Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Dec 20.
Published in final edited form as: Epilepsia. 2010 Feb 3;51(8):1456–1467. doi: 10.1111/j.1528-1167.2009.02491.x

Impaired Maturation of Cortical GABAA Receptor Expression in Pediatric Epilepsy

Laura A Jansen 1,2, Lindsey D Peugh 1, William H Roden 1, Jeffrey G Ojemann 1,3
PMCID: PMC3243731  NIHMSID: NIHMS341252  PMID: 20132297

SUMMARY

Purpose

Expression of the protein subunits that make up the GABAA receptor pentamer is known to change during postnatal brain development in animal models. In the present study, analysis of cortical GABAA subunit expression was performed in control human tissue obtained from infancy through adolescence, and was compared to that from similarly aged children with intractable focal epilepsy.

Methods

Twenty frozen pediatric control and 25 epileptic neocortical specimens were collected. The membrane fractions were isolated and subjected to quantitative Western blot analysis. Subunit expression was correlated with clinical factors including age, pathology, and medication exposure.

Results

In control cortical samples, α1 and γ2 GABAA receptor subunits exhibited low expression in infancy which increased over the first several years of life and then stabilized through adolescence. In contrast, α4 subunit expression was higher in infants than in older children. The level of the chloride transporter KCC2 increased markedly with age, while that of NKCC1 decreased. These patterns were absent in the epileptic children, both in those with focal cortical dysplasia and in those with cortical gliosis. While there was marked variability in GABAA receptor subunit expression amongst the epileptic children, identifiable patterns of subunit expression were found in each individual child.

Discussion

Maturation of cortical GABAA receptor subunit expression continues over the first several years of postnatal human development. Intractable focal epilepsy in children is associated with disruption of this normal developmental pattern. These findings have significant implications for the treatment of children with medications that modulate GABAA receptor function.

Keywords: GABAA Receptor, Epilepsy, Development, Focal Cortical Dysplasia, Human, Cerebral Cortex

Introduction

In the adult, γ-Aminobutyric Acid (GABA) mediates the majority of fast inhibitory neurotransmission in the central nervous system through its activation of the GABAA receptor. GABAA receptors are heteropentameric ligand-gated ion channels that can be composed from 6 α, 3 β, 4 γ, δ, ε, π, θ, and 3 ρ subunits (Jones-Davis and Macdonald, 2003). In addition to the traditional “phasic” form of fast inhibition mediated by synaptic GABAA receptors, primarily of the α1β2/3γ2 composition, extrasynaptic “tonic” GABAA currents also serve to regulate neuronal excitability. The tonic GABAA current is a continuous inhibitory current mediated by high-affinity, extrasynaptic GABAA receptors that are activated by low ambient levels of GABA. Based on rodent and human studies, tonic GABAA current in the neocortex is expected to be mediated primarily by receptors of the α4β2/3δ and α5β2/3γ2 subunit compositions (Farrant and Nusser, 2005; Glykys and Mody, 2007).

The subunit composition of neuronal GABAA receptors changes during prenatal and postnatal development. This has significant implications for the use of GABAergic pharmacologic agents in the treatment of epilepsy in infants and children. Developmental regulation of GABAA receptor subunit expression has been well studied in rat neocortex. While there is significant variability amongst the findings of investigators using different technical approaches, the consensus indicates that α1 subunit expression increases from birth through around postnatal day 30 (Laurie et al., 1992; Fritschy et al., 1994; Roberts and Kellogg, 2000; Yu et al., 2006). This change is associated with an increase in high-affinity binding of the α1 selective agonist zolpidem (Roberts and Kellogg, 2000) and an increase in the rate of GABAA current decay (Dunning et al., 1999). In contrast, expression of α2 and α5 subunits appears to be lower in the adult than in the newborn rat cortex (Laurie et al., 1992; Fritschy et al., 1994; Yu et al., 2006). In the few studies that have been reported investigating GABAA receptor subunit expression in cortical neurons during postnatal human development, increased α1 expression has also been seen in the adult as compared with the infant, along with an increase in benzodiazepine binding (Reichelt et al., 1991; Brooks-Kayal and Pritchett, 1993; Andersen et al., 2002; Kanaumi et al., 2006). However, the time course of this increase in α1 expression and the expression of other key GABAA receptor subunits in postnatal human development have not previously been defined.

In mature neurons, GABAA receptor channel opening allows influx of Cl−, leading to membrane hyperpolarization and decreased excitability. In contrast, due to elevated intracellular Cl− concentrations, GABAA receptor activation in immature neurons instead produces efflux of Cl− and membrane depolarization (Ben-Ari, 2002; Dzhala et al., 2005). This excitatory action of GABA has a number of critically important roles in brain development, including regulation of neuronal migration and synapse formation (Ben-Ari et al., 2007). The increased intracellular Cl− concentration in immature neurons is due to relatively high levels of the NKCC1 sodium-potassium-chloride cotransporter, which pumps chloride into the cell, as compared with the KCC2 potassium-chloride cotransporter, which pumps chloride out of the cell. This developmental switch has been demonstrated in both rat and human cortex (Dzhala et al., 2005), although its precise time course in humans has not been yet been thoroughly investigated.

In the present study, we examine protein expression of the predominant subunits mediating phasic (α1, γ2) and tonic (α4, α5, δ) GABAA receptor currents in human cortex during postnatal development. Expression of the chloride transporters NKCC1 and KCC2 was also investigated. These results were then compared with those from similarly aged children with intractable epilepsy due to focal cortical dysplasia (FCD) or cortical gliosis. Finally, correlation was sought between GABAA subunit expression patterns in the epileptic children and clinical variables.

Methods

Pediatric cortical brain specimens

Children with intractable focal epilepsy were evaluated and underwent brain surgery at Seattle Children’s Hospital in Seattle, WA. Informed consent for the use of a portion of the resected tissue for research purposes was obtained from each subject or their legal guardian under the guidance of the hospital’s Institutional Review Board. After excision, the fresh tissue was frozen in liquid nitrogen and stored at −80° C. Control frozen autoptic brain tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland.

Western Blot Analysis

Rabbit polyclonal antibodies against human GABAA receptor subunits α1, α4, α5, δ, and γ2 were obtained from Novus Biologicals (Littleton, CO). NKCC1 antibody was obtained from Aviva Systems Biology (San Diego, CA), KCC2 antibody was obtained from Abcam (Cambridge, MA), and GFAP antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Frozen cortical tissue (100–200 mg) was separated from underlying white matter and homogenized in 5 mM Tris/HCl (pH 7.4) with 0.32 M sucrose. The homogenates were centrifuged at 3000×g for 5 min at 4 °C. The supernatant was then centrifuged at 40,000×g for 1 h at 4 °C. The resultant membrane pellets were resuspended in 50 mM Tris/HCl (pH 7.4), using 100 μL per 100 mg original tissue weight. Protein concentrations were determined using a Bradford protein assay. For individual Western blot experiments, 30 or 60 μg of protein per lane was electrophoretically separated and transferred to an Immobilon-FL PVDF membrane (Millipore, Billerica, MA). The membrane was blocked with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE), incubated in primary antibodies overnight at 4 °C, and then incubated in goat anti-rabbit infrared dye-labeled secondary antibody (Alexa Fluor 680, Molecular Probes, Inc., Eugene, OR) for 2 h at room temperature. Infrared fluorescence was used for signal detection and quantitation (Odyssey Infrared Imaging System, LI-COR Biosciences). Binding of mouse monoclonal antibodies against β-tubulin (Novus Biologicals), visualized with IRDye 800 goat anti-mouse IgG (LI-COR), was used as a loading control.

