Recurrent neonatal seizures increase tonic inhibition and respond to enhancers of δ-containing GABAA receptors

Abstract

About one-third of neonatal seizures do not respond to the first-line anticonvulsant phenobarbital, which activates phasic inhibition and whose effectiveness decreases over time. Whether enhancing tonic inhibition can treat refractory seizures or status epilepticus in neonates remains uncertain. We evaluated the effect of recurrent seizure-like events (SLE) on α5- and δ-GABAA receptor (α5- and δ-GABAAR) subunit expression and tonic inhibition in neonatal C57BL/6J mice (P6-9, both sexes) using acute brain slices. We investigated the impact of THIP (gaboxadol) on neonatal behavioral seizures, neuronal apoptosis, and neurodegeneration in vivo. We found neonatal neocortical expression of α5- and δ-GABAAR subunits. Blocking α5-GABAARs with L-655,708 did not affect acute neonatal SLE, whereas enhancing δ-GABAARs with THDOC, a neurosteroid, reduced them. The α5- and δ-GABAAR membrane expression increased after 8 hours of neonatal SLE and correlated with increased δ-mediated conductance but not α5-mediated conductance. Enhancing tonic inhibition was more effective in reducing recurrent neonatal SLE (8 hours) compared with early treatment. Increasing tonic inhibition reduced the duration, severity, and number of kainic acid-induced in vivo neonatal behavioral seizures without increasing neurodegeneration or apoptosis. We conclude that recurrent neonatal seizures increase tonic inhibition. Therefore, enhancing tonic inhibition may be a treatment strategy for prolonged neonatal status epilepticus.

Keywords: Cell biology; Epilepsy; Ion channels; Neuroscience; Seizures.

Figure 1. The α5- and δ-GABA_AR subunits are expressed in the neonatal neocortex.

(A) Representative pseudocolored images of the δ subunit (top) and α5 subunit (bottom) in the neonatal neocortex. Columns: DAPI (yellow), NeuN (cyan), subunit (magenta), and composite. P8 mice. (B and C) Representative pseudocolored images of somatic neuronal δ (B) and α5 subunit (C) expression. Left: Z-stack (30 μm slice, 2 μm steps) from each group. Right: Imaris 3D reconstruction highlighting shape and overlap. Rows: YFP-expressing neurons (cyan), subunit (magenta), and composite. P12. Scale bars, 25 μm. YFP, yellow fluorescence protein.

Figure 2. Inhibiting the α5-containing GABA_AR does not worsen seizure-like events in the neonatal neocortex.

(A) Seizure-like events induced by Low-Mg²⁺ artificial CSF (aCSF) recorded in the neocortical layer IV/V of acute brain slices (P8) using an extracellular field electrode before and after L-655 at 50 nM, 1 μM, and 10 μM. The corresponding Fast Fourier Transform (FFT) power area calculated every 30 seconds is displayed below the traces. (B) SLE FFT power ratios (L-655/baseline) in the neonatal neocortex using different L-655 concentrations. One-way ANOVA, F(2, 17) = 1.185, P = 0.33, n = 6, 9, 5 recordings, respectively. (C) Number of SLE events during L-655 (y axis) versus baseline (x axis) under different L-655 concentrations. Circles: individual slices. Mean ± 95% CI. The line represents a slope of 1.

Figure 3. Enhancing tonic inhibition with the neurosteroid analog THDOC reduces neonatal seizure-like events.

(A) Seizure-like events induced by Low-Mg²⁺ aCSF in the neocortex layer IV/V recorded using an extracellular field electrode in acute brain slices (P8), with the corresponding FFT power area calculated every 30 seconds for THDOC 100 nM, 1 μM, and 10 μM. (B) FFT power ratios in the neonatal neocortex using different THDOC concentrations. One-way ANOVA, F(2,28) = 6.253, P = 0.006, Tukey’s post hoc test is shown in the graph; n = 13, 11, 7, respectively. (C) Number of SLE during THDOC (y axis) versus baseline (x axis). Circles: individual slices. Mean ± 95% CI. The line represents a slope of 1.

Figure 4. Recurrent neonatal seizure-like events for 8 hours increase the membrane expression of α5- and δ-GABA_AR subunits.

