GABA tonic currents and glial cells are altered during epileptogenesis in a mouse model of Dravet syndrome

Abstract

Dravet Syndrome (DS) is a rare autosomic encephalopathy with epilepsy linked to Na_v1.1 channel mutations and defective GABAergic signaling. Effective therapies for this syndrome are lacking, urging a better comprehension of the mechanisms involved. In a recognized mouse model of DS, we studied GABA tonic current, a form of inhibition largely neglected in DS, in brain slices from developing mice before spontaneous seizures are reported. In neurons from the temporal cortex (TeCx) and CA1 region, GABA tonic current was reduced in DS mice compared to controls, while in the entorhinal cortex (ECx) it was not affected. In this region however allopregnanonole potentiation of GABA tonic current was reduced in DS mice, suggesting altered extrasynaptic GABA_A subunits. Using THIP as a selective agonist, we found reduced δ subunit mediated tonic currents in ECx of DS mice. Unexpectedly in the dentate gyrus (DG), a region with high δ subunit expression, THIP-evoked currents in DS mice were larger than in controls. An immunofluorescence study confirmed that δ subunit expression was reduced in ECx and increased in DG of DS mice. Finally, considering the importance of neuroinflammation in epilepsy and neurodevelopmental disorders, we evaluated classical markers of glia activation. Our results show that DS mice have increased Iba1 reactivity and GFAP expression in both ECx and DG, compared to controls. Altogether we report that before spontaneous seizures, DS mice develop significant alterations of GABA tonic currents and glial cell activation. Understanding all the mechanisms involved in these alterations during disease maturation and progression may unveil new therapeutic targets.

Keywords: GFAP - glial fibrillary acidic protein; Iba-1; Nav1.1 channel; SCN1A encephalopathy; delta subunits; extrasynaptic GABA receptor A.

Figure 1

GABA tonic currents are altered in DS mice during epileptogenesis. (A) Whole-cell recordings showing representative GABA tonic currents (I-tonic) evoked by blocking all GATs with nipecotic acid in perfusion (not indicated) and unmasked with a GABA_A receptor antagonist (gray continuous lines; bicuculline or picrotoxin; see “Materials and methods” Section) in layer V pyramidal neuron of brain slices from 15 to 19 days old DS mice (Na_v1.1^+/-) and same age littermate wild type controls (wt). Dotted lines indicate the I-tonic before and after GABA_A receptor antagonist (gray bars; see “Materials and methods” Section). Green bars mark allopregnanolone (Allo) application. Scale bars, 1 min, 50 pA. (B) Box chart summarizing GABA I-tonic in wt and DS mice from TeCx (n = 9 cells from three mice for wt and 11 cells from five mice for DS mice, p = 0.042, two sample t-test), CA1 (n = 4 cells from four mice for wt and five cells from three mice for DS mice, p = 0.025, two sample t-test) and ECx (n = 7 cells from four mice for wt and nine cells from four mice for DS mice, p = 0.399, two sample t-test). (C) Histogram summarizing the relative GABA I-tonic potentiation in ECx (mean ± SEM; same cells as in B; p = 0.037, two sample t-test) induced by ALLO in wt and Na_v1.1^+/- mice. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 2

GABA_AR δ subunit response and expression are altered in DS mice. (A) Whole-cell recordings showing representative tonic currents (I-tonic) evoked by THIP (not indicated) and evaluated with GABA_A receptor antagonist bicuculline (gray continuous lines; see “Materials and methods” Section) in layer V pyramidal neuron of brain slices containing ECx and DG from 15 to 19 days old DS mice (Na_v1.1^+/-) and same age littermate wild type (wt) controls. Scale bars, 30 s, 50 pA. (B) Box chart summarizing I-tonic in DS and wt mice from ECx (n = 10 cells from four mice for wt and 10 cells from three mice for DS mice, p = 6.783 E⁻⁴, two sample t-test) and DG (n = 9 cells from five mice for wt and nine cells from five mice for DS mice, p = 0.017, two sample t-test). (C) Confocal fluorescence images of δ subunit expression in ECx and DG from 19 to 21 days old DS mice and same age littermate wt controls. (D) Histogram summarizing the relative fluorescence in DS compared to wt mice from ECx (26 and 29 fields from three wt and three DS mice, respectively, p = 0.003, two sample t test) and DG (27 and 38 fields from 4 wt and 4 DS mice, respectively, p = 0.00001, two sample t-test). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 3

Glial cell markers GFAP and Iba1 are increased in DS mice. (A,C) Confocal fluorescence images of GFAP and Iba1 immunofluorescence in ECx and DG from 19 to 21 days old DS mice (Na_v1.1^+/-) and same age littermate wild type (wt) controls. (B) Histogram (mean ± SEM) summarizing the relative GFAP fluorescence in DS mice compared to wt mice from ECx (n = 3 mice PN 19–21, 6 ECx, 18 fields for WT and DS mice p < 0.001, one sample t-test) and DG (n = 3 mice PN 19–21, six DG, 18 fields for WT and DS mice p < 0.001, one sample t-test). (D) Histogram (mean ± SEM) summarizing number of Iba1 positive cells/μ² in DS compared to WT mice from ECx (n = 4 mice PN 19–21, eight Ecx, 24 fields for WT and DS mice p = 0.00817, two sample t-test) and DG (n = 4 mice PN 19–21, eight DG, 24 fields for WT and DS mice p = 0.0038237, two sample t-test). **p ≤ 0.01, ***p ≤ 0.001.