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: GABA reversal potentials in Scn1b-/- neurons are significantly depolarized compared to WT. GABA-evoked currents at different holding potentials were recorded using the gramicidin perforated patch clamp method from cortical layer II/III and hippocampal CA1 and CA3 neurons in slices from P13-19 Scn1b WT or -/- mice. (A,B) Representative GABA currents evoked in a layer II/III pyramidal cell in a cortical slice of a P17 Scn1b WT (A) or -/- (B) mouse at holding potentials varying from -90 mV to -30 mV. (C) Comparison of the current-voltage relationships (I-V curves) obtained from Scn1b WT and -/- mice. Values are pooled from cortical and hippocampal neurons. (D) Comparison of reversal potentials for GABA-mediated currents in Scn1b WT and -/- neurons. Each value is representative of 6–7 individual mice.

Fig 4: Bumetanide suppresses GABA-evoked firing in P14-18 Scn1b−/− neurons. (A) Representative traces showing GABA-evoked responses at different membrane holding potentials after establishing gramicidin perforated patch recording. At membrane potentials more depolarized than −60 mV (−20 and −40 mV shown here), Scn1b−/− neurons fired spontaneously. Puff application of GABA suppressed spontaneous firing (A-I and A-II). In contrast, at membrane potentials more hyperpolarized than −60 mV, application of GABA induced AP firing (A-III and A-IV). (B) Representative example of the time course of the effect of 20 µM bumetanide on GABA-induced firing in a P16 Scn1b−/− CA1 pyramidal cell. At a holding potential of −70 mV, puff application of GABA induced repetitive firing in a subset of neurons. Bath application of 20 µM bumetanide resulted in the time dependent inhibition of GABA-evoked firing (B-I to B-IV). 30 min wash with ACSF partially reversed the GABA-evoked firing (B-V and B-VI). Each trace is representative of results from 4 individual mice.

Fig 5: 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.