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. 2020 Nov;7(11):2137-2149.
doi: 10.1002/acn3.51205. Epub 2020 Sep 26.

Excitatory and inhibitory neuron defects in a mouse model of Scn1b-linked EIEE52

Jacob M Hull  1 , Heather A O'Malley, Chunling Chen  2 , Yukun Yuan  2 , Nicholas Denomme  2 , Alexandra A Bouza  2 , Charles Anumonwo  2 , Luis F Lopez-Santiago  2 , Lori L Isom  1   2
Affiliations

Excitatory and inhibitory neuron defects in a mouse model of Scn1b-linked EIEE52

Jacob M Hull et al. Ann Clin Transl Neurol. 2020 Nov.

Abstract

Objective: Human variants in voltage-gated sodium channel (VGSC) α and β subunit genes are linked to developmental and epileptic encephalopathies (DEEs). Inherited, biallelic, loss-of-function variants in SCN1B, encoding the β1/β1B subunits, are linked to early infantile DEE (EIEE52). De novo, monoallelic variants in SCN1A (Nav1.1), SCN2A (Nav1.2), SCN3A (Nav1.3), and SCN8A (Nav1.6) are also linked to DEEs. While these VGSC-linked DEEs have similar presentations, they have diverse mechanisms of altered neuronal excitability. Mouse models have suggested that Scn2a-, Scn3a-, and Scn8a-linked DEE variants are, in general, gain of function, resulting in increased persistent or resurgent sodium current (INa ) and pyramidal neuron hyperexcitability. In contrast, Scn1a-linked DEE variants, in general, are loss-of-function, resulting in decreased INa and hypoexcitability of fast-spiking interneurons. VGSC β1 subunits associate with Nav1.1, Nav1.2, Nav1.3, and Nav1.6 and are expressed throughout the brain, raising the possibility that insults to both pyramidal and interneuron excitability may drive EIEE52 pathophysiology.

Methods: We investigated excitability defects in pyramidal and parvalbumin-positive (PV +) interneurons in the Scn1b-/- model of EIEE52. We also used Scn1bFL/FL mice to delete Scn1b in specific neuronal populations.

Results: Scn1b-/- cortical PV + interneurons were hypoexcitable, with reduced INa density. Scn1b-/- cortical pyramidal neurons had population-specific changes in excitability and impaired INa density. Scn1b deletion in PV + neurons resulted in 100% lethality, whereas deletion in Emx1 + or Camk2a + neurons did not affect survival.

