copyright

Abstract 

Aim

Dysmorphic neurons and balloon cells in focal cortical dysplasia (FCD) reportedly show immaturity and abnormal differentiation with neuronal and glial components. Although normal-looking neurons (NL-neurons) in FCD are major constituent elements, their biological characteristics have never been identified. The aim of this study was to investigate maturation of NL-neurons with the focus on neuronal developmental lineage.

Methods

Eighteen FCD surgical specimens and controls were examined immunohistochemically using the antibodies for nestin, mammalian achaete-scute complex homolog 1 (Mash1), prospero-related homeobox 1 (Prox1), neuron-specific beta-III tubulin (Tuj1) and microtubule-associated protein 2 (MAP2) of neuronal lineage, glutamic acid decarboxylase (GAD), calretinin (CR) and calbindin (CB) of interneuron markers, and glial fibrillary-acidic protein (GFAP) of glial cell marker. Additionally, we performed fluorescent-double staining with these markers, and semi-quantitative analysis.

Results

NL-neurons in FCD had both mature and immature components, without interneuron components. NL-neurons in FCD showed abnormal maturation with the combined expression of MAP2 and Mash1/Prox1. Prox1-containing cell distribution in the deep layer was different from that of Mash1-containing cells in the superficial area. The MAP2-containing cell concentration decreased in the order of type I-A, I-B, II-A and II-B, but the Tuj1-containing cell concentration increased.

Conclusion

These findings may reflect differences in neuronal function and expression timing in developmental stages. From the standpoint of molecular expression, abnormal maturation of NL-neurons may initiate synaptic dysfunction, resulting in intractable seizures of FCD.

Keywords: Focal cortical dysplasia, Maturation, Normal-looking neuron

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1. Introduction 

Focal cortical dysplasia (FCD) is recognized as the major cause of epilepsy in childhood. FCD was identified as a pathologic substrate associated with epilepsy in 1971.¹ The FCD pathology is characteristically no laminar formation of the cortex with disoriented and occasionally dysmorphic and/or balloon cells.², ³ FCD is pathologically divided into four subtypes.² Interestingly, each subtype has predominant location, mentality, epileptic condition, imaging and surgical outcome.⁴, ⁵ The most severe type might be type II-B, although it shows the best surgical outcome.⁴, ⁵

The FCD pathogenesis is thought to be mainly embryonic developmental insults and results in forming dysplastic lesions with abnormal neuronal migration and differentiation.⁶ Balloon cells in FCD type II-B are commonly identified with various stage markers for neuronal maturation; nestin, β-tubulin III (Tuj1), vimentin, and microtubule-associated protein 2 (MAP2), neurofilament, peripherin, and α-internexin.⁷ On the other hand, most balloon cells have the combination of neuronal and glial components, and expression of both MAP2 and glial fibrillary-acidic protein (GFAP).⁸ Dysmorphic cells reportedly express mammalian achaete-scute homolog-1 (Mash1),⁹ which is a helix loop-helix transcription factor expressed in progenitors.¹⁰ In the early embryonic brain, Mash1-containing cells are recognized in the neocortical subventricular zone, and at the next stage, prospero-related homeobox 1 (Prox1)-expressed cells can be fated to be a neuronal precursor.¹¹ These facts indicate that dysmorphic neurons and balloon cells in FCD remain immature or undifferentiated. Many normal-looking neurons (NL-neurons) are usually observable in FCD lesions, but occasionally dysmorphic neurons or balloon cells. In the present study, we attempted to confirm that NL-neurons in FCD were in the immature stages of neuronal development.

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2. Materials and methods 

2.1. FCD surgical tissue preparation 

Eighteen patients with drug-resistant epilepsy having FCD on imagings participated in this study. Their clinical features are summarized in Table 1. Studied samples were part of the tissues removed for therapeutic reasons after careful assessment of the epileptogenic areas, determined by analysis of seizure semiology, electroencephalography, magnetic resonance imaging, fluorodeoxyglucose positron emission tomography and ictal or interictal single photon emission computed tomography. After resection, hematoxylin and eosin (HE) and Klüver-Barrera (KB) stainings were done. The tissues diagnosed as FCD with no hippocampal sclerosis by two individual neuropathologists were divided into 4 groups by Palmini's classification; type I shows abnormal cortical lamination in the absence of dysmorphic neurons with giant or immature neurons (type I-B) or neither (type I-A), while type II has architectural abnormalities with dysmorphic neurons in the presence of balloon cells (type II-B) or in the absence of those cells (type II-A).² For age-matched controls, we used brain tissues of 5 healthy controls, who died suddenly and unexpectedly of pneumonia and traffic accidents at 1, 1.5, 6, 8 and 8.5 years of age, without epilepsy and neuropathological changes. Brain tissues of 23 and 29 weeks gestation were used to confirm nestin-, Tuj1-, Mash1- and Prox1-immunoreactivities. Additionally, we added 3 tuberous sclerosis complex and 4 hemimegalencephaly patients as disease controls. Informed consent to use the removed tissues for this study was obtained from patients and controls or their parents. This study was approved by the ethical committee of our institute and hospital.

