Relative roles of HCN4 and synaptic excitation in pyramidal neuron firing rates in a hyperactive Rheb-mTOR condition

Abstract

Objective: De novo somatic mutations in mTOR pathway genes during fetal development lead to focal malformation of cortical development (FMCD) and epilepsy. This FMCD is characterized by misplacement of enlarged pyramidal neurons displaying increased mTOR activity. Whether these neurons display enhanced excitability has remained an open question. Several lines of evidence in experimental murine FMCD suggest that FMCD neurons display a complex set of alterations, including dendrite hypertrophy associated with decreased rheobase but opposing changes in excitability due to the abnormal expression of hyperpolarization-activated cyclic nucleotide-gated isoform 4 (HCN4) channels. However, there is very little information on synaptic excitatory activity and how these different alterations are integrated to shape their firing rate, particularly at the onset of seizures at approximately postnatal day 21 (P21) and after the establishment of recurrent seizures (at P28). Methods: To address these questions, we generated FMCD by using in utero electroporation to express a constitutively active Rheb (RhebS16H), an mTOR canonical activator, in layer 2/3 cortical pyramidal neurons, and assessed its effects on pyramidal cell excitability at P21 and P28 using acute brain slices in vitro. Results: At both time points, RhebS16H neurons exhibited cytomegaly along with increased dendritic complexity and abnormal HCN current expression. However, they displayed distinct changes in the properties of spines and excitatory postsynaptic currents (EPSCs). Specifically, there was an increase in EPSC amplitude at both ages, but whereas no change in frequency was observed at P21, a decrease in frequency was seen at P28. Experimental data-driven multicompartment single-cell computational models investigating dysmorphic neuronal excitability at P28 predicted that HCN4 channels, rather than excitatory synaptic inputs, predominantly contribute to the firing of RhebS16H neurons. Significance: Overall, these experimental and computational findings advance our understanding of the mechanisms of excitability of RhebS16H neurons, with implications for FMCD-related seizure activity due to mTOR hyperactivity.