(A) Western blot showing enrichment of the membrane-bound Na⁺/K⁺ ATPase in the membrane column using the fractionation kit. (B) Sample Western blots of the α5- and δ-GABA_AR subunits from acute brain slices incubated in aCSF (Ctrl). Same lysate as A. (C) Sample as in B but from acute brain slices incubated in Low-Mg²⁺ aCSF. (D) Example amido black total protein stain of δ and α5 subunit lanes showing uniform loading in aCSF (T, total; M, membrane). Expected MW for each subunit is around 55 kDa (dashed boxes). (E) Left, ratio of the membrane to total δ subunit in aCSF- and Low-Mg²⁺–treated slices. aCSF: 0.88 [0.70, 1.06], Low-Mg²⁺: 1.20 [0.99, 1.40], unpaired t test, n = 6 animals, 6 blots. Right, ratio of membrane to total α5 subunit in aCSF- and Low-Mg²⁺–treated slices. aCSF: 0.76 [0.57, 0.95], Low-Mg²⁺: 1.15 [0.85, 1.45], unpaired t test, n = 6 mice, 6 blots. Circles: lysate ratios. Lines: mean ± 95% CI.

Figure 5. The total and δ-GABA_AR–mediated tonic conductance increases in the neocortex after 8 hours of recurrent neonatal seizure-like events.

Representative whole-cell voltage-clamp recordings of a neocortical pyramidal cell (layer V) without prior seizure incubation (Ctrl, left) and after 8 hours of SLE (Low-Mg²⁺, right) during perfusion of aCSF (A), THIP 1 μM (B), and THIP 10 μM (C), V_h –70 mV. Top lines indicate drug perfusions; right lines indicate fits to the all-points histogram under each condition. (D) Example of spontaneous inhibitory postsynaptic currents in aCSF during control and SLE conditions at 8 hours. (E) Tonic inhibitory conductances in the neocortex under aCSF (Ctrl: 4.10 pS/pF [3.08, 5.11], SLE: 6.78 pS/pF [4.68, 8.88], unpaired t test, n = 8, 8), THIP 1 μM (Ctrl: 13.2 pS/pF [10.5, 15.9], SLE: 18.9 pS/pF [15.3, 22.5], unpaired t test, n = 8, 9), and THIP 10 μM (Ctrl: 44.4 pS/pF [35.5, 53.3], SLE: 61.3 pS/pF [51.3, 71.3], unpaired t test, n = 9, 9). Note the increase in tonic current with THIP (different y axis). (F) Spontaneous inhibitory postsynaptic current amplitude (Ctrl: 66.2 pA [47.4, 85.1], SLE: 57.9 pA [35.9, 80.0], P = 0.51, unpaired t test) and frequency (Ctrl: 3.21 Hz [2.25, 4.17], SLE: 1.85 Hz [0.83, 2.87], unpaired t test), n = 8, both groups. Not pictured: Rise time (Mann-Whitney test, P = 0.44) and decay (unpaired t test, P = 0.58). White circles: individual slices. Black lines: mean ± 95% CI.

Figure 6. The α5-GABA_AR–mediated currents increase after recurrent neonatal seizure-like events, but their proportion remains the same.

Representative whole-cell voltage-clamp recordings of a neocortical pyramidal cell (layer V) without seizure incubation (Ctrl, top) and after 8 hours of Low-Mg²⁺ (SLE, below), during perfusion of aCSF (A) or THIP 10 μM (B) before the application of L-655 (200 nM), V_h –70 mV. Top line indicates various drug perfusions; right lines indicate fits to the all-points histograms under each condition (black = α5-mediated current blocked by L-655, gray = net and residual currents). (C) α5-mediated tonic conductances with no THIP (Ctrl: 4.32 pS/pF [3.02, 5.63], SLE: 6.93 pS/pF [3.09, 10.8], P = 0.182, unpaired t test, n = 9, 10) and α5-mediated tonic conductances under 10 μM THIP (Ctrl: 11.3 pS/pF [8.61, 14.1], SLE: 23.9 pS/pF [13.7, 34.1], unpaired t test, n = 6, 5). (D) Percent conductance mediated by α5-containing GABA_ARs with and without THIP (control: 61.2% [49, 73.5] and 25.5% [13.0, 37.9], SLE: 48.7% [38.3, 59.1] and 24.8% [20.2, 29.3], 1-way ANOVA, F(3, 26) = 12, P < 0.0001; Šídák’s multiple comparisons test; n = 9, 6, 10, 5, respectively). Circles: individual recordings. Black lines: mean ± 95% CI.

