Hyperexcitability in human MECP2 null neuronal networks manifests as calcium-dependent reverberating super bursts

Abstract

Rett syndrome (RTT) patients show abnormal developmental trajectories including loss of language and repetitive hand movements but also have signs of cortical hyperexcitability such as seizures. RTT is predominantly caused by mutations in MECP2 and can be modelled in vitro using human stem cell-derived neurons. MECP2 null excitatory neurons are smaller in soma size and have reduced synaptic connectivity but are also hyperexcitable, due to higher input resistance, which increases the chance to evoke action potentials with a given depolarized current. Few studies examine how single neuron activity integrates into neuronal networks during human development. Paradoxically, networks of MECP2 null neurons show a decrease in the frequency of bursting patterns consistent with synaptic hypoconnectivity, but no hyperexcitable network events have been reported. Here, we show that MECP2 null neurons have an increase in the frequency of a network event described as reverberating super bursts (RSBs) relative to isogenic controls. RSBs can be mistakenly called as a single long duration burst by standard burst detection algorithms. However, close examination revealed an initial large amplitude network burst followed by high frequency repetitive low amplitude mini-bursts. Using a custom burst detection algorithm, we unfolded the multi-burst structure of RSBs revealing that MECP2 null networks increased the total number of bursts relative to isogenic controls. Application of the Ca2+ chelator EGTA-AM selectively eliminated RSBs and rescued the network burst phenotype relative to the isogenic controls. Our results indicate that during early development, MECP2 null neurons are hyperexcitable and produce hyperexcitable networks. This may predispose them to the emergence of hyper-synchronic states that potentially translate into seizures. Network hyperexcitability is dependent on asynchronous neurotransmitter release driven by pre-synaptic Ca2+ and can be rescued by EGTA-AM to restore typical network dynamics. HIGHLIGHTSO_LIReverberating super-bursts (RSBs) follow a stereotypic form of a large initial network burst followed by several smaller amplitude high-frequency mini-bursts. C_LIO_LIRSBs occur more often in MECP2 null excitatory networks. C_LIO_LIMECP2 null excitatory networks with increased RSBs show a hyperexcitable network burst phenotype relative to isogenic controls. C_LIO_LIThe calcium chelator, EGTA-AM, decreases RSBs and rescues the dynamics of MECP2 null hyperexcitable networks. C_LI