Differences in cortical versus subcortical GABAergic signaling: a candidate mechanism of electroclinical uncoupling of neonatal seizures

Abstract

Electroclinical uncoupling of neonatal seizures refers to electrographic seizure activity that is not clinically manifest. Uncoupling increases after treatment with Phenobarbital, which enhances the GABA(A) receptor (GABA(A)R) conductance. The effects of GABA(A)R activation depend on the intracellular Cl(-) concentration ([Cl(-)](i)) that is determined by the inward Cl(-) transporter NKCC1 and the outward Cl(-) transporter KCC2. Differential maturation of Cl(-) transport observed in cortical versus subcortical regions should alter the efficacy of GABA-mediated inhibition. In perinatal rat pups, most thalamic neurons maintained low [Cl(-)](i) and were inhibited by GABA. Phenobarbital suppressed thalamic seizure activity. Most neocortical neurons maintained higher [Cl(-)](i), and were excited by GABA(A)R activation. Phenobarbital had insignificant anticonvulsant responses in the neocortex until NKCC1 was blocked. Regional differences in the ontogeny of Cl(-) transport may thus explain why seizure activity in the cortex is not suppressed by anticonvulsants that block the transmission of seizure activity through subcortical networks.

Figure 1. Thalamus has lower [Cl⁻]_i than the neocortex in early post-natal development

A) Two-photon imaging of Clomeleon in the ventroposterior thalamus and neocortex (layer IV/V) at P10; overlay of multiple planes. Left panel: YFP fluorescence. Right panel: neuronal somata pseudo-colored to a single value according to [Cl⁻]_i averaged over the soma. B) Histogram of [Cl⁻]_i from the neurons depicted in A. C) Differences in [Cl⁻]_i between thalamus and neocortex during development (* indicates statistical significance, Table 1; n=15 mice). D) E_cl calculated using the Nernst equation at 33 °C for each slice. Mean±SEM

Figure 2. The thalamus is inhibited while the neocortex is depolarized during early development

A) MUA recordings from ventroposterior thalamus (left, P7) and neocortex (right, P9) in the presence of 2 mM kynurenic acid. High-pass filtered at 50 Hz. B) MUA frequency plotted from the same traces indicated above; peak function fit indicated by red line. C) Isoguvacine to control ratio (ISO/CON) of MUA frequency in the thalamus (●) and neocortex (▲) at P3-4 (blank) and P7-8 (filled). D) Individual thalamus and neocortex responses to 10 μM isoguvacine at P3-4, P7-8 and P9.

Figure 3. Spontaneous thalamo-cortical epileptiform activity during early development is stable over time

A) Left panel: Combined thalamo-cortical slice imaged in the recording chamber. Right panel: Schematic representation of the thalamo-cortical slice and the typical position of the recording electrodes (D: dorsal, L: lateral). B) Simultaneous extracellular recording from neocortex layer IV/V (top, ■) and ventroposterior thalamus (bottom, ●) in low-Mg²⁺ (P9). C) Higher magnifications of ictal events depicted in dashed boxes in A. D) Top: First 4 neocortical ictal events from A; bottom: Signal power calculated every 5 sec, showing increase power during epileptiform events. E) Entire trace signal power calculated every 10 min from neocortex (top) and thalamus (bottom). EEG band (1–160Hz). F) Ratio of each 10 min to the first 10 min during an 80 min recording. No statistical differences between each 10 min segment (NEO: F(8,35)=0.20, p=0.99; THA: F(8,35)=0.55, p=0.81; n=5 slices; One-way ANOVA).

Figure 4. Effects of epileptiform activity on [Cl⁻]_i in the thalamo-cortical slices