Table 1. Clinicopathological profile of FCD patients.

Case	Sex	Age at surgery	Age at seizure onset	Seizure	Intelligence	FCD location on imaging	Palmini's classification	Other neuropathology	Immunohistochemistry of NL-neurons
									MAP2	GFAP	MBP	Nestin	Tuj1	Mash1	Prox1
1	F	8M	1m	CPS	47 (DQ)	P+O	I-A	n.s.c.	+	−	−	−	++	+	+
2	M	2Y	2m	CPS+GTC	100 (IQ)	P	I-A	Gliosis	++	−	−	−	+	+	−
3	F	3Y	3m	CPS	50 (DQ)	F	I-A	EN	++	−	−	−	+	+	−
4	M	6Y	4m	CPS	33 (DQ)	F	I-A	EN	++	−	−	−	+	+	+
5	M	7Y	11m	CPS	58 (IQ)	F	I-A	EN	++	−	−	−	++	+	+
6	M	6M	1m	CPS	40 (DQ)	P	I-B	EN, gliosis	−	−	−	−	++	++	+
7	M	2Y	20d	CPS	IS (DQ)	F	I-B	CAL, EN	++	−	−	−	+	−	+
8	F	3Y	3m	CPS	15 (DQ)	P	I-B	Gliosis	++	−	−	−	++	+	−
9	F	10Y	4m	CPS	45 (IQ)	F	I-B	EN, gliosis	++	−	−	−	+	+	−
10	F	12Y	11m	CPS	43 (IQ)	F	I-B	Gliosis	++	−	−	−	+	+	−
11	F	3Y	6m	CPS+GTC	40 (DQ)	F+P	II-A	CAL, EN, gliosis	+	−	−	−	++	+	−
12	F	5Y	2y 9m	CPS	81 (IQ)	F	II-A	Gliosis	++	−	−	−	+	+	+
13	M	6Y	3d	CPS	16 (DQ)	T+P+O	II-A	n.s.c.	+	−	−	−	++	+	−
14	M	7Y	7m	CPS	50 (IQ)	P	II-A	CAL, EN, gliosis	++	−	−	−	++	+	+
15	M	10Y	1y 11m	CPS+GTC	22 (IQ)	F+T+P	II-A	CAL, EN	++	−	−	−	++	+	+
16	M	3Y	2y 9m	CTC	15 (DQ)	T+P+O	II-B	CAL, EN	+	−	−	−	++	+	+
17	M	8Y	3m	CPS+GTC	30 (IQ)	F+P	II-B	CAL, EN, gliosis	+	−	−	−	++	+	−
18	F	19Y	9m	CPS+GTC	25 (IQ)	P	II-B	CAL. EN, gliosis	++	−	−	−	++	−	−
TSC 1	F	7M		CPS+GTC	65 (DQ)	Diffuse cerebral cortex		Cortical tuber	++	−	−	−	+	+	−
TSC 2	F	4Y		CPS+GTC	50 (DQ)	Diffuse cerebral cortex		Cortical tuber	++	−	−	−	+	+	−
TSC 3	M	21Y		CPS+GTC	Under normal	Diffuse cerebral cortex		Cortical tuber	++	−	−	−	+	−	−
HME 1	F	3M		CPS+GTC	30 (DQ)	rt-hemisphere		PMG, CAL, EN	++	−	−	−	+	+	−
HME 2	F	3M		CPS+GTC	35 (DQ)	rt-hemisphere		PMG, CAL, EN	++	−	−	−	+	+	−
HME 3	M	3M		CPS+GTC	30 (DQ)	lt-hemisphere		PMG, CAL, EN	++	−	−	−	+	−	−
HME 4	M	6M		CPS+GTC	50 (DQ)	lt-hemisphere		PMG, CAL, EN	++	−	−	−	−	−	–
Control		23–29GW		n.d.	n.d.	n.d.	No	n.s.c.	−	−	−	+	++	++	++
		1–12M		No	Normal	No	No	n.s.c.	+	−	−	−	+	−	−
		1–8Y		No	Normal	No	No	n.s.c.	++	−	−	−	−	−	−

