Abstracts

This abstract has been invited to present during the Basic Science Poster Highlights poster session

Rationale: Pathogenic gain of function mutations in the SCN8A (Na_v1.6) sodium channel can result in developmental epileptic encephalopathy-13 (DEE-13). DEE-13 in its severest forms is characterized by epileptic encephalopathy and intractable seizures, with seizure onset in early infancy. Na_v1.6 is highly expressed prenatally, suggesting that the impact of this mutation on early neural network formation is critical to disease pathogenesis. However, modeling human prenatal neural network development and activity is challenging. One promising new platform is human brain organoids (or simply organoids), in vitro 3D brain-like structures created from either human embryonic or induced pluripotent stem cells (hESCs or hiPSCs). Organoids recapitulate many structural features of the human brain and are ideally suited to model early brain development. We have generated a “fusion” organoid model in which excitatory neuron-predominant cortex-like (Cx) or hippocampal-like (Hc), and inhibitory interneuron-predominant ganglionic eminence-like (GE) populations integrate, yielding an ideal platform for modeling neural circuit assembly. The fusion organoids contain proportions of excitatory/inhibitory neurons and generate characteristic neural oscillations at frequencies seen in typical cortex and hippocampus in vivo (Samarasinghe et. al., Nature Neuroscience, https://rdcu.be/cMksf)._x000D_
Methods: Brain organoids were generated from p.SCN8A mutant or isogenic control hiPSCs using established laboratory protocols. Organoids were fused at age day 56.  Fused organoids were infected with AAV-1-GcAMP at age day ~110 and subject to two-photon microscopy at age ~124 days. Ca2+ data were analyzed using Matlab algorithms. Extracellular recordings were performed on day 120-130 fused organoids and oscillatory activity was quantified using Igor Pro. Immunohistochemical (IHC) analyses were performed using standard laboratory protocols to establish cell-type expression and organoid cytoarchitecture. _x000D_
Results: DEE-13 cortex fusion organoids harboring p.SCN8A mutations showed evidence of overt hyperexcitatbility by both Ca2+ indicator imaging and extracellular recordings of local field potentials (LFPs). In contrast, hippocampal organoids harboring the identical pathogenic variant did not demonstrate overt hyperexcitatbility, but unlike matched isogenic controls, the mutants were unable to generate sharp wave ripple complexes and did not generate the stereotyped patterns of theta-gamma phase amplitude coupling seen in controls. IHC of DEE-13 hippocampal organoids revealed a relative reduction of astrocytes and GAD65/SST+ interneurons. Similar changes were not seen in cortex. _x000D_
Conclusions: These data suggest that (1) hippocampus and cortex fusion organoids generate complex and distinct network activities, (2) that p.SCN8A variants differentially impact cortex versus hippocampus with cortex demonstrating overt hyperexcitability and hippocampus showing changes in networks associated with learning and memory , and (3) that hippocampal neural network changes may be a result of altered astrocytic and interneuron expression._x000D_
Funding: This work was supported by NINDS K08NS119747, Simons-BTI 717153, and Cure 20204012 (PI, Samarasinghe)