Data analysis

Fluorescence intensity was measured for each band of interest using Odyssey Software Version 2.1 (LI-COR) after subtraction of background fluorescence. The values obtained from each lane were then corrected for protein loading based on β-tubulin expression. Protein levels were graphed relative to the expression of a simultaneously measured reference sample on each blot. Graphing and curve-fitting were carried out using Excel (Microsoft) and Origin 8 software (OriginLab Corporation, Northampton, MA). Statistical analysis was performed using InStat (GraphPad Software, Inc., San Diego, CA).

Results

Cortical Specimens

Frozen postmortem control cortical tissue was obtained from 20 children aged 6 weeks to 20 years with non-neurologic causes of death. The postmortem interval prior to freezing averaged 6.0 hours (range 1–15 hours). One control specimen consisted of normal frontal cortex unavoidably resected in the course of craniopharyngioma surgery. Table 1 lists the available clinical details for each control specimen.

Table 1.

Control Specimens

Research Number Age (years) Sex Brain Region Cause of Death PMI (hours)
M2112 0.1 M Occipital Tricuspid Atresia 2
U227 0.1 F Temporal SIDS 5
U4589 0.4 M Occipital Spinal Muscular Atrophy, Type 1 6
U4629 0.5 M Parietal Spinal Muscular Atrophy, Type 1 3
U1325 0.5 F Occipital SIDS 1
U1281 0.6 M Occipital SIDS 6
U1897 0.9 M Occipital Spinal Muscular Atrophy, Type 1 3
U1547 1.7 M Parietal Asthma 10
U1864 2.5 F Temporal Laryngitis & Bronchiolitis 8
U1791 2.8 F Temporal Drowning 12
U446 3.9 F Temporal Chondrodysplasia Punctata 1
439 4.0 M Frontal n/a (Craniopharyngioma surgery) n/a
U4907 4.8 F Frontal Asthma 15
U4898 7.7 M Occipital Drowning 12
U1860 8.0 M Temporal Cardiac Arrhythmia 5
U4787 12.9 M Occipital Asthma 15
U1670 13.3 M Occipital Asphyxia by hanging 5
U4638 15.1 F Temporal Chest injuries 5
U1465 17.5 M Frontal Multiple injuries-hit and run 4
U1475 20.3 M Occipital Multiple injuries-car accident 3
Open in a new tab

Frozen cortical tissue was also collected from 25 children aged 3 months to 17 years with intractable epilepsy due to focal cortical dysplasia (FCD) or cortical gliosis, a subset of those who underwent resective surgery at Seattle Children’s Hospital between 2004 and 2009. Table 2 and Supplemental Table 1 contain clinical details for each specimen, including age at surgery, age of seizure onset, brain region analyzed, results of pathologic analysis, seizure frequency and types, anticonvulsant drug exposure at the time of surgery, previous anticonvulsant drug exposure, and surgical outcome. The tissue selected for analysis corresponded to the area of greatest abnormality on pre- or intra-operative EEG recording and pathologic analysis. Sixteen of the resections had pathologic diagnoses of FCD, which are areas of abnormal cortical development divided into four subtypes based on the classification of Palmini et al. (Palmini et al., 2004): FCD 1A, isolated architectural abnormalities such as dyslamination and columnar disorganization; FCD 1B, architectural abnormalities plus giant or immature neurons; FCD 2A, architectural abnormalities with dysmorphic neurons but without balloon cells; and FCD 2B, architectural abnormalities with dysmorphic neurons and balloon cells, which are developmentally immature cells with both neuronal and glial characteristics. The majority of the FCD specimens analyzed in this study (11 of 16) were of the FCD 2A subtype (Table 2). Nine of the resected tissues analyzed had pathologic diagnoses of cortical gliosis due to previous insults such as tumor surgery, stroke, traumatic brain injury, or mesial temporal sclerosis (Supplemental Table 1). Most of the children in this series had multiple daily seizures and had failed to respond to numerous anticonvulsant medications, and surgical outcomes were generally quite good, with 19 of the 25 (76%) becoming seizure-free after surgery.

Table 2.

Epilepsy Focal Cortical Dysplasia Specimens

Research Number Age at Surgery (years) Age of Onset (years) Sex Brain Region FCD Subtype EEG Sz. Freq. Seizure Types AED at Surgery Previous AED Engel Class
427 0.3 0.01 M Temporal 2A >90/day MYO, TON, IS LEV, CLZ, ZON, PHB, MDZ ACTH I
426 0.6 0.2 F Temporal 2A >20/day CPS, IS PHB, ZON, DIAZ ACTH, CBZ, TPM I
428 0.7 0.3 F Parietal 2A 3/day IS LEV, ZON, LTG ACTH I
404 1.7 0.3 M Temporal 2A >40/day CPS, A-ABS, GTC LEV, PHB, ZON, PHT CBZ, TPM, KETO I
429 3.6 1.2 M Temporal 2A >50/day ATON, TON, IS TPM, VPA ZON, ACTH, DIAZ, LTG III
441 5.3 3 F Occipital 2A 10/hour CPS LTG, OXC ZON, CBZ I
483 6.4 4.4 F Frontal 2B >20/day CPS LEV, TPM CBZ, ZON, LTG, VPA, CLZ, DIAZ, PHT I
533 7.5 6.2 F Frontal 2A 2–7/wk CPS ZON, LEV CBZ I
437 8.0 4 F Temporal 2A 4–5/day CPS, GTC LTG, OXC, DIAZ, LRZ ZON, PHB, PHT, GBP, MDZ II
484 8.9 3 M Frontal 2B 2/week CPS TPM, CLR, ETHO OXC, LEV, LTG, ZON, VPA, LRZ, CBZ, PREG IV
419 9.0 0.8 F Temporal 1B 2–7/day CPS, SPS OXC, VPA LEV, PHT, PHB, CBZ, GBP, TPM I
457 9.4 2.4 F Frontal 2B 3–10/hr GTC, SPS VPA, PHT, MDZ TPM, CBZ, DIAZ, TPM, LTG, LEV, LRZ I
518 10.4 8.7 F Temporal 1A 8/wk SPS, CPS OXC, LTG, DIA LEV III
502 10.8 1.2 M Temporal 2A 1–4/day CPS, GTC ZON, LTG, CLZ PHB, OXC, VPA, CBZ, DIAZ, IVIG I
540 14.2 9.2 F Temporal 2A 1/day CPS, GTC LEV, OXC, LTG TPM I
450 16.0 9.4 M Temporal 2A 7/wk CPS, SPS CBZ, LEV PHT, OXC I
Open in a new tab

