Fig 2: GABAergic currents in cortical and hippocampal neurons of Scn1b−/− mice remain inward/depolarizing after the second postnatal week. Whole cell recordings of bicuculline-sensitive inward currents were made from P13-19 Scn1b−/− brain slices under similar conditions as described in Fig. 2. (A–D) Representative recordings of spontaneous synaptic inward currents in a cortical layer II/III pyramidal cell of a P17 Scn1b−/− mouse in the absence (A) and presence (B) of 10 µM bicuculline. Bicuculline blocked all large amplitude, inward synaptic events (C) and significantly decreased the frequency of synaptic responses (D). Each value is representative of 10 individual mice. (E–H) Representative recordings of spontaneous synaptic inward currents in a CA3 pyramidal cell of a P18 Scn1b−/− mouse in the absence (E) and presence (F) of 10 µM bicuculline. Bicuculline blocked all large amplitude, inward synaptic events (G) and significantly decreased the frequency of synaptic responses. (H) Arrows in E indicate large amplitude inward currents corresponding to GDPs. Each value is representative of 7–10 individual mice.

Fig 3: GABAergic currents in cortical and hippocampal neurons of Scn1b WT mice are outward/hyperpolarizing after the second postnatal week. Whole cell bicuculline-sensitive inward currents were recorded from cortical layer II/III and hippocampal CA1 or CA3 neurons of P13-19 Scn1b WT mice using a low Cl– containing, K-gluconate-based pipette solution at a holding potential of −70 mV. (A,B) Representative recordings of spontaneous synaptic inward currents in a cortical layer II/III pyramidal cell of a P17 Scn1b WT mouse in the absence (A) and presence (B) of 10 µM bicuculline. Bicuculline had no significant effect on the amplitudes of synaptic events (C), but significantly increased the frequency of synaptic responses (D). Each value is representative of 5–10 individual mice.

Fig 4: Hippocampal and cortical neurons in brain slices from P4-7 Scn1b WT and −/− mice display excitatory GABAergic spontaneous synaptic currents and synchronous recurrent giant inward currents. (A–D) Spontaneous postsynaptic currents in cortical layer II/III and hippocampal CA1 or CA3 pyramidal cells from P4-7 Scn1b WT or −/− mice were recorded at a holding potential of −70 mV using the whole-cell patch-clamp recording technique, a low Cl−-containing, K-gluconate-based internal solution, and ACSF external solution. (A,B) Representative recording of spontaneous postsynaptic currents in a hippocampal CA3 pyramidal cell from a P7 WT mouse in the absence (A) and presence (B) of 10 µM bicuculline. (C) Bicuculline blocked large amplitude inward currents and shifted the curve of cumulative amplitude distribution to the left. (D) Bicuculline reduced the frequency of synaptic currents and shifted the curve of cumulative inter-event interval distribution to the right. Similar results were seen in hippocampal CA1 and cortical layer II/III neurons in slices from both Scn1b WT and −/− mice. Each value is representative of 5–6 individual mice. (E–H) Spontaneous GDP-related giant inward currents were recorded from Scn1b WT or −/− hippocampal CA3 pyramidal cells at a holding potential of −70 mV using a NMDG-Cl-based pipette solution. (E) Representative currents from P6 WT brain. (F) Giant currents are sensitive to block by bicuculline. (G) Block by bicuculline is reversible. (H) Giant inward currents are sensitive to block by glutamate receptor antagonists CNQX and APV. Each trace is representative of 7 CA3 neurons from 4 individual WT mice.

Fig 5: Excitation-contraction coupling in ventricular CMs from Scn1b-null mice.(A) Representative example of excitation-contraction (E-C) coupling recording. Top: ICa triggered by voltage clamp depolarization; middle: whole-cell Ca2+ transient; bottom: Ca2+ transient time profile. (B) I-V curve shows dramatically decrease of ICa in CMs from Scn1b-null mice. (C) Ca2+ transient amplitude did not change in CMs from Scn1b-null mice compared with WT. (D) E-C coupling gain (ratio between the Ca2+ transient amplitude and the ICa) decreased in CMs from Scn1b-null mice. * P < 0.05; ** P < 0.01; *** P < 0.001. WT vs. Scn1b-null by Student’s t test. N, number of mice; n, number of cells. Data represent mean ± SEM.