Small Fluorescent Probes Show iGluRs are in the Synapses of Transfected Neurons under Basal Conditions

Abstract

We imaged transiently transfected live glutamate receptors (iGluRs), namely AMPAR and NMDAR, with super-resolution in three-dimension, labeled with differently sized fluorophores. We used small organic fluorescent dyes (∼ 4 nm), small quantum dots (sQD, ∼10 nm in diameter), or big (commercial) quantum dots (bQD, ∼ 20 nm in diameter). The iGluRs were imaged along with a synaptic protein, Homer1c, using fiducial markers that eliminate stage drift, and under conditions where the receptor cross-linking is monitored or eliminated. With small probes under basal conditions, we find that both AMPAR and NMDAR are predominantly within the synapse (∼84-95%), in contrast to bQDs, which show only 5-10% within the synapse. The results can likely be explained by cross-linking and inhibited mobility of the iGluRs labeled with bQDs, but not with sQDs or organic fluorophores. Hence, this data indicates that there is not a highly diffusible pool of extrasynaptic iGluRs in the plasma membrane, as has been widely reported. In addition, within a synapse, the distribution is non-homogeneous, perhaps due to the non-homogeneous distribution of glutamate.