Interpretation: This work suggests that SCN1B-linked DEE variants impact both excitatory and inhibitory neurons, leading to the increased severity of EIEE52 relative to other DEEs.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
PV‐dependent Cre deletion of Scn1b results in early lethality while Emx1 and or Camk2a‐dependent Cre deletion does not. A. Kaplan–Meier survival curve of Scn1bFl / Fl/PV‐Cre mice (median 25 days, N = 62) compared to Scn1b −/− mice (median of 18 days, N = 60, P < 0.0001, Mantel–Cox test), Scn1bFl/Fl/CamK2a‐Cre and Scn1bFl/Fl/Emx1‐Cre mice, (no deaths, N = 21 and N = 15, respectively, both P < 0.0001, Mantel–Cox test).
Figure 2
Figure 2
Scn1b deletion results in cortical PV + neuron hypoexcitability. (A) Representative images of tdTom + cells (left), anti‐PV labeling (middle), and merge (right). Most tdTom + cells (89%) are also detected with anti‐PV antibodies (upper arrow). Few tdTom + neurons display minimal to no labeling with anti‐PV antibodies (lower arrow). Scale bar = 100 μm. (B) Pie charts demonstrating the extent of labeling specificity (left) and labeling efficiency (right). (C) Representative voltage traces from whole‐cell recordings of neurons positive for red fluorescence (n/N = 22/8) from brain slices of Scn1b+/+/PV‐Cre/tdTom (Scn1b +/+/PV in text) or Scn1b ‐/‐/PV‐Cre/tdTom (Scn1b ‐/‐/PV in text) mice at P14‐20. (D–F) Characteristic features of FS interneurons were present in all tdTom‐labeled neurons recorded relative to layer 5 pyramidal neurons (see Fig. 4 for additional details) including low spike frequency adaptation (D.), short AP half width (E.), and large AHP magnitudes (F). (G) Representative voltage traces of Scn1b +/+/PV (black) or Scn1b ‐/‐/PV mice (red). (H) Current injection vs. APs fired in 1‐s of recordings above. Asterisks indicate P value for area under the curve (***P < 0.005, n/N = 11/4 Scn1b +/+/PV 11/4 Scn1b ‐/‐/PV. (E) Maximum firing rate for neurons in B (***P < 0.005). (I‐K) Average resting membrane potential (F) and input resistance (G) from recordings in G. See Table 1 for numerical data and biophysical properties.
Figure 3
Figure 3
Scn1b deletion reduces INa density in dissociated cortical PV + neurons (A) Representative INa traces from whole‐cell recordings from cortical tdTom + neurons of Scn1b +/+/PV and Scn1b ‐/‐/PV mice. Current elicited by depolarizing steps from −120 mV to 30 mV from a holding potential of −120 mV (traces up to peak are shown for visualization of smaller nested currents). (B) Current–voltage relationship for recordings as in A (n/N = 10/3 Scn1b +/+/PV, 8/3 Scn1b ‐/‐/PV). (C) Peak INa density at −25 mV from recordings in B. Asterisks indicate P value (**P < 0.01). (D) Normalized voltage dependence of steady‐state activation and inactivation of recordings in B. See Table 2 for numerical values
Figure 4
Figure 4
Scn1b deletion results in complex pyramidal neuron excitability defects. A., E, and I. Representative voltage traces from whole‐cell recordings of layer 5 (A), subiculum (E) and layer 6 (I) pyramidal neurons in acute brain slices of Scn1b +/+ (black) or Scn1b ‐/‐ mice (red) B., F., J., Current injection vs. APs fired in 1‐s of recordings above. Firing at low current injection quantified as area under the curve up to 50 pA. Depolarization block is quantified as AP count at highest current injection divided by max AP count (with AP failure defined as max voltage < 0 mV, right asterisks). Layer 5 (B) pyramidal neurons show no change in firing at low current injections or degree of depolarization block (n/N = 12/7 Scn1b +/+, 14/7 Scn1b ‐/‐). Subicular (F) and Layer 6 pyramidal neurons (J) and show increased firing at low current amplitudes (left asterisks indicate p‐value) in Scn1b +/+ vs. Scn1b ‐/‐ mice (n/N = 25/12 Scn1b+/+, 24/13 Scn1b‐/‐ subiculum; n/N = 20/10 Scn1b +/+, 21/9 Scn1b ‐/‐ layer 6, Welch’s t‐test). C., G., K. RMP is not affected by Scn1b deletion in layer 5 (C) but is depolarized in subicular (G) and layer 6 pyramidal neurons (K). D., H., L. Input resistance is unaffected by Scn1b deletion in layer 5 (D) but is increased in subicular (H) and layer 6 pyramidal neurons (L). See Table 3 for quantification of biophysical properties. Asterisks indicate p‐values (*P < 0.05, ***P < 0.005, and ****P < 0.0001)
Figure 5
Figure 5
Scn1b deletion results in reduced transient and persistent INa in layer 6 pyramidal neurons. (A) Representative INa traces from nucleated patches from cortical layer 6 pyramidal neurons of Scn1b +/+ and Scn1b −/− mice in acute brain slices. Current elicited by depolarizing steps from −120 mV to 30 mV from a holding potential of −120 mV. (B) Current–voltage relationship for nucleated patches as in A (n/N = 10/10 Scn1b +/+, 10/9 Scn1b −/−). (C) Peak INa density at −20 mV from recordings in B. Welch’s t‐test. Asterisks indicate P value (*P < 0.05). (D) Normalized voltage dependence of steady state activation and inactivation of recordings in B. (E) Representative persistent INa recorded in intact neurons in Scn1b +/+ and Scn1b −/− cortical layer 6 pyramidal neurons in acute brain slices using a 150 mV/3s voltage ramp starting from a holding potential of −120 mV. Traces are the average of four recordings from the same neuron. F. Persistent INa at −20 mV (n/N = 9/4 Scn1b +/+, 7/3 Scn1b ‐/‐). (G) Voltage dependence of activation for data in F. Asterisks indicate P value (*P < 0.05 and ***P < 0.005).
Figure 6
Figure 6
Scn1b deletion impairs inhibitory neurotransmission. (A) Representative sIPSC traces from whole‐cell recordings from layer 6 pyramidal neurons of Scn1b +/+ (black) and Scn1b ‐/‐ (red) mice. (B) Cumulative probability plots of sIPSC amplitude (n/N = 12/4 Scn1b +/+, 12/4 Scn1b ‐/‐) as in A. Inset bar graphs show mean sIPSC amplitude. (C) Cumulative probability plot of interevent intervals as in A. Top asterisks indicate P‐value for comparing cumulative probabilities (****P < 0.0001, Kolmogorov–Smirnov test). Inset bar graphs shown mean sIPSC frequency (1/IEI). Asterisk indicates P‐value (***P < 0.005, Welch’s t‐test).
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