Abbreviations for Table 2 and Supplemental Table 1: AED = antiepileptic drug; A-ABS = atypical absence seizures; MYO = myoclonic seizures; ATON = atonic seizures; CLON = clonic seizures; TON = tonic seizures; GTC = generalized tonic-clonic seizures; IS = infantile spasms; SPS = simple partial seizures; CPS = complex partial seizures; MTS = mesial temporal sclerosis; ACTH = adrenocorticotropic hormone; CBZ = carbamazepine; CEL = celontin; CLR = clorazepate; CLZ = clonazepam; DIA = diamox; DIAZ = diazepam; ETH = ethosuximide; ETHO = ethotoin; FLB = felbamate; GBP = gabapentin; IVIG = intravenous immunoglobulin; KETO = ketogenic diet; LEV = levetiracetam; LRZ = lorazepam; LTG = lamotrigine; MDZ = midozolam; OXC = oxcarbazepine; PHB = phenobarbital; PHT = phenytoin; PREG = pregabalin; PYR = pyridoxine; TPM = topiramate; VGT = vigabatrin; VNS = vagal nerve stimulator; VPA = valproic acid; ZON = zonisamide. Engel Class I = seizure free; Class II = > 85% reduction in seizures; Class III = > 50% reduction in seizures; Class IV = no significant improvement.(Engel, 1993)

GABAA Receptor Subunit Expression in Normal Postnatal Human Development

The protein expression level of several GABAA receptor subunits was analyzed by quantitative Western blotting of cortical membranes from the control autopsy specimens. The α1, α4, α5, δ, and γ2 subunits were selected for analysis because they are the predominant ones contributing to cortical phasic (α1β2/3γ2) and tonic (α4β2/3δ, α5β2/3γ2) GABAA receptor currents (Glykys and Mody, 2007). Details regarding the specific antibodies used are provided in Supplemental Table 2, including suppliers, catalog numbers, dilutions, and measured and expected molecular weights of the bands of interest. Figure 1A shows a representative control blot demonstrating lower α1 and γ2 expression in infants as compared with older children and adolescents. Expression of the α4 subunit was high early in infancy and decreased with age (Fig. 1B). In contrast, the α5 and δ subunits did not show a clear age-related expression pattern (Fig. 1C).

Figure 1.

Figure 1

Open in a new tab

GABAA receptor alpha 1 and gamma 2 subunit expression increases with age in control human cortical samples, while alpha 4 subunit expression decreases with age. (A, B) Representative Western blots of control cortical membranes from children aged 0.1 to 20.3 years probed with antibodies against the GABAA alpha 1, gamma 2, or alpha 4 subunits followed by fluorescent secondary antibody. Antibody staining of β-tubulin is shown as a control for protein loading. (C) GABAA receptor alpha 5 and delta subunit expression does not change significantly with age in control human cortical samples, as shown in this representative Western blot.

In Figure 2, the average quantitated expression levels of the developmentally-regulated α1, γ2, and α4 subunits are plotted against age for each of the twenty control cortical samples analyzed. The expression of the α1 and γ2 subunits increases logarithmically over the first 5 to 6 years of life, and then plateaus through adolescence (Fig. 2A, C). Conversely, expression of the α4 subunit decreases over the first several years of life before leveling off (Fig. 2E). Analysis of α1, γ2, and α4 cortical expression levels during the first 5 years of life plotted on a semilogarithmic scale definitively demonstrates the significant relationship present during this period (Fig. 2B, D, F).

Figure 2.

Figure 2

Open in a new tab

Human cortical GABAA receptor alpha 1 and gamma 2 subunit expression continues to increase over the first 5 to 6 years of life before reaching a plateau. Alpha 4 subunit expression decreases during this same time period. The panels on the left depict the relative alpha 1 (A), gamma 2 (C), and alpha 4 (E) expression levels for each of the 20 control cortical specimens analyzed as a function of age. The graphs in panels (B), (D), and (F) expand the data from the first 5 years of life by plotting subunit expression versus the logarithm of age, which was then subjected to linear regression analysis. The Pearson correlation coefficients (R) and associated P values are shown. Each point represents the average of the fluorescence intensities of the bands of interest from each specimen expressed relative to the intensity of a simultaneously measured sample from an 8 year old control subject after correcting for differences in protein loading as determined by β-tubulin expression. Data were obtained from 16 separate Western blots performed using different combinations of the 20 control specimens.

Chloride Transporter Expression in Normal Postnatal Human Development

It has been shown that expression of NKCC1, which transports chloride into the cell, is highest in human cortex around the time of birth, while that of KCC2, which extrudes chloride from the cell, is low at birth and increases into adulthood (Dzhala et al., 2005). These changes in relative NKCC1 and KCC2 levels result in the transition from excitatory, depolarizing GABAergic responses in immature neurons to inhibitory, hyperpolarizing responses in mature neurons (Ben-Ari, 2002). We sought to replicate these results in our control human cortical samples and to better define the time course of this developmental switch. Blots using the KCC2 antibody routinely demonstrated strong labeling of two separate bands, one at the expected monomer size of approx. 130 kDa, and another of >250 kDa, which represents oligomers of KCC2. The mature, functional form of KCC2 is oligomeric, which is detected along with the monomeric form in Western blots using standard detergents and sulfhydryl-reducing agents (Ludwig et al., 2003; Blaesse et al., 2006; Aronica et al., 2007). As shown in a representative Western blot (Fig. 3A) and quantitatively (Fig. 3B), the ratio of NKCC1 to KCC2 expression is very high in the youngest infants, decreases rapidly until around 2 years of age, and then remains low into adulthood. These results suggest that in humans, immature excitatory GABAergic responses may persist in cortical neurons through the first two years of life.

Figure 3.

Figure 3

Open in a new tab

The relative expression of the chloride transporters NKCC1 and KCC2 changes dramatically with age in control human cortical samples. (A) Representative Western blot of control cortical membranes from children aged 0.1 to 20.3 years probed with antibodies against NKCC1 or KCC2 followed by fluorescent secondary antibody. KCC2 appears as a doublet consisting of monomer and dimer forms. (B) The ratio of NKCC1 to KCC2 expression is plotted as a function of age for each of the 20 analyzed control cortical specimens. Data were obtained from 14 separate Western blots performed using different combinations of the 20 control specimens.

GABAA Receptor Subunit Expression in Intractable Pediatric Epilepsy

We next sought to determine if the observed developmental maturation of GABAA receptor subunit expression observed in control pediatric cortex was also present in resected epileptogenic cortex. The first group analyzed had pathologic diagnoses of focal cortical dysplasia (FCD). As shown by representative Western blots (Fig. 4A, B) and summary data from all 16 FCD specimens analyzed (Fig. 4C, D), no age-related pattern of GABAAα 1, γ2, α5, α4, or δ subunit expression was found. In contrast to the control specimens, some of the youngest infants with FCD had high levels of α1 and γ2 expression, while some older children had high levels of α4 expression. In several of the older children, α1 and γ2 expression were barely detectable. However, clear patterns of coordinated subunit expression were seen for each individual patient, with concordance between levels of α1, γ2, and α5, as well as between α4 and δ (Fig. 4C, D). The strong linear relationships between α1 and γ2, α5 and γ2, and α4 and δ expression are demonstrated graphically in Fig. 4E, F, and G. A significant linear correlation was also found in our control subjects between α1 and γ2 levels, as well as between α4 and δ (data not shown).

Figure 4.

