Abstracts

Rationale: A common clinical manifestation in pediatric patients with Dravet Syndrome (DS) is delay of developmental milestones. Similarly, DS mouse models resulting from mutations in Scn1a or Scn1b, encoding the voltage-gated sodium channel Nav1.1 or 1/ 1B subunits, respectively, demonstrate developmental delay. The mechanisms underlying these effects remain unclear. Here, we investigated a potential developmental delay in the maturation of GABAergic inhibition in the Scn1b null DS mouse model. These mice exhibit severe, spontaneous seizures beginning at approximately postnatal day (P) P10 and die by ~P21. Thus far, two DS patients with homozygous, functional null mutations in SCN1B have recently been reported, making this null mouse model important to our understanding of DS. We have shown that giant depolarization potentials (GDPs), a response mediated by the excitatory action of -aminobutyric acid (GABA) during early brain development, are still observable in layer II/III of the neocortex and the hippocampal CA3 region in Scn1b null, but not WT, slices from P16-21 mice. These observations suggest that developmental maturation of GABAergic function in cortical and hippocampal neurons may be delayed due to Scn1b loss-of-function. Methods: To test this, whole-cell patch-clamp recordings were made from hippocampal CA3 or cortical layer II/III pyramidal cells in slices prepared from P5-7 or P16-19 WT or Scn1b null mice under conditions of low intracellular chloride concentration ([Cl-]i) and a holding potential of -70 mV. Results: At P5-7, pyramidal cells in hippocampal CA3 or cortical layer II/III in slices from both WT and Scn1b null mice showed synchronized, recurrent giant inward currents (corresponding to GDPs), which were sensitive to the GABAA receptor antagonist bicuculline. As expected, neurons in slices from P16-21 WT mice did not exhibit any detectable bicuculline-sensitive, spontaneous inward current. Bicuculline did not affect the amplitudes, but significantly increased the frequency of spontaneous glutamatergic synaptic events, suggesting an increase in glutamate release due to disinhibition of GABAA receptors by bicuculline. Thus, the GABAergic function in WT neurons was mature and inhibitory in nature at P16-19. In contrast, both cortical layer II/III and hippocampal CA3 pyramidal cells in slices from Scn1b null mice at P16-19 still showed prominent bicuculline-sensitive, spontaneous inward currents. Application of bicuculline blocked these large amplitude inward currents and significantly reduced the frequency of synaptic events, suggesting that the observed inward currents are mediated by GABAA receptors. Conclusions: The presence of bicuculline-sensitive inward currents in neurons in P16-19 Scn1b null slices under our specific recording conditions suggests that GABAergic function may remain immature and excitatory. We propose that loss-of-function mutations in Scn1b may delay developmental maturation of GABAergic inhibition in central neurons, and may contribute to seizures in DS. Supported by NIH grant NS076752 to LLI.