M, male; F, female; y, year(s); m, month(s); d, days; CPS, complex partial seizure; GTC, generalized tonic-clonk seizure; IQ, intelligent quotient; DQ, developmental quotient; P, parietal lobe; F, frontal lobe; O, occipital lobe; T, temporal lobe; CAL, calcification(s); EN, ectopic neuron(s) in the white matter; n.d.: not described; n.s.c., no significant changes; PMG: polymicrogyria; ++, observed immunopositive neurons; +, few immunopositive neurons; −, no immunopositive neurons; TSC; tuberous sclerosis complex; HME, hemimegalencephaly.

2.2. Normal-looking neurons 

We defined normal-looking (NL) neurons with no giant cell, no dysmorphic neuron and no balloon cell, using HE and KB staining. Many NL-neurons were observed in a FCD lesion, although the characteristic marker cells of FCD were few in number in the lesion. NL-neurons were small and round, or showed pyramidal neuron-like features.

2.3. Immunohistochemistry 

The serial sections were deparaffinized, rehydrated and pre-treated in 0.3% H₂O₂ for 20min to remove endogeneous peroxidase activity. Heat-induced epitope retrieval was performed in sodium citrate buffer solution (pH 6.0) by warming up to 95°C in a microwave oven for 10min. Following blocking with 2% bovine albumin in phosphate-buffered saline (PBS), the sections were incubated at 4°C for overnight with the antibodies. We used monoclonal antibodies against MAP2 (dilution of 1:100; Sigma, St. Louis, MO), Tuj1 (1:1000; R&D Systems Inc., Minneapolis, MN), nestin (1:100; Chemicon International, Temecula, CA), glutamic acid dehydroxylase (GAD) (1:1000; Biomole International, Plymouth Meeting, PA), calretinin (CR) (1:1000; Sigma), calbindin (CB) (1:1000; Sigma) and GFAP (1:1000; Sigma), as well as the polyclonal antibodies against Prox1 (1:500; Sigma) and Mash1 (1:1000; kind gift of Dr. J.E. Johnson). As secondary antibodies, horseradish peroxidase-labeled mouse-and-rabbit-IgG sera were used (Nichirei, Tokyo, Japan). The antigen–antibody complex was visualized with aminoethyl carbazole (Nichirei, Tokyo, Japan). Slide-mounted sections were counterstained with 0.2% methyl green or hematoxylin. The stained sections were observed with a microscope (BX50; Olympus, Tokyo, Japan), digitized with a DXM1200F digital camera system (Nikon, Tokyo, Japan), and the neuronal developmental lineage of NL-neurons in FCD was evaluated.

2.4. Double labeling 

To confirm the relationship between the neuron and its immaturity, double-labeling observation was performed with MAP2, Mash1, Tuj1, GFAP and myelin basic protein (MBP; 1:200; DAKO Corporation, Carpinteria, CA), as well as GAD, CR and CB. Alexafluor-488- and -568-conjugated secondary antibodies (Invitrogen Corporation, Carlsbad, CA) were applied for 1h at room temperature, and sections were mounted in a VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA). The fluorescent-stained sections were examined under a fluorescent microscope system (BX51 and BH2-RFL-T; Olympus) and digitized by a DP70 system (Olympus).

2.5. Counts of immunopositive cells and their comparison between FCD subtypes 

Various immunopositivities of NL-neurons were observed in all cases, and the number of cells was counted in each of 5 fields at a magnification of 200 times. The number was corrected per 100 neurons in each case as the immunopositive cell concentration. Four subtypes were analyzed.

For statistical analysis, the ANOVA test was used for comparison between the subtypes. Post hoc comparisons were done with Fisher's protected least-significant difference test at a significance level of p<0.05.

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3. Results 

All FCD lesions showed abnormal lamination of the cortices and many ectopic neurons in the white matter (Fig. 1A). Our FCD series was composed of five type I-A, five type I-B, five type II-A and three type II-B. The immunohistochemistry is summarized in Table 1.

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• Fig. 1. 

  Immunohistochemistry of focal cortical dysplasia. In the FCD lesion of Case 15 (type II-A) in Table 1, cortical neurons are distributed at random (A). Large and dysmorphic neurons are mainly observed in the deep area (B). Tuj1-immunopositive premature neurons are within the same distribution of MAP2-immunopositive neurons (B and C). Interestingly, Mash1-immunopositive cells localize in the superficial area (D), whereas Prox1-immunopositive cells are in the deep area and white matter (E). Insets in C, D and E are large magnifications of each rectangular region. Cx, cortex; WM, white matter; A, Klüver–Barrera staining; B, MAP2-immunohistochemistry (IHC); C, Tuj1-IHC; D, Mash1-IHC; E, Prox1-IHC. Scale bar is 500μm.

