Learning differentially increases the synaptic levels of AMPA receptor subunits (GluA1‐GluA4) in the rodent hippocampus: a single‐synapse approach

Abstract Background Decades of experimental work support the idea that synapses are the anatomical substrate for experience‐dependent plasticity in the brain. Changes in the synaptic strength underlie learning and memory via the accumulation of glutamate AMPA receptors (AMPAR) at the surface of excitatory synapses. AMPAR are tetrameric receptors constituted by multiple combination of four subunits: GluA1 GluA2, GluA3, and GluA4. However, the relative contribution of each AMPAR subunit on learning and memory is relatively unexplored. Method Here, we introduce Fluorescence Analysis of Single‐Synapse Potentiation induced by Learning (FASS‐PiL), a flow cytometry‐based method to quantify surface levels of all four GluA‐AMPAR subunits, in parallel, in isolated synaptosomes after a learning episode in rodents. We evaluated surface levels of all four AMPAR subunits in synaptosomes of mice trained in a learning protocol commonly used for studying episodic memory in the hippocampus (e.g., Object Location Memory (OLM) and Object Recognition Memory (ORM) tasks). Briefly, after exploring novel objects in a novel environment for 10 min, mice were returned to their home cage for 60 min. After hippocampus dissection, synaptosome isolation, and immunostaining, samples were analyzed via flow cytometry. Result We first demonstrated that FASS‐PiL is a simple and sensitive method to track all four GluAs at the synaptosome surface (each GluA subunit was paired with the presynaptic marker Neurexin‐1beta to focus on synaptosome particles containing both pre‐ and postsynaptic compartments). Notably, by profiling hundreds of events, our data showed that the OLM training increases the number of hippocampal synaptosomes expressing high levels of GluA1 and GluA2, but not GluA3 and GluA4 (vs control animals). Conclusion Our results indicate that plasticity‐related mechanisms underlying learning are AMPA‐subunit‐specific in hippocampal synapses. Our approach could provide the basis for protocols to study behavioral‐relevant mechanisms of plasticity directly at the synapse, with single‐event resolution.