ObjectiveDravet syndrome (DS) is a severe, early-onset epilepsy with an increased incidence of sudden death. Evidence of interictal breathing deficits in DS suggest that alterations in subcortical projections to brainstem nuclei may exist, which might be driving comorbidities in DS. The aim of this study was to determine if a subcortical structure, the bed nucleus of the stria terminalis (BNST) in the extended amygdala, is activated by seizures, exhibits changes in excitability, and expresses any alterations in neurons projecting to a brainstem nucleus associated with respiration, stress response and homeostasis.MethodsExperiments were conducted using F1 mice generated by breeding 129.Scn1a+/- mice with wildtype C57BL/6J mice. Immunohistochemistry was performed to quantify neuronal c-fos activation in DS mice after observed spontaneous seizures. Whole cell patch clamp and current clamp electrophysiology recordings were conducted to evaluate changes in intrinsic and synaptic excitability in the BNST.ResultsSpontaneous seizures in DS mice significantly enhanced neuronal c-fos expression in the BNST. Further, the BNST had altered AMPA/NMDA postsynaptic receptor composition and showed changes in spontaneous neurotransmission, with greater excitation and decreased inhibition. BNST to parabrachial nucleus (PBN) projection neurons exhibited intrinsic excitability in wildtype mice, while these projection neurons were hypoexcitable in DS mice.SignificanceThe findings suggest that there is altered excitability in neurons of the BNST, including BNST to PBN projection neurons, in DS mice. These alterations could potentially be driving comorbid aspects of DS outside of seizures, including respiratory dysfunction and sudden death.SIGNIFICANCE STATEMENTDravet syndrome (DS) is an early-onset epilepsy with an increased risk of sudden death. We determined that there are alterations in a subcortical nucleus, the bed nucleus of the stria terminalis (BNST) of the extended amygdala, in a murine DS model. The BNST is involved in stress, anxiety, feeding, and respiratory function. We found enhanced activation in the BNST after seizures and alterations in basal synaptic neurotransmission–with enhanced spontaneous excitatory and decreased spontaneous inhibitory postsynaptic events. Evaluating those neurons that project to the parabrachial nucleus (PBN), a nucleus with multiple homeostatic roles, we found them to be hypoexcitable in DS. Alterations in BNST to brainstem projections could be implicated in comorbid aspects of DS, including respiratory dysfunction and sudden death.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in refractory epilepsy patients. Accumulating evidence from recent human studies and animal models suggests that seizure-related respiratory arrest may be important for initiating cardiorespiratory arrest and death. Prior evidence suggests that apnea onset can coincide with seizure spread to the amygdala and that stimulation of the amygdala can reliably induce apneas in epilepsy patients, potentially implicating amygdalar regions in seizure-related respiratory arrest and subsequent postictal hypoventilation and cardiorespiratory death. This study aimed to determine if an extended amygdalar structure, the dorsal bed nucleus of the stria terminalis (dBNST), is involved in seizure-induced respiratory arrest (S-IRA) and death using DBA/1 mice, a mouse strain which has audiogenic seizures (AGS) and a high incidence of postictal respiratory arrest and death. The presence of S-IRA significantly increased c-Fos expression in the dBNST of DBA/1 mice. Furthermore, disruption of synaptic output from the dBNST via viral-induced tetanus neurotoxin (TeNT) significantly improved survival following S-IRA in DBA/1 mice without affecting baseline breathing or hypercapnic (HCVR) and hypoxic ventilatory response (HVR). This disruption in the dBNST resulted in changes to the balance of excitatory/inhibitory (E/I) synaptic events in the downstream brainstem regions of the lateral parabrachial nucleus (PBN) and the periaqueductal gray (PAG). These findings suggest that the dBNST is a potential subcortical forebrain site necessary for the mediation of S-IRA, potentially through its outputs to brainstem respiratory regions.

Objective: Dravet syndrome (DS) is a developmental and epileptic encephalopathy with an increased incidence of sudden death. Evidence of interictal breathing deficits in DS suggests that alterations in subcortical projections to brainstem nuclei may exist, which might be driving comorbidities in DS. The aim of this study was to determine if a subcortical structure, the bed nucleus of the stria terminalis (BNST) in the extended amygdala, is activated by seizures, exhibits changes in excitability, and expresses any alterations in neurons projecting to a brainstem nucleus associated with respiration, stress response and homeostasis.Methods: Experiments were conducted using F1 mice generated by breeding 129.Scn1a +/− mice with wildtype C57BL/6J mice. Immunohistochemistry was performed to quantify neuronal c-fos activation in DS mice after observed spontaneous seizures. Whole cell patch clamp and current clamp electrophysiology recordings were conducted to evaluate changes in intrinsic and synaptic excitability in the BNST.Results: Spontaneous seizures in DS mice significantly enhanced neuronal c-fos expression in the BNST. Further, the BNST had altered AMPA/NMDA postsynaptic receptor composition and showed changes in spontaneous neurotransmission, with greater excitation and decreased inhibition. BNST to parabrachial nucleus (PBN) projection neurons exhibited intrinsic excitability in wildtype mice, while these projection neurons were hypoexcitable in DS mice. Significance:The findings suggest that there is altered excitability in neurons of the BNST, including BNST to PBN projection neurons, in DS mice. These alterations could potentially be driving comorbid aspects of DS outside of seizures, including respiratory dysfunction and sudden death.

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