A) Extracellular field potential recording in neocortex layer IV/V in the thalamo-cortical slice preparation from P10 CLM-1 mouse. Epileptiform discharges were induced by low-Mg²⁺ ACSF. Examples of two-photon confocal imaging of YFP (left) in neocortical neurons pseudo-colored to represent [Cl⁻]_i in control (1), 20 min (2) and 40 min (3) after onset of epileptiform discharges. B) Examples of two-photon imaging of YFP (left) in neurons in the ventro-posterior thalamus, pseudo-colored to represent [Cl⁻]_i in control (1), 20 min (2) and 40 min (3) after onset of epileptiform discharges. Epileptiform discharges were induced and recorded as assayed in part A. C–D) Corresponding changes of [Cl⁻]_i in control and during epileptiform activity. Distribution of [Cl⁻]_i (bin size 5 mM) in neocortex (C; 150 neurons) and thalamus (D; 227 neurons) in control (1), 20 min (2) and 40 min (3) after onset of epileptiform discharges. Gauss fits yielded corresponding means and standard deviations of 20 ± 0.6 mM (1), 26.3 ± 0.2 mM (2) and 43.4 ± 2.3 mM (3) in the neocortex (C) and 10 ± 0.2 mM (1), 11.1 ± 0.2 mM (2) and 13.2 ± 0.3 mM (3) in the thalamus. E) Summary effects of epileptiform activity on intracellular chloride accumulation (mean ± s.e.; *indicates statistical significance) in the neocortex (268 neurons from n = 3 slices at P9-10) and thalamus (348 neurons from n = 2 slices at P9-10).

Figure 5. Phenobarbital reduces the power of epileptiform activity in thalamus but not in the neocortex

A) Simultaneous extracellular recording from a thalamo-cortical slice in low-Mg²⁺ (P9). Dashed boxes indicated higher magnification segments in B (□ neocortex, ○ thalamus). B) Higher magnification of the initial segment of two ictal events: (1) control and (2) during 100 μM phenobarbital. C) Signal power (EEG band) determined every 30 seconds during the recording depicted in A. D) Linear correlation of the ictal signal power between thalamus and neocortex. Power was normalized to the maximum ictal event obtained from C. Control (△), phenobarbital (▲). Line represents linear regression (CON[black]: r=0.87±0.09, p<0.001; n=35. PB[gray] r=0.92±0.15, p<0.001, n=9). The linear regression slopes are statistically different (CON: 0.58; PB: 1.40; p<0.001). E) Effect of phenobarbital on individual recordings (* indicates p<0.001; ns indicates p=0.838). Filled symbol indicates mean±SEM.

Figure 6. Phenobarbital is ineffective in the electrically disconnected neocortex

A) Extracellular recording of spontaneous epileptiform activity from the rat neocortex (P8) in low-Mg²⁺. Dashed boxes indicate higher magnification segments during control (□) and 100 μM phenobarbital (■) in the lower panels. B) Signal power (EEG band) determined every 30 seconds during the recording depicted in A. C) Effect of phenobarbital on individual recordings, filled symbol indicates mean±SEM (p=0.224, paired t-test). D) Same as A but recorded in a P11 Clomeleon mice. E) Higher magnification of events depicted in the middle panel (control □; phenobarbital ■). F) Effect of phenobarbital on individual recordings, filled symbol indicates mean±SEM (p=0.106, paired t-test).

Figure 7. Phenobarbital decreases epileptiform activity in the disconnected thalamus

A) Extracellular recording of spontaneous epileptiform activity from the electrically disconnected rat thalamus (P10) in low-Mg²⁺ plus 100 μM 4-AP. Dashed boxes indicate higher magnification segments during control (1) and 100 μM phenobarbital (2) in the lower panels. B) Fast Fourier Transform of the segments depicted in the lower panels of A. C) Signal power (EEG band) determined every 30 seconds during the recording depicted in A. D) Phenobarbital to control ratio of all slices recorded. Filled symbol indicates mean±SEM (n=6; p=<0.001, one sample t-test ).

Figure 8. Co-application of phenobarbital and bumetanide decreases the epileptiform activity in the neocortex

A) Simultaneous extracellular recording from a thalamo-cortical slice in low-Mg²⁺ (P10). Dashed boxes indicated higher magnification segments in B (□ neocortex, ○ thalamus). B) Higher magnification of two ictal events: (1) control and (2) during 100 μM phenobarbital and 10 μM bumetanide. C) Signal power (EEG band) determined every 30 seconds during the recording depicted in A. Line indicates the presence of 100 μM phenobarbital and 10 μM bumetanide. D) Effect of phenobarbital on individual recordings (*, indicates p=0.010 for neocortex; p=0.011 for thalamus; paired t-test). Filled symbol indicates mean±SEM.