In age-matched controls, MAP2- or Tuj1-containing cells were confirmed in the cortex, but Mash1-, Prox1- and nestin-containing cells were not (Table 1 and Fig. 2D1–D5, E1–E5). However, their immunopositivities were confirmed in fetal brains (Table 1 and Fig. 2C1–C5). On the contrary, all FCD patients had diffusely Tuj1- and MAP2-containing neurons in the cortices and white matter (Fig. 1, Fig. 2). Tuj1-containing neurons were fewer than those of MAP2-containing neurons. Mash1-containing neurons distributed in the superficial area of the cortex and white matter (Fig. 1D). Interestingly, Prox1-containing neurons distributed in the deep area of the cortex and white matter (Fig. 1E). Nestin-containing cells were never observed (Fig. 2A2). GAD-, CR- and CB-containing cells, which were interneurons, diffusely distributed in the cortex, and became fewer in number (data not shown) as previously reported.¹², ¹³

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• Fig. 2. 

  Immunohistochemistry of neocortices of various malformed and normal developing brains. Normal-looking neuron of focal cortical dysplasia (FCD) expresses MAP2 (A1), Tuj1 (A3), Mash1 (A4) and Prox1 (A5), but not Nestin (A2). In tuberous sclerosis (TSC), MAP2 is expressed (B1) and Tuj1 is faintly positive (B3, arrows), but Nestin, Mash1 and Prox1 are negative (B2, B4, and B5). In early fetus brain, Nestin (C2), Tuj1 (C3), Mash1 (C4, arrows) and Prox1 (C5, arrows) are positive immunoreactivities, whereas MAP2 is negative (C1). In 1-month-old brain, MAP2 (D1) and Tuj1 (D3) are faintly immunostained (arrows), whereas Nestin (D2), Mash1 (D4) and Prox1 (D5) are negative. In childhood, MAP2 (E1) is only immunopositive. Scale bars are 50μm.

From the double staining, FCD revealed MAP2-containing cells which had no glial features (Fig. 3A–F), but showed Mash1-immunoreactivity (Fig. 3G–I). Tuj1-containing cells also had Mash1-immunoreactivity (Fig. 3J–L). However, Mash1-containing cells did not have GAD-, CR- or CB-immunoreactivities (data not shown).

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• Fig. 3. 

  Normal-looking neurons in focal cortical dysplasia and their abnormal maturation. MAP2-containing cells have no glial components of astrocyte (A–C) or oligodendrocyte (D–F). MAP2-containing cells have Mash1-immunoreactivities (G–I). Many double-labeled cells with MAP2 and Mash1 are visible (G). Tuj1-containing cells have Mash1-immunoreactivities (J–L, arrows in L). All figures of the superficial area of Case 15 (type II-A) are shown in Table 1. A, D and G, MAP2; B, GFAP; C, merged figure of A and B; E, MBP; F, merged figure of D and E; H and K, Mash1; I, merged figure of G and H; J, Tuj1; L, merged figure of J and K. Scale bars indicate 25μm.

The number of immunopositive cells was estimated per 100 neurons after counting in 5 different fields. The numbers of MAP2-containing cells were 77.4±10.5 (mean±standard deviation) in control, 35.8±13.2 in type I-A, 23.0±10.1 in type I-B, 16.0±7.6 in type II-A, and 10.2±5.0 in type II-B (Fig. 4). The numbers of Tuj1-containing cells were 2.4±1.8 in control, 12.5±15.6 in type I-A, 13.1±9.4 in type I-B, 21.3±15.7 in type II-A, and 33.1±6.1 in type II-B. There were almost no Nestin-containing cells in all subtypes. The numbers of Mash1-containing cells were zero in control, 14.4±15.7 in type I-A, 12.3±7.4 in type I-B, 13.4±13.8 in type II-A, and 12.0±17.9 in type II-B. The numbers of Prox1-containing cells were 0.2±0.4 in control, 3.2±4.6 in type I-A, 1.1±1.2 in type I-B, 5.8±11.9 in type II-A, and 3.1±2.8 in type II-B. Statistical analysis revealed a significant difference of MAP2-containing cell concentration between each subtype (p<0.001 or p<0.05) and the Tuj1-containing cell concentration between types I and II (p<0.001). The Prox1-containing cell concentration showed a significant difference between types I-B and II-A (p<0.05). There were no differences of Mash1-containing cell concentration between each subtype.

