Abstract:
Aims: The postsynaptic density (PSD) is a dense region of protein that lies beneath the postsynaptic membrane of excitatory glutamatergic synapses. Understanding the molecular architecture of the PSD scaffolding proteins may reveal whether structural changes in architecture are correlated with synaptic plasticity. Of particular interest are two N-terminal isoforms of SAP97 (alpha and betaSAP97) that are known to differentially regulate synaptic plasticity, potentially by localising surface glutamate receptors to different synaptic compartments (Li et al., 2011; Waites et al., 2009). However, the ~200 nm resolution limit of traditional optical microscopy has greatly complicated detailed study of protein arrangements within the densely packed PSD. Methods: We have applied a single molecule localisation method of super resolution imaging known as dSTORM (Baddeley et al., 2011) to image the distribution of PSD proteins in cultured rat hippocampal neurons. Results: Super resolution imaging of Homer, Bassoon, Shank, PSD-95, and alpha-actinin reveals physically discernible distributions that are not resolvable with conventional optical microscopy. A comparison between neurons transiently overexpressing alpha or betaSAP97 isoforms shows differences in synapse size, with alphaSAP97 expressing PSDs larger than those expressing alphaSAP97 (median: alpha = 0.18um 2 ; beta = 0.10um 2 ). We also observe differences in the distribution of surface AMPA receptors between the two populations. Conclusions: Our data reveal that different isoforms of the same PSD protein can differentially affect synaptic architecture, which may dictate diverse functional roles in synaptic plasticity.
