Rationale:
Sudden Unexpected Death in Epilepsy (SUDEP) is the most common cause of death in refractory epilepsy patients, yet SUDEP underlying mechanisms are unclear. Seizure-related respiratory dysfunction following cardiac failure has been observed in SUDEP patients, and identifying the neurocircuitry involved would provide insight into potential neuromodulatory targets. Our lab has shown that the Bed Nucleus of the Stria Terminalis (BNST) increased c-fos activation after seizures with respiratory failure, and blocking BNST improves seizure mortality. Therefore, the BNST may play a critical role in seizure-related respiratory dysfunction through modulation of respiratory brainstem circuitry.
Methods:
Male and female DBA1 seizure primed mice were used in an audiogenic seizure model (AGS). Fiber photometry (FP) was used to monitor BNST calcium-dependent neural activity in vivo, and neural activity quantified by the change in fluorescence normalized to an isosbestic control (dF/F). BNST activity was recorded during hypercapnia (5 and 7% Co2) and hypoxia (8% O2) concurrently with whole body plethysmography (WBP) to measure hypercapnic and hypoxic ventilatory response (HCVR and HVR), and during seizure induced respiratory arrest. A virally mediated Targeted Recombination in Active Population (TRAP) strategy was used to selectively target BNST neurons active during AGS induced respiratory arrest. The TRAP virus was paired with a cre-dependent viral vector expressing fluorescent synaptophysin to identify functional projections of seizure-activated BNST neurons. The TRAP virus was also paired with a cre-dependent channel rhodopsin to optogenetically modulate BNST terminals on end terminals of neurons in brainstem respiratory regions . Intrinsic excitability of seizure-activated BNST neurons was recorded compared to non-activated neurons using whole-cell patch clamp.
Results:
During gas exposure, mice had the expected HCVR (p=0.001) and HVR (p=0.0027), respiratory response was correlated to FP. As measured by FP, BNST in vivo activity decreased during hypercapnia but showed no response during hypoxia (n=4 mice, 3 trials/mouse). BNST activity was drastically increased during AGS preceding respiratory failure and death. BNST activity was drastically increased during AGS preceding respiratory failure and death. Seizure-activated BNST neurons formed synaptophysin tagged synapses in the respiratory brain regions of nucleus tractus solitarius (NTS), parabrachial nucleus of the pons (PBN), and periaqueduct gray (PAG) (n=4 mice). Seizure-activated BNST neurons had higher resting membrane potential compared to non-activated BNST neurons (p=0.03). Preliminary data shows that optogenetic stimulation of BNST seizure-activated terminals on NTS neurons did not produce either inhibitory or excitatory currents in NTS neurons (n=1 mouse, 5 cells), but work characterizing other respiratory brainstem regions is still ongoing.
Conclusions:
Overall, our work suggests that seizures activate BNST neurons that project to respiratory brainstem regions, and that BNST activity is altered by ventilatory challenges, suggesting this maybe a potential modulation target for SUDEP.
Funding: NIH NINDS R01NS133169