Figure 4

Open in a new tab

GABAA receptor subunit expression in pediatric epilepsy due to focal cortical dysplasia does not display the age-related progression seen in control cortex, but instead reveals coordinated expression patterns unique to each patient. (A, B) Representative Western blots of cortical membranes from children with FCD aged 0.3 to 16.0 years probed with antibodies against GABAA alpha 1, gamma 2, or alpha 5 subunits (A) or against the alpha 4 or delta subunits (B) followed by fluorescent secondary antibody. (C, D) Plots of relative GABAA receptor subunit expression versus age for each of the 16 FCD specimens analyzed. The FCD subtype of each specimen is indicated on the top axis. Each point represents the average of the fluorescence intensities of the bands of interest from each specimen expressed relative to the intensity of a simultaneously measured sample from an 8 year old subject with FCD after correcting for differences in protein loading as determined by β-tubulin expression. Data were obtained from 16 separate Western blots performed using different combinations of the 16 FCD specimens. (E, F, G) Relative expression levels are plotted for alpha 1 versus gamma 2 (E), alpha 5 versus gamma 2 (F), and alpha 4 versus delta (G) for each of the FCD specimens analyzed, which were then subjected to linear regression analysis. The Pearson correlation coefficients (r) and associated P values are shown.

We assessed whether the loss of the normal pattern of developmental maturation was unique to FCD by also analyzing resected epileptogenic cortical specimens with pathologic diagnoses of gliosis. Significant non-age related variability in GABAA receptor subunit expression was also found in these patients, although to a somewhat lesser extent than in the FCD group. Individual coordinated subunit expression patterns were again seen, with a significant linear correlation between α1 and γ2 expression (Supplemental Figure 1).

One possible explanation for variations in subunit expression is the relative degree of neuronal loss and corresponding gliosis in each specimen, as GABAA receptors have been demonstrated in astrocytes in addition to neurons (Fraser et al., 1995). We addressed this possibility by assessing the expression of glial fibrillary acidic protein (GFAP) in the cytosolic fractions isolated from our control, FCD, and gliosis specimens (Supplemental Figure 2). The level of GFAP expression was not correlated with the expression of α1 or any other subunit analyzed, indicating that epilepsy-related astrocyte proliferation does not explain the GABAA receptor subunit expression results seen in the epileptic specimens.

Chloride Transporter Expression in Intractable Pediatric Epilepsy

As shown above, expression of the chloride transporters NKCC1 and KCC2 is normally developmentally regulated, and their relative levels may determine whether activation of neuronal GABAA receptors has excitatory or inhibitory consequences. Similar to what was seen with the GABAA receptor subunits, the normal developmental patterns of NKCC1 and KCC2 expression were absent from the epileptic specimens. Some of the young infants exhibited relatively high KCC2 and low NKCC1 expression, more characteristic of normal mature cortex, while some older children demonstrated very low KCC2 or high NKCC1 levels, more characteristic of normal immature cortex (Fig. 5A, B, C). Since NKCC1 is present in cerebral astrocytes as well as in neurons (Chen and Sun, 2005), and its expression levels thereby influenced by the degree of cortical astrocytosis, NKCC1 levels were also compared with that of GFAP and were not found to be significantly correlated (P = 0.24).

Figure 5.

Figure 5

Open in a new tab

Chloride transporter expression in pediatric epilepsy due to focal cortical dysplasia does not display the age-related progression seen in control cortex. (A) Representative Western blot of cortical membranes from children with FCD aged 0.3 to 16.0 years probed with antibodies against NKCC1 or KCC2 followed by fluorescent secondary antibody. (B, C) Plots of relative NKCC1 (B) and KCC2 (C) expression versus age for each of the FCD specimens analyzed. The FCD subtype of each specimen is indicated on the top axis. Each point represents the average of the fluorescence intensities of the bands of interest from each specimen expressed relative to the intensity of a simultaneously measured sample from an 8 year old subject with FCD after correcting for differences in protein loading as determined by β-tubulin expression. Data were obtained from 10 separate Western blots performed using different combinations of the FCD specimens.

Correlation of GABAA receptor subunit and chloride transporter expression in FCD with clinical factors

We next questioned whether clinical variables that have been shown to modulate receptor or transporter expression in experimental epilepsy models might play a significant role in our results. We investigated the possible contributions of age, seizure frequency, duration of epilepsy, exposure to benzodiazepines or barbiturates at the time of surgery, patient sex, brain region (temporal versus extratemporal), and FCD subtype using multiple linear regression analysis. Given the highly correlated receptor subunit expression patterns in the individual epilepsy specimens demonstrated above, the relationship of clinical factors with α1 expression as a representation of α1/α5/γ2 levels was assessed, while correlation with α4 was assessed as a representation of α4/δ levels. None of these clinical variables alone or in combination were found to be significantly related to GABAAα 1, GABAAα 4, or KCC2 expression levels in our samples (P > 0.05).

Visual assessment of the data from Fig. 4C made apparent that the epileptic specimens with the highest α1/γ2/α5 expression were either very young or of the FCD 2B subtype. Therefore, the multiple regression analysis described above was repeated after converting the “age” and “FCD subtype” variables to the single binary variable “age less than one year or FCD 2B” versus “age greater than one year, not FCD 2B”. In that analysis, the binary variable combining age and FCD subtype was significantly associated with both α1 (P = 0.02) and α4 (P = 0.01) expression levels. When expression of these subunits was assessed in direct comparison to age-matched children without epilepsy, it became clear that in the very young and in the FCD 2B specimens, α1 and γ2 expression was similar to or higher than in controls, while in older FCD type 1 and 2A specimens, α1 and γ2 expression was substantially reduced compared to controls (Fig. 6A–D). Specifically, α1 or γ2 levels were less than 75% of age matched controls in 7 of 8 non-2B specimens from children older than one year of age, in comparison to 0 of 3 non-2B specimens from children less than one year of age and 0 of 3 FCD 2B specimens. A similar partitioning was seen in KCC2 levels, with decreased expression in older non-2B samples and increased or unchanged expression in the very young and in the FCD 2B samples (Fig. 6A–D). In contrast, α4 and δ subunit expression levels were elevated in all subtypes of epileptic FCD specimens as compared with age-matched controls (Fig. 6A–D). This elevation was most pronounced in the older non-2B specimens.

Figure 6.

Figure 6

Open in a new tab

GABAA receptor subunit and chloride transporter expression in pediatric FCD as compared with age-matched controls. (A) Graph of average protein expression levels for GABAA receptor subunits and chloride transporters ± SEM as a percentage of simultaneously measured age-matched controls. Shown are averages from infants < 1 yr with FCD 2A (n = 3), children aged > 1 yr with FCD types 1 or 2A (n = 8), and children with FCD 2B (n = 3). *P < 0.05, one-way ANOVA with post hoc Tukey’s test. (B, C, D) Representative Western blots of cortical membranes from epileptic children with FCD run simultaneously with age-matched control samples.

