GluA3 role in shaping synaptic plasticity and neuronal structure

AMPA-type glutamate receptors (AMPARs) are composed of GluA1-GluA4 subunits, which ultimately mediate fast synaptic transmission. Among AMPARs subunits, the physiopathological role of GluA3 remains mostly unveiled. Here, to get a grasp on its function, we downregulated GluA3 through an shRNA based approach in both an in vitro and in vivo model. Primary hippocampal rat neurons were infected with either shRNA-GRIA3 or a scrambleRNA at DIV10, and at DIV20 we performed biochemical and molecular evaluations. GluA3 downregulation selectively reduced the postsynaptic enrichment of GluN2B-containing NMDA receptors, which suggests lateralization to extrasynaptic sites. Live calcium imaging revealed a significant reduction in calcium transients, which is independent of glutamate release as indicated by glutamate imaging analyses. In addition, high frequency single spine stimulation led to aberrant calcium accumulation at dendritic spines. Moreover, dendritic arbor complexity was significantly reduced. Transcriptomic analysis via RNA-seq showed the disregulated expression of key activity-related genes (e.g., CAMK2A, MAPK1), as well as pathways related to protein trafficking and neuronal development. Notably, the impact of GluA3 silencing was time-dependent, with earlier silencing (DIV3) producing stronger effects than DIV10, while later silencing (DIV14) was no longer detrimental. To further validate the contribution of GluA3 to brain function, we are currently moving to an in vivo model. Considering the time-dependent role of GluA3 in primary hippocampal neurons, we are downregulating its expression in the dorsal CA1 (dCA1) region of the hippocampus in both adolescent and adult mice. These animals will undergo behavioral assessments combined with fiber photometry recordings, as well as dendritic arborization analyses. Altogether, our findings reveal that GluA3-containing AMPARs are key modulators of postsynaptic calcium signaling and neuronal differentiation; they orchestrate NMDAR localization and activation of the CaMKII-ERK pathway. Additionally, in vivo investigations will provide a substantial contribution to the understanding of its role in complex brain functions.