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• Fig. 4. 

  Distribution of immunopositive cells in each subtype.

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4. Discussion 

Although the examined cases were few, our results indicated that NL-neurons in FCD had various developmental components with immature components, Mash1, Prox1 and Tuj1, but without interneuron components. MAP2- and Tuj1-containing cells were distributed differently in every FCD subtype, although MAP2-containing cell concentration significantly decreased in all subtypes, compared with control. This phenomenon may reflect pathogenetic differences between FCD subtypes. Type I-A, I-B, II-A and II-B show gradually severe clinical and pathological features.⁴, ⁵ In this order, the MAP2-containing cell concentration decreased, but the Tuj1-containing cell concentration increased. This may indicate that many NL-neurons in type II-B are immature.

Dysmorphic neurons in FCD have been described as immature and undifferentiated, having both neuronal and glial components.¹⁴ The focus of the present study was the maturation of NL-neurons in FCD. The biological characteristics of NL-neurons in FCD have not been described, although they are major FCD constituent elements. NL-neurons in FCD show morphologically neuronal immaturity or abnormal migration.¹⁵ Recent molecular neuroscience has explained mammalian neuronal lineage in detail.¹⁶ In the first stage of neuronal development, nestin expresses in undifferentiated neuroepithelial cells.¹⁷ As nestin, Mash1 also appears in neuronal progenitors in the neocortical ventricular zone.¹¹, ¹⁸ Prox1, which expresses in the neocortical subventricular zone, characterizes secondary precursor neurons.¹¹, ¹⁹ Both Mash1- and Prox1-containing cells are mitotically active and have transient proliferative potential.¹¹ After the disappearance of Mash1 and/or Prox1, Tuj1 (the earliest marker of postmitotic neuron) and MAP2 (the marker of mature neuron) express in the cortical plate. In the current study, Mash1 and Prox1 were never observed in age-matched controls. Thus, these transcriptional factors disappeared in the postnatal cortex. On the basis of our results, it may be speculated that NL-neurons in FCD can be characterized not only by their developmental immaturity but also by expression of neuronal mature-markers such as MAP2. In NL-neurons in FCD, both transcription factors may have irregular functions, while Mash1 and Prox1 downstream genes are currently unknown. Although Mash1 obviously induces a neuronal trait and plays an important role in neuronal maturation, further study is warranted to understand the molecular mechanism of the mixture of various developmental stage markers in NL-neurons in FCD. Moreover, type II-A has a relatively larger number of Prox1-containing cells than the other types. This may reflect neuronal undifferentiation and lead to neuronal dysmorphism.

Mash1 expresses in both projection neurons and interneurons. To clarify which type of neuron Mash1 expressed in FCD, we checked the colocalizations of Mash1 and MAP2/Tuj1, and Mash1 and GAD/CR/CB, interneuron markers. Mash1 colocalized with MAP2 and Tuj1, but not with interneuron markers. This indicates that Mash1-containing cells have immature projection neurons. One can speculate that Mash1-containing cells of FCD may be projection neurons and originate from neuronal precursors in the neocortical ventricular zone.

Interestingly, Prox1-labeled cells exist in the neocortical subventricular zone, the starting point for radial migration of predictable pyramidal neurons.¹¹ The Prox1-containing cell distribution in the deep area of the cortex and white matter, was different from that of Mash1-containing cells, predominantly in the superficial area of the cortex. This may reflect that both labeled cells have a different function and birth timing.

In summary, epileptogenesis of FCD has been discussed in terms of morphological abnormalities and dysfunction of synapse, as well as the dysregulation of neuronal inhibition associated with abnormal distribution of interneurons.²⁰, ²¹ Here, we provided data on direct evidence for the dysmaturation of NL-neurons in FCD from the standpoint of molecular expression. This maturational abnormality may initiate synaptic dysfunction, resulting in characteristic symptoms of FCD and intractable seizures.

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Acknowledgements 

We are indebted to Dr. J.E. Johnson, University of Texas Southwestern Medical Center for providing the antibody for Mash1, to Dr. A. Kakita, University of Niigata, for the pathological diagnosis, and to Mr. S. Kumagai, National Center of Neurology and Psychiatry, for technical assistance. This study was supported by grants from the Ministries of Health, Welfare and Labor, and Education, Culture, Sports, Science and Technology of Japan.

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