Probing synaptic distribution and arrangement of native surface AMPAR in mouse brain slices with 3D super-resolution microscopy

AMPARs are ion channels gated by the neurotransmitter Glutamate and are found in majority of excitatory neurons. Their numbers, arrangement, and diffusion on the cell-membrane of neurons act as important indicators of synaptic plasticity. Single molecule localization microscopy (SMLM) techniques have been used to extensively study the distribution of surface AMPARs and their clustering in synaptic nano-domains. However, most of these studies have been limited to dissociated neuronal cultures and hence lack the ability to probe surface AMPARs in brain tissue with intact circuitry, and as a function of aging and pathology. In this work, we utilize a recently developed small probe, CAM2, to specifically label native, surface AMPARs (specifically, GluA2-4 subunits) in mouse brain slices. This approach avoids artefacts arising due to protein overexpression and modification. We use a custom-built microscope to perform 2-color 3D dSTORM imaging of AMPARs as well as a post-synaptic density protein Homer1. By reducing light scattering caused by refractive-index mismatch, correcting for instrument-induced aberrations using a deformable mirror and reducing out-of-focus fluorescence background by using HILO illumination, we are able to achieve <20 nm (lateral) and 90 nm (axial) localization precision in 30 μm thick, fixed Thy1-YFP-H mouse brain slices. This allows us to visualize AMPAR clustering in synaptic nanodomains and their distribution in synaptic and extra-synaptic sites in the cortex, as well as the more densely crowded hippocampus. Addition of ratiometric imaging capability to our microscope will allow us to look at one more color, such as a pre-synaptic density protein, Bassoon, improving the accuracy of AMPAR classification as synaptic vs extra-synaptic.