Calcium-Dependent Hyperexcitability in Human Stem Cell-Derived Rett Syndrome Neuronal Networks

Background: Mutations in predominantly cause Rett syndrome and can be modeled in vitro using human stem cell-derived neurons. Patients with Rett syndrome have signs of cortical hyperexcitability, such as seizures. Human stem cell-derived null excitatory neurons have smaller soma size and reduced synaptic connectivity but are also hyperexcitable due to higher input resistance. Paradoxically, networks of null neurons show a decrease in the frequency of network bursts consistent with a hypoconnectivity phenotype. Here, we examine this issue.

Methods: We reanalyzed multielectrode array data from 3 isogenic cell line pairs recorded over 6 weeks ( = 144). We used a custom burst detection algorithm to analyze network events and isolated a phenomenon that we termed reverberating super bursts (RSBs). To probe potential mechanisms of RSBs, we conducted pharmacological manipulations using bicuculline, EGTA-AM, and DMSO on 1 cell line ( = 34).

Results: RSBs, often misidentified as single long-duration bursts, consisted of a large-amplitude initial burst followed by several high-frequency, low-amplitude minibursts. Our analysis revealed that null networks exhibited increased frequency of RSBs, which produced increased bursts compared with isogenic controls. Bicuculline or DMSO treatment did not affect RSBs. EGTA-AM selectively eliminated RSBs and rescued network burst dynamics.

Conclusions: During early development, null neurons are hyperexcitable and produce hyperexcitable networks. This may predispose them to the emergence of hypersynchronic states that potentially translate into seizures. Network hyperexcitability depends on asynchronous neurotransmitter release that is likely driven by presynaptic Ca and can be rescued by EGTA-AM to restore typical network dynamics.