PMC

GABA_A 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 GABA_A 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) GABA_A 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.

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.

GABA_A receptor subunit and chloride transporter expression in pediatric FCD as compared with age-matched controls. (A) Graph of average protein expression levels for GABA_A 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.

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.

Human cortical GABA_A 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.

GABA_A 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 GABA_A 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 GABA_A 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.