Memory formation is thought to depend on the recruitment of specific neuronal ensembles, termed engrams, together with activity-dependent synaptic plasticity. N6-methyladenosine (m6A), the most abundant internal modification of mammalian messenger RNA, has been implicated in neuronal function and memory; however, its contribution to the formation and plasticity of defined memory-associated neuronal ensembles remains unclear.
To investigate the role of m6A in these processes, we pharmacologically inhibited METTL3, the major m6A methyltransferase, using the selective catalytic inhibitor STM2457. This approach reduces METTL3-dependent m6A deposition while avoiding direct depletion of the METTL3 protein. Activity-dependent neuronal recruitment was monitored in primary hippocampal neurons using the robust activity marking (RAM) system. In parallel, single-spine structural plasticity was examined in organotypic hippocampal slices using two-photon glutamate uncaging.
STM2457-treated primary hippocampal neurons exhibited increased RAM labelling under unstimulated conditions, indicating altered basal activity-dependent gene expression or neuronal ensemble recruitment. Following network activation, which typically boosts the number of RAM-positive neurons, a limited increase was observed in STM2457-treated neurons. Using glutamate uncaging protocol which induces spine enlargement in organotypic hippocampal slices, we found that STM2457 treatment impaired the sustained spine enlargement, indicating a failure to express structural long-term potentiation at individual dendritic spines.
Together, our data provided novel insights into the role of METTL3-dependent m6A methylation in activity-dependent recruitment of hippocampal neuronal ensembles, as well as for sustained structural plasticity at the single-spine level. The unexpected increase in basal RAM labelling, combined with impaired structural long-term potentiation, further suggests that m6A plays a critical role in maintaining the distinction between baseline neuronal activity and synaptic strengthening during memory encoding.