Chronic subthreshold intermittent theta burst stimulation promotes structural axon initial segment plasticity in cortical neurons

Repetitive transcranial magnetic stimulation (rTMS) is used widely in neuroscience to study and alter neural plasticity. The cellular mechanisms underlying the effect of rTMS on the brain remain unclear but is primarily thought to act via activity-dependent synaptic plasticity mechanisms. Here we investigated whether chronic repetitive magnetic stimulation in vitro and in vivo can induce another form of activity-dependent neural plasticity, axon initial segment (AIS) plasticity. Cortical neurons isolated from postnatal wild-type mice were stimulated with 6 hours of sham, repetitive magnetic stimulation in the form of intermittent theta-burst stimulation (iTBS), or 15 mM potassium chloride, with changes to AIS location and length measured +0 hours and +24 hours post-stimulation. In addition, adult transgenic mice expressing green fluorescent protein at the AIS received daily sham or iTBS over the primary motor cortices for 7 consecutive days and processed for microscopy 3 hours after the last stimulation. Analysis of neurons stimulated in vitro showed that chronic iTBS caused bidirectional and time-dependent shifts to the AIS position relative to the soma and a delayed shortening of the AIS length at +24 hours. In the adult mice, 7 consecutive days of daily iTBS decreased AIS lengths in layers 2/3 and 5 pyramidal neurons. Our findings provide in vitro and in vivo evidence that rTMS induces neuronal plasticity outside of the synapse, which may contribute to the long-lasting effect of rTMS on the brain with repeated stimulation protocols.