Authors :
Presenting Author: Paula Sullivan, BS – UC Davis
Shruti Shah, BS – UC Davis
Ammara Rehman, BS – UC Davis
Mandar Patil, MS – UC Davis
Roy Ben-Shalom, PhD – University of California, Davis
Caren Armstrong, MD PhD – UC Davis
Rationale:
KCNT1 encodes a sodium-activated potassium channel involved in regulating neuronal excitability. Pathogenic variants cause autosomal dominant nocturnal frontal lobe epilepsy or a severe developmental and epileptic encephalopathy (DEE) with poor genotype-phenotype correlation even within families. No targeted treatments currently exist for this severe disorder. Here, we used high-density microelectrode array (HD-MEA) chips to record activity from wild-type (WT) and mice with a KCNT1 gain of function mutation. The HD-MEA is an in vitro system used to evaluate native circuit architecture. Developing a robust in vitro assay for KCNT1 variants on the HD-MEA could allow for both patient and variant-specific effects on neuronal network activity to be determined, as well as for preemptive evaluation of specific pharmacologic agents for those patients. Methods:
Dissociated neurons from p0-p2 WT or heterozygous KCNT1 G269S variant (KCNT1+/G269S, equivalent to human G288S gain of function variant) mouse pups were cultured directly on Maxwell HD-MEA chips and network activity evaluated at div 7, 14, 21, and 28. To confirm findings in cortical columns, acute brain slices of WT or KCNT1+/G269S motor cortex were plated and recorded on the HD-MEA chips and activity evaluated. To facilitate the mechanistic understanding and future in silico modeling of different variants in KCNT1+/G269S, patch clamp recordings of layer V pyramidal cells were performed in both WT and KCNT1+/G269S mice and intrinsic properties evaluated.
Results:
Dissociated cultures from KCNT1+/G269S had increased baseline network activity (4.5Hz firing rate at day 28 vs 2.0Hz in WT) and increased frequency of bursting compared with WT cultures (120 vs 60 bursts in WT). Preliminary slice experiments confirm increased firing rate in KCNT1+/G269S compared with WT (0.75 vs 0.6Hz in WT). When comparing layer V pyramidal cells from WT and KCNT1+/G269S, input resistance was lower (91 vs 169Mohm in WT), sag was smaller (-0.5 vs -7.4mV in WT), rheobase was higher (250pA vs 100pA in WT), action potential peak was higher (73 vs 33mV in WT), and afterhyperpolarization smaller (-11 vs -15mV in WT) in KCNT1+/G269S mice.
Conclusions:
Taken together, the results indicate specific abnormalities in cortical neurons of KCNT1+/G269S mice which underlie differences in activity in both dissociated neurons and cortical slices on the HD-MEA. In summary, these experiments provide preliminary evidence that an ex vivo model of KNCT1 variants can be used to probe excitability on the HD-MEA chip, highlighting the potential to use HD-MEA in model systems for developing future tailored treatment strategies for KCNT1.
Funding: UC Davis Department of Neurology