Discussion

Our study demonstrates pronounced changes in cortical GABAA receptor subunit expression over the first several years of postnatal human development. These changes coincide with a developmental switch in the expression of the neuronal chloride transporters NKCC1 and KCC2. Based on this data, expression of synaptic α1β2/3γ2 GABAA receptors is expected to be very low in infants and young children, while that of extrasynaptic α4β2/3δ GABAA receptors is expected to be relatively high. This would lead to increased influence of tonic GABAA receptor currents over phasic currents, similar to the situation in migrating embryonic cortical neurons (Ma and Barker, 1995; Owens et al., 1999). Furthermore, the predominance of NKCC1 over KCC2 neuronal chloride transporter expression in this same age group may result in elevated intracellular chloride concentrations and depolarizing responses to GABAA receptor activation. These findings have substantial implications for the treatment of seizures or sedation in infants and young children with GABAergic agents such as benzodiazepines or barbiturates, which may be less effective or even counterproductive in this age group as compared with older children and adults (Painter et al., 1999; Silverstein et al., 2008).

Caution should however be exercised in attempting to translate our experimental data to clinical practice. Determination of GABAA subunit and chloride transporter expression was done using cortical homogenates from autopsy specimens and therefore does not provide cell- or network-specific information. Perimortem and postmortem factors may also have had unanticipated effects. We tried to minimize this by choosing specimens from children with non-neurologic causes of death and by using tissue with the shortest available postmortem intervals. There was no significant correlation found between expression of any of the proteins analyzed and postmortem interval (data not shown). We were also able to obtain a control surgical specimen from a child undergoing tumor surgery who was nearly exactly age-matched with one of our autopsy specimens, and found no significant difference when comparing expression levels of the five GABAA receptor subunits and the two chloride transporters between the two (P > 0.05, two-tailed paired t-test).

Postnatal structural development of the human brain shows regional differences, as synaptic density does not reach peak values in frontal cortex until over a year of age, while peak values in occipital and temporal cortex are reached by 6 months (Huttenlocher and Dabholkar, 1997). We chose our control samples from multiple cortical regions rather than a single one to better reflect which areas were obtained in the epilepsy samples. However, we did not detect a significant correlation between cortical region and subunit or transporter expression levels in our control samples (data not shown). The vast majority of both our control and epileptic samples were from more posterior areas (occipital, temporal, and parietal cortex) as compared with frontal cortex (17/20 control; 19/25 epileptic), and all samples of less than 4 years of age were from these more posterior regions, so that spatial developmental factors are less likely to have been prominent. Therefore, despite limitations inherent to using human tissue, the robust age-related trends demonstrated here are likely to have clinical implications, and emphasize the importance of the consideration of postnatal developmental changes in the study of any pediatric neurologic condition.

The next important finding demonstrated here is the distinct absence of the normal developmental maturation of GABAA receptor subunit and chloride transporter expression patterns in epileptogenic cortex resected from children with intractable epilepsy. The non-age related variation in expression levels was most prominent in resections containing areas of focal cortical dysplasia. Despite the marked inter-patient variability, the highly correlated expression of the α1, α5, and γ2 subunits in individual patients, in addition to the correlation between expression of the α4 and δ subunits, was quite pronounced. This makes functional sense, in that the α1 and α5 subunits generally pair with the γ2 subunit, while the α4 subunit pairs with δ. Moreover, these correlated expression patterns could lead to relative over- or underproduction of GABAA receptors that are sensitive to enhancement by benzodiazepines (α1β2/3γ2, α5β2/3γ2) versus those that are insensitive to benzodiazepines but more sensitive to enhancement by neurosteroids or increases in ambient GABA levels (α4β2/3δ). Work by others has demonstrated significant inter-patient variability in GABAA receptor subunit mRNA expression in hippocampal dentate granule cells (DGC) isolated from children with intractable epilepsy. A strong correlation between α1 and γ2 levels was also seen in that study (Porter et al., 2005), while GABAAα 4 and δ subunit levels were coordinately regulated in adult DGC (Brooks-Kayal et al., 1999). Combined with our current results, this suggests that at least some of the factors that regulate GABAA receptor subunit expression in hippocampal DGC may also function in cortical neurons.

Previous studies of GABAA receptor expression and function in focal cortical dysplasia have produced varying results. Single-cell PCR studies of dysplastic and heterotopic neurons in resected tissue from older children and adults with FCD revealed a reduction in α1 subunit mRNA (Crino et al., 2001). However, in an immunohistochemical study which included two adults with cortical dysplasia, no difference in α1 immunoreactivity was identified (Loup et al., 2000). A recent electrophysiologic study of GABAA receptor currents in dissociated neurons from children with FCD revealed pharmacologic properties suggestive of lower α1 subunit expression in cytomegalic neurons in regions of “severe” cortical dysplasia (Palmini FCD types 2A and 2B plus hemimegalencephaly), as compared with non-dysplastic epileptogenic cortex and regions of “mild” cortical dysplasia (Andre et al., 2008). In our own pharmacologic study of GABAA receptor properties in four infants less than a year of age with FCD 2A, responses to benzodiazepines and barbiturates were unchanged compared with age-matched controls (Jansen et al., 2008), consistent with the preserved α1 and γ2 subunit expression levels in this group demonstrated in the current study. Some studies have reported a reduction in inhibitory postsynaptic current frequency in dysplastic neurons (Calcagnotto et al., 2005), while others have found no change (Cepeda et al., 2003; Cepeda et al., 2005). The disparities in the findings of these studies of the neuronal GABAergic properties of regions of FCD are reflected in the inter-patient variation in GABAA receptor subunit expression seen in our current study.

Chloride transporter expression has also been previously studied in FCD. NKCC1 immunoreactivity has been reported to be increased in dysplastic neurons from children and adults with FCD, while KCC2 expression was characterized by a shift from neuropil to intrasomatic localization (Aronica et al., 2007; Munakata et al., 2007; Sen et al., 2007). Correspondingly, a depolarizing shift in the GABA reversal potential has been identified in immature-appearing neurons from regions of FCD (Cepeda et al., 2007). Fourteen of the 16 FCD samples in our present study demonstrated either elevated NKCC1 or depressed KCC2 protein expression levels when compared with age-matched controls, but the effect this would have on the GABA reversal potential of individual neurons is difficult to predict.

Several clinical factors have been reported to influence GABAA receptor subunit expression or function in animal models, including age and pathology as described above, as well as sex (Galanopoulou, 2008), exposure to benzodiazepines or barbiturates (Raol et al., 2005), brain region (Yu et al., 2006), epilepsy duration (Tsunashima et al., 1997), and seizure frequency (Mathern et al., 1997). We found no significant correlation between these variables and the expression levels seen in our study. Several factors may have prevented us from detecting a correlation, including a relatively small patient population and marked predominance of FCD 2A over other FCD subtypes.

Cell loss is a potentially important confounding variable, and we have attempted to address this issue in several ways. First, all receptor subunit and transporter expression levels were normalized to the expression of β-tubulin, which would be expected to label microtubules from neurons as well as glial cells, thus serving as an indicator of total cellular density. Second, we assessed expression of GFAP in the cytosolic fractions of our specimens as an indicator of astrocyte density (given that tissues undergoing neuronal death exhibit corresponding astrocytosis), and found that this was not correlated with the observed receptor subunit expression patterns. Finally, although cell loss could account for overall low levels of GABAA receptor subunit expression, it would not account for the observation that several samples with very low α1, α5, and γ2 subunit expression had high levels of α4 and δ subunit expression.