Figure 7. Enhancing tonic inhibition is more effective after recurrent neonatal seizure-like events.

Top: Seizure-like events induced by Low-Mg²⁺ aCSF in the neocortex, layer IV/V (A), and CA1 stratum pyramidale (B) recorded with an extracellular field electrode in acute brain slices (P8). Below: FFT power area from top traces calculated every 30 seconds. The upper group was recorded with no prior Low-Mg²⁺ incubation, while the lower group was after 8 hours of incubation. (C) FFT power ratios versus seizure incubation duration in the neonatal neocortex and CA1 region. Linear regression, n = 37 neocortices (slope = –0.037), n = 41 CA1 regions (slope = –0.039). Dashed lines ± 95% CI. (D) THIP’s effect between slices at 0, 3, and after 8 hours in Low-Mg²⁺. Neocortex: 0 hours: 0.88 [0.75, 1.0], 3 hours: 0.68 [0.45, 0.92], 8 hours: 0.53 [0.34, 0.72]; 1-way ANOVA, F(2, 15) = 6.009, P = 0.012; n = 7, 6, 5 respectively. CA1 region: 0 hours: 0.91 [0.74, 1.08], 3 hours: 0.66 [0.45, 0.87], 8 hours: 0.57 [0.38, 0.76]; 1-way ANOVA, F(2, 17) = 4.604, P = 0.025, n = 7, 8, 5 respectively. (E) Diazepam’s (DZP) effect on slices after 8 hours in Low-Mg²⁺. Neocortex: 1.01 [0.98, 1.33], P = 0.88, 1-sample t test, n = 10; CA1 region: 1.05 [0.93, 1.17], P = 0.35, 1-sample t test, n = 10. (F) Number of SLE during THIP (y axis) versus baseline (x axis) in the pup neocortex and CA1 region at 0, 3, and 8 hours of Low-Mg²⁺. The line represents a slope of 1. Circles: individual slices. Black lines: mean ± 95% CI.

Figure 8. THIP (gaboxadol) reduces behavioral seizure frequency and severity in vivo in neonatal mice.

(A) In vivo seizure protocol (P8–9). (B) Behavioral seizure severity over time (Racine scale) between kainic acid (KA), KA+THIP, and THIP alone over 90 minutes. Two-way ANOVA: time, F(3.70, 59.2) = 28.75, P < 0.0001; treatment: F(2,16) = 167.7, P < 0.0001; time × treatment interaction F(18, 144) = 12.1, P < 0.0001; Tukey’s post hoc test values that are significant between KA vs. KA+THIP are displayed. Comparisons with THIP are not pictured. n (mice) = 6 KA, 7 KA+THIP, 6 THIP. (C–E) Behavioral seizure severity (Racine scale) every 10 minutes in KA, KA+THIP, and THIP. (F) Number of convulsive seizures by stage between KA and KA+THIP groups (unpaired t test, n = same as above per group). Circles indicate an average of mice in B and individual mice in F. Mean ± 95% CI.

Figure 9. THIP does not cause neuronal apoptosis or degeneration in the neonatal brain in vivo.

(A) Representative pseudocolored images of the neocortex from each group. Rows: KA, KA+THIP, vehicle plus THIP (Veh+THIP). All images were obtained from mice after 5 hours of behavioral seizures. (B) Number of cleaved caspase-3–positive (CC3⁺) neurons after 5 hours of behavioral seizures. One-way ANOVA, F(2,33) = 2.22, P = 0.124. Mean ± 95% CI. (C) Same as A but with Fluoro-Jade C (FJC) staining. (D) Number of FJC⁺ neurons after 5 hours of behavioral seizures. One-way ANOVA, F(2, 33) = 3.69, P = 0.036, Tukey’s post hoc test. Data analysis conducted on log-transformed data. N = 12 slices in all groups, KA = 6 mice, KA+THIP = 7 mice, Veh+THIP = 6 mice. Circles: individual brain slice. CC3, mean ± 95% CI; FJC, geometric mean ± 95% CI. Scale bar, 25 μm.