Abstracts

Rationale: Dravet syndrome is a developmental and epileptic encephalopathy characterized by seizures, developmental delays, gait abnormalities and elevated risk of sudden unexpected death in epilepsy. Most patient cases are caused by de novo loss-of-function mutations in the gene SCN1A, causing a haploinsufficiency of the alpha subunit of the voltage-gated sodium channel Na_V1.1. Within the brain, decreased Na_V1.1 function is hypothesized to reduce GABAergic inhibitory neurotransmission, driving neuronal network hyperexcitability and subsequent pathology. We have developed a human in vitro model of Dravet syndrome using differentiated neurons from human induced-pluripotent stem cells (iPSCs), plating neuronal cultures on high-density microelectrode arrays (HD-MEAs) for network, single cell, and subcellular resolution recordings. This model enables studies of Dravet syndrome pathophysiology from human neurons in vitro and evaluation of therapeutics on relevant electrophysiological endpoints.


Methods: GABAergic cortical neurons (SCN1A^+/- and SCN1A^+/+), glutamatergic neurons and astrocytes were differentiated from human iPSCs and plated on HD-MEAs. Electrophysiological recordings were performed weekly over a span of 3-months. Each HD-MEA contained 26,400 electrodes, with 17.5 µm resolution. Arrays were scanned for neuronal activity and recording performed from detected neurons. Analysis of the recordings measured features of single neuron firing, network activity, and network bursting.


Results: Over time the cultures developed increased spiking activity and synchronous network bursting. Recordings were processed through a spike sorting pipeline for curation of single unit activity and to assess the effects of pharmacological treatments. We found that SCN1A^+/- exhibited increased network synchrony and excitability compared with SCN1A^+/+ culture. Potentiation of Na_V1.1 reduced network excitability through increasing spike rate of putative GABAergic neurons.


Conclusions: These results demonstrated that human neurons assayed in vitro across months recapitulate increased network synchronous firing observed in the Dravet syndrome patients. Potentiation of Na_V1.1 in human iPSC-derived neurons resulted in decreased firing synchrony in neuronal networks through increased GABAergic neuron activity. Our results support the use of human neurons and HD-MEAs as viable high-throughput electrophysiological platform to enable therapeutic discovery.


Funding: NA