An additional consideration is the role of ongoing seizures in promoting alterations in subunit expression patterns. Prolonged seizure activity, such as occurs in status epilepticus, has been shown to alter GABAA receptor subunit trafficking in hippocampal DGC such that surface expression of γ2 subunits is decreased, while that of δ subunits is unchanged (Goodkin et al., 2008). We have partially assessed for this by seeking correlation between EEG-determined seizure frequency and subunit expression levels, although possible effects of prolonged seizures occurring shortly before the time of surgical resection have not been fully investigated.

One pattern did become apparent, as epileptic infants less than a year of age and children with FCD 2B (FCD with balloon cells) exhibited unchanged or increased α1 and γ2 levels compared with age matched controls, while children over a year of age with FCD type 1 or 2A exhibited substantial decreases in α1 and γ2 levels. Levels of the KCC2 transporter exhibited the same pattern. The differential response in infants may correspond to the experimental finding that immature rats subjected to status epilepticus develop increases in hippocampal DGC α1 expression, while older rats demonstrate decreased DGC α1 levels after the same insult (Brooks-Kayal et al., 1998; Zhang et al., 2004; Raol et al., 2006). Conversely, α4 and δ subunit expression was elevated in all of our epileptic FCD samples when directly compared with age matched controls. Further investigation is underway to assess the pharmacologic consequences of these alterations in composition of the GABAA receptor population in pediatric FCD.

In summary, this study defines the time course of the postnatal developmental maturation of human cortical GABAA receptor subunit and chloride transporter expression. This transformation continues over the first several years of life, which has potential therapeutic implications for the use of GABAergic agents in infants and young children. Furthermore, we show that this normal developmental maturation is absent in epileptogenic regions of focal cortical dysplasia or gliosis. Instead, these regions display individual expression patterns that may predict better clinical responsiveness to benzodiazepines (such as diazepam or clonazepam), neurosteroids (such as ganaxolone), drugs that increase ambient GABA levels (such as vigabatrin), or agents that block chloride uptake by NKCC1 (such as bumetanide). Hopefully, further elucidation of this process will promote the goal of rational and successful targeting of anticonvulsant therapy in pediatric epilepsy.

Supplementary Material

Supplementary Figure 1

Supplemental Figure 1. GABAA receptor subunit expression in pediatric epilepsy due to cortical gliosis. (A) Representative Western blot of cortical membranes from children with epilepsy and gliosis aged 3.3 to 17.4 years probed with antibodies against GABAA alpha 1 or gamma 2 subunits followed by fluorescent secondary antibody. (B) Plot of relative GABAA receptor subunit expression versus age for each of the 9 gliosis specimens analyzed. Each point represents the average of the fluorescence intensities of the bands of interest from each specimen expressed relative to the intensity of a simultaneously measured sample from an 8 year old subject with epilepsy and gliosis after correcting for differences in protein loading as determined by β-tubulin expression. Data were obtained from 4 separate Western blots performed using 9 gliosis specimens. (C) Relative expression levels are plotted for alpha 1 versus gamma 2 for each of the gliosis specimens analyzed, which were then subjected to linear regression analysis. The Pearson correlation coefficient (r) and associated P value are shown.

Supplementary Figure 2

Supplemental Figure 2. Variations in GABAA receptor subunit expression in pediatric epilepsy are not due to the degree of astrocytosis. (A, B, C) Representative Western blots of the cytosolic fractions isolated from control samples (A), children with epilepsy due to FCD (B), or epilepsy due to cortical gliosis (C) probed with antibodies against GFAP followed by fluorescent secondary antibody. (D, E, G) Relative expression levels are plotted for the GABAA alpha 1 subunit versus GFAP for the control (D), FCD (E), and gliosis (F) specimens analyzed, which were then subjected to linear regression analysis. The Pearson correlation coefficients (r) and associated P values are shown.

Supplementary Table 1
Supplementary Table 2

Acknowledgments

This work was supported by National Institutes of Health Grant K08 NS052454 and the Child Neurology Society (L.A.J). We thank Laura Lee, Elizabeth Limbacher, Nadine Nielsen, Katherine Pearce, and Eileen Reichert for assistance in obtaining informed consent, members of the Seattle Children’s Hospital Epilepsy Center for clinical evaluations, and Dr. Robert Hevner and members of the Pathology Department at Seattle Children’s for pathologic reviews. A special thank you goes to the children and families contributing to this study.

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Footnotes

None of the authors has any conflict of interest to disclose.

References

  1. Andersen DL, Eckert AL, Tsai VW, Burke CJ, Tannenberg AE, Dodd PR. GABA(A) receptor sites in the developing human foetus. Brain Res Dev Brain Res. 2002;139:107–119. doi: 10.1016/s0165-3806(02)00534-5. [DOI] [PubMed] [Google Scholar]
  2. Andre VM, Cepeda C, Vinters HV, Huynh M, Mathern GW, Levine MS. Pyramidal cell responses to gamma-aminobutyric acid differ in type I and type II cortical dysplasia. J Neurosci Res. 2008;86:3151–3162. doi: 10.1002/jnr.21752. [DOI] [PubMed] [Google Scholar]
  3. Aronica E, Boer K, Redeker S, Spliet WG, van Rijen PC, Troost D, Gorter JA. Differential expression patterns of chloride transporters, Na+-K+-2Cl--cotransporter and K+-Cl--cotransporter, in epilepsy-associated malformations of cortical development. Neuroscience. 2007;145:185–196. doi: 10.1016/j.neuroscience.2006.11.041. [DOI] [PubMed] [Google Scholar]
  4. Ben-Ari Y. Excitatory actions of gaba during development: the nature of the nurture. Nat Rev Neurosci. 2002;3:728–739. doi: 10.1038/nrn920. [DOI] [PubMed] [Google Scholar]
  5. Ben-Ari Y, Gaiarsa JL, Tyzio R, Khazipov R. GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol Rev. 2007;87:1215–1284. doi: 10.1152/physrev.00017.2006. [DOI] [PubMed] [Google Scholar]
  6. Blaesse P, Guillemin I, Schindler J, Schweizer M, Delpire E, Khiroug L, Friauf E, Nothwang HG. Oligomerization of KCC2 correlates with development of inhibitory neurotransmission. J Neurosci. 2006;26:10407–10419. doi: 10.1523/JNEUROSCI.3257-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brooks-Kayal AR, Pritchett DB. Developmental changes in human gamma-aminobutyric acidA receptor subunit composition. Ann Neurol. 1993;34:687–693. doi: 10.1002/ana.410340511. [DOI] [PubMed] [Google Scholar]
  8. Brooks-Kayal AR, Shumate MD, Jin H, Lin DD, Rikhter TY, Holloway KL, Coulter DA. Human neuronal gamma-aminobutyric acid(A) receptors: coordinated subunit mRNA expression and functional correlates in individual dentate granule cells. J Neurosci. 1999;19:8312–8318. doi: 10.1523/JNEUROSCI.19-19-08312.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brooks-Kayal AR, Shumate MD, Jin H, Rikhter TY, Coulter DA. Selective changes in single cell GABA(A) receptor subunit expression and function in temporal lobe epilepsy. Nat Med. 1998;4:1166–1172. doi: 10.1038/2661. [DOI] [PubMed] [Google Scholar]
  10. Calcagnotto ME, Paredes MF, Tihan T, Barbaro NM, Baraban SC. Dysfunction of synaptic inhibition in epilepsy associated with focal cortical dysplasia. J Neurosci. 2005;25:9649–9657. doi: 10.1523/JNEUROSCI.2687-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cepeda C, Andre VM, Flores-Hernandez J, Nguyen OK, Wu N, Klapstein GJ, Nguyen S, Koh S, Vinters HV, Levine MS, Mathern GW. Pediatric cortical dysplasia: correlations between neuroimaging, electrophysiology and location of cytomegalic neurons and balloon cells and glutamate/GABA synaptic circuits. Dev Neurosci. 2005;27:59–76. doi: 10.1159/000084533. [DOI] [PubMed] [Google Scholar]
  12. Cepeda C, Andre VM, Wu N, Yamazaki I, Uzgil B, Vinters HV, Levine MS, Mathern GW. Immature neurons and GABA networks may contribute to epileptogenesis in pediatric cortical dysplasia. Epilepsia. 2007;48(Suppl 5):79–85. doi: 10.1111/j.1528-1167.2007.01293.x. [DOI] [PubMed] [Google Scholar]
  13. Cepeda C, Hurst RS, Flores-Hernandez J, Hernandez-Echeagaray E, Klapstein GJ, Boylan MK, Calvert CR, Jocoy EL, Nguyen OK, Andre VM, Vinters HV, Ariano MA, Levine MS, Mathern GW. Morphological and electrophysiological characterization of abnormal cell types in pediatric cortical dysplasia. J Neurosci Res. 2003;72:472–486. doi: 10.1002/jnr.10604. [DOI] [PubMed] [Google Scholar]
  14. Chen H, Sun D. The role of Na-K-Cl co-transporter in cerebral ischemia. Neurol Res. 2005;27:280–286. doi: 10.1179/016164105X25243. [DOI] [PubMed] [Google Scholar]
  15. Crino PB, Duhaime AC, Baltuch G, White R. Differential expression of glutamate and GABA-A receptor subunit mRNA in cortical dysplasia. Neurology. 2001;56:906–913. doi: 10.1212/wnl.56.7.906. [DOI] [PubMed] [Google Scholar]
  16. Dunning DD, Hoover CL, Soltesz I, Smith MA, O’Dowd DK. GABA(A) receptor-mediated miniature postsynaptic currents and alpha-subunit expression in developing cortical neurons. J Neurophysiol. 1999;82:3286–3297. doi: 10.1152/jn.1999.82.6.3286. [DOI] [PubMed] [Google Scholar]
  17. Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire E, Jensen FE, Staley KJ. NKCC1 transporter facilitates seizures in the developing brain. Nat Med. 2005;11:1205–1213. doi: 10.1038/nm1301. [DOI] [PubMed] [Google Scholar]
  18. Engel J., Jr . Surgical Treatment of the Epilepsies. Raven Press; New York, NY: 1993. [Google Scholar]
  19. Farrant M, Nusser Z. Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. Nat Rev Neurosci. 2005;6:215–229. doi: 10.1038/nrn1625. [DOI] [PubMed] [Google Scholar]
  20. Fraser DD, Duffy S, Angelides KJ, Perez-Velazquez JL, Kettenmann H, MacVicar BA. GABAA/benzodiazepine receptors in acutely isolated hippocampal astrocytes. J Neurosci. 1995;15:2720–2732. doi: 10.1523/JNEUROSCI.15-04-02720.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fritschy JM, Paysan J, Enna A, Mohler H. Switch in the expression of rat GABAA-receptor subtypes during postnatal development: an immunohistochemical study. J Neurosci. 1994;14:5302–5324. doi: 10.1523/JNEUROSCI.14-09-05302.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Galanopoulou AS. Dissociated gender-specific effects of recurrent seizures on GABA signaling in CA1 pyramidal neurons: role of GABA(A) receptors. J Neurosci. 2008;28:1557–1567. doi: 10.1523/JNEUROSCI.5180-07.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Glykys J, Mody I. Activation of GABAA receptors: views from outside the synaptic cleft. Neuron. 2007;56:763–770. doi: 10.1016/j.neuron.2007.11.002. [DOI] [PubMed] [Google Scholar]
  24. Goodkin HP, Joshi S, Mtchedlishvili Z, Brar J, Kapur J. Subunit-specific trafficking of GABA(A) receptors during status epilepticus. J Neurosci. 2008;28:2527–2538. doi: 10.1523/JNEUROSCI.3426-07.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Huttenlocher PR, Dabholkar AS. Regional differences in synaptogenesis in human cerebral cortex. J Comp Neurol. 1997;387:167–178. doi: 10.1002/(sici)1096-9861(19971020)387:2<167::aid-cne1>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  26. Jansen LA, Peugh LD, Ojemann JG. GABA(A) receptor properties in catastrophic infantile epilepsy. Epilepsy Res. 2008;81:188–197. doi: 10.1016/j.eplepsyres.2008.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jones-Davis DM, Macdonald RL. GABA(A) receptor function and pharmacology in epilepsy and status epilepticus. Curr Opin Pharmacol. 2003;3:12–18. doi: 10.1016/s1471-4892(02)00015-2. [DOI] [PubMed] [Google Scholar]
  28. Kanaumi T, Takashima S, Iwasaki H, Mitsudome A, Hirose S. Developmental changes in the expression of GABAA receptor alpha 1 and gamma 2 subunits in human temporal lobe, hippocampus and basal ganglia: an implication for consideration on age-related epilepsy. Epilepsy Res. 2006;71:47–53. doi: 10.1016/j.eplepsyres.2006.05.019. [DOI] [PubMed] [Google Scholar]
  29. Laurie DJ, Wisden W, Seeburg PH. The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. J Neurosci. 1992;12:4151–4172. doi: 10.1523/JNEUROSCI.12-11-04151.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Loup F, Wieser HG, Yonekawa Y, Aguzzi A, Fritschy JM. Selective alterations in GABAA receptor subtypes in human temporal lobe epilepsy. J Neurosci. 2000;20:5401–5419. doi: 10.1523/JNEUROSCI.20-14-05401.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ludwig A, Li H, Saarma M, Kaila K, Rivera C. Developmental up-regulation of KCC2 in the absence of GABAergic and glutamatergic transmission. Eur J Neurosci. 2003;18:3199–3206. doi: 10.1111/j.1460-9568.2003.03069.x. [DOI] [PubMed] [Google Scholar]
  32. Ma W, Barker JL. Complementary expressions of transcripts encoding GAD67 and GABAA receptor alpha 4, beta 1, and gamma 1 subunits in the proliferative zone of the embryonic rat central nervous system. J Neurosci. 1995;15:2547–2560. doi: 10.1523/JNEUROSCI.15-03-02547.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mathern GW, Bertram EH, 3rd, Babb TL, Pretorius JK, Kuhlman PA, Spradlin S, Mendoza D. In contrast to kindled seizures, the frequency of spontaneous epilepsy in the limbic status model correlates with greater aberrant fascia dentata excitatory and inhibitory axon sprouting, and increased staining for N-methyl-D-aspartate, AMPA and GABA(A) receptors. Neuroscience. 1997;77:1003–1019. doi: 10.1016/s0306-4522(96)00516-7. [DOI] [PubMed] [Google Scholar]
  34. Munakata M, Watanabe M, Otsuki T, Nakama H, Arima K, Itoh M, Nabekura J, Iinuma K, Tsuchiya S. Altered distribution of KCC2 in cortical dysplasia in patients with intractable epilepsy. Epilepsia. 2007;48:837–844. doi: 10.1111/j.1528-1167.2006.00954.x. [DOI] [PubMed] [Google Scholar]
  35. Owens DF, Liu X, Kriegstein AR. Changing properties of GABA(A) receptor-mediated signaling during early neocortical development. J Neurophysiol. 1999;82:570–583. doi: 10.1152/jn.1999.82.2.570. [DOI] [PubMed] [Google Scholar]
  36. Painter MJ, Scher MS, Stein AD, Armatti S, Wang Z, Gardiner JC, Paneth N, Minnigh B, Alvin J. Phenobarbital compared with phenytoin for the treatment of neonatal seizures. N Engl J Med. 1999;341:485–489. doi: 10.1056/NEJM199908123410704. [DOI] [PubMed] [Google Scholar]
  37. Palmini A, Najm I, Avanzini G, Babb T, Guerrini R, Foldvary-Schaefer N, Jackson G, Luders HO, Prayson R, Spreafico R, Vinters HV. Terminology and classification of the cortical dysplasias. Neurology. 2004;62:S2–8. doi: 10.1212/01.wnl.0000114507.30388.7e. [DOI] [PubMed] [Google Scholar]
  38. Porter BE, Zhang G, Celix J, Hsu FC, Raol YH, Telfeian A, Gallagher PR, Coulter DA, Brooks-Kayal AR. Heterogeneous GABAA receptor subunit expression in pediatric epilepsy patients. Neurobiol Dis. 2005;18:484–491. doi: 10.1016/j.nbd.2004.12.010. [DOI] [PubMed] [Google Scholar]
  39. Raol YH, Zhang G, Budreck EC, Brooks-Kayal AR. Long-term effects of diazepam and phenobarbital treatment during development on GABA receptors, transporters and glutamic acid decarboxylase. Neuroscience. 2005;132:399–407. doi: 10.1016/j.neuroscience.2005.01.005. [DOI] [PubMed] [Google Scholar]
  40. Raol YH, Zhang G, Lund IV, Porter BE, Maronski MA, Brooks-Kayal AR. Increased GABA(A)-receptor alpha1-subunit expression in hippocampal dentate gyrus after early-life status epilepticus. Epilepsia. 2006;47:1665–1673. doi: 10.1111/j.1528-1167.2006.00640.x. [DOI] [PubMed] [Google Scholar]
  41. Reichelt R, Hofmann D, Fodisch HJ, Mohler H, Knapp M, Hebebrand J. Ontogeny of the benzodiazepine receptor in human brain: fluorographic, immunochemical, and reversible binding studies. J Neurochem. 1991;57:1128–1135. doi: 10.1111/j.1471-4159.1991.tb08270.x. [DOI] [PubMed] [Google Scholar]
  42. Roberts AA, Kellogg CK. Synchronous postnatal increase in alpha1 and gamma2L GABA(A) receptor mRNAs and high affinity zolpidem binding across three regions of rat brain. Brain Res Dev Brain Res. 2000;119:21–32. doi: 10.1016/s0165-3806(99)00146-7. [DOI] [PubMed] [Google Scholar]
  43. Sen A, Martinian L, Nikolic M, Walker MC, Thom M, Sisodiya SM. Increased NKCC1 expression in refractory human epilepsy. Epilepsy Res. 2007;74:220–227. doi: 10.1016/j.eplepsyres.2007.01.004. [DOI] [PubMed] [Google Scholar]
  44. Silverstein FS, Jensen FE, Inder T, Hellstrom-Westas L, Hirtz D, Ferriero DM. Improving the treatment of neonatal seizures: National Institute of Neurological Disorders and Stroke workshop report. J Pediatr. 2008;153:12–15. doi: 10.1016/j.jpeds.2008.01.041. [DOI] [PubMed] [Google Scholar]
  45. Tsunashima K, Schwarzer C, Kirchmair E, Sieghart W, Sperk G. GABA(A) receptor subunits in the rat hippocampus III: altered messenger RNA expression in kainic acid-induced epilepsy. Neuroscience. 1997;80:1019–1032. doi: 10.1016/s0306-4522(97)00144-9. [DOI] [PubMed] [Google Scholar]
  46. Yu ZY, Wang W, Fritschy JM, Witte OW, Redecker C. Changes in neocortical and hippocampal GABAA receptor subunit distribution during brain maturation and aging. Brain Res. 2006;1099:73–81. doi: 10.1016/j.brainres.2006.04.118. [DOI] [PubMed] [Google Scholar]
  47. Zhang G, Raol YH, Hsu FC, Coulter DA, Brooks-Kayal AR. Effects of status epilepticus on hippocampal GABAA receptors are age-dependent. Neuroscience. 2004;125:299–303. doi: 10.1016/j.neuroscience.2004.01.040. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure 1

Supplemental Figure 1. GABAA receptor subunit expression in pediatric epilepsy due to cortical gliosis. (A) Representative Western blot of cortical membranes from children with epilepsy and gliosis aged 3.3 to 17.4 years probed with antibodies against GABAA alpha 1 or gamma 2 subunits followed by fluorescent secondary antibody. (B) Plot of relative GABAA receptor subunit expression versus age for each of the 9 gliosis specimens analyzed. Each point represents the average of the fluorescence intensities of the bands of interest from each specimen expressed relative to the intensity of a simultaneously measured sample from an 8 year old subject with epilepsy and gliosis after correcting for differences in protein loading as determined by β-tubulin expression. Data were obtained from 4 separate Western blots performed using 9 gliosis specimens. (C) Relative expression levels are plotted for alpha 1 versus gamma 2 for each of the gliosis specimens analyzed, which were then subjected to linear regression analysis. The Pearson correlation coefficient (r) and associated P value are shown.

NIHMS341252-supplement-Supplementary_Figure_1.tif (645.3KB, tif)
Supplementary Figure 2

Supplemental Figure 2. Variations in GABAA receptor subunit expression in pediatric epilepsy are not due to the degree of astrocytosis. (A, B, C) Representative Western blots of the cytosolic fractions isolated from control samples (A), children with epilepsy due to FCD (B), or epilepsy due to cortical gliosis (C) probed with antibodies against GFAP followed by fluorescent secondary antibody. (D, E, G) Relative expression levels are plotted for the GABAA alpha 1 subunit versus GFAP for the control (D), FCD (E), and gliosis (F) specimens analyzed, which were then subjected to linear regression analysis. The Pearson correlation coefficients (r) and associated P values are shown.

NIHMS341252-supplement-Supplementary_Figure_2.tif (969.8KB, tif)
Supplementary Table 1
NIHMS341252-supplement-Supplementary_Table_1.pdf (23.6KB, pdf)
Supplementary Table 2
NIHMS341252-supplement-Supplementary_Table_2.doc (42.5KB